SMART MODE:

NORMAL GENOMIC

• Hemenway CS, Halligan BW, Gould GC, Levy LS
• Identification and analysis of a third mouse Polycomb gene, MPc3.
• Gene. 2000; 242: 31-40
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• A new mouse Polycomb (Pc) gene, MPc3, has been identified. The MPc3 protein contains the highly conserved chromodomain and carboxy-terminal COOH box of other known Pc proteins from diverse species. Like other Pc proteins, MPc3 physically interacts with the RING finger proteins RING1A and dinG/RING1B. MPc3 maps to the distal arm of mouse chromosome 11 (11E2), a region that contains other known Pc genes in addition to several disease loci that may be linked to abnormal Pc gene function.

• Jay D, Garcia EJ, Lara JE, Medina MA, de la Luz Ibarra M
• Determination of a cAMP-dependent protein kinase phosphorylation site in the C-terminal region of human endothelial actin-binding protein.
• Arch Biochem Biophys. 2000; 377: 80-4
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• Three different C-terminal regions of human endothelial actin-binding protein-280 (ABP-280 or ABP; nonmuscle filamin) were subcloned and efficiently expressed in the Escherichia coli BL21 (DE3) system as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As predicted by the aminoacid sequence one of the fragments, a 109-kDa peptide (residues 1671-2647), contained a calpain cleavage site and two potential cAMP-dependent protein kinase (PKA) phosphorylation sites (serine 2152 and threonine 2336). A second fragment, a 74-kDa peptide (residues 1671-2331), contained a calpain cleavage site and one of the three presumptive PKA phosphorylation sites (serine 2152). The third fragment, a 48-kDa peptide (residues 2223-2647), contained only one of the PKA sites (threonine 2336). Phosphorylation of these truncated peptides indicated that only the fragments containing serine 2152 incorporated phosphate after PKA treatment. Site-directed mutagenesis analysis confirmed that serine 2152 is the unique substrate for PKA in the C-terminal region of ABP. The functional significance of phosphorylation of this residue, which belongs to a serine-proline motif, is discussed.

• Wilkes DE, Otto JJ
• Molecular cloning of profilin from Tetrahymena thermophila.
• Gene. 2000; 246: 295-301
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• The actin-binding protein profilin was isolated from Tetrahymena thermophila by affinity chromatography, and the peptide sequence was determined for part of the protein. The cDNA sequence was obtained by using the peptide sequence, reverse transcription-PCR and 5' and 3' RACE. The cDNA coded for a profilin of 16680Da, which made it among the largest known profilins, and it had a predicted isoelectric point of 8.27. The deduced amino acid sequence was divergent from other profilins, having more than 26% identity only with profilin from Tetrahymena pyriformis. The sequence contained insertions that are also present in profilins from Tetrahymena pyriformis and Trypanosoma brucei. There appeared to be only a single profilin gene and one transcript from this gene.

• Kostyukova A, Maeda K, Yamauchi E, Krieger I, Maeda Y
• Domain structure of tropomodulin: distinct properties of the N-terminal and C-terminal halves.
• Eur J Biochem. 2000; 267: 6470-5
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• The structure of tropomodulin, the unique capping protein for the pointed end (the slow-growing end) of an actin filament, was studied. An improved Escherichia coli expression system for chicken E-tropomodulin was established and tropomodulin was prepared, Tmod (N39), in which 15 amino acid residues from the original C-terminus are deleted at the DNA level. This expression and purification system accidentally co-produces an 11-kDa fragment with the original N-terminus (N11). By applying limited proteolysis to Tmod (N39), a 20-kDa C-terminal fragment (C20) was obtained. The limited proteolysis data, as well as the fluorescence spectrometry and CD analyses of Tmod (N39), C20 and N11, revealed that tropomodulin is an alpha-helical protein that consists of two distinct domains. The C-terminal half (20 kDa) is resistant to proteolysis, which suggests that this domain is tightly folded. In contrast, the N-terminal half is susceptible to proteolysis, indicating that in solution this half is likely to be extended or to form a highly flexible structure. Cross-linking experiments with glutaraldehyde indicated that Tmod (N39) and N11 can form complexes with tropomyosin, whereas C20 cannot. This confirms the previous report that the site(s) of interaction with tropomyosin resides in the N-terminal 11-kDa region of tropomodulin.

• Muckian C, Hillmann A, Kenny D, Shields DC
• A novel variant of the platelet glycoprotein Ibalpha macroglycopeptide region lacks any copies of the "perfect" 13 amino acid repeat.
• Thromb Haemost. 2000; 83: 513-4

• He X, Li Y, Schembri-King J, Jakes S, Hayashi J
• Identification of actin binding protein, ABP-280, as a binding partner of human Lnk adaptor protein.
• Mol Immunol. 2000; 37: 603-12
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• Human Lnk (hLnk) is an adaptor protein with multiple functional domains that regulates T cell activation signaling. In order to identify cellular Lnk binding partners, a yeast two-hybrid screening of human spleen cDNA library was carried out using human hLnk as bait. A polypeptide sequence identical to the C-terminal segment of the actin binding protein (ABP-280) was identified as a hLnk binding protein. The expressed hLnk and the FLAG tagged C-terminal 673 amino acid residues of ABP-280 or the endogenous ABP-280 in COS-7 cells could be co-immunoprecipitated using antibodies either to hLnk, FLAG or ABP-280, respectively. Furthermore, immunofluorescence confocal microscope showed that hLnk and ABP-280 co-localized at the plasma membrane and at juxtanuclear region of COS-7 cells. In Jurkat cells, the endogenous hLnk also associates with the endogenous ABP-280 indicating that the association of these two proteins is physiological. The interacting domains of both proteins were mapped using yeast two-hybrid assays. Our results indicate that hLnk binds to the residues 2006-2454 (repeats 19-23C) of ABP-280. The domain in hLnk that associates with ABP-280 was mapped to an interdomain region of 56 amino acids between pleckstrin homology and Src homology 2 domains. These results suggest that hLnk may exert its regulatory role through its association with ABP-280.

• Liu S, Ginsberg MH
• Paxillin binding to a conserved sequence motif in the alpha 4 integrin cytoplasmic domain.
• J Biol Chem. 2000; 275: 22736-42
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• alpha(4)beta(1) integrin-mediated cell adhesion results in increased cell migration, reduced cell spreading, and focal adhesion formation relative to other beta(1) integrins. Paxillin, a signaling adapter protein, binds tightly to the alpha(4) cytoplasmic domain and is implicated in alpha(4) integrin signaling. We now report the mapping of a paxillin-binding site in the alpha(4) cytoplasmic domain and an assessment of its role in the alpha(4) tail-specific integrin functions. By using truncation mutants and a peptide competition assay, we found that a region of 9 amino acid residues (Glu(983)-Tyr(991)) within the alpha(4) cytoplasmic domain contains a minimal sequence sufficient for paxillin binding. Alanine scanning of this region implicated Tyr(991) and Glu(983) as critical residues. The role of these residues was confirmed by introducing these Ala substitutions into the full-length alpha(4) tail sequence. Y991A or E983A substitution disrupted the interaction of alpha(4) integrins with paxillin. These same two point mutations reversed the effects of the alpha(4) tail on cell spreading. The key features of the identified paxillin-binding sequence are present in all alpha(4) integrins sequenced to date, including that from Xenopus laevis. The maintenance of this sequence motif suggests that paxillin binding is an evolutionarily conserved function of alpha(4) integrins.

• Critchley DR
• Focal adhesions - the cytoskeletal connection.
• Curr Opin Cell Biol. 2000; 12: 133-9
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• Cellular contacts with the extracellular matrix are regulated by the Rho family of GTPases through their effects on both the actin and the microtubule cytoarchitecture. Recent genetic, biochemical and structural data have highlighted the role played by a subset of actin-binding proteins in coupling integrins to cytoskeletal actin and in assembling signalling complexes that are important for cell motility and cell proliferation.

• Zent R, Fenczik CA, Calderwood DA, Liu S, Dellos M, Ginsberg MH
• Class- and splice variant-specific association of CD98 with integrin beta cytoplasmic domains.
• J Biol Chem. 2000; 275: 5059-64
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• CD98 is a type II transmembrane protein involved in neutral and basic amino acid transport and in cell fusion events. CD98 was implicated in the function of integrin adhesion receptors by its capacity to reverse suppression of integrin activation by isolated integrin beta(1A) domains. Here we report that CD98 associates with integrin beta cytoplasmic domains with a unique integrin class and splice variant specificity. In particular, CD98 interacted with the ubiquitous beta(1A) but not the muscle-specific splice variant, beta(1D), or leukocyte-specific beta(7) cytoplasmic domains. The ability of CD98 to associate with integrin cytoplasmic domains correlated with its capacity to reverse suppression of integrin activation. The association of CD98 with integrin beta(1A) cytoplasmic domains may regulate the function and localization of these membrane proteins.

• Menon B, Sudhakaran PR
• Actin binds to the cytoplasmic tail of alpha 1 subunit of integrin in hepatocytes.
• Indian J Biochem Biophys. 2000; 37: 81-5
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• alpha 1 beta 1-Integrin is a common receptor for laminin and collagen IV on hepatocytes. The interactions of intracellular domain of integrins with cytoplasmic elements are critical in the initiation and transduction of signals. In order to understand the nature of cytoplasmic components that can interact with cytoplasmic domain of alpha 1 integrin, cytoplasmic extracts of monolayers of rat hepatocytes were subjected to chromatography over an affinity column prepared by coupling a 60-mer synthetic cytoplasmic tail of alpha 1 subunit. SDS-PAGE analysis of the eluate showed the presence of a 47 kDa protein. Dot-Blot assay using radio-iodinated 47 kDa protein showed the binding of the protein to 60-mer C tail in a concentration dependent manner. Immunoblot analysis using specific antibodies showed that the 47 kDa protein is actin.

• Arregui C, Pathre P, Lilien J, Balsamo J
• The nonreceptor tyrosine kinase fer mediates cross-talk between N-cadherin and beta1-integrins.
• J Cell Biol. 2000; 149: 1263-74
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• Cadherins and integrins must function in a coordinated manner to effectively mediate the cellular interactions essential for development. We hypothesized that exchange of proteins associated with their cytoplasmic domains may play a role in coordinating function. To test this idea, we used Trojan peptides to introduce into cells and tissues peptide sequences designed to compete for the interaction of specific effectors with the cytoplasmic domain of N-cadherin, and assayed their effect on cadherin- and integrin-mediated adhesion and neurite outgrowth. We show that a peptide mimicking the juxtamembrane (JMP) region of the cytoplasmic domain of N-cadherin results in inhibition of N-cadherin and beta1-integrin function. The effect of JMP on beta1-integrin function depends on the expression of N-cadherin and is independent of transcription or translation. Treatment of cells with JMP results in the release of the nonreceptor tyrosine kinase Fer from the cadherin complex and its accumulation in the integrin complex. A peptide that mimics the first coiled-coil domain of Fer prevents Fer accumulation in the integrin complex and reverses the inhibitory effect of JMP. These findings suggest a new mechanism through which N-cadherin and beta1-integrins are coordinately regulated: loss of an effector from the cytoplasmic domain of N-cadherin and gain of that effector by the beta1-integrin complex.

• Li M, Bermak JC, Wang ZW, Zhou QY
• Modulation of dopamine D(2) receptor signaling by actin-binding protein (ABP-280).
• Mol Pharmacol. 2000; 57: 446-52
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• Proteins that bind to G protein-coupled receptors have recently been identified as regulators of receptor anchoring and signaling. In this study, actin-binding protein 280 (ABP-280), a widely expressed cytoskeleton-associated protein that plays an important role in regulating cell morphology and motility, was found to associate with the third cytoplasmic loop of dopamine D(2) receptors. The specificity of this interaction was originally identified in a yeast two-hybrid screen and confirmed by protein binding. The functional significance of the D(2) receptor-ABP-280 association was evaluated in human melanoma cells lacking ABP-280. D(2) receptor agonists were less potent in inhibiting forskolin-stimulated cAMP production in these cells. Maximal inhibitory responses of D(2) receptor activation were also reduced. Further yeast two-hybrid experiments showed that ABP-280 association is critically dependent on the carboxyl domain of the D(2) receptor third cytoplasmic loop, where there is a potential serine phosphorylation site (S358). Serine 358 was replaced with aspartic acid to mimic the effects of receptor phosphorylation. This mutant (D(2)S358D) displayed compromised binding to ABP-280 and coupling to adenylate cyclase. PKC activation also generated D(2) receptor signaling attenuation, but only in ABP-containing cells, suggesting a PKC regulatory role in D(2)-ABP association. A mechanism for these results may be derived from a role of ABP-280 in the clustering of D(2) receptors, as determined by immunocytochemical analysis in ABP-deficient and replete cells. Our results suggest a new molecular mechanism of modulating D(2) receptor signaling by cytoskeletal protein interaction.

• Van Troys M, Dewitte D, Verschelde JL, Goethals M, Vandekerckhove J, Ampe C
• The competitive interaction of actin and PIP2 with actophorin is based on overlapping target sites: design of a gain-of-function mutant.
• Biochemistry. 2000; 39: 12181-9
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• We studied the effect of mutations in an alpha-helical region of actophorin (residues 91-108) on F-actin and PIP(2) binding. As in cofilin, residues in the NH(2)-terminal half of this region are involved in F-actin binding. We show here also that basic residues in the COOH-terminal half of the region participate in this interaction whereby we extend the previously defined actin binding interface [Lappalainen, P., et al. (1997) EMBO J. 16, 5520-5530]. In addition, we demonstrate that some of the lysines in this alpha-helical region in actophorin are implicated in PIP(2) binding. This indicates that the binding sites of F-actin and PIP(2) on actophorin overlap, explaining the mutually exclusive binding of these ligands. The Ca(2+)-dependent F-actin binding of a number of actophorin mutants (carrying a lysine to glutamic acid substitution at the COOH-terminal positions of the actin binding helical region) may mimic the behavior of members of the gelsolin family. In addition, we show that PIP(2) binding, but not actin binding, of actophorin is strongly enhanced by a point mutation that leads to a reinforcement of the positive electrostatic potential of the studied alpha-helical region.

• Huang CJ, Chen YH, Ting LP
• Hepatitis B virus core protein interacts with the C-terminal region of actin-binding protein.
• J Biomed Sci. 2000; 7: 160-8
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• Hepatitis B viral core protein is present in the nucleus and cytoplasm of infected hepatocytes. There is a strong correlation between the intrahepatic distribution of core protein and the viral replication state and disease activity in patients with chronic hepatitis. To understand the role of core protein in the pathogenesis of HBV, we used a yeast two-hybrid system to search for cellular proteins interacting with the carboxyl terminus of core protein, as this region is involved in a number of important functions in the viral replication cycle including RNA packaging and DNA synthesis. A cDNA encoding the extreme C-terminal region of human actin-binding protein, ABP-276/278, was identified. This interaction was further confirmed both in vitro and in vivo. In addition, the extreme C-terminal region of ABP-276/278 interacted with the nearly full-length HBV core protein. Since this region is present in both the core and the precore proteins, it is likely that both core and precore proteins of HBV can interact with the C-terminal region of ABP-276/278. The minimal region of ABP-276/278 which interacted with the HBV core protein was the C-terminal 199 amino acid residues which correspond to part of the 23rd repeat, the entire 24th repeat and the intervening hinge II region in ABPs. The potential functional outcome of ABP interaction in HBV replication and its contribution to the pathological changes seen in patients with chronic HBV infection are discussed.

• Thompson TG et al.
• Filamin 2 (FLN2): A muscle-specific sarcoglycan interacting protein.
• J Cell Biol. 2000; 148: 115-26
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• Mutations in genes encoding for the sarcoglycans, a subset of proteins within the dystrophin-glycoprotein complex, produce a limb-girdle muscular dystrophy phenotype; however, the precise role of this group of proteins in the skeletal muscle is not known. To understand the role of the sarcoglycan complex, we looked for sarcoglycan interacting proteins with the hope of finding novel members of the dystrophin-glycoprotein complex. Using the yeast two-hybrid method, we have identified a skeletal muscle-specific form of filamin, which we term filamin 2 (FLN2), as a gamma- and delta-sarcoglycan interacting protein. In addition, we demonstrate that FLN2 protein localization in limb-girdle muscular dystrophy and Duchenne muscular dystrophy patients and mice is altered when compared with unaffected individuals. Previous studies of filamin family members have determined that these proteins are involved in actin reorganization and signal transduction cascades associated with cell migration, adhesion, differentiation, force transduction, and survival. Specifically, filamin proteins have been found essential in maintaining membrane integrity during force application. The finding that FLN2 interacts with the sarcoglycans introduces new implications for the pathogenesis of muscular dystrophy.

• Trask TM, Trask BC, Ritty TM, Abrams WR, Rosenbloom J, Mecham RP
• Interaction of tropoelastin with the amino-terminal domains of fibrillin-1 and fibrillin-2 suggests a role for the fibrillins in elastic fiber assembly.
• J Biol Chem. 2000; 275: 24400-6
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• Alignment of tropoelastin molecules during the process of elastogenesis is thought to require fibrillin-containing microfibrils. In this study, we have demonstrated that amino-terminal domains of two microfibrillar proteins, fibrillin-1 and fibrillin-2, interact with tropoelastin in solid phase binding assays. The tropoelastin-binding site was localized to a region beginning at the glycine-rich and proline-rich regions of fibrillin-2 and fibrillin-1, respectively, and continuing through the second 8-cysteine domain. Characterization of the binding requirements using the fibrillin-2 construct found that a folded, secondary structure was necessary for binding. Furthermore, binding between tropoelastin and fibrillin was mediated by ionic interactions involving the lysine side chains of tropoelastin. The importance of the lysine side chains was corroborated by the finding that the fibrillin-2 construct did not bind to mature elastin, whose lysine side chains have been modified to form cross-links. Interestingly, there was no interaction between the fibrillin constructs and tropoelastin in solution phase, suggesting that binding of tropoelastin to a solid substrate exposes a cryptic binding site. These results suggest that fibrillin plays an important role in elastic fiber assembly by binding tropoelastin and perhaps facilitating side chain alignment for efficient cross-linking.

• van der Ven PF, Obermann WM, Lemke B, Gautel M, Weber K, Furst DO
• Characterization of muscle filamin isoforms suggests a possible role of gamma-filamin/ABP-L in sarcomeric Z-disc formation.
• Cell Motil Cytoskeleton. 2000; 45: 149-62
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• Filamin, also called actin binding protein-280, is a dimeric protein that cross-links actin filaments in the cortical cytoplasm. In addition to this ubiquitously expressed isoform (FLN1), a second isoform (ABP-L/gamma-filamin) was recently identified that is highly expressed in mammalian striated muscles. A monoclonal antibody was developed, that enabled us to identify filamin as a Z-disc protein in mammalian striated muscles by immunocytochemistry and immunoelectron microscopy. In addition, filamin was identified as a component of intercalated discs in mammalian cardiac muscle and of myotendinous junctions in skeletal muscle. Northern and Western blots showed that both, ABP-L/gamma-filamin mRNA and protein, are absent from proliferating cultured human skeletal muscle cells. This muscle specific filamin isoform is, however, up-regulated immediately after the induction of differentiation. In cultured myotubes, ABP-L/gamma-filamin localises in Z-discs already at the first stages of Z-disc formation, suggesting that ABP-L/gamma-filamin might play a role in Z-disc assembly.

• Feng S, Christodoulides N, Resendiz JC, Berndt MC, Kroll MH
• Cytoplasmic domains of GpIbalpha and GpIbbeta regulate 14-3-3zeta binding to GpIb/IX/V.
• Blood. 2000; 95: 551-7
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• Shear stress causes the platelet glycoprotein (Gp) Ib/IX/V to bind to von Willebrand factor, resulting in platelet adhesion. GpIb/IX/V also functions to stimulate transmembranous signaling, leading to platelet activation and the expression of a ligand-receptive GpIIb-IIIa complex. The highly conserved cytoplasmic domain of GpIbalpha binds directly to a dimeric 14-3-3 adapter protein zeta isoform. To explore structural determinants of GpIb/IX/V binding to 14-3-3zeta, the authors examined 14-3-3zeta interactions with GpIbalpha and GpIbbeta in heterologous cells and platelets. Truncations of GpIbalpha at amino acid 542 or 594, or deletions of residues 542 through 590, inhibited binding of 14-3-3zeta. Deletion of GpIbalpha from Trp(570) to Ser(590) eliminated 14-3-3zeta binding, and deletion of the sequence from Arg(542)-Trp(570) enhanced binding of 14-3-3zeta to GpIbalpha. All GpIbalpha mutations that eliminated GpIbalpha binding to the GST-14-3-3zeta fusion protein also eliminated GpIbbeta binding to the fusion protein. Forskolin treatment of Chinese hamster ovary cells expressing wild-type GpIbalpha/beta/IX resulted in the phosphorylation of GpIbbeta associated with enhanced binding of GpIbbeta to GST-14-3-3zeta fusion protein and increased 14-3-3zeta coimmunoprecipitated with GpIbalpha. When intact human platelets aggregated in response to 90 dynes/cm(2) shear stress, 14-3-3zeta disassociated from GpIbalpha. Prostacyclin treatment of platelets inhibited shear stress-induced aggregation and the release of 14-3-3zeta from GpIbalpha. These data demonstrate that amino acid residues in the cytoskeletal interaction domains of GpIbalpha regulate 14-3-3zeta binding to GpIbalpha/beta/IX, and suggest that protein kinase A-dependent phosphorylation of GpIbbeta enhances 14-3-3zeta binding to the GpIb/IX/V complex in human platelets. (Blood. 2000;95:551-557)

• Valmu L, Fagerholm S, Suila H, Gahmberg CG
• The cytoskeletal association of CD11/CD18 leukocyte integrins in phorbol ester-activated cells correlates with CD18 phosphorylation.
• Eur J Immunol. 1999; 29: 2107-18
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• Leukocyte adhesion is a regulated process, which involves CD11/CD18 leukocyte integrins. CD11/CD18 acidity may be regulated intracellularly, and the CD18 polypeptide has previously been shown to become phosphorylated on serine and threonine after phorbol ester activation of T cells. Increased adhesiveness is believed to be mediated by regulating the overall avidity of cellular contact. CD11/CD18 integrins have earlier been reported to interact with several cytoskeletal proteins. We have now studied the involvement of the CD18 phosphorylation in cytoskeletal associations. We have investigated the distribution of phosphorylated CD18 between soluble, cytoskeletal and nuclear fractions of T cell detergent lysates. A significant amount of phosphorylated CD18 polypeptides was observed to fraction along with the cytoskeleton, while the majority of the cell surface CD18 molecules remained in the soluble fraction. Putative candidates for this altered cytoskeletal binding of CD11/CD18 were shown to be talin and filamin, which were observed to bind to CD18 cytoplasmic peptides and co-precipitate with CD18. The importance of the CD18 cytoplasmic domain in the regulation of the leukocyte adhesion was further strengthened by inhibition of phorbol ester-induced T cell adhesion with a phosphorylated lipopeptide corresponding to the cytoplasmic portion of the CD18. These results indicate that the induced CD18 phosphorylation and the altered cytoskeletal binding of the phosphorylated integrin complex may contribute to the increased avidity of CD11/CD18-mediated leukocyte adhesion.

• Hindson VJ, Ashworth JL, Rock MJ, Cunliffe S, Shuttleworth CA, Kielty CM
• Fibrillin degradation by matrix metalloproteinases: identification of amino- and carboxy-terminal cleavage sites.
• FEBS Lett. 1999; 452: 195-8
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• Fibrillin molecules form the structural framework of elastic fibrillin-rich microfibrils of the extracellular matrix. We have investigated the proteolysis of recombinant fibrillin molecules by five matrix metalloproteinases. Cleavage sites were defined at the carboxy-terminal end of the fibrillin-1 proline-rich region and the corresponding fibrillin-2 glycine-rich region (exon 10), and within exon 49 towards the carboxy-terminus of fibrillin-1. Cleavage at these sites is predicted to disrupt the structure and function of the fibrillin-rich microfibrils.

• Goldmann WH, Teodoridis JM, Sharma CP, Hu B, Isenberg G
• Fragments from actin binding protein (ABP-280; filamin) insert into reconstituted lipid layers.
• Biochem Biophys Res Commun. 1999; 259: 108-12
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• Previous computer analyses suggested two possible lipid binding sites, residues 49-71 and 131-155, of the primary amino acid sequence on ABP-280 (filamin), which could facilitate membrane attachment/insertion. We expressed these regions as fusion proteins with schistosomal GST and investigated their interaction with mixtures of zwitterionic (dimyristoyl-l-alpha-phosphatidylcholine, DMPC) and anionic (dimyristoyl-l-alpha-phosphatidylglycerol, DMPG) phospholipids in reconstituted lipid bilayers by differential scanning calorimetry (DSC). Using vesicles of mixed DMPC/DMPG with increasing fusion protein concentrations, we established in calorimetric assays a decrease of the main chain transition enthalpy, DeltaH, and a shift in chain melting temperature. This is indicative of the insertion of these fragments into the hydrophobic region of lipid membranes. We confirmed these findings by the film balance technique using lipid monolayers (DMPG). The binding judged from both methods was of moderate affinity.

• Calderwood DA, Zent R, Grant R, Rees DJ, Hynes RO, Ginsberg MH
• The Talin head domain binds to integrin beta subunit cytoplasmic tails and regulates integrin activation.
• J Biol Chem. 1999; 274: 28071-4
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• The beta subunit cytoplasmic domains of integrin adhesion receptors are necessary for the connection of these receptors to the actin cytoskeleton. The cytoplasmic protein, talin, binds to beta integrin cytoplasmic tails and actin filaments, hence forming an integrin-cytoskeletal linkage. We used recombinant structural mimics of beta(1)A, beta(1)D and beta(3) integrin cytoplasmic tails to characterize integrin-binding sites within talin. Here we report that an integrin-binding site is localized within the N-terminal talin head domain. The binding of the talin head domain to integrin beta tails is specific in that it is abrogated by a single point mutation that disrupts integrin localization to talin-rich focal adhesions. Integrin-cytoskeletal interactions regulate integrin affinity for ligands (activation). Overexpression of a fragment of talin containing the head domain led to activation of integrin alpha(IIb)beta(3); activation was dependent on the presence of both the talin head domain and the integrin beta(3) cytoplasmic tail. The head domain of talin thus binds to integrins to form a link to the actin cytoskeleton and can thus regulate integrin function.

• Li MG et al.
• Filamin is required for ring canal assembly and actin organization during Drosophila oogenesis.
• J Cell Biol. 1999; 146: 1061-74
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• The remodeling of the actin cytoskeleton is essential for cell migration, cell division, and cell morphogenesis. Actin-binding proteins play a pivotal role in reorganizing the actin cytoskeleton in response to signals exchanged between cells. In consequence, actin-binding proteins are increasingly a focus of investigations into effectors of cell signaling and the coordination of cellular behaviors within developmental processes. One of the first actin-binding proteins identified was filamin, or actin-binding protein 280 (ABP280). Filamin is required for cell migration (Cunningham et al. 1992), and mutations in human alpha-filamin (FLN1; Fox et al. 1998) are responsible for impaired migration of cerebral neurons and give rise to periventricular heterotopia, a disorder that leads to epilepsy and vascular disorders, as well as embryonic lethality. We report the identification and characterization of a mutation in Drosophila filamin, the homologue of human alpha-filamin. During oogenesis, filamin is concentrated in the ring canal structures that fortify arrested cleavage furrows and establish cytoplasmic bridges between cells of the germline. The major structural features common to other filamins are conserved in Drosophila filamin. Mutations in Drosophila filamin disrupt actin filament organization and compromise membrane integrity during oocyte development, resulting in female sterility. The genetic and molecular characterization of Drosophila filamin provides the first genetic model system for the analysis of filamin function and regulation during development.

• Wixler V et al.
• Identification of novel interaction partners for the conserved membrane proximal region of alpha-integrin cytoplasmic domains.
• FEBS Lett. 1999; 445: 351-5
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• The alpha3Abeta1 integrin is a laminin receptor with a broad specificity for different laminin isoforms. Furthermore, it regulates the function of other integrins, like alpha2beta1, alpha5beta1 and alpha6Abeta1. In a yeast two hybrid screen of a human placenta cDNA library, we identified cDNAs coding for four different proteins that strongly interact with the conserved region of the cytoplasmic domain of the alpha3A integrin subunit. In addition to the cDNA for nucleotide exchange factor Mss4 and the putative tumour suppressor protein BIN1, two novel cDNAs were identified. Association analysis with different integrin subunits revealed them as cDNAs that encode binding proteins which react with a broad spectrum of alpha subunits. The conserved membrane proximal region of the alpha3A chain was identified as the binding site for all four proteins. They, therefore, may be involved in the regulation of general functions of integrins.

• Kenny D, Morateck PA, Fahs SA, Warltier DC, Montgomery RR
• Cloning and expression of canine glycoprotein Ibalpha.
• Thromb Haemost. 1999; 82: 1327-33
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• The interaction of the glycoprotein (GP) Ib-IX-V complex with von Willebrand factor (vWF) is critical in initiation of haemostasis and thrombosis through platelet adhesion to damaged endothelium. The binding site for vWF resides within the GPIbalpha subunit of the complex. To further define the physiological function of platelet GPIbalpha we cloned and expressed the canine GPIbalpha cDNA. A canine platelet cDNA library was constructed and screened with a randomly primed 32P-labeled 1041-base-pair restriction fragment of the human GPIbalpha cDNA. Analysis of 23 clones demonstrated that the canine GPIbalpha cDNA is 2530 nucleotides in length and includes a short 5' untranslated segment of 42 nucleotides followed by a signal peptide of 16 amino acids, a mature peptide of 645 amino acids and a 3' noncoding region of 455 nucleotides. A single intron of 142 nucleotides, 6 nucleotides upstream from the ATG translation initiation codon was identified in the canine gene in a similar location to that present in the human gene. Chinese hamster ovary cells that stably express human GPIbbeta and GPIX were transfected with the canine GPIbalpha cDNA. Canine GPIbalpha was expressed on the surface of these cells and bound vWF in the presence of botrocetin. The binding of vWF was inhibited by an anti-vWF human monoclonal antibody known to inhibit vWF binding to GPIbalpha. The results of this investigation will allow the development of reagents to study the physiological function of GPIbalpha in an animal model.

• Gu M, Xi X, Englund GD, Berndt MC, Du X
• Analysis of the roles of 14-3-3 in the platelet glycoprotein Ib-IX-mediated activation of integrin alpha(IIb)beta(3) using a reconstituted mammalian cell expression model.
• J Cell Biol. 1999; 147: 1085-96
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• We have reconstituted the platelet glycoprotein (GP) Ib-IX-mediated activation of the integrin alpha(IIb)beta(3) in a recombinant DNA expression model, and show that 14-3-3 is important in GPIb-IX signaling. CHO cells expressing alpha(IIb)beta(3) adhere poorly to vWF. Cells expressing GPIb-IX adhere to vWF in the presence of botrocetin but spread poorly. Cells coexpressing integrin alpha(IIb)beta(3) and GPIb-IX adhere and spread on vWF, which is inhibited by RGDS peptides and antibodies against alpha(IIb)beta(3). vWF binding to GPIb-IX also activates soluble fibrinogen binding to alpha(IIb)beta(3) indicating that GPIb-IX mediates a cellular signal leading to alpha(IIb)beta(3) activation. Deletion of the 14-3-3-binding site in GPIbalpha inhibited GPIb-IX-mediated fibrinogen binding to alpha(IIb)beta(3) and cell spreading on vWF. Thus, 14-3-3 binding to GPIb-IX is important in GPIb-IX signaling. Expression of a dominant negative 14-3-3 mutant inhibited cell spreading on vWF, suggesting an important role for 14-3-3. Deleting both the 14-3-3 and filamin-binding sites of GPIbalpha induced an endogenous integrin-dependent cell spreading on vWF without requiring alpha(IIb)beta(3), but inhibited vWF-induced fibrinogen binding to alpha(IIb)beta(3). Thus, while different activation mechanisms may be responsible for vWF interaction with different integrins, GPIb-IX-mediated activation of alpha(IIb)beta(3) requires 14-3-3 interaction with GPIbalpha.

• Kaapa A, Peter K, Ylanne J
• Effects of mutations in the cytoplasmic domain of integrin beta(1) to talin binding and cell spreading.
• Exp Cell Res. 1999; 250: 524-34
• Display abstract

• Integrins are transmembrane proteins linking the extracellular matrix or certain cell-cell contacts to the cytoskeleton. To study integrin-cytoskeleton interactions we wanted to relate talin-integrin interaction to integrin function in cell spreading and formation of focal adhesions. For talin-binding studies we used fusion proteins of glutathione S-transferase and the cytoplasmic domain of integrin beta(1) (GST-cytobeta(1)) expressed in bacteria. For functional studies chimeric integrins containing the extracellular and transmembrane parts of beta(3) linked to the cytoplasmic domain of beta(1) were expressed in CHO cells as a dimer with the alpha(IIb) subunit. Point mutations in the amino acid sequence N(785)PIY(788) of beta(1) disrupted both the integrin-talin interaction and the ability of the integrin to mediate cell spreading. COOH-terminal truncation of beta(1) at the amino acid position 797 disrupted its ability to mediate cell spreading, whereas the disruption of talin binding required deletion of five more amino acids (truncation at position 792). A synthetic peptide from this region of beta(1) (W(780)DTGENPIYKSAV(792)) bound to purified talin and inhibited talin binding to GST-cytobeta(1). The ability of the mutants to mediate focal adhesion formation or to codistribute to focal adhesions formed by other integrins correlated with their ability to mediate cell spreading. These results confirm the previous finding that a talin-binding site in the integrin beta(1) tail resides at or close to the central NPXY motif and suggest that the integrin-talin interaction is necessary but not sufficient for integrin-mediated cell spreading.

• Pfaff M, Du X, Ginsberg MH
• Calpain cleavage of integrin beta cytoplasmic domains.
• FEBS Lett. 1999; 460: 17-22
• Display abstract

• We showed previously that the calcium-dependent protease, calpain, cleaves the cytoplasmic domain of the integrin beta3 subunit. To investigate whether susceptibility to calpain is a common feature of all integrin beta subunits, and to map calpain cleavage sites in different integrin beta tails, we treated recombinant cytoplasmic domains of integrin beta1A, beta1D, beta2, beta3 and beta7 subunits with purified calpain in vitro. We found that the cytoplasmic domains of all these integrin chains were cleaved by calpain. HPLC followed by mass spectrometry was used to identify calpain cleavage sites. These sites were clustered in the C-terminal half of the integrin beta cytoplasmic domains in regions flanking the two NXXY motifs, suggesting the possibility that the structural framework provided by these motifs is recognized by calpain. We used the knowledge of these cleavage sites to develop cleavage site-specific antibodies and to demonstrate cleavage of the beta1A cytoplasmic domain in intact platelets stimulated with calcium ionophore or thrombin. Thus susceptibility to calpain cleavage is common to integrin beta subunits, can be induced in intact cells, and appears to favor regions surrounding two conserved NXXY motifs.

• Hayakawa K et al.
• Characterization of the myosin light chain kinase from smooth muscle as an actin-binding protein that assembles actin filaments in vitro.
• Biochim Biophys Acta. 1999; 1450: 12-24
• Display abstract

• In addition to its kinase activity, myosin light chain kinase has an actin-binding activity, which results in bundling of actin filaments [Hayakawa et al., Biochem. Biophys. Res. Commun. 199, 786-791, 1994]. There are two actin-binding sites on the kinase: calcium- and calmodulin-sensitive and insensitive sites [Ye et al., J. Biol. Chem. 272, 32182-32189, 1997]. The calcium/calmodulin-sensitive, actin-binding site is located at Asp2-Pro41 and the insensitive site is at Ser138-Met213. The cyanogen bromide fragment, consisting of Asp2-Met213, is furnished with both sites and is the actin-binding core of myosin light chain kinase. Cross-linking between the two sites assembles actin filaments into bundles. Breaking of actin-binding at the calcium/calmodulin-sensitive site by calcium/calmodulin disassembles the bundles.

• Tsujioka M, Machesky LM, Cole SL, Yahata K, Inouye K
• A unique talin homologue with a villin headpiece-like domain is required for multicellular morphogenesis in Dictyostelium.
• Curr Biol. 1999; 9: 389-92
• Display abstract

• Molecules involved in the interaction between the extracellular matrix, cell membrane and cytoskeleton are of central importance in morphogenesis. Talin is a large cytoskeletal protein with a modular structure consisting of an amino-terminal membrane-interacting domain, with sequence similarities to members of the band 4.1 family, and a carboxy-terminal region containing F-actin-binding and vinculin-binding domains [1] [2]. It also interacts with the cytoplasmic tail of beta integrins which, on the external face of the membrane, bind to extracellular matrix proteins [3]. The possible roles of talin in multicellular morphogenesis in development remain largely unexplored. In Dictyostelium, a eukaryotic microorganism capable of multicellular morphogenesis, a talin homologue (TALA) has previously been identified and shown to play an important role in cell-to-substrate adhesion and maintenance of normal elastic properties of the cell [4] [5] [6]. Here, we describe a second talin homologue (TALB) that is required for multicellular morphogenesis in the development of Dictyostelium. Unlike any other talin characterised to date, it contains an additional carboxy-terminal domain homologous to the villin headpiece.

• Kim IF, Mohammadi E, Huang RC
• Isolation and characterization of IPP, a novel human gene encoding an actin-binding, kelch-like protein.
• Gene. 1999; 228: 73-83
• Display abstract

• The kelch family of proteins is defined by a 50 amino-acid repeat that has been shown to associate with actin. Here we describe the cloning and initial characterization of IPP, a novel human gene that predicts a kelch family protein homologous to the mouse Ipp gene, a previously described kelch family member. A 3kb IPP cDNA clone was isolated from a human placenta library using a probe derived from Ipp. Restriction mapping and Southern blot analysis show that IPP comprises eight exons spanning more than 47kb of genomic DNA. Fluorescence in situ hybridization maps the gene to chromosome 1p32-1p34. Northern blot analysis reveals transcripts of 1.4, 2.2, 5. 0, and 7.3kb. The 1.4 and 2.2kb messages are found exclusively in testis, while the 5.0 and 7.3kb messages are expressed at varying levels in ovary, placenta, small intestine, spleen, testis, and thymus. The IPP cDNA clone contains a 1752bp open reading frame that predicts a 584 amino-acid, 66kDa protein. Sequence analysis indicates an N-terminal POZ protein-protein interaction domain and a C-terminal kelch repeat domain consisting of six tandemly arranged repeats. Cosedimentation assays performed with these domains expressed as glutathione S-transferase fusion proteins demonstrate an actin-binding activity mediated specifically by the kelch repeat domain of IPP.

• Leinweber BD, Fredricksen RS, Hoffman DR, Chalovich JM
• Fesselin: a novel synaptopodin-like actin binding protein from muscle tissue.
• J Muscle Res Cell Motil. 1999; 20: 539-45
• Display abstract

• A novel actin binding protein has been isolated from chicken gizzard muscle. When isolated, a pair of proteins with apparent molecular weights of 79 kDa and 103 kDa are obtained; both proteins have a pI near 9.3. Peptide mapping indicates that these proteins are related. Antibodies against this protein cross-reacted with proteins from other smooth muscle containing tissues as well as skeletal and heart muscle. Traces of cross-reactive material were also detected in brain and kidney tissue. The affinity of this protein for actin is ca. 1x10(6) M(-1). Interestingly, this actin binding protein is a potent actin-bundling agent. A partial sequence analysis confirmed that there were no previously reported homologues in smooth muscle. However, considerable homology was found with the protein synaptopodin that is found in nervous tissue and kidney but is absent from muscle tissue. It is likely that the new protein is a member of the synaptopodin family. We call the smooth muscle actin binding protein fesselin.

• Leung CL, Sun D, Zheng M, Knowles DR, Liem RK
• Microtubule actin cross-linking factor (MACF): a hybrid of dystonin and dystrophin that can interact with the actin and microtubule cytoskeletons.
• J Cell Biol. 1999; 147: 1275-86
• Display abstract

• We cloned and characterized a full-length cDNA of mouse actin cross-linking family 7 (mACF7) by sequential rapid amplification of cDNA ends-PCR. The completed mACF7 cDNA is 17 kb and codes for a 608-kD protein. The closest relative of mACF7 is the Drosophila protein Kakapo, which shares similar architecture with mACF7. mACF7 contains a putative actin-binding domain and a plakin-like domain that are highly homologous to dystonin (BPAG1-n) at its NH(2) terminus. However, unlike dystonin, mACF7 does not contain a coiled-coil rod domain; instead, the rod domain of mACF7 is made up of 23 dystrophin-like spectrin repeats. At its COOH terminus, mACF7 contains two putative EF-hand calcium-binding motifs and a segment homologous to the growth arrest-specific protein, Gas2. In this paper, we demonstrate that the NH(2)-terminal actin-binding domain of mACF7 is functional both in vivo and in vitro. More importantly, we found that the COOH-terminal domain of mACF7 interacts with and stabilizes microtubules. In transfected cells full-length mACF7 can associate not only with actin but also with microtubules. Hence, we suggest a modified name: MACF (microtubule actin cross-linking factor). The properties of MACF are consistent with the observation that mutations in kakapo cause disorganization of microtubules in epidermal muscle attachment cells and some sensory neurons.

• De Cristofaro R, De Candia E
• Thrombin interaction with platelet GpIb: structural mapping and effects on platelet activation (review).
• Int J Mol Med. 1999; 3: 363-71
• Display abstract

• Platelet glycoprotein Ib (GpIb) is an integral platelet membrane glycoprotein which plays a major role in haemostasis, being involved in both von Willebrand factor (vWF) and alpha-thrombin high affinity binding. Such interactions contribute to the early adhesion of platelets to exposed subendothelium and to the process of platelet activation. Glycoprotein Ib belongs to the so called (LRR) family of proteins, characterized by a structural motif consisting of the presence of one or more tandem LRRs, flanked by conserved sequences. Several experimental strategies have recently documented the involvement of the thrombin domain referred to as 'heparin binding site' in the binding to GpIb. This review is aimed at reporting on the structural mapping of both alpha-thrombin and GpIb domains involved in such interaction and on possible roles of thrombin-GpIb binding on the mechanisms supporting the platelet activation.

• Bouton MC, Thurieau C, Guillin MC, Jandrot-Perrus M
• Characteristics of the interaction between thrombin exosite 1 and the sequence 269-287 [correction of 269-297] of platelet glycoprotein Ibalpha.
• Thromb Haemost. 1998; 80: 310-5
• Display abstract

• The interaction between GPIb and thrombin promotes platelet activation elicited via the hydrolysis of the thrombin receptor and involves structures located on the segment 238-290 within the N-terminal domain of GPIbalpha and the positively charged exosite 1 on thrombin. We have investigated the ability of peptides derived from the 269-287 sequence of GPIbalpha to interact with thrombin. Three peptides were synthesized, including Ibalpha 269-287 and two scrambled peptides R1 and R2 which are comparable to Ibalpha 269-287 with regards to their content and distribution of anionic residues. However, R2 differs from both Ibalpha 269-287 and R1 by the shifting of one proline from a central position to the N-terminus. By chemical cross-linking, we observed the formation of a complex between 125I-Ibalpha 269-287 and alpha-thrombin that was inhibited by hirudin, the C-terminal peptide of hirudin, sodium pyrophosphate but not by heparin. The complex did not form when gamma-thrombin was substituted for alpha-thrombin. Ibalpha 269-287 produced only slight changes in thrombin amidolytic activity and inhibited thrombin binding to fibrin. R1 and R2 also formed complexes with alpha-thrombin, modified slightly its catalytic activity and inhibited its binding to fibrin. Peptides Ibalpha 269-287 and R1 inhibited platelet aggregation and secretion induced by low thrombin concentrations whereas R2 was without effect. Our results indicate that Ibalpha 269-287 interacts with thrombin exosite 1 via mainly electrostatic interactions, which explains why the scrambled peptides also interact with exosite 1. Nevertheless, the lack of effect of R2 on thrombin-induced platelet activation suggests that proline 280 is important for thrombin interaction with GPIb.

• Steinmetz MO et al.
• A distinct 14 residue site triggers coiled-coil formation in cortexillin I.
• EMBO J. 1998; 17: 1883-91
• Display abstract

• We have investigated the process of the assembly of the Dictyostelium discoideum cortexillin I oligomerization domain (Ir) into a tightly packed, two-stranded, parallel coiled-coil structure using a variety of recombinant polypeptide chain fragments. The structures of these Ir fragments were analyzed by circular dichroism spectroscopy, analytical ultracentrifugation and electron microscopy. Deletion mapping identified a distinct 14 residue site within the Ir coiled coil, Arg311-Asp324, which was absolutely necessary for dimer formation, indicating that heptad repeats alone are not sufficient for stable coiled-coil formation. Moreover, deletion of the six N-terminal heptad repeats of Ir led to the formation of a four- rather than a two-helix structure, suggesting that the full-length cortexillin I coiled-coil domain behaves as a cooperative folding unit. Most interestingly, a 16 residue peptide containing the distinct coiled-coil 'trigger' site Arg311-Asp324 yielded approximately 30% helix formation as monomer, in aqueous solution. pH titration and NaCl screening experiments revealed that the peptide's helicity depends strongly on pH and ionic strength, indicating that electrostatic interactions by charged side chains within the peptide are critical in stabilizing its monomer helix. Taken together, these findings demonstrate that Arg311-Asp324 behaves as an autonomous helical folding unit and that this distinct Ir segment controls the process of coiled-coil formation of cortexillin I.

• Zhang W, Han SW, McKeel DW, Goate A, Wu JY
• Interaction of presenilins with the filamin family of actin-binding proteins.
• J Neurosci. 1998; 18: 914-22
• Display abstract

• Mutations in presenilin genes PS1 and PS2 account for approximately 50% of early-onset familial Alzheimer's disease (FAD). The PS1 and PS2 genes encode highly homologous transmembrane proteins related to the Caenorhabditis elegans sel-12 and spe-4 gene products. A hydrophilic loop region facing the cytoplasmic compartment is likely to be functionally important because at least 14 mutations in FAD patients have been identified in this region. We report here that the loop regions of PS1 and PS2 interact with nonmuscle filamin (actin-binding protein 280, ABP280) and a structurally related protein (filamin homolog 1, Fh1). Overexpression of PS1 appears to modify the distribution of ABP280 and Fh1 proteins in cultured cells. A monoclonal antibody recognizing ABP280 and Fh1 binds to blood vessels, astrocytes, neurofibrillary tangles, neuropil threads, and dystrophic neurites in the AD brain. Detection of ABP280/Fh1 proteins in these structures suggests that these presenilin-interacting proteins may be involved in the development of AD and that interactions between presenilins and ABP280/Fh1 may be functionally significant. The ABP280 gene is located on the human X chromosome, whereas the newly identified Fh1 gene maps to human chromosome 3. These results provide a new basis for understanding the function of presenilin proteins and further implicate cytoskeletal elements in AD pathogenesis.

• Meyer SC, Sanan DA, Fox JE
• Role of actin-binding protein in insertion of adhesion receptors into the membrane.
• J Biol Chem. 1998; 273: 3013-20
• Display abstract

• The goal of this study was to determine whether actin-binding protein (ABP) regulates membrane composition. ABP-deficient and ABP-containing cells were transfected with the cDNAs coding for glycoprotein (GP) Ib-IX, a platelet receptor that interacts with ABP. Most of the GP Ib-IX remained inside the ABP-deficient cells. When ABP was present, functional GP Ib-IX was inserted into the membrane. GP Ib-IX lacking the domain that interacts with ABP also showed increased membrane insertion in ABP-expressing cells. Furthermore, a fragment of ABP that lacks the dimerization and GP Ib-IX-binding sites restored the spreading of the cells and increased the amount of GP Ib-IX in the membrane. Finally, expression of ABP also increased endogenous beta1 integrin in the membrane. These results indicate that 1) ABP maintains the properties of the cell such that adhesion receptors can be efficiently expressed in the membrane; 2) increased receptor expression is accompanied by increased ability of the cell to spread; and 3) ABP exerts its effect by a mechanism that does not appear to involve direct cross-linking of actin filaments or direct interaction with receptors.

• Kitaguchi T, Murata M, Ambo H, Ikeda Y
• Characterization of cDNA encoding full-length mouse platelet glycoprotein IX.
• Blood Coagul Fibrinolysis. 1998; 9: 381-5
• Display abstract

• Glycoprotein (GP) IX is a platelet membrane 20 kD protein, which is associated with GPIbalpha, GPIb beta, and GPV to form GPIb/IX/V complex. GPIb/IX/V complex is a major receptor for von Willebrand factor, which mediates platelet adhesion and aggregation under high shear stress conditions. The relevance of this receptor for hemostasis has been implicated by a congenital bleeding disorder lacking the receptor, Bernard-Soulier syndrome. All subunits for the human receptor have been cloned and characterized. However, the function of GPIX is still elusive. To obtain further information of GPIX, we have determined a cDNA sequence of mouse GPIX (811 bp). The deduced amino-acid sequence (177aa) was 71% identical to the human GPIX protein. All cysteine residues in extracytoplasmic domain and putative N-linked glycosylation site (Asn44) were conserved. Mouse GPIX contained a leucine-rich glycoprotein sequence composed of 24 amino acids, as did human GPIX.

• Glogauer M, Arora P, Chou D, Janmey PA, Downey GP, McCulloch CA
• The role of actin-binding protein 280 in integrin-dependent mechanoprotection.
• J Biol Chem. 1998; 273: 1689-98
• Display abstract

• To survive in a mechanically active environment, cells must adapt to variations of applied membrane tension. A collagen-coated magnetic bead model was used to apply forces directly to the actin cytoskeleton through integrin receptors. We demonstrate here that by a calcium-dependent mechanism, human fibroblasts reinforce locally their connection with extracellular adhesion sites by inducing actin assembly and by recruiting actin-binding protein 280 (ABP-280) into cortical adhesion complexes. ABP-280 was phosphorylated on serine residues as a result of force application. This phosphorylation and the force-induced actin reorganization were largely abrogated by inhibitors of protein kinase C. In a human melanoma cell line that does not express ABP-280, actin accumulation could not be induced by force, whereas in stable transfectants expressing ABP-280, force-induced actin accumulation was similar to human fibroblasts. Cortical actin assembly played a role in regulating the activity of stretch-activated, calcium-permeable channels (SAC) since sustained force application desensitized SAC to subsequent force applications, and the decrease in stretch sensitivity was reversed after treatment with cytochalasin D. ABP-280-deficient cells showed a > 90% increase in cell death compared with ABP-280 +ve cells after force application. We conclude that ABP-280 plays an important role in mechanoprotection by reinforcing the membrane cortex and desensitizing SACs.

• Retta SF et al.
• beta1-integrin cytoplasmic subdomains involved in dominant negative function.
• Mol Biol Cell. 1998; 9: 715-31
• Display abstract

• The beta1-integrin cytoplasmic domain consists of a membrane proximal subdomain common to the four known isoforms ("common" region) and a distal subdomain specific for each isoform ("variable" region). To investigate in detail the role of these subdomains in integrin-dependent cellular functions, we used beta1A and beta1B isoforms as well as four mutants lacking the entire cytoplasmic domain (beta1TR), the variable region (beta1COM), or the common region (beta1 deltaCOM-B and beta1 deltaCOM-A). By expressing these constructs in Chinese hamster ovary and beta1 integrin-deficient GD25 cells (Wennerberg et al., J Cell Biol 132, 227-238, 1996), we show that beta1B, beta1COM, beta1 deltaCOM-B, and beta1 deltaCOM-A molecules are unable to support efficient cell adhesion to matrix proteins. On exposure to Mn++ ions, however, beta1B, but none of the mutants, can mediate cell adhesion, indicating specific functional properties of this isoform. Analysis of adhesive functions of transfected cells shows that beta1B interferes in a dominant negative manner with beta1A and beta3/beta5 integrins in cell spreading, focal adhesion formation, focal adhesion kinase tyrosine phosphorylation, and fibronectin matrix assembly. None of the beta1 mutants tested shows this property, indicating that the dominant negative effect depends on the specific combination of common and B subdomains, rather than from the absence of the A subdomain in the beta1B isoform.

• Shojaee N, Patton WF, Chung-Welch N, Su Q, Hechtman HB, Shepro D
• Expression and subcellular distribution of filamin isotypes in endothelial cells and pericytes.
• Electrophoresis. 1998; 19: 323-32
• Display abstract

• Two principal forms of the actin binding protein, filamin, are expressed in mammalian cells: nonmuscle and muscle isotypes (FLN-1 and FLN-2). A protein that copurifies with an alpha-naphthyl acetate hydrolyzing esterase from human omentum microvessel endothelial cells (EC) is isolated by nondenaturing electrophoresis, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and electroblotting. The purified protein is subjected to in situ trypsin cleavage, reversed-phase high performance liquid chromatography (HPLC) and automated Edman degradation. Six peptide fragments from the protein are identified to have 60-66% identity with nonmuscle filamin (ABP-280). Two of these peptides are 100% identical to a previously sequenced human muscle filamin fragment. Polyclonal antibody is produced using a 16-residue synthetic peptide corresponding to a structural beta-sheet region of muscle filamin. Compared with a variety of vascular cells evaluated, retinal pericytes express an abundance of both muscle and non-muscle filamin isotypes. Pericytes contain at least 10 times more muscle filamin than human umbilical vein EC and at least three times the amount expressed in human omentum microvessel and bovine pulmonary artery EC. Differential detergent fractionation indicates that both filamin isotypes are primarily localized in the cytosol and membrane/organelle fractions of pericytes. Another actin crosslinking protein, alpha-actinin, is primarily found in the cytosol and cytoskeletal fractions. The dynamic regulation of actin microfilament organization in pericytes may be controlled in part by the two filamin isotypes, which in turn may contribute to pericyte contractility.

• Sakurai Y et al.
• The cDNA cloning and molecular characterization of a snake venom platelet glycoprotein Ib-binding protein, mamushigin, from Agkistrodon halys blomhoffii venom.
• Thromb Haemost. 1998; 79: 1199-207
• Display abstract

• The entire cDNA sequences of a novel snake venom platelet glycoprotein (GP) Ib-binding protein (BP) composed of an alpha/beta heterodimeric structure, termed mamushigin, from Agkistrodon halys blomhoffii were determined, that include the leader peptides (21/23 amino acid residues) and mature subunits (136/123 amino acid residues). The mature subunits of mamushigin are 37.5% identical, and showed a high degree of similarity (37.7-67.5% identity) with the respective subunits of group VII C-type lectins (19). The sequences of the leader peptides of the mamusigin subunits showed the highest similarity (alpha-73.9/beta-82.6%) with those of factor IX/X-BP from Trimeresurus flavoviridis, and the cleavage site residue in both proteins was the same Ala(-1). The GPIb-binding specificity of mamushigin is strongly supported by several lines of evidence, but mamushigin can directly aggregate normal platelets, similar to alboaggregin-B (AL-B) (1). This differs from other GPIb-BP's. In mamushigin-treated platelets, serotonin was not released, and flow cytometric analysis using a monoclonal antibody PAC-1 totally excluded platelet GPIIb/IIIa activation. Mamushigin enhanced platelet aggregation at low-shear stress, and this effect totally disappeared in the presence of GPIb-receptor blockers specific for von Willebrand factor binding, but not by GPIIb/IIIa-receptor blockers. At high-shear stress, mamushigin blocked platelet aggregation in a dose-dependent manner, as seen with other GPIb-BP's. This paper, therefore, describes the cDNA cloning and molecular characterization of mamushigin which has a different effect on platelet aggregation under different shear stress.

• Kenny D, Jonsson OG, Morateck PA, Montgomery RR
• Naturally occurring mutations in glycoprotein Ibalpha that result in defective ligand binding and synthesis of a truncated protein.
• Blood. 1998; 92: 175-83
• Display abstract

• The platelet GPIb-V-IX complex is the receptor for the initial binding of von Willebrand factor (vWF) mediating platelet adhesion. The complex is composed of four membrane-spanning glycoproteins (GP): GPIbalpha, GPIbbeta, GPIX, and GPV. Bernard-Soulier syndrome results from a qualitative or quantitative defect in one or more components of the platelet membrane GPIb-V-IX complex. We describe the molecular basis of a novel Bernard-Soulier syndrome variant in two siblings in whom GPIbalpha was not detected on the platelet surface but that was present in a soluble form in plasma. DNA sequence analysis showed that the affected individuals were compound heterozygotes for two mutations. One, inherited from a maternal allele, a T777 --> C point mutation in GPIbalpha converting Cys65 --> Arg within the second leucine rich repeat, the other, a single nucleotide substitution (G2078 --> A) for the tryptophan codon (TGG) causing a nonsense codon (TGA) at residue 498 within the transmembrane region of GPIbalpha, inherited from a mutant paternal allele. The Bernard-Soulier phenotype was observed in siblings who were compound heterozygotes for these two mutations. Although GPIbalpha was not detected on the surface of the patient's platelets, soluble GPIbalpha could be immunoprecipitated from plasma. When plasmids encoding GPIbalpha containing the Cys65 --> Arg mutation were transiently transfected into Chinese hamster ovary (CHO) cells stably expressing the GPbeta-IX complex (CHObetaIX), the expression of GPIbalpha was similar to the wild-type (WT) GPIbalpha, but did not bind vWF. When plasmids encoding GPIbalpha containing the Trp498 --> stop were transiently transfected into CHObetaIX, the surface expression of GPIbalpha was barely detectable compared with the WT GPIbalpha. Thus, this newly described compound heterozygous defect produces Bernard-Soulier syndrome by a combination of synthesis of a nonfunctional protein and of a truncated protein that fails to insert into the platelet membrane and is found circulating in plasma.

• Sharma P et al.
• Interaction of the SR CaATPase with the cytoplasmic region of phospholamban.
• Biochem Soc Trans. 1998; 26: 228-228

• Rietzler M, Bittner M, Kolanus W, Schuster A, Holzmann B
• The human WD repeat protein WAIT-1 specifically interacts with the cytoplasmic tails of beta7-integrins.
• J Biol Chem. 1998; 273: 27459-66
• Display abstract

• Integrins of the beta7 subfamily, alpha4 beta7 and alphaE beta7, contribute to lymphocyte homing and to the development of protective or autoreactive immune responses at mucosal sites. The beta subunits of integrins are considered important for regulation of stimulated cell adhesion and adhesion-dependent signal transduction. Using a yeast interaction trap screen, a human WD repeat protein, termed WAIT-1, was isolated that interacts with the integrin beta7 cytoplasmic tail and is homologous to mouse EED and Drosophila ESC proteins. WAIT-1 also binds to the cytoplasmic domains of alpha4 and alphaE but not to those of integrin beta1, beta2, and alphaL subunits. Association of WAIT-1 and beta7-integrin was confirmed by coprecipitation from transiently transfected 293 cells. The binding site for WAIT-1 was mapped to a short membrane-proximal region of the beta7 cytoplasmic tail with Tyr-735 being of critical importance. Northern blot analysis revealed multiple WAIT-1-related transcripts with differential expression in circulating leukocytes, tissue-resident cells of diverse origin, and lymphoid malignancies. These results suggest that WAIT-1, together with the recently identified RACK1, may define a novel subfamily of WD repeat proteins that interact with distinct subsets of integrin cytoplasmic tails and may act as specific regulators of integrin function.

• Katsutani S et al.
• Cloning and characterization of the gene encoding the murine glycoprotein V: the conserved thrombin-cleavable protein on platelet surface.
• Thromb Res. 1998; 92: 43-51
• Display abstract

• Glycoprotein V (GPV) is a platelet membrane protein present as a subunit of the GPIb/V/IX complex, a major receptor for von Willebrand factor, and is specifically cleaved by thrombin. In this study, we have cloned and characterized murine GPV gene. The entire coding sequence of murine GPV consisted of 1704 nucleotides and coded 567 amino acids, which were 70% identical with human GPV. Fifteen leucine-rich tandem repeats were present and the consensus sequence of the repeats was completely matched with that of human GPV. The thrombin-cleavage site was also conserved exactly at the same position. In Northern blot, murine GPV mRNA was specifically expressed in murine platelets, bone marrow cells and megakaryocytic cell lines. In the survey of other organs, GPV was not expressed at all. These results demonstrate that GPV is highly conserved, thrombin-cleavable protein beyond the species, and is a specific protein in the platelet-megakaryocyte lineage.

• Lin Y, Kishi H, Nakamura A, Takagi T, Kohama K
• N-terminal myosin-binding fragment of talin.
• Biochem Biophys Res Commun. 1998; 249: 656-9
• Display abstract

• Talin, an actin-binding protein from smooth muscle, is shown to bind to myosin in such a way that it stimulates the ATPase activity of myosin irrespective of the phosphorylation state of myosin. The binding site is shown to be localized at the N-terminal, 47 KDa fragment. The position of the actin-binding site at the C terminal suggests that talin may work as a crosslinker between myosin and actin.

• Crommie D, Hemler ME
• Beta1 integrin cytoplasmic domain regulates the constitutive conformation detected by MAb 15/7, but not the ligand-induced conformation.
• J Cell Biochem. 1998; 71: 63-73
• Display abstract

• The anti-integrin beta1 MAb 15/7 sometimes may be a reporter of integrin activation or ligand occupancy. However, certain beta1 tail deletions eliminate ligand binding despite inducing maximal constitutive 15/7 expression [Puzon-Mclaughlin et al. (1 996): J Biol Chem 271:16580-16585]. Here we describe beta1 tail mutations (e.g., double point mutations [D759L/F763L, F766L/E769L], or replacement of the beta1 tail by the beta5 tail) that prevent rather than induce constitutive appearance of the 15/7 epitope. Despite variable losses of constitutive 15/7 epitope, these mutants all retained a similar inducible 15/7 epitope component as seen upon incubation with GRGDSP peptide ligand. In addition, constitutive 15/7 expression did not correlate with integrin localization into focal adhesions. In conclusion, we show for the first time for a fully functional integrin that specific mutations within the beta1 tail can down-regulate the constitutive appearance of an extracellular conformation defined by MAb 15/7. Because this regulation occurs away from the ligand binding site and does not correlate with responsiveness to integrin ligand, cell adhesion, or localization into focal adhesions, a novel type of conformational regulation is suggested.

• Afshar-Kharghan V, Lopez JA
• Bernard-Soulier syndrome caused by a dinucleotide deletion and reading frameshift in the region encoding the glycoprotein Ib alpha transmembrane domain.
• Blood. 1997; 90: 2634-43
• Display abstract

• We investigated the molecular genetic and biosynthetic basis of Bernard-Soulier syndrome in a severely affected white woman. Flow cytometric analysis showed a severe deficiency of glycoprotein (GP) Ib, GP IX, and GP V on the surface of her platelets. Similarly, GP Ib alpha was undetectable by immunoblot analysis of platelet lysates. Surprisingly, a large quantity of a 70-kD protein (which probably represents a GP Ib alpha degradation product) was found in the patient's plasma in much greater quantities than in the plasma of an unaffected individual. To analyze the molecular lesion responsible for the disorder, we amplified and sequenced gene segments corresponding to the entire coding regions of the GP Ib alpha, GP Ib beta, and GP IX genes. The patient was homozygous for a specific GP Ib alpha allele that contained two tandem VNTR repeats in the region encoding the macroglycopeptide (C variant) and three differences from the published GP Ib alpha gene sequence. Two mutations were unlikely to be involved in the disorder: the substitution of a single base (T --> C) in the second nucleotide of exon 2, which is in the 5' untranslated region of the GP Ib alpha transcript, and a silent mutation in the third base of the codon for Arg342 (A --> G) that does not change the amino acid sequence. The third mutation was a deletion of the last two bases of the codon for Tyr492 (TAT). This mutation causes a frameshift that alters the GP Ib alpha amino acid sequence, beginning within its transmembrane region. The mutant polypeptide contains 81 novel amino acids and is 38 amino acids shorter than its wild-type counterpart. The new sequence changes the hydrophobic nature of the transmembrane domain and greatly decreases the net positive charge of what had been the cytoplasmic domain. The deletion mutation was introduced into the GP Ib alpha cDNA, alone and in combination with the 5' mutation, and expressed in Chinese hamster ovary (CHO) cells. The deletion alone severely reduced GP Ib alpha expression on the cell surface. Expression was not decreased further by addition of the 5' mutation, confirming that the deletion was the cause of the Bernard-Soulier phenotype. Stable cell lines expressing the mutant polypeptide secreted large amounts of the polypeptide into the medium, suggesting that the mutant anchors poorly in the plasma membrane. Nevertheless, a fraction of the mutant was able to associate with GP Ib beta, as demonstrated by their coimmunoprecipitation with a GP Ib beta antibody.

• Fisher PR, Noegel AA, Fechheimer M, Rivero F, Prassler J, Gerisch G
• Photosensory and thermosensory responses in Dictyostelium slugs are specifically impaired by absence of the F-actin cross-linking gelation factor (ABP-120).
• Curr Biol. 1997; 7: 889-92
• Display abstract

• Chemotactic aggregation of starving amoebae of Dictyostelium discoideum leads to formation of a motile, multicellular organism - the slug - whose anterior tip controls its phototactic and thermotactic behaviour. To determine whether proteins that regulate the in vitro assembly of actin are involved in these responses, we tested phototaxis and thermotaxis in mutant slugs in which the gene encoding one of five actin-binding proteins had been disrupted. Of the proteins tested - severin, alpha-actinin, fimbrin, the 34 kD actin-bundling protein and the F-actin cross-linking gelation factor (ABP-120) - only ABP-120 proved essential for normal phototaxis and thermotaxis in the multicellular slugs. The related human protein ABP-280 is required for protein phosphorylation cascades initiated by lysophosphatidic acid and tumor necrosis factor alpha. The repeating segments constituting the rod domains of ABP-120 and ABP-280 may be crucial for the function of both proteins in specific signal transduction pathways by mediating interactions with regulatory proteins.

• Fucini P, McCoy AJ, Gomez-Ortiz M, Schleicher M, Noegel AA, Stewart M
• Crystallization and preliminary X-Ray diffraction characterization of a dimerizing fragment of the rod domain of the Dictyostelium gelation factor (ABP-120).
• J Struct Biol. 1997; 120: 192-5
• Display abstract

• We have expressed in Escherichia coli a construct corresponding to sequence repeats 5 and 6 of the rod domain of the actin-binding protein Dictyostelium gelation factor (ABP-120). We have obtained orthorhombic P212121 crystals of the protein with a = 43.5 A, b = 103.2 A, c = 124.4 A. These crystals diffract past 2.2 A resolution using synchrotron radiation and are suitable for high-resolution structural analysis. ABP-120 is a key component of the Dictyostelium cytoskeleton, where it functions to crosslink F-actin filaments into networks. This crosslinking function of ABP-120 depends crucially on the formation of dimeric molecules that contain an actin-binding site on each chain, and this dimerization is brought about through interactions between repeating sequence modules in the rod domain. Because the construct we have expressed retains the ability to dimerize, it should enable us to establish the precise manner in which these sequence repeats interact with one another in the intact molecule.

• Meyer SC et al.
• Identification of the region in actin-binding protein that binds to the cytoplasmic domain of glycoprotein IBalpha.
• J Biol Chem. 1997; 272: 2914-9
• Display abstract

• Actin-binding protein (ABP-280) is a component of the submembranous cytoskeleton and interacts with the glycoprotein (GP) Ibalpha subunit of the GP Ib-IX complex in platelets. In the present studies, we have identified the binding site for GP Ibalpha in ABP-280. A melanoma cell line lacking ABP-280 was stably transfected with the cDNAs coding for GP Ib-IX, then transiently transfected with cDNA coding for various carboxyl-truncates of ABP-280. Immunocapture assays and co-immunoprecipitation experiments from detergent-lysed cells showed that deletion of the carboxyl-terminal repeats 20-24 of ABP-280 had no effect on GP Ib-IX binding, but deletion of residues 2099 through 2136 within repeat 19 abolished binding. In the yeast two-hybrid system, an ABP-280 fragment comprising repeats 17-19 bound GP Ibalpha. Deletion from either end abolished binding. Individual or multiple repeats of ABP-280 were expressed as fusion protein in bacteria and purified; structural folding was evaluated, and binding to GP Ib-IX was assessed. Binding depended on the presence of repeats 17-19. None of the individual repeats were able to bind to GP Ib-IX. These findings demonstrate that residues 1850-2136 comprising repeats 17-19 contain the binding site for GP Ib-IX.

• Krutzsch H, Williams SB, McKeown LP, Gralnick HR
• Structure-function studies on the inhibition of binding of alpha-thrombin to platelet GpIb alpha by the peptide D-Y-Y-P-E and the artifactual inhibitory activity of C-terminal E in this peptide.
• Thromb Res. 1997; 88: 333-5

• Zieger B, Hashimoto Y, Ware J
• Alternative expression of platelet glycoprotein Ib(beta) mRNA from an adjacent 5' gene with an imperfect polyadenylation signal sequence.
• J Clin Invest. 1997; 99: 520-5
• Display abstract

• Glycoprotein (GP) Ib is a major component of the platelet membrane receptor for von Willebrand factor, designated the GP Ib-IX-V complex. GP Ib is composed of two subunits (GP Ib(alpha) and GP Ib(beta)) each synthesized from separate genes. The 206 amino acid precursor of GP Ib(beta) is synthesized from a 1.0-kb mRNA expressed by megakaryocytes and was originally characterized from cDNA clones of human erythroleukemic (HEL) cell mRNA, a cell line exhibiting megakaryocytic-like properties. The cell line CHRF-288-11 also exhibits megakaryocytic-like properties, but synthesizes two related GP Ib(beta) mRNA species of 3.5 and 1.0 kb. We performed cDNA cloning experiments to identify the origin of the 3.5-kb transcript and determine its relationship to the 1.0-kb GP Ib(beta) mRNA found in megakaryocytes, platelets, and HEL cells. Our cloning experiments demonstrate that the longer transcript results from a nonconsensus polyadenylation recognition sequence, 5'AACAAT3', within a separate gene located upstream to the platelet GP Ib(beta) gene. In the absence of normal polyadenylation the more 5' gene uses the polyadenylation site within its 3' neighbor, the platelet GP Ib(beta) gene. This newly identified 5' gene contains an open reading frame encoding 369 amino acids with a high degree of sequence similarity to an expanding family of GTP-binding proteins.

• Hayashi T et al.
• Corrected DNA sequence of the platelet glycoprotein IX gene.
• Thromb Haemost. 1997; 77: 1034-5

• Tachikawa M, Nakagawa H, Terasaki AG, Mori H, Ohashi K
• A 260-kDa filamin/ABP-related protein in chicken gizzard smooth muscle cells is a new component of the dense plaques and dense bodies of smooth muscle.
• J Biochem (Tokyo). 1997; 122: 314-21
• Display abstract

• A 260-kDa protein, termed cgABP260, which localized in the dense plaques and dense bodies of smooth muscle cells, was found in a low-salt alkaline extract of chicken gizzard smooth muscle. An antibody against cgABP260 was used to screen a chicken gizzard cDNA library, and the nucleotide sequence of the partial cDNA encoding this protein was determined. Comparison of predicted amino acid sequences revealed that the protein had significant homology with human ABP-280 and chicken retina filamin [Barry, C.P. et al. (1993) J. Biol. Chem. 268, 25577-25586], but despite the high homology, cgABP260 was immunologically distinguishable from filamin. Immunoblot analysis showed that an anti-cgABP260 antibody reacted exclusively with the cgABP260 band of smooth, skeletal, and cardiac muscle tissues. By indirect immunofluorescence, the membrane-affinity-purified antibody against cgABP260 intensely stained the dense plaques of the isolated smooth muscle cells. Immunoelectron microscopy showed that immunogold particles representing cgABP260 were found abundantly on the dense plaques and less abundantly on the dense bodies. Its amino acid sequence, molecular size, immunological reactivity, and localization in smooth muscle thus indicated that cgABP260 is a new component of the dense plaques and dense bodies of smooth muscle cells.

• Liu G et al.
• Cytoskeletal protein ABP-280 directs the intracellular trafficking of furin and modulates proprotein processing in the endocytic pathway.
• J Cell Biol. 1997; 139: 1719-33
• Display abstract

• Furin catalyzes the proteolytic maturation of many proproteins within the trans-Golgi network (TGN)/endosomal system. Furin's cytosolic domain (cd) directs both the compartmentalization to and transit between its manifold processing compartments (i.e., TGN/biosynthetic pathway, cell surface, and endosomes). Here we report the identification of the first furin cd sorting protein, ABP-280 (nonmuscle filamin), an actin gelation protein. The furin cd was used as bait in a yeast two-hybrid screen to identify ABP-280 as a furin-binding protein. Binding analyses in vitro and coimmunoprecipitation studies in vivo showed that furin and ABP-280 interact directly and that ABP-280 tethers furin molecules to the cell surface. Quantitative analysis of both ABP-280-deficient and genetically replete cells showed that ABP-280 modulates the rate of internalization of furin but not of the transferrin receptor, a cycling receptor. However, although ABP-280 directs the rate of furin internalization, the efficiency of sorting of the endoprotease from the cell surface to early endosomes is independent of expression of ABP-280. By contrast, efficient sorting of furin from early endosomes to the TGN requires expression of ABP-280. In addition, ABP-280 is also required for the correct localization of late endosomes (dextran bead uptake) and lysosomes (LAMP-1 staining), demonstrating a pleiotropic role for this actin binding protein in the organization of cellular compartments and directing protein traffic. Finally, and consistent with the trafficking studies on furin, we showed that ABP-280 modulates the processing of furin substrates in the endocytic but not the biosynthetic pathways. The novel roles of ABP-280 and the cytoskeleton in the sorting of furin in the TGN/ endosomal system and the formation of proprotein processing compartments are discussed.

• Hajkova L, Bjorkegren Sjogren C, Korenbaum E, Nordberg P, Karlsson R
• Characterization of a mutant profilin with reduced actin-binding capacity: effects in vitro and in vivo.
• Exp Cell Res. 1997; 234: 66-77
• Display abstract

• We are investigating structure-function relationships in profilin and actin by site-specific mutagenesis using a yeast, Saccharomyces cerevisiae, expression system to produce wild-type and mutant proteins. This paper shows that deleting proline 96 and threonine 97, which are located close to the major actin binding site on profilin, did not significantly alter the interaction between profilin and phosphatidylinositol 4,5-bisphosphate, nor did it affect the profilin:poly(L-proline) interaction. The mutant protein, however, had a lower capacity to bind to actin in vitro than wild-type profilin, though it showed a slightly increased profilin-enhanced nucleotide exchange on the actin. When microinjected into Swiss 3T3 mouse fibroblasts or porcine aortic endothelial cells, the mutant profilin did not change the organization of the microfilament system like the wild-type profilin did. This provides further evidence that profilin controls microfilament organization in the cell by interacting directly with actin.

• Puzon-McLaughlin W, Yednock TA, Takada Y
• Regulation of conformation and ligand binding function of integrin alpha5beta1 by the beta1 cytoplasmic domain.
• J Biol Chem. 1996; 271: 16580-5
• Display abstract

• We have studied the role of the cytoplasmic domain in the conformation and affinity modulation of the integrin beta1. Expression of a conformation-dependent anti-beta1 antibody 15/7 correlates with activation in wild-type beta1. Truncation of 16 carboxyl-terminal residues in the cytoplasmic domain (the 762t beta1 mutant) induces constitutive expression of the 15/7 epitope at a high level (which probably reflects a major conformational change of the extracellular domain) but does not activate ligand binding. The dissociation of epitope expression and affinity suggests that the epitope expression reflects the conformation of nonligand binding sites of the extracellular domain of beta1 but does not necessarily reflect that of the ligand binding sites. Indeed we discovered that the 15/7 epitope is in fact located in the nonligand binding region of beta1 (within residues 354-425). The 762t mutant has apparently normal alpha/beta association, suggesting that the overexpression of the 15/7 epitope is not due to alpha/beta dissociation. The data suggest that the carboxyl-terminal 16 residues of the beta1 cytoplasmic domain are critical for properly modulating conformation and affinity of beta1 integrins.

• Pfaff M, Reinhardt DP, Sakai LY, Timpl R
• Cell adhesion and integrin binding to recombinant human fibrillin-1.
• FEBS Lett. 1996; 384: 247-50
• Display abstract

• Fibrillin-1 is a major constituent of tissue microfibrils that occur in most connective tissues, either in close association with or independent of elastin. To test possible cell-adhesive functions of this protein, we used recombinant human fibrillin-1 polypeptides produced in a mammalian expression system in cell attachment and solid-phase integrin binding assays. Fibrillin-1 polypeptides containing the single RGD sequence located in the fourth 8-cysteine domain, mediated distinct cell adhesion of a variety of cell lines and bound to purified integrin alphaVbeta3. Integrins alphaIIbbeta3, alpha5beta1, alpha2beta1 and alpha1beta1 did not interact with any of the recombinant fibrillin-1 peptides. Our results indicate a novel role for fibrillin-1 in cellular interactions mediated via an RGD motif that is appropriately exposed for recognition by integrin alphaVbeta3.

• Bikle DD, Munson S, Komuves L
• Zipper protein, a B-G protein with the ability to regulate actin/myosin 1 interactions in the intestinal brush border.
• J Biol Chem. 1996; 271: 9075-83
• Display abstract

• We recently identified a 28-kDa protein in the intestinal brush border that resembled tropomyosin in terms of size, homology, and alpha helical content. This protein contained 27 heptad repeats, nearly all of which began with leucine, leading to its name zipper protein. Subsequent analysis, however, indicated that both a 49-kDa and a 28-kDa immunoreactive protein existed in intestinal brush-border extracts. Using 5'-rapid amplification of cDNA ends analysis, we extended the N-terminal sequence of zipper protein to the apparent translation start site. This additional sequence contained a putative transmembrane domain and two potential tryptic cleavage sites C-terminal to the transmembrane domain which would release a 28-kDa cytoplasmic protein if utilized. The additional sequence was highly homologous to members of the B-G protein family, a family with no known function. Immunoelectron microscopy showed that zipper protein was confined to the membrane of the microvillus where it was in close association with brush-border myosin 1 (BBM1). Recombinant zipper protein (28-kDa cytoplasmic portion) blocked the binding of actin to BBM1 and inhibited actin-stimulated BBM1 ATPase activity. In contrast, zipper protein had no effect on endogenous or K/EDTA-stimulated BBM1 ATPase activity. Furthermore, zipper protein displaced tropomyosin from binding to actin, suggesting that these homologous proteins bind to the same sites on the actin molecule. We conclude that zipper protein is a transmembrane protein of the B-G family localized to the intestinal epithelial cell microvillus. The extended cytoplasmic tail either in the intact molecule or after tryptic cleavage may participate in regulating the binding and, thus, activation of BBM1 by actin in a manner similar to tropomyosin.

• Vargas M, Sansonetti P, Guillen N
• Identification and cellular localization of the actin-binding protein ABP-120 from Entamoeba histolytica.
• Mol Microbiol. 1996; 22: 849-57
• Display abstract

• Several actin-binding proteins participate in the morphological changes that occur during amoeboid movement. The gene encoding one of these proteins, the gelation factor ABP-120, was identified and characterized from trophozoites of Entamoeba histolytica. The sequence contains 2574 nucleotides, with an open reading frame of 858 amino acids, giving a protein of 93 kDa belonging to the spectrin family. The N-terminal domain of ABP-120 from E. histolytica revealed a consensus site for actin binding homologous to the actin-binding sites of ABP-120 of Dictyostelium discoideum, alpha-actinin and spectrin. Analysis of the central domain revealed the presence of four repeats of a 73-amino-acid motif constituting 31% of the protein. In addition, a stretch of 105 amino acids was highly divergent when compared with the C-terminal domain of D. discoideum ABP-120. This sequence showed short motifs that are homologous to microtubule-binding domains. We found that ABP-120 from E. histolytica binds to F-actin. In addition, upon motility of the parasite, this protein localized in the pseudopod and the uroid region, implying a role for ABP-120 in movement and capping of surface receptors in E. histolytica.

• Danyluk J, Carpentier E, Sarhan F
• Identification and characterization of a low temperature regulated gene encoding an actin-binding protein from wheat.
• FEBS Lett. 1996; 389: 324-7
• Display abstract

• A cDNA corresponding to a putative actin-binding protein was cloned from a cold-acclimated wheat cDNA library. The cDNA, designated Wcor719, encodes a polypeptide of 142 amino acids with a calculated molecular mass of 15.8 kDa and a pI of 4.27. The protein has the two conserved domains identified as actin and phosphatidylinositol 4,5-biphosphate (PIP2) binding sites found in members of the cofilin family. Northern analyses revealed that Wcor719 transcript accumulation is rapid and strongly up-regulated by low temperature. This accumulation was greater in the tolerant winter wheat and rye species compared to the less tolerant ones. The rapidity of transcript induction and the significant homology with actin-binding proteins (ABP) from different organisms suggest that the product of this gene might be involved in the dynamic reorganization of the cytoskeleton during low temperature acclimation. It may also serve as a key factor in the signal transduction pathway during cold acclimation.

• Baatout S
• Profilin: an update.
• Eur J Clin Chem Clin Biochem. 1996; 34: 575-7

• Lebart MC, Casanova D, Benyamin Y
• Actin interaction with purified dystrophin from electric organ of Torpedo marmorata: possible resemblance with filamin-actin interface.
• J Muscle Res Cell Motil. 1995; 16: 543-52
• Display abstract

• We have purified dystrophin from Torpedo marmorata electric tissue by means of alkaline extraction in conjunction with an affinity chromatography column using anti-peptide antibodies. Using solution (cosedimentation) and solid phase experiments (sedimentation with Sepharose filamentous actin and ELISA), we have demonstrated that purified dystrophin is able to bind filamentous and monomeric actin. Using ELISA coupled with biotin labelled peptides and taking advantage of strong affinity binding of streptavidin-biotin complex, we have identified two exposed sequences of the actin molecule implicated in dystrophin binding: fragment 40-113, further restricted to peptide 75-106 and peptide 360-372. In a previous study, we have shown that fragment 40-113 displays binding site(s) for filamin but probably not for alpha-actinin. Moreover, we have recently reported that alpha-actinin and filamin display divergent behaviours towards conformational changes of actin. In this study, we have demonstrated that, similarly to filamin, dystrophin binding is insensitive to the locking of actin in a monomeric conformation. Taken together, these results lead us to favour the idea that dystrophin could share properties in common with filamin in its binding of actin.

• Sharma CP, Ezzell RM, Arnaout MA
• Direct interaction of filamin (ABP-280) with the beta 2-integrin subunit CD18.
• J Immunol. 1995; 154: 3461-70
• Display abstract

• beta 2-Integrins mediate leukocyte extravasation into inflamed tissues, phagocytosis, and target cell killing, functions that require an intact actin cytoskeleton. Previous studies have focused on elucidating interactions of the cytoplasmic tails of integrins with the cytoskeleton at focal contacts in stationary cells. As integrins are also located at other types of cell-substratum junctions, such as the leading edge of migrating cells, additional cytosolic proteins abundant at these sites may also interact with integrins. In this study, we have identified the actin-binding protein, filamin (ABP-280), as a major cytoskeletal protein that binds to the cytoplasmic tail of the beta 2-integrin subunit CD18. Filamin bound to cytoplasmic CD18 directly and specifically, co-immunoprecipitated with beta 2-integrins in detergent cell lysate and co-immunolocalized with cross-linked beta 2-integrins in intact cells. The filamin binding site in CD18 was localized to the N-terminal (amino acids (aa) 724 to 747) but not to the C-terminal (aa 743 to 769) half of cytoplasmic CD18. Filamin did not bind to the major alpha-actinin binding site (aa 733 to 742), however, suggesting that these two cytoskeletal proteins bind to distinct but overlapping sites. Given the conservation of the filamin binding region among beta-integrin subunits, these findings suggest the presence of similar associations between filamin and other integrins. These associations may be important in the spreading and extension of lamellipodia at the leading edge during cell movement and, if interrupted, may contribute to the dramatic decrease in cell locomotion observed in genetic deletions involving filamin or integrins.

• Lopez JA, Weisman S, Sih T, Li CQ, Sanan DA
• Association of GP Ib with actin-binding protein does not require GP IX.
• Blood Coagul Fibrinolysis. 1994; 5: 479-85
• Display abstract

• GP IX is necessary for optimal expression of the GP Ib-IX complex on the surface of transfected cells, and presumably also on the surface of the platelet. The authors investigated whether increasing complex association with the cytoskeleton is one mechanism by which GP IX exerts its effect. CHO and L cell lines that express high levels of GP Ib were used to determine whether GP Ib (GPIb alpha and GPIb beta) associated with the cytoskeleton. GP Ib in these cells was found in the insoluble cytoskeletal fraction from Triton X-100 lysates in a proportion similar to that found in cells expressing the full complex. As in platelets and cells expressing the full complex, the association of GP Ib with the cytoskeleton was shown to be mediated by actin-binding protein (ABP). This was demonstrated by the observation that a monoclonal antibody against GPIb alpha precipitated ABP from GP Ib-expressing cells, and polyclonal anti-ABP antibodies specifically coprecipitated GP Ib. In addition, colocalization of the two components in intact cells was demonstrated by confocal microscopy. These data indicate that the association of GP Ib with the cytoskeleton is independent of GP IX, which therefore must increase surface expression of the complex by another mechanism.

• Patrosso MC et al.
• The exon-intron organization of the human X-linked gene (FLN1) encoding actin-binding protein 280.
• Genomics. 1994; 21: 71-6
• Display abstract

• We have determined the exon-intron organization of the human X-linked gene (FLN1) encoding actin-binding protein 280 (filamin), a ubiquitous protein that plays an important role in the mechanochemical activities of cells through its association with actin filaments and membrane components. The gene is composed of 47 exons spanning approximately 26 kb. The first and part of the second exon are untranslated. The actin-binding domain at the N-terminus is encoded by exons 2 to 5. The 96-amino-acid repeats corresponding to the elongated rod backbone of the protein are encoded by the remaining 42 exons: size, location, and boundaries of the exons cannot be easily correlated with the repeated structure, while sequences interrupting the repeats (the two hinge segments preceding repeats 16 and 24 and the 8-amino-acid (aa) segment interrupting the 15th repeat) were encoded by separate exons, suggesting that they may be recent additions to the X-linked protein. The 8-aa segment is encoded by exon 29, which is alternatively spliced.

• Tempel M, Goldmann WH, Isenberg G
• Computer analyses suggest interactions of non-muscle filamin with lipid membranes.
• FEBS Lett. 1994; 350: 169-72
• Display abstract

• It is concluded from structure predictions of the primary amino acid sequence by computer analyses that two segments of non-muscle filamin could facilitate lipid membrane attachment or anchoring. Residues 49-71 of the amino-terminal may attach to phospholipid membranes, and residues 131-155 may anchor in the hydrophobic region of lipid membranes.

• Lange U, Steiner S, Grolig F, Wagner G, Philippsen P
• Cloning and sequencing of a gene coding for an actin binding protein of Saccharomyces exiguus.
• Biochim Biophys Acta. 1994; 1217: 214-8
• Display abstract

• The actin binding protein Abp1p of the yeast Saccharomyces cervisiae is thought to be involved in the spatial organisation of cell surface growth. It contains a potential actin binding domain and an SH-3 region, a common motif of many signal transduction proteins [1]. We have cloned and sequenced an ABP1 homologous gene of Saccharomyces exiguus, a yeast which is only distantly related to S. cerevisiae. The protein encoded by this gene is slightly larger than the respective S. cerevisiae protein (617 versus 592 amino acids). The two genes are 67.4% identical and the deduced amino acid sequences share an overall identity of 59.8%. The most conserved regions are the 148 N-terminal amino acids containing the potential actin binding site and the 58 C-terminal amino acids including the SH3 domain. In addition, both proteins contain a repeated motif of unknown function which is rich in glutamic acids with the sequence EEEEEEEAPAPSLPSR in the S. exiguus Abp1p.

• Schenkels LC, Schaller J, Walgreen-Weterings E, Schadee-Eestermans IL, Veerman EC, Nieuw Amerongen AV
• Identity of human extra parotid glycoprotein (EP-GP) with secretory actin binding protein (SABP) and its biological properties.
• Biol Chem Hoppe Seyler. 1994; 375: 609-15
• Display abstract

• In this paper the identity of the salivary protein EP-GP (extra-parotid glycoprotein) is reported, also apparent in other human secretions. Immunochemical and biochemical analysis demonstrated that EP-GP is similar to the secretory actin-binding protein (SABP), also known as gross cystic disease fluid protein-15 (GCDFP-15) and prolactin-inducible protein (PIP). The molecular mass and charge microheterogeneity of EP-GP, also observed for SABP, was shown to be predominantly caused by the carbohydrate moiety. In addition, evidence was given that EP-GP is not related to the lipocalin Von Ebner's gland protein (human; VEGh). The biological significance of EP-GP and its homologues is not clear. EP-GP bound to actin and fibrinogen as described for SABP and GCDFP-15. However, the affinity for these proteins does not appear to have any direct physiological role in the mucosal secretions. On the other hand, EP-GP binds to several bacteria. By electron microscopy the ultrastructural localization is demonstrated of EP-GP to the cell wall of both Streptococcus salivarius HB and its cell appendage-lacking mutant Streptococcus salivarius HB-C12. Concerning this finding we hypothesize on the possible functional aspects of this enigmatic protein EP-GP.

• Lebart MC et al.
• Characterization of the actin binding site on smooth muscle filamin.
• J Biol Chem. 1994; 269: 4279-84
• Display abstract

• We have isolated an NH2-terminal fragment of filamin (M(r) = 70,000) after digestion with Staphylococus aureus V8 protease. This fragment was shown to interact with filamentous actin in cosedimentation assays. Using cross-reactive anti-peptides antibodies directed against the strongly conserved 27-mer sequence of alpha-actinin, already implicated as an actin binding site (Kuhlman, P. A., Hemmings, L., and Critchley, D. R. (1992) FEBS Lett. 304, 201-206), we obtained evidence suggesting that the homologous sequence of filamin (121-147 sequence) is the major element in the interaction with actin. In particular, we used enzyme-linked immunosorbent assay experiments, in conjunction with a synthetic peptide approach, and found that the hydrophobic part of the 27-mer peptide (141-147 sequence) is largely involved in actin binding. Thus, the filamin sequence 121-147 (or the alpha-actinin sequence 108-134) and the actin counterpart composed of residues 112-125 and 360-372 (we have already implicated) could constitute the main interface between actin and these cytoskeletal proteins. However, the divergent behavior of filamin and alpha-actinin toward conformational changes of actin argues in favor of distinctive interfaces. Finally, the ionic strength dependence of the filamin-actin interaction, in contrast to that with alpha-actinin, strongly suggests that, besides hydrophobic interactions conferred by the 27-mer sequence, more hydrophilic region(s) of filamin participate(s) in the binding.

• Pollard TD, Quirk S
• Profilins, ancient actin binding proteins with highly divergent primary structures.
• Soc Gen Physiol Ser. 1994; 49: 117-28

• Brown KD, Zinkowski RP, Hays SE, Binder LI
• Actin-binding protein is a component of bovine erythrocytes.
• Cell Motil Cytoskeleton. 1993; 24: 100-8
• Display abstract

• Actin-binding protein (ABP) is a well-characterized polypeptide capable of crosslinking filamentous actin. To date, this polypeptide has been shown to exist in a number of tissues and cultured cell lines. This report shows that by using a panel of three monoclonal antibodies for immunoblotting and immunofluorescence analysis, that ABP is present in bovine erythrocytes. Moreover, the data obtained suggest that this protein is a component of the erythrocyte membrane skeleton. Additionally, bovine erythrocyte ABP is shown to possess both an apparent molecular weight and an isoelectric point identical to that of bovine smooth muscle filamin, implying that these two polypeptides are identical.

• Goldmann WH, Isenberg G
• Analysis of filamin and alpha-actinin binding to actin by the stopped flow method.
• FEBS Lett. 1993; 336: 408-10
• Display abstract

• We ascertained by the stopped flow method the overall association rate constant, k+1, of filamin and alpha-actinin to fluorescently labelled filamentous actin of approximately 1.3 x 10(6) M-1.s-1 and approximately 1.0 x 10(6) M-1.s-1 as well as the overall dissociation rate constant, k-1, of approximately 0.6 s-1 and approximately 0.4 s-1, respectively. The overall equilibrium constant, K, for filamin and alpha-actinin to actin deduced from the relation K = k+1/k-1 agree well with published data.

• Maestrini E et al.
• Mapping of two genes encoding isoforms of the actin binding protein ABP-280, a dystrophin like protein, to Xq28 and to chromosome 7.
• Hum Mol Genet. 1993; 2: 761-6
• Display abstract

• ABP-280 is a ubiquitous actin binding protein present in the cytoskeleton of many different cell types. ABP-280 was mapped to distal Xq28, 50-60 kb downstream of the Green Colour Pigment (GCP) genes. To establish if ABP-280 may be a candidate for one of the muscle disease localized by linkage analysis to distal Xq28 we looked for alternative forms of ABP-280 mRNA. Several different ABP-280 mRNAs were indeed identified: two are X-linked and are produced by alternative splicing of a small exon of 24 nucleotides. At least one additional gene encoding a RNA more than 70% identical to ABP-280 in the 1700 bp sequenced has also been found. It was mapped to chromosome 7. While both forms of the X-linked ABP-280 are ubiquitous, the gene on chromosome 7 is highly expressed only in skeletal muscle and heart. The two genes were therefore excellent candidates for the X-linked and for the autosomal dominant form of the Emery-Dreifuss Muscular Dystrophy (EDMD) both of which have been described. So far, however we were unable to demonstrate mutations in the coding region or affecting the alternative splicing of the X-linked form of ABP-280, in several patients studied, and we think that it is quite unlikely that this is the gene responsible for EDMD.

• Barry CP, Xie J, Lemmon V, Young AP
• Molecular characterization of a multi-promoter gene encoding a chicken filamin protein.
• J Biol Chem. 1993; 268: 25577-86
• Display abstract

• We report the cloning and sequencing of cDNA encoding a chicken filamin protein. The 2,567 amino acid protein contains an NH2-terminal 267 amino acid actin-binding domain followed by a series of 24 repeating units that are each approximately 95 amino acids in length. The overall primary structure of filamin closely resembles that of human actin-binding protein (ABP). However, filamin lacks a 24-amino-acid insertion prior to repeat 16 that is contained within ABP. This region of human ABP is a site of calpain cleavage and is thought to confer flexibility on the molecule. Hence, it is possible that the properties of actin gels formed with either human ABP or filamin reflect the presence or absence of this insertion. Filamin is encoded within a multi-promoter transcription unit. A downstream filamin promoter (Fil1) resembles those of certain housekeeping genes and has a putative binding site for the transcription factor E2F. Thus, transcription from this promoter may be linked to the cell cycle. A second filamin promoter (Fil2) is located at least 8 kilobases upstream from the Fil1 promoter. This structural arrangement suggests that regulation of filamin gene expression is likely to be complex.

• Mejean C, Lebart MC, Boyer M, Roustan C, Benyamin Y
• Localization and identification of actin structures involved in the filamin-actin interaction.
• Eur J Biochem. 1992; 209: 555-62
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• The interface between gizzard filamin and skeletal muscle actin was located on the actin monomer. Conserved sequences 105-120 and 360-372, in the actin subdomain 1 near the myosin binding sites, were involved in this interaction. The corresponding peptides for these sequences were each found to bind filamin and compete in the actin-filamin interaction. When these two peptides were used together in the presence of filamin and filamentous actin, they dissociated sedimentable complexes formed by these two proteins.

• Karinch AM, Zimmer WE, Goodman SR
• The identification and sequence of the actin-binding domain of human red blood cell beta-spectrin.
• J Biol Chem. 1990; 265: 11833-40
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• The junctions of the red blood cell membrane skeleton are formed by interactions between spectrin and actin protofilaments. A spectrin tryptic peptide of 16.5-kDa apparent molecular mass (based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis) which binds to F-actin in cosedimentation experiments has been identified. The peptide has been partially purified by gel filtration, anion-, and cation exchange chromatography. Intact spectrin heterodimer causes half-maximal inhibition of the 16.5-kDa peptide/F-actin interaction at a concentration of 5 microM. Comparison of the two-dimensional iodopeptide maps of the 16.5-kDa peptide with maps of alpha- and beta-spectrin, demonstrate that the peptide is generated from the beta subunit. It shows no significant relationship to the peptide maps of the beta-spectrin domains I-IV. Protein sequencing indicated that this actin-binding domain represents a stretch of amino acids at the N terminus of the beta subunit from alanine 47 probably through lysine 186. The sequence derived molecular weight of this actin-binding domain is 16,290 g/mol. The sequence presented represents the region of greatest homology among the spectrin supergene family (spectrin, dystrophin, alpha-actinin).

• Noegel AA, Rapp S, Lottspeich F, Schleicher M, Stewart M
• The Dictyostelium gelation factor shares a putative actin binding site with alpha-actinins and dystrophin and also has a rod domain containing six 100-residue motifs that appear to have a cross-beta conformation.
• J Cell Biol. 1989; 109: 607-18
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• The 120-kD gelation factor and alpha-actinin are among the most abundant F-actin cross-linking proteins in Dictyostelium discoideum. Both molecules are homodimers and have extended rod-like configurations that are respectively approximately 35 and 40 nm long. Here we report the complete cDNA sequence of the 120-kD gelation factor which codes for a protein of 857 amino acids. Its calculated molecular mass is 92.2 kD which is considerably smaller than suggested by its mobility in SDS-PAGE. Analysis of the sequence shows a region that is highly homologous to D. discoideum alpha-actinin, chicken fibroblast alpha-actinin, and human dystrophin. This conserved domain probably represents an actin binding site that is connected to the rod-forming part of the molecule via a highly charged stretch of amino acids. Whereas the sequence of alpha-actinin (Noegel, A., W. Witke, and M. Schleicher. 1987. FEBS [Fed. Eur. Biochem. Soc.] Lett. 221:391-396) suggests that the extended rod domain of the molecule is based on four spectrin-like repeats with high alpha-helix potential, the rod domain of the 120-kD gelation factor is constructed from six 100-residue repeats that have a high content of glycine and proline residues and which, in contrast to alpha-actinin, do not appear to have a high alpha-helical content. These repeats show a distinctive pattern of regions that have high beta-sheet potential alternating with short zones rich in residues with a high potential for turns. This observation suggests that each 100-residue motif has a cross-beta conformation with approximately nine sheets arranged perpendicular to the long axis of the molecule. In the high beta-potential zones every second residue is often hydrophobic. In a cross-beta structure, this pattern would result in one side of the domain having a surface rich in hydrophobic side chains which could account for the dimerization of the 120-kD gelation factor subunits.

• Weihing RR
• Actin-binding and dimerization domains of HeLa cell filamin.
• Biochemistry. 1988; 27: 1865-9
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• HeLa cell filamin is a linear, bivalent, homodimer of high molecular weight subunits (Mr 250,000 that may cross-link actin filaments in vivo into supramolecular structures such as networks and bundles. We used millimolar Ca protease from chicken breast muscle to cleave the subunit into smaller fragments that we mapped with respect to the overall structure of the dimer. The enzyme cleaves HeLa filamin into a larger (Mr 192,000) and a smaller (Mr 104,000) fragment; the smaller fragment is the precursor of a still smaller (Mr 92,000) fragment. Only the larger fragment bound to actin in a cosedimentation test, suggesting that it contains the actin-binding region of the subunit. Digestion of HeLa filamin that had been cross-linked with dimethyl adipimidate produced a good yield of the Mr 192,000 fragment but a poor yield of the Mr 104,000/92,000 fragments. Since native filamins are head-to-head dimers, it was expected that cross-linking would proceed most readily at the dimerization site and, therefore, it appears that the Mr 192,000 fragment is cleaved from cross-linked filamin because it is distal to the dimerization region, while the Mr 104,000/92,000 fragments are absent because they lie at the dimerization region and were cross-linked to a form that was not identifiable by sodium dodecyl sulfate electrophoresis.

• Weihing RR
• The filamins: properties and functions.
• Can J Biochem Cell Biol. 1985; 63: 397-413
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• The filamins are a group of homologous proteins defined by their high native molecular weight (500,000), their amino acid compositions, their cross-reactivity to antibodies to heterologous filamins, their localization to actin networks and bundles in situ, and their ability to cross-link actin filaments in vitro into three-dimensional networks and bundles. Native filamins contain two subunits (relative mass = 250 000). Each subunit carries at least one actin-binding site and formation of bivalent dimers is therefore believed to explain filamin's ability to cross-link actin filaments. Formation of networks in vitro (corresponding to formation of macroscopic gels) has been analyzed using the theory of Flory. As predicted, a sharp transition to gel (at the critical gelation concentration of filamin) is observed when actin is mixed with increasing concentrations of filamin and the critical gelation concentration is found to vary inversely with the length of actin filaments. However, the measured values of the critical gelation concentration are all higher (2- to 14-fold) than predicted by the theory and the prediction that the critical concentration varies directly with the actin concentration was verified with only one of two techniques used. Filamin's length (160-190 nm) and flexibility (1000-fold greater than actin filaments) may make it especially well fitted to cross-link actin filaments into three-dimensional networks when present in low molar ratios (1:200 to 1:50) relative to actin. At higher molar ratios (greater than 1:20) it also cross-links actin filaments into bundles. Assuming that filamin actually helps organize supramolecular structures inside cells (not yet tested directly), then its concentration relative to actin may help determine whether networks or bundles are formed. Other factors that may influence its localization and function inside cells include competition with other actin-binding proteins (such as myosin and tropomyosin) for binding sites on actin and phosphorylation, which may alter its ability to bind to actin.

• Weihing RR, Franklin JS
• Striated paracrystals that contain HMWP, the homolog of actin-binding protein and filamin from HeLa cells.
• Cell Motil. 1983; 3: 535-43
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• HMWP (high molecular weight protein), a high molecular weight actin binding protein, was previously isolated from HeLa cells; its physical properties, amino acid composition, and intracellular localization indicated its homology with actin-binding protein and filamin [Weihing, 1982, 1983]. We now report the identification of HMWP in striated paracrystals. Purified HMWP is incubated at 25 degrees C and subjected to negative staining with uranyl acetate. Examination by electron microscopy reveals long, striated paracrystals formed from filaments a few nanometers in diameter that lie parallel to the long axis of the paracrystal. At intervals of about 200 nm, the filaments are crossed by granular aggregates, accounting for the striated appearance. Treatment of the paracrystals with an affinity-purified antibody to HMWP decorates the filaments; such decorations are not observed if nonimmune goat IgG or phosphate-buffered saline are substituted for the antibody. Electron microscopic and electrophoretic analysis of paracrystals sedimented onto grids by centrifugation at 864 g reveals that the grids are covered with paracrystals and the major polypeptide present on grids centrifuged in parallel is HMWP. Taken together, these data indicate that the filaments of the paracrystals contain elongated molecules of HMWP. Additional experiments are needed to decide if the paracrystals form by self-association between HMWP molecules or by association with one or more of the minor polypeptides that remain in the purified HMWP.

• Davies PJ, Wallach D, Willingham M, Pastan I, Lewis MS
• Self-association of chicken gizzard filamin and heavy merofilamin.
• Biochemistry. 1980; 19: 1366-72
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• Filamin is a high molecular weight (subunit Mr 250 000) actin-binding protein isolated from smooth muscle. The protein forms a gel when mixed with solutions of F-actin. A proteolytic fragment of filamin, heavy merofilamin (subunit Mr 240 000), generated by the action of Ca2+-activated protease binds to actin but does not produce gelation. We have studied the self-association properties of filamin and heavy merofilamin by direct examination in the electron microscope and by equilibrium sedimentation distribution studies in the ultracentrifuge. Filamin self-associates reversibly to form dimers; the free energy of dimerization is approximately 7 kcal/mol. Further association to form tetramer and multimer appears to be irreversible. Warming of filamin solutions accelerates aggregation. Heavy merofilamin does not appear to self-associate but is entirely monomeric. These studies suggest that filamin produces gelation of F-actin by binding to actin and then self-associating to cross-link actin filaments into a gel.

• Shizuta Y, Shizuta H, Gallo M, Davies P, Pastan I
• Purification and properties of filamin, and actin binding protein from chicken gizzard.
• J Biol Chem. 1976; 251: 6562-7
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• Filamin, a protein recently identified in chicken gizzard (Wang, K., Ash, F., and Singer, S. J. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 4483-4486), has been purified free of other components and its molecular properties have been examined. Filamin has a sedimentation constant (S020,w) of 8.86 S and a partial specific volume of 0.734 ml/g. Sedimentation equilibrium experiments give a value of 498,000 for the molecular weight of native filamin. From these data a frictional ratio of 2.32 has been calculated. On sodium dodecyl sulfate gel electrophoresis, filamin migrates as a single protein band with an estimated molecular weight of 240,000. Filamin is a soluble protein and under a variety of conditions tested does not by itself form filaments or precipitate from solution. However, filamin binds to rabbit skeletal muscle F-actin, and the complex is readily sedimented by centrifugation to yield a gelatinous pellet containing actin and filamin. These results indicate that filamin is a dimeric protein with a moderate degree of asymmetry that binds to actin. The results also suggest that the distribution of filamin in cells is derived from its interaction with polymerized actin.

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