Identification of integrin collagen receptors on human melanoma cells

Integrin receptors may mediate the adhesion of cells to a number of extracellular matrix components. We found that the attachment of human melanoma cells to collagen types I and IV was blocked by antibodies to the integrin beta 1 subunit but not by peptides containing the Arg-Gly-Asp sequence. Ligand affinity chromatography was used to search for integrin-related receptors which mediate adhesion to native collagens. Detergent extracts of surface 125I-iodinated melanoma cells were chromatographed on type I or IV collagen-Sepharose columns. Bound material was eluted and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. EDTA, but not Arg-Gly-Asp peptides, eluted a mixture of two integrin-related heterodimeric complexes. Each complex contained the integrin beta 1 chain with Mr of 110,000 and a distinct alpha chain with Mr of either 200,000 or 150,000. Immunoprecipitation with specific monoclonal antibodies identified the complexes as very late activation antigen (VLA)-1 (alpha 1 beta 1) and VLA-2 (alpha 2 beta 1), respectively. The binding of these receptors to collagen appeared to be specific because they failed to be retained on fibronectin- or laminin-Sepharose columns. Immunofluorescent staining of cells on collagen substrates with antibodies to VLA-1 and VLA-2 localized these complexes in vinculin-positive adhesion plaques. In contrast, the receptor complexes were not detected in adhesion plaques of cells attached to fibronectin- or laminin-coated substrates. These results indicate that melanoma cells express at least two different integrin-related collagen-binding receptor complexes that appear to mediate cell adhesion to collagen.     

The binding of heparin to type IV collagen: domain specificity with identification of peptide sequences from the alpha 1(IV) and alpha 2(IV) which preferentially bind heparin

Three distinctive heparin-binding sites were observed in type IV collagen by the use of rotary shadowing: in the NC1 domain and at distances 100 and 300 nm from the NC1 domain. Scatchard analysis indicated different affinities for these sites. Electron microscopic analysis of heparin-type IV collagen interaction with increasing salt concentrations showed the different affinities to be NC1 greater than 100 nm greater than 300 nm. The NC1 domain bound specifically to chondroitin/dermatan sulfate side chains as well. This binding was observed at the electron microscope and in solid-phase binding assays (where chondroitin sulfate could compete for the binding of [3H]heparin to NC1-coated substrata). The triple helix-rich, rod-like domain of type IV collagen did not bind to chondroitin/dermatan sulfate side chains. In solid-phase binding assays only heparin could compete for the binding of [3H]heparin to this domain. In order to more precisely map potential heparin-binding sites in type IV collagen, we chemically synthesized 17 arginine- and lysine-containing peptides from the alpha 1(IV) and alpha 2(IV) chains. Three peptides from the known sequence of the alpha 1(IV) and alpha 2(IV) chains were shown to specifically bind heparin: peptide Hep-I (TAGSCLRKFSTM), from the alpha 1(NC1) chain, peptide Hep-II (LAGSCLARFSTM), a peptide corresponding to the same sequence in peptide Hep-I from the alpha 2 (NC1) chain, and peptide Hep-III (GEFYFDLRLKGDK) which contained an interruption of the triple helical sequence of the alpha 1(IV) chain at about 300 nm from the NC1 domain, were demonstrated to bind heparin in solid-phase binding assays and compete for the binding of [3H]heparin to type IV collagen-coated substrata. Therefore, each of these peptides may represent a potential heparin-binding site in type IV collagen. The mapping of the binding of heparin or related structures, such as heparan sulfate proteoglycan, to specific sequences of type IV collagen could help the understanding of several structural and functional properties of this basement membrane protein as well as interactions with other basement membrane and/or cell surface-associated macromolecules.     

Identification of a heparin binding site and the biological activities of the laminin alpha1 chain carboxy-terminal globular domain

The carboxy-terminal globular domain (G-domain) of the laminin alpha1 chain has been shown to promote heparin binding, cell adhesion, and neurite outgrowth. In this study, we defined the potential sequences originating from the G-domain of laminin alpha1 chain which possess these functional activities. A series of peptides were synthesized from the G-domain, termed LG peptides (LG-1 to LG-6) and were tested for their various biological activities. In the direct [3H] heparin binding assays, LG-6 (residues 2,335-2,348: KDFLSIELVRGRVK) mediated high levels of [3H]heparin binding, and this peptide also directly promoted cell adhesion and spreading, including B16F10, M2, HT1080, and PC12 cells. The peptide LG-6 also promoted the neurite outgrowth of PC12 cells, mouse granule cells, and chick telencephalic cells. An anti-peptide LG-6 antibody inhibited laminin-1 and peptide LG-6-mediated cell adhesion and neurite outgrowth. Furthermore, an anti-integrin alpha2 antibody also inhibited the cell adhesion activity. These results suggest that peptide LG-6 plays a functional role as a heparin binding site in the G-domain of the laminin alpha1 chain, and this sequence was thus concluded to play a crucial role in regulating cell adhesion and spreading and neurite out-growth which is related to integrin alpha2.     

Identification and characterization of a tumor cell receptor for CSVTCG, a thrombospondin adhesive domain

We have previously shown that peptides derived from the thrombospondin sequence, CSVTCG, promoted tumor cell adhesion. To further investigate this observation, the CSVTCG-tumor cell adhesion receptor from A549 human lung adenocarcinoma cells was isolated and characterized. A single protein peak was isolated by CSVTCG affinity chromatography which also analyzed as a single peak by anion exchange chromatography. The purified protein had a pI of 4.7 and analyzed on SDS-gels as a single band of M(r) = 50,000 under nonreducing conditions and as two protein bands of M(r) = 50,000, and 60,000 under reducing conditions. Purified CSVTCG binding protein (CBP) bound either CSVTCG- or TSP- Sepharose but showed little interaction with either VCTGSC- or BSA- Sepharose. CBP was cell surface exposed. CSVTCG derivatized with [125I] Bolton-Hunter reagent was taken up by cells in a dose-dependent manner and the cell association was inhibited with a monospecific polyclonal anti-CBP antibody. Examination of the cell proteins crosslinked to labeled CSVTCG by SDS-gel electrophoresis revealed one band that comigrated with purified CPB. Using an in vitro binding assay, purified CBP bound mannose, galactose, and glucosamine-specific lectins. CBP bound TSP saturably and reversibly. The binding was Ca+2/Mg+2 ion dependent and inhibited with fluid phase TSP and anti-CBP. Little or no binding was observed on BSA, fibronectin, GRGES, and GRGDS. Heparin, but not lactose, inhibited binding. Anti-CBP IgG and anti-CSVTCG peptide IgG inhibited A549 cell spreading and adhesion on TSP but not on fibronectin and laminin. These results indicate that CBP and the CSVTCG peptide domain of TSP can mediate TSP-promoted tumor cell adhesion.     

一种特异性识别小细胞肺癌细胞的小分子肽

应用“一个珠子一个化合物”的组合化学肽库技术,筛选得到特异性识别小细胞肺癌细胞(DMS53)的小分子肽.初次筛选共得到32个与DMS53阳性结合的珠子,经氨基酸序列分析后发现,含有cNGRXXXc或cXNGRXXc肽链结构的序列共有10个.再次合成三种有代表性的小分子肽,发现cFNGRQQc与DMS53的结合率明显高于其他小分子肽.选择cFNGRQQc作进一步的细胞特异性研究,发现cFNGRQQc与DMS53的粘附特异性明显高于其他细胞系,对cFNGRQQc的结构分析显示,-NGR-及六肽长度对小分子肽与DMS53细胞的粘附非常重要.用抗整合素、E-cadherin、NCAM及ICAM的抗体或多肽阻断小分子肽与DMS53细胞表面的相应受体结合,未见明显的阻断效应.小分子肽与DMS53细胞表面的结合位点有待于进一步证实.     

The chain register in heterotrimeric collagen peptides affects triple helix stability and folding kinetics

Collagen type IV is a highly specialized form of collagen found only in basement membranes, where it provides mechanical stability and structural integrity to tissues and organs, and binding sites for cell adhesion. In its ubiquitous form, collagen type IV consists of two alpha1 chains and one alpha2 chain, whose internal alignment within the triple helix seems to exert a strong influence on the binding affinity to alpha1beta1 integrin receptor. This has been assessed recently using two synthetic collagen peptides that contain the cell adhesion epitope of collagen type IV and are assembled into the most plausible alpha1alpha2alpha1' and alpha2alpha1alpha1' registers. In the present study, the effects of the chain register on the stability of the triple helix and the folding kinetics of these collagen peptides were investigated by CD spectroscopy and microcalorimetry. The results revealed a multi-domain structural organization for both trimers, with an unexpected strong effect of the chain alignment on the conformational stability. Molecular dynamics simulations served to rationalize more properly the experimental results.     

Perpetuation of immunological memory through common MHC-I binding modes of peptidomimic and antigenic peptides

Understanding the molecular mechanisms of immunological memory assumes importance in vaccine design. We had earlier hypothesized a mechanism for the maintenance of immunological memory through the operation of a network of idiotypic and anti-idiotypic antibodies (Ab2). Peptides derived from an internal image carrying anti-idiotypic antibody are hypothesized to facilitate the perpetuation of antigen specific T cell memory through similarity in peptide-MHC binding as that of the antigenic peptide. In the present work, the existence of such peptidomimics of the antigen in the Ab2 variable region and their similarity of MHC-I binding was examined by bioinformatics approaches. The analysis employing three known viral antigens and one tumor-associated antigen shows that peptidomimics from Ab2 variable regions have structurally similar MHC-I binding patterns as compared to antigenic peptides, indicating a structural basis for memory perpetuation.     

Non-natural cell surface receptors: synthetic peptides capped with N-cholesterylglycine efficiently deliver proteins into Mammalian cells

Protein toxins such as shiga toxin and cholera toxin penetrate into cells by binding small molecule-based cell surface receptors localized to cholesterol and sphingolipid-rich lipid raft subdomains of cellular plasma membranes. Molecular recognition between these toxins and their receptors triggers endocytic protein uptake through endogenous membrane trafficking pathways. We report herein the synthesis of functionally related non-natural cell surface receptors comprising peptides capped with N-cholesterylglycine as the plasma membrane anchor. The peptide moieties of these receptors were based on high-affinity epitopes of anti-hemaglutinin antibodies (anti-HA), anti-Flag antibodies, and a moderate-affinity Strep Tag II peptide ligand of the streptavidin protein from Streptomyces avidini. These non-natural receptors were directly loaded into plasma membranes of Jurkat lymphocytes to display peptides from lipid rafts on the cell surface. Molecular recognition between these receptors and added cognate anti-HA, anti-Flag, or streptavidin proteins resulted in rapid clathrin-mediated endocytosis; fluorescent target proteins were completely internalized within 4-12 h of protein addition. Analysis of protein uptake by epifluorescence microscopy and flow cytometry revealed intracellular fluorescence enhancements of 100-fold to 200-fold (10 microM non-natural receptor) with typically >99% efficiency. This method enabled intracellular delivery of a functional Escherichia coli beta-galactosidase enzyme conjugated to Protein A from Staphylococcus aureus. We termed this novel delivery strategy "synthetic receptor targeting", which is an efficient method to enhance macromolecular uptake by decorating mammalian cells with chemically defined synthetic receptors that access the molecular machinery controlling the organization of cellular plasma membranes.     

Recognition properties of peptides hydropathically complementary to residues 356-375 of the c-raf protein

Two peptides with hydropathic complementarity to residues 356-375 of the c-raf protein were synthesized to determine if they recognize the raf-(356-375) peptide as well as the entire protein. One peptide was deduced from the complementary mRNA for the raf protein corresponding to residues 356-375, whereas the other was deduced solely from the amino acid sequence of the 20-mer segment using a computer program able to generate peptide sequences with hydropathic complementarity to a given sequence. Specific binding of both peptides to the raf 20-mer segment was demonstrated when either the raf 20-mer peptide or the complementary peptides were immobilized on a column. Binding affinities were in the millimolar-micromolar range. Identical binding properties were observed with peptides synthesized with either all D- or all L-amino acids, suggesting a lack of conformational dependence. Binding was also unaffected by the presence of 8 M urea or detergents, was dependent on solvent characteristics of pH and ionic strength, and was abolished by the presence of competing peptides in the eluting buffer. Recognition between raf complementary peptides was accompanied by spectral changes in the far and near UV region, as monitored by circular dichroism. Proteolytic degradation was retarded by the binding of these peptides. Once immobilized on a column, these peptides proved useful for the isolation by affinity chromatography of a recombinant c-raf protein from an Escherichia coli crude cell extract.     

ICAM-2 peptides mediate lymphocyte adhesion by binding to CD11a/CD18 and CD49d/CD29 integrins.

Three fifteen-amino-acid polypeptides designated peptides 1, 2 and 3 were synthesised as likely candidates for mimicking the role of ICAM-2 as a ligand. The ability of each peptide to bind lymphoid cells was tested. Peptide 2 largely mediated cell attachment of unstimulated cells and this binding was only marginally increased by stimulating the cells with phorbol dibutyrate (P(Bu)2). Peptide 3 mediated minimal spontaneous cell attachment, but this binding was significantly enhanced following P(Bu)2 stimulation. Peptide 1 had no effect on cell attachment with or without stimulation. The cell attachment to peptide 2 was both temperature- and cation-dependent. Studies using specific monoclonal antibodies showed that with unstimulated cells, anti-VLA-4 alpha(CD49d) or beta chain (CD29) antibodies (KD4-13 and 4B4) and anti-CD18 (1B4) each partially inhibited the cell binding. Monoclonal antibodies against CD54 (ICAM-1; 84H10 or LB2), MHC class 1 (W6/32) and control mouse IgG had no effect. When anti-CD29 and anti-CD18 monoclonal antibodies were used concurrently, there was almost complete inhibition of the cell attachment. These observations indicated that cell adhesion via ICAM-2 is mediated: (i) predominantly by peptide 2 in unstimulated and P(Bu)2-stimulated cells, and also, to some extent, by peptide 3 in P(Bu)2-stimulated cells and (ii) by binding to both CD11/CD18 and CD49d/CD29 integrins.     

Alterations in cell surface carbohydrate composition of a human colon carcinoma cell line affect adhesion to extracellular matrix components.

The adhesion of HT29 human colon adenocarcinoma cells to different extracellular matrix components was studied. While treatment of the cells with sialidase had no detectable effect on binding to laminin and fibronectin, attachment to collagen IV was decreased. However, additional removal of beta-(1-4)-bound galactose led to significantly reduced binding to all of the substrates, including fibronectin and laminin. Tunicamycin treatment, monitored by lectin-induced aggregation, drastically diminished cell adhesion to laminin and fibronectin, whereas cell binding to collagen IV was not affected. Arg-Gly-Asp (RGD)-related peptides were used to study the adhesion to collagen IV. The results show that a serine-containing RGD-related peptide GRGDSP has virtually no effect on colon carcinoma cell adhesion to type IV collagen. In contrast, when serine was substituted for threonine (GRGDTP) adhesion to collagen IV was strongly inhibited. After incubation of sialidase-treated cells with the threonine-containing peptide adhesion was almost totally blocked. These results demonstrate the existence of both RGD-dependent and carbohydrate-based mechanisms for metastatic human HT29 cell binding to collagen IV.     

Coordinate role for cell surface chondroitin sulfate proteoglycan and alpha 4 beta 1 integrin in mediating melanoma cell adhesion to fibronectin

Cellular recognition and adhesion to the extracellular matrix (ECM) has a complex molecular basis, involving both integrins and cell surface proteoglycans (PG). The current studies have used specific inhibitors of chondroitin sulfate proteoglycan (CSPG) synthesis along with anti-alpha 4 integrin subunit monoclonal antibodies to demonstrate that human melanoma cell adhesion to an A-chain derived, 33-kD carboxyl-terminal heparin binding fragment of human plasma fibronectin (FN) involves both cell surface CSPG and alpha 4 beta 1 integrin. A direct role for cell surface CSPG in mediating melanoma cell adhesion to this FN fragment was demonstrated by the identification of a cationic synthetic peptide, termed FN-C/H-III, within the fragment. FN-C/H-III is located close to the amino terminal end of the fragment, representing residues #1721-1736 of intact FN. FN-C/H-III binds CSPG directly, can inhibit CSPG binding to the fragment, and promotes melanoma cell adhesion by a CSPG-dependent, alpha 4 beta 1 integrin-independent mechanism. A scrambled version of FN-C/H-III does not inhibit CSPG binding or cell adhesion to the fragment or to FN-C/H-III, indicating that the primary sequence of FN-C/H-III is important for its biological properties. Previous studies have identified three other synthetic peptides from within this 33-kD FN fragment that promote cell adhesion by an arginyl-glycyl-aspartic acid (RGD) independent mechanism. Two of these synthetic peptides (FN-C/H-I and FN-C/H-II) bind heparin and promote cell adhesion, implicating cell surface PG in mediating cellular recognition of these two peptides. Additionally, a third synthetic peptide, CS1, is located in close proximity to FN-C/H-I and FN-C/H-II and it promotes cell adhesion by an alpha 4 beta 1 integrin-dependent mechanism. In contrast to FN-C/H-III, cellular recognition of these three peptides involved contributions from both CSPG and alpha 4 integrin subunits. Of particular importance are observations demonstrating that CS1-mediated melanoma cell adhesion could be inhibited by interfering with CSPG synthesis or expression. Since CS1 does not bind CSPG, the results suggest that CSPG may modify the function and/or activity of alpha 4 beta 1 integrin on the surface of human melanoma cells. Together, these results support a model in which the PG and integrin binding sites within the 33-kD fragment may act in concert to focus these two cell adhesion receptors into close proximity on the cell surface, thereby influencing initial cellular recognition events that contribute to melanoma cell adhesion on this fragment.     

Design of peptides with high affinities for heparin and endothelial cell proteoglycans

Proteoglycan-binding peptides were designed based on consensus sequences in heparin-binding proteins: XBBXBX and XBBBXXBX, where X and B are hydropathic and basic residues, respectively. Initial peptide constructs included (AKKARA)(n) and (ARKKAAKA)(n) (n = 1-6). Affinity coelectrophoresis revealed that low M(r) peptides (600-1,300) had no affinities for low M(r) heparin, but higher M(r) peptides (2,000-3,500) exhibited significant affinities (K(d) congruent with 50-150 nM), which increased with peptide M(r). Affinity was strongest when sequence arrays were contiguous and alanines and arginines occupied hydropathic and basic positions, but inclusion of prolines was disruptive. A peptide including a single consensus sequence of the serglycin proteoglycan core protein bound heparin strongly (K(d) congruent with 200 nM), likely owing to dimerization through cysteine-cysteine linkages. Circular dichroism showed that high affinity heparin-binding peptides converted from a charged coil to an alpha-helix upon heparin addition, whereas weak heparin-binding peptides did not. Higher M(r) peptides exhibited high affinities for total endothelial cell proteoglycans (K(d) congruent with 300 nM), and approximately 4-fold weaker affinities for their free glycosaminoglycan chains. Thus, peptides including concatamers of heparin-binding consensus sequences may exhibit strong affinities for heparin and proteoglycans. Such peptides may be applicable in promoting cell-substratum adhesion or in the design of drugs targeted to proteoglycan-containing cell surfaces and extracellular matrices.     

Peptide and small molecule microarray for high throughput cell adhesion and functional assays

A novel class of chemical microchips consisting of glass microscope slides was prepared for the covalent attachment of small molecule ligands and peptides through site-specific oxime bond or thiazolidine ring ligation reaction. Commercially available microscope slides were thoroughly cleaned and derivatized with (3-aminopropyl)triethoxysilane (APTES). The amino slides were then converted to glyoxylyl derivatives via two different routes: (1) coupling of Fmoc-Ser followed by deprotection and oxidation, or (2) coupling with protected glyoxylic acid and final deprotection with HCl. Biotin or peptide ligands derivatized at the carboxyl terminus with a 4,7,10-trioxa-1,13-tridecanediamine succinimic acid linker and an amino-oxy group or a 1,2-amino-thiol group (e.g., cysteine with a free N(alpha)-amino group) were printed onto these slides using a DNA microarray spotter. After chemical ligation, the microarray of immobilized ligands was analyzed with three different biological assays: (1) protein-binding assay with fluorescence detection, (2) functional phosphorylation assay using [gamma(33)P]-ATP and specific protein kinase to label peptide substrate spots, and (3) adhesion assay with intact cells. In the cell adhesion assay, not only can we determine the binding specificity of the peptide against different cell lines, we can also determine functional cell signaling of attached cells using immunofluorescence techniques in situ on the microchip. This chemical microchip system enables us to rapidly analyze the functional properties of numerous ligands that we have identified from the "one-bead one-compound" combinatorial library method.     

A novel peptide sequence in perlecan domain IV supports cell adhesion, spreading and FAK activation.

Perlecan/HSPG2 is a large, multi-domain, multifunctional heparan sulfate proteoglycan with a wide tissue distribution. With the exception of its unique domain I, each of perlecan's other four domains shares sequence similarity to other protein families including low density lipoprotein (LDL) receptor, laminin alpha chain, neural cell adhesion molecule (NCAM), immunoglobulin (Ig) superfamily members, and epidermal growth factor (EGF). Previous studies demonstrated that glycosaminoglycan-bearing perlecan domain I supports early chondrogenesis and growth factor delivery. Other sites in the core protein interact with other matrix molecules and support cell adhesion, although the peptide sequences involved remain unidentified. To identify novel functional motifs within perlecan, we used a bioinformatics approach to predict regions likely to be on the exterior of the folded protein. Unique hydrophilic sequences of about 18 amino acids were selected for testing in cell adhesion assays. A novel peptide sequence (TWSKVGGHLRPGIVQSG) from an immunoglobulin (Ig) repeat in domain IV supported rapid cell adhesion, spreading and focal adhesion kinase (FAK) activation when compared to other peptides, a randomly scrambled sequence of the domain IV peptide or a negative control protein. MG-63 human osteosarcoma cells, epithelial cells and multipotent C(3)H10T1/2 cells, but not bone marrow cells, rapidly, i.e., within 30 min, formed focal adhesions and assembled an actin cytoskeleton on domain IV peptide. Cell lines differentially adhered to the domain IV peptide, suggesting adhesion is receptor specific. Adhesion was divalent cation independent and heparin sensitive, a finding that may explain some previously poorly understood observations obtained with intact perlecan. Collectively, these studies demonstrate the feasibility of using bioinformatics-based strategies to identify novel functional motifs in matrix proteins such as perlecan.     

Conjugated platinum(IV)-peptide complexes for targeting angiogenic tumor vasculature

The integrins alpha vbeta3 and alpha vbeta5 and the membrane-spanning surface protein aminopeptidase N (APN) are highly expressed in tumor-induced angiogenesis, making them attractive targets for therapeutic intervention. Both integrins and APN recognize a broad range of peptides containing RGD (Arg-Gly-Asp) and NGR (Asn-Gly-Arg) motifs, respectively. Here, we describe the design, synthesis, and characterization of a series of mono- and difunctionalized platinum(IV) complexes in which a conjugated peptide motif, containing RGD, (CRGDC)c, (RGDfK)c, or NGR, is appended as a "tumor-homing device" to target tumor endothelial cells selectively over healthy cells. Platinum(IV)-peptide complexes with nonspecific amino acids or peptide moieties were prepared as controls. Concentration-response curves of these compounds were evaluated against primary proliferating endothelial cells and tumor cell lines and compared to those of cisplatin, a well-described platinum-based chemotherapeutic agent. The Pt(IV)-RGD conjugates were highly and specifically cytotoxic to cell lines containing alpha vbeta3 and alpha vbeta5, approaching the activity of cisplatin. The Pt(IV)-NGR complexes were less active than Pt(IV)-RGD-containing compounds but more active than nonspecific Pt-peptide controls. Integrin alpha vbeta3 mediated, at least in part, the anti-proliferative effect of a Pt(IV)-RGD conjugate, as demonstrated by a decreased inhibitory response when endothelial cells were either (1) incubated with an excess of alpha vbeta3/alpha vbeta5-specific RGD pentapeptides or (2) transfected with RNAi for beta 3, but not beta 1, integrins. These results suggest a rational approach to improved chemotherapy with Pt(IV)-peptide conjugates by selective drug delivery to the tumor compartment.     

Regulation of tumor cell chemotaxis by type IV collagen is mediated by a Ca(2+)-dependent mechanism requiring CD47 and the integrin alpha(V)beta(3)

Studies from our laboratories demonstrated that synthetic peptides from the non-collagenous (NC-1) domain of the alpha3 (IV) chain of type IV collagen (COL IV) enhanced tumor cell adhesion (Han, J., Ohno, N., Monboisse, J. C., Pasco, S., Borel, J. P., and Kefalides, N. A. (1997) J. Biol. Chem. 272, 20395-20401). We have isolated the receptors for the alpha3(IV)185-203 peptide from melanoma and prostate tumor cells and identified them as CD47/integrin-associated protein and the integrin alpha(V)beta(3) (Shahan, T. A., Ziaie, Z., Pasco, S., Fawzi, A., Bellon, G., Monboisse, J. C., and Kefalides, N. A. (1999) Cancer Res. 59, 4584-4590). In the present study we have examined the effect of CD47 and the integrin alpha(V)beta(3) on in vitro tumor cell chemotaxis and Ca(2+)(i) modulation in response to COL IV, from the anterior lens capsule (ALC-COL IV) and peptides from its NC-1 domain. COL IV as well as the alpha3(IV) peptide promoted tumor cell chemotaxis with an immediate increase in intracellular [Ca(2+)]. Treating tumor cells with CD47 and integrin alpha(V)beta(3)-reactive antibodies reduced chemotaxis as well as the rise in [Ca(2+)](i) in response to ALC-COL IV or the alpha3(IV)185-203 peptide but not to Engelbreth-Holm-Swarm-COL IV or fibronectin. The alpha3(IV)185-203 synthetic peptide stimulated an increase in calcium from intracellular stores exclusively, whereas ALC-COL IV, Engelbreth-Holm-Swarm-COL IV, and fibronectin stimulated Ca(2+) flux from both internal and external stores. Furthermore, treatment of the cells with Ca(2+) chelator bis-(O-aminophenoxyl)ethane-N,N,N',N'-tetraaceticacid- acetomethoxy ester inhibited chemotaxis toward both ALC-COL IV and the alpha3(IV)185-203 peptide. These data indicate that CD47 and integrin alpha(V)beta(3) regulate tumor cell chemotaxis in response to COL IV and the alpha3(IV)185-203 peptide through a Ca(2+)-dependent mechanism.     

The PPFLMLLKGSTR motif in globular domain 3 of the human laminin-5 alpha3 chain is crucial for integrin alpha3beta1 binding and cell adhesion

Laminin-5 regulates various cellular functions, including cell adhesion, spreading, and motility. Here, we expressed the five human laminin alpha3 chain globular (LG) domains as monomeric, soluble fusion proteins, and examined their biological functions and signaling. Recombinant LG3 (rLG3) protein, unlike rLG1, rLG2, rLG4, and rLG5, played roles in cell adhesion, spreading, and integrin alpha3beta1 binding. More significantly, we identified a novel motif (PPFLMLLKGSTR) in the LG3 domain that is crucial for these responses. Studies with the synthetic peptides delineated the PPFLMLLKGSTR peptide within LG3 domain as a major site for both integrin alpha3beta1 binding and cell adhesion. Substitution mutation experiments suggest that the Arg residue is important for these activities. rLG3 protein- and PPFLMLLKGSTR peptide-induced keratinocyte adhesion triggered cell signaling through FAK phosphorylation at tyrosine-397 and -577. To our knowledge, this is the first report demonstrating that the PPFLMLLKGSTR peptide within the LG3 domain is a novel motif that is capable of supporting integrin alpha3beta1-dependent cell adhesion and spreading.