Secondary literature sources for the Int_alpha domain

For each of the hand selected primary papers associated with the Int_alpha domain, 100 neighbouring papers are retrieved from PubMed. If one of these neighbouring papers is referenced by more than two primary papers, it is shown in the table below.

Laminin-binding integrins in tumor progression and metastasis.

Ziober BL, Lin CS, Kramer RH
Semin Cancer Biol. 1996; 7: 119-28

Interactions between tumor cells and extracellular matrix occur at several points during the metastatic cascade. Epithelial tumors, which represent nearly 90% of human neoplasia, must invade their underlying basement membrane to enter the interstitial stroma. For distant metastasis, malignant cells must penetrate basement membranes to gain access to blood vessels and organ parenchyma. Integrin receptors that bind to multiple laminin isoforms appear to mediate tumor cell adhesion to basement membranes before and during invasion. It is notable that changes in several laminin-binding integrins occur during tumor progression. These changes may include increased or decreased expression, or changes in distribution from a polarized to a dispersed pattern. Integrins not only mediate cell adhesion and motility but also transduce important downstream signaling events that regulate cell growth, survival, and gene expression. During tumor progression, the development of variant cells with changes in integrin expression and the associated signaling pathways could result in cells with a highly invasive and metastatic phenotype.

Modulation of cell proliferation by the integrin cytoplasmic domain.

Zheng DQ, Fornaro M, Bofetiado CJ, Tallini G, Bosari S, Languino LR
Kidney Int. 1997; 51: 1434-40

Integrin adhesion receptors modulate cell functions, including cell proliferation and survival. The beta 1C integrin, an alternatively spliced form of beta 1A, containing a unique cytoplasmic domain sequence, inhibits cell growth in vitro. In vivo, the expression of beta 1C correlates with a benign, nonproliferative phenotype in epithelial cells. The studies discussed in this article indicate that modulation of cell proliferation, in normal or pathological conditions, might be achieved by the regulated expression of variant integrin subunits.

[Expression of alpha 1-4 integrins in hypertrophic scar fibroblasts]

Zhao Y, He Q, Niu X
Zhonghua Zheng Xing Wai Ke Za Zhi. 2000; 16: 37-9

OBJECTIVE: To determine the expression levels of alpha 1-4 integrins in hypertrophic scar fibroblasts and investigate the role that alpha 1-4 integrins may play in the process of hypertrophic scar formation. METHODS: The membrane protein was extracted from hypertrophic scar and normal skin tissues. The content of alpha 1-4 integrins of the membrane protein was detected by immunoblotting method. The locations of alpha 1-4 integrin expression were examined by immunohistochemical stain. RESULTS: 1. Higher concentration of alpha 1-4 integrins was identified in the membrane protein extracted from hypertrophic scar than from normal skin. 2. In normal skin, the expression of alpha 1, alpha 2 and alpha 3 integrins located on the epidermal cell surface but not on the dermal fibroblast surface. In the hypertrophic scar, not only did the epidermal cell surface express alpha 1-3 integrins, but also the fibroblast surface expressed alpha 1-4 integrins. CONCLUSIONS: The expressions of hypertrophic scar fibroblast surface integrins are subjected not only to cell-extracellular matrix adhesion, but also to intracellular/extracellular signal transduction. Integrins may play an important role in regulating much of fibroblast behavior, including growth, differentiation, phenotype, and in scar tissue remodelling.

Role of focal adhesion kinase in signaling by the extracellular matrix.

Zhao JH, Guan JL
Prog Mol Subcell Biol. 2000; 25: 37-55

Integrin activation by R-ras.

Zhang Z, Vuori K, Wang H, Reed JC, Ruoslahti E
Cell. 1996; 85: 61-9

Expression of a constitutively active R-ras converted two cell lines that grow in suspension into highly adherent cells. There was little change in cell surface expression of integrins, but attachment to surfaces coated with the integrin ligands was greatly enhanced. Cells transfected with activated R-ras bound integrin ligands from solution with higher affinities and assembled severalfold more fibronectin matrix than control transfectants. Introduction of a dominant negative R-ras into adherent cells reduced the adhesiveness of the cells, indicating that endogenous R-ras can control the ligand-binding activity of integrins. These results provide a mechanism for the modulation of integrin ligand-binding activity as well as novel function for R-ras.

Alpha 3 beta 1 adhesion to laminin-5 and invasin: critical and differential role of integrin residues clustered at the boundary between alpha 3 N-terminal repeats 2 and 3.

Zhang XP, Puzon-McLaughlin W, Irie A, Kovach N, Prokopishyn NL, Laferte S, Takeuchi K, Tsuji T, Takada Y
Biochemistry. 1999; 38: 14424-31

Integrin/ligand interaction is a therapeutic target for many diseases. We previously reported that residues critical for ligand binding are clustered in N-terminal repeat 3 (in the predicted 2-3 loop) of alpha 4, alpha 5 and alpha IIb. Here we have localized residues critical for ligand binding in the alpha 3 subunit of integrin alpha 3 beta 1 with distinct ligand specificity (laminin-5). We identified an alpha 3 epitope common to several function-blocking anti-alpha 3 antibodies at the boundary between repeats 1 and 2 (residues 75-80). We found that swapping the predicted 4-1 loop (residues 153-165) at the boundary between repeats 2 and 3 with the corresponding alpha 4 sequence and mutating Thr-162 and Gly-163 residues in this predicted loop block laminin-5 binding. Thr-162 and Gly-163 and the antibody epitope are separated in the primary structure; however, they are close to each other in the proposed beta-propeller model. Mutating residues recently reported to block (Tyr-186 and Trp-188) or enhance (Asp-122) laminin-5 binding to alpha 3 beta 1 [Krukonis, E. S., Dersch, P., Eble, J. A., and Isberg, R. R.(1998) J. Biol. Chem. 273, 31837-31843] did not affect laminin-5 binding under the assay conditions used. Thr-162 and Gly-163 are not critical for adhesion to invasin, indicating that laminin-5 and invasin may use different recognition mechanisms, and that mutation of Thr-162 and Gly-163 does not drastically affect the integrity of alpha 3 beta 1. These results suggest that residues critical for ligand binding may be similarly (but not identically) located in repeat 3 of the alpha subunit regardless of ligand specificity.

Amino acid residues in the PSI domain and cysteine-rich repeats of the integrin beta2 subunit that restrain activation of the integrin alpha(X)beta(2).

Zang Q, Springer TA
J Biol Chem. 2001; 276: 6922-9

The leukocyte integrin alpha(X)beta(2) (p150,95) recognizes the iC3b complement fragment and functions as the complement receptor type 4. alpha(X)beta(2) is more resistant to activation than other beta(2) integrins and is inactive in transfected cells. However, when human alpha(X) is paired with chicken or mouse beta(2), alpha(X)beta(2) is activated for binding to iC3b. Activating substitutions were mapped to individual residues or groups of residues in the N-terminal plexin/semaphorin/integrin (PSI) domain and C-terminal cysteine-rich repeats 2 and 3. These regions are linked by a long range disulfide bond. Substitutions in the PSI domain synergized with substitutions in the cysteine-rich repeats. Substitutions T4P, T22A, Q525S, and V526L gave full activation. Activation of binding to iC3b correlated with exposure of the CBR LFA-1/2 epitope in cysteine-rich repeat 3. The data suggest that the activating substitutions are present in an interface that restrains the human alpha(X)/human beta(2) integrin in the inactive state. The opening of this interface is linked to structural rearrangements in other domains that activate ligand binding.

The top of the inserted-like domain of the integrin lymphocyte function-associated antigen-1 beta subunit contacts the alpha subunit beta -propeller domain near beta-sheet 3.

Zang Q, Lu C, Huang C, Takagi J, Springer TA
J Biol Chem. 2000; 275: 22202-12

We find that monoclonal antibody YTA-1 recognizes an epitope formed by a combination of the integrin alpha(L) and beta(2) subunits of LFA-1. Using human/mouse chimeras of the alpha(L) and beta(2) subunits, we determined that YTA-1 binds to the predicted inserted (I)-like domain of the beta(2) subunit and the predicted beta-propeller domain of the alpha(L) subunit. Substitution into mouse LFA-1 of human residues Ser(302) and Arg(303) of the beta(2) subunit and Pro(78), Thr(79), Asp(80), Ile(365), and Asn(367) of the alpha(L) subunit is sufficient to completely reconstitute YTA-1 reactivity. Antibodies that bind to epitopes that are nearby in models of the I-like and beta-propeller domains compete with YTA-1 monoclonal antibody for binding. The predicted beta-propeller domain of integrin alpha subunits contains seven beta-sheets arranged like blades of a propeller around a pseudosymmetry axis. The antigenic residues cluster on the bottom of this domain in the 1-2 loop of blade 2, and on the side of the domain in beta-strand 4 of blade 3. The I domain is inserted between these blades on the top of the beta-propeller domain. The antigenic residues in the beta subunit localize to the top of the I-like domain near the putative Mg(2+) ion binding site. Thus, the I-like domain contacts the bottom or side of the beta-propeller domain near beta-sheets 2 and 3. YTA-1 preferentially reacts with activated LFA-1 and is a function-blocking antibody, suggesting that conformational movements occur near the interface it defines between the LFA-1 alpha and beta subunits.

Integrins as a primary signal transduction molecule regulating monocyte immediate-early gene induction.

Yurochko AD, Liu DY, Eierman D, Haskill S
Proc Natl Acad Sci U S A. 1992; 89: 9034-8

Integrins are cell surface receptors found on monocytes that facilitate adhesion to both cellular and extracellular substrates. These integrins are thought to be involved in the selective gene induction observed after monocyte adhesion to various extracellular matrices. To investigate this hypothesis, we stimulated monocytes with monoclonal antibodies to different integrin receptors to specifically mimic the integrin receptor-ligand interactions. Engagement of the common beta chain of the beta 1 subfamily of integrins resulted in expression of the inflammatory mediator genes, interleukin 1 beta, interleukin 1 receptor antagonist, and monocyte adherence-derived inflammatory gene 6 (MAD-6), whereas engagement of the common beta chain of the beta 2 family did not. Furthermore, to characterize integrin-mediated gene induction, we examined the ability of antibodies to the alpha chain of integrin receptors to regulate gene expression. Engagement of the very late antigen 4 (VLA-4) receptor resulted in induction of all the mediator genes. Receptor crosslinking was required because individual Fab fragments were unable to stimulate gene induction whereas the divalent F(ab')2 fragment and the whole IgG molecule could. Interleukin 1 beta secretion was dependent on the anti-integrin antibody used. Some antibodies required a second signal and, for others, direct engagement was sufficient for protein production. In conclusion, engagement of integrin receptors regulated the production of both inflammatory mediator mRNA and protein. These results suggest that integrin-dependent recognition and adherence may provide the key signals for initiation of the inflammatory response during monocyte diapedesis.

The integrin alpha(9)beta(1) binds to a novel recognition sequence (SVVYGLR) in the thrombin-cleaved amino-terminal fragment of osteopontin.

Yokosaki Y, Matsuura N, Sasaki T, Murakami I, Schneider H, Higashiyama S, Saitoh Y, Yamakido M, Taooka Y, Sheppard D
J Biol Chem. 1999; 274: 36328-34

The integrin alpha(9)beta(1) mediates cell adhesion to tenascin-C and VCAM-1 by binding to sequences distinct from the common integrin-recognition sequence, arginine-glycine-aspartic acid (RGD). A thrombin-cleaved NH(2)-terminal fragment of osteopontin containing the RGD sequence has recently been shown to also be a ligand for alpha(9)beta(1). In this report, we used site-directed mutagenesis and synthetic peptides to identify the alpha(9)beta(1) recognition sequence in osteopontin. alpha(9)-transfected SW480, Chinese hamster ovary, and L-cells adhered to a recombinant NH(2)-terminal osteopontin fragment in which the RGD site was mutated to RAA (nOPN-RAA). Adhesion was completely inhibited by anti-alpha(9) monoclonal antibody Y9A2, indicating the presence of a non-RGD alpha(9)beta(1) recognition sequence within this fragment. Alanine substitution mutagenesis of 13 additional conserved negatively charged amino acid residues in this fragment had no effect on alpha(9)beta(1)-mediated adhesion, but adhesion was dramatically inhibited by either alanine substitution or deletion of tyrosine 165. A synthetic peptide, SVVYGLR, corresponding to the sequence surrounding Tyr(165), blocked alpha(9)beta(1)-mediated adhesion to nOPN-RAA and exposed a ligand-binding-dependent epitope on the integrin beta(1) subunit on alpha(9)-transfected, but not on mock-transfected cells. These results demonstrate that the linear sequence SVVYGLR directly binds to alpha(9)beta(1) and is responsible for alpha(9)beta(1)-mediated cell adhesion to the NH(2)-terminal fragment of osteopontin.

Direct extracellular contact between integrin alpha(3)beta(1) and TM4SF protein CD151.

Yauch RL, Kazarov AR, Desai B, Lee RT, Hemler ME
J Biol Chem. 2000; 275: 9230-8

Previously we established that the alpha(3)beta(1) integrin shows stable, specific, and stoichiometric association with the TM4SF (tetraspannin) protein CD151. Here we used a membrane impermeable cross-linking agent to show a direct association between extracellular domains of alpha(3)beta(1) and CD151. The alpha(3)beta(1)-CD151 association site was then mapped using chimeric alpha(6)/alpha(3) integrins and CD151/NAG2 TM4SF proteins. Complex formation required an extracellular alpha(3) site (amino acids (aa) 570-705) not previously known to be involved in specific integrin contacts with other proteins and a region (aa 186-217) within the large extracellular loop of CD151. Notably, the anti-CD151 monoclonal antibody TS151r binding epitope, previously implicated in alpha(3) integrin association, was mapped to the same region of CD151 (aa 186-217). Finally, we demonstrated that both NH(2)- and COOH-terminal domains of CD151 are located on the inside of the plasma membrane, thus confirming a long suspected model of TM4SF protein topology.

The role of the amino-terminal beta-barrel domain of the alpha and beta subunits in the yeast F1-ATPase.

Yao B, Mueller DM
J Bioenerg Biomembr. 1999; 31: 95-104

The crystal structure of mitochondrial F1-ATPase indicates that the alpha and beta subunits fold into a structure defined by three domains: the top beta-barrel domain, the middle nucleotide-binding domain, and the C-terminal alpha-helix bundle domain (Abrahams et al., 1994); Bianchet et al., 1998). The beta-barrel domains of the alpha and beta subunits form a crown structure at the top of F1, which was suggested to stabilize it (Abrahams et al. 1994). In this study, the role of the beta-barrel domain in the alpha and beta subunits of the yeast Saccharomyces cerevisiae F1, with regard to its folding and assembly, was investigated. The beta-barrel domains of yeast F1alpha and beta subunits were expressed individually and together in Escherichia coli. When expressed separately, the beta-barrel domain of the beta subunit formed a large aggregate structure, while the domain of the alpha subunit was predominately a monomer or dimer. However, coexpression of the beta-barrel domain of alpha subunit with the beta-barrel domain of beta subunit, greatly reduced the aggregation of the beta subunit domain. Furthermore, the two domains copurified in complexes with the major portion of the complex found in a small molecular weight form. These results indicate that the beta-barrel domain of the alpha and beta subunits interact specifically with each other and that these interactions prevent the aggregation of the beta-barrel domain of the beta subunit. These results mimic in vivo results and suggest that the interactions of the beta-barrel domains may be critical during the folding and assembly of F1.

Integrin transmembrane signaling and cytoskeletal control.

Yamada KM, Miyamoto S
Curr Opin Cell Biol. 1995; 7: 681-9

Integrins are remarkably multifunctional: they mediate cell adhesion and migration, orchestrate organization of the actin-based cytoskeleton, and activate signal transduction pathways. Recent studies have identified a variety of steps and hierarchies in these intracellular cytoskeletal and signaling responses, laying the groundwork for future studies on specificity and coordination with responses to growth factors.

Folding and function of I domain-deleted Mac-1 and lymphocyte function-associated antigen-1.

Yalamanchili P, Lu C, Oxvig C, Springer TA
J Biol Chem. 2000; 275: 21877-82

In those integrins that contain it, the I domain is a major ligand recognition site. The I domain is inserted between beta-sheets 2 and 3 of the predicted beta-propeller domain of the integrin alpha subunit. We deleted the I domain from the integrin alpha(M) and alpha(L) subunits to give I-less Mac-1 and lymphocyte function-associated antigen-1 (LFA-1), respectively. The I-less alpha(M) and alpha(L) subunits were expressed in association with the wild-type beta(2) subunit on the surface of transfected cells and bound to all the monoclonal antibodies mapped to the putative beta-propeller and C-terminal regions of the alpha(M) and alpha(L) subunits, suggesting that the folding of these domains is independent of the I domain. I-less Mac-1 bound to the ligands iC3b and factor X, but this binding was reduced compared with wild-type Mac-1. In contrast, I-less Mac-1 did not bind to fibrinogen or denatured bovine serum albumin. Binding to iC3b and factor X by I-less Mac-1 was inhibited by the function-blocking antibody CBRM1/32, which binds to the beta-propeller domain of the alpha(M) subunit. I-less LFA-1 did not bind its ligands intercellular adhesion molecule-1 and -3. Thus, the I domain is not essential for the folding, heterodimer formation, and surface expression of Mac-1 and LFA-1 and is required for binding to some ligands, but not others.

Model for the three-dimensional structure of vitronectin: predictions for the multi-domain protein from threading and docking.

Xu D, Baburaj K, Peterson CB, Xu Y
Proteins. 2001; 44: 312-20

The structure of vitronectin, an adhesive protein that circulates in high concentrations in human plasma, was predicted through a combination of computational methods and experimental approaches. Fold recognition and sequence-structure alignment were performed using the threading program PROSPECT for each of three structural domains, i.e., the N-terminal somatomedin B domain (residues 1-53), the central region that folds into a four-bladed beta-propeller domain (residues 131-342), and the C-terminal heparin-binding domain (residues 347-459). The atomic structure of each domain was generated using MODELLER, based on the alignment obtained from threading. Docking experiments between the central and C-terminal domains were conducted using the program GRAMM, with limits on the degrees of freedom from a known inter-domain disulfide bridge. The docked structure has a large inter-domain contact surface and defines a putative heparin-binding groove at the inter-domain interface. We also docked heparin together with the combined structure of the central and C-terminal domains, using GRAMM. The predictions from the threading and docking experiments are consistent with experimental data on purified plasma vitronectin pertaining to protease sensitivity, ligand-binding sites, and buried cysteines.

Structure-function of the putative I-domain within the integrin beta 2 subunit.

Xiong YM, Zhang L
J Biol Chem. 2001; 276: 19340-9

The central region (residues 125-385) of the integrin beta(2) subunit is postulated to adopt an I-domain-like fold (the beta(2)I-domain) and to play a critical role in ligand binding and heterodimer formation. To understand structure-function relationships of this region of beta(2), a homolog-scanning mutagenesis approach, which entails substitution of nonconserved hydrophilic sequences within the beta(2)I-domain with their homologous counterparts of the beta(1)I-domain, has been deployed. This approach is based on the premise that beta(1) and beta(2) are highly homologous, yet recognize different ligands. Altogether, 16 segments were switched to cover the predicted outer surface of the beta(2)I-domain. When these mutant beta(2) subunits were transfected together with wild-type alpha(M) in human 293 cells, all 16 beta(2) mutants were expressed on the cell surface as heterodimers, suggesting that these 16 sequences within the beta(2)I-domain are not critically involved in heterodimer formation between the alpha(M) and beta(2) subunits. Using these mutant alpha(M)beta(2) receptors, we have mapped the epitopes of nine beta(2)I-domain specific mAbs, and found that they all recognized at least two noncontiguous segments within this domain. The requisite spatial proximity among these non-linear sequences to form the mAb epitopes supports a model of an I-domain-like fold for this region. In addition, none of the mutations that abolish the epitopes of the nine function-blocking mAbs, including segment Pro(192)-Glu(197), destroyed ligand binding of the alpha(M)beta(2) receptor, suggesting that these function-blocking mAbs inhibit alpha(M)beta(2) function allosterically. Given the recent reports implicating the segment equivalent to Pro(192)-Glu(197) in ligand binding by beta(3) integrins, these data suggest that ligand binding by the beta(2) integrins occurs via a different mechanism than beta(3). Finally, both the conformation of the beta(2)I-domain and C3bi binding activity of alpha(M)beta(2) were dependent on a high affinity Ca(2+) binding site (K(d) = 105 &mgr;m), which is most likely located within this region of beta(2).

Expression of integrin and organization of F-actin in epithelial cells depends on the underlying surface.

Wu XY, Cornell-Bell A, Davies TA, Simons ER, Trinkaus-Randall V
Invest Ophthalmol Vis Sci. 1994; 35: 878-90

PURPOSE. To evaluate the role of ionic interactions in the cell surface expression of integrins and the organization of F-actin. Understanding these interactions will allow the development of surfaces for prosthetic purposes that will promote the normal expression of adhesion proteins. METHODS. Hema (hydroxyethylmethacrylate) hydrogels were used to mimic the charges present on extracellular matrix proteins. The surfaces were modified by the addition of amines (N,N-dimethylaminoethylmethacrylate; NDAM) or carboxyl moieties (methacrylic acid). The effects of ionic interactions on cellular spreading and on the expression of proteins were examined by modification of the stoichiometrically defined amounts of positive and negative charges on the Hemas. Changes in intracellular pH and the distribution and localization of protein were monitored using fluorescent markers, spectrofluorometry, and confocal laser scanning microscopy, respectively. The immunohistochemical studies were confirmed by flow cytometric analysis. RESULTS. The data indicate that although cells adhered to all the surfaces, the number of cells possessing adhesion receptors is significantly greater on surfaces with amine functionalities. Cell seeding and plating efficiency after 2 hours were identical on all surfaces. The intracellular pH of epithelial cells grown on surfaces containing NDAM, a tertiary amine, was higher than that of cells grown on Hemas containing only methacrylic acid. Lamellipodial extensions and an extensive actin network were present on surfaces containing 5% NDAM. The alpha 6 subunit was localized along the lateral cell membranes. The alpha 2 and 3 subunits were present along cell membranes and at lamellipodial extensions. Cells cultured on surfaces containing only methacrylic acid did not spread. Actin filaments were not detected, and alpha 6 was negligible on these surfaces. CONCLUSIONS. This is a novel approach to understanding cell-substrate interactions, and one that allows quantitative evaluation of the response of cells to defined surfaces. The organization of F-actin is altered by the substrates containing only carboxyl moieties. The distribution of integrin subunits is also altered by the substrate. These results indicate that epithelial cell spreading and protein expression may be regulated by ionic interactions.

Signaling networks regulating beta1 integrin-mediated adhesion of T lymphocytes to extracellular matrix.

Woods ML, Shimizu Y
J Leukoc Biol. 2001; 69: 874-80

T-cell recognition of foreign antigen and migration to specific anatomic sites in vivo involves transient adhesive contacts between beta1 integrins expressed on T cells and cell surface proteins or extracellular-matrix components. Engagement of the CD3-T-cell receptor (CD3-TCR) complex initiates a complex signaling cascade involving coordinated regulation and recruitment of tyrosine and lipid kinases to specific regions or microdomains in the plasma membrane. Although considerable attention has been focused on the signaling events by which the CD3-TCR complex regulates transcriptional events in the nucleus, CD3-TCR signaling also rapidly enhances integrin-mediated adhesion without increasing surface expression of integrins. Recent studies suggest that CD3-TCR signaling to beta1 integrins involves coordinated recruitment and activation of the Tec family tyrosine kinase Itk by src family tyrosine kinases and phosphatidylinositol 3-kinase. These signaling events that regulate integrin-mediated T-cell adhesion share both common and distinct features with the signaling pathways regulating interleukin-2 gene transcription.

Signaling from the matrix to the cytoskeleton: role of cell surface proteoglycans in matrix assembly.

Woods A, Couchman JR
Kidney Int Suppl. 1996; 54: 647-647

Integrin modulation by lateral association.

Woods A, Couchman JR
J Biol Chem. 2000; 275: 24233-6

The LIM-only protein DRAL/FHL2 binds to the cytoplasmic domain of several alpha and beta integrin chains and is recruited to adhesion complexes.

Wixler V, Geerts D, Laplantine E, Westhoff D, Smyth N, Aumailley M, Sonnenberg A, Paulsson M
J Biol Chem. 2000; 275: 33669-78

LIM proteins contain one or more double zinc finger structures (LIM domains) mediating specific contacts between proteins that participate in the formation of multiprotein complexes. We report that the LIM-only protein DRAL/FHL2, with four and a half LIM domains, can associate with alpha(3A), alpha(3B), alpha(7A), and several beta integrin subunits as shown in yeast two-hybrid assays as well as after overexpression in human cells. The amino acid sequence immediately following the conserved membrane-proximal region in the integrin alpha subunits or the C-terminal region with the conserved NXXY motif of the integrin beta subunits are critical for binding DRAL/FHL2. Furthermore, the DRAL/FHL2 associates with itself and with other molecules that bind to the cytoplasmic domain of integrin alpha subunits. Deletion analysis of DRAL/FHL2 revealed that particular LIM domains or LIM domain combinations bind the different proteins. These results, together with the fact that full-length DRAL/FHL2 is found in cell adhesion complexes, suggest that it is an adaptor/docking protein involved in integrin signaling pathways.

Integrin-mediated signaling in human neutrophil functioning.

Williams MA, Solomkin JS
J Leukoc Biol. 1999; 65: 725-36

Integrins are important signal transducers for virtually all neutrophil functions. Although a variety of signals ultimately result in integrin activation, the intracellular targets of integrin-initiated signals are poorly delineated to date. Polymorphonuclear (PMN) leukocyte responses to inflammation are dependent on both the stimulants and the extracellular environment encountered. Integrin ligation, by cell-cell or cell-matrix interactions, activates a variety of signaling cascades. These events dictate the nature of PMN responses to the encountered stimulus. The complex system of effector molecule recruitment and permissive signaling by integrins serves to strictly regulate PMN functions such as cell adhesion, motility, oxidant production, and protein synthesis. Moreover, there is evidence that cross-talk between integrins exists to prime integrin populations for subsequent functioning. This review summarizes the current understanding of signaling mechanisms for integrin priming and activation. In this connection, the role of specific signaling molecules in key PMN functions are examined.

Calcium-cell cycle regulator, differentiator, killer, chemopreventor, and maybe, tumor promoter.

Whitfield JF, Bird RP, Chakravarthy BR, Isaacs RJ, Morley P
J Cell Biochem Suppl. 1995; 22: 74-91

Ca2+ and Ca(2+)-binding proteins are involved in running the cell cycle. Ca2+ spikes and signals from integrin-activated focal adhesion complexes and Ca2+ receptors on the cell surface along with cyclic AMP begin the cycle of cyclin-dependent protein kinases (PKs). These transiently expressed PKs stimulate the coordinate expression of DNA-replicating enzymes, activate replication enzymes, inactivate replication suppressors (e.g., retinoblastoma susceptibility protein), activate the replicator complexes at the end of the G1 build-up, and when replication is complete they and a Ca2+ spike trigger mitotic prophase. Another Ca2+ surge at the end of metaphase triggers the destruction of the prophase-stimulating PKs and starts anaphase. Ca2+ finally stimulates cytoplasmic division (cytokinesis). However, Ca2+ does more than this in epithelial cells, such as those lining the colon, and skin keratinocytes. These cells also need Ca2+, integrin signals, and only a small amount (e.g., 0.05-0.1 mM) of external Ca2+ to start DNA replication. Signals from their surface Ca2+ receptors trigger a combination of differentiation and apoptosis ("diffpoptosis") when external Ca2+ concentration reaches their setpoints. The skin's steep, upwardly directed, Ca2+ gradient has a low concentration in the basal layer to allow stem and precursor keratinocytes to proliferate, and higher concentrations in the suprabasal layers to trigger the differentiation-apoptosis ("diffpoptosis") mechanism that converts granular cells into protective, hard-shelled, dead corneocytes. A similar Ca2+ gradient may exist in the colon crypt allowing the stem cell and its amplifying transit or precursor offspring to cycle in the lower parts of the crypt, while stopping proliferation and stimulating terminal differentiation in the upper crypt and flat mucosa. Raising the amount of Ca2+ in fecal water above a critical level reduces proliferation and thus colorectal carcinogenesis in normal rats and some high-risk humans. But during carcinogenesis the Ca2+ sensors malfunction or their signals become ineffective: high Ca2+ does not stop, and may even stimulate, the proliferation of initiated mutants. Therefore, Ca2+ may either not affect, or even promote, the growth of epithelial cells in carcinogen-initiated rat colon and human adenoma patients. Clearly, a much greater understanding of how Ca2+ controls the proliferation and differentiation of epithelial cells and why initiated cells lose their responsiveness to Ca2+ are needed to assess the drawbacks and advantages of using Ca2+ as a chemopreventor.

Regulation of extracellular matrix degradation by cell-extracellular matrix interactions.

Werb Z, Tremble P, Damsky CH
Cell Differ Dev. 1990; 32: 299-306

An appropriate balance of extracellular matrix synthesis and degradation is required for normal morphogenesis and maintenance of tissue architecture. Extracellular matrix molecules and their receptors, as well as proteinases and their inhibitors, are all involved in matrix remodeling. In this report we show that signal transduction through extracellular matrix receptors regulates matrix remodeling.

Integrin signaling in leukocytes: lessons from the alpha6beta1 integrin.

Wei J, Shaw LM, Mercurio AM
J Leukoc Biol. 1997; 61: 397-407

The adhesive interactions of leukocytes with basement membrane components and other extracellular matrix (ECM) proteins are mediated largely by the integrin family of receptors. These interactions can be regulated by various effector molecules including chemokines, growth factors, and other cell surface proteins by a mechanism termed inside-out signaling of integrin function. On engagement by their ligands, integrins activate a wide range of signaling pathways that regulate a broad array of leukocyte functions such as chemotaxis, cytokine responsiveness, phagocytosis, and gene expression. This review highlights recent advances in the understanding of integrin signaling in leukocytes with an emphasis on our own work on the alpha6beta1 integrin, the major laminin receptor expressed by these cells.

Biochemical analysis of integrin-mediated Shc signaling.

Wary KK, Dans M, Mariotti A, Giancotti FG
Methods Mol Biol. 1999; 129: 35-49

Purification and immunological analysis of phospholipase D from castor bean endosperm.

Wang X, Dyer JH, Zheng L
Arch Biochem Biophys. 1993; 306: 486-94

Phospholipase D (EC 3.1.4.4) has been implicated in diverse cellular processes, but its physiological role is not well established in plants. In order to develop immunological and molecular biology approaches to address the problem, we report here the immunological analysis and N-terminal amino acid sequence of a cytosolic phospholipase D from castor bean (Ricinus communis L.). The enzyme was purified to apparent homogeneity from germinating castor bean endosperm. The specific activity of the purified enzyme was enhanced by approximately 670-fold with an overall yield of 4%. Its molecular mass was estimated at 92 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of this enzyme was KLVENIEETVGFGKG. Polyclonal antibodies were raised against the purified enzyme. The antibodies inhibited the activity of transphosphatidylation more than that of hydrolysis of phospholipase D. The differential effect on the two activities of this enzyme implies that different active sites on this enzyme may be involved in the two reactions. Immunoblot analyses showed that the amounts of phospholipase D protein relative to the total endosperm proteins increased during the first 5 days of germination. The antibodies cross-reacted to proteins from several tested plant species, and those proteins had molecular masses similar to that of castor bean phospholipase D. These results indicate that the expression of phospholipase D in castor bean changes according to growth stages and that phospholipase D enzymes of different plant species are structurally related.

Integrin signaling: tyrosine phosphorylation events in focal adhesions.

Vuori K
J Membr Biol. 1998; 165: 191-9

[Integrins and reproduction]

Vinatier D
Contracept Fertil Sex. 1995; 23: 327-34

Integrins are a large family of cell surface proteins that serve as receptors, involved in cell-cell and cell-matrix interactions. These receptors are of fundamental importance in the organization of tissues and organs. Integrins participate in a complex apparatus anchoring cells to their surrounding and transducing signals into the cells. These signals regulate many important aspects of cell behaviour, including growth, differentiation and phenotype. Their roles in reproductive performance begin to be explored. It seems that all stages of reproduction involve integrins. During gametogenesis integrins let the gamete to receive the external informations. During fertilization integrins seem dramatically involved. Integrins are expressed in human endometrium. Endometrium integrin molecules appear to be regulated within the cycling endometrium and disruption of integrin expression may be associated with decreased uterine receptivity and infertility. Integrins are present on trophoblaste. A specific integrin type is expressed on invasive trophoblast. Low or absent expression of this specific type should be encountered during pre eclampsia. Probably the knowledge of these integrins will provide to the clinicians new diagnosis tools evaluating the quality of endometrium and of embryos.

The important role of integrins in reproductive processes.

Vinatier D
Eur J Obstet Gynecol Reprod Biol. 1995; 62: 263-4

[Integrins and integrin-associated molecules: targets for the development of antimetastatic therapies]

Velasco-Velazquez MA, Molina-Guarneros JA, Mendoza-Patino N, Sullivan Lopez J, Mandoki JJ
Rev Invest Clin. 1999; 51: 183-93

Integrins are receptors that mediate cell adhesion and the formation of signaling complex. Changes in the expression of integrins are required during the following steps in the generation of metastases: a) angiogenesis; b) detachment from the primary tumor; c) tumor cell-platelet interaction; d) adhesion to vascular endothelium and e) proliferation. There is a correlation between invasive capability and changes in the expression of some proteins that are clustered in focal adhesion sites, as FAK, CD82, CD9 or CD63. Both, integrin blocking (using antibodies or RGD containing peptides), as well as induced changes in the expression of integrin-associated molecules, are able to inhibit formation of metastases. Discovery and characterization of molecules that regulate the adhesive capability of tumor cells, will lead to development of antimetastasic therapies. In the search of tumor dissemination inhibitors, integrins and some integrin-associated molecules are important pharmacological targets.

Lymphocyte adhesion mediated by integrins.

van Kooyk Y, Figdor CG
Res Immunol. 1993; 144: 709-22

Function and interactions of integrins.

van der Flier A, Sonnenberg A
Cell Tissue Res. 2001; 305: 285-98

Integrins are heterodimeric cell adhesion molecules that link the extracellular matrix to the cytoskeleton. The integrin family in man comprises 24 members, which are the result of different combinations of 1 of 18 alpha- and 1 of 8 beta-subunits. Alternative splicing of mRNA of some alpha- and beta-subunits and postranslational modifications of integrin subunits further increase the diversity of the integrin family. In their capacity as adhesion receptors that organize the cytoskeleton, integrins play an important role in controlling various steps in the signaling pathways that regulate processes as diverse as proliferation, differentiation, apoptosis, and cell migration. The intracellular signals that lead to these effects may be transduced via cytoplasmic components, which have been identified as integrin-binding proteins in yeast two-hybrid screens and which could mediate the coupling of integrins to intracellular signaling pathways. In this review an overview is given of the function and ligand-binding properties of integrins as well as of proteins that associate with integrins and may play a role in their signaling function.

Purification and characterization of phospholipase D (PLD) from rice (Oryza sativa L.) and cloning of cDNA for PLD from rice and maize (Zea mays L.).

Ueki J, Morioka S, Komari T, Kumashiro T
Plant Cell Physiol. 1995; 36: 903-14

Phospholipase D (PLD) was purified to high homogeneity from rice bran (Oryza sativa L.). Two peaks of PLD activity were resolved by Mono Q anion-exchange chromatography. The molecular mass of PLD in both peaks was 82 kDa on SDS-PAGE and 78 kDa in gel filtration. Antibodies raised against the protein in one of the peaks precipitated the enzyme activities in both peaks. Enzymatic characteristics of PLD in the two peaks were identical except for a difference of 0.1 in the isoelectric points. Sequence analysis covering more than 10% of the amino acids of the proteins and peptide mapping did not detect any difference in the primary structure of the proteins. A cDNA for PLD was isolated from rice and it encoded a protein of 812 residues. The N-terminal sequences of purified PLDs matched the deduced amino acid sequence starting from residue 47. A Northern blot showed this gene was expressed in leaves, roots, developing seeds and cultured cells, and a Southern blot detected a single band of rice genomic DNA hybridizing to the cDNA. A cDNA for PLD was also isolated from maize. The similarity of the deduced amino acid sequences of PLD was 90% between rice and maize, 73% between the cereals and castor bean.

Integrins: a review of their structure and mechanisms of ligand binding.

Tuckwell DS, Weston SA, Humphries MJ
Symp Soc Exp Biol. 1993; 47: 107-36

Adhesive interactions between cells and between cells and extracellular matrices play key roles in determining spatiotemporal positioning, influencing site-specific gene expression, and dictating proliferation rate. In addition, aberrant adhesion contributes to various aspects of disease pathology. These phenotypic effects of adhesion are mediated initially by the recognition of adhesive components of the extracellular matrix by membrane-intercalated receptor molecules and ultimately by the transduction of chemical and physical signals to the cell interior. Cell-cell and cell-matrix interactions are highly complex, since they involve the interfacing of surface membrane structures with each other or with three-dimensional aggregates of glycoproteins and proteoglycans, and it is this complexity that provides the necessary versatility for cells to react appropriately to either gross or subtle changes in their environment. Reagents with the ability to modulate adhesion could have many types of use: They could be employed to dissect the role of cell migration in development, provide insight into how adhesion might regulate gene expression and cell phenotype, and they could have widespread therapeutic applications in the treatment of thrombosis, inflammation and cancer. The quest to develop such reagents has necessitated the elucidation of the mechanisms of cell adhesion, and in particular the identification of the molecules involved and their modes of interaction. This article reviews the state of this quest; in particular, the molecular basis of ligand binding by integrin receptors.

A secondary structure model of the integrin alpha subunit N-terminal domain based on analysis of multiple alignments.

Tuckwell DS, Humphries MJ, Brass A
Cell Adhes Commun. 1994; 2: 385-402

The integrins are alpha/beta heterodimeric proteins which mediate cell-matrix and cell-cell interactions. Current data indicate that the N-terminal moiety of the alpha subunit is involved in ligand binding. This region of the receptor is made up of a seven-fold repeated sequence of unknown structure which contains EF-hand-like putative divalent cation-binding sites. Recent studies have shown that multiple sequence alignments can be analysed to yield secondary structure predictions. Therefore, to obtain a model structure for the integrin alpha subunit N-terminal domain repeat, a large alignment of the seven repeats from sixteen integrin sequences was generated. Two methods of analysis were used: First, Chou and Fasman and Garnier, Osguthorpe and Robson predictions were carried out for individual sequences and the consensus predictions derived. Consensus hydrophobicity and chain flexibility data were also used to provide additional data. Second, sites of conservation and variation were analysed by a computer program STAMA (STructure After Multiple Alignment) to yield a secondary structure prediction. The two analyses gave essentially the same predicted structure: undefined region, loop, alpha-helix, beta-strand, divalent cation-binding loop, beta-strand, putative turn, loop, beta-strand. This is the first model structure to be presented for an integrin domain. Its implications for integrin function are discussed.

Molecular and cellular biology of integrins.

Tuckwell DS, Humphries MJ
Crit Rev Oncol Hematol. 1993; 15: 149-71

Ligand binding and affinity modulation of integrins.

Tozer EC, Hughes PE, Loftus JC
Biochem Cell Biol. 1996; 74: 785-98

Integrins are cell adhesion receptors that mediate cell-cell and cell-extracellular matrix interactions. The extracellular domains of these receptors possess binding sites for a diverse range of protein ligands. Ligand binding is divalent cation dependent and involves well-defined motifs in the ligand. Integrins can dynamically regulate their affinity for ligands (inside-out signaling). This ability to rapidly modulate their affinity state is key to their involvement in such processes as cell migration and platelet aggregation. This review will focus on two aspects of integrin function: first, on the molecular basis of ligand-integrin interactions and, second, on the underlying mechanisms controlling the affinity state of integrins for their ligands.

Structure-function analysis of the integrin beta 7 subunit: identification of domains involved in adhesion to MAdCAM-1.

Tidswell M, Pachynski R, Wu SW, Qiu SQ, Dunham E, Cochran N, Briskin MJ, Kilshaw PJ, Lazarovits AI, Andrew DP, Butcher EC, Yednock TA, Erle DJ
J Immunol. 1997; 159: 1497-505

Beta 7 integrins serve special roles in mucosal immunity. Alpha 4 beta 7-mediated adhesion to mucosal addressin cell adhesion molecule-1 (MAdCAM-1) directs lymphocyte homing to the gut, and alpha E beta 7 mediates binding of lymphocytes to E-cadherin on epithelial cells. Since alpha 4 beta 7 mediates adhesion to MAdCAM-1 but alpha 4 beta 1 does not, we used beta 7/beta 1 chimeras to directly assess the importance of specific regions of beta 7 in MAdCAM-1 binding. We found a region of beta 7 (residues 46-386) that accounts for specificity of alpha 4 beta 7 binding to MAdCAM-1. We also used human/mouse and human/rat chimeric beta 7 subunits to map epitopes recognized by fifteen anti-beta 7 mAbs. Six of seven Abs that block adhesion to MAdCAM-1 and E-cadherin (Fib 21, 22, 27, 30, 504; Act-1) mapped to amino acid residues 176-250. Residues 176-250 lie within the region of beta 7 that specifies MAdCAM-1 binding and also within a region that has a predicted structure homologous to the metal ion-dependent adhesion site (MIDAS) domains of the integrin subunits alpha L and alpha M. Three new Abs that recognize beta 7 in the presence of Mn2+, but not Ca2+, and promote adhesion to MAdCAM-1, mapped to amino acids 46-149. One blocking and five other Abs mapped to other regions (amino acids 387-725). We conclude that a MIDAS-like domain serves a critical role in beta 7 integrin-mediated adhesion.

Alpha 6 integrin distribution in human embryonic and adult tissues.

Terpe HJ, Stark H, Ruiz P, Imhof BA
Histochemistry. 1994; 101: 41-9

Alpha 6 integrin is an adhesion molecule that connects cells with extracellular matrix molecules of the laminin family. The laminin interaction seems to be essential for cell differentiation during embryogenesis and for the subsequent maintenance of tissue integrity in the adult. Alpha 6 integrin can also interact with laminin-independent cellular ligands and in this way plays a role in homing of leucocytes. Furthermore, in cancer biology alpha 6 integrin has an important role in metastasis and as a possible new prognostic factor; exact knowledge of alpha 6 integrin distribution in normal human tissues is therefore a crucial element. By immuno-histochemical methods we have screened alpha 6 integrin expression of representative human tissues from the adult and the embryonic organism. All tested epithelia were alpha 6 integrin positive, except for the endocrine cells of the pancreas and the adrenal glands. Heterogeneous staining was found on non-epithelial tissues. Strong staining was evident in peripheral nerves (Schwann cells), germ and Sertoli cells, endothelia, and smooth muscle cells of the myometrium. Weak staining was found in nerve cells of the stratum granulosum, the microglia, Kupffer's cells and stromal cells of the ovary. All fibroblasts, striated muscle cells and astrocytes were negative. The tissue distribution of alpha 6 integrin and the semi-quantitative estimation of their expression level should provide a better understanding of alpha 6 integrin function under normal and pathological conditions, in particular in tumour progression.

Integrin function and regulation in development.

Tarone G, Hirsch E, Brancaccio M, De Acetis M, Barberis L, Balzac F, Retta F, Botta C, Altruda F, Silengo L
Int J Dev Biol. 2000; 44: 725-31

Integrins are a large family of membrane receptors, consisting of alpha and beta subunits, that play a pivotal role in the interaction of cells with the extracellular matrix. Such interaction regulates the organization of cells in organs and tissues during development as well as cell differentiation and proliferation. We have shown that unfertilized oocytes express integrins that might be important during fertilization. We also analyzed nervous system and muscle tissue development showing that integrin expression is precisely regulated during organization of these tissues. The results indicate that two distinct integrin alpha subunits mediate the outgrowth of processes in nerve and glial cells. Alpha1 integrin, a laminin receptor, is up-regulated by nerve growth factor and other differentiation stimuli and is involved in neurite extension by nerve cells. In contrast, process extension by glial cells is likely to involve the alphaV integrin. Moreover, the latter integrin subunit is also transiently expressed in muscle of the embryo body where it localizes predominantly at developing myotendinous junctions. After birth this integrin disappears and is substituted by the alpha7 subunit. At the same time, important changes also occur in the expression of the associated beta subunit. In fact, the beta1A isoform which is expressed in fetal muscles, is substituted by beta1D. These isoforms are generated by alternative splicing and differ in only a few amino acid residues at the COOH terminus of the protein. This region of the molecule is exposed at the cytoplasmic face of the plasma membrane and is connected to the actin filaments. Our results show that beta1D, which is expressed only in striated muscle tissues, binds to both cytoskeletal and extracellular matrix proteins with an affinity higher than beta1A. Thus, beta1D provides a stronger link between the cytoskeleton and extracellular matrix necessary to support mechanical tension during muscle contraction. These results indicate that cells can regulate their interactions with the extracellular matrix by changing their expression of alpha integrin subunits and thus ligand specificity, or by more subtle changes involving alternative usage of different cytoplasmic domains. The important role of both alpha and beta integrin subunit cytoplasmic domains during development is further illustrated by the analysis of targeted mutations which we have generated by homologous recombination in mice.

PTEN gene and integrin signaling in cancer.

Tamura M, Gu J, Tran H, Yamada KM
J Natl Cancer Inst. 1999; 91: 1820-8

Integrins are major adhesion- and signaling-receptor proteins that mediate cell migration and invasion. They also trigger a variety of signal transduction pathways and regulate cytoskeletal organization, specific gene expression, growth control, and apoptosis (programmed cell death). Consequently, integrins are thought to play important roles in embryonic development and in the biology of cancers. The functions of integrins can be negatively regulated by the recently discovered tumor suppressor PTEN, a protein with homology to protein tyrosine phosphatases and tensin. The PTEN gene is mutated in a wide range of human cancers. PTEN inhibits cell migration and invasion by directly dephosphorylating two key tyrosine-phosphorylated proteins, thereby antagonizing interactions of integrins with the extracellular matrix and integrin-triggered signaling pathways. Other studies demonstrate important roles for PTEN in dephosphorylating a key signal transduction lipid. In the absence of PTEN, this lipid signal transduction pathway can protect tumor cells from apoptosis. Thus, PTEN appears to be a unique tumor suppressor-with both lipid phosphatase and protein tyrosine phosphatase activities-that negatively regulates cell interactions with the extracellular matrix and that maintains cell sensitivity to apoptosis, e.g., after loss of cell contact with the extracellular matrix. The complex signal transduction pathways regulated by PTEN are described in this review. PTEN and the signaling pathways it regulates may provide novel targets for potential therapy.

Identification of a regulatory region of integrin beta 1 subunit using activating and inhibiting antibodies.

Takada Y, Puzon W
J Biol Chem. 1993; 268: 17597-601

Members of the beta 1 integrin subfamily recognize multiple ligands such as fibronectin, laminin, and collagen and mediate cell-cell and cell-extracellular matrix interactions. beta 1 subunit may play a central role in regulating beta 1 integrin avidity. Here we have identified a small region of beta 1 subunit (residues 207-218) that is critical for the binding of both activating (8A2, A1A5, and TS2/16) and inhibiting (4B4, 4B5, 13, AIIB2, and P4C10) monoclonal antibodies against human beta 1 using interspecies chimeric beta 1 and site-directed mutagenesis. Chicken beta 1 that has human sequence within residues 207-218 (CH mutant) is recognized by all the human specific antibodies listed above. The region 207-218 is located between the two putative ligand binding sites (residues 120-182 and 220-231), and the amino acid sequence of the region involves a predicted bend structure. The other anti-beta 1 antibodies that do not affect cell attachment to ligands (K20, 102DF5, LM442, and LM534) recognized the carboxyl-terminal regions of extracellular domain of beta 1 (residues 426-587 for K20 and 588-708 for 102DF5, LM442, and LM534, respectively). Our data suggest a potential mechanism for the avidity regulation of beta 1 integrin through conformational changes of beta 1 subunit.

Emerging paradigms of integrin ligand binding and activation.

Sugimori T, Griffith DL, Arnaout MA
Kidney Int. 1997; 51: 1454-62

Adhesion of cells to each other or to the extracellular matrix provides essential signals that regulate many cellular functions including cell migration, proliferation, differentiation and apoptosis. The integrin superfamily orchestrates many of these complex adhesive events through regulated interactions with a large variety of ligands. Crystallization of some ligands and of a ligand-binding integrin domain, reviewed here, together with extensive mutagenesis studies are beginning to shed light on the inner workings of these receptors.

Integrins, angiogenesis and vascular cell survival.

Stromblad S, Cheresh DA
Chem Biol. 1996; 3: 881-5

The interactions between integrins and the extracellular matrix have been identified as important regulators of vascular cell survival, proliferation and invasion during the complex process of blood vessel formation by angiogenesis.

Control of normal mammary epithelial phenotype by integrins.

Streuli CH, Edwards GM
J Mammary Gland Biol Neoplasia. 1998; 3: 151-63

Mammary epithelial cells contact a specialized extracellular matrix in vivo known as the basement membrane. Interactions with extracellular matrix are mediated through integrins. These cell surface receptors are involved with the formation of adhesion complexes, which link the extracellular matrix with the actin-based cytoskeleton, and are also associated with components of growth factor signaling pathways. Differentiation of breast epithelia into lactational cells requires appropriate hormones and integrin-mediated interactions with basement membrane. Integrins may regulate the ability of lactogenic hormones to trigger their intracellular signaling pathways.

Leukocyte integrins.

Stewart M, Thiel M, Hogg N
Curr Opin Cell Biol. 1995; 7: 690-6

Lymphocytes, monocytes and granulocytes, which are collectively known as 'leukocytes', circulate primarily within the vascular system and lymphoid tissue but are found in essentially all tissues of the body. This mobile lifestyle necessitates the constant making and breaking of adhesive contacts with targets in their immediate environment. The adhesion receptors termed integrins, which are expressed in abundance by leukocytes, are well suited to carry out the transient interactions in which these cells engage. Major advances in the leukocyte integrin field this year have been the realization of the extensive roles for alpha 4 integrins in leukocyte function, the solution of the crystal structure of an I domain and its identification as a major ligand-binding site, and the initial understanding of how divalent cations may function in an active integrin.

[Cell membrane binding components for constituents of the extracellular matrix: structural mediator of the epithelial matrix interaction in the gastrointestinal tract]

Stallmach A, Matthes H, Riecken EO
Z Gastroenterol. 1991; 29: 398-403

Growth, migration, differentiation and metabolic functions of the epithelium in the gastrointestinal tract are regulated by the extracellular matrix. Different cell membrane binding components including the integrins for constituents of the extracellular matrix are expressed in the epithelial cells. These cell membrane binding components may be structural mediators of cell-matrix interaction in the gastrointestinal tract. A characterization of this interaction is of great importance, not only to understand physiological processes, such as epithelial migration and differentiation, but also for the pathogenesis of healing processes (ulcer healing), immunologically mediated processes (adhesion of immunocompetent cells), and especially for tumor pathology (invasion and metastasis). Gastrointestinal diseases are characterized by alterations in the expression of cell membrane binding components for different constituents of the extracellular matrix. In chronic inflammatory bowel disease, a changed expression can be identified on epithelial cells and, in especially malignant transformation of epithelial cells resulted in a pathologic expression of cell adhesion molecules. Oncogenes may modify the expression and function of these cell membrane binding components in the course of malignant transformation. In animal models, it was possible to reduce the frequency of tumor invasion and metastasis and to achieve longer survival times by blocking the cell membrane binding components on malignant cells. An increasing understanding of the role of cell membrane binding components in the epithelium-matrix interaction will certainly also be translated in the future into new therapeutic concepts.

The sensation and regulation of interactions with the extracellular environment: the cell biology of lymphocyte adhesion receptors.

Springer TA
Annu Rev Cell Biol. 1990; 6: 359-402

An extracellular beta-propeller module predicted in lipoprotein and scavenger receptors, tyrosine kinases, epidermal growth factor precursor, and extracellular matrix components.

Springer TA
J Mol Biol. 1998; 283: 837-62

An abundant, widely dispersed, extracellular sequence repeat that contains a consensus YWTD motif is shown here to occur in groups of six contiguous repeats. Thirteen lines of evidence, including experimental and computational data, predict with p<3x10(-9) that the repeats do not form tandem domains, but rather each group of six repeats folds into a compact beta-propeller structure. The six beta-sheets are arranged about a 6-fold pseudosymmetry axis, and each repeat contributes loops to the faces surrounding the pseudosymmetry axis. Seven different endocytic receptors that contain from one to eight YWTD beta-propeller domains act as lipoprotein, vitellogenin, and scavenger receptors. In the low density lipoprotein receptor (LDLR), the many mutations in familial hypercholesterolaemia that map to the YWTD domain can now be interpreted. In the extracellular matrix component nidogen, the YWTD domain functions to bind laminin. Three YWTD domains and interspersed fibronectin type III (FN3) domains constitute almost the entire extracellular domain of the sevenless and c-ros receptor tyrosine kinases. YWTD domains often are bounded by epidermal growth factor (EGF) modules, including in the EGF precursor itself. YWTD beta-propellers have a circular folding pattern that brings neighboring modules into close proximity, and may have important consequences for the architecture of multi-domain proteins.

Integrins and their ligands.

Sonnenberg A
Curr Top Microbiol Immunol. 1993; 184: 7-35

Integrins are expressed on almost every cell type and are responsible for the linkage of the extracellular matrix with the cytoskeleton. In this review I have focused on the intra- and extracellular proteins that bind to integrins. Although many integrins bind to the same extracellular ligand, they mostly recognize different sites on these ligands. Some integrins interact with the same site but then there are requirements for different additional sequences to obtain high affinity. By modulating the expression and activity of integrins in the plasma membrane, cells can adapt their capacity of binding to the matrix. How integrins become activated is as yet not clear, but interaction with other proteins or lipids may be critical. Binding to ligands could also be modulated by alternative splicing of mRNAs for ligand binding sites in the extracellular domain. In Drosophila, the mRNA for the extracellular domain of the PS2 integrin is spliced near a site implicated in ligand binding. In humans, however, there are no indications that alternative splicing contributes to the regulation of function of the extracellular domain of integrins. The only splice variant of the extracellular domain of an integrin identified so far concerns are alpha subunit of the alpha IIb beta 3 complex, but the splicing occurs in a region that has not been implicated in cell adhesion. There is also no evidence as yet that integrin function can be modulated by alternative splicing of mRNA for the cytoplasmic domain of integrin subunits. However, the loss of function seen with some deletion mutants of the cytoplasmic domains of integrin subunits suggests that such a mechanism may well exist. In a different way the binding capacity of a given cell can be influenced by regulating the expression of its ligand or by alternative mRNA splicing of sequences encoding the cell binding domain in their ligands. In the case of fibronectin, the mRNA for one of the integrin binding sites is subject to alternative splicing. The mRNAs for the three chains of laminin appear not to be subject to alternative splicing but, by combining different variant chains of laminin, isoforms can be generated which may have different affinities for integrins. Binding of cells to the matrix therefore does not only depend on the expression and activity of the correct integrin but also of the correct variant of the ligand.(ABSTRACT TRUNCATED AT 400 WORDS)

Allostery and proteolysis: two novel modes of regulating integrin function.

Smith JW
Matrix Biol. 1997; 16: 173-8

Integrins are involved in transmitting signals between the cytoplasm and the extracellular matrix. Importantly, the transfer of information is bi-directional: signals flow inside-out and outside-in. Here, I discuss two potential modes by which integrin function is likely to be regulated. It is hypothesized that the integrin cytoplasmic tails are proteolytic substrates, and that cleavage of the cytoplasmic domain regulates the ligand binding affinity of integrins. It is also hypothesized that the ligand binding site is allosterically regulated by separate divalent ion binding sites that independently control ligand association and dissociation rate. Both hypotheses are suggested by reports in the literature and can be tested experimentally.

Mapping the collagen-binding site in the I domain of the glycoprotein Ia/IIa (integrin alpha(2)beta(1)).

Smith C, Estavillo D, Emsley J, Bankston LA, Liddington RC, Cruz MA
J Biol Chem. 2000; 275: 4205-9

The I domain present within the alpha2 chain of the integrin alpha(2)beta(1) (GPIa/IIa) contains the principal collagen-binding site. Based on the crystal structure of the alpha2-I domain, a hypothetical model was proposed in which collagen binds to a groove on the upper surface of the I domain (Emsley, J., King, S. L., Bergelson, J. M., and Liddington, R. C. (1997) J. Biol. Chem. 272, 28512-28517). We have introduced point mutations into 13 residues on the upper surface of the domain. Recombinant mutant proteins were assayed for binding to monoclonal antibodies 6F1 and 12F1, to collagen under static conditions, and for the ability to retain adhesive activity under flow conditions. The mutations to residues surrounding the metal ion-dependent adhesion site that caused the greatest loss of collagen binding under both static and flow conditions are N154S in the betaA-alpha1 turn, N190D in the betaB-betaC turn, D219R in the alpha3-alpha4 turn, and E256V and H258V in the betaD-alpha5 turn. Mutation in one of the residues that coordinate the metal binding, S155A, completely lost the adhesive activity under flow but bound normally under static conditions, whereas the mutation Y285F had the converse effect. We conclude that the upper surface of the domain, including the metal ion-dependent adhesion site motif, defines the collagen recognition site.

Integrin-mediated calcium signaling and regulation of cell adhesion by intracellular calcium.

Sjaastad MD, Nelson WJ
Bioessays. 1997; 19: 47-55

Integrins are ubiquitous trans-membrane adhesion molecules that mediate the interaction of cells with the extracellular matrix (ECM). Integrins link cells to the ECM by interacting with the cell cytoskeleton. In cases such as leukocyte binding, integrins mediate cell-cell interactions and cell-ECM interactions. Recent research indicates that integrins also function as signal transduction receptors, triggering a number of intracellular signaling pathways that regulate cell behavior and development. A number of integrins are known to stimulate changes in intracellular calcium levels, resulting in integrin activation. Although changes in intracellular calcium regulate a vast number of cellular functions, this review will discuss the stimulation of calcium signaling by integrins and the role of intracellular calcium in the regulation of integrin-mediated adhesion.

Feedback regulation of cell-substratum adhesion by integrin-mediated intracellular Ca2+ signaling.

Sjaastad MD, Angres B, Lewis RS, Nelson WJ
Proc Natl Acad Sci U S A. 1994; 91: 8214-8

Integrin binding to extracellular matrix (ECM) regulates cell migration and gene expression in embryogenesis, metastasis, would healing, and the inflammatory response. In many cases, binding of integrins to ECM triggers intracellular signaling pathways. The regulatory roles of intracellular signaling mechanisms in these events are poorly understood. Using single-cell analysis, we demonstrate that beads coated with peptide containing Arg-Gly-Asp (RGD), an integrin recognition motif found in many ECM proteins, elicit a rapid transient increase in intracellular calcium in Madin-Darby canine kidney (MDCK) epithelial cells. Also, significantly more beads bind to responding cells than to nonresponders. Several independent methods that inhibit RGD-induced Ca2+ signaling decrease both the number of beads bound and the strength of adhesion to an RGD-coated substratum. These results indicate that intracellular Ca2+ signaling participates in a positive feedback loop that enhances integrin-mediated cell adhesion.

Cell adhesion and migration properties of beta 2-integrin negative polymorphonuclear granulocytes on defined extracellular matrix molecules. Relevance for leukocyte extravasation.

Sixt M, Hallmann R, Wendler O, Scharffetter-Kochanek K, Sorokin LM
J Biol Chem. 2001; 276: 18878-87

Regulated adhesion of leukocytes to the extracellular matrix is essential for transmigration of blood vessels and subsequent migration into the stroma of inflamed tissues. Although beta(2)-integrins play an indisputable role in adhesion of polymorphonuclear granulocytes (PMN) to endothelium, we show here that beta(1)- and beta(3)-integrins but not beta(2)-integrin are essential for the adhesion to and migration on extracellular matrix molecules of the endothelial cell basement membrane and subjacent interstitial matrix. Mouse wild type and beta(2)-integrin null PMN and the progranulocytic cell line 32DC13 were employed in in vitro adhesion and migration assays using extracellular matrix molecules expressed at sites of extravasation in vivo, in particular the endothelial cell laminins 8 and 10. Wild type and beta(2)-integrin null PMN showed the same pattern of ECM binding, indicating that beta(2)-integrins do not mediate specific adhesion of PMN to the extracellular matrix molecules tested; binding was observed to the interstitial matrix molecules, fibronectin and vitronectin, via integrins alpha(5)beta(1) and alpha(v)beta(3), respectively; to laminin 10 via alpha(6)beta(1); but not to laminins 1, 2, and 8, collagen type I and IV, perlecan, or tenascin-C. PMN binding to laminins 1, 2, and 8 could not be induced despite surface expression of functionally active integrin alpha(6)beta(1), a major laminin receptor, demonstrating that expression of alpha(6)beta(1) alone is insufficient for ligand binding and suggesting the involvement of accessory factors. Nevertheless, laminins 1, 8, and 10 supported PMN migration, indicating that differential cellular signaling via laminins is independent of the extent of adhesion. The data demonstrate that adhesive and nonadhesive interactions with components of the endothelial cell basement membrane and subjacent interstitium play decisive roles in controlling PMN movement into sites of inflammation and illustrate that beta(2)-integrins are not essential for such interactions.

Regulation of human osteoblast integrin expression by orthopedic implant materials.

Sinha RK, Tuan RS
Bone. 1996; 18: 451-7

Integrins are transmembrane proteins that mediate attachment to extracellular matrix (ECM) proteins. Integrins act as ECM receptors and are believed to play a role in signal transduction and gene regulation, potentially influencing several cellular functions, including differentiation, growth, and migration. Several integrins have previously been found in bone cells in vivo and in vitro. Because the biologic fixation of orthopedic implants depends on optimal cell interactions at the interface, we examined whether integrins are involved in the attachment of osteoblasts to implant materials. In this study, we have used immunohistochemistry to examine the expression of integrins in primary human osteoblasts cultured on tissue culture polystyrene (PS), and rough and polished disks of the orthopedic alloys titanium-aluminum-vanadium (Ti) and cobalt-chrome-molybdenum (CC). The integrin subunits, alpha 2, alpha 3, alpha 4, alpha 5, alpha v, alpha 6, beta 1 and beta 3, were expressed by primary human osteoblasts cultured on PS coated with various ECM molecules. However, alpha 5 and alpha 6 were notably absent in cells attached to the alloys. Also, alpha 3 was not present on rough Ti, polished CC, or rough CC, and beta 3 was not expressed by cells on rough CC. Thus, the nature of the metal alloy appeared to influence the expression of particular integrins. As a result, the ability of cells to adhere to and receive messages from the extracellular matrix may also be influenced by the substratum. These differences may explain previously observed differences in degree of cell attachment to these metals.

Role of integrins in cellular responses to mechanical stress and adhesion.

Shyy JY, Chien S
Curr Opin Cell Biol. 1997; 9: 707-13

Mechanical stresses are important environmental cues for both normal cellular functions and pathophysiological changes in conditions such as cardiac hypertrophy and atherosclerosis. There is increasing evidence that mechanotransduction processes in response to mechanical stresses share many common features with processes in cell adhesion, such as an increase in tyrosine phosphorylation of proteins in the focal adhesion sites. Recent findings suggest that integrins may function as mechanotransducers in cells.

Integrins in the immune system.

Shimizu Y, Rose DM, Ginsberg MH
Adv Immunol. 1999; 72: 325-80

Intracellular signaling pathways and the regulation of cell adhesion.

Shimizu Y
Hum Cell. 1996; 9: 175-80

Adhesion molecules play an essential role in the host immune response by mediating the adhesive interactions that are essential for immune cell trafficking and activation. Integrins are one family of adhesion receptors that leukocytes utilize to interact with other cells and with components of the extracellular matrix. Since leukocytes rapidly alternate between adhesive and nonadhesive states, the functional activity of integrins expressed on leukocytes is carefully and precisely regulated. Resting T lymphocytes express integrin receptors, but they mediate minimal cell adhesion. However, activation of the T cell results within minutes in increased integrin functional activity that occurs without a change in the level of integrin expression on the cell surface. Increased integrin-mediated adhesion appears to be a general response of T cells to activation, since a diverse array of activation stimuli are capable of inducing this rapid increase in integrin functional activity. We have used DNA-mediated gene transfer and site-directed mutagenesis to elucidate the intracellular signaling pathways that regulate integrin-mediated cell adhesion. Our studies have revealed two important general themes. First, the lipid kinase phosphatidylinositol 3-kinase (PI 3-K) plays a role in integrin regulation mediated by many regulators of integrin function. Second, there are cell-specific differences in the signaling pathways that regulate integrin function. These studies illustrate the complex nature of the signaling pathways that regulate lymphocyte adhesion.

Computational design of an integrin I domain stabilized in the open high affinity conformation.

Shimaoka M, Shifman JM, Jing H, Takagi J, Mayo SL, Springer TA
Nat Struct Biol. 2000; 7: 674-8

We have taken a computational approach to design mutations that stabilize a large protein domain of approximately 200 residues in two alternative conformations. Mutations in the hydrophobic core of the alphaMbeta2 integrin I domain were designed to stabilize the crystallographically defined open or closed conformers. When expressed on the cell surface as part of the intact heterodimeric receptor, binding of the designed open and closed I domains to the ligand iC3b, a form of the complement component C3, was either increased or decreased, respectively, compared to wild type. Moreover, when expressed in isolation from other integrin domains using an artificial transmembrane domain, designed open I domains were active in ligand binding, whereas designed closed and wild type I domains were inactive. Comparison to a human expert designed open mutant showed that the computationally designed mutants are far more active. Thus, computational design can be used to stabilize a molecule in a desired conformation, and conformational change in the I domain is physiologically relevant to regulation of ligand binding.

Structure/function analysis of the integrin beta 1 subunit by epitope mapping.

Shih DT, Edelman JM, Horwitz AF, Grunwald GB, Buck CA
J Cell Biol. 1993; 122: 1361-71

Monoclonal antibodies (mAbs) have been produced against the chicken beta 1 subunit that affect integrin functions, including ligand binding, alpha subunit association, and regulation of ligand specificity. Epitope mapping of these antibodies was used to identify regions of the subunit involved in these functions. To accomplish this, we produced mouse/chicken chimeric beta 1 subunits and expressed them in mouse 3T3 cells. These chimeric subunits were fully functional with respect to heterodimer formation, cell surface expression, and cell adhesion. They differed in their ability to react with a panel anti-chicken beta 1 mAbs. Epitopes were identified by a loss of antibody binding upon substitution of regions of the chicken beta 1 subunit by homologous regions of the mouse beta 1 subunit. The identification of the epitope was confirmed by a reciprocal exchange of chicken and mouse beta 1 domains that resulted in the gain of the ability of the mouse subunit to interact with a particular anti-chicken beta 1 mAb. Using this approach, we found that the epitopes for one set of antibodies that block ligand binding mapped toward the amino terminal region of the beta 1 subunit. This region is homologous to a portion of the ligand-binding domain of the beta 3 subunit. In addition, a second set of antibodies that either block ligand binding, alter ligand specificity, or induce alpha/beta subunit dissociation mapped to the cysteine rich repeats near the transmembrane domain of the molecule. These data are consistent with a model in which a portion of beta 1 ligand binding domain rests within the amino terminal 200 amino acids and a regulatory domain, that affects ligand binding through secondary changes in the structure of the molecule resides in a region of the subunit, possibly including the cysteine-rich repeats, nearer the transmembrane domain. The data also suggest the possibility that the alpha subunit may exert an influence on ligand specificity by interacting with this regulatory domain of the beta 1 subunit.

Airway epithelial integrins: why so many?

Sheppard D
Am J Respir Cell Mol Biol. 1998; 19: 349-51

In vivo functions of integrins: lessons from null mutations in mice.

Sheppard D
Matrix Biol. 2000; 19: 203-9

The integrin family (Hynes, R.O., 1992. Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69, 11-25) is composed of at least 24 heterodimers formed from eight beta subunits and 18 alpha subunits. Thus far, mice expressing null mutations of seven of the eight beta subunits and 13 of the 18 known alpha subunits have been generated, With only a few exceptions, the phenotypes of each of the knockout lines are quite distinct. Studies utilizing integrin knockout mice and cells derived from these mice have provided considerable and sometimes surprising insights into unique functions of individual members of this family.

Identification and characterization of novel airway epithelial integrins.

Sheppard D
Am Rev Respir Dis. 1993; 148: 3842-3842

Integrins are heterodimeric glycoproteins that mediate cell-to-matrix and some cell-to-cell interactions. Recent evidence underscores the important roles of these receptors in signaling machines that transduce positional information into complex changes in cell behavior. As such, integrins have been shown to play critical roles in cell growth, differentiation, and migration. Most cells express multiple members of this family, but the integrin repertoire of any given cell appears to be highly tissue- and cell-type-specific. We have used the homology-based polymerase chain reaction to identify known and novel integrin subunits in airway epithelial cells. With this technique we have identified three novel integrin subunits that participate in the formation of at least four novel integrin heterodimers. The best characterized of these, alpha v beta 6, is a receptor for the extracellular matrix protein fibronectin, and appears to be expressed only in terminally differentiated mucosal epithelial cells. The novel alpha subunit, alpha 9, forms a heterodimer with the known beta subunit, beta 1, in some epithelial cell lines. Elucidatation of the specific roles these receptors play in airway health and disease will likely provide unique insights into both the biology of integrins and the biology of the airway epithelium.

Cell migration: regulation of force on extracellular-matrix-integrin complexes.

Sheetz MP, Felsenfeld DP, Galbraith CG
Trends Cell Biol. 1998; 8: 51-4

Cell migration relies upon forces generated by the cell. Recent studies have provided new insights into the processes by which cells generate and regulate the forces applied to extracellular matrix (ECM)-bound integrins and have led us to the working model described here. In this model, ECM binding to integrins in the front of lamellipodia causes those integrins to attach to the rearward-moving cytoskeleton. Integrin-cytoskeleton attachments in the front are strengthened as a result of ECM rigidity, enabling the cell to pull itself forward. The reduction in contact area at the rear compared with that at the lamellipodium concentrates the traction forces in the rear on fewer integrin-ECM bonds, facilitating release. In such a model, cell pathfinding and motility can be influenced by ECM rigidity.

Perspectives series: cell adhesion in vascular biology. Integrin signaling in vascular biology.

Shattil SJ, Ginsberg MH
J Clin Invest. 1997; 100: 1-5

Signaling networks linking integrins and rho family GTPases.

Schwartz MA, Shattil SJ
Trends Biochem Sci. 2000; 25: 388-91

Integrins and Rho family GTPases function coordinately to mediate adhesion-dependent events in cells. Recently, it has also become apparent that integrins regulate Rho GTPases and vice versa. Integrins and GTPases might therefore be organized into complex signaling cascades that regulate cell behavior.

Integrins: emerging paradigms of signal transduction.

Schwartz MA, Schaller MD, Ginsberg MH
Annu Rev Cell Dev Biol. 1995; 11: 549-99

Integrins receive signals from other receptors that lead to activation of ligand binding (inside-out signaling) and matrix assembly. Upon binding ligands, they also activate intracellular signaling pathways. These signals converse with pathways that are initiated by soluble ligands to regulate cell functions. In this way, cell adhesion is coordinated with other events to orchestrate complex cellular behavior.

Integrating with integrins.

Schwartz MA, Ingber DE
Mol Biol Cell. 1994; 5: 389-93

Our central claim is that signaling by integrins provides a mechanism by which signals generated in response to adhesion, soluble hormones, and mechanical forces can interact. Such interactions permit cells to integrate these different classes of external stimuli and hence to orchestrate an efficient response. This integrating function of integrins is likely to be essential for much of development and physiology, as well as complex pathologies such as cancer. Understanding in detail how these signals are transduced and processed is likely to be an important area of research in the near future.

Integrins, oncogenes, and anchorage independence.

Schwartz MA
J Cell Biol. 1997; 139: 575-8

Signaling by integrins: implications for tumorigenesis.

Schwartz MA
Cancer Res. 1993; 53: 1503-6

Matrix in signal transduction and growth factor modulation.

Schuppan D, Ruhl M
Braz J Med Biol Res. 1994; 27: 2125-41

The extracellular matrix (ECM) is indispensable for the survival of multicellular organisms. It provides the adherent cells with crucial clues for migration, proliferation and differentiation. These clues are transmitted to the interior of the cell by ECM receptors like the integrins. Signaling by the ECM occurs by induction of assembly and disassembly of cytoskeletal structures or by modulation of classical signal transduction pathways such as activation of phosphatidylinositol-proteases, growth factors and cytokines that are specifically bound to its constituents and thereby stored, localized and modulated in terms of their biological activities. Finally, both the quantity and the quality of growth factor signaling appear to be dependent on the temporal and spatial activation of ECM receptors, supporting the requirement of a crosstalk between matrix and growth factor receptors.

Osteoblast integrin adhesion and signaling regulate mineralization.

Schneider GB, Zaharias R, Stanford C
J Dent Res. 2001; 80: 1540-4

Integrin adhesion and signaling events may contribute to the progressive differentiation of the osteoblast and to the initiation of a mineralized matrix. The purpose of our study was to begin to analyze the role of integrin receptors, in particular alpha2beta1, alpha5beta1, and alphaVbeta3, regarding mediation of the initiation of a mineralized matrix. Integrin-perturbation assays were conducted in microdot cultures of UMR-106-01 Bone Sialoprotein (BSP) osteoblast cells. For phenotypic analysis, we performed bright-field microscopy and Aliziran Red S staining to analyze effects on mineralization initiation. Mineralization was reduced significantly (P < 0.001) following the addition of alpha5- or beta1-integrin subunit antibody by approximately 20% and 45%, respectively--alphaVbeta3 integrin by nearly 65%, and alpha2beta1 integrin by nearly 95%. This effect was reversible following the removal of the antiintegrin antibody. These results suggest that integrin adhesion and signaling events may contribute to the ability of this cell line to mediate the initiation of the mineralization phenotype biologically.

Modulation of epithelial morphogenesis and cell fate by cell-to-cell signals and regulated cell adhesion.

Schmidt JW, Piepenhagen PA, Nelson WJ
Semin Cell Biol. 1993; 4: 161-73

Cell-cell and cell-extracellular matrix (ECM) interactions control many developmental decisions of epithelial cell fate and morphogenesis. Protein tyrosine kinases are one class of regulatory molecules that have been implicated in the modulation of these processes. Several protein tyrosine kinases co-localize with cell-cell (cadherin) and cell-ECM (integrin) adhesion molecules at specific adhesion domains of epithelial cells. Protein tyrosine kinases may regulate epithelial development by modulating cell-cell and cell-ECM interactions and by relaying signals initiated by these interactions to other cellular components that determine cell structure and function.

Integrin signalling and tyrosine phosphorylation: just the FAKs?

Schlaepfer DD, Hunter T
Trends Cell Biol. 1998; 8: 151-7

The integrin family of transmembrane receptors have long been recognized for their structural roles in linking extracellular matrix proteins with the cellular actin cytoskeleton to regulate cell shape, cell migration and tissue architecture. Lately, it has become clear that integrin receptors can initiate intracellular signals that synergize with those from growth-factor receptor protein-tyrosine kinases in modulating cell growth. This review describes our current knowledge of integrin-stimulated tyrosine phosphorylation events and downstream signalling pathways, with emphasis on the recent progress made in understanding the molecular pathways linking the integrin receptors with mitogen-activated protein (MAP) kinase cascades.

Adhesion-growth factor interactions during differentiation: an integrated biological response.

Sastry SK, Horwitz AF
Dev Biol. 1996; 180: 455-67

Integrin cytoplasmic domains: mediators of cytoskeletal linkages and extra- and intracellular initiated transmembrane signaling.

Sastry SK, Horwitz AF
Curr Opin Cell Biol. 1993; 5: 819-31

The integrin cytoplasmic domains are generally short amino acid sequences that are highly divergent among alpha-subunits and partially conserved among beta-subunits. Interest in them has increased over the past few years as they have been shown to mediate several kinds of transmembrane signaling events as well as structural connections between the extracellular matrix and the cytoskeleton.

Focal adhesions: a nexus for intracellular signaling and cytoskeletal dynamics.

Sastry SK, Burridge K
Exp Cell Res. 2000; 261: 25-36

Role of alpha V integrins and vitronectin in human melanoma cell growth.

Sanders LC, Felding-Habermann B, Mueller BM, Cheresh DA
Cold Spring Harb Symp Quant Biol. 1992; 57: 233-40

Regulation of integrin function.

Sanchez-Mateos P, Cabanas C, Sanchez-Madrid F
Semin Cancer Biol. 1996; 7: 99-109

Cells communicate with their environment through several kinds of cell surface receptor. One of the most important families of cell adhesion receptors are the integrins, which include receptors that mediate cell-cell as well as cell-extracellular matrix interactions. A distinctive feature of integrins is their variable adhesive competence that is reversibly modified depending on the state of cell differentiation and/or activation or in response to environmental signals. The acquisition of adhesive function by integrins may be a consequence of conformational changes in these receptors that result in an increased ligand binding affinity. In addition, cells can control integrin-mediated adhesion through other mechanisms, including receptor clustering and association to cytoskeleton, phenomena that regulate the avidity of integrins for ligand molecules without altering their monovalent affinity. These phenomena have collectively been designated as 'post-receptor occupancy events'. These two interesting aspects of the regulation of integrin function are reviewed.

Leukocyte integrins: structure, function and regulation of their activity.

Sanchez-Madrid F, Corbi AL
Semin Cell Biol. 1992; 3: 199-210

Adhesion is a crucial requirement for the correct regulation of immune and inflammatory responses. In the immune system, leukocytes can interact with each other and with vascular endothelium as well as with extracellular matrix components, changing rapidly and transiently from circulating non-adherent to adherent states. Most of these interactions are mediated by integrins. This review will focus mainly on the structure and function of integrins expressed by leukocytes. The mechanisms for regulating the functional activity of these adhesion receptors, as well as the intracellular signals transduced through integrins, are described.

Anchorage dependence, integrins, and apoptosis.

Ruoslahti E, Reed JC
Cell. 1994; 77: 477-8

Integrins as signaling molecules and targets for tumor therapy.

Ruoslahti E
Kidney Int. 1997; 51: 1413-7

Adhesion molecules include ligands and receptors. Together they provide cells with anchorage and traction for migration, and the receptors also mediate signals that control cell polarity, survival, growth, differentiation and gene expression. Integrins are a major group of versatile adhesion receptors that serve both adhesive and signaling functions. They possess shared and unique specifics both outside and inside the cell. Many of the integrins share an affinity toward the RGD recognition sequence in their extracellular matrix ligands, but are still capable of distinguishing different RGD-containing proteins. The shared signaling pathways are likely to include changes in intracellular Ca2+ and PIP2 concentrations, and the activation of protein kinase C and focal adhesion kinase. Examples of integrin-specific signaling include that the alpha v beta 3 integrin (vitronectin receptor) can potentiate the effects of insulin and certain other growth factors and that the alpha 5 beta 1 integrin (fibronectin receptor) supports cell survival in serum-free cultures by up-regulating the anti-apoptosis protein Bcl-2. Another integrin function is that some integrins, in particular alpha 5 beta 1, are necessary for fibronectin matrix formation. Overexpression of alpha 5 beta 1, which results in the assembly of additional fibronectin matrix, reduces tumorigenicity of cultured tumor cells. Systemic treatment of tumor-bearing mice with an artificially generated fibronectin matrix suppresses metastasis. These and other findings indicate that the ligand binding and signaling functions of integrins offer targets for new therapeutic approaches.

Integrin signaling and matrix assembly.

Ruoslahti E
Tumour Biol. 1996; 17: 117-24

Cell adhesion and migration are controlled by the level of alpha 5 beta 1 integrin (fibronectin receptor) and by the amount of fibronectin matrix around the cell; adhesion is promoted at all levels of integrin expression and matrix assembly, whereas high levels of either or both curb cell migration. The alpha 5 beta 1 integrin appears to be a growth-suppressing integrin, and it protects cells from apoptosis when growth factors are absent. In contrast, the alpha v beta 3 integrin (vitronectin receptor) cooperates with certain growth factors, potentiating their effect on cells. These effects of alpha 5 beta 1 and alpha v beta 3 depend on signaling pathways specific for these integrins; integrins have both common and specific pathways for signaling into the cell. Moreover, integrins require activation from the inside of the cell to be able to bind their ligand outside the cell. High alpha 5 beta 1 expression and abundant matrix formation also suppress tumorigenicity in vivo, whereas perturbing the function of alpha 5 beta 1 with peptides that block its ligand binding suppresses experimental metastasis. Effecting these changes in tumor cells by gene therapy or pharmacological approaches may provide useful new cancer therapies.

RGD and other recognition sequences for integrins.

Ruoslahti E
Annu Rev Cell Dev Biol. 1996; 12: 697-715

Proteins that contain the Arg-Gly-Asp (RGD) attachment site, together with the integrins that serve as receptors for them, constitute a major recognition system for cell adhesion. The RGD sequence is the cell attachment site of a large number of adhesive extracellular matrix, blood, and cell surface proteins, and nearly half of the over 20 known integrins recognize this sequence in their adhesion protein ligands. Some other integrins bind to related sequences in their ligands. The integrin-binding activity of adhesion proteins can be reproduced by short synthetic peptides containing the RGD sequence. Such peptides promote cell adhesion when insolubilized onto a surface, and inhibit it when presented to cells in solution. Reagents that bind selectively to only one or a few of the RGD-directed integrins can be designed by cyclizing peptides with selected sequences around the RGD and by synthesizing RGD mimics. As the integrin-mediated cell attachment influences and regulates cell migration, growth, differentiation, and apoptosis, the RGD peptides and mimics can be used to probe integrin functions in various biological systems. Drug design based on the RGD structure may provide new treatments for diseases such as thrombosis, osteoporosis, and cancer.

Integrins and the myocardium.

Ross RS, Borg TK
Circ Res. 2001; 88: 1112-9

Extracellular matrix provides a structural, chemical, and mechanical substrate that is essential in cardiac development, growth, and responses to pathophysiological signals. Transmembrane receptors termed integrins provide a dynamic interaction of environmental cues and intracellular events. Integrins orchestrate multiple functions in the intact organism including organogenesis, regulation of gene expression, cell proliferation, differentiation, migration, and death. They are expressed in all cellular components of the cardiovascular system, including the vasculature, blood, cardiac myocytes and nonmuscle cardiac cells. The focus of this review will be on the role of integrins in the myocardium. We will provide background on integrin structure and function, discuss how the expression of integrins is critical to the form and function of the developing and postnatal myocardium, and review the known data on integrins as signaling molecules in the heart. Finally, we will offer insights to the future research directions into this important family of extracellular matrix receptors in the myocardium.

Extracellular matrix-dependent tissue-specific gene expression in mammary epithelial cells requires both physical and biochemical signal transduction.

Roskelley CD, Desprez PY, Bissell MJ
Proc Natl Acad Sci U S A. 1994; 91: 12378-82

Extracellular matrix (ECM) profoundly influences the growth and differentiation of the mammary gland epithelium, both in culture and in vivo. Utilizing a clonal population of mouse mammary epithelial cells that absolutely requires an exogenous ECM for function, we developed a rapid assay to study signal transduction by ECM. Two components of the cellular response to a basement membrane overlay that result in the expression of the milk protein beta-casein were defined. The first component of this response involves a rounding and clustering of the cells that can be physically mimicked by plating the cells on a nonadhesive substratum. The second component is biochemical in nature, and it is associated with beta 1 integrin clustering and increased tyrosine phosphorylation. The second component is initiated in a morphology-independent manner, but the proper translation of this biochemical signal into a functional response requires cell rounding and cell clustering. Thus, physical and biochemical signal transduction events contribute to the ECM-dependent regulation of tissue-specific gene expression in mouse mammary epithelial cells.

Signal transduction by cell adhesion receptors in leukocytes.

Rosales C, Juliano RL
J Leukoc Biol. 1995; 57: 189-98

Leukocytes usually pass through the blood stream as nonadherent cells, but during an immune response and inflammation, they become adherent in order to migrate through tissues. Three families of adhesion molecules, the immunoglobulin family, the selectins, and the integrins, participate in interactions between leukocytes and tissues. Near sites of inflammation, leukocytes initially interact with the endothelium via selectins, causing them to slow down and to roll along the walls of blood vessels. Next, chemoattractans induce the activation of integrins on leukocytes. Finally, activated integrins mediate leukocyte migration through the endothelium into the inflamed site. Interactions of leukocytes with other cells and various extracellular matrix (ECM) proteins lead to different functional cell responses, including changes in growth, behavior, and differentiation. Many of these interactions are mediated by integrins, which "integrate" ECM protein signals with the cytoskeleton and which also act as true receptors that generate biochemical signals within the cell. Changes in pH, cytoplasmic Ca2+ concentration, phosphorylation, and gene induction have all been observed after integrin engagement. Adhesion-mediated gene induction in monocytes is perhaps the best example that integrins initiate signaling cascades in the cell to deliver information from the ECM all the way to the nucleus.

Adhesion receptors in health and disease.

Rojas AI, Ahmed AR
Crit Rev Oral Biol Med. 1999; 10: 337-58

Cell adhesion molecules have been recognized to play a major role in a variety of physiological and pathological phenomena. They determine the specificity of cell-cell binding and the interactions between cells and extracellular matrix proteins. Some of them may also function as receptors that trigger intracellular pathways and participate in cellular processes like migration, proliferation, differentiation, and cell death. The receptors that mediate adhesion between epithelial cells that are discussed in this review include integrins, selectins, the immunoglobulin superfamily members, and cadherins. The intent of this review is to inform the reader about recent advances in cellular and molecular functions of certain receptors, specifically those that are considered important in cell adhesion. We have deliberately not provided all-inclusive detailed information on every molecule, but instead, have presented a generalized overview in order to give the reader a global perspective. This information will be useful in enhancing the reader's understanding of the molecular pathology of diseases and recognizing the potential role of these receptors and ligands as therapeutic agents.

PDGF-regulated rab4-dependent recycling of alphavbeta3 integrin from early endosomes is necessary for cell adhesion and spreading.

Roberts M, Barry S, Woods A, van der Sluijs P, Norman J
Curr Biol. 2001; 11: 1392-402

Background: It has been postulated that the regulation of integrin vesicular traffic facilitates cell migration by internalizing integrins at the rear of the cell and transporting them forward within vesicles for exocytosis at the leading edge to form new contacts with the extracellular matrix. The rab family of GTPases control key targeting events in the endo/exocytic pathway; therefore, these GTPases may be involved in the regulation of cell-matrix contact assembly.Results: The endo/exocytic cycle of alphavbeta3 and alpha5beta1 integrins was studied using mouse 3T3 fibroblast cell lines. In serum-starved cells, internalized integrins were transported through rab4-positive, early endosomes and arrived at the rab11-positive, perinuclear recycling compartment approximately 30 min after endocytosis. From the recycling compartment, integrins were recycled to the plasma membrane in a rab11-dependent fashion. Following treatment with PDGF, alphavbeta3 integrin, but not alpha5beta1, was rapidly recycled directly back to the plasma membrane from the early endosomes via a rab4-dependent mechanism without the involvement of rab11. This rapid recycling pathway directed alphavbeta3 to numerous small puncta distributed evenly across the dorsal surface of the cell, and the integrin only became localized into focal complexes at later times following PDGF addition. Interestingly, inhibition of PDGF-stimulated alphavbeta3 recycling using dominant-negative rab4 mutants compromised cell adhesion and spreading on vitronectin (a ligand for alphavbeta3), but adhesion to fibronectin (a ligand for alpha5beta1 and alphavbeta3) was unchanged.Conclusions: We propose that growth factor-regulated, rab4-dependent recycling of alphavbeta3 integrin from early endosomes to the plasma membrane is a critical upstream event in the assembly of cell-matrix contacts.

Signal transduction through integrins: a central role for focal adhesion kinase?

Richardson A, Parsons JT
Bioessays. 1995; 17: 229-36

The integrins are receptors for proteins of the extracellular matrix, both providing a physical link to the cytoskeleton and transducing signals from the extracellular matrix. Activation of integrins leads to tyrosine and serine phosphorylation of a number of proteins, elevation of cytosolic calcium levels, cytoplasmic alkalinization, changes in phospholipid metabolism and, ultimately, changes in gene expression. The recently discovered focal adhesion kinase localizes to focal contacts, which are sites of integrin clustering, and focal adhesion kinase can physically associate with integrins in vitro. As integrins lack intrinsic catalytic activity, focal adhesion kinase is a candidate for a signaling molecule that is recruited by integrins in order to trigger the generation of intracellular second messengers. Thus, focal adhesion kinase may play a central role in signal transduction through integrins.

Normal Ligand Binding and Signaling by CD47 (Integrin-associated Protein) Requires a Long Range Disulfide Bond between the Extracellular and Membrane-spanning Domains.

Rebres RA, Vaz LE, Green JM, Brown EJ
J Biol Chem. 2001; 276: 34607-16

CD47 is a unique member of the Ig superfamily with a single extracellular Ig domain followed by a multiply membrane-spanning (MMS) domain with five transmembrane segments, implicated in both integrin-dependent and -independent signaling cascades. Essentially all functions of CD47 require both the Ig and MMS domains, raising the possibility that interaction between the two domains is required for normal function. Conservation of Cys residues among CD47 homologues suggested the existence of a disulfide bond between the Ig and MMS domains that was confirmed by chemical digestion and mapped to Cys(33) and Cys(263). Subtle changes in CD47 conformation in the absence of the disulfide were suggested by decreased binding of two anti-Ig domain monoclonal antibodies, decreased SIRPalpha1 binding, and reduced CD47/SIRPalpha1-mediated cell adhesion. Mutagenesis to prevent formation of this disulfide completely disrupted CD47 signaling independent of effects on ligand binding, as assessed by T cell interleukin-2 secretion and Ca(2+) responses. Loss of the disulfide did not affect membrane raft localization of CD47 or its association with alpha(v)beta(3) integrin. Thus, a disulfide bond between the Ig and MMS domains of CD47 is required for normal ligand binding and signal transduction.

Integrins and laminins in tissue remodeling.

Quaranta V, Plopper GE
Kidney Int. 1997; 51: 1441-6

Critical residues for ligand binding in an I domain-like structure of the integrin beta1 subunit.

Puzon-McLaughlin W, Takada Y
J Biol Chem. 1996; 271: 20438-43

Several integrin alpha subunits have an inserted sequence of about 200 residues (the I or A domain) that is critical for ligand interactions. The presence of an I domain-like structure within the integrin beta subunit has been proposed based on the similarity of the hydropathy profiles and the homology of sequences between the alpha and beta subunits. This study was designed to determine whether the region of the beta1 subunit that includes residues 101-335 has the characteristics of an I domain. We found novel critical residues for ligand binding (Ser-132, Asn-224, Asp-226, Glu-229, Asp-233, Asp-267, and Asp-295, in addition to the previously reported Asp-130) using site-directed mutagenesis. The critical residues for ligand binding are located in several of loop structures of the region (or in a potential loop between an alpha helix and a beta strand), which have been predicted using multiple secondary structure prediction methods. The data suggest that the beta subunit has multiple disrupted critical oxygenated residues for ligand binding similar to those found in the alpha I domain.

Morphological control of cell growth and viability.

Price LS
Bioessays. 1997; 19: 941-3

Integrin-mediated cell adhesion and subsequent cell spreading are essential for the growth and survival of many cell types. While integrin engagement is known to activate various signalling pathways, the role that cell spreading plays in the control of growth and survival is not clear. Using a novel technique, however, Chen et al. demonstrate that the effect of cell spreading on growth and survival is not a consequence of increased area of contact with the extracellular matrix, supporting the hypothesis that regulation through changes in cell tension and architecture play a key role.

beta-propeller repeats and a PDZ domain in the tricorn protease: predicted self-compartmentalisation and C-terminal polypeptide-binding strategies of substrate selection.

Ponting CP, Pallen MJ
FEMS Microbiol Lett. 1999; 179: 447-51

Prokaryotic proteases demonstrate a variety of substrate-selection strategies that prevent uncontrolled protein degradation. Proteasomes and ClpXP-like proteases form oligomeric structures that exclude large substrates from central solvated chambers containing their active sites. Monomeric prolyl oligopeptidases have been shown to contain beta-propeller structures that similarly reduce access to their catalytic residues. By contrast, Tsp-like enzymes contain PDZ domains that are thought to specifically target C-terminal polypeptides. We have investigated the sequence of Thermoplasma acidophilum tricorn protease using recently-developed database search methods. The tricorn protease is known to associate into a 20 hexamer capsid enclosing an extremely large cavity that is 37 nm in diameter. It is unknown, however, how this enzyme selects its small oligopeptide substrates. Our results demonstrate the presence in tricorn protease of a PDZ domain and two predicted six-bladed beta-propeller domains. We suggest that the PDZ domain is involved in targeting non-polar C-terminal peptides, similar to those generated by the T. acidophilum proteasome, whereas the beta-propeller domains serve to exclude large substrates from the tricorn protease active site in a similar manner to that previously indicated for prolyl oligopeptidase.

Integrins in tumour development and spread.

Pignatelli M, Stamp G
Cancer Surv. 1995; 24: 113-27

The study of integrin receptor expression and function in carcinomas will undoubtedly increase our understanding of the malignant process and clarify the relative importance of the numerous alterations that are identified. Whether this will lead to direct clinical applications is not clear at present, but it will certainly alter our approach to the evaluation of cancer treatment. The more immediate search for prognostic indicators and metastatic site preference in tumours expressing a particular integrin profile is partly hampered by the small numbers of cases studied in most series, where observed trends in tumour subsets barely achieve significance. Perhaps the next phase should be to evaluate promising approaches in large multicentre studies to establish the relative importance of these trends in tumour prognosis and response to treatment, preferably by developing reagents that will permit the demonstration of integrins in routinely processed archival material from human tumour specimens. Future developments in therapy may depend on the knowledge that may emerge from such work.

Integrins as promiscuous signal transduction devices.

Petty HR, Todd RF 3rd
Immunol Today. 1996; 17: 209-12

Recent studies suggest physical and functional interactions of glycosylphosphatidylinositol (GPI)-linked proteins (CD14, CD16b and CD87) with leukocyte beta 2 integrins. As discussed in this article, it now appears that beta 2 integrins relay proinflammatory information from GPI-anchored membrane receptors to the cytoplasm via exodomain interactions.

"RKKH" peptides from the snake venom metalloproteinase of Bothrops jararaca bind near the metal ion-dependent adhesion site of the human integrin alpha(2) I-domain.

Pentikainen O, Hoffren AM, Ivaska J, Kapyla J, Nyronen T, Heino J, Johnson MS
J Biol Chem. 1999; 274: 31493-505

Integrin alpha(1)beta(1) and alpha(2)beta(1) are the major cellular receptors for collagen, and collagens bind to these integrins at the inserted I-domain in their alpha subunit. We have previously shown that a cyclic peptide derived from the metalloproteinase domain of the snake venom protein jararhagin blocks the collagen-binding function of the alpha(2) I-domain. Here, we have optimized the structure of the peptide and identified the site where the peptide binds to the alpha(2) I-domain. The peptide sequence Arg-Lys-Lys-His is critical for recognition by the I-domain, and five negatively charged residues surrounding the "metal ion-dependent adhesion site" (MIDAS) of the I-domain, when mutated, show significantly impaired binding of the peptide. Removal of helix alphaC, located along one side of the MIDAS and suggested to be involved in collagen-binding in these I-domains, does not affect peptide binding. This study supports the notion that the metalloproteinase initially binds to the alpha(2) I-domain at a location distant from the active site of the protease, thus blocking collagen binding to the adhesion molecule in the vicinity of the MIDAS, while at the same time leaving the active site free to degrade nearby proteins, the closest being the beta(1) subunit of the alpha(2)beta(1) cell-surface integrin itself.

[Integrins-mediated signal transduction]

Pei XT
Sheng Li Ke Xue Jin Zhan. 1995; 26: 53-6

New aspects of integrin signaling in cancer.

Parise LV, Lee J, Juliano RL
Semin Cancer Biol. 2000; 10: 407-14

Members of the integrin family of cell adhesion receptors influence several important aspects of cancer cell behavior, including motility and invasiveness, cell growth, and cell survival. Engagement of integrins with extracellular matrix (ECM) proteins can activate members of the Rho-family of small GTPases; conversely, Rho- and Ras-family proteins can influence the ability of integrins to bind their ligands. These events impinge on the control of cell motility, and ultimately on invasive and metastatic behavior. Integrin engagement with ECM also has important effects on cell survival, particularly for cells of epithelial origin. In some cases, specific integrins have selective effects on the efficiency of signal transduction in cell survival pathways. Copyright 2000 Academic Press.

The role of membrane lipids in regulation of integrin functions.

Pande G
Curr Opin Cell Biol. 2000; 12: 569-74

Recent evidence suggests that the biochemical and physical organization of lipid molecules in the plasma membrane can affect integrin-mediated cellular functions. The nature and mechanism of integrin-lipid interactions are unknown, but it is clear that they play specific roles in modulating the properties of integrins and integrin-associated proteins. A better knowledge of integrin functions, especially in the lipid milieu of plasma membranes, is necessary for the understanding of the phenomena that are regulated by integrins.

Experimental support for a beta-propeller domain in integrin alpha-subunits and a calcium binding site on its lower surface.

Oxvig C, Springer TA
Proc Natl Acad Sci U S A. 1998; 95: 4870-5

Integrins are large, heterodimeric surface molecules of wide importance in cell adhesion. The N-terminal half of all integrin alpha-subunits contains seven weak sequence repeats of approximately 60 amino acids that are important in ligand binding and have been predicted to fold cooperatively into a single beta-propeller domain with seven beta-sheets. We provide evidence supporting this model with a mouse mAb to human Mac-1 (alphaM beta2, CD11b/CD18). This antibody, CBRM1/20, binds to amino acid residues that are in different repeats and are 94 residues apart in the primary structure in the loop between strands 1 and 2 of beta-sheet 5 and in the loop between strands 3 and 4 of beta-sheet 6. The 1-2 loops of beta-sheets 5-7 in integrins have EF hand-like Ca2+-binding motifs. CBRM1/20 binds to Mac-1 in the presence of Ca2+ or Sr2+ with an EC50 of 0.2 mM. Mg2+ or Mn2+ cannot substitute. Antibodies to other epitopes on the Mac-1 beta-propeller domain bind in the absence of calcium. mAb CBRM1/20 does not block ligand binding. Thus, the region on the lower surface of the beta-propeller domain to which mAb CBRM1/20 binds does not bind ligand and, furthermore, cannot bind other integrin domains, such as those of the beta-subunit.

Conformational changes in tertiary structure near the ligand binding site of an integrin I domain.

Oxvig C, Lu C, Springer TA
Proc Natl Acad Sci U S A. 1999; 96: 2215-20

For efficient ligand binding, integrins must be activated. Specifically, a conformational change has been proposed in a ligand binding domain present within some integrins, the inserted (I) domain [Lee, J., Bankston, L., Arnaout, M. & Liddington, R. C. (1995) Structure (London) 3, 1333-1340]. This proposal remains controversial, however, despite extensive crystal structure studies on the I domain [Lee, J., Bankston, L., Arnaout, M. & Liddington, R. C. (1995) Structure (London) 3, 1333-1340; Liddington, R. & Bankston, L. (1998) Structure (London) 6, 937-938; Qu, A. & Leahy, D. J. (1996) Structure (London) 4, 931-942; and Baldwin, E. T., Sarver, R. W., Bryant, G. L., Jr., Curry, K. A., Fairbanks, M. B., Finzel, B. C. , Garlick, R. L., Heinrikson, R. L., Horton, N. C. & Kelly, L. L. (1998) Structure (London) 6, 923-935]. By defining the residues present in the epitope of a mAb against the human Mac-1 integrin (alphaMbeta2, CD11b/CD18) that binds only the active receptor, we provide biochemical evidence that the I domain itself undergoes a conformational change with activation. This mAb, CBRM1/5, binds the I domain very close to the ligand binding site in a region that is widely exposed regardless of activation as judged by reactivity with other antibodies. The conformation of the epitope differs in two crystal forms of the I domain, previously suggested to represent active and inactive receptor. Our data suggests that conformational differences in the I domain are physiologically relevant and not merely a consequence of different crystal lattice interactions. We also demonstrate that the transition between the two conformational states depends on species-specific residues at the bottom of the I domain, which are proposed to be in an interface with another integrin domain, and that this transition correlates with functional activity.

Adhesion modulation by antiadhesive molecules of the extracellular matrix.

Orend G, Chiquet-Ehrismann R
Exp Cell Res. 2000; 261: 104-10

[Multiplicity and purification of animal phospholipase D]

Okamura S, Yamashita S
Seikagaku. 1995; 67: 1290-5

Interaction of bacteria and bacterial toxins with intestinal epithelial cells.

Nusrat A, Sitaraman SV, Neish A
Curr Gastroenterol Rep. 2001; 3: 392-8

The epithelium of the intestinal tract is a key barrier between the external environment and the internal body environment. Intestinal epithelial cells are targets for luminal bacteria and viruses and must discriminate between pathogenic and nonpathogenic commensal organisms. Pathogenic bacteria and their secreted products influence epithelial cell function and induce diarrhea by numerous mechanisms that range from an effect on epithelial cell-cell associations to intracellular signal transduction pathways. These effects lead to an inflammatory response and an influx of neutrophils into the epithelium. Infiltrating neutrophils, in turn, signal to epithelial cells, induce a secretory response, and perpetuate the diarrhea. Conversely, commensal bacteria have the ability to suppress inflammatory responses by inhibiting specific intracellular signal transduction pathways. Some of these diverse host pathogenic responses are addressed in this review.

Crystal structure of the alpha1beta1 integrin I-domain: insights into integrin I-domain function.

Nolte M, Pepinsky RB, Venyaminov SYu, Koteliansky V, Gotwals PJ, Karpusas M
FEBS Lett. 1999; 452: 379-85

The alpha1beta1 integrin is a major cell surface receptor for collagen. Ligand binding is mediated, in part, through a 200 amino acid inserted 'I'-domain contained in the extracellular part of the integrin alpha chain. Integrin I-domains contain a divalent cation binding (MIDAS) site and require cations to interact with integrin ligands. We have determined the crystal structure of recombinant I-domain from the rat alpha1beta1 integrin at 2.2 A resolution in the absence of divalent cations. The alpha1 I-domain adopts the dinucleotide binding fold that is characteristic of all I-domain structures that have been solved to date and has a structure very similar to that of the closely related alpha2beta1 I-domain which also mediates collagen binding. A unique feature of the alpha1 I-domain crystal structure is that the MIDAS site is occupied by an arginine side chain from another I-domain molecule in the crystal, in place of a metal ion. This interaction supports a proposed model for ligand-induced displacement of metal ions. Circular dichroism spectra determined in the presence of Ca2+, Mg2+ and Mn2+ indicate that no changes in the structure of the I-domain occur upon metal ion binding in solution. Metal ion binding induces small changes in UV absorption spectra, indicating a change in the polarity of the MIDAS site environment.

Tyrosine phosphorylation of p130Cas in cell adhesion and transformation.

Nojima Y, Mimura T, Morino N, Hamasaki K, Furuya H, Sakai R, Nakamoto T, Yazaki Y, Hirai H
Hum Cell. 1996; 9: 169-74

Integrins comprise the major class of receptors used by cells to interact with the extracellular matrix. Integrin/matrix interactions play a critical role in a variety of biological processes, including embryonic development, wound healing, tumor metastasis, cell growth and differentiation. It is now evident that integrins can transduce biochemical signals across the plasma membrane to the cell interior. Protein tyrosine phosphorylation has attracted much attention as an important regulator for integrin-mediated signal transduction. Recently, we have identified a novel signaling molecule, p130Cas, which participates in integrin-mediated signal transduction. p130Cas was originally identified as a protein hyperphosphorylated in cells expressing transforming gene products p47v-crk (v-Crk) and p60v-crk (v-Src). In this brief review, we will discuss about a role of p130Cas in signal transduction triggered by cell adhesion and transformation.

Signaling mechanisms and the activation of leukocyte integrins.

Newton RA, Thiel M, Hogg N
J Leukoc Biol. 1997; 61: 422-6

Myeloid cells and lymphocytes primarily circulate in the vascular system but move into the tissues in response to inflammatory signals. Such a mobile lifestyle necessitates the continual making and breaking of cell and cell matrix contacts; the receptors known as the integrins are well suited as mediators of this transient adhesiveness. In general, leukocyte integrins are not constitutively active but become adhesive in response to signaling through other membrane receptors. Candidate receptors for receiving the activating signals are the seven membrane-spanning receptors that are found on all leukocytes. The identity of the signals, however, that are responsible for triggering integrin adhesion in vivo and for promoting directed leukocyte movement into tissues remains incompletely resolved.

Integrin adhesion receptors: structure, function and implications for biomedicine.

Newham P, Humphries MJ
Mol Med Today. 1996; 2: 304-13

Over the past decade, multi-disciplinary approaches have led to the discovery and characterization of several classes of adhesion molecules. Under normal conditions, these molecules provide support for cells, regulate cell migration and contain information that cells use when sensing their environment. In disease, adhesive function is frequently compromised and results in tissue disorder, aberrant cell migration and dysregulation of signalling pathways. The integrins are a major family of adhesion receptors produced by most cell types and are a means by which the cell senses its immediate environment and responds to changes in extracellular matrix composition. Recent years have seen major advances in our understanding of integrin-ligand interactions, and have revealed a structurally dynamic family of receptors capable of translating information into and out of the cell.

[Integrin signal transduction via FAK in osteoblasts--early differentiation triggered by osteopontin]

Nemoto A, Uemura T
Seikagaku. 1999; 71: 140-5

Mechanical induction of beta 1-integrin-mediated calcium signaling in a hepatocyte cell line.

Nebe B, Rychly J, Knopp A, Bohn W
Exp Cell Res. 1995; 218: 479-84

Mechanical stress influences growth, differentiation, and gene expression in a variety of cell types. It is believed that via extracellular matrix the mechanical stimulus is transmitted to integrin receptors which thus play a key role in transducing signals into the cell interior. Here we demonstrate that incubation of suspended hepatocytes with specific antibodies to beta 1-integrin subunits followed by a short-term mechanical stimulation is sufficient to induce a rise in intracellular Ca2+. The results indicate that mechanical loading of individual integrin subunits activates Ca(2+)-specific signal pathways.

[Integrins, structure and function]

Nakstad B
Tidsskr Nor Laegeforen. 1993; 113: 595-9

Integrins are molecules of central importance in physiological and pathological processes. Integrins are an important factor in intercellular adhesion processes and cell migration leading to normal architecture of tissue, in inflammatory and tumour cell migration and in reparatory processes of damaged tissue. The integrin designation is derived from the role of these substances in integrating processes within and outside the cell. Therefore these membrane proteins play a role in cell growth, differentiation and gene expression. The role played by integrins in cell signal transduction and inflammation is discussed in this article. During the past few years we have seen an explosion of research in this field, which may lead to therapeutic benefits in a few years time.

The molecular effects of oncogenesis on cell-extracellular matrix adhesion (Review).

Nadav L, Katz BZ
Int J Oncol. 2001; 19: 237-46

Cell-extracellular matrix adhesive interactions provide a key regulatory mode of cellular behavior. The molecular basis of adhesion-mediated signaling responses has been under investigation over the past few years. Tyrosine phosphorylation initiated by cell adhesion plays a crucial role in regulating adhesion-mediated signaling and cytoskeletal rearrangement. Oncogenesis involves aberrant interactions between cells and the extracellular matrix. The mechanisms that underline the functions of oncogenes and tumor suppressors often involve modulation of specific tyrosine phosphorylated cytoplasmic proteins, thereby affecting directly adhesion-mediated signaling. The constitutive kinase activity of oncogenes such as v-Src and BCR/Abl hyper-phosphorylates cytoskeletal and signaling molecules, and modulates the functions of integrins, the predominant family of extracellular matrix receptors. The tumor suppressor gene PTEN was recently identified as a key regulator of adhesion-mediated signaling. This review summarizes the direct effects of oncogenes, tumor suppressor genes and their products on the adhesive responses of cells. Understanding of the molecular basis of these effects may provide the means to develop novel therapeutics to control pathological processes associated with aberrant cell-extracellular matrix interactions.

Structural principles for the propeller assembly of beta-sheets: the preference for seven-fold symmetry.

Murzin AG
Proteins. 1992; 14: 191-201

Twisted beta-sheets, packed face to face, may be arranged in circular formation like blades of a propeller or turbine. This beta-propeller fold has been found in three proteins: that in neuraminidase consists of six beta-sheets while those in methylamine dehydrogenase and galactose oxidase are composed of seven beta-sheets. A model for multisheet packing in the beta-propeller fold is proposed. This model gives both geometrical parameters of the beta-propellers composed of different numbers of sheets and patterns of residue packing at their sheet-to-sheet interfaces. All the known beta-propeller structures have been analyzed, and the observed geometries and residue packing are found to be in good agreement with those predicted by models. It is shown that unusual seven-fold symmetry is preferable to six- or eight-fold symmetry for propeller-like multi-sheet assembly. According to the model, a six-beta-sheet propeller has to have predominantly small residues in the beta-strands closed to its six-fold axis, but no strong sequence constraints are necessary for a seven-fold beta-propeller.

[Integrins: their structures, functions and gene expressions in the central nervous system. Acta anat.]

Murase S, Hayashi Y
Kaibogaku Zasshi. 1997; 72: 433-50

More than ten years have passed since integrin was shown to function in cellular attachment. To date integrin research has been one of major fields in cell biology. Integrin, which functions as an integrator of both extra- and intracellular skeletal molecules, is regarded as one of the essential molecules for cellular signal transduction as well. Thus, integrin appears to be essential and indispensable for many cellular phenomena. Although every type of cell is thought to express a few kinds of integrin molecules, their expression and functional roles in neurons remain to be determined. Both intensive and extensive researches should reveal one by one how integrins are involved in the neural network formation in development, neuronal plasticity and regeneration, higher function of CNS, and also neuronal degeneration in both inflammation and degenerative diseases.

Review: How was metazoan threshold crossed? The hypothetical Urmetazoa.

Muller WE
Comp Biochem Physiol A Mol Integr Physiol. 2001; 129: 433-60

The origin of Metazoa remained - until recently - the most enigmatic of all phylogenetic problems. Sponges [Porifera] as 'living fossils', positioned at the base of multicellular animals, have been used to answer basic questions in metazoan evolution by molecular biological techniques. During the last few years, cDNAs/genes coding for informative proteins have been isolated and characterized from sponges, especially from the marine demosponges Suberites domuncula and Geodia cydonium. The analyses of their deduced amino acid sequences allowed a molecular biological resolution of the monophyly of Metazoa. Molecules of the extracellular matrix/basal lamina, with the integrin receptor, fibronectin and galectin as prominent examples, cell-surface receptors (tyrosine kinase receptors), elements of nerve system/sensory cells (metabotropic glutamate receptor), homologs/modules of an immune system [immunoglobulin-like molecules, SRCR- and SCR-repeats, cytokines, (2-5)A synthetase], as well as morphogens (myotrophin) classify the Porifera as true Metazoa. As 'living fossils', provided with simple, primordial molecules allowing cell-cell and cell-matrix adhesion, as well as processes of signal transduction as known in a more complex manner from higher Metazoa, sponges also show peculiarities. Tissues of sponges are rich in telomerase activity, suggesting a high plasticity in the determination of cell lineages. It is concluded that molecular biological studies with sponges as models will not only help to understand the evolution to the Metazoa but also the complex, hierarchical regulatory network of cells in higher Metazoa [reviewed in Progress in Molecular Subcellular Biology, vols. 19, 21 (1998) Springer Verlag]. The hypothetical ancestral animal, the Urmetazoa, from which the metazoan lineages diverged (more than 600 MYA), may have had the following characteristics: cell adhesion molecules with intracellular signal transduction pathways, morphogens/growth factors forming gradients, a functional immune system, and a primordial nerve cell/receptor system.

Cloning and expression of a divergent integrin subunit beta 8.

Moyle M, Napier MA, McLean JW
J Biol Chem. 1991; 266: 19650-8

Rabbit and human cDNA clones have been identified that encode a novel integrin beta subunit. The sequences that encode this subunit, which has been designated as beta 8, were isolated initially from rabbit placental cDNA libraries using an oligonucleotide probe derived from a highly conserved region of integrin beta subunit sequences. The rabbit clone was used to isolate human beta 8 cDNA clones from human placental and MG-63 osteosarcoma cell libraries. The putative beta 8 polypeptides, which comprise 769 and 768 residues in human and rabbit, respectively, show a high degree of inter-species conservation (approximately 90% identity). In contrast, beta 8 is distinct from the other integrin beta subunits. At the amino acid level human beta 8 ranges from 31 to 37% identity with human beta 1-7. The domain structure of beta 8 is typical of the integrin beta subunits. Human beta 8 has a 42-residue N-terminal signal peptide, a large extracellular domain (approximately 639 residues) that contains four cysteine-rich repeats, a transmembrane domain (approximately 30 residues), and a C-terminal cytoplasmic domain (approximately 58 residues). There are several structural features that are unique to the beta 8 polypeptide, as compared with the other integrin beta subunits. Six of the 56 cysteine residues that are conserved within the extracellular domains of beta 1, beta 2, beta 3, beta 5, beta 6, and the beta subunit from Drosophila are absent in the beta 8 polypeptide. Also, the cytoplasmic domain of the beta 8 subunit shares no homology with the cytoplasmic regions of any of the other integrin beta subunits. Northern analysis demonstrated an approximately 8-kilobase beta 8 mRNA in rabbit placenta, kidney, brain, ovary, and uterus. PCR analysis revealed that beta 8 mRNA is also present in several transformed human cell lines. The beta 8 polypeptide has been transiently expressed in 293 human embryonic kidney cells. A polyclonal antipeptide antibody specific for beta 8 and a polyclonal antibody that recognizes alpha v epitopes were used to show that beta 8 can complex with the endogenous alpha v subunit in 293 cells and that the resulting integrin is expressed as a cell surface complex.

Molecular basis of ligand recognition by integrin alpha 5beta 1. I. Specificity of ligand binding is determined by amino acid sequences in the second and third NH2-terminal repeats of the alpha subunit.

Mould AP, Askari JA, Humphries MJ
J Biol Chem. 2000; 275: 20324-36

The NH(2)-terminal portion (putative ligand-binding domain) of alpha subunits contains 7 homologous repeats, the last 3 or 4 of which possess divalent cation binding sequences. These repeats are predicted to form a seven-bladed beta-propeller structure. To map ligand recognition sites on the alpha(5) subunit we have taken the approach of constructing and expressing alpha(V)/alpha(5) chimeras. Although the NH(2)-terminal repeats of alpha(5) and alpha(V) are >50% identical at the amino acid level, alpha(5)beta(1) and alpha(V)beta(1) show marked differences in their ligand binding specificities. Thus: (i) although both integrins recognize the Arg-Gly-Asp (RGD) sequence in fibronectin, the interaction of alpha(5)beta(1) but not of alpha(V)beta(1) with fibronectin is strongly dependent on the "synergy" sequence Pro-His-Ser-Arg-Asn; (ii) alpha(5)beta(1) binds preferentially to RGD peptides in which RGD is followed by Gly-Trp (GW) whereas alpha(V)beta(1) has a broader specificity; (iii) only alpha(5)beta(1) recognizes peptides containing the sequence Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA). Therefore, amino acid residues involved in ligand recognition by alpha(5)beta(1) can potentially be identified in gain-of-function experiments by their ability to switch the ligand binding properties of alpha(V)beta(1) to those of alpha(5)beta(1). By introducing appropriate restriction enzyme sites, or using site-directed mutagenesis, parts of the NH(2)-terminal repeats of alpha(V) were replaced with the corresponding regions of the alpha(5) subunit. Chimeric subunits were expressed on the surface of Chinese hamster ovary-B2 cells (which lack endogenous alpha(5)) as heterodimers with hamster beta(1). Stable cell lines were generated and tested for their ability to attach to alpha(5)beta(1)-selective ligands. Our results demonstrate that: (a) the first three NH(2)-terminal repeats contain the amino acid sequences that determine ligand binding specificity and the same repeats include the epitopes of function blocking anti-alpha subunit mAbs; (b) the divalent cation-binding sites (in repeats 4-7) do not confer alpha(5)beta(1)- or alpha(V)beta(1)-specific ligand recognition; (c) amino acid residues Ala(107)-Tyr(226) of alpha(5) (corresponding approximately to repeats 2 and 3) are sufficient to change all the ligand binding properties of alpha(V)beta(1) to those of alpha(5)beta(1); (d) swapping a small part of a predicted loop region of alpha(V) with the corresponding region of alpha(5) (Asp(154)-Ala(159)) is sufficient to confer selectivity for RGDGW and the ability to recognize RRETAWA.

From adhesion to signalling: roles of integrins in the biology of human melanoma.

Mortarini R, Anichini A
Melanoma Res. 1993; 3: 87-97

Integrins are cell surface heterodimers which act as regulators of adhesion and as signal transducers in normal and neoplastic cells. The expression and function of integrins are subject to change during the neoplastic transformation of melanocytes and the progression of melanoma. The integrin profile of human melanoma is also characterized by marked inter- and intratumour heterogeneity. These processes influence the interaction of melanoma cells with extracellular matrix (ECM) components and with other cell types that express integrin ligands. Integrins on melanoma cells not only act as mediators of adhesive interactions but also act as signalling molecules. The signal transducing function of integrins plays a role in a number of biological responses of melanoma cells to ECM-derived stimuli, including production of proteolytic enzymes, invasion of basement membranes, expression of genes and proliferation.

Structure and regulation of phospholipase D.

Morris AJ, Engebrecht J, Frohman MA
Trends Pharmacol Sci. 1996; 17: 182-5

Regulatory mechanisms underlying T cell integrin receptor function.

Mobley JL, Reynolds PJ, Shimizu Y
Semin Immunol. 1993; 5: 227-36

Adhesion molecules allow lymphocytes to interact with and respond to the extracellular environment. Since these interactions must be essentially transient in nature, the function of lymphocyte adhesion molecules must be precisely regulated. Studies of integrin receptors vividly illustrate the various mechanisms by which the function of these adhesion molecules can be regulated. These include: (1) activation-dependent changes in functional activity; (2) changes in levels of expression due to differentiation events; (3) cell-specific differences in integrin binding; and (4) differential binding to distinct ligands by the same integrin. These mechanisms provide highly precise and specific modes of regulating lymphocyte interactions with a wide variety of potential counter-receptors and ligands.

Integrin function: molecular hierarchies of cytoskeletal and signaling molecules.

Miyamoto S, Teramoto H, Coso OA, Gutkind JS, Burbelo PD, Akiyama SK, Yamada KM
J Cell Biol. 1995; 131: 791-805

Integrin receptors play important roles in organizing the actin-containing cytoskeleton and in signal transduction from the extracellular matrix. The initial steps in integrin function can be analyzed experimentally using beads coated with ligands or anti-integrin antibodies to trigger rapid focal transmembrane responses. A hierarchy of transmembrane actions was identified in this study. Simple integrin aggregation triggered localized transmembrane accumulation of 20 signal transduction molecules, including RhoA, Rac1, Ras, Raf, MEK, ERK, and JNK. In contrast, out of eight cytoskeletal molecules tested, only tensin coaccumulated. Integrin aggregation alone was also sufficient to induce rapid activation of the JNK pathway, with kinetics of activation different from those of ERK. The tyrosine kinase inhibitors herbimycin A or genistein blocked both the accumulation of 19 out of 20 signal transduction molecules and JNK- and ERK-mediated signaling. Cytochalasin D had identical effects, whereas three other tyrosine kinase inhibitors did not. The sole exception among signaling molecules was the kinase pp125FAK which continued to coaggregate with alpha 5 beta 1 integrins even in the presence of these inhibitors. Tyrosine kinase inhibition also failed to block the ability of ligand occupancy plus integrin aggregation to trigger transmembrane accumulation of the three cytoskeletal molecules talin, alpha-actinin, and vinculin; these molecules accumulated even in the presence of cytochalasin D. However, it was necessary to fulfill all four conditions, i.e., integrin aggregation, integrin occupancy, tyrosine kinase activity, and actin cytoskeletal integrity, to achieve integrin-mediated focal accumulation of other cytoskeletal molecules including F-actin and paxillin. Integrins therefore mediate a transmembrane hierarchy of molecular responses.

Fibronectin and integrins in cell adhesion, signaling, and morphogenesis.

Miyamoto S, Katz BZ, Lafrenie RM, Yamada KM
Ann N Y Acad Sci. 1998; 857: 119-29

Fibronectin and integrins play crucial roles in a variety of morphogenetic processes, in which they mediate cell adhesion, migration, and signal transduction. They induce hierarchical transmembrane organization of cytoskeletal and signaling molecules into multimolecular complexes of more than 30 proteins. Organization of these complexes is a synergistic process dependent on integrin aggregation and occupancy, as well as tyrosine phosphorylation. Integrins also cooperate with growth-factor receptors to enhance signaling. Fibronectin and integrins induce a variety of downstream effects, including enhanced transcription factor activity, induction of over 30 genes (> half novel), and altered expression of over 100 proteins. Fibronectin and integrins therefore trigger a hierarchy of signaling responses involved in regulating processes crucial for normal morphogenesis, including cell adhesion, migration, and specific gene expression.

Developmental regulation of alphav integrins produces functional changes in astrocyte behavior.

Milner R, Relvas JB, Fawcett J, ffrench-Constant C
Mol Cell Neurosci. 2001; 18: 108-18

To examine the role of the extracellular matrix in regulating astrocyte behavior we previously characterized alphav integrin expression on postnatal astrocytes in vitro and found that they express alphavbeta5 and alphavbeta8. Here we show that differentiation of immature cells into astrocytes is accompanied by developmental regulation of alphav integrins, downregulation of alphavbeta1 and alphavbeta8, and upregulation of alphavbeta5. In addition, using two previously described astrocyte cell lines, we found that the neurite-permissive A7 cell line expressed high levels of alphavbeta1 in addition to alphavbeta5 and alphavbeta8, while the neurite-inhibitory Neu7 cell line expressed only alphavbeta5. To examine integrin function we generated clones of the Neu7 cell line expressing alphavbeta1 or alphavbeta3 in addition to alphavbeta5. This showed that the parent Neu7 cells migrated more slowly than the A7 cells on fibronectin and vitronectin, but that Neu7 cells expressing alphavbeta1 or alphavbeta3 integrins showed enhanced migration on fibronectin and vitronectin, respectively. These results show that alphav integrin expression is regulated during astrocyte development and confirm an instructive role in cell migration for alphavbeta1 in embryonic cells and alphavbeta3 in astroglial tumors.

Therapeutic approaches for sticky situations. IBC Conference on Integrins: applying multidisciplinary approaches for targeted therapies. Cambridge, MA, USA, 29-30 January 1998.

Mierke DF
Mol Med Today. 1998; 4: 194-5

Distribution of integrin cell adhesion receptors on normal bronchial epithelial cells and lung cancer cells in vitro and in vivo.

Mette SA, Pilewski J, Buck CA, Albelda SM
Am J Respir Cell Mol Biol. 1993; 8: 562-72

The interactions of bronchial epithelial cells with the basement membrane control cell morphology, differentiation, and proliferation in addition to having a major role in malignant transformation. Since these interactions are mediated by the integrin family of cell adhesion receptors, we characterized the integrin repertoire and adhesive properties of normal human bronchial epithelial cells in culture and cell lines derived from nine lung carcinomas using subunit-specific monoclonal antibodies. In addition, the integrin repertoire of three of the transformed cell lines was reexamined after the cells formed tumor nodules in immunodeficient mice. Bronchial epithelial cells in culture expressed multiple integrin subunits with the capability of binding to collagen and laminin (alpha 2, alpha 3, and alpha 6) and at least two subunits that are capable of mediating adhesion to fibronectin (alpha 3 and an alpha v-containing integrin). The alpha v beta 3 vitronectin receptor was not present. This distribution closely mimicked that seen by bronchial epithelial cells in situ. Cell lines derived from transformed pulmonary epithelial cells showed great heterogeneity with respect to integrin expression--some showing fewer, some greater, and some the same types of integrins as nontransformed epithelial cells. Only slight changes in integrin expression were seen in tumor cells propagated in immunodeficient mice. Although the adhesion characteristics of the transformed cells mirrored their adhesion receptor profile, no correlation between integrin profile and the ability to grow in SCID mice was observed. This study defines the integrin repertoire of human bronchial epithelial cells and sets the stage for future investigations exploring how the regulation and signal transduction mechanisms of these receptors might affect important pulmonary processes such as bronchial cell differentiation, wound healing, and malignant transformation.

Integrins minireview series.

McDonald JA
J Biol Chem. 2000; 275: 21783-21783

Integrin alpha- and beta 4-subunit-domain homologues in cyanobacterial proteins.

May AP, Ponting CP
Trends Biochem Sci. 1999; 24: 12-3

[Subclassification, molecular structure, function and ligand in integrin superfamily]

Matsuura N, Takada Y
Nippon Rinsho. 1995; 53: 1623-30

Integrins are the major family of cell surface receptors that mediate adhesion to the extracellular matrix and sometimes cell-cell adhesive interactions. These integrin-mediated adhesive interactions are involved in the regulation of many cellular functions, including embryonic development, tumor cell growth and metastasis, programmed cell death, hemostasis, inflammation, immune reaction, bone reabsorption, etc. Integrins are composed of alpha and beta transmembrane subunits selected from among 16 alpha and 8 beta subunits that heterodimerize to produce more than 20 different receptors which bind specific ligands. Ligand binding sites have been clarified by chimera integrin protein in some integrins. Integrins link to intracellular cytoskeletal complexes and bundles of actin filaments. There have been many reports about intracellular signaling pathways activated by integrin-ligand interactions.

Growth factor regulation of integrin-mediated cell motility.

Matsumoto K, Ziober BL, Yao CC, Kramer RH
Cancer Metastasis Rev. 1995; 14: 205-17

Cell motility, a primary component of tumor cell invasion, is a continuum of sequential events in which the cell extends pseudopodia, forms nascent attachments, assembles and contracts the cytoskeleton, and finally, as it translocates forward, disengages distal adhesions. What triggers cells to move? Substratum contact mediated by integrin adhesion receptors is important, but other signals such as chemokinetic factors appear to be required for continued crawling. It is now apparent that integrins do not simply bind cells to matrix in a Velcro-like fashion, but also are potent signaling molecules. Initial engagement of integrins induces their condensation into focal contacts, forming anchors to the extracellular matrix and discrete signal-transducing complexes on the cytoplasmic surface. A number of growth factors, through either autocrine or paracrine pathways, can activate the cellular machinery that mobilizes the cell. Thus, these two classes of receptors--the integrin receptors that bind specific extracellular adhesion molecules, and growth factor receptors that bind their respective ligands--can regulate cell locomotion. Not surprisingly, there is 'cross-talk' between integrin and growth factor receptors that occurs through their common intracellular signaling pathways. In this way, each receptor type can either amplify or attenuate the other's signal and downstream response. An example of growth factor-induced motility is the epithelial-mesenchymal transition induced by hepatocyte growth factor/scatter factor (HGF/SF). When bound to its receptor, the c-met proto-oncogene product, HGF/SF induces a phenotypic conversion that appears to be an important aspect of tumor progression in malignant carcinomas. The motogenic response produced by HGF/SF in carcinoma cells occurs in discrete steps in which integrins and focal adhesion kinase (p125FAK) are first recruited to focal contacts. This is rapidly followed by cell spreading, disruption of focal adhesions and cell-cell contacts, and, finally, cell crawling. The precise mechanism by which growth factors such as HGF/SF and its receptor induce this motogenic response and modulate integrin function has not been clearly defined but appears to involve several signaling pathways. Understanding the process by which growth factor and integrin receptors interact and regulate motility may suggest novel targets for therapeutic intervention.

Identification of integrins in cultured corneal fibroblasts and in isolated keratocytes.

Masur SK, Cheung JK, Antohi S
Invest Ophthalmol Vis Sci. 1993; 34: 2690-8

PURPOSE. The integrins are a family of transmembrane glycoproteins that function in attachment of cells to one another and to the extracellular matrix. When cell--cell and cell--matrix interactions are altered, the population of integrins may change. In particular, removing cells from their normal environment may be used as a model of wounding. The current study reports the identification of the integrins expressed at the cell surface of noncultured keratocytes and of cultured corneal fibroblasts, which are derived from keratocytes grown in primary culture. METHODS. For integrin identification, the surface proteins of keratocytes and cultured corneal fibroblasts were labeled with biotin, and the integrins were immunoprecipitated using anti-integrin antibodies. Attachment assays determined (1) the extracellular matrix preference of the cultured corneal fibroblasts and (2) the effects of function-perturbing antibodies against the fibronectin receptor (alpha 5 beta 1) or against other beta 1-containing integrins. RESULTS. The integrins of noncultured keratocytes were present as heterodimeric alpha, beta surface proteins that were immunoprecipitated by anti-beta 1, anti-alpha v, anti-alpha 6, anti-alpha 3, anti-alpha 1, and anti-beta 3. Furthermore, when the keratocytes were placed in culture, the integrin pattern changed. The classic fibronectin receptor, alpha 5 beta 1, is then expressed along with additional integrins that bind to fibronectin. Using attachment assays, we determined that the cultured corneal fibroblasts prefer fibronectin to collagen, vitronectin, or laminin as extracellular matrix substrate. In addition, function-perturbing antibodies against the fibronectin receptor (alpha 5 beta 1) or against beta 1 inhibit attachment of cultured corneal fibroblasts to fibronectin. CONCLUSIONS. Receptors for fibronectin and other extracellular matrix molecules are expressed at the cell surface in cultured corneal fibroblasts, and are in position to play a significant functional role as seen in attachment to extracellular matrix.

Integrin receptor signaling: the propagation of an alpha-helix model.

Marcantonio EE, David FS
Matrix Biol. 1997; 16: 179-84

Upon ligand binding to integrin receptors, a transmembrane conformation change occurs, which is required for the engagement of the actin cytoskeleton. Integrin receptor latency clearly involves the proximal portions of the alpha and beta cytoplasmic domains. Several experiments suggest that these two regions, which are highly conserved among integrins, may be associated, and this association is the structural basis for latency. We propose that ligand binding leads to a disruption of this association, which allows for the folding of the proximal beta cytoplasmic domain. Thus, in this model, the alpha chain association keeps the beta unfolded, and ligand binding leads to the propagation of an alpha helix from the transmembrane domain through the proximal beta cytoplasmic domain, leading to signal transduction.

Modulation of integrins expression during human osteoblasts "in vitro" differentiation.

Manduca P, Pistone M, Sanguineti C, Lu K, Stringa E
Boll Soc Ital Biol Sper. 1993; 69: 699-704

We here report the modulation of the adhesion of cultured human osteoblasts on Laminin during the acquisition of differentiated phenotype. We also show that interference with the differentiation program caused by treatment with Retinoic acid of the cultures, causes changes of the capability to adhere to Laminin and type I Collagen. The younger or dedifferentiated cells have lower capability to bind to Laminin or Collagen. The maturation associated changes are specific for the adhesion to the above substrata and do not involve the adhesion to FN or plastic. The alpha subunit(s) of the integrin receptor(s) for these proteins is likely to be responsible for the modulation adhesion to Laminin and Collagen.

Regulation of apoptosis by integrin receptors.

Malik RK
J Pediatr Hematol Oncol. 1997; 19: 541-5

[Integrins: a family of cell adhesion receptors]

Lustig L, Denduchis B
Medicina (B Aires). 1993; 53: 357-63

Integrins are a family of cell adhesion molecules (CAM's) that mediate the communication between the intracellular and the extracellular compartments. The growing interest in CAM's is due to the essential role they play in cell-cell and cell-matrix recognition processes. These receptors are formed by a non-covalently associated glycoprotein complex of two distinct polypeptide chains, called, alpha and beta. The association of different subunits results in the formation of, at least, 16 different integrins that provide cells with a great versatility in their adhesion properties. An integrin molecule comprises a cytoplasmic domain that interacts with the cytoskeleton, a transmembranous domain and an extracellular domain that binds to one or more ligands. beta 1, beta 2 and beta 3, are the best characterized integrin subfamilies; they are expressed, in different amounts, in epithelial and endothelial cells, leukocytes, fibroblasts and platelets. b1 integrins are essentially involved in cell-extracellular matrix interactions and beta 2 subfamily in leukocyte-leukocyte and leukocyte-endothelial cell communications. The integrin subfamily beta 3 mediates the adhesion of platelets with fibrinogen and other ligands. During embryonic development, integrins in association with other CAM's, play an essential role in cell migration and morphogenesis. Moreover, in processes like inflammation, wound healing and thrombosis, integrins and other CAM's mediate the interactions among the injured tissue and circulating cells. In two genetic diseases like the leukocyte adhesion deficiency and the Glanzmann's thrombasthenia an impairment in leukocyte-endothelial cell interactions and platelet aggregation is detected, due to deficiencies or abnormalities in beta 2 or beta 3 integrin subfamilies, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Association of the membrane proximal regions of the alpha and beta subunit cytoplasmic domains constrains an integrin in the inactive state.

Lu C, Takagi J, Springer TA
J Biol Chem. 2001; 276: 14642-8

The adhesiveness of integrins is regulated through a process termed "inside-out" signaling. To understand the molecular mechanism of integrin inside-out signaling, we generated K562 stable cell lines that expressed LFA-1 (alpha(L)beta(2)) or Mac-1 (alpha(M)beta(2)) with mutations in the cytoplasmic domain. Complete truncation of the beta(2) cytoplasmic domain, but not a truncation that retained the membrane proximal eight residues, resulted in constitutive activation of alpha(L)beta(2) and alpha(M)beta(2), demonstrating the importance of this membrane proximal region in the regulation of integrin adhesive function. Furthermore, replacement of the alpha(L) and beta(2) cytoplasmic domains with acidic and basic peptides that form an alpha-helical coiled coil caused inactivation of alpha(L)beta(2). Association of these artificial cytoplasmic domains was directly demonstrated. By contrast, replacement of the alpha(L) and beta(2) cytoplasmic domains with two basic peptides that do not form an alpha-helical coiled coil activated alpha(L)beta(2). Induction of ligand binding by the activating cytoplasmic domain mutations correlated with the induction of activation epitopes in the extracellular domain. Our data demonstrate that cytoplasmic, membrane proximal association between integrin alpha and beta subunits, constrains an integrin in the inactive conformation.

Epitope mapping of antibodies to the C-terminal region of the integrin beta 2 subunit reveals regions that become exposed upon receptor activation.

Lu C, Ferzly M, Takagi J, Springer TA
J Immunol. 2001; 166: 5629-37

The cysteine-rich repeats in the stalk region of integrin beta subunits appear to convey signals impinging on the cytoplasmic domains to the ligand-binding headpiece of integrins. We have examined the functional properties of mAbs to the stalk region and mapped their epitopes, providing a structure-function map. Among a panel of 14 mAbs to the beta(2) subunit, one, KIM127, preferentially bound to alpha(L)beta(2) that was activated by mutations in the cytoplasmic domains, and by Mn(2+). KIM127 also bound preferentially to the free beta(2) subunit compared with resting alpha(L)beta(2). Activating beta(2) mutations also greatly enhanced binding of KIM127 to integrins alpha(M)beta(2) and alpha(X)beta(2). Thus, the KIM127 epitope is shielded by the alpha subunit, and becomes reexposed upon receptor activation. Three other mAbs, CBR LFA-1/2, MEM48, and KIM185, activated alpha(L)beta(2) and bound equally well to resting and activated alpha(L)beta(2), differentially recognized resting alpha(M)beta(2) and alpha(X)beta(2), and bound fully to activated alpha(M)beta(2) and alpha(X)beta(2). The KIM127 epitope localizes within cysteine-rich repeat 2, to residues 504, 506, and 508. By contrast, the two activating mAbs CBR LFA-1/2 and MEM48 bind to overlapping epitopes involving residues 534, 536, 541, 543, and 546 in cysteine-rich repeat 3, and the activating mAb KIM185 maps near the end of cysteine-rich repeat 4. The nonactivating mAbs, 6.7 and CBR LFA-1/7, map more N-terminal, to subregions 344-432 and 432-487, respectively. We thus define five different beta(2) stalk subregions, mAb binding to which correlates with effect on activation, and define regions in an interface that becomes exposed upon integrin activation.

alpha1 Integrin cytoplasmic domain is involved in focal adhesion formation via association with intracellular proteins.

Loster K, Vossmeyer D, Hofmann W, Reutter W, Danker K
Biochem J. 2001; 356: 233-40

Integrins are heterodimeric adhesion receptors consisting of alpha- and beta-subunits capable of binding extracellular matrix molecules as well as other adhesion receptors on neighbouring cells. These interactions induce various signal transduction pathways in many cell types, leading to cytoskeletal reorganization, phosphorylation and induction of gene expression. Integrin ligation leads to cytoplasmic protein-protein interactions requiring both integrin cytoplasmic domains, and these domains are initiation points for focal adhesion formation and subsequent signal transduction cascades. In previous studies we have shown that the very short cytoplasmic alpha1 tail is required for post-ligand events, such as cell spreading as well as actin stress-fibre formation. In the present paper we report that cells lacking the cytoplasmic domain of the alpha1 integrin subunit are unable to form proper focal adhesions and that phosphorylation on tyrosine residues of focal adhesion components is reduced on alpha1beta1-specific substrates. The alpha1 cytoplasmic sequence is a specific recognition site for focal adhesion components like paxillin, talin, alpha-actinin and pp125FAK. It seems to account for alpha1-specific signalling, since when peptides that mimic the cytoplasmic domain of alpha1 are transferred into cells, they influence alpha1beta1-specific adhesion, presumably by competing for binding partners. For alpha1 integrin/protein binding, the conserved Lys-Ile-Gly-Phe-Phe-Lys-Arg motif and, in particular, the two lysine residues, are important.

Integrin-mediated signal transduction.

Longhurst CM, Jennings LK
Cell Mol Life Sci. 1998; 54: 514-26

Integrins, expressed on virtually every cell type, are proteins that mediated cellular interactions with components of the extracellular matrix (ECM) and cell surface integral plasma membrane proteins. In addition, integrins interact with the cytoskeleton and through this process participate in cell migration, tissue organization, cell growth, haemostasis, inflammation, target recognition of lymphocytes and the differentiation of many cell types. Signals generated from ligand-integrin interactions are propagated via the integrin cytoplasmic tails to signal transduction pathways within the cell (outside-in signalling). Information from within the cell can also be transmitted to the outside via integrin affinity modulation (inside-out signalling). Protein tyrosine phosphorylation has a central role in integrin-initiated cell signalling, leading to cytoskeletal organization and focal adhesion formation. This review will examine the current understanding of integrin function, focusing on the intracellular consequences of integrin-ligand interaction.

New insights into integrin-ligand interaction.

Loftus JC, Liddington RC
J Clin Invest. 1997; 100: 7781-7781

Cell adhesion in vascular biology. New insights into integrin-ligand interaction.

Loftus JC, Liddington RC
J Clin Invest. 1997; 99: 2302-6

Chondrocyte integrin expression and function.

Loeser RF
Biorheology. 2000; 37: 109-16

The extracellular matrix (ECM) is an "information rich" environment and interactions between the chondrocyte and ECM regulate many biological processes important to cartilage homeostasis and repair including cell attachment, growth, differentiation, and survival. The integrin family of cell surface receptors appears to play a major role in mediating cell-matrix interactions that are important in regulating these processes. Chondrocytes have been found to express several members of the integrin family which can serve as receptors for fibronectin (alpha 5 beta 1), types II and VI collagen (alpha 1 beta 1, alpha 2 beta 1, alpha 10 beta 1), laminin (alpha 6 beta 1), and vitronectin and osteopontin (alpha V beta 3). Integrin expression can be regulated by growth factors including IGF-I and TGF-beta. By providing a link between the ECM and the cytoskeleton, integrins may be important transducers of mechanical stimuli. Integrin binding stimulates intracellular signaling which can affect gene expression and regulate chondrocyte function. Further studies are needed to more clearly define the role of integrins in cartilage.

A model for the structure of the P domains in the subtilisin-like prohormone convertases.

Lipkind GM, Zhou A, Steiner DF
Proc Natl Acad Sci U S A. 1998; 95: 7310-5

The proprotein convertases are a family of at least seven calcium-dependent endoproteases that process a wide variety of precursor proteins in the secretory pathway. All members of this family possess an N-terminal proregion, a subtilisin-like catalytic module, and an additional downstream well-conserved region of approximately 150 amino acid residues, the P domain, which is not found in any other subtilase. The pro and catalytic domains cannot be expressed in the absence of the P domains; their thermodynamic instability may be attributable to the presence of large numbers of negatively charged Glu and Asp side chains in the substrate binding region for recognition of multibasic residue cleavage sites. Based on secondary structure predictions, we here propose that the P domains consist of 8-stranded beta-barrels with well-organized inner hydrophobic cores, and therefore are independently folded components of the proprotein convertases. We hypothesize further that the P domains are integrated through strong hydrophobic interactions with the catalytic domains, conferring structural stability and regulating the properties and activity of the convertases. A molecular model of these interdomain interactions is proposed in this report.

Evidence that the integrin beta3 and beta5 subunits contain a metal ion-dependent adhesion site-like motif but lack an I domain.

Lin EC, Ratnikov BI, Tsai PM, Gonzalez ER, McDonald S, Pelletier AJ, Smith JW
J Biol Chem. 1997; 272: 14236-43

The amino-terminal domain of each integrin beta subunit is hypothesized to contain an ion binding site that is key to cell adhesion. A new hypothesis regarding the structure of this site is suggested by the crystallization of the I domains of the integrin alphaL and alphaM subunits (Lee, J.-O., Rieu, P., Arnaout, M. A., and Liddington, R. (1995) Cell 80, 631-638; Qu, A., and Leahy, D. J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10277-10281). In those proteins, an essential metal ion is bound by a metal ion-dependent adhesion site (MIDAS). The MIDAS is presented at the apex of a larger protein module called an I domain. The metal ligands in the MIDAS can be separated into three distantly spaced clusters of oxygenated residues. These three coordination sites also appear to exist in the integrin beta3 and beta5 subunits. Here, we examined the putative metal binding site within beta3 and beta5 using site-directed mutagenesis and ligand binding studies. We also investigated the fold of the domain containing the putative metal binding site using the PHD structural algorithm. The results of the study point to the similarity between the integrin beta subunits and the MIDAS motif at two of three key coordination points. Importantly though, the study failed to identify a residue in either beta subunit that corresponds to the second metal coordination group in the MIDAS. Moreover, structural algorithms indicate that the fold of the beta subunits is considerably different than the I domains. Thus, the integrin beta subunits appear to present a MIDAS-like motif in the context of a protein module that is structurally distinct from known I domains.

Structural features of GPI-specific phospholipase D revealed by proteolytic fragmentation and Ca2+ binding studies.

Li JY, Hollfelder K, Huang KS, Low MG
J Biol Chem. 1994; 269: 28963-71

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is abundant in plasma and is potentially capable of degrading the anchor utilized by many cell surface proteins. The goal of this work was to study structural features of the GPI-PLD that might be involved in regulation of its activity. Trypsin cleaved the 100-110 kDa GPI-PLD polypeptide into three major fragments (two of approximately 40 kDa and a carboxyl-terminal fragment of 30 kDa) which were relatively resistant to further proteolysis. Pretreatment of the GPI-PLD with chelators resulted in complete degradation. During the cleavage process the GPI-PLD enzymatic activity increased approximately 3-4-fold but no other major change in its properties (e.g. inhibition by chelators and lipids, thermal stability, oligomerization, etc.) was observed. Intact or trypsinized GPI-PLD bound 45Ca2+ (approximately 5.5 ions/molecule GPI-PLD; Kd approximately 16.1 microM as determined by equilibrium dialysis) which could not be blocked by the addition of other divalent metal ions. However, inhibition of enzymatic activity by divalent cation chelators appeared to involve removal of bound Zn2+ rather than Ca2+. A metal analysis of GPI-PLD revealed approximately 5 and 10 atom/molecule of calcium and zinc, respectively. The data suggest that the predicted integrin E-F hand-like sites in GPI-PLD are functional but not directly involved in enzymatic activity.

The structural basis of dynamic cell adhesion: heads, tails, and allostery.

Liddington RC, Bankston LA
Exp Cell Res. 2000; 261: 37-43

Paracrine signaling in the endometrium: integrins and the establishment of uterine receptivity.

Lessey BA, Arnold JT
J Reprod Immunol. 1998; 39: 105-16

The importance of cell shape and polarity have long been recognized. In the endometrium, a complex association of different cell types undergo cyclic renewal, differentiation and eventually apoptosis and shedding, with the sole purpose of allowing implantation of the nascent embryo. Many of these physiologic processes depend on the timely expression of cell adhesion molecules which maintain tissue architecture by mediating cell-cell and cell-substratum attachments. One family of cell adhesion molecules are the integrins, cell surface glycoproteins composed of heterodimeric alpha and beta subunits that serve as receptors for the extracellular matrix. We have studied the endometrium as a unique site of integrin expression that appear to be under both endocrine and paracrine control. In addition, it is suspected that the engagement of integrins by extracellular matrix on the embryo results in signal transduction leading to the transcription and translation of genes critical for implantation. While the role of integrins in endometrial function is not yet clearly defined, these interesting molecules represent excellent markers of normal and abnormal states of receptivity and may provide clues to the regulation and mechanics of implantation in general.

Integrins and reproduction revisited.

Lessey BA
Eur J Obstet Gynecol Reprod Biol. 1995; 62: 264-5

Extracellular matrix signaling from the cellular membrane skeleton to the nuclear skeleton: a model of gene regulation.

Lelievre S, Weaver VM, Bissell MJ
Recent Prog Horm Res. 1996; 51: 417-32

It is well established that cells must interact with their microenvironment and that such interaction is crucial for coordinated function and homeostasis. However, how cells receive and integrate external signals leading to gene regulation is far from understood. It is now appreciated that two classes of cooperative signals are implicated: a soluble class including hormones and growth factors and a class of insoluble signals emanating from the extracellular matrix (ECM) directly through contact with the cell surface. Using 3-dimensional culture systems and transgenic mice, we have been able to identify some of the elements of this ECM-signaling pathway responsible for gene regulation in rodent mammary gland differentiation and involution. Our major observations are 1) the requirement for a laminin-rich basement membrane; 2) the existence of a cooperative signaling pathway between basement membrane and the lactogenic hormone prolactin (PRL);3) the importance of beta 1-integrins and bHLH transcription factor(s) and the presence of DNA response elements (exemplified by BCE-1, located on a milk protein gene, beta-casein); and 4) the induction of mammary epithelial cell programmed cell death following degradation of basement membrane. We hypothesize that this cooperative signaling between ECM and PRL may be achieved through integrin- and laminin-directed restructuring of the cytoskeleton leading to profound changes in nuclear architecture and transcription factor localization. We postulate that the latter changes allow the prolactin signal to activate transcription of the beta-casein gene. To further understand the molecular mechanisms underlying ECM and hormonal cooperative signaling, we are currently investigating ECM regulation of a "solid-state" signaling pathway including ECM fiber proteins, plasma membrane receptors, cytoskeleton, nuclear matrix and chromatin. We further postulate that disruption of such a pathway may be implicated in cell disorders including transformation and carcinogenesis.

The regulation of integrin function by Ca(2+).

Leitinger B, McDowall A, Stanley P, Hogg N
Biochim Biophys Acta. 2000; 1498: 91-8

Integrins are metalloproteins whose receptor function is dependent on the interplay between Mg(2+) and Ca(2+). Although the specificity of the putative divalent cation binding sites has been poorly understood, some issues are becoming clearer and this review will focus on the more recent information. The MIDAS motif is a unique Mg(2+)/Mn(2+) binding site located in the integrin alpha subunit I domain. Divalent cation bound at this site has a structural role in coordinating the binding of ligand to the I domain containing integrins. The I-like domain of the integrin beta subunit also has a MIDAS-like motif but much less is known about its cation binding preferences. The N-terminal region of the integrin alpha subunit has been modelled as a beta-propeller, containing three or four 'EF hand' type divalent cation binding motifs for which the function is ill defined. It seems certain that most integrins have a high affinity Ca(2+) site which is critical for alphabeta heterodimer formation, but the location of this site is unknown. Finally intracellular Ca(2+) fluxes activate the Ca(2+) requiring enzyme, calpain, which regulates cluster formation of leucocyte integrins.

Integrin I domains and their function.

Leitinger B, Hogg N
Biochem Soc Trans. 1999; 27: 826-32

Structure and function of leukocyte integrins.

Larson RS, Springer TA
Immunol Rev. 1990; 114: 181-217

Collagen and collagenase gene expression in three-dimensional collagen lattices are differentially regulated by alpha 1 beta 1 and alpha 2 beta 1 integrins.

Langholz O, Rockel D, Mauch C, Kozlowska E, Bank I, Krieg T, Eckes B
J Cell Biol. 1995; 131: 1903-15

The reorganization of extracellular matrix (ECM) is an important function in many biological and pathophysiological processes. Culture of fibroblasts in a three-dimensional collagenous environment represents a suitable system to study the underlying mechanisms resulting from cell-ECM interaction, which leads to reprogramming of fibroblast biosynthetic capacity. The aim of this study was to identify receptors that transduce ECM signals into cellular events, resulting in reprogramming of connective tissue metabolism. Our data demonstrate that in human skin fibroblasts alpha 1 beta 1 and alpha 2 beta 1 integrins are the major receptors responsible for regulating ECM remodeling: alpha 1 beta 1 mediates the signals inducing downregulation of collagen gene expression, whereas the alpha 2 beta 1 integrin mediates induction of collagenase (MMP-1). Applying mAb directed against different integrin subunits resulted in triggering the heterodimeric receptors and enhancing the normal biochemical response to receptor ligation. Different signal transduction inhibitors were tested for their influence on gel contraction, expression of alpha 1(I) collagen and MMP-1 in fibroblasts within collagen gels. Ortho-vanadate and herbimycin A displayed no significant effect on any of these three processes. In contrast, genistein reduced lattice contraction, and completely inhibited induction of MMP-1, whereas type I collagen down-regulation was unaltered. Calphostin C inhibited only lattice contraction. Taken together, these data indicate a role of tyrosine-specific protein kinases in mediating gel contraction and induction of MMP-1, as well as an involvement of protein kinase C in the contraction process. The data presented here indicate that different signaling pathways exist leading to the three events discussed here, and that these pathways do not per se depend upon each other.

Mechano-sensing and mechano-reaction of soft connective tissue cells.

Lambert ChA, Nusgens BV, Lapiere ChM
Adv Space Res. 1998; 21: 1081-91

One main function of the connective tissues is to provide cells with a mechanically resistant attachment support required for survival, division and differentiation. All cells contain membrane-anchored attachment proteins able to recognize specific chemical motifs in the extracellular macromolecules forming the supporting scaffold, made of various types of collagen, adhesive glycoproteins, elastin, proteoglycans, etc... These cell-matrix interactions are mainly mediated by receptors of the integrins family, heterodimeric molecules made of an extracellular domain connected through a transmembrane sequence to an intracytoplasmic tail. Upon recognition of the extracellular ligand, the clustering and activation of the integrins result in the recruitment of a complex of proteins and formation of the focal adhesion plaque, containing both cytoskeletal and catalytic signaling molecules. Activation results in polymerization of actin and formation of stress fibers. These structures establish a physical link between the extracellular matrix components and the cytoskeleton through the integrins providing a continuous path acting as a mechanotransducer. This connection is used by the cells to perform their mechanical functions as adhesion, migration and traction. In vitro experimental models using fibroblasts in a collagen gel demonstrate that cells are in mechanical equilibrium with their support which regulates their replicative and biosynthetic phenotype. The present review discusses the molecular structures operating in the transmission of the mechanical messages from the support to the connective tissue cells, and their effect on the cellular machinery. We present arguments for investigating these mechanisms in understanding the perception of reduced gravity and the resulting reaction leading to microgravity induced pathologies.

Integrins and matrix molecules in salivary gland cell adhesion, signaling, and gene expression.

Lafrenie RM, Yamada KM
Ann N Y Acad Sci. 1998; 842: 42-8

Integrins play crucial roles in embryonic and adult cell adhesion, migration, morphogenesis, growth, and differentiation in many cell systems, including human salivary gland cells. Integrins function by binding through their extracellular domain to a specific peptide recognition site in a ligand, and then transmitting information to the cytoplasm by way of their cytoplasmic tails. By this transmembrane signaling process, integrins can mediate assembly of adhesion sites and organization of the actin-containing cytoskeleton by forming supermolecular complexes of cytoskeletal and signaling molecules. The specific steps in the assembly of these complexes as well as novel mechanisms for synergy between integrin and growth factor signaling pathways are still being determined. integrin-mediated interactions also have major effects on gene expression. For example, integrin-mediated adhesion to fibronectin by the HSG salivary gland cell line significantly alters the pattern of proteins synthesized and genes expressed. In fact, at least five transcription factors are activated, and over 30 genes (many of them novel) are found to be induced by such integrin-mediated interactions by salivary gland cells. The roles of integrins, in collaborative interactions with growth factors and signaling pathways, and in the induction of novel genes during salivary gland development, should provide fruitful areas of research for many years.

Single subunit chimeric integrins as mimics and inhibitors of endogenous integrin functions in receptor localization, cell spreading and migration, and matrix assembly.

LaFlamme SE, Thomas LA, Yamada SS, Yamada KM
J Cell Biol. 1994; 126: 1287-98

The ability of single subunit chimeric receptors containing various integrin beta intracellular domains to mimic and/or inhibit endogenous integrin function was examined. Chimeric receptors consisting of the extracellular and transmembrane domains of the small subunit of the human interleukin-2 receptor connected to either the beta 1, beta 3, beta 3B, or beta 5 intracellular domain were transiently expressed in normal human fibroblasts. When expressed at relatively low levels, the beta 3 and beta 5 chimeras mimicked endogenous ligand-occupied integrins and, like the beta 1 chimera (LaFlamme, S. E., S. K. Akiyama, and K. M. Yamada. 1992. J. Cell Biol. 117:437), concentrated with endogenous integrins in focal adhesions and sites of fibronectin fibril formation. In contrast, the chimeric receptor containing the beta 3B intracellular domain (a beta 3 intracellular domain modified by alternative splicing) was expressed diffusely on the cell surface, indicating that alternative splicing can regulate integrin receptor distribution by an intracellular mechanism. Furthermore, when expressed at higher levels, the beta 1 and beta 3 chimeric receptors functioned as dominant negative mutants and inhibited endogenous integrin function in localization to fibronectin fibrils, fibronectin matrix assembly, cell spreading, and cell migration. The beta 5 chimera was a less effective inhibitor, and the beta 3B chimera and the reporter lacking an intracellular domain did not inhibit endogenous integrin function. Comparison of the relative levels of expression of the transfected beta 1 chimera and the endogenous beta 1 subunit indicated that in 10 to 15 h assays, the beta 1 chimera can inhibit cell spreading when expressed at levels approximately equal to the endogenous beta 1 subunit. Levels of chimeric receptor expression that inhibited cell spreading also inhibited cell migration, whereas lower levels were able to inhibit alpha 5 beta 1 localization to fibrils and matrix assembly. Our results indicate that single subunit chimeric integrins can mimic and/or inhibit endogenous integrin receptor function, presumably by interacting with cytoplasmic components critical for endogenous integrin function. Our results also demonstrate that beta intracellular domains, expressed in this context, display specificity in their abilities to mimic and inhibit endogenous integrin function. Furthermore, the approach that we have used permits the analysis of intracellular domain function in the processes of cell spreading, migration and extracellular matrix assembly independent of effects due to the rest of integrin dimers. This approach should prove valuable in the further analysis of integrin intracellular domain function in these and other integrin-mediated processes requiring the interaction of integrins with cytoplasmic components.

Integrin cytoplasmic domains as connectors to the cell's signal transduction apparatus.

LaFlamme SE, Homan SM, Bodeau AL, Mastrangelo AM
Matrix Biol. 1997; 16: 153-63

Integrins mediate the bidirectional transfer of signals across the plasma membrane. Integrin cytoplasmic domains provide one pathway linking integrin engagement with the cell's signal transduction apparatus. Recent structure-function studies have defined regions of beta cytoplasmic domains required for integrin function and have identified distinct roles for individual alpha cytoplasmic domains in regulating cell behavior. Newly identified proteins that bind to integrin alpha and beta cytoplasmic domains have provided new insights and new questions into the mechanisms involved in integrin signaling.

[Integrins]

Lafage MH, Alexandre C
Rev Rhum Ed Fr. 1994; 61: 575-81

Signaling by integrin receptors.

Kumar CC
Oncogene. 1998; 17: 1365-73

Adhesive interactions are critical for the proliferation, survival and function of all cells. Integrin receptors as the major family of adhesion receptors have been the focus of study for more than a decade. These studies have tremendously enhanced our understanding of the integrin-mediated adhesive interactions and have unraveled novel integrin functions in cell survival mechanisms and in the activation of divergent signaling pathways. The signals from integrin receptors are integrated from those originating from growth factor receptors in order to organize the cytoskeleton, stimulate cell proliferation and rescue cells from matrix detachment-induced programmed cell death. These functions are critical in the regulation of multiple processes such as tissue development, inflammation, angiogenesis, tumor cell growth and metastasis and programmed cell death.

Functions of alpha3beta1 integrin.

Kreidberg JA
Curr Opin Cell Biol. 2000; 12: 548-53

alpha3beta1 integrin is a laminin receptor with apparently diverse functions. In epithelial cells it acts as a receptor for the basement membrane, whereas in neuronal and possibly tumor cells it mediates migration. Interactions of alpha3beta1 integrin with tetraspanin proteins may provide clues to how it transduces signals that affect cell behavior.

Signal transduction by integrins: increased protein tyrosine phosphorylation caused by clustering of beta 1 integrins.

Kornberg LJ, Earp HS, Turner CE, Prockop C, Juliano RL
Proc Natl Acad Sci U S A. 1991; 88: 8392-6

The integrin family of cell adhesion receptors mediates many of the interactions between cells and the extracellular matrix. Because the extracellular matrix has profound influences on cell behavior, it seems likely that integrins transduce biochemical signals across the cell membrane. The nature of these putative signals has, thus far, remained elusive. Antibody-mediated clustering of integrin receptors was used to mimic the integrin clustering process that occurs during formation of adhesive contacts. Human epidermal carcinoma (KB) cells were incubated with an anti-beta 1 integrin monoclonal antibody for 30 min on ice followed by incubation at 37 degrees C with anti-rat IgG. This treatment, which induced integrin clustering, stimulated the phosphorylation on tyrosine residues of a 115- to 130-kDa complex of proteins termed pp130. When integrins were clustered in the presence of the phosphatase inhibitor sodium orthovanadate, pp130 showed a substantial increase in phosphorylation compared to the case in which integrins were clustered in the absence of vanadate. Maximal pp130 phosphorylation was observed 10-20 min after initiation of integrin clustering in the absence of vanadate or after 5-10 min in its presence. These time courses roughly parallel the formation of integrin clusters on the cell surface as observed by fluorescence microscopy. pp130 phosphorylation depended on the amount of anti-integrin antibody present. Additionally, the tyrosine phosphorylation of pp130 showed specificity since it was stimulated by antibodies to the integrin alpha 3 and beta 1 subunits but not by antibodies to other integrin alpha subunits or to nonintegrin cell surface proteins. Immunoprecipitation experiments clearly demonstrated that pp130 is not itself a beta 1 integrin. It is postulated, therefore, that the integrin-stimulated tyrosine phosphorylation of pp130 may reflect part of an important signal transduction process between the extracellular matrix and the cell interior.

Regulation of integrin function by inside-out signaling mechanisms.

Kolanus W, Zeitlmann L
Curr Top Microbiol Immunol. 1998; 231: 33-49

Integrins and inside-out signal transduction: converging signals from PKC and PIP3.

Kolanus W, Seed B
Curr Opin Cell Biol. 1997; 9: 725-31

Recent studies have identified molecules that interact with integrins and appear to participate in the signaling pathways that regulate integrin adhesiveness. Clues provided by studies of these molecules point to the integration by integrins of signal transduction pathways implicated in cell division and activation.

Integrin mediation of type II cell adherence to provisional matrix proteins.

Kim HJ, Ingbar DH, Henke CA
Am J Physiol. 1996; 271: 27786-27786

Lung injury causes alveolar type I epithelial cell death, basement membrane denudation, and alveolar flooding with serum fibronectin and fibrinogen. For successful restoration of normal architecture, the epithelium must be regenerated from progenitor type II alveolar cells. Using adhesion assays, we examined whether type II alveolar cells adhere to the provisional matrix proteins fibronectin, fibrinogen, and fibrin, and whether integrins mediate this adherence. Rat type II cells adhered to fibronectin, vitronectin, fibrinogen, and fibrin. Synthetic RGD (arginine-glycine-aspartic acid) peptide blocked this adhesion. Flow cytometry and Western analysis indicated that type II cells expressed beta 1- and alpha v beta 3-integrins. Anti-beta 1-and anti-alpha v beta 3-integrin antibodies blocked type II cell adhesion to fibronectin and to fibronectin and fibrinogen, respectively. In summary, type II cells adhered to fibronectin, fibrinogen, and fibrin, and adhesion was partially mediated by integrins. This study provides the first evidence of type II cell adhesion to fibrin gels and vitronectin, beta 1- and alpha v beta 3-integrin mediation of type II cell adhesion, and the presence of the alpha v beta 3-integrin on type II epithelial cells.

The role of the I domain in ligand binding of the human integrin alpha 1 beta 1.

Kern A, Briesewitz R, Bank I, Marcantonio EE
J Biol Chem. 1994; 269: 22811-6

We report here the analysis of potential ligand binding domains within the human integrin alpha 1 subunit, a known collagen/laminin receptor. This integrin is effectively blocked by the mouse monoclonal antibody 1B3.1. A truncated version of the alpha 1 subunit lacking the NH2-terminal half of the extracellular domain is not recognized by monoclonal antibody 1B3.1. Furthermore, we have isolated a cDNA containing the I domain from chicken alpha 1 bearing significant homology to the human and rat alpha 1 sequences. Replacing the human I domain with its chicken counterpart led to the surface expression of a functional heterodimer with endogenous mouse beta 1 on NIH 3T3 cells. However, 1B3.1 does not bind to the chicken/human chimera, demonstrating that the human alpha 1 I domain is required for epitope recognition. Mutation of Asp253 within the I domain to alanine resulted in surface expression of an alpha beta heterodimer recognized by 1B3.1 but with markedly reduced binding to collagen IV or laminin. Since a previously reported mutation of a homologous Asp in the Mac-1 I domain has similar consequences, these results suggest a central role for the I domain in ligand recognition for all integrin alpha subunits containing this domain.

[Cell adhesion and proteases]

Kawashima S
Tanpakushitsu Kakusan Koso. 1997; 42: 2255-62

Integrins in morphogenesis and signaling.

Katz BZ, Yamada KM
Biochimie. 1997; 79: 467-76

Integrins are a family of heterodimeric transmembrane receptors that provide a physical and biochemical bridge between components of the extracellular matrix and the intracellular physiological environment. Binding of integrins to their ligands results in the formation of cytoplasmic multi-protein assemblies composed of both cytoskeletal and signaling molecules. The composition and activity of these assemblies is regulated by the nature of integrin-ligand interactions, as well as by intracellular regulators that include tyrosine kinases and phosphatases, PKC, and small GTPases. Integrin-mediated cellular physiological responses include the activation of signal transduction, cytoskeletal rearrangements, and co-regulation of growth factor activities. These responses, combined with integrin-mediated cell adhesion, play a major role in tissue morphogenesis and developmental processes.

Integrin-ligand interactions: scratching the surface.

Kanazashi SI, Sharma CP, Arnaout MA
Curr Opin Hematol. 1997; 4: 67-74

Integrins provide cells with critical means of communication with their microenvironments. By linking events at the cell surface to a dynamic cytoskeleton-signaling complex, integrins enable cells to rapidly modify their mechanical and genetic machinery in response to environmental cues. This review highlights major advances made in understanding the structural basis of integrin-ligand interactions and their regulation.

Interaction between collagen and the alpha(2) I-domain of integrin alpha(2)beta(1). Critical role of conserved residues in the metal ion-dependent adhesion site (MIDAS) region.

Kamata T, Liddington RC, Takada Y
J Biol Chem. 1999; 274: 32108-11

A docking model of the alpha(2) I-domain and collagen has been proposed based on their crystal structures (Emsley, J., King, S., Bergelson, J., and Liddington, R. C. (1997) J. Biol. Chem. 272, 28512-28517). In this model, several amino acid residues in the I-domain make direct contact with collagen (Asn-154, Asp-219, Leu-220, Glu-256, His-258, Tyr-285, Asn-289, Leu-291, Asn-295, and Lys-298), and the protruding C-helix of alpha(2) (residues 284-288) determines ligand specificity. Because most of the proposed critical residues are not conserved, different I-domains are predicted to bind to collagen differently. We found that deleting the entire C-helix or mutating the predicted critical residues had no effect on collagen binding to whole alpha(2)beta(1), with the exception that mutating Asn-154, Asp-219, and His-258 had a moderate effect. We performed further studies and found that mutating the conserved surface-exposed residues in the metal ion-dependent adhesion site (MIDAS) (Tyr-157 and Gln-215) significantly blocks collagen binding. We have revised the docking model based on the mutagenesis data. In the revised model, conserved Tyr-157 makes contact with collagen in addition to the previously proposed Asn-154, Asp-219, His-258, and Tyr-285 residues. These results suggest that the collagen-binding I-domains (e.g. alpha(1), alpha(2), and alpha(10)) bind to collagen in a similar fashion.

Adhesion molecules in cancer: the role of integrins.

Juliano RL, Varner JA
Curr Opin Cell Biol. 1993; 5: 812-8

It has been known for some time that cell adhesion receptors, including members of the integrin family, play an important role in the biology of tumors. Until recently, most of the emphasis in this area of research has concerned the functioning of integrins as adhesive molecules in the invasive and metastatic behavior of malignant cells. Now it has become clear that integrins can function as true receptors capable of transducing signals to the cell interior. Tyrosine phosphorylation seems to be a key aspect of integrin-mediated signal transduction, and a new tyrosine kinase has been described that seems to be important in this process. Evidence is accumulating that integrin-mediated signals can induce gene expression and affect transit through the cell cycle. Thus, the role of integrins in cancer seems not only to involve cell adhesion events, but may also involve the regulation of tumor cell growth and differentiation.

Signal transduction from the extracellular matrix.

Juliano RL, Haskill S
J Cell Biol. 1993; 120: 577-85

Integrin signals and tumor growth control.

Juliano RL
Princess Takamatsu Symp. 1994; 24: 118-24

Integrins can trigger signals by activation of cytoplasmic tyrosine kinases, including pp125FAK. Preliminary evidence suggests that serine/threonine kinases such as ERKs may also be activated via integrins. Thus, there seems to be at least partial overlap between RTK signaling pathways and integrin signaling. In tumor cells, ectopic expression or over-expression of certain integrins such as alpha 5/beta 1 can result in reduced tumorigenesis. Presumably the effects of integrins on tumor growth are mediated by the integrin signaling pathway(s) involving FAK and ERKs. However, the precise mechanisms involved have not yet been elucidated.

Signal transduction by integrins and its role in the regulation of tumor growth.

Juliano R
Cancer Metastasis Rev. 1994; 13: 25-30

It has become clear that integrins have effects on tumorigenesis that are in addition to their role in mediating cell adhesion during invasive and metastatic processes. Integrins can transduce signals from the extracellular matrix to cell interior, via tyrosine kinases, and possibly by other mechanisms as well. These integrin mediated signals contribute to control of gene expression, to regulation of anchorage-dependence, to cell cycle transit, and to the control of tumor growth.

Cooperation between soluble factors and integrin-mediated cell anchorage in the control of cell growth and differentiation.

Juliano R
Bioessays. 1996; 18: 911-7

Recently it has become clear that integrins and other adhesive receptors play an important role in the control of cell growth and differentiation. In various cell types, anchorage to the extracellular matrix via integrins strongly influences the ability of the cell to respond to soluble mitogens or to differentiation factors. Thus adhesive receptors must generate signals that influence cell behavior. Some of the pathways of adhesion receptor signaling are now beginning to be worked out, but there is still much to learn. In particular, the mechanistic basis for the cooperation between anchorage signals and signals from soluble growth and differentiation factors remains ill-defined. This review will examine some of the current information linking adhesion receptors to control of mitogenesis and differentiation.

Integrins: a role as cell signalling molecules.

Jones JL, Walker RA
Mol Pathol. 1999; 52: 208-13

Integrins form the major family of proteins that mediates cell-matrix interactions. As well as an adhesive function, it is increasingly apparent that integrins can transduce messages via classic signalling pathways and impact upon such fundamental cellular processes as proliferation, apoptosis, differentiation, and motility. Dysregulation of these processes are a feature of many malignancies. Altered integrin expression has been observed in many human tumours, and perturbation of integrin function or expression in experimental systems has demonstrated that altered integrin signalling may directly contribute to the development of the malignant phenotype.

Adhesion receptors and cell invasion: mechanisms of integrin-guided degradation of extracellular matrix.

Ivaska J, Heino J
Cell Mol Life Sci. 2000; 57: 16-24

The integrins are a large family of heterodimeric cell adhesion receptors mediating cell-matrix and cell-cell adhesion. They seem to play a central role in cell migration and invasion and are therefore essential in processes such as healing of tissue injuries and the progression of human cancer. Integrins function in cell invasion by mediating cell movement on matrix molecules and also by regulating the expression of matrix-degrading enzymes, namely the matrix metalloproteinases. Here we review recent findings on the mechanisms by which integrins regulate matrix degradation. A novel, multistep model of integrin-guided collagen degradation is proposed.

Multiple loop structures critical for ligand binding of the integrin alpha4 subunit in the upper face of the beta-propeller mode 1.

Irie A, Kamata T, Takada Y
Proc Natl Acad Sci U S A. 1997; 94: 7198-203

A non-I-domain integrin, alpha4beta1, recognizes vascular cell adhesion molecule 1 (VCAM-1) and the IIICS portion of fibronectin. To localize regions of alpha4 critical for ligand binding, we swapped several predicted loops within or near the putative ligand-binding site of alpha4 (which spans repeats 2-5 of the seven N-terminal repeats) with the corresponding regions of alpha5. Swapping residues 112-131 in repeat 2, or residues 237-247 in repeat 4, completely blocked adhesion to immobilized VCAM-1 and connecting segment 1 (CS-1) peptide. However, swapping residues 40-52 in repeat 1, residues 151-164 in repeat 3, or residues 282-288 (which contain a putative cation binding motif) in repeat 5 did not affect or only slightly reduced adhesion to these ligands. The binding of several function-blocking antibodies is blocked by swapping residues 112-131, 151-164, and 186-191 (which contain previously identified residues critical for ligand binding, Tyr-187 and Gly-190). These results are consistent with the recently published beta-propeller folding model of the integrin alpha4 subunit [Springer, T. A. (1997) Proc. Natl. Acad. Sci. USA 94, 65-72], in which seven four-stranded beta-sheets are arranged in a torus around a pseudosymmetric axis. The regions of alpha4 critical for ligand binding are adjacent to each other and are located in the upper face, the predicted ligand-binding site, of the beta-propeller model, although they are not adjacent in the primary structure.

[Molecular mechanisms of integrin-ligand interaction]

Irie A, Kamata T, Takada Y
Tanpakushitsu Kakusan Koso. 1997; 42: 1679-86

Hepatocyte-matrix interactions.

Iredale JP, Arthur MJ
Gut. 1994; 35: 729-32

Integrins, tensegrity, and mechanotransduction.

Ingber DE
Gravit Space Biol Bull. 1997; 10: 49-55

Physical forces, such as those due to gravity, play an important role in tissue development and remodeling. Yet, little is known about how individual cells sense mechanical signals or how they transduce them into a chemical response. Rather than listing the numerous signal pathways that have been found to be sensitive to mechanical stimulation, we need to place potential molecular signaling mechanisms within the context of the entire cell. The model presented is based on the concept that cells use tensegrity architecture to organize their cytoskeleton and stabilize their form. Studies with stick and string tensegrity cell models predict that living cells are hard-wired to respond immediately to external mechanical stresses. This hard-wiring exists in the form of discrete cytoskeletal filament networks that mechanically couple specific cell surface receptors, such as integrins, to nuclear matrix scaffolds and to potential transducing molecules that physically associate with the cytoskeleton. If these signaling molecules do function in a "solid-state", then mechanical stresses may be transduced into biochemical responses through force-dependent changes in cytoskeletal geometry or through local alterations in thermodynamic or kinetic parameters. Changes in cytoskeletal tension (prestress) also may play a role in signal amplification and adaptation. Recent experimental results are described which provide direct support for the tensegrity theory.

Integrins as mechanochemical transducers.

Ingber D
Curr Opin Cell Biol. 1991; 3: 841-8

A recent resurgence of interest in mechanical forces and cell shape as biological regulators has revealed extracellular matrix as the site at which forces are transmitted both to and from cells. at the same time, great advances have been made in terms of defining cell-surface integrin receptors as transmembrane molecules that mediate cell attachment and physically interlink extracellular matrix with the intracellular cytoskeleton. Convergence of these two lines of research has begun to elucidate the molecular mechanism by which cells sense physical forces and transduce mechanical signals into a biochemical response.

Molecular cloning of the rat integrin alpha 1-subunit: a receptor for laminin and collagen.

Ignatius MJ, Large TH, Houde M, Tawil JW, Barton A, Esch F, Carbonetto S, Reichardt LF
J Cell Biol. 1990; 111: 709-20

Integrin heterodimers mediate a variety of adhesive interactions, including neuronal attachment to and process outgrowth on laminin. We report here the cloning and primary sequence of an M-200 kD integrin alpha subunit that associates with the integrin beta 1 subunit to form a receptor for both laminin and collagen. Similarities in ligand-binding specificity, relative molecular mass and NH2-terminal sequence make this a strong candidate for the rat homologue of the alpha subunit of the human integrin VLA-1. The full-length rat alpha 1 cDNAs encode a protein containing a purative signal sequence and a mature polypeptide of 1,152 amino acids, with extracellular, transmembrane and cytoplasmic domains. Several structural features are conserved with other integrin alpha chains, including (a) a sequence motif repeated seven times in the NH2-terminal half; (b) potential Ca2+/Mg2+ binding sites in repeats 5, 6, and 7, and (c) alignment of at least 14 of 23 cysteine residues. This rat alpha 1 sequence also contains a 206-amino acid I domain, inserted between repeats 2 and 3, that is homologous to I domains found in the same position in the alpha subunits of several integrins (VLA-2, Mac-1, LFA-1, p150). The rat alpha 1 and human VLA-2 apha subunits share greater than 50% sequence identity in the seven repeats and I domain, suggesting that these sequence identities may underlie some of their similar ligand-binding specificities. However, the rat integrin alpha 1 subunit has several unique features, including a 38-residue insert between two Ca2+/Mg2+ binding domains, and a divergent 15-residue cytoplasmic sequence, that may potentially account for unique functions of this integrin.

Genetic analyses of cell-matrix interactions in development.

Hynes RO
Curr Opin Genet Dev. 1994; 4: 569-74

The extracellular matrix and its cell surface receptors are thought to be important in development. Recent applications of targeted mutagenesis in mice have begun to test hypotheses based on in vitro data and patterns of expression. 'Knockout' mutations of matrix molecules and integrins reveal complexities arising from multiple receptors and ligands.

Dynamic aspects of adhesion receptor function--integrins both twist and shout.

Humphries MJ, Mould AP, Tuckwell DS
Bioessays. 1993; 15: 391-7

The recognition of extracellular molecules by cell surface receptors is the principal mechanism used by cells to sense their environment. Consequently, signals transduced as a result of these interactions make a major contribution to the regulation of cellular phenotype. Historically, particular emphasis has been placed on elucidating the intracellular consequences of growth factor and cytokine binding to cells. In addition to these interactions, however, cells are usually in intimate contact with a further source of complex structural and functional information, namely immobilised extracellular matrix and/or cell surface adhesion proteins. A key question in recent years has been whether cells use the myriad of adhesion protein-receptor interactions purely for structural and migratory function, or whether these interactions also make a more varied contribution to cell phenotype. Here we review dynamic aspects of the function of one major class of adhesion receptor, the integrins. In particular, we focus on the evidence for shape changes in integrin molecules, the mechanisms responsible for regulating ligand binding, and the signals transduced following integrin occupancy.

Integrin cell adhesion receptors and the concept of agonism.

Humphries MJ
Trends Pharmacol Sci. 2000; 21: 29-32

Most cells are adherent and rely on adhesive interactions to regulate their shape, motility and growth. These interactions are critical for tissue integrity and homeostasis but they also contribute to many of the most common diseases in humans. The integrins are a key family of cell-surface receptors that mediate the downstream consequences of cell adhesion and are therefore prime targets for the development of therapeutic agents. In addition to their adhesive activity, integrins also exhibit several other classical features of signalling receptors. Sufficient evidence is now available to pose the question of whether integrins should be classified as true signalling receptors; this article both reviews this evidence and attempts to identify remaining gaps.

Towards a structural model of an integrin.

Humphries MJ
Biochem Soc Symp. 1999; 65: 63-78

Integrins are currently viewed as the principal family of extracellular matrix receptors. The interactions mediated by integrins are responsible for certain typical properties of adhesive cells, such as attachment and migration, but these molecules are also recognized to contribute to intracellular signalling processes, either by transducing signals themselves or by enabling and/or coordinating signalling via other receptor systems. As yet, the structural basis of integrin function is unknown, although detailed computer-based predictions have suggested working models for integrin tertiary structure. In this chapter, I will review this information and discuss recent studies examining the molecular basis of integrin regulation using stimulatory and inhibitory monoclonal antibodies (mAbs). Through the use of sensitive isolated integrin-binding assays, stimulatory mAbs have been found to function either by inducing shape changes in integrins or by selectively recognizing and stabilizing active and ligand-occupied conformations of integrins, while blocking mAbs were found to be allosteric inhibitors of ligand binding that report specific ligand engagement events. This information has improved our understanding of the composition of the integrin ligand-binding pocket and the structural basis of integrin activation.

Integrin activation: the link between ligand binding and signal transduction.

Humphries MJ
Curr Opin Cell Biol. 1996; 8: 632-40

A major function of the integrin family of receptors is to provide a physical connection between extracellular adhesion proteins and intracellular cytoskeletal/signalling molecules. These linkages are dynamic and are influenced in a bidirectional manner by changes in the microenvironment of the plasma membrane that occur both inside and outside of cells. The mechanisms employed by integrins to transduce information are complex, but a series of recent studies has clarified their molecular basis. In particular, explanations for the interdependence of ligand binding, occupancy by divalent cations and receptor conformation have been obtained, and some of the key sites responsible for each property have been localized within the integrin heterodimer. These insights now permit a better visualization of the intricate molecular switch that controls the adhesive phenotype.

Integrin structure.

Humphries MJ
Biochem Soc Trans. 2000; 28: 311-39

The integrins are a family of alpha,beta heterodimeric receptors that mediate dynamic linkages between extracellular adhesion molecules and the intracellular actin cytoskeleton. Integrins are expressed by all multicellular animals, but their diversity varies widely among species; for example, in mammals, 19 alpha and 8 beta subunit genes encode polypeptides that combine to form 25 different receptors, whereas the Drosophila and Caenorhabditis genomes encode only five and two integrin alpha subunits respectively. Thousands of studies over the last two decades have investigated the molecular, cellular and organismal basis of integrin function. Gene deletion has demonstrated essential roles for almost all integrins, with the defects suggesting widespread contributions to both the maintenance of tissue integrity and the promotion of cellular migration. Integrin-ligand interactions are now considered to provide physical support for cells in order to maintain cohesion, to permit the generation of traction forces to enable movement, and to organize signalling complexes to modulate differentiation and cell fate. Animal-model studies have also shown that integrins contribute to the progression of many common diseases, and have implicated them as potential therapeutic targets. The use of anti-integrin monoclonal antibodies and ligand-mimetic peptides has validated this suggestion for inflammatory, neoplastic, traumatic and infectious conditions. Thus, to understand more about the mechanisms underlying tissue organization and cellular trafficking, and to identify approaches for regulating these processes in disease, there is intense interest in determining the molecular basis of integrin function. It is important to state at the outset that the tertiary structure of the integrin dimer is unknown. Our current understanding of the molecular basis of integrin function is therefore compiled from the results of a large number of studies that have employed a wide range of complementary technologies.

Integrin affinity modulation.

Hughes PE, Pfaff M
Trends Cell Biol. 1998; 8: 359-64

Integrin cell-adhesion receptors mediate interactions between cells and the extracellular matrix. Dynamic regulation of integrin adhesive function is termed 'activation' or 'inside-out' signalling. Activation is key to integrin function in processes as diverse as cell migration, the organization of the extracellular matrix and platelet aggregation. Consequently, there has been an intense effort to elucidate the molecular mechanism of integrin activation. This has resulted in the recent identification of novel cytoplasmic partners for integrins and the emerging characterization of the signal-transduction pathways that regulate integrin 'inside-out' signalling. Here, the authors review the recent developments that have provided us with an increased understanding of the basis of integrin activation.

Breaking the integrin hinge. A defined structural constraint regulates integrin signaling.

Hughes PE, Diaz-Gonzalez F, Leong L, Wu C, McDonald JA, Shattil SJ, Ginsberg MH
J Biol Chem. 1996; 271: 6571-4

Integrins are heterodimeric (alpha, beta) cell adhesion receptors. We demonstrate that point mutations in the cytoplasmic domains of both the alpha and beta subunits promote constitutive signaling by the integrin alphaIIbbeta3. By generating charge reversal mutations, we show these "activating" mutations may act by disrupting a potential salt bridge between the membrane-proximal portions of the alpha and beta subunit cytoplasmic domains. Thus, the modulation of specific interactions between the alpha and beta subunit cytoplasmic domains may regulate transmembrane signaling through integrins. In addition, these activating mutations induce dominant alterations in cellular behavior, such as the assembly of the extracellular matrix. Consequently, somatic mutations in integrin cytoplasmic domains could have profound effects in vivo on integrin-dependent functions such as matrix assembly, cell migration, and anchorage-dependent cell growth and survival.

Characterization of the interaction between integrins and recombinant human osteopontin.

Hu DD, Hoyer JR, Smith JW
Ann N Y Acad Sci. 1995; 760: 312-4

Structural and functional studies with antibodies to the integrin beta 2 subunit. A model for the I-like domain.

Huang C, Zang Q, Takagi J, Springer TA
J Biol Chem. 2000; 275: 21514-24

To establish a structure and function map of the beta2 integrin subunit, we mapped the epitopes of a panel of beta2 monoclonal antibodies including function-blocking, nonblocking, and activating antibodies using human/mouse beta2 subunit chimeras. Activating antibodies recognize the C-terminal half of the cysteine-rich region, residues 522-612. Antibodies that do not affect ligand binding map to residues 1-98 and residues 344-521. Monoclonal antibodies to epitopes within a predicted I-like domain (residues 104-341) strongly inhibit LFA-1-dependent adhesion. These function-blocking monoclonal antibodies were mapped to specific residues with human --> mouse knock-out or mouse --> human knock-in mutations. Combinatorial epitopes involving residues distant in the sequence provide support for a specific alignment between the beta-subunit and I domains that was used to construct a three-dimensional model. Antigenic residues 133, 332, and 339 are on the first and last predicted alpha-helices of the I-like domain, which are adjacent on its "front." Other antigenic residues in beta2 and in other integrin beta subunits are present on the front. No antigenic residues are present on the "back" of the domain, which is predicted to be in an interface with other domains, such as the alpha subunit beta-propeller domain. Most mutations in the beta2 subunit in leukocyte adhesion deficiency are predicted to be buried in the beta2 subunit I-like domain. Two long insertions are present relative to alpha-subunit I-domains. One is tied down to the back of the I-like domain by a disulfide bond. The other corresponds to the "specificity-determining loop" defined in beta1 and beta3 integrins and contains the antigenic residue Glu(175) in a disulfide-bonded loop located near the "top" of the domain.

Folding of the beta-propeller domain of the integrin alphaL subunit is independent of the I domain and dependent on the beta2 subunit.

Huang C, Springer TA
Proc Natl Acad Sci U S A. 1997; 94: 3162-7

We have studied the folding during biosynthesis of the lymphocyte function-associated antigen 1 (LFA-1) alphaL subunit using mAb to epitopes that map to seven different regions within the amino acid sequence. The N-terminal portion of alphaL is predicted to contain a beta-propeller domain, consisting of seven beta-sheets, and an I domain that is predicted to be inserted between beta-sheet 2 and beta-sheet 3 of the beta-propeller. The I domain of alphaL folds before association with the beta2 subunit, as shown by immunoprecipitation of the unassociated alphaL subunit by mAbs specific for four different sequence elements within the I domain. By contrast, the beta-propeller domain is not folded in unassociated alphaL after a chase of as long as 12 h after synthesis, but does fold upon association with beta2. This is shown with mAbs to regions of alphaL, that precede and follow the I domain in the primary structure. A mAb that maps near the junction of the C terminus of the I domain with the beta-propeller domain suggests that this region is partially folded before subunit association. The results show that the I domain and beta-propeller domains fold independently of one another, and suggest that the beta-propeller domain bears an interface for association with the beta subunit.

Integrins, adhesion, and cardiac remodeling.

Hsueh WA, Law RE, Do YS
Hypertension. 1998; 31: 176-80

Integrins are heterodimeric cell surface receptors that mediate a cell's ability to perceive its environment, respond to changed in its environment, and alter its environment. When activated, these receptors form focal adhesions, which are areas of close attachment of the cells to extracellular matrix proteins in which colocalization of cytoskeletal proteins, intracellular signaling molecules, and growth factor receptors occurs. In cardiac fibroblasts, integrins mediate cell growth and adhesion. Growth factors such as angiotensin II regulate DNA synthesis, protooncogene expression, extracellular matrix production, adhesion, and other actions of cardiac fibroblasts, many of which require integrin activation. In addition to controlling growth factor and hemodynamic effects, regulation ofintegrin activity may be useful to affect cardiac fibrosis and the remodeling process.

Integrin signaling and cell growth control.

Howe A, Aplin AE, Alahari SK, Juliano RL
Curr Opin Cell Biol. 1998; 10: 220-31

Integrins contribute to cell growth by providing a physical linkage between cytoskeletal structures and the extracellular matrix, and also by participating in various signal transduction processes. The interaction of integrins with matrix ligands can generate signals in and of itself, and can also modulate signals instigated by soluble factors such as peptide mitogens. Cellular events affected by integrin-mediated signaling include motility, cell division, differentiation and programmed cell death. Elucidation of how integrin-mediated cell adhesion controls cell growth is likely to be of fundamental importance in understanding complex biological processes, such as tissue morphogenesis and tumor progression.

Phosphatidylinositol-glycan-specific phospholipase D is an amphiphilic glycoprotein that in serum is associated with high-density lipoproteins.

Hoener MC, Brodbeck U
Eur J Biochem. 1992; 206: 747-57

Phosphatidylinositol (PtdIns)-glycan-specific phospholipase D was purified from bovine and human serum by phase separation in Triton X-114 and by chromatography on DEAE-cellulose, octyl-Sepharose, concanavalin-A-Sepharose, and hydroxyapatite. The purification of the two enzymes was approximately 1200-fold with a recovery of 3-5%. Bovine serum contained about 40 micrograms/ml of PtdIns-glycan-specific phospholipase D, about 10 times more than the amount determined in human serum. PtdIns-glycan-specific phospholipase D is also present in mammalian cerebrospinal fluid and in mammalian milk but to a much lesser extent than in serum. Enzyme from bovine and human serum displayed amphiphilic properties as revealed by sucrose density gradient centrifugation and gel filtration in the absence and presence of detergent. On density gradient centrifugation, both enzymes sedimented with an apparent sedimentation coefficient of about 6.0 S in the presence of 0.1% Triton X-100, and formed aggregates up to 14.5 S in the absence of detergent. Upon gel filtration, the bovine and human enzymes migrated with a Stokes' radius of 6.5 nm and 6.6 nm, respectively, in the presence of Triton X-100. In the absence of Triton X-100, both enzymes gave a Stokes' radius of 8.8 nm. Serial centrifugation of serum at increasing NaBr concentrations revealed that the majority of the enzyme is contained in the high-density lipoprotein fraction. PtdIns-glycan-specific phospholipase D from bovine and human serum contained 27 and 28 N-acetylglucosamine residues, respectively. Treatment with N-glycosidase F decreased the apparent molecular mass of the bovine and human enzyme from 115 and 123 kDa to 91 and 87 kDa, respectively. Sequence analysis of peptides derived from PtdIns-glycan-specific phospholipase D of bovine serum by CNBr cleavage gave 100% identity to the sequence published for the bovine liver enzyme while there was 83% similarity and 74% identity to the sequence of peptides obtained from the human serum enzyme.

The role of alpha and beta chains in ligand recognition by beta 7 integrins.

Higgins JM, Cernadas M, Tan K, Irie A, Wang J, Takada Y, Brenner MB
J Biol Chem. 2000; 275: 25652-64

Integrins alpha(E)beta(7) and alpha(4)beta(7) are involved in localization of leukocytes at mucosal sites. Although both alpha(E)beta(7) and alpha(4)beta(7) utilize the beta(7) chain, they have distinct binding specificities for E-cadherin and mucosal addressin cell adhesion molecule-1 (MAdCAM-1), respectively. We found that mutation of the metal ion-dependent adhesion site (MIDAS) in the alpha(E) A-domain (D190A) abolished E-cadherin binding, as did mutation F298A on the A-domain surface near the MIDAS cleft. A docking model of the A-domain with E-cadherin domain 1 indicates that coordination of the alpha(E) MIDAS metal ion by E-cadherin Glu(31) and a novel projection of Phe(298) into a hydrophobic pocket on E-cadherin provide the basis for the interaction. The location of the binding site on the alpha(E) A-domain resembles that on other integrins, but its structure appears distinctive and particularly adapted to recognize the tip of E-cadherin, a unique integrin ligand. Additionally, mutation of the beta(7) MIDAS motif (D140A) abolished alpha(E)beta(7) binding to E-cadherin and alpha(4)beta(7)-mediated adhesion to MAdCAM-1, and alpha(4) chain mutations that abrogated binding of alpha(4)beta(1) to vascular cell adhesion molecule-1 and fibronectin similarly reduced alpha(4)beta(7) interaction with MAdCAM-1. Thus, although specificity can be determined by the integrin alpha or beta chain, common structural features of both subunits are required for recognition of dissimilar ligands.

Integrins and cell structure: powerful determinants of heart development and heart function.

Hescheler J, Fleischmann BK
Cardiovasc Res. 2000; 47: 645-7

The leukocyte beta 1 integrins.

Hemler ME, Lobb RR
Curr Opin Hematol. 1995; 2: 61-7

We summarize publications appearing in the past year on the blood cell beta 1 integrins VLA-1 through -6, including characterization of their ligand interactions, activation epitopes, signaling mechanisms, cellular distribution, and in vivo relevance. These studies extend our understanding of the beta 1 integrins, and continue to underscore their importance in leukocyte biology, which derives from their central role in mechanisms of cellular adhesion, growth, and differentiation.

Integrin associated proteins.

Hemler ME
Curr Opin Cell Biol. 1998; 10: 578-85

Integrin cytoplasmic domains may interact directly with serveral different cytoskeletal proteins and intracellular signaling molecules. Also, integrins interact directly with other transmembrane structures, including transmembrane-4 superfamily (TM4SF) proteins. New evidence suggests that TM4SF proteins may act as linkers between extracellular integrin alpha chain domains and intracellular signaling molecules, such as phosphatidylinositol 4-kinase and protein kinase C.

Generation by limited proteolysis of a catalytically active 39-kDa protein from the 115-kDa form of phosphatidylinositol-glycan-specific phospholipase D from bovine serum.

Heller M, Butikofer P, Brodbeck U
Eur J Biochem. 1994; 224: 823-33

It has been suggested previously that small amounts of the mature 115-kDa form of phosphatidylinositol (PtdIns)-glycan-specific phospholipase D from bovine serum may exist as a 47-kDa form which can also be generated in vitro by treatment with proteases. In this study, we investigated the possible proteolytic processing by trypsin of partially purified PtdIns-glycan- specific phospholipase D from bovine serum and found that tryptic digestion caused an apparent activation of the enzyme when assayed in the presence of 0.1% (mass/vol.) Triton X-100. Trypsin cleaved the 115-kDa form of PtdIns-glycan-specific phospholipase D into three major polypeptides with molecular masses of 33, 39, and 47 kDa. Under non-denaturing conditions, the polypeptides remained tightly but noncovalently associated with each other. However, in the presence of 6 M urea, the polypeptides could be separated by anion-exchange chromatography. After renaturation, PtdIns-glycan-specific phospholipase D activity was found to be associated with a 39-kDa fragment. Based on its size and its amino acid sequence, the active-site-containing fragment consisted of approximately 275 residues of the N-terminal region of PtdIns-glycan-specific phospholipase D. The active 39-kDa fragment hydrolyzed the PtdIns-glycan-anchors of solubilized acetylcholinesterase from bovine erythrocytes and variant surface glycoprotein from blood stream trypanosomes. However, this fragment was inactive on membrane-associated acetylcholinesterase and PtdIns.

Signal transduction: multiple pathways, multiple options for therapy.

Heldin CH
Stem Cells. 2001; 19: 295-303

Many aspects of cell behavior, such as growth, motility, differentiation, and apoptosis, are regulated by signals cells receive from their environment. Such signals are important, e.g., during embryonal development, wound healing, hematopoiesis, and in the regulation of the immune response, and may come from interactions with other cells or components of the extracellular matrix, or from binding of soluble signaling molecules to specific receptors at the cell membrane. Hereby different signaling pathways are initiated inside the cell. Perturbations of such signaling pathways are seen in several types of diseases, e.g., cancer, inflammatory conditions, and atherosclerosis. Thus, antagonists of several signaling pathways have potential clinical utility. Several such compounds are currently used or are in clinical trials; others are currently being analyzed in animal models.

Integrin-type extracellular matrix receptors in cancer and inflammation.

Heino J
Ann Med. 1993; 25: 335-42

The integrins are a large family of cell adhesion receptors, involved in cell-cell and cell-matrix interactions. At present, 20 different integrin heterodimers are known. Integrins participate in a complex apparatus anchoring cells to their surroundings and transducting signals into the cells. These signals regulate many important aspects of cell behaviour, including growth, differentiation, and phenotype. This is an overview of the molecular and cellular biology of the integrin-type extracellular matrix receptors. Integrins may play a central role in the healing process of tissue injuries, and in many diseases, especially in human cancer.

The collagen receptor integrins have distinct ligand recognition and signaling functions.

Heino J
Matrix Biol. 2000; 19: 319-23

Distinct collagen subtypes are recognized by specific cell surface receptors. Two of the best known collagen receptors are members of the integrin family and are named alpha1beta1 and alpha2beta1. Integrin alpha1beta1 is abundant on smooth muscle cells, whereas the alpha2beta1 integrin is the major collagen receptor on epithelial cells and platelets. Many cell types, such as fibroblasts, osteoblasts, chondrocytes, endothelial cells, and lymphocytes may concomitantly express both of the receptors. We have studied the cell biology of these integrins at two levels. First, we have analyzed their ligand binding mechanism and specificity. Second, we have studied their signaling function inside three-dimensional collagen gels. This mini-review summarizes our most recent results. In conclusion, our data indicate that alpha1beta1 and alpha2beta1 integrins have differences in their ligand binding specificity. Furthermore, the two receptors are connected to distinct signaling pathways and their ligation may lead to opposite cellular responses.

[Integrin story]

Hayashi M, Miyamoto Y
Tanpakushitsu Kakusan Koso. 1999; 44: 130-5

Integrin binding and cell spreading on extracellular matrix act at different points in the cell cycle to promote hepatocyte growth.

Hansen LK, Mooney DJ, Vacanti JP, Ingber DE
Mol Biol Cell. 1994; 5: 967-75

This study was undertaken to determine the importance of integrin binding and cell shape changes in the control of cell-cycle progression by extracellular matrix (ECM). Primary rat hepatocytes were cultured on ECM-coated dishes in serum-free medium with saturating amounts of growth factors (epidermal growth factor and insulin). Integrin binding and cell spreading were promoted in parallel by plating cells on dishes coated with fibronectin (FN). Integrin binding was separated from cell shape changes by culturing cells on dishes coated with a synthetic arg-gly-asp (RGD)-peptide that acts as an integrin ligand but does not support hepatocyte extension. Expression of early (junB) and late (ras) growth response genes and DNA synthesis were measured to determine whether these substrata induce G0-synchronized hepatocytes to reenter the growth cycle. Cells plated on FN exhibited transient increases in junB and ras gene expression (within 2 and 8 h after plating, respectively) and synchronous entry into S phase. Induction of junB and ras was observed over a similar time course in cells on RGD-coated dishes, however, these round cells did not enter S phase. The possibility that round cells on RGD were blocked in mid to late G1 was confirmed by the finding that when trypsinized and replated onto FN-coated dishes after 30 h of culture, they required a similar time (12-15 h) to reenter S phase as cells that had been spread and allowed to progress through G1 on FN. We have previously shown that hepatocytes remain viable and maintain high levels of liver-specific functions when cultured on these RGD-coated dishes.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein kinase mediators of integrin signal transduction.

Hannigan GE, Dedhar S
J Mol Med. 1997; 75: 35-44

Protein kinases are important mediators of signal transduction initiated by soluble growth factors and cytokines. Cellular interactions with the extracellular matrix are mediated largely by members of the integrin class of cell adhesion molecules, which also subsume signal transduction functions required for cell growth, differentiation, and survival. Here we review the involvement of protein kinases in mediating integrin intracellular signal transduction and the possible role for these molecules in regulating integrin adhesion. Although in most cases mechanistic details are incomplete, the emerging theme of protein kinases mediating cross-talk between growth factor receptor and integrin signalling systems provides a timely backdrop against which to present new developments in this area. The contribution of the actin cytoskeleton to integrin signal transduction is discussed, with respect to the concept of 'solid-state' signalling providing a mechanism for imposing order on the protein-protein interactions which underlie signal discrimination. Moreover, we review evidence that dysregulated integrin signalling contributes to pathological processes including arthritis, thrombasthenia, leucocyte adhesion deficiencies, and tumour angiogenesis and invasion.

Tissue architecture: the ultimate regulator of epithelial function?

Hagios C, Lochter A, Bissell MJ
Philos Trans R Soc Lond B Biol Sci. 1998; 353: 857-70

The architecture of a tissue is defined by the nature and the integrity of its cellular and extracellular compartments, and is based on proper adhesive cell-cell and cell-extracellular matrix interactions. Cadherins and integrins are major adhesion-mediators that assemble epithelial cells together laterally and attach them basally to a subepithelial basement membrane, respectively. Because cell adhesion complexes are linked to the cytoskeleton and to the cellular signalling pathways, they represent checkpoints for regulation of cell shape and gene expression and thus are instructive for cell behaviour and function. This organization allows a reciprocal flow of mechanical and biochemical information between the cell and its microenvironment, and necessitates that cells actively maintain a state of homeostasis within a given tissue context. The loss of the ability of tumour cells to establish correct adhesive interactions with their microenvironment results in disruption of tissue architecture with often fatal consequences for the host organism. This review discusses the role of cell adhesion in the maintenance of tissue structure and analyses how tissue structure regulates epithelial function.

Integrin-ligand interactions: a year in review.

Haas TA, Plow EF
Curr Opin Cell Biol. 1994; 6: 656-62

Many cell-cell and cell-matrix interactions depend upon the engagement of specific ligands by members of the integrin family of cell-adhesion receptors. In concert with the identification of new integrins, the number of integrin ligands continues to expand dramatically. The diversity of the integrin ligands bridges many areas of cell and molecular biology. Ligand recognition by integrins requires not only the presence of the cognate primary sequence within an appropriate secondary structure, but also the correct tertiary and quaternary structure of the ligand. Presentation of an 'activated' ligand sequence to specific contact sites within the integrin under specified divalent-cation conditions is necessary for a productive and high-affinity interaction.

Cell adhesion: the molecular basis of tissue architecture and morphogenesis.

Gumbiner BM
Cell. 1996; 84: 345-57

A variety of cell adhesion mechanisms underlie the way that cells are organized in tissues. Stable cell interactions are needed to maintain the structural integrity of tissues, and dynamic changes in cell adhesion participate in the morphogenesis of developing tissues. Stable interactions actually require active adhesion mechanisms that are very similar to those involved in tissue dynamics. Adhesion mechanisms are highly regulated during tissue morphogenesis and are intimately related to the processes of cell motility and cell migration. In particular, the cadherins and the integrins have been implicated in the control of cell movement. Cadherin mediated cell compaction and cellular rearrangements may be analogous to integrin-mediated cell spreading and motility on the ECM. Regulation of cell adhesion can occur at several levels, including affinity modulation, clustering, and coordinated interactions with the actin cytoskeleton. Structural studies have begun to provide a picture of how the binding properties of adhesion receptors themselves might be regulated. However, regulation of tissue morphogenesis requires complex interactions between the adhesion receptors, the cytoskeleton, and networks of signaling pathways. Signals generated locally by the adhesion receptors themselves are involved in the regulation of cell adhesion. These regulatory pathways are also influenced by extrinsic signals arising from the classic growth factor receptors. Furthermore, signals generated locally be adhesion junctions can interact with classic signal transduction pathways to help control cell growth and differentiation. This coupling between physical adhesion and developmental signaling provides a mechanism to tightly integrate physical aspects of tissue morphogenesis with cell growth and differentiation, a coordination that is essential to achieve the intricate patterns of cells in tissues.

Extracellular matrix and its receptors during development.

Gullberg D, Ekblom P
Int J Dev Biol. 1995; 39: 845-54

Extracellular matrix (ECM) components are essential for morphogenesis of virtually all tissues. The ECM interacts with the cell surface by binding to specific receptors. The first family of receptors for the ECM that was identified was the integrin family. Integrins are composed of an alpha and a beta-chain, both of which are single pass transmembrane proteins. In muscle cells the dystroglycan complex forms another important receptor system for ECM. It is a complex composed of many proteins. Recent studies have shown that dystroglycan is expressed by embryonic epithelial cells as well. The nature of constituents of the dystroglycan complex is well known for muscle, whereas the detailed composition of the dystroglycan complex in embryonic epithelium is not yet well known. We here review the evidence that binding of ECM to integrins and the dystroglycan complex could be essential for muscle and epithelial cell development and function. It is likely that integrins and the dystroglycan complex have distinct roles during development. It will be an interesting task to study the signal transduction pathways elicited by the interactions between ECM and the two receptor systems during muscle and epithelial morphogenesis.

Role of two conserved glycine residues in the beta-propeller domain of the integrin alpha4 subunit in VLA-4 conformation and function.

Guerrero-Esteo M, Ruiz-Velasco N, Munoz M, Teixido J
FEBS Lett. 1998; 429: 123-8

The N-terminal region of the alpha integrin subunits is predicted to fold into a beta-propeller domain. Using K562 alpha4 transfectants we show that mutations at alpha4 subunit residues Gly130 and Gly190 affect the conformation of this domain causing a reduction in the recognition of alpha4 by anti-alpha4 antibodies which map to the beta-propeller. The improper alpha4 conformation also led to an altered association with the beta1 subunit, and to a lack of alpha4beta1 adhesion to VCAM-1 and CS-1/fibronectin, as well as an abolishment of anti-alpha4- and anti-beta1-dependent homotypic aggregation. The total conservation of Gly130 and Gly190 among integrin alpha subunits suggests their importance in the correct folding of their respective beta-propeller domains, and thus, in the adhesive activity of the integrins.

Signal transduction in cell-matrix interactions.

Guan JL, Chen HC
Int Rev Cytol. 1996; 168: 81-121

Cell-matrix interactions play critical roles in a variety of physiological and pathological processes by regulating cell migration, proliferation, and differentiation. Integrin family cell-adhesion receptors have been identified as the major receptors for various extracellular matrix proteins and shown to be capable of transducing biochemical signals across the plasma membrane to regulate cellular functions. Protein tyrosine phosphorylation has been implicated in a central role in integrin-initiated signal transduction. Other signaling pathways have also been shown to be activated by cell-matrix interactions. This chapter reviews our current understanding of the key molecular components and their interactions in the integrin-signaling pathways and examines the role of various signaling pathways and molecular players in the context of cellular functions regulated by cell-matrix interactions.

Role of focal adhesion kinase in integrin signaling.

Guan JL
Int J Biochem Cell Biol. 1997; 29: 1085-96

Integrins are the major cell surface receptors for extracellular matrix molecules, which play critical roles in a variety of biological processes. Focal adhesion kinase has recently been established as a key component of the signal transduction pathways triggered by integrins. Aggregation of FAK with integrins and cytoskeletal proteins in focal contacts has been proposed to be responsible for FAK activation and autophosphorylation by integrins in cell adhesion. This may be achieved by FAK interaction with talin or other cytoskeletal proteins that in turn associate with the cytoplasmic domain of integrin beta subunits. Autophosphorylation of FAK at Y397 leads to its association with Src, resulting in activation of both kinases. The activated FAK/Src complex acts on potential substrates tensin, paxillin and p130cas. Besides cytoskeletal regulation, FAK phosphorylation and/or binding to paxillin and p130cas may trigger downstream activation of MAP kinase by the adoptor protein Crk. Src association with FAK may also lead to its phosphorylation of other sites on FAK, including a binding site for Grb2. Cell adhesion-dependent association of FAK and Grb2 may provide a mechanism by which MAP kinase is activated in cell adhesion. PI 3-kinase has also been shown to bind FAK in a cell adhesion-dependent manner at the major autophosphorylation site Y397. This association could lead to activation of PI 3-kinase and its downstream effectors. Recent results from a number of different approaches have shown that integrin signaling through FAK leads to increased cell migration on fibronectin as well as potentially regulating cell proliferation and survival.

Focal adhesion kinase in integrin signaling.

Guan JL
Matrix Biol. 1997; 16: 195-200

Focal adhesion kinase (FAK) has recently been established as a key component of the signal transduction pathways triggered by integrins. Aggregation of integrins and the cytoskeletal proteins tensin, paxillin and talin is proposed to be responsible for FAK activation and autophosphorylation by integrins in cell adhesion. Activation and autophosphorylation of FAK lead to its binding to a number of intracellular signaling molecules, including Src, Grb2 and PI 3-kinase. FAK/Src association activates both kinases, which act on the potential substrates tensin, paxillin and p130cas. Besides cytoskeletal regulation, FAK phosphorylation of paxillin and p130cas could also lead to MAP kinase pathway by adaptor proteins such as Crk and Nck. Recent studies indicated that integrin signaling through FAK causes increased cell migration and potentially regulates cell proliferation and survival. Future challenges will include clarifying the roles of signaling pathways downstream of FAK in cell migration and cell cycle regulation.

Desmosomes and hemidesmosomes: structure and function of molecular components.

Green KJ, Jones JC
FASEB J. 1996; 10: 871-81

Desmosomes and hemidesmosomes are the major cell surface attachment sites for intermediate filaments at cell-cell and cell-substrate contacts, respectively. The transmembrane molecules of the desmosome belong to the cadherin family of calcium-dependent adhesion molecules, whereas those in the hemidesmosome include the integrin class of cell matrix receptors. In each junction, the cytoplasmic domains of certain transmembrane junction components contain unusually long carboxy-terminal tails not found in those family members involved in linkage of actin to the cell surface. These domains are thought to be important for the regulation of junction assembly and specific attachment of intermediate filaments via associated adapter proteins. Recent developments have suggested the exciting possibility that these junctions, in addition to playing an important structural function in tissue integrity, are both acceptors and affectors of cell signaling pathways. Many desmosomal and hemidesmosomal constituents are phosphoproteins and in certain cases the function of specific phosphorylation sites in regulating protein-protein interactions is being uncovered. In addition, a more active role in transmitting signals that control morphogenesis during development and possibly even regulate cell growth and differentiation are being defined for cytoplasmic and membrane components of these junctions.

Drosophila PS1 integrin is a laminin receptor and differs in ligand specificity from PS2.

Gotwals PJ, Fessler LI, Wehrli M, Hynes RO
Proc Natl Acad Sci U S A. 1994; 91: 11447-51

We have expressed Drosophila position-specific (PS) integrins on the surfaces of Schneider S2 cells and tested for adhesion and spreading on various matrix molecules. We report that PS1 integrin is a laminin receptor and that PS1 and PS2 integrins promote cell spreading on two different Drosophila extracellular matrix molecules, laminin and tiggrin, respectively. The differing ligand specificities of these two integrins, combined with data on the in vivo expression patterns of the integrins and their ligands, lead to a model for the structure of integrin-dependent attachments in the pupal wings and embryonic muscles of Drosophila.

Divalent cations stabilize the alpha 1 beta 1 integrin I domain.

Gotwals PJ, Chi-Rosso G, Ryan ST, Sizing I, Zafari M, Benjamin C, Singh J, Venyaminov SY, Pepinsky RB, Koteliansky V
Biochemistry. 1999; 38: 8280-8

Recent structural and functional analyses of alpha integrin subunit I domains implicate a region in cation and ligand binding referred to as the metal ion-dependent adhesion site (MIDAS). Although the molecular interactions between Mn2+ and Mg2+ and the MIDAS region have been defined by crystallographic analyses, the role of cation in I domain function is not well understood. Recombinant alpha 1 beta 1 integrin I domain (alpha1-I domain) binds collagen in a cation-dependent manner. We have generated and characterized a panel of antibodies directed against the alpha1-I domain, and selected one (AJH10) that blocks alpha 1 beta 1 integrin function for further study. The epitope of AJH10 was localized within the loop between the alpha 3 and alpha 4 helices which contributes one of the metal coordination sites of the MIDAS structure. Kinetic analyses of antibody binding to the I domain demonstrate that divalent cation is required to stabilize the epitope. Denaturation experiments demonstrate that cation has a dramatic effect on the stabilization of the I domain structure. Mn2+ shifts the point at which the I domain denatures from 3.4 to 6.3 M urea in the presence of the denaturant, and from 49.5 to 58.6 degrees C following thermal denaturation. The structural stability provided to the alpha1-I domain by divalent cations may contribute to augmented ligand binding that occurs in the presence of these cations.

Cell adhesion molecules: selectins and integrins.

Gonzalez-Amaro R, Sanchez-Madrid F
Crit Rev Immunol. 1999; 19: 389-429

Cell adhesion molecules play a key role in different physiological and pathological phenomena. Selectins comprise a family of three members (E-, P-, and L-selectin) that are differentially expressed by leukocytes and endothelial cells, and are involved in the early steps of leukocyte extravasation. Although many different putative ligands of selectins have been described, their physiological relevance has not been elucidated in all cases. Selectins and some of their counter-receptors function also as signal-transducing receptors, significantly contributing to leukocyte and endothelial cell activation. Integrins represent a large family of adhesion receptors that are widely expressed and mainly interact with extracellular matrix components. The affinity and avidity of integrins can be modulated by different mechanisms triggered either from the extracellular milieu or through intracellular signals. Integrins show a close relationship with cytoskeleton and exert an important role as signal-transducing receptors, activating different biochemical pathways, mainly the mitogen-activated protein (MAP) kinase cascade. In addition to their adhesive function, these receptors modulate key intracellular phenomena, including cell activation, proliferation, and apoptosis. Because selectins and integrins are involved in leukocyte extravasation and the inflammatory response, different strategies are in progress for the blockade of these molecules and, therefore, for therapy of inflammatory diseases.

Fibronectin and integrins in development.

Glukhova MA, Thiery JP
Semin Cancer Biol. 1993; 4: 241-9

Extracellular matrix glycoprotein fibronectin and integrins, transmembrane receptors for extracellular matrix proteins, have been implicated in morphogenesis, cytodifferentiation and organogenesis. During development, embryonic cells move on or through fibronectin-rich matrices, which support migration and contain the guidance cues that direct the cells along their migratory pathways. Integrins are involved in different aspects of the movement, including recognition of the pathway, transient attachment to the substrate and cytoskeleton rearrangement in the moving cells as well as organization of the matrix during migratory pathway formation. Differentiated tissues exhibit a characteristic and often rather limited set of integrins. On the contrary, during development, tissue repertoire of integrins changes, and integrins that are absent from the adult differentiated tissue may appear transiently and be expressed only during certain developmental stages. These periods of transient expression coincide with cell movement, differentiation and maturation of cells and can be correlated with changes of environment, i.e. neighboring cells, composition and structure of the extracellular matrix. In this article we discuss the recent data on the roles of fibronectin-integrin interactions in directed cell migration in embryos and on the distribution and possible functions of integrins in developing tissues.

Integrin-extracellular matrix interactions in connective tissue remodeling and osteoblast differentiation.

Globus RK, Moursi A, Zimmerman D, Lull J, Damsky C
ASGSB Bull. 1995; 8: 19-28

The differentiaton of bone cells is a complex multistep process. Bone is somewhat unusual in that it is very actively and continually remodeled in the adult and that maintenance of its mass in the mature organism is exquisitely sensitive to mechanical as well as chemical signals. Bone is also unique because it consists of a very large amount of extracellular matrix (ECM) that is mineralized. The integrin family of ECM receptors has been shown to play an important role in tissue morphogenesis in several systems. Our studies on the regulation of matrix remodeling enzymes by integrins in rabbit synovial fibroblasts show that two b1 integrin fibronectin (FN) receptor complexes (alpha 5 beta 1 and alpha 4 beta 1) cooperate in detecting subtle changes in the composition of the ECM. As a result of signal transduction by these integrins, the levels of mRNA and protein for several members of the metalloproteinase family are regulated in these cells. We have also used antibody and RGD peptide perturbation studies to determine the significance of cell/ECM interactions to normal osteogenesis. We found that interactions between the cell binding domain of FN and integrins are required for both normal morphogenesis and gene expression in cultured osteoblasts that differentiate to form bone-like tissue in culture. These data lead us to propose that beta 1 integrins play an important role in osteoblast differentiation as well as in bone remodeling.

Integrins: dynamic regulation of ligand binding.

Ginsberg MH
Biochem Soc Trans. 1995; 23: 439-46

Integrins: role in cell adhesion and communication.

Gille J, Swerlick RA
Ann N Y Acad Sci. 1996; 797: 93-106

Adhesive interactions are crucial for the integrity and function of all cells and tissues. As one of the major families of cell adhesion receptors, the integrins have been the focus of scientific interest for more than a decade. The resulting studies have tremendously enhanced the understanding of integrin-mediated adhesive interactions and have unveiled novel integrin functions in the cytoskeletal organization of microfilaments and in the activation of diverse signaling pathways. These functions are critically involved in the regulation of multiple processes, such as tissue development, inflammation, tumor cell growth and metastasis, and programmed cell death. The global view of integrin receptor biology has radically changed and has become much more subtle and elaborate. The enormous complexity of integrin function is determined by the heterodimeric formation of more than 20 functional integrin receptors, the cell type-specific distribution, the receptor activation state, the presence of different activation and deactivation signals, and the subsequent employment of distinct cytoskeletal and signaling complexes within a more dimensional network of time and space. This article summarizes the structural and functional properties of the integrin receptors and emphasizes some of the major achievements made in the past to enhance the understanding of integrin biology.

Integrin-mediated adhesion and signaling in tumorigenesis.

Giancotti FG, Mainiero F
Biochim Biophys Acta. 1994; 1198: 47-64

Assembly and mechanosensory function of focal contacts.

Geiger B, Bershadsky A
Curr Opin Cell Biol. 2001; 13: 584-92

Focal contacts, focal complexes and related extracellular matrix adhesions are used by cells to explore their environment. These sites act as mechanosensory 'devices', where internal contractile forces or externally applied force can regulate the assembly of the adhesion site and trigger adhesion-dependent signaling involving Rho-family small G-proteins and other signaling pathways. The molecular mechanisms underlying these processes are discussed.

Recent insights into ligand binding, activation and signalling by integrin adhesion receptors.

Garratt AN, Humphries MJ
Acta Anat (Basel). 1995; 154: 34-45

In recent years, analyses of the structure and function of membrane-intercalated adhesion molecules have shown them to play key roles in determining cellular phenotype. As expected, adhesion has an important role in regulating cellular positioning, but there is also compelling evidence that information transduced via adhesion molecules affects the differentiation status of cells. Cell surface adhesion molecules can be classified into a number of gene families, including immunoglobulins, cadherins, selectins, proteoglycans, and integrins. All of these types of molecule are co-expressed on most cells, and therefore the overall contribution of adhesion to cell phenotype is likely to be a net effect of the individual contributions of each of these groups. In this review, we will focus on the role of the integrins, which appear to be particularly important mediators of cell migration and adhesion-dependent intracellular signalling. A great deal is now known about the extracellular faces of integrins, including their structure and ligand-binding mechanisms, and in recent years, our knowledge of integrin-dependent signalling via cytoplasmic domains has improved considerably. An emerging picture is one of a dynamic family of receptors than can be expressed in different states of activation. Alterations in activity are apparently mediated by conformational changes that can be induced from both outside and inside cells. In turn, these changes in activity have concomitant consequences for adhesion and signalling.

Integrin-fibronectin interactions at the cell-material interface: initial integrin binding and signaling.

Garcia AJ, Boettiger D
Biomaterials. 1999; 20: 2427-33

Integrin receptors mediate cell adhesion to extracellular matrices and provide signals that direct proliferation and differentiation. Integrin binding involves receptor-ligand interactions at the cell-substrate interface and assembly and reorganization of structural and signaling elements at the cytoplasmic face. Using a cross-linking/extraction/reversal method to quantify bound integrins, we demonstrate that the density of alpha5beta1 integrin-fibronectin bonds increases linearly with ligand density, as predicted by simple receptor-ligand equilibrium. This linear relationship is consistent with linear increases in cell adhesion strength with receptor and ligand surface densities. Furthermore, we show that phosphorylation of FAK, a tyrosine kinase involved in early integrin-mediated signaling, increases linearly with the number of integrin-Fn bonds. These linear relationships suggest the absence of cooperative effects in the initial stages of mechanical coupling and adhesion-mediated signaling.

Integrins.

Gailit JO, Clark RA
Adv Dermatol. 1993; 8: 129-52

Redistribution and dysfunction of integrins in cultured renal epithelial cells exposed to oxidative stress.

Gailit J, Colflesh D, Rabiner I, Simone J, Goligorsky MS
Am J Physiol. 1993; 264: 14957-14957

Tubular obstruction by detached renal tubular epithelial cells is a major cause of oliguria in acute renal failure. Viable renal tubular cells can be recovered from urine of patients with acute tubular necrosis, suggesting a possible defect in cell adhesion to the basement membrane. To study this process of epithelial cell desquamation in vitro, we investigated the effect of nonlethal oxidative stress on the integrin adhesion receptors of the primate kidney epithelial cell line BS-C-1. Morphological and functional studies of cell adhesion properties included the following: interference reflection microscopy, intravital confocal microscopy and immunocytochemistry, flow cytometric analysis of integrin receptor abundance, and cell-matrix attachment assay. High levels of the integrin subunits alpha 3, alpha v, and beta 1 were detected on the cell surface by fluorescence-activated cell sorting (FACS) analysis, as well as lower levels of alpha 1, alpha 2, alpha 4, alpha 5, alpha 6, and beta 3. Exposure of BS-C-1 cells to nonlethal oxidative stress resulted in the disruption of focal contacts, disappearance of talin from the basal cell surface, and in the redistribution of integrin alpha 3-subunits from predominantly basal location to the apical cell surface. As measured in a quantitative cell attachment assay, oxidative stress decreased BS-C-1 cell adhesion to type IV collagen, laminin, fibronectin, and vitronectin. Defective adhesion was not associated with a loss of alpha 3-, alpha 4-, or alpha v-integrin subunits from the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Beta propellers: structural rigidity and functional diversity.

Fulop V, Jones DT
Curr Opin Struct Biol. 1999; 9: 715-21

Recently solved structures and proposed models have helped to reveal the structural characteristics of the beta-propeller fold, as well as the features that contribute to its high rigidity and stability. Possible strategies for identifying beta-propeller proteins in newly characterised sequences are helping to overcome the problems of predicting the beta-propeller fold from amino acid sequences.

Prolyl oligopeptidase: an unusual beta-propeller domain regulates proteolysis.

Fulop V, Bocskei Z, Polgar L
Cell. 1998; 94: 161-70

Prolyl oligopeptidase is a large cytosolic enzyme that belongs to a new class of serine peptidases. The enzyme is involved in the maturation and degradation of peptide hormones and neuropeptides, which relate to the induction of amnesia. The 1.4 A resolution crystal structure is presented here. The enzyme contains a peptidase domain with an alpha/beta hydrolase fold, and its catalytic triad (Ser554, His680, Asp641) is covered by the central tunnel of an unusual beta propeller. This domain makes prolyl oligopeptidase an oligopeptidase by excluding large structured peptides from the active site. In this way, the propeller protects larger peptides and proteins from proteolysis in the cytosol. The structure is also obtained with a transition state inhibitor, which may facilitate drug design to treat memory disorders.

The integrin-mediated cyclic strain-induced signaling pathway in vascular endothelial cells.

Frangos SG, Knox R, Yano Y, Chen E, Di Luozzo G, Chen AH, Sumpio BE
Endothelium. 2001; 8: 1-10

The irregular distribution of plaque in the vasculature results from the interaction of local hemodynamic forces with the vessel wall. One well-characterized force is cyclic circumferential strain, the repetitive pulsatile pressure distention on the arterial wall. This review summarizes current research, which has aimed to elicit the signal transduction pathway by which cyclic strain elicits functional and structural responses in endothelial cells; specifically, it summarizes the signaling pathway that begins with the reorganization of integrins. One method by which these extracellular matrix receptors affect signal transduction is through their ability to initiate the process of phosphorylation on tyrosine residues of cytoplasmic protein kinases, including focal adhesion kinase. The strain-induced pathway appears to also involve ras and the mitogen-activated protein kinase family of enzymes, and preliminary data suggests a role for src as well. Ultimately, it is the regulation of gene expression through the modulation of transcription factors that allows endothelial cells to respond to changes in local hemodynamics.

Normal and abnormal nephrogenesis.

Fouser L, Avner ED
Am J Kidney Dis. 1993; 21: 64-70

During the past decade, exciting advances in the fields of cell and molecular biology have provided new insight into the processes of normal and abnormal nephron induction and renal morphogenesis. Although the specific molecular signals that control renal mesenchymal-epithelium inductive interaction remain unknown, recent data suggest that postinductive nephrogenesis may be regulated by the overall balance of a number of local autocrine and/or paracrine growth factor systems. Alterations in the critical balance of regulatory factors might produce a variety of hypoplastic and dysplastic nephropathies or hyperplastic lesions such as tubular cysts. Additional studies demonstrate that extracellular matrix components and cell surface integrins have important regulatory roles in ureteric bud development and branching. Perturbations in matrix or integrin expression due to altered gene activity or toxin exposure would be expected to produce a variety of renal abnormalities ranging from failure of nephron induction (aplasia) to focal disruptions of differentiation (segmental dysplasia). Finally, several groups of genes encoding transcriptional regulatory proteins have been identified that appear to regulate aspects of cell proliferation, pattern formation, and segment-specific differentiation during normal and abnormal nephrogenesis. Future studies will elucidate the roles that specific genes and proteins play in renal development and will ultimately reveal the manner in which their dysregulation or dysfunction causes a variety of developmental renal disorders.

Alternatively spliced variants: a new view of the integrin cytoplasmic domain.

Fornaro M, Languino LR
Matrix Biol. 1997; 16: 185-93

A large number of studies have underscored a major role for the integrin alpha beta cytoplasmic domains in the modulation of cell functions. Cytoplasmic domain variants of the beta 1, beta 3, beta 4, alpha 3, alpha 6 and alpha 7 subunits have been described. These molecules are generated by alternative splicing events and are expressed in a cell- or tissue-type specific manner. Some of these variants (beta 1C, beta 1D, alpha 6A and alpha 7A) are predominantly expressed upon differentiation and have been shown to be regulated during development. The studies on the structure-function relationship of the integrin variant subunits, published between 1989 and now, will be reviewed here for the first time. The results demonstrate that differences in the cytoplasmic domain do not affect either the alpha beta heterodimer formation or the ligand specificity. Instead, alternatively spliced integrin cytoplasmic domains appear to be essential modulators of receptor localization, cell proliferation and migration, as well as phosphorylation of signaling molecules. These observations lead to the current hypothesis that cell-type specific regulation of alternatively spliced integrin cytoplasmic domains may provide a highly specialized mechanism to control cell growth and intracellular signaling pathways.

Modulation of adhesion-dependent cAMP signaling by echistatin and alendronate.

Fong JH, Ingber DE
Biochem Biophys Res Commun. 1996; 221: 19-24

We measured intracellular cAMP levels in cells during attachment and spreading on different extracellular matrix (ECM) proteins. Increases in cAMP were observed within minutes when cells attached to fibronectin, vitronectin, and a synthetic RGD-containing fibronectin peptide (Petite 2000), but not when they adhered to another integrin alpha nu beta 3 ligand, echistatin. Because echistatin also inhibits bone resorption, we measured the effects of adding another osteoporosis inhibitor, alendronate, in this system. Alendronate inhibited the cAMP increase induced by ligands that primarily utilize integrin alpha nu beta 3 (vitronectin, Peptite 2000), but not by fibronectin which can also use integrin alpha 5 beta 1. These results show that cell adhesion to ECM can increase intracellular cAPM levels and raise the possibility that inhibitors of osteoporosis may act, in part, by preventing activation of this pathway by integrins.

Interplay between an AAA module and an integrin I domain may regulate the function of magnesium chelatase.

Fodje MN, Hansson A, Hansson M, Olsen JG, Gough S, Willows RD, Al-Karadaghi S
J Mol Biol. 2001; 311: 111-22

In chlorophyll biosynthesis, insertion of Mg(2+) into protoporphyrin IX is catalysed in an ATP-dependent reaction by a three-subunit (BchI, BchD and BchH) enzyme magnesium chelatase. In this work we present the three-dimensional structure of the ATP-binding subunit BchI. The structure has been solved by the multiple wavelength anomalous dispersion method and refined at 2.1 A resolution to the crystallographic R-factor of 22.2 % (R(free)=24.5 %). It belongs to the chaperone-like "ATPase associated with a variety of cellular activities" (AAA) family of ATPases, with a novel arrangement of domains: the C-terminal helical domain is located behind the nucleotide-binding site, while in other known AAA module structures it is located on the top. Examination by electron microscopy of BchI solutions in the presence of ATP demonstrated that BchI, like other AAA proteins, forms oligomeric ring structures. Analysis of the amino acid sequence of subunit BchD revealed an AAA module at the N-terminal portion of the sequence and an integrin I domain at the C terminus. An acidic, proline-rich region linking these two domains is suggested to contribute to the association of BchI and BchD by binding to a positively charged cleft at the surface of the nucleotide-binding domain of BchI. Analysis of the amino acid sequences of BchI and BchH revealed integrin I domain-binding sequence motifs. These are proposed to bind the integrin I domain of BchD during the functional cycle of magnesium chelatase, linking porphyrin metallation by BchH to ATP hydrolysis by BchI. An integrin I domain and an acidic and proline-rich region have been identified in subunit CobT of cobalt chelatase, clearly demonstrating its homology to BchD. These findings, for the first time, provide an insight into the subunit organisation of magnesium chelatase and the homologous colbalt chelatase. Copyright 2001 Academic Press.

Anti-integrin drugs developed to treat inflammation.

Featherstone C
Lancet. 1996; 347: 1106-1106

Inside-out signaling through integrins.

Faull RJ, Ginsberg MH
J Am Soc Nephrol. 1996; 7: 1091-7

Rapid and yet precisely controlled changes in cell adhesion are a hallmark of a number of basic physiological processes. Many of these processes directly impact upon nephrology, including: (1) cell migration, which occurs during leukocyte infiltration into inflammatory sites (e.g., in glomerulonephritis, interstitial nephritis, and renal transplant rejection) and during embryogenesis (e.g., in development of the genitourinary system): and (2) platelet aggregation at sites of clot formation (e.g., in glomerulonephritis). The integrins are a family of adhesion molecules whose function is controlled by the cells that express them, and this properly makes them eminently suitable receptors for those situations that demand flexibility of the cellular adhesive phenotype. The control of integrin function occurs via regulatory signals that originate within the cell cytoplasm and are then transmitted to the external ligand-binding domain of the receptor. The generation and transmission of these "inside-out" signals are the subjects of intensive research that will be summarized in this review article.

Integrins--the glue of life.

Etzioni A
Lancet. 1999; 353: 341-3

Integrin-mediated activation of Cdc42 controls cell polarity in migrating astrocytes through PKCzeta.

Etienne-Manneville S, Hall A
Cell. 2001; 106: 489-98

We describe here a signal transduction pathway controlling the establishment of mammalian cell polarity. Scratching a confluent monolayer of primary rat astrocytes leads to polarization of cells at the leading edge. The microtubule organizing center, the microtubule cytoskeleton, and the Golgi reorganize to face the new free space, and directed cell protrusion and migration specifically occur perpendicularly to the scratch. We show here that the interaction of integrins with extracellular matrix at the newly formed cell front leads to the activation and polarized recruitment of Cdc42, which in turn recruits and activates a cytoplasmic mPar6/PKCzeta complex. Localized PKCzeta activity, acting through the microtubule motor protein dynein, is required for all aspects of induced polarity in these cells.

How do integrins integrate? The role of cell adhesion receptors in differentiation and development.

Erle DJ, Pytela R
Am J Respir Cell Mol Biol. 1992; 6: 459-60

Alpha 5 integrin is a critical component of adhesion plaques in myogenesis.

Enomoto MI, Boettiger D, Menko AS
Dev Biol. 1993; 155: 180-97

We investigated the distribution and expression of alpha 5 beta 1 and alpha 3 beta 1 integrin in differentiating myogenic cells in culture. The myogenic cells expressed both alpha 5 and alpha 3 integrin subunits with the same molecular sizes as those expressed by chicken embryo fibroblasts (CEF). However, the ratio of total alpha 5 to alpha 3 was threefold higher in the muscle cultures than that in CEF cultures. A new method is described whereby adhesion plaque-associated integrin was cross-linked to its extracellular matrix ligand on the substrate using a nonpenetrating cross-linker, BS3, and integrin not involved in substrate adhesion as well as cytoskeletal proteins were removed with a zwitterionic detergent. alpha 5 and beta 1 integrin, but not alpha 3 could be cross-linked to fibronectin at adhesion plaques throughout myogenesis in culture. Alpha 5 beta 1 integrin was found only at the edge of myoblasts 4 hr after plating but became distributed under their entire surface by 1 day in culture. When the muscle cells became elongated, a morphology they express after the initiation of terminal differentiation, and as they began to fuse, alpha 5 was found redistributed in small adhesion plaques along the lateral edges of the postmitotic myocytes and early myotubes. In mature myotubes, which are large multinucleated branched structures, alpha 5 beta 1 integrin was localized to parallel streaks underneath their entire substrate surface. Throughout the different stages of myogenesis, vinculin colocalized with alpha 5 beta 1 integrin in adhesion plaques, but alpha-actinin only colocalized to the adhesion plaques in myoblasts, not in myotubes. These studies suggest that alpha 5 beta 1 integrin through its dynamic interaction with both fibronectin and the cytoskeleton is important for both the signals which initiate the differentiation process and for subsequent morphological and structural changes during the differentiation process.

Structural basis of collagen recognition by integrin alpha2beta1.

Emsley J, Knight CG, Farndale RW, Barnes MJ, Liddington RC
Cell. 2000; 101: 47-56

We have determined the crystal structure of a complex between the I domain of integrin alpha2beta1 and a triple helical collagen peptide containing a critical GFOGER motif. Three loops on the upper surface of the I domain that coordinate a metal ion also engage the collagen, with a collagen glutamate completing the coordination sphere of the metal. Comparison with the unliganded I domain reveals a change in metal coordination linked to a reorganization of the upper surface that together create a complementary surface for binding collagen. Conformational changes propagate from the upper surface to the opposite pole of the domain, suggesting both a basis for affinity regulation and a pathway for signal transduction. The structural features observed here may represent a general mechanism for integrin-ligand recognition.

Crystal structure of the I domain from integrin alpha2beta1.

Emsley J, King SL, Bergelson JM, Liddington RC
J Biol Chem. 1997; 272: 28512-7

We have determined the high resolution crystal structure of the I domain from the alpha-subunit of the integrin alpha2beta1, a cell surface adhesion receptor for collagen and the human pathogen echovirus-1. The domain, as expected, adopts the dinucleotide-binding fold, and contains a metal ion-dependent adhesion site motif with bound Mg2+ at the top of the beta-sheet. Comparison with the crystal structures of the leukocyte integrin I domains reveals a new helix (the C-helix) protruding from the metal ion-dependent adhesion site face of the domain which creates a groove centered on the magnesium ion. Modeling of a collagen triple helix into the groove suggests that a glutamic acid side chain from collagen can coordinate the metal ion, and that the C-helix insert is a major determinant of binding specificity. The binding site for echovirus-1 maps to a distinct surface of the alpha2-I domain (one edge of the beta-sheet), consistent with data showing that virus and collagen binding occur by different mechanisms. Comparison with the homologous von Willebrand factor A3 domain, which also binds collagen, suggests that the two domains bind collagen in different ways.

The integrin superfamily and the eye.

Elner SG, Elner VM
Invest Ophthalmol Vis Sci. 1996; 37: 696-701

Integrins may have many different roles in the eye, from influencing ocular development, cell migration in wound healing or pathologic processes, and modulating inflammation and thrombosis in ocular tissue. With the variety of integrins expressed on ocular cells, great versatility and redundancy are afforded in integrin interactions. Elucidation of the functional roles of specific integrins may answer important questions regarding tumor invasion, metastasis, and ocular development. The roles of integrins in inflammation is a rapidly advancing field that soon may lead to anti-inflammatory pharmacologic agents. Potent integrin antagonists, such as cyclic RGD peptides isolated from viper venom, may prove to be useful as anti-inflammatory, anti-metastatic, or anti-thrombotic agents for the treatment of ocular disease.

Integrin-mediated adhesive properties of uroepithelial cells are inhibited by treatment with bacterial toxins.

Elgavish A, Pattanaik A, Lloyd K, Reed R
Am J Physiol. 1994; 266: 15529-15529

Gram-negative bacteria are a dominant cause of urinary tract infection, and their ability to produce toxins is an important virulence attribute. Cellular mechanisms triggered by the production of toxins in the lower urinary tract have not been completely defined. Ureteral epithelial cells (UT; A. Elgavish, Infect. Immun. 61: 3304-3312, 1993) have served as an in vitro model to explore the possibility that bacterial toxins act on UT by affecting integrin-mediated adhesive properties. The effect of treatment with lipopolysaccharides (LPS) from three strains of the gram-negative Escherichia coli [055:B5 (LPS-1), 0111:B4 (LPS-4), and 0127:B8 (LPS-5)] and lipoteichoic acids from two gram-positive bacteria, Streptococcus faecalis (LT-2) and Bacillus subtilis (LT-3), were examined. LPS-5 inhibited markedly UT attachment to collagen and fibronectin. LPS-4 had no effect, whereas LPS-1 inhibited UT attachment to collagen but not to fibronectin. The fact that LPS-5 and LT-2 inhibited an Arg-Gly-Asp sequence-sensitive component of UT attachment to fibronectin is consistent with the possibility that these toxins acted via a mechanism involving typical fibronectin receptors. UT spreading was inhibited markedly by LPS-1, LT-2, and LT-3, whereas LPS-4 and LPS-5 had no effect. Because clustering of integrins is a crucial step in integrin-mediated signal transduction, the possibility that toxins inhibited spreading by affecting clustering was tested. Treatment with LT-2, which inhibited spreading dramatically, abolished completely a UT cell population containing more than five to eight beta 1- or beta 4-subunit-containing integrin clusters.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell signalling by integrins and immunoglobulin receptors in primed neutrophils.

Edwards SW
Trends Biochem Sci. 1995; 20: 362-7

Neutrophils use a variety of cell-surface receptors for attachment to surfaces, such as the endothelium or opsonized pathogens. During inflammation or infection, chemoattractants or cytokines bind to neutrophils and upregulate or 'prime' their responsiveness. Priming can increase the number of some receptors expressed on the cell surface but can also change receptor function, so that the receptor has altered ligand-binding properties or becomes linked to new intracellular signalling systems.

Signalling in extracellular-matrix-mediated control of epithelial cell phenotype.

Edwards G, Streuli C
Biochem Soc Trans. 1995; 23: 464-8

Interactions with an appropriate ECM are crucial for normal cell behaviour. The basement membrane contributes to both the tissue-specific lactational function and survival of mammary epithelial cells, possibly through a similar beta 1-integrin-dependent mechanism. Of current interest is whether the downstream intracellular signals diverge, with milk expression regulated through cell shape and the Jak-Stat pathway and survival mediated through controls on the Bcl-2/Bax and Ice checkpoints.

Integrin-mediated muscle cell spreading. The role of protein kinase c in outside-in and inside-out signaling and evidence of integrin cross-talk.

Disatnik MH, Rando TA
J Biol Chem. 1999; 274: 32486-92

Muscle cell survival depends upon the presence of various integrins with affinities for different extracellular matrix proteins. The absence of either alpha(5) or alpha(7) integrins leads to degenerative disorders of skeletal muscle, muscular dystrophies. To understand the cell survival signals that are mediated by integrin engagement with matrix proteins, we studied the early signaling events initiated by the attachment of muscle cells to fibronectin, an interaction that is mediated primarily by alpha(5) integrins. Cells that express alpha(5) integrin rapidly spread on fibronectin, and this process is associated with the phosphorylation of focal adhesion kinase (FAK). Cells deficient in alpha(5) integrin failed to spread or promote FAK phosphorylation when plated on fibronectin. For alpha(5)-expressing cells, both spreading and FAK phosphorylation could be blocked by inhibitors of protein kinase C (PKC), indicating that PKC is necessary for this "outside-in signaling" mediated by alpha(5) integrin. Surprisingly, activators of PKC could promote spreading and FAK phosphorylation in alpha(5)-deficient muscle cells plated on fibronectin. This PKC-induced cell spreading appeared to be due to activation of alpha(4) integrins ("inside-out signaling") since it could be blocked by peptides that specifically inhibit alpha(4) integrin binding to fibronectin. A model of integrin signaling in muscle cells is presented in which there is a positive feedback loop involving PKC in both outside-in and inside-out signaling, and the activation of this cycle is essential for cell spreading and downstream signaling to promote cell survival. In addition, the data indicate a cross-talk that occurs between integrins in which the outside-in signaling via one integrin can promote the activation of another integrin via inside-out signaling.

Purification and N-terminal sequence analysis of Streptomyces chromofuscus phospholipase D.

Dinh TT, McClure GD, Kennerly DA
Int Arch Allergy Immunol. 1995; 107: 69-71

Partially purified commercial phospholipase D (PLD) was fractionated by dye-ligand affinity chromatography and nondenaturing polyacrylamide gel electrophoresis (PAGE). Active material migrated as three bands on SDS-PAGE. The two higher-abundance species were shown to have identical N-terminal sequences, while the third band was present in much smaller amounts and had a distinct sequence. Cloning Streptomyces chromofuscus PLD will allow the construction of stable transfectants of mast cell lines permitting regulated expression of PLD.

Ligand recognition by the I domain-containing integrins.

Dickeson SK, Santoro SA
Cell Mol Life Sci. 1998; 54: 556-66

Seven of the integrin alpha subunits described to date, alpha 1, alpha 2, alpha L, alpha X, alpha d, alpha M and alpha E, contain a highly conserved I (or A) domain of approximately 200 amino acid residues inserted near the amino-terminus of the subunit. As the result of a variety of independent experimental approaches, a large body of data has recently accumulated that indicates that the I domains are independent, autonomously folding domains capable of directly binding ligands that play a necessary and important role in ligand binding by the intact integrins. Recent crystallographic studies have elucidated the structures of recombinant alpha M and alpha L I domains and also delineated a novel divalent cation-binding motif within the I domains (metal ion-dependent adhesion site, MIDAS) that appears to mediate the divalent cation binding of the I domains and the I domain-containing integrins to their ligands.

Determinants of ligand binding specificity of the alpha(1)beta(1) and alpha(2)beta(1) integrins.

Dickeson SK, Mathis NL, Rahman M, Bergelson JM, Santoro SA
J Biol Chem. 1999; 274: 32182-91

The alpha(1)beta(1) and alpha(2)beta(1) integrins are cell surface collagen receptors. Cells expressing the alpha(1)beta(1) integrin preferentially adhere to collagen IV, whereas cells expressing the alpha(2)beta(1) integrin preferentially adhere to collagen I. Recombinant alpha(1) and alpha(2) integrin I domains exhibit the same collagen type preferences as the intact integrins. In addition, the alpha(2) integrin I domain binds echovirus 1; the alpha(1) I domain does not. To identify the structural components of the I domains responsible for the varying ligand specificities, we have engineered several alpha(1)/alpha(2) integrin I domain chimeras and evaluated their virus and collagen binding activities. Initially, large secondary structural components of the alpha(2) I domain were replaced with corresponding regions of the alpha(1) I domain. Following analysis in echovirus 1 and collagen binding assays, chimeras with successively smaller regions of alpha(1) I were constructed and analyzed. The chimeras were analyzed by ELISA with several different alpha(2) integrin monoclonal antibodies to assess their proper folding. Three different regions of the alpha(1) I domain, when present in the alpha(2) I domain, conferred enhanced collagen IV binding activity upon the alpha(2) I domain. These include the alpha3 and alpha5 helices and a portion of the alpha6 helix. Echovirus 1 binding was lost in a chimera containing the alphaC-alpha6 loop; higher resolution mapping identified Asn(289) as playing a critical role in echovirus 1 binding. Asn(289) had not been implicated in previous echovirus 1 binding studies. Taken together, these data reveal the existence of multiple determinants of ligand binding specificities within the alpha(1) and alpha(2) integrin I domains.

Integrins: alternative splicing as a mechanism to regulate ligand binding and integrin signaling events.

de Melker AA, Sonnenberg A
Bioessays. 1999; 21: 499-509

Integrins are a family of transmembrane proteins composed of heterodimers of alpha and beta subunits. With their extracellular domain they bind extracellular matrix proteins or other cell surface molecules, and their cytoplasmic domain binds to cytoskeletal and signaling proteins. Thus, they are in an ideal position to transfer information from the extracellular environment to the interior of the cell and vice versa. For several integrin subunits, alternative splicing of mRNA leads to variations in the sequence of both extracellular and cytoplasmic domains. Many integrin splice variants have specific expression patterns, but for some time, functional differences between these variants were not evident. Recent experiments using transfected cell lines and gene targeting of specific splice variants have contributed significantly to our understanding of the function of these splice variants. The results indicate that alternative splicing is a mechanism to subtly regulate the ligand binding and signaling activity of integrins.

Biochemical and functional characteristics of integrins: a new family of adhesive receptors present in hematopoietic cells.

Dejana E, Lauri D
Haematologica. 1990; 75: 1-6

Glycosylphosphatidylinositol-phospholipase D: a tool for glycosylphosphatidylinositol structural analysis.

Deeg MA, Davitz MA
Methods Enzymol. 1995; 250: 630-40

Cleavage by the GPI-PLD provides definitive evidence of a minimal GPI structure: glucosamine-phosphatidylinositol. Unlike the case for PI-PLC, cleavage by the GPI-PLD is unaffected by acylation of the inositol ring. Thus the GPI-PLD provides an excellent simple enzymatic tool for analyzing the basic core structure of GPI anchors.

Midportion antibodies stimulate glycosylphosphatidylinositol-specific phospholipase D activity.

Deeg MA, Bowen RF
Arch Biochem Biophys. 1999; 370: 278-84

Limited information is known regarding the regulation, structural features, and functional domains of glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD, EC 3. 1.4.50). Previous studies demonstrated that trypsin cleavage of GPI-PLD at or near Arg325 and/or Arg589 in bovine serum GPI-PLD was associated with an increase in enzymatic activity. Since the Arg325 is predicted to be in a region between the catalytic domain and predicted beta-propeller structure in the C-terminal portion of GPI-PLD (T. A. Springer, Proc. Natl. Acad. Sci. USA 94, 65-72, 1997), we hypothesized that this connecting region is important for catalytic activity. Trypsin cleavage of human serum GPI-PLD, which has an Arg325 but lacks the Arg589 present in bovine serum GPI-PLD, also increased GPI-PLD activity. Peptide-specific antibodies to residues 275-296 (anti-GPI-PLD(275)) and a monoclonal antibody, 191, with an epitope encompassing Arg325, also stimulated GPI-PLD activity. Pretreating human GPI-PLD with trypsin demonstrated that anti-GPI-PLD(275) only stimulated the activity of intact GPI-PLD. These results suggest that trypsin activation and anti-GPI-PPLD(275) may have similar effects on GPI-PLD. Consistent with this is the observation that both manipulations decreased the affinity of GPI-PLD for mixed micelle substrates. These results indicate that the midportion region of GPI-PLD is important in regulating enzymatic activity.

Integrin cytoplasmic interactions and bidirectional transmembrane signalling.

Dedhar S, Hannigan GE
Curr Opin Cell Biol. 1996; 8: 657-69

Integrins are heterodimeric integral plasma membrane proteins containing extracellular, transmembrane, and cytoplasmic domains. These highly versatile receptors mediate not only cell adhesion and migration, but also the bidirectional transfer of information across the plasma membrane. The cytoplasmic domains of integrins are required for the transduction of this bidirectional information, and have recently been shown to participate in direct interactions with some novel cytoplasmic proteins, such as an ankyrin repeat containing serine/threonine protein kinase (integrin-linked kinase) and beta3 endonexin. New evidence also suggests that, via interactions with focal adhesion kinase, the integrin cytoplasmic domains can coordinate actin cytoskeletal organization and responses to growth factors. The elucidation of the signal transduction pathways activated by integrins is an intense area of investigation that has shown that integrins have some unique properties as signal transducing receptors.

Integrins and signal transduction.

Dedhar S
Curr Opin Hematol. 1999; 6: 37-43

Integrins are dynamic cell-surface receptors that provide a physical link between the extracellular matrix and the cell cytoskeleton. The interaction of integrins with extracellular matrix ligands also results in the generation of intracellular signals that regulate cell survival and growth, cell differentiation, and cell motility. The signaling pathways regulated by integrins are both diverse and complex, and involve cross-talk among multiple pathways. An added level of complexity resides in the findings that intracellular signals generated by a variety of agonists can modulate cell adhesion by regulating integrin-matrix interactions. The identification and functional characterization of integrin receptor-proximal components involved in this fascinating "inside-out" and "outside-in" signal transduction remains an area of intense investigation. This review focuses on recent advances in this area.

Integrin mediated signal transduction in oncogenesis: an overview.

Dedhar S
Cancer Metastasis Rev. 1995; 14: 165-72

Signal transduction via the beta 1 integrins is a required intermediate in interleukin-1 beta induction of alkaline phosphatase activity in human osteosarcoma cells.

Dedhar S
Exp Cell Res. 1989; 183: 207-14

In this report data are presented which demonstrate that the induction of an osteoblastic phenotype by interleukin-1 beta (IL-1 beta) requires an intermediate step involving signal transduction via the beta 1 family of integrin glycoproteins. Recombinant human IL-1 beta inhibits human osteosarcoma cell proliferation, stimulates integrin expression, and induces alkaline phosphatase activity, a marker of osteoinductive and osteoblastic phenotype. The approximately 10-fold stimulation of expression of the beta 1 integrins occurs rapidly (within 20 to 40 h), whereas the alkaline phosphatase activity is not induced until at least 5 days after the addition of IL-1 beta. To determine whether the early stimulation of integrin expression is required for the subsequent expression of alkaline phosphatase activity, polyclonal as well as monoclonal antibodies directed against the alpha 5 and beta 1 integrin subunits were added to cultures at the same time as IL-1 beta. These antibodies inhibited by 55 to 82% the longer term induction of the osteoblastic differentiation marker, alkaline phosphatase activity, but did not however affect the IL-1 beta-induced stimulation of integrin expression or the inhibition of cell proliferation. In addition, at the concentrations used, there was no effect of the antibodies on cell attachment. These data suggest that the stimulation of integrin expression by IL-1 beta, and the resulting enhanced integrin-extracellular matrix interactions, is a required intermediate event in the IL-1 beta regulation of osteoblastic cell differentiation. The data also suggest that the integrins are capable of signal transduction resulting in altered gene expression, and may also play a crucial role in modulating cytokine-mediated effects on cell differentiation.

Cell-substrate interactions and signaling through ILK.

Dedhar S
Curr Opin Cell Biol. 2000; 12: 250-6

Interactions between cells and the extracellular matrix (ECM) result in the regulation of cell growth, cell differentiation and cell migration. These interactions are mediated by integrins and growth factor receptors and intracellular effectors that couple these receptors to downstream components are key to the transduction of ECM signals. This review summarizes recent advances in our understanding of signal transduction via integrins, focusing on the role of integrin-linked kinase in some of these pathways. Research into this interesting protein is uncovering novel aspects of coordinated signaling by the ECM and growth factors.

Integrins and mechanotransduction of the vascular myogenic response.

Davis MJ, Wu X, Nurkiewicz TR, Kawasaki J, Davis GE, Hill MA, Meininger GA
Am J Physiol Heart Circ Physiol. 2001; 280: 142733-142733

This review summarizes what is currently known about the role of integrins in the vascular myogenic response. The myogenic response is the rapid and maintained constriction of a blood vessel in response to pressure elevation. A role for integrins in this process has been suggested because these molecules form an important mechanical link between the extracellular matrix and the vascular smooth muscle cytoskeleton. We briefly summarize evidence for a general role of integrins in mechanotransduction. We then describe the integrin subunit combinations known to exist in smooth muscle and the vascular wall matrix proteins that may interact with these integrins. We then discuss the effects of integrin-specific peptides and antibodies on vascular tone and on calcium entry mechanisms in vascular smooth muscle. Because integrin function is linked to the cytoskeleton, we discuss evidence for the role of the cytoskeleton in determining myogenic responsiveness. Finally, we analyze evidence that integrin-linked signaling pathways, such as those involving protein tyrosine phosphorylation cascades and mitogen-activated protein kinases, are required for myogenic tone.

Integrins interact with focal adhesions through multiple distinct pathways.

David FS, Zage PE, Marcantonio EE
J Cell Physiol. 1999; 181: 74-82

Integrin signaling involves oligomerization and a transmembrane conformational change induced by receptor occupancy. Previous work has shown that subsets of focal adhesion-associated proteins are recruited to integrins as a result of clustering, ligand binding, or both. However, it is unclear whether these discrete subsets reflect the differential binding of cytoplasmic proteins to the integrin or whether a single protein or set of proteins binds the integrin and is differentially activated by receptor occupancy or clustering. To address this question, we made mutations of the beta1 integrin cytoplasmic domain in the context of a single subunit chimera and studied their activation of various known integrin-mediated signaling pathways. We show here that the indirect association of the integrin with actin is distinct from its interactions with both preformed focal adhesions and FAK. Therefore, multiple independent signaling pathways exist from the integrin to the focal adhesion, which may reflect the association of independent factors with the integrin beta1 cytoplasmic domain.

Anti-apoptotic action of macrophage stimulating protein (MSP).

Danilkovitch-Miagkova A, Leonard EJ
Apoptosis. 2001; 6: 183-90

MSP is a serum protein belonging to the plasminogen-related kringle domain protein family. In addition to macrophages, epithelial cells are also MSP targets. MSP is a multifunctional factor regulating cell adhesion and motility, growth and survival. MSP mediates its biological activities by activating a transmembrane receptor tyrosine kinase called RON in humans or SKT in mice. MSP can protect epithelial cells from apoptosis by activating two independent signals in the PI3-K/AKT or the MAPK pathway. The MAPK pathway mediates the MSP antiapoptotic effect only if additional signaling pathways are activated through adhesion. This indicates that MSP receptors and integrins, the receptors mediating cell-matrix-dependent adhesion, can collaborate in promotion of cell survival. This adhesion-dependent pathway, which is essential for the MAPK-mediated anti-apoptotic effect, remains to be identified. A hypothesis that Stat3 might represent a key component of the adhesion-induced anti-apoptotic pathway is presented in this review.

Fibronectin, integrins, and growth control.

Danen EH, Yamada KM
J Cell Physiol. 2001; 189: 1-13

Cell proliferation is controlled not only by soluble mitogens but also by components of the extracellular matrix (ECM) such as fibronectin, to which cells adhere via the integrin family of transmembrane receptors. Input from both growth factor receptors and integrins is required to stimulate progression through the G1 phase of the cell cycle, via induction of G1 cyclins and suppression of inhibitors of the G1 cyclin-dependent kinases. Extensive crosstalk takes place between integrin and growth factor receptor signaling pathways, and mitogenic signaling is weak and transient in the absence of integrin-mediated cell adhesion. In normal untransformed cells, all of the important mitogenic signal transduction cascades, namely those downstream of the Ras and Rho family small GTPases and the phosphoinositide 3-OH kinase-PKB/Akt pathway, are regulated by integrin-mediated cell adhesion. As a result, these cells are anchorage-dependent for growth. In contrast, constitutive activity of each of these pathways has been reported in cancer cells, which not only reduces their mitogen dependence but also allows these cells to grow in an anchorage-independent fashion.

Signal transduction by integrin receptors for extracellular matrix: cooperative processing of extracellular information.

Damsky CH, Werb Z
Curr Opin Cell Biol. 1992; 4: 772-81

Adhesion receptors allow cells to interact with a dynamic and information-rich environment of extracellular matrix molecules. The integrin family of adhesion receptors transduces signals from the extracellular matrix that regulate growth, gene expression and differentiation, as well as cell shape, motility and cytoskeletal architecture. Recent data support the hypothesis that integrins transduce signals cooperatively with other classes of adhesion receptors or with growth factor receptors. Furthermore, the ability of integrins to interact with the cytoskeleton appears to be fundamental to their mechanism for signal transduction.

The solid state environment orchestrates embryonic development and tissue remodeling.

Damsky CH, Moursi A, Zhou Y, Fisher SJ, Globus RK
Kidney Int. 1997; 51: 1427-33

Cell interactions with extracellular matrix and with other cells play critical roles in morphogenesis during development and in tissue homeostasis and remodeling throughout life. Extracellular matrix is information-rich, not only because it is comprised of multifunctional structural ligands for cell surface adhesion receptors, but also because it contains peptide signaling factors, and proteinases and their inhibitors. The functions of these groups of molecules are extensively interrelated. In this review, three primary cell culture models are described that focus on adhesion receptors and their roles in complex aspects of morphogenesis and remodeling: the regulation of proteinase expression by fibronectin and integrins in synovial fibroblasts; the regulation of osteoblast differentiation and survival by fibronectin, and the regulation of trophoblast differentiation and invasion by integrins, cadherins and immunoglobulin family adhesion receptors.

Extracellular matrix-integrin interactions in osteoblast function and tissue remodeling.

Damsky CH
Bone. 1999; 25: 95-6

Previous work with cultured primary cells, from our group and from other laboratories, has shown that signals from extracellular matrix, transduced by integrins, play critical roles in regulating gene expression, tissue-specific differentiation, and survival of primary osteoblasts and fibroblasts. This summary will focus on our most recent work, dealing with the role of cell-extracellular matrix interactions and focal adhesion kinase in regulating cell survival in osteoblasts and fibroblasts, and the role of beta1 integrins in tissue organization and remodeling in bone.

Understanding the sequence determinants of conformational switching using protein design.

Dalal S, Regan L
Protein Sci. 2000; 9: 1651-9

An important goal of protein design is to understand the forces that stabilize a particular fold in preference to alternative folds. Here, we describe an extension of earlier studies in which we successfully designed a stable, native-like helical protein that is 50% identical in sequence to a predominantly beta-sheet protein, the B1 domain of Streptococcal IgG-binding protein G. We report the characteristics of a series of variants of our original design that have even higher sequence identity to the B1 domain. Their properties illustrate the extent to which protein stability and conformation can be modulated through careful manipulation of key amino acid residues. Our results have implications for understanding conformational change phenomena of central biological importance and in probing the malleability of the sequence/structure relationship.

The pharmacology of the integrins.

Cox D, Aoki T, Seki J, Motoyama Y, Yoshida K
Med Res Rev. 1994; 14: 195-228

Bi-directional signal transduction by integrin receptors.

Coppolino MG, Dedhar S
Int J Biochem Cell Biol. 2000; 32: 171-88

The integrin family of cell surface glycoproteins functions primarily as receptors for extracellular matrix ligands. There are now many well characterized integrin-ligand interactions which are known to influence many aspects of cell behaviour including cell morphology, cell adhesion, cell migration as well as cellular proliferation and differentiation. However, in fulfilling these functions, integrins are not simple adhesion receptors that physically mediate connections across the plasma membrane. Rather, integrin function itself is highly regulated, largely through the formation of specific associations with both structural and regulatory components within cells. It is these intracellular interactions which allow integrin function to effect many biochemical signalling pathways and therefore to impinge upon complex cellular activities. Recently, much research has focused on elucidating the molecular mechanisms which control integrin function and the molecular processes which transduce integrin-mediated signalling events. In this review, we discuss progress in the field of integrin signal transduction including, where applicable, potential therapeutic applications arising from the research.

Scatter factors and invasive growth.

Comoglio PM, Boccaccio C
Semin Cancer Biol. 2001; 11: 153-65

Scatter factors are unequivocal signals governing a genetic program that includes cell detachment, repulsion, protection from apoptosis, invasiveness of extracellular matrices and proliferation. This pleiomorphic response is defined as 'invasive growth'. Under physiological conditions, it leads to morphogenic cell movements through the matrix, and--primarily--to ordered building of epithelial tubules. Dysfunctions in invasive growth cause enhanced proliferation, uncontrolled migration into surrounding tissues, and failure to differentiate, events that foster tumour growth and invasiveness. Scatter factors act through tyrosine kinase receptors that belong to the Met oncogene family. Here we discuss how alterations of these receptors or of their signal transduction pathways are responsible for cancer onset and progression towards metastasis. Copyright 2001 Academic Press.

1997 keystone symposium on signal transduction by cell adhesion receptors.

Clark EA, Hynes RO
Biochim Biophys Acta. 1997; 1333: 916-916

Structural and biologic properties of integrin-mediated cell adhesion.

Cheresh DA
Clin Lab Med. 1992; 12: 217-36

The integrins are a family of proteins that mediate cellular adhesion mechanisms involved in development, differentiation, wound healing, immune recognition, and inflammation. In addition, these adhesion receptors also potentiate disease states including cancer, thrombosis, and inflammatory diseases. To understand the role of integrins in these processes, it is important to define their structural and functional properties. It is also imperative to understand the distribution of integrins on normal and malignant cell types. Integrins are regulated on the cell surface, and these events likely promote specific cellular adhesion mechanisms. This is of particular concern among tumor cells, which not only attach to the extracellular matrix but must invade and migrate through it. Integrins are surface-expressed proteins that mediate a linkage to the cytoskeleton within the cell, and thus cell morphology and traction during migration. Thus, by understanding the structural basis of integrin--ligand and integrin--cytoskeleton interactions we will gain insight into the molecular basis of cancer, thrombosis, wound healing, and inflammation.

The involvement of the cell matrix receptors, or VLA integrins, in the morphogenetic behavior of normal and malignant cells is gradually being uncovered.

Cassiman JJ
Cancer Genet Cytogenet. 1989; 41: 19-32

The VLA-Integrins are members of a family of cell surface receptors that recognize Arg-Gly-Asp containing ligands and that allow cells to bind extracellular matrix molecules such as fibronectin, laminin, and collagens. These interactions reorganize the topographic distribution of the receptors at the cell surface and are relayed to the cytoplasm and even to the cell nucleus. Coordinated organization of the intracellular cytoskeleton, cell spreading on the matrix, and movement of the cells along concentration gradients of these matrix molecules are the result of these interactions. Evidence exists that the migration of embryonic cells, platelet activation on endothelial defects, and the invasion of fibroblasts, lymphoid cells, and epithelial cells during wound healing are facilitated by Integrin-matrix interactions. Moreover, deficiency in the synthesis of a particular Integrin subunit or the inappropriate expression of one or a few members of this family might contribute fundamentally to invasion and metastasis of malignant cells. Although extensive information is available on the structure of the common beta 1 subunit and on the function of the fibronectin receptor, in particular, the other five members of the very late antigen family, their respective alpha subunits and their function as cell surface receptors remain poorly understood. The rationale for having at least six different receptors on some cells, how they might collaborate, and to what end remains unknown. Finally, the interaction of these cell surface receptors and the modulation of their affinity for their ligand by other cell surface or extracellular matrix components such as heparan sulfate, gangliosides a.o. have been barely touched upon. It can be predicted that these molecules and their interaction with the extracellular matrix will provide a key to the understanding of many normal and malignant morphogenetic cellular properties.

Integrin-mediated signal transduction pathways.

Cary LA, Han DC, Guan JL
Histol Histopathol. 1999; 14: 1001-9

Integrins serve as adhesion receptors for extracellular matrix proteins and also transduce biochemical signals into the cell. They regulate a variety of cellular functions, including spreading, migration, proliferation and apoptosis. Many signaling pathways downstream of integrins have been identified and characterized and are discussed here. In particular, integrins regulate many protein tyrosine kinases and phosphatases, such as FAK and Src, to coordinate many of the cell processes mentioned above. The regulation of MAP kinases by integrins is important for cell growth or other functions, and the putative roles of Ras and FAK in these pathways are discussed. Phosphatidylinositol lipids and their modifying enzymes, particularly PI 3-kinase, are strongly implicated as mediators of integrin-regulated cytoskeletal changes and cell migration. Similarly, actin cytoskeleton regulation by the Rho family of GTPases is coordinated with integrin signaling to regulate cell spreading and migration, although the exact relationship between these pathways is not clear. Finally, intracellular pH and calcium fluxes by integrins are suggested to affect a variety of cellular proteins and functions.

Isolation of leukocyte response integrin: a novel RGD-binding protein involved in regulation of phagocytic function.

Carreno MP, Gresham HD, Brown EJ
Clin Immunol Immunopathol. 1993; 69: 43-51

We have described previously an adhesive protein on neutrophils (PMN) which recognizes fibrinogen, fibronectin (Fn), von Willebrandt's factor, vitronectin, collagen, and synthetic peptides containing the Arg-Gly-Asp (RGD) sequence (Gresham et al., J. Cell Biol. 108, 1935-1943, 1989). We have called this oligospecific receptor the leukocyte response integrin (LRI). Engagement of LRI leads to both increased ingestion via PMN IgG Fc receptors and to adhesion and chemotaxis to certain extracellular matrix proteins. Now, we have purified an RGD-binding receptor from DMSO-differentiated HL-60 cells (dHL-60) by peptide affinity chromatography which has the biochemical, immunologic, and functional characteristics of LRI. The purified protein contains two bands of 135 and 90 kDa under nonreducing conditions SDS-PAGE. Immunologic characterization of the dHL-60 RGD receptor showed that, by Western blot and ELISA, the lower M(r) band was recognized by mAb 7G2, raised against placental beta 3, which is known to inhibit LRI function. However, despite this functional and immunologic cross-reactivity with beta 3, the receptor was not recognized efficiently by a polyclonal antibody to placental RGD-binding proteins, predominantly alpha v beta 3. Moreover, polyclonal antibody raised to the dHL-60 receptor (Ab1) did not react with placental RGD-binding proteins. By immunoprecipitation or ELISA, we demonstrated that the purified RGD-binding receptor was not alpha IIb beta 3 or alpha v beta 3 and did not contain the integrin chains alpha 4, beta 2, or beta 7. Functionally, Ab1 totally inhibited Fn-stimulated ingestion by PMN. Moreover, Ab1 inhibited phagocytosis stimulated by the peptide KGAGDV, which is the most specific ligand for LRI currently known, and Ab1 inhibited the binding of KGAGDV-coated microspheres to PMN and monocytes. FACS analysis with Ab1 showed staining of monocytes, PMN, and lymphocytes but not platelets or erythrocytes. We conclude that LRI is a novel RGD-binding receptor which exists on leukocytes and which shares an antigenic epitope(s) with beta 3. This receptor recognizes multiple RGD-containing ligands and can mediate signal transduction for adhesion, chemotaxis, and activation of increased phagocytic potential by PMN and monocytes.

Integrins and actin filaments: reciprocal regulation of cell adhesion and signaling.

Calderwood DA, Shattil SJ, Ginsberg MH
J Biol Chem. 2000; 275: 22607-10

Actin-membrane interaction in focal adhesions.

Burridge K, Nuckolls G, Otey C, Pavalko F, Simon K, Turner C
Cell Differ Dev. 1990; 32: 337-42

Focal adhesions are regions of the plasma membrane where cells in tissue culture adhere strongly to the underlying extracellular matrix, and which at their cytoplasmic face serve to anchor bundles of actin microfilaments. They provide an experimental model for studying the links between the cytoskeleton and the extracellular matrix. Members of the integrin family of extracellular matrix receptors are prominent components, spanning the membrane in focal adhesions, but there is evidence that other membrane components are also needed for these structures to form. A number of proteins are concentrated at the cytoplasmic face of focal adhesions. Recent efforts have sought to determine the links between actin and the integrin cytoplasmic domains. Using in vitro binding assays, two potential bridges between actin and integrin have been identified. One involves talin, which has recently been shown to bind actin directly. The other involves the actin-binding protein, alpha-actinin, which has been found to interact with several integrins. The physiological significance of these two potential bridges between actin and integrin remains to be determined in vivo.

Focal adhesions, contractility, and signaling.

Burridge K, Chrzanowska-Wodnicka M
Annu Rev Cell Dev Biol. 1996; 12: 463-518

Focal adhesions are sites of tight adhesion to the underlying extracellular matrix developed by cells in culture. They provided a structural link between the actin cytoskeleton and the extracellular matrix and are regions of signal transduction that relate to growth control. The assembly of focal adhesions is regulated by the GTP-binding protein Rho. Rho stimulates contractility which, in cells that are tightly adherent to the substrate, generates isometric tension. In turn, this leads to the bundling of actin filaments and the aggregation of integrins (extracellular matrix receptors) in the plane of the membrane. The aggregation of integrins activates the focal adhesion kinase and leads to the assembly of a multicomponent signaling complex.

Extracellular matrix and the implantation cascade in pigs.

Burghardt RC, Bowen JA, Newton GR, Bazer FW
J Reprod Fertil Suppl. 1997; 52: 151-64

The structural and functional alterations of uterine epithelial cells that permit the apical-apical union of conceptus and uterine epithelium are complex and are likely to involve many different adhesion molecules with distinct but inter-related functions. A number of changes in the molecular composition at the apical surface of uterine epithelial cells associated with the transition from the pre-receptive to the receptive state in the pig uterus are reviewed. Molecules that function in the adhesion cascade resulting in implantation are represented by a variety of adhesion systems. However, integrins are probably the dominant adhesion systems because their capacity to mediate adhesion is linked to their activation by engaging other surface molecules.

Drosophila PS2 integrin mediates RGD-dependent cell-matrix interactions.

Bunch TA, Brower DL
Development. 1992; 116: 239-47

Integrins are a family of transmembrane glycoproteins that mediate cell-matrix and cell-cell interactions. We have transfected cultured Drosophila cells with genes that express the Drosophila PS2 integrin. We demonstrate that this integrin is expressed on the surface of the cells and can mediate cell spreading on an undefined component of fetal calf serum or on the purified vertebrate matrix molecules vitronectin and fibronectin. Additionally, PS2 integrin can cause cell spreading on RGD peptide. The spreading on matrix components or RGD peptide can be inhibited by soluble RGD peptide and is dependent on divalent cations.

Integrin, a transmembrane glycoprotein complex mediating cell-substratum adhesion.

Buck CA, Horwitz AF
J Cell Sci Suppl. 1987; 8: 231-50

A monoclonal antibody, CSAT, which inhibits the adhesion of chick cells to substrata coated with fibronectin, laminin and vitronectin, has been used to identify a cell surface receptor required for cell-substratum adhesion. This receptor, termed integrin, is found on the ventral surface of cells in close contact adhesion sites, at the periphery of adhesion plaques and beneath stress fibres. It is a heterodimer consisting of non-covalently linked alpha and beta subunits. Integrin binds directly to laminin, fibronectin and vitronectin with dissociation constants in the micromolar range. The binding of integrin to matrix molecules is sensitive to peptides carrying the cell-binding sequence Arg-Gly-Asp and requires heteromeric integrity. Integrin also binds directly to the cytoskeleton-associated protein talin. Thus, integrin has the properties of a transmembrane molecule capable of bringing extracellular matrix and cytoskeleton-associated molecules in proper juxtaposition to form adhesion structures. The integrin beta subunit is phosphorylated following Rous sarcoma virus transformation. Phosphorylation alters the ability of the receptor to bind extracellular matrix molecules as well as talin, suggesting a mechanism for the alteration of cellular adhesive and morphological properties following malignant transformation. A major phosphorylation site is on the cytoplasmic domain of the beta subunit. Synthetic peptides homologous with this region of integrin inhibit integrin-talin binding. The gene for the beta subunit of integrin has been sequenced. Its structure is consistent with the membrane-spanning properties of the receptor. Integrin is structurally and serologically related to adhesion receptors from mammalian tumour cells, fibroblasts, platelets and lymphocytes. It appears to be a member of a supergene family of receptors involved in cellular adhesive interactions. Antibody and peptide inhibition experiments have suggested a role for integrin and integrin-like molecules in cell migration, neurite extension, neural differentiation, histogenesis and embryonic development in Drosophila. Thus, integrin appears representative of a set of evolutionarily conserved, biologically important adhesive molecules.

Integrins as mediators of morphogenesis in Drosophila.

Brown NH, Gregory SL, Martin-Bermudo MD
Dev Biol. 2000; 223: 1-16

Integrins hold Drosophila together.

Brown NH
Bioessays. 1993; 15: 383-90

The Drosophila position-specific (PS) integrins are members of the integrin family of cell surface receptors and are thought to be receptors for extracellular matrix components. Each PS integrin consists of an alpha subunit, alpha PS1 or alpha PS2, and a beta PS subunit. Mutations in the beta PS subunit and the alpha PS2 subunit have been characterised and reveal that the PS integrins have an essential role in the adhesion of different cell layers to each other. The PS integrins are especially required for the function of the cell-matrix-cell junctions, where the muscles attach to the epidermis and where one surface of the developing wing adheres to the other. These junctions are similar to vertebrate focal adhesions and hemidesmosomes, which also contain integrins. Integrin-mediated cell to cell adhesion via the extracellular matrix provides a way for tissues to adhere to each other without intermingling of their cells.

Where the outside meets the inside: integrins as activators and targets of signal transduction cascades.

Brown E, Hogg N
Immunol Lett. 1996; 54: 189-93

In fibroblasts, signaling through the adhesion receptors known as integrins synergizes with other cellular stimulators such as the growth factors. There is currently great interest in the details of the ensuing 'outside in' signal transduction mechanisms, and the focal adhesion kinase in particular, has been a focus of attention. Less is understood of signalling through integrins on leukocytes which also perform a costimulator role. The activity of these leukocyte integrins is not constitutive but is initiated via signalling through other receptors, termed 'inside out' signalling. These signals cause movement and clustering of integrins in the membrane leading to strengthened adhesion between cells.

Molecular evolution of integrins: genes encoding integrin beta subunits from a coral and a sponge.

Brower DL, Brower SM, Hayward DC, Ball EE
Proc Natl Acad Sci U S A. 1997; 94: 9182-7

The integrin family of cell surface receptors is strongly conserved in higher animals, but the evolutionary history of integrins is obscure. We have identified and sequenced cDNAs encoding integrin beta subunits from a coral (phylum Cnidaria) and a sponge (Porifera), indicating that these proteins existed in the earliest stages of metazoan evolution. The coral betaCn1 and, especially, the sponge betaPo1 sequences are the most divergent of the "beta1-class" integrins and share a number of features not found in any other vertebrate or invertebrate integrins. Perhaps the greatest difference from other beta subunits is found in the third and fourth repeats of the cysteine-rich stalk, where the generally conserved spacings between cysteines are highly variable, but not similar, in betaCn1 and betaPo1. Alternatively spliced cDNAs, containing a stop codon about midway through the full-length translated sequence, were isolated from the sponge library. These cDNAs appear to define a boundary between functional domains, as they would encode a protein that includes the globular ligand-binding head but would be missing the stalk, transmembrane, and cytoplasmic domains. These and other sequence comparisons with vertebrate integrins are discussed with respect to models of integrin structure and function.

Role of the PS integrins in Drosophila development.

Brower DL, Brabant MC, Bunch TA
Immunol Cell Biol. 1995; 73: 558-64

The PS1 and PS2 integrins of Drosophila are heterodimers of alphaPS1betaPS and alphaPS2betaPS subunits, respectively, with very strong structural similarity to vertebrate integrins. Cell transfection experiments indicate that the PS integrins are receptors for extracellular matrix components and are functionally analogous to vertebrate integrins. Matrix ligands from Drosophila tissues have been identified for PS1 and PS2 integrins, using transformed cells and a cell-spreading assay. Mutations in all three subunit genes have been identified, and the phenotypes of mutants indicate that PS integrins are required for the proper morphogenesis of a number of embryonic tissues. Using methods to produce genetic mosaics and transformation of integrin transgenes into whole animals, integrin functions in adult morphogenesis also have been examined. In the pupal wing, integrins are critically required to maintain the connection between dorsal and ventral epithelia, and recent results suggest that in early pupal development, the integrins are acting as specific receptors, as opposed to a non-specific cell-matrix glue.

Expression of native and truncated forms of the human integrin alpha 1 subunit.

Briesewitz R, Epstein MR, Marcantonio EE
J Biol Chem. 1993; 268: 2989-96

We report here the molecular cloning of cDNAs encoding for the human integrin alpha 1 subunit. The sequence is characteristic of an I domain containing integrin alpha subunit, with a high degree of homology to the rat integrin alpha 1 subunit, including complete identity of the transmembrane and cytoplasmic domains between the two species. The human cDNA directs the expression in mouse NIH 3T3 cells of authentic human alpha 1 protein as demonstrated by the reactivity of this subunit with two human-specific anti-alpha 1 monoclonal antibodies. This exogenous integrin specifically binds to type IV collagen in a Mg(2+)-dependent fashion. We have expressed in both transient systems and in stable cell lines truncated, soluble forms of the human alpha 1 subunit combined with truncated, soluble forms of beta 1 subunits. Although soluble beta 1 subunit was found in the media when the corresponding cDNA was used, the secretion of the soluble alpha 1 subunit was found to be dependent on dimerization with soluble beta 1. Co-transfection of truncated human alpha 1 cDNA with truncated forms of either the human or avian beta 1 cDNA led to efficient secretion of alpha 1.beta 1 heterodimers. These soluble heterodimers specifically bind to collagen IV in a manner similar to their full-length counterparts. Biosynthetic studies using stably expressing cell lines demonstrate that the soluble heterodimers and the native heterodimers are formed independently, strongly suggesting that the transmembrane or cytoplasmic domains of alpha and beta subunits are involved in the assembly of native heterodimers.

Restricted distribution of integrin beta 6 mRNA in primate epithelial tissues.

Breuss JM, Gillett N, Lu L, Sheppard D, Pytela R
J Histochem Cytochem. 1993; 41: 1521-7

Integrins are cell adhesion receptors that mediate cell-extracellular matrix and cell-cell interactions. Each integrin consists of two glycoprotein subunits (alpha and beta). We have previously described a novel integrin beta-subunit, beta 6, which is expressed in cultured epithelial cells. beta 6 can associate with alpha v to form the fibronectin-binding heterodimer alpha v beta 6. Here we report the tissue distribution of beta 6 integrin mRNA determined by in situ hybridization of a beta 6 cRNA probe with representative frozen tissue sections from a rhesus monkey tissue bank. We detected beta 6 mRNA exclusively in epithelial cells. However, beta 6 mRNA expression varied greatly among different epithelia. High levels of beta 6 mRNA were found only in two very specialized epithelial cell types: a portion of the kidney tubule epithelium, termed macula densa, and the endometrial epithelium of secretory phase uterus. In the endometrium, beta 6 expression was highest in the differentiated epithelium of functional layer glands, suggesting that beta 6 expression can be regulated in a differentiation-dependent manner. beta 6 expression may also depend on the stage in the estrous cycle, since we found much lower beta 6 mRNA levels in a specimen of proliferative phase endometrium. Epithelium in several other tissues, including salivary gland ducts, gall bladder, and epididymis, contained detectable levels of beta 6 mRNA, albeit much lower than in macula densa and endometrium. In other epithelia, including skin and lung, beta 6 mRNA was undetectable. Taken together, these results suggest that in normal adult primates beta 6 expression is regulated in a cell type-specific manner, restricted to a few epithelial tissues.

Functional consequences of integrin gene mutations in mice.

Bouvard D, Brakebusch C, Gustafsson E, Aszodi A, Bengtsson T, Berna A, Fassler R
Circ Res. 2001; 89: 211-23

Integrins are cell-surface receptors responsible for cell attachment to extracellular matrices and to other cells. The application of mouse genetics has significantly increased our understanding of integrin function in vivo. In this review, we summarize the phenotypes of mice carrying mutant integrin genes and compare them with phenotypes of mice lacking the integrin ligands.

Specific tracheal migration is mediated by complementary expression of cell surface proteins.

Boube M, Martin-Bermudo MD, Brown NH, Casanova J
Genes Dev. 2001; 15: 1554-62

Migration of the Drosophila tracheal cells relies on cues provided by nearby cells; however, little is known about how these signals specify a migratory path. Here we investigate the role of cell surface proteins in the definition of such a pathway. We have found that the PS1 integrin is required in the tracheal cells of the visceral branch, whereas the PS2 integrin is required in the visceral mesoderm; both integrins are necessary for the spreading of the visceral branch over its substratum. This is the first identification of a cell surface molecule with expression restricted to a subset of tracheal cells that all migrate in a given direction. We have also found that expression of PS1 in the visceral branch is regulated by the genes that direct tracheal cell migration, showing that integrin expression is part of the cell-fate program that they specify. These results support a model in which signal transduction determines the tracheal migratory pathways by regulating the expression of cell surface proteins, which in turn interact with surface molecules on the surrounding cell population.

Chick integrin alpha V subunit molecular analysis reveals high conservation of structural domains and association with multiple beta subunits in embryo fibroblasts.

Bossy B, Reichardt LF
Biochemistry. 1990; 29: 10191-8

We have cloned and characterized a chick homologue of the human vitronectin receptor alpha subunit (alpha v) whose primary sequence is 83% identical with its human counterpart but less than 40% identical with any other known integrin alpha subunit. Comparison of the chick and human sequences reveals several highly conserved regions, including the cytoplasmic domain. The putative ligand binding domain contains alpha v-specific residues that may contribute to ligand binding specificity. These are concentrated in three regions that are located before and between the first three Ca2+ binding domains. Polyclonal antibodies raised against two peptides deduced from the putative cytoplasmic and extracellular domains of the chick alpha v sequence recognize specifically integrin heterodimers in chick embryo fibroblasts. At least three putative beta subunits coimmunoprecipitate with the chick alpha v subunit. In addition to a protein with the same molecular weight as beta 3 (94K), protein bands of Mr 84K and 110K are also coprecipitated. By successive immunodepletions, we demonstrate that this latter Mr 110K subunit is beta 1, which appears to be one of the alpha v-associated subunits in chick embryo fibroblasts.

Tyrosine phosphorylation of 40 kDa proteins in osteoblastic cells after mechanical stimulation of beta1-integrins.

Bierbaum S, Notbohm H
Eur J Cell Biol. 1998; 77: 60-7

Using a method for the mechanical stimulation of cells which was adapted from one developed by Wang and Ingber employing magnetic microbeads [Wang, N. D., D. E. Ingber: Control of cytoskeletal mechanics by extracellular matrix, cell shape, and mechanical tension. Biophys. J. 66, 2181-2189 (1994)], mechanical stress could be applied to specific receptors on the cell surface. To achieve this, ferromagnetic microbeads coated with different ligands were magnetized after adhesion to the cells. The beads were then 'twisted' using a second magnetic field oriented perpendicular to the magnetizing one. Contrary to most current methods, it was possible to confer the strain without deforming the cell as a whole, thus being able to observe the individual reactions of transmembrane receptors to mechanical stress. An increase in tyrosine phosphorylation of proteins migrating at approximately 40 kDa could be observed as a reaction to stress on the beta1-subunits of the integrin family, while stress to other transmembrane molecules like the transferrin or low density lipoprotein receptors with no connection to the cytoskeleton did not give this reaction. Fibroblastic cells showed, contrary to osteoblastic cells, no reaction to stress applied on transmembrane proteins.

Neutrophil activation by adhesion: mechanisms and pathophysiological implications.

Berton G, Yan SR, Fumagalli L, Lowell CA
Int J Clin Lab Res. 1996; 26: 160-77

Neutrophil adhesion plays an essential role in the formation of an inflammatory exudate. Moreover, adhesion activates selective neutrophil functions and regulates the cell response to additional stimuli. In this review we summarize the information available on adhesion molecules involved in neutrophil adhesion to endothelial cells and extracellular matrix proteins and the experimental approaches which have been developed to block neutrophil adhesion and neutrophil mediated tissue damage. We also address the mechanisms of activation of selective neutrophil functions by adhesion molecules and, in particular the mechanisms of signal transduction by neutrophil integrins. On the basis of recent results obtained in our and other laboratories we propose a model hypothesizing mechanisms of signaling by neutrophil integrins involved in regulation of selective functions.

Integrin signalling in neutrophils and macrophages.

Berton G, Lowell CA
Cell Signal. 1999; 11: 621-35

Integrins have been characterized extensively as adhesion receptors capable of transducing signals inside the cell. In myelomonocytic cells, integrin-mediated adhesive interactions regulate different selective cell responses, such as transmigration into the inflammatory site, cytokine secretion, production or reactive oxygen intermediates, degranulation and phagocytosis. In the last few years, great progress has been made in elucidating mechanisms of signal transduction by integrins in neutrophils and macrophages. This review summarises the current information on the role of integrins in regulating myelomonocytic cell functions and highlights the signalling pathways activated by integrin engagement in these cells. Also, exploiting the current knowledge of mechanisms of integrin signal transduction in other cell types, we propose a model to explain how integrins transduce signals inside neutrophils and macrophages, and how signaling pathways leading to regulation of selective cell functions may be coordinated.

Integrins: structure and functions.

Berman AE, Kozlova NI
Membr Cell Biol. 2000; 13: 207-44

Integrins are cell surface transmembrane glycoproteins which perform receptor functions in cell interactions with the extracellular matrix and cell-cell contacts. Another function of integrins is transduction of signals which mediate the effects of the matrix on the physiological activity of cells (motility, proliferation, differentiation, etc.). Contemporary views on the molecular structure of integrins and their involvement in the organization of the cytoskeleton and control of gene activity are presented. Biochemical mechanisms, by which the signal functions of integrins are realized, are discussed. The data on the participation of integrins in oncogenic cell transformation, metastasis, and apoptosis are analyzed.

Integrins as receptors for virus attachment and cell entry.

Bergelson JM, Finberg RW
Trends Microbiol. 1993; 1: 287-8

Multiple roles for integrins during development.

Beauvais-Jouneau A, Thiery JP
Biol Cell. 1997; 89: 5-11

Interactions between cells and extracellular matrix play a crucial role during development by controlling tissue remodelling and cell migration. Integrins are the main family of cell surface receptors for extracellular matrix. The knockout of integrin genes in mouse embryos has provided new insights into the function of these receptors during embryonic development and morphogenesis. The lethality observed either during embryonic life or after birth suggests that many integrins are essential.

Integrins and human intestinal cell functions.

Beaulieu JF
Front Biosci. 1999; 4: 31021-31021

Integrins are a large family of cell-surface receptors involved in cell adhesion to the extracellular matrix. In epithelia, it is mainly the integrins belonging to the beta1 and beta4 classes that bind to basement membrane molecules such as the laminins and the type IV collagens. Beta1 and beta4 integrins regulate the assembly of adhesive junctions as well as the activation of various signaling pathways leading to the modulation of gene expression. In this review, I will discuss what is currently known about integrins in human intestinal epithelial cells. The interest in the intestinal cell model to analyze cell-matrix interactions will be delineated and the recent experimental evidence showing that these interactions can regulate cell proliferation and differentiation will be presented.

Are changes in integrin affinity and conformation overemphasized?

Bazzoni G, Hemler ME
Trends Biochem Sci. 1998; 23: 30-4

The activation of integrin-type adhesion receptors might result in the increased affinity of the receptor for ligand. In addition, the activated receptor might display new epitopes, which are increasingly monitored in clinical settings. Here, we highlight examples of integrin 'activation' that is not accompanied by enhanced ligand binding. Also, we emphasize that the dominant integrin conformational changes occur not with 'activation', but after integrins have already bound ligand.

Involvement of integrins in cell survival.

Bates RC, Lincz LF, Burns GF
Cancer Metastasis Rev. 1995; 14: 191-203

Apoptosis is a regulated process of cell death by which cells actively participate in their own destruction. In multicellular organisms, the balance between cell proliferation and apoptosis provides homeostatic control, and a regulatory failure of either event can contribute to oncogenesis. The extracellular matrix (ECM) is known to play a regulatory role in cellular growth and differentiation, but only more recently has it been recognized as a regulator of apoptosis. In these processes the major transmitters of ECM-derived signals to the cell are members of the integrin family, although the mechanical process of cell spreading also plays a role. Both in vivo and in vitro the loss of adhesion to specific components of the ECM can lead to cell death, and such apoptosis can be induced experimentally by blocking integrin binding. Heterotypic and homotypic cell-cell adhesion can also protect from adhesion-dependent apoptosis and there is evidence to suggest that this too in integrin mediated. In addition, some integrin mediated signaling appears to promote apoptosis. The downstream mechanisms of integrin signaling causing cell death have not been greatly explored, but there is evidence from two different systems that the induction of ICE transcription and nuclear translocation of p53 are candidate processes. Alterations in integrin expression or signaling therefore are likely to contribute to tumor development by enabling escape from apoptosis. Also, the recognition of the importance of cell-cell adhesion in tumor cell survival offers the potential of developing improved drug regimes for the treatment of malignancy.

Role of integrins in enterocyte migration.

Basson MD
Clin Exp Pharmacol Physiol. 1998; 25: 280-5

1. Enterocyte motility depends critically on cell-matrix interactions. Although still incompletely understood, these appear critically dependent upon integrin-mediated cell adhesion. 2. In addition to providing a mechanism for cell adhesion and traction, the integrins are likely to serve as true receptors for the matrix across which cell motility occurs, initiating signals by both mechanical and chemical means that alter cell phenotype and proliferation as well as cell motility. 3. Sound rationale now exists to postulate that soluble growth factors within the extracellular milieu regulate intestinal mucosal healing not only directly but also indirectly by modulating integrin expression and organization.

Is protein folding hierarchic? II. Folding intermediates and transition states.

Baldwin RL, Rose GD
Trends Biochem Sci. 1999; 24: 77-83

The folding reactions of some small proteins show clear evidence of a hierarchic process, whereas others, lacking detectable intermediates, do not. Evidence from folding intermediates and transition states suggests that folding begins locally, and that the formation of native secondary structure precedes the formation of tertiary interactions, not the reverse. Some notable examples in the literature have been interpreted to the contrary. For these examples, we have simulated the local structures that form when folding begins by using the LINUS program with nonlocal interactions turned off. Our results support a hierarchic model of protein folding.

Structure/function of the beta-barrel domain of F1-ATPase in the yeast Saccharomyces cerevisiae.

Bakhtiari N, Lai-Zhang J, Yao B, Mueller DM
J Biol Chem. 1999; 274: 16363-9

The first 90 amino acids of the alpha- and beta-subunits of mitochondrial F1-ATPase are folded into beta-barrel domains and were postulated to be important for stabilizing the enzyme (Abrahams, J. P., Leslie, A. G., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628). The role of the domains was studied by making chimeric enzymes, replacing the domains from the yeast Saccharomyces cerevisiae enzyme with the corresponding domains from the enzyme of the thermophilic bacterium Bacillus PS3. The enzymes containing the chimeric alpha-, beta-, or alpha- and beta-subunits were not functional. However, gain-of-function mutations were obtained from the strain containing the enzyme with the chimeric PS3/yeast beta-subunit. The gain-of-function mutations were all in codons encoding the beta-barrel domain of the beta-subunit, and the residues appear to map out a region of subunit-subunit interactions. Gain-of-function mutations were also obtained that provided functional expression of the chimeric PS3/yeast alpha- and beta-subunits together. Biochemical analysis of this active chimeric enzyme indicated that it was not significantly more thermostable or labile than the wild type. The results of this study indicate that the beta-barrel domains form critical contacts (distinct from those between the alpha- and beta-subunits) that are important for the assembly of the ATP synthase.

[The normal functions of integrins and their involvement in pathology]

Badescu M, Mustata T, Badescu L
Rev Med Chir Soc Med Nat Iasi. 1998; 102: 21-6

Ordered cell-cell and cell-matrix adhesive interactions are a fundamental feature of all multicellular organisms. Numerous studies in recent years have confirmed that both types of cell adhesion are mediated by cell surface receptors known as "adhesion molecules". These receptors can be divided into a limited number of families. Knowledge gained from basic research into cell adhesion is now being applied to clinical problems, and some of these have been summarized here. Integrins are heterodimeric proteins mediating cell-cell and cell-extracellular matrix adhesive connections and signal transduction across the plasma membrane. The important roles of integrins are in Leukocyte Adhesion Deficiency, in viral diseases, neural development and cancer. Suggestive data now points to roles in functions characterized in part by morphological rearrangements, such as learning and memory, and injury responses.

RhoA Inactivation by p190RhoGAP Regulates Cell Spreading and Migration by Promoting Membrane Protrusion and Polarity.

Arthur WT, Burridge K
Mol Biol Cell. 2001; 12: 2711-20

The binding of extracellular matrix proteins to integrins triggers rearrangements in the actin cytoskeleton by regulating the Rho family of small GTPases. The signaling events that mediate changes in the activity of Rho proteins in response to the extracellular matrix remain largely unknown. We have demonstrated in previous studies that integrin signaling transiently suppresses RhoA activity through stimulation of p190RhoGAP. Here, we investigated the biological significance of adhesion-dependent RhoA inactivation by manipulating p190RhoGAP signaling in Rat1 fibroblasts. The inhibition of RhoA activity that is induced transiently by adhesion was antagonized by expression of dominant negative p190RhoGAP. This resulted in impaired cell spreading on a fibronectin substrate, reduced cell protrusion, and premature assembly of stress fibers. Conversely, overexpression of p190RhoGAP augmented cell spreading. Dominant negative p190RhoGAP elevated RhoA activity in cells on fibronectin and inhibited migration, whereas overexpression of the wild-type GAP decreased RhoA activity, promoted the formation of membrane protrusions, and enhanced motility. Cells expressing dominant negative p190RhoGAP, but not control cells or cells overexpressing the wild-type GAP, were unable to establish polarity in the direction of migration. Taken together, these data demonstrate that integrin-triggered RhoA inhibition by p190RhoGAP enhances spreading and migration by regulating cell protrusion and polarity.

Effect of the tertiary structure alteration by ligation on the interface contacts between subunits of hemoglobin.

Arata Y
Biochim Biophys Acta. 1995; 1247: 24-34

In order to obtain knowledge of molecular mechanism underlying the cooperative ligand binding to hemoglobin (Hb), atomic contacts between subunits in the hypothetical intermediate quaternary structures have been investigated using the atomic coordinates of O2-liganded subunits' within the T-state. The atomic coordinates are obtained by energy minimization of hypothetical atomic coordinate sets in which an O2 molecule is placed near the iron atom of a deoxy subunit. In an 'O2-liganded beta-subunit', drastic atomic displacements occur at the distal side amino-acid residues E11Val and E12Leu in order to produce sufficient space for the O2 molecule, while this kind of atomic displacement cannot be seen in an 'O2-liganded alpha-subunit'. Therefore the features of structural alterations at heme surroundings by O2 ligation are markedly different between alpha- and beta-subunits. When an alpha-subunit in deoxy Hb is replaced by the corresponding 'O2-liganded subunit', tight inter-subunit contacts between alpha 1FG4(92)Arg and the two residues beta 2C3(37)Trp and beta 2C6(40)Arg strengthen greatly, while, when a beta-subunit is replaced by a corresponding O2-liganded one, serious steric hindrances occur between beta 2FG4(97)His and alpha 1CD2(44)Pro. The characteristics of structural alteration confirm that the effect of O2-ligation at alpha-subunit is transmitted to the other subunit through the alpha FG4Arg. However, the role of FG4His in beta-subunit is not as clear as that of the alpha FG4Arg.

Cell adhesion molecules, signal transduction and cell growth.

Aplin AE, Howe AK, Juliano RL
Curr Opin Cell Biol. 1999; 11: 737-44

Signals from dynamic cellular interactions between the extracellular matrix and neighboring cells ultimately input into the cellular decision-making process. These interactions form the basis of anchorage-dependent growth. Recent advances have provided the mechanistic details behind the ability of integrins, and other cell adhesion molecules (CAMs), to regulate both early signal transduction events initiated by soluble factors and downstream events more proximally involved in cell cycle progression. These actions appear to depend on the ability of CAMs to initiate the formation of organized structures that permit the efficient flow of information.

Signal transduction and signal modulation by cell adhesion receptors: the role of integrins, cadherins, immunoglobulin-cell adhesion molecules, and selectins.

Aplin AE, Howe A, Alahari SK, Juliano RL
Pharmacol Rev. 1998; 50: 197-263

Fibronectin and integrins in invasion and metastasis.

Akiyama SK, Olden K, Yamada KM
Cancer Metastasis Rev. 1995; 14: 173-89

The adhesive glycoprotein fibronectin and integrin receptors appear to play important roles in the progression of metastatic disease. Fibronectin is a multifunctional extracellular glycoprotein that has at lest two independent cell adhesion regions with different receptor specificities. The cell adhesive region in the central portion of fibronectin is comprised of at least two minimal amino acid sequences--an Arg-Gly-Asp (RGD) sequence and a Pro-His-Ser-Arg-Asn (PHSRN) sequence--which function in synergy. Another cell adhesive region is located near the carboxy-terminus in the alternatively spliced IIICS module. The critical minimal sequences for this region Leu-Asp-Val (LDV) and Arg-Glu-Asp-Val (REDV) which function in an additive rather than synergistic fashion. Integrins are heterodimeric, transmembrane cell adhesion receptors for fibronectin and other extracellular matrix molecules. Several different integrins bind to fibronectin. The alpha 5 beta 1 fibronectin-specific integrin binds to the central RGD/PHSRN site. The alpha 4 beta 1 integrin binds to the IIICS site. Fibronectin-integrin interactions are important in tumor cell migration, invasion, and metastasis. In addition to promoting cell adhesion to the extracellular matrix, these proteins may also function in chemotaxis and control of proliferation. Peptide and antibody inhibitors of fibronectin and integrin functions have been shown to be effective inhibitors of metastasis, and are potentially important reagents for the study and control of cancer.

Integrins in cell adhesion and signaling.

Akiyama SK
Hum Cell. 1996; 9: 181-6

Cell adhesive interactions play important roles during many normal physiological processes such as embryonic development and wound repair, and also during the progression of diseases such as cancer. Cell adhesion is mediated by the specific interactions of cell surface receptors with extracellular glycoproteins. The best characterized cell adhesion receptors are the integrins. Integrins comprise a family of more than 23 noncovalent, heterodimeric complexes consisting of an alpha and a beta subunit. Each subunit is a glycoprotein with a large, globular extracellular domain and a transmembrane domain. Most integrins have relatively small cytoplasmic domains consisting of fewer than 60 amino acids. Although many integrins can bind fibronectin, the alpha 5, beta 1, integrin is the major fibronectin receptor on most cells. This integrin mediates such cellular responses to fibronectin substrates as adhesion, migration, assembly of extracellular matrix, and signal transduction. Integrin ligands, such as fibronectin, are not passive adhesive molecules but are active participants in the cell adhesive process that leads to signal transduction. The best characterized integrin ligand is fibronectin. Fibronectin is a multifunctional glycoprotein comprised of three different types of homologous repeating units (termed type I, type II, and type III). Fibronectin has at least two independent cell adhesive regions: one located near the center of the polypeptide chain in the ninth and tenth type III modules binds to the alpha 5 beta 1 integrin. The biological function of the central cell adhesive region requires two critical amino acid sequences--an Arg-Gly-Asp (RGD) sequence and a Pro-His-Ser-Arg-Asn (PHSRN) sequence, which function in synergy--for optimal binding to the alpha 5 beta 1 integrin. Furthermore, the spacing between the crucial RGD and PHSRN sequences is also important for activity, suggesting the sequences themselves are necessary, but not sufficient, to account for the cell adhesive activity of fibronectin. One of the manifestations of integrin-mediated signal transduction including protein tyrosine phosphorylation. One cytoplasmic protein that is phosphorylated in response to cell adhesion is the focal adhesion kinase known as pp125FAK or FAK. The beta 1, beta 3, and beta 5 integrin intracellular domains are sufficient to initiate signal transduction pathways. Furthermore, alternative splicing can regulate the ability of beta integrin intracellular domains to participate in signal transduction. Other intracellular responses to cell adhesion include stimulation of migration, the assembly of an F-actin cytoskeleton and specialized structures called focal contacts, changes of cytoplasmic pH and calcium ion concentration, and modulation of proliferation and gene expression. Such varied modes of signal transduction are probably differentially controlled by a mechanism that requires either integrin receptor clustering alone, ligand occupancy in addition to clustering, or clustering and/or ligand occupancy plus tyrosine kinase activity for different responses. The examination of the fundamental mechanisms important for adhesion of cultured human cells and the resultant signaling processes has the potential of providing an understanding of molecular mechanisms involved in complex physiological processes and serving the basis for the development of novel therapeutic agents for the treatment of human disease.

Improved purification and biochemical characterization of phospholipase D from cabbage.

Abousalham A, Riviere M, Teissere M, Verger R
Biochim Biophys Acta. 1993; 1158: 1-7

Phospholipase D (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4) was purified from cabbage leaves. The two step purification procedure involved hydrophobic chromatography on Octyl-Sepharose followed by a Mono-Q/FPLC-column with a total yield of 23% and a purification factor of 1000. A zymographic assay was used to detection of PL D activities at various stages of purification under non denaturing PAGE. The molecular mass was determined to be 90 kDa using the SDS/PAGE method, and 90,200 Da as calculated from the amino acid analysis. The isoelectric point of the enzyme is acidic (pI = 4.7). The amino-acid composition and 29 residues of the NH2-terminal amino-acid sequence were determined.