NORMAL GENOMIC

• Park SY, Kim SY, Jung MY, Bae DJ, Kim IS
• Epidermal growth factor-like domain repeat of stabilin-2 recognizesphosphatidylserine during cell corpse clearance.
• Mol Cell Biol. 2008; 28: 5288-98
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• Exposure of phosphatidylserine (PS) on the cell surface occurs earlyduring apoptosis and serves as a recognition signal for phagocytes.Clearance of apoptotic cells by a membrane PS receptor is one of thecritical anti-inflammatory functions of macrophages. However, the PSbinding receptors and their recognition mechanisms have not been fullyinvestigated. Recently, we reported that stabilin-2 is a PS receptor thatmediates the clearance of apoptotic cells, thus releasing theanti-inflammatory cytokine, transforming growth factor beta. In thisstudy, we showed that epidermal growth factor (EGF)-like domain repeats(EGFrp) in stabilin-2 can directly and specifically recognize PS. TheEGFrps also competitively impaired apoptotic cell uptake by macrophages inin vivo models. We also showed that calcium ions are required forstabilin-2 to mediate phagocytosis via EGFrp. Interestingly, at least fourtandem repeats of EGF-like domains were required to recognize PS, and thesecond atypical EGF-like domain in EGFrp was critical forcalcium-dependent PS recognition. Considering that PS itself is animportant target molecule for both apoptotic cells and nonapoptotic cellsduring various cellular processes, our results should help elucidate themolecular mechanism by which apoptotic cell clearance in the human bodyoccurs and also have implications for targeting PS externalization ofnonapoptotic cells.

• Kwon HJ, Lagace TA, McNutt MC, Horton JD, Deisenhofer J
• Molecular basis for LDL receptor recognition by PCSK9.
• Proc Natl Acad Sci U S A. 2008; 105: 1820-5
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• Proprotein convertase subtilisin/kexin type 9 (PCSK9) posttranslationallyregulates hepatic low-density lipoprotein receptors (LDLRs) by binding toLDLRs on the cell surface, leading to their degradation. The binding siteof PCSK9 has been localized to the epidermal growth factor-like repeat A(EGF-A) domain of the LDLR. Here, we describe the crystal structure of acomplex between PCSK9 and the EGF-A domain of the LDLR. The binding sitefor the LDLR EGF-A domain resides on the surface of PCSK9'ssubtilisin-like catalytic domain containing Asp-374, a residue for which again-of-function mutation (Asp-374-Tyr) increases the affinity of PCSK9toward LDLR and increases plasma LDL-cholesterol (LDL-C) levels in humans.The binding surface on PCSK9 is distant from its catalytic site, and theEGF-A domain makes no contact with either the C-terminal domain or theprodomain. Point mutations in PCSK9 that altered key residues contributingto EGF-A binding (Arg-194 and Phe-379) greatly diminished binding to theLDLR's extracellular domain. The structure of PCSK9 in complex with theLDLR EGF-A domain defines potential therapeutic target sites for blockingagents that could interfere with this interaction in vivo, therebyincreasing LDLR function and reducing plasma LDL-C levels.

• Chlenski A et al.
• Neuroblastoma angiogenesis is inhibited with a folded synthetic moleculecorresponding to the epidermal growth factor-like module of thefollistatin domain of SPARC.
• Cancer Res. 2004; 64: 7420-5
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• Secreted protein acidic and rich in cysteine (SPARC) is a multifunctionalmatricellular glycoprotein. In vitro, SPARC inhibits the proliferation andmigration of endothelial cells stimulated by growth factors and inducesendothelial cell apoptosis. We previously showed that SPARC also inhibitsangiogenesis in vivo and impairs the growth of the pediatric tumorneuroblastoma (NB). SPARC comprises three domains that are independentlyfolded by a complex pattern of disulfide bonds and have a high degree ofstructural conservation. In this study, separate modules of the SPARCdomains were synthesized as cysteine-linked peptides and tested for theirability to inhibit angiogenesis. Peptide FS-E, representing the epidermalgrowth factor (EGF)-like module of the follistatin (FS) domain, did notcause endothelial cell apoptosis but strongly inhibited basic fibroblastgrowth factor (bFGF)-induced endothelial cell migration with an ED(50) =10 pmol/L. In vivo, peptide FS-E blocked bFGF-stimulated angiogenesis andneovascularization induced by NB cells. The EGF-like conformation wasessential for peptide FS-E function because reduction of its two disulfidebonds completely abrogated peptide activity. Peptides FS-K and EC-N,corresponding to part of the Kazal module of the FS domain and theconserved alpha-helix in the extracellular calcium-binding domain,respectively, had minimal to no inhibitory activity. Our data show thatthe EGF-like module of the SPARC FS domain is angiosuppressive, and itsstructural conformation is critical for antiangiogenic activity.

• Balzar M et al.
• Epidermal growth factor-like repeats mediate lateral and reciprocalinteractions of Ep-CAM molecules in homophilic adhesions.
• Mol Cell Biol. 2001; 21: 2570-80
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• Ep-CAM is a new type of cell adhesion molecule (CAM) which does notstructurally resemble the members of the four major families (cadherins,integrins, selectins, and CAMs of the immunoglobulin superfamily) andmediates Ca(2+)-independent, homophilic adhesions. The extracellulardomain of Ep-CAM consists of a cysteine-rich region, containing two typeII epidermal growth factor (EGF)-like repeats, followed by a cysteine-poorregion. We generated mutated Ep-CAM forms with various deletions in theextracellular domain. These deletion mutants, together with monoclonalantibodies recognizing different epitopes in the extracellular domain,were used to investigate the role of the EGF-like repeats in the formationof intercellular contacts mediated by Ep-CAM molecules. We establishedthat both EGF-like repeats are required for the formation ofEp-CAM-mediated homophilic adhesions, including the accumulation of Ep-CAMmolecules at the cell-cell boundaries, and the anchorage of the Ep-CAMadhesion complex to F-actin via alpha-actinin. Deletion of either EGF-likerepeat was sufficient to inhibit the adhesion properties of the molecule.The first EGF-like repeat of Ep-CAM is required for reciprocalinteractions between Ep-CAM molecules on adjacent cells, as wasdemonstrated with blocking antibodies. The second EGF-like repeat wasmainly required for lateral interactions between Ep-CAM molecules. Lateralinteractions between Ep-CAM molecules result in the formation oftetramers, which might be the first and necessary step in the formation ofEp-CAM-mediated intercellular contacts.

• Buchner G et al.
• Identification of a new EGF-repeat-containing gene from human Xp22: acandidate for developmental disorders.
• Genomics. 2000; 65: 16-23
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• Epidermal growth factor (EGF) repeat-containing proteins constitute anexpanding family of proteins involved in several cellular activities suchas blood coagulation, fibrinolysis, cell adhesion, and neural andvertebrate development. By using a bioinformatic approach, we haveidentified a new member of this family named MAEG (MAM- and EGF-containinggene; HGMW-approved gene symbol and gene name). Sequence analysisindicates that MAEG encodes a secreted protein characterized by thepresence of five EGF repeats, three of which display a Ca(2+)-bindingconsensus sequence. In addition, a MAM domain is also present at theC-terminus of the predicted protein product. The human and murinefull-length cDNAs were identified and mapped to human Xp22 and to themouse syntenic region. Northern analysis indicates that MAEG is expressedearly during development. Taken together, these data render MAEG acandidate for human and murine developmental disorders.

• Saharinen J, Hyytiainen M, Taipale J, Keski-Oja J
• Latent transforming growth factor-beta binding proteins(LTBPs)--structural extracellular matrix proteins for targeting TGF-betaaction.
• Cytokine Growth Factor Rev. 1999; 10: 99-117
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• Growth factors of the transforming growth factor-beta family are potentregulators of the extracellular matrix formation, in addition to theirimmunomodulatory and regulatory roles for cell growth. TGF-beta s aresecreted from cells as latent complexes containing TGF-beta and itspropeptide, LAP (latency-associated peptide). In most cells LAP iscovalently linked to an additional protein, latent TGF-beta bindingprotein (LTBP), forming the large latent complex. LTBPs are required forefficient secretion and correct folding of TGF-beta s. The secreted largelatent complexes associate covalently with the extracellular matrix viathe N-termini of the LTBPs. LTBPs belong to the fibrillin-LTBP family ofextracellular matrix proteins, which have a typical repeated domainstructure consisting mostly of epidermal growth factor (EGF)-like repeatsand characteristic eight cysteine (8-Cys) repeats. Currently fourdifferent LTBPs and two fibrillins have been identified. LTBPs containmultiple proteinase sensitive sites, providing means to solubilize thelarge latent complex from the extracellular matrix structures. LTBPs arenow known to exist both as soluble molecules and in association with theextracellular matrix. An important consequence of this is LTBP-mediateddeposition and targeting of latent, activatable TGF-beta intoextracellular matrices and connective tissues. LTBPs have a dual function,they are required both for the secretion of the small latent TGF-betacomplex as well as directing bound latent TGF-beta to extracellular matrixmicrofibrils. However, it is not known at present whether LTBPs arecapable of forming microfibrils independently, or whether they are a partof the fibrillin-containing fibrils. Most LTBPs possess RGD-sequences,which may have a role in their interactions with the cell surface. Atleast LTBP-1 is chemotactic to smooth muscle cells, and is involved invascular remodelling. Analyses of the expressed LTBPs have revealedconsiderable variations throughout the molecules, generated both byalternative splicing and utilization of multiple promoter regions. Thesignificance of this structural diversity is mostly unclear at present.

• Pinkas-Kramarski R et al.
• ErbB tyrosine kinases and the two neuregulin families constitute aligand-receptor network.
• Mol Cell Biol. 1998; 18: 6090-101
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• The recently isolated second family of neuregulins, NRG2, shares itsprimary receptors, ErbB-3 and ErbB-4, and induction of mammary celldifferentiation with NRG1 isoforms, suggesting functional redundancy ofthe two growth factor families. To address this possibility, we analyzedreceptor specificity of NRGs by using an engineered cellular system. Theactivity of isoform-specific but partly overlapping patterns ofspecificities that collectively activate all eight ligand-stimulatableErbB dimers was revealed. Specifically, NRG2-alpha [corrected], likeNRG1-beta [corrected], emerges as a narrow-specificity ligand, whereasNRG2-beta [corrected] is a pan-ErbB ligand that binds with differentaffinities to all receptor combinations, including those containingErbB-1, but excluding homodimers of ErbB-2. The latter protein, however,displayed cooperativity with the direct NRG receptors. Apparently,signaling by all NRGs is funneled through the mitogen-activated proteinkinase (MAPK). However, the duration and potency of MAPK activation dependon the identity of the stimulatory ligand-receptor ternary complex. Weconclude that the NRG-ErbB network represents a complex and nonredundantmachinery developed for fine-tuning of signal transduction.

• Rahmatullah M, Schroering A, Rothblum K, Stahl RC, Urban B, Carey DJ
• Synergistic regulation of Schwann cell proliferation by heregulin andforskolin.
• Mol Cell Biol. 1998; 18: 6245-52
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• A peptide corresponding to the epidermal growth factor homology domain ofbeta-heregulin stimulated autophosphorylation of the heregulin receptorserbB2 and erbB3 in Schwann cells and activation of the mitogen-activatedprotein (MAP) kinases ERK1 and ERK2. Heregulin-dependent activation ofPAK65, a component of the stress-activated signaling pathway, ribosomal S6kinase, and a cyclic AMP (cAMP) response element binding protein (CREB)kinase, identified as p95(RSK2), was also observed. Receptorphosphorylation and activation of these kinases in response to heregulinoccurred in the absence of forskolin stimulation and were not augmented incells treated with forskolin, a direct activator of adenylyl cyclase.Schwann cell proliferation in response to heregulin was observed only whenthe cells were also exposed to an agent that elevates cAMP levels. In theabsence of heregulin, elevation of cAMP levels failed to stimulate Schwanncell proliferation. Forskolin significantly enhanced heregulin-stimulatedexpression of cyclin D and phosphorylation of the retinoblastoma geneproduct. In cells treated with both heregulin and forskolin there was asustained accumulation of phospho-CREB, which was not observed in cellstreated with either agent alone. Heregulin and forskolin synergisticallyactivated transcription of a cyclin D promoter construct. These resultsdemonstrate that heregulin-stimulated activation of MAP kinase is notsufficient to induce maximal Schwann cell proliferation. Expression ofcritical cell cycle regulatory proteins and cell division requireactivation of both heregulin and cAMP-dependent processes.

• Sunnerhagen M, Olah GA, Stenflo J, Forsen S, Drakenberg T, Trewhella J
• The relative orientation of Gla and EGF domains in coagulation factor X isaltered by Ca2+ binding to the first EGF domain. A combined NMR-smallangle X-ray scattering study.
• Biochemistry. 1996; 35: 11547-59
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• Coagulation factor X is a serine protease containing three noncatalyticdomains: an N-terminal gamma-carboxyglutamic acid (Gla)1 domain followedby two epidermal growth factor (EGF)-like domains. The isolated N-terminalEGF domain binds Ca2+ with a Kd of 10(-3) M. When linked to the Gladomain, however, its Ca2+ affinity is increased 10-fold. In this paper, wepresent the NMR solution structure of the factor X Gla-EGF domain pairwith Ca2+ bound to the EGF domain, as well as small angle X-ray scattering(SAXS) data on the Gla-EGF domain pair with and without Ca2+. Our resultsshow that Ca2+ binding to the EGF domain makes the Gla and EGF domainsfold toward each other using the Ca2+ site as a hinge. Presumably, asimilar mechanism may be responsible for alterations in the relativeorientation of protein domains in many other extracellular proteinscontaining EGF domains with the consensus for Ca2+ binding. The results ofthe NMR and SAXS measurements reported in this paper confirm our previousresult that the Gla domain is folded also in its apo state when linked tothe EGF domain [Sunnerhagen, M., et al. (1995) Nat. Struct. Biol. 2,504-509]. Finally, our study clearly demonstrates the powerful combinationof NMR and SAXS in the study of modular proteins, since this enablesreliable evaluation of both short-range (NMR) and long-range interactions(SAXS).

• Hrabal R, Komives EA, Ni F
• Structural resiliency of an EGF-like subdomain bound to its targetprotein, thrombin.
• Protein Sci. 1996; 5: 195-203
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• The thrombin-bound structures of native peptide fragments from the fifthEGF-like domain of thrombomodulin were determined by use of NMR andtransferred NOE spectroscopy. The bound peptides assume an EGF-likestructure of an antiparallel beta-sheet, a novel structural motif observedfor a bound peptide in protein-peptide complexes. There is a remarkablestructural resiliency of this structure motif manifested in its ability toaccommodate a different number of residues within the disulfide loop.Docking experiments revealed that the key contacts with thrombin arehydrophobic interactions between the side chains of residues Ile 414 andIle 424 of thrombomodulin and a hydrophobic pocket on the thrombinsurface. Residues Leu 415, Phe 419, and Ile 420, which would have beenburied in intact EGF-like domains, are unfavorably exposed in the complexof thrombin with the EGF-like thrombomodulin fragment, thus providing arationale for the enhancement of binding affinity upon the deletion of Ile420. The unique beta-sheet structures of the bound peptides are specifiedby the presence of disulfide bridges in the peptides because acorresponding linear thrombomodulin fragment folds into a sheet structurewith a different backbone topology. The different bound conformations forthe linear and the cyclized peptides indicate that side-chain interactionswithin a specific environment may dictate the folding of bound peptides inprotein-peptide complexes.

• Riese DJ et al.
• The epidermal growth factor receptor couples transforming growthfactor-alpha, heparin-binding epidermal growth factor-like factor, andamphiregulin to Neu, ErbB-3, and ErbB-4.
• J Biol Chem. 1996; 271: 20047-52
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• The epidermal growth factor (EGF) family hormones amphiregulin (AR),transforming growth factor-alpha (TGF-alpha), and heparin-binding EGF-likegrowth factor (HB-EGF) are thought to play significant roles in thegenesis or progression of a number of human malignancies. However, theability of these ligands to activate all four erbB family receptors hasnot been evaluated. Therefore, we have assessed the stimulation of erbBfamily receptor tyrosine phosphorylation by these hormones in a panel ofmouse Ba/F3 cell lines expressing the four erbB family receptors, singlyand in pairwise combinations. We also measured the stimulation ofinterleukin-3-independent survival or proliferation in this panel of Ba/F3cell lines to compare the patterns of erbB family receptor coupling tophysiologic responses induced by these peptides. EGF, TGF-alpha, AR, andHB-EGF all stimulated qualitatively similar patterns of erbB familyreceptor tyrosine phosphorylation and coupling to physiologic responses.Therefore, EGF, TGF-alpha, AR, and HB-EGF are functionally identical inthis model system and behave differently from the EGF family hormonesbetacellulin and neuregulins.

• Goji J, Nakamura H, Ito H, Mabuchi O, Hashimoto K, Sano K
• Expression of c-ErbB2 in human neuroblastoma tissues, adrenal medullaadjacent to tumor, and developing mouse neural crest cells.
• Am J Pathol. 1995; 146: 660-72
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• We have examined the expression of c-ErbB2 in primary neuroblastomatissues, mouse neural crest-derived tissues, and human adrenal glandadjacent to neuroblastoma tissue and of age-matched controls. c-ErbB2expression was observed in approximately 60% of cases analyzed, and therewere two staining patterns; one showed focal and cytoplasmic and the othershowed diffuse and membrane staining patterns. The expression of c-ErbB2in neuroblastoma tissues was confirmed by reverse transcription polymerasechain reaction and Western blot analysis. Diffuse and membrane staining ofc-ErbB2 was well correlated with high urinary catecholamine secretion. Inmouse tissues, cytoplasmic expression of c-ErbB2 was observed in immatureperipheral neurons and adrenomedullary cells. In mature neurons, theimmunoreactivity was confined to the plasma membrane. These resultssuggest that the expression of c-ErbB2 in neuroblastoma reflects thephenotype of developing peripheral neurons. Postnatal human and mouseadrenomedullary cells lacked c-ErbB2 immunoreactivity, although apparentlynormal adrenomedullary cells adjacent to neuroblastoma tissues showedstrong cytoplasmic expression of c-ErbB2. It is not known whether thephenotypic conversion of adjacent adrenal medullary cells had occurredbefore or after tumor progression at present.

• Chang JY, Stafford DW, Straight DL
• The roles of factor VII's structural domains in tissue factor binding.
• Biochemistry. 1995; 34: 12227-32
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• Factor VIIa binds to tissue factor in one of the initial steps of bloodclotting. In order to determine the role of the various domains of thefactor VII molecule in this interaction, we made several chimeric factorVII proteins using recombinant DNA techniques. The molecules have factorIX domains substituted into factor VII and vice versa. The domainsexchanged were the 4-carboxyglutamic acid plus aromatic stack domain(gla), the first epidermal growth factor-like domain (Egf-1), the secondepidermal growth factor-like domain (Egf-2), and the catalytic domain.Using tissue factor-coated microtiter wells, competition binding studieswith 125I-labeled factor VIIa indicated factor VIIa's Kd is 4.2 nM.Employing the same microtiter plate assay, koff and kon were determinedand yielded a Kd of 1.5 nM. The results of competitive binding experimentsand activation assays using chimeric proteins indicated the interactionbetween factor VIIa and tissue factor involves direct contact betweentissue factor and factor VIIa's Egf-1 domain and catalytic domain. On theother hand, the gla and Egf-2 domains, while necessary for optimalbinding, may merely impart structure to the rest of the molecule. However,either one or both of the latter domains might contribute a relativelysmall amount of energy to direct binding.

• Han X, Kasahara N, Kan YW
• Ligand-directed retroviral targeting of human breast cancer cells.
• Proc Natl Acad Sci U S A. 1995; 92: 9747-51
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• We explored the feasibility of designing retroviral vectors that cantarget human breast cancer cells with characteristic receptors vialigand-receptor interaction. The ecotropic Moloney murine leukemia virusenvelope was modified by insertion of sequences encoding human heregulin.Ecotropic virus, which normally does not infect human cells, whenpseudotyped with the modified envelope protein now crosses species toinfect human breast cancer cell lines that overexpress HER-2 (humanepidermal growth factor receptor; also called ERBB2) and HER-4 (alsocalled ERBB4), while human breast cancer cell lines expressing low levelsof these receptors remain resistant to infection. Since about 20% of humanbreast cancers overexpress HER-2 and some of breast cancer cell linesoverexpress both HER-2 and HER-4, cell-specific targeting of retroviralvectors may provide a different approach for in vivo gene therapy of thistype of breast cancer.

• Kurachi S et al.
• Regulatory mechanism of human factor IX gene: protein binding at theLeyden-specific region.
• Biochemistry. 1995; 34: 14270-14270

• Schulze B, Mann K, Battistutta R, Wiedemann H, Timpl R
• Structural properties of recombinant domain III-3 of perlecan containing aglobular domain inserted into an epidermal-growth-factor-like motif.
• Eur J Biochem. 1995; 231: 551-6
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• A fragment comprising approximately domain III-3 of the basement membraneheparan sulfate proteoglycan perlecan was prepared in recombinant formfrom kidney cell clones. This fragment was predicted to contain acysteine-free globular domain inserted within anepidermal-growth-factor(EGF)-like motif (L4 module) and three additionalEGF-like motifs (LE module) without large inserts. This prediction wasconfirmed by electron microscopy, which demonstrated a globule joined to avery short rod-like segment. The globule was selectively destroyed bypepsin, which also demonstrated that its insertion into an EGF-like motifdid not prevent the typical disulfide connections known for such motifs.Yet the globule was more stable against neutral proteinases. The fragmentshowed a distinct content (55-60%) of alpha helical and beta structure anda partially reversible melting of the conformation in 6 M guanidine.Antibodies raised against recombinant domain III-3 demonstrated a completecross-reaction with tissue-derived perlecan but not with laminin and adistinct basement membrane staining of tissue sections. Most of theepitopes were lost after reduction and alkylation. Together the datademonstrated a proper folding of recombinant domain III-3 similar to itsstructure in the native protein and provided the first structural evidencefor a novel globular protein motif L4 based on an EGF-like scaffold.

• Bacus SS et al.
• Medullary carcinoma is associated with expression of intercellularadhesion molecule-1. Implication to its morphology and its clinicalbehavior.
• Am J Pathol. 1994; 145: 1337-48
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• The histological hallmarks for the diagnosis of medullary breast cancerare circumscription, syncytial architecture, diffuse inflammatoryinfiltrate, and highly atypical nuclei. The biological and prognosticimplication is a lower propensity to metastasize. We studied 19 medullarycarcinomas for expression of the intercellular adhesion molecule-1 andlymphocyte-function-associated antigen-1, Neu differentiation factor,tumor necrosis factor-alpha, and the expression of HER-2/neu, HER-4, andHER-3 receptors. Our study revealed that all of the 19 medullarycarcinomas expressed the intercellular adhesion molecule-1 and lymphocytefunction associated antigen. Eighteen of 19 cancers expressed Neudifferentiation factor and tumor necrosis factor-alpha. All medullarycancers expressed the HER-2/neu receptor, however, in the majority of thecases, the staining was confined to the cytoplasm. Only 4 of 12 cancersexpressed HER-4 and none of the eight medullary cancers tested expressedHER-3. By comparison, in a control group of infiltrating ductalcarcinomas, expression of intercellular adhesion molecule-1, lymphocytefunction associated antigen-1, and Neu differentiation factor was positivein about 25 to 30% of the cases, HER-4 was expressed in 75% and HER-3 in95% of the cases. Taken together, our observations suggest that theexpression of intercellular adhesion molecule-1, lymphocyte functionassociated antigen, Neu differentiation factor, and tumor necrosisfactor-alpha as factors that may affect the special morphology and thebiological behavior that characterizes medullary carcinomas.

• Shum L, Reeves SA, Kuo AC, Fromer ES, Derynck R
• Association of the transmembrane TGF-alpha precursor with a protein kinasecomplex.
• J Cell Biol. 1994; 125: 903-16
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• A variety of growth factors including transforming growth factor-alpha(TGF-alpha) are synthesized as transmembrane precursors. The shortcytoplasmic domain of the transmembrane TGF-alpha precursor lacks anyapparent motif associated with signal transduction. However, the sequenceconservation of this cytoplasmic domain and its abundance of cysteineresidues, reminiscent of the cytoplasmic domains of CD4 and CD8, suggest abiological function. In this study, we showed that transmembrane TGF-alphawas rapidly internalized after interaction with a specific antibody andthat this internalization was greatly decreased when the COOH-terminal 31amino acids were removed. Chemical cross-linking experiments revealed twoassociated proteins of 86 and 106 kD which coimmunoprecipitated with theTGF-alpha precursor. The association of p86 was dependent on the presenceof the COOH-terminal cytoplasmic 31 amino acids of the TGF-alphaprecursor, whereas p106 still remained associated when this segment wasdeleted. In addition, p106 was tyrosine-phosphorylated and exposed on thecell surface. The protein complex associated with transmembrane TGF-alphadisplayed kinase activities towards tyrosine, serine, and threonineresidues. These activities were not associated with transmembraneTGF-alpha when the COOH-terminal segment was truncated. The association ofa protein kinase complex with transmembrane TGF-alpha may provide thebasic elements for a "reverse" mode of signaling through the cytoplasmicdomain of this growth factor, which may lead to two-directionalcommunication during ligand-receptor interaction.

• Chegini N, Zhao Y, McLean FW
• Expression of messenger ribonucleic acid and presence of immunoreactiveproteins for epidermal growth factor (EGF), transforming growth factoralpha (TGF alpha) and EGF/TGF alpha receptors and 125I-EGF binding sitesin human fallopian tube.
• Biol Reprod. 1994; 50: 1049-58
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• Reverse transcription polymerase chain reaction (RT-PCR) revealed that theFallopian tubes express epidermal growth factor (EGF), transforming growthfactor (TGF alpha), and EGF receptor (EGF-R) mRNA. The RT-PCR product wasverified by restriction enzyme digestion analysis. Immunohistochemically,EGF, TGF alpha, and EGF-R were localized in Fallopian tubes by use ofspecific antibodies to human EGF, mature fragments of human TGF alpha, andmonoclonal antibodies to the extracellular binding domain of EGF-R. Thetubal epithelial cells were the primary site of immunoreactive EGF, TGFalpha, and EGF-R, which were present to a lesser extent in the stromalcells, smooth muscle cell layers, fibroblasts of serosal tissue, andarterial endothelial and smooth muscle cells. Using antibodies generatedagainst the amino and carboxy termini of TGF alpha precursor produced asimilar cellular distribution to that observed for mature TGF alpha. Theintensity of immunoreactive TGF alpha with these antibodies was similar tothat seen with EGF. The ciliated and nonciliated epithelial cells in theampullary and isthmus regions immunostained with similar intensity forEGF, TGF alpha, and EGF-R. The immunostaining for EGF, TGF alpha, andEGF-R was cycle-dependent, was considerably higher during lateproliferative and early-to-mid-secretory phases than during earlyproliferative and late secretory phases of the menstrual cycle, and wasreduced during the postmenopausal period. Specimens obtained 5-12 yr aftertubal ligation immunostained for EGF, TGF alpha, and EGF-R similarly tosections from unligated tubes taken during the same phase of the cycle.Quantitative autoradiography of 125I-EGF binding generated a patternsimilar to that of immunostaining for EGF-R binding. Net grain density/100microns 2 calculated for different cell types indicated that theepithelial cells had a significantly higher grain density than did othertubal cell types (p < 0.05) without the cycle dependency seen in theimmunohistochemical study. In summary, the results demonstrate that thehuman Fallopian tube expresses mRNA and contains immunoreactive proteinsfor EGF, TGF alpha, and EGF-R as well as binding sites for 125I-EGF. Thecycle dependency and lower immunostaining in postmenopausal tubes suggesta potential regulation of their expression by ovarian steroids. Theresults imply the importance of EGF/TGF alpha in a variety of tubalbiochemical and physiological functions and possibly early embryonicdevelopment.

• no M, Raab G, Lau K, Abraham JA, Klagsbrun M
• Purification and characterization of transmembrane forms ofheparin-binding EGF-like growth factor.
• J Biol Chem. 1994; 269: 31315-21
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• Heparin-binding epidermal growth factor-like growth factor (HB-EGF), whosecDNA has a predicted 208-codon open reading frame, is synthesized as amembrane-spanning precursor that is processed to release mature mitogenicproteins of approximately 73-87 amino acids in length. Previous work hasfocused on the structural and biological properties of secreted HB-EGF. Inthis study, human recombinant transmembrane HB-EGF, produced by expressionof HB-EGF1-208 cDNA in a baculovirus system, has been isolated, purified,and characterized structurally and biologically. Two isoforms oftransmembrane HB-EGF (HB-EGFTM) were purified from membrane fractions ofinfected insect cells by a combination of heparin affinity chromatographyand reversed-phase high performance liquid chromatography. The isoformdesignated as HB-EGFTM-1, a 21.5-kDa protein, yielded no N-terminalsequence, suggesting that it is N-terminally blocked. However,HB-EGFTM-II, a 24-kDa protein, was N-terminally sequenced and found to beinitiated at Asp63 in the 208-amino acid residue primary translationproduct. This N terminus is the same as that determined for a 18-kDaisoform of secreted HB-EGF purified from the conditioned medium of insectcells expressing HB-EGF1-149 cDNA and is also identical to the N terminusof the longest form of secreted HB-EGF initially purified from humanmacrophage-like U-937 cell conditioned medium. HB-EGFTM-II cross-reactedon a Western blot with an antibody directed against the 16 C-terminalamino acids of the cytoplasmic tail of HB-EGF, indicating that it containsa putative transmembrane domain. HB-EGFTM-II was bioactive and stimulatedthe proliferation of BALB/c 3T3 cells and smooth muscle cells and themotility of smooth muscle cells, albeit with approximately 10-25% of thespecific activity of secreted HB-EGF isoforms. We concluded thattransmembrane HB-EGF is bioactive when isolated, consistent with thepossibility of its functioning as a juxtacrine growth factor when stilltethered to the cell.

• Carraway KL 3rd et al.
• The erbB3 gene product is a receptor for heregulin.
• J Biol Chem. 1994; 269: 14303-6
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• ErbB3 is a member of the epidermal growth factor (EGF) receptor subfamilyof receptor tyrosine kinases and is believed to be a receptor for anunknown ligand. We have tested the possibility that heregulin, a growthfactor possessing an EGF-like domain, is a ligand for ErbB3. We have foundthat the iodinated recombinant EGF-like domain of heregulin-beta 1(125I-rHRG beta 1(177-244) bound specifically to insect cell-expressedbovine ErbB3 with a dissociation constant of 0.85 nM. Moreover, 125I-rHRGbeta 1(177-244) bound to NIH3T3 fibroblasts stably transfected with bovineerbB3 with a dissociation constant of 60 pM, but did not bind to parentalcells. 125I-rHRG beta 1(177-244) could be chemically cross-linked to a170-180 kDa protein in erbB3-transfected fibroblasts, and the cross-linkedproduct could be immunoprecipitated with antibodies specific for ErbB3.Finally, rHRG beta 1 stimulated the tyrosine phosphorylation of both ErbB3and endogenous p185erbB2/neu in transfectants but not in parental cells.We conclude that ErbB3 is a receptor for HRG and is capable of mediatingHRG-stimulated tyrosine phosphorylation of itself and p185erbB2/neu incells that express both receptors.

• Mroczkowski B, Reich M
• Identification of biologically active epidermal growth factor precursor inhuman fluids and secretions.
• Endocrinology. 1993; 132: 417-25
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• A biologically active form of the epidermal growth factor (EGF) precursorhas been detected in human fluids and secretions. The secreted proteinidentified in human urine and milk has an apparent molecular mass of160-170 kilodaltons and exhibits an affinity for the glycosaminoglycanheparin. More importantly, the secreted EGF precursor is capable ofactivating the intrinsic tyrosyl kinase activity of the EGF receptor. Ourresults demonstrate that the soluble form of the precursor is generatedfrom the membrane-anchored form by a processing step that takes place atthe cell surface and involves truncation of the cytoplasmic andtransmembrane domains of the intact EGF precursor. The findings supportthe hypothesis that the secreted 160- to 170-kilodalton EGF glycoproteinthat accumulates in urine and milk is proteolytically derived from theplasma membrane-spanning precursor expressed in the kidney and mammarygland.

• Weidner KM, Sachs M, Birchmeier W
• The Met receptor tyrosine kinase transduces motility, proliferation, andmorphogenic signals of scatter factor/hepatocyte growth factor inepithelial cells.
• J Cell Biol. 1993; 121: 145-54
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• Depending on the target cells and culture conditions, scatterfactor/hepatocyte growth factor (SF/HGF) mediates several distinctactivities, i.e., cell motility, proliferation, invasiveness, tubularmorphogenesis, angiogenesis, or cytotoxicity. A small isoform of SF/HGFencoded by a natural splice variant, which consists of the NH2-terminalhairpin structure and the first two kringle domains but not the proteasehomology region, induces cell motility but not mitogenesis. Two types ofSF/HGF receptors have recently been discovered in epithelial cells, thehigh affinity c-Met receptor tyrosine kinase, and low affinity/highcapacity binding sites, which are probably located on heparan sulfateproteoglycans. In the present study, we have addressed the questionwhether the various biological activities of SF/HGF are transduced intocells by a single type of receptor. We have here examined MDCK epithelialcells transfected with a hybrid cDNA encoding the ligand binding domain ofthe nerve growth factor (NGF) receptor and the membrane-spanning andtyrosine kinase domains of the Met receptor. We demonstrate that allbiological effects of SF/HGF upon epithelial cells such as the inductionof cell motility, proliferation, invasiveness, and tubular morphogenesiscan now be triggered by the addition of NGF. Thus, it is likely that allknown biological signals of SF/HGF are transduced through the receptortyrosine kinase encoded by the c-Met protooncogene.

• Gerwin BI
• Overexpression of the p185erB-2 tyrosine kinase growth factor receptor:control or chaos.
• Am J Respir Cell Mol Biol. 1992; 6: 357-8

• Saris CJ, Domen J, Berns A
• The pim-1 oncogene encodes two related protein-serine/threonine kinases byalternative initiation at AUG and CUG.
• EMBO J. 1991; 10: 655-64
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• The pim-1 gene is frequently found activated by proviral insertion inmurine T cell lymphomas. Overexpression of pim-1 in lymphoid cells bytransgenesis formally proved its oncogenic potential. The pim-1 cDNAsequence predicts that both murine and human pim-1 encode a 34 kd proteinwith homology to protein kinases. In this study, we show that the murinepim-1 gene encodes a 44 kd protein in addition to the predicted 34 kdprotein. The 44 kd protein is an amino-terminal extension of the 34 kdprotein and is synthesized by alternative translation initiation at anupstream CUG codon. Contrary to previous findings by others, we provideevidence that both murine and human pim-1 gene products areprotein-serine/threonine kinases. Murine 44 kd and 34 kd pim-1 proteinsexhibit comparable in vitro kinase activity and are both mainlycytoplasmic, but they differ in in vivo association state and half-life.

• Konstas AG, Marshall GE, Lee WR
• Immunogold localisation of laminin in normal and exfoliative iris.
• Br J Ophthalmol. 1990; 74: 450-7
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• Immunoelectron microscopic studies of exfoliative iris tissue (sevenspecimens) revealed the presence of laminin in the fibrillar component ofexfoliation material. The immunogold label was uniformly distributed onthe exfoliation fibres. Deposition of laminin labelled exfoliationmaterial in the dilator muscle was a noteworthy feature, as was anapparent depletion of laminin in the basement membranes of ostensiblyunaffected vessels. In control iris tissue (five enucleated eyes) lamininwas identified in the basement membrane round vascular contractile cells,but not beneath the endothelium.

• Isackson PJ, Dunbar JC, Bradshaw RA
• Role of glandular kallikreins as growth factor processing enzymes:structural and evolutionary considerations.
• J Cell Biochem. 1987; 33: 65-75
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• Hormones and growth factors are generally released from larger precursorsby limited proteolysis. The causative agents remain poorly defined withrespect to location and properties. One subset of proteases, the glandularkallikreins, have been implicated in a few cases, in part because of theirspecific association with mature forms of some hormones. However, limiteddistribution and low copy number in some species cast doubt on thishypothesis, and they may well play other physiological functions thatremain to be elucidated.

• Bell GI et al.
• Human epidermal growth factor precursor: cDNA sequence, expression invitro and gene organization.
• Nucleic Acids Res. 1986; 14: 8427-46
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• Complementary DNA clones encoding the human kidney epidermal growth factor(EGF) precursor have been isolated and sequenced. They predict thesequence of a 1,207 amino acid protein which contains EGF flanked bypolypeptide segments of 970 and 184 residues at its NH2- and COOH-termini,respectively. The structural organization of the human EGF precursor issimilar to that previously described for the mouse protein and there is66% identity between the two sequences. Transfection of COS-7 cells withthe human EGF precursor cDNA linked to the SV40 early promoter indicatethat it can be synthesized as a membrane protein with its NH2-terminusexternal to the cell surface. The human EGF precursor gene isapproximately 110 kilobase pairs and has 24 exons. Its exon-intronorganization revealed that various domains of the EGF precursor areencoded by individual exons. Moreover, 15 of the 24 exons encode proteinsegments that are homologous to sequences in other proteins. Exonduplication and shuffling appear to have played an important role indetermining the present structure of this protein.

• Gray A, Dull TJ, Ullrich A
• Nucleotide sequence of epidermal growth factor cDNA predicts a128,000-molecular weight protein precursor.
• Nature. 1983; 303: 722-5
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• Epidermal growth factor (EGF) has a profound effect on the differentiationof specific cells in vivo, and has been shown to be a potent mitogenicfactor for a variety of cultured cells, of both ectodermal and mesodermalorigin (see ref. 1 for review). This 53-amino acid polypeptide of knownsequence contains six cysteine residues, which are thought to form threeintrachain disulphide bonds. Urogastrone, a polypeptide bearinganti-gastric secretory activity isolated from human urine, which ispresumably synthesized in submandibular and Brunner's glands, sharesextensive sequence homology (70%) with EGF and may represent the human EGFequivalent. Here we present the sequence of a mouse EGF cDNA clone, whichsuggests that EGF is synthesized as a large protein precursor of 1,168amino acids. Our data indicate that the discrepancy between EGF levels inmale and female mouse submaxillary glands (MSGs) is due to different EGFmRNA levels in these tissues, and suggest that precursor EGF processingmay differ from that described previously for other polypeptide hormones.