Synthesis of heterotrimeric collagen peptides containing the alpha1beta1 integrin recognition site of collagen type IV.

Collagen type IV provides a biomechanically stable scaffold into which the other constituents of basement membranes are incorporated, but it also plays an important role in cell adhesion. This occurs with collagen type IV mainly via the alpha1beta1 integrin, and the proposed epitope involved in this type of collagen/integrin interaction corresponds to a non-sequential R/Xaa/D motif, where the arginine and aspartate residues are provided by the alpha2 and alpha1 chains of the collagen molecule, respectively. Since the stagger of the three alpha chains in native collagen type IV is still unknown and different alignments of the chains lead to different spatial epitopes, two heterotrimeric collagen peptides containing the natural 457-469 sequences of the cell adhesion site were synthesized in which the single chains were assembled via disulfide bonds into the two most plausible alpha1alpha2alpha1' and alpha2alpha1alpha1' registers. The differentiated triple-helical stabilities of the two heterotrimers suggest a significant structural role of the chain register in collagen, although the binding to alpha1beta1 integrin is apparently less affected as indicated by preliminary experiments.     

Structural basis of collagen recognition by integrin alpha2beta1

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

Human basement membrane collagen (type IV). The amino acid sequence of the alpha 2(IV) chain and its comparison with the alpha 1(IV) chain reveals deletions in the alpha 1(IV) chain

The cDNA and protein sequences of the N-terminal 60% of the alpha 2(IV) chain of human basement membrane collagen have been determined. By repeated primer extension with synthetic oligodeoxynucleotides and mRNA from either HT1080 cells or human placenta overlapping clones were obtained which cover 3414 bp. The derived protein sequence allows for the first time a comparison and alignment of both alpha chains of type IV collagen from the N terminus. This alignment reveals an additional 43 amino acid residues in the alpha 2(IV) chain as compared to the alpha 1(IV) chain. 21 of these additional residues form a disulfide-bridged loop within the triple helix which is unique among all known collagens.     

Identification of a tetrapeptide recognition sequence for the alpha 2 beta 1 integrin in collagen

The alpha 2 beta 1 integrin serves as either a specific cell surface receptor for collagen or as both a collagen and laminin receptor depending upon the cell type. Recently we established that the alpha 2 beta 1 integrin binds to a site within the alpha 1 (I)-CB3 fragment of type I collagen (Staatz, W. D., Walsh, J. J., Pexton, T., and Santoro, S. A. (1990) J. Biol. Chem. 265, 4778-4781). To define the alpha 2 beta 1 recognition sequence further we have prepared an overlapping set of synthetic peptides which completely spans the 148-amino acid alpha 1(I)-CB3 fragment and tested the peptides for ability to inhibit cell adhesion to collagen and laminin substrates. The minimal active recognition sequence defined by these experiments is a tetrapeptide of the sequence Asp-Gly-Glu-Ala (DGEA) corresponding to residues 435-438 of the type I collagen sequence. The DGEA-containing peptides effectively inhibited alpha 2 beta 1-mediated Mg2(+)-dependent adhesion of platelets, which use the alpha 2 beta 1 integrin as a collagen-specific receptor, to collagen but had no effect on alpha 5 beta 1-mediated platelet adhesion to fibronectin or alpha 6 beta 1-mediated platelet adhesion to laminin. In contrast, with T47D breast adenocarcinoma cells, which use alpha 2 beta 1 as a collagen/lamin receptor, adhesion to both collagen and laminin was inhibited by DGEA-containing peptides. Deletion of the alanine residue or substitution of alanine for either the glutamic or aspartic acid residues in DGEA-containing peptides resulted in marked loss of inhibitory activity. These results indicate that the amino acid sequence DGEA serves as a recognition site for the alpha 2 beta 1 integrin complex on platelets and other cells.     

Structure of the integrin alpha2beta1-binding collagen peptide

We have determined the 1.8 Å crystal structure of a triple helical integrin-binding collagen peptide (IBP) with sequence (Gly-Pro-Hyp)₂-Gly-Phe-Hyp-Gly-Glu-Arg-(Gly-Pro-Hyp)₃. The central GFOGER hexapeptide is recognised specifically by the integrins α2β1, α1β1, α10β1 and α11β1. These integrin/collagen interactions are implicated in a number of key physiological processes including cell adhesion, cell growth and differentiation, and pathological states such as thrombosis and tumour metastasis. Comparison of the IBP structure with the previously determined structure of an identical collagen peptide in complex with the integrin α2-I domain (IBP^c) allows the first detailed examination of collagen in a bound and an unbound state. The IBP structure shows a direct and a water-mediated electrostatic interaction between Glu and Arg side-chains from adjacent strands, but no intra-strand interactions. The interactions between IBP Glu and Arg side-chains are disrupted upon integrin binding. A comparison of IBP and IBP^c main-chain conformation reveals the flexible nature of the triple helix backbone in the imino-poor GFOGER region. This flexibility could be important to the integrin-collagen interaction and provides a possible explanation for the unique orientation of the three GFOGER strands observed in the integrin-IBP^c complex crystal structure.     

The receptor for the basement membrane glycoprotein entactin is the integrin alpha 3/beta 1.

Entactin is a glycoprotein found in basement membranes in complex with laminin, and purified entactin can promote the attachment and spreading of cells. We report here the isolation and identification of the plasma membrane receptor for entactin from PC-3 human prostate carcinoma cells which attach and spread on entactin. The receptor was isolated by affinity chromatography on mouse recombinant entactin-Sepharose of 125I surface-labeled octyl glucoside cell extracts. The receptor, which consisted of two polypeptides of relative molecular masses of 150 and 116 kDa, bound to the entactin-Sepharose matrix in the presence of CaCl2, MgCl2, and MnCl2, and was eluted with EDTA, but not with Arg-Gly-Asp-containing peptides. Utilizing anti-integrin antibodies, the heterodimeric receptor was identified as the integrin alpha 3 beta 1. Purified alpha 3 beta 1 bound to entactin Sepharose in a divalent cation-dependent manner and liposomes prepared with fractions eluted from the entactin-Sepharose matrix, as well as purified alpha 3 beta 1 also bound to entactin. Liposomes prepared with other integrins such as alpha 2 beta 1 did not bind to entactin. Antibody inhibition assays demonstrated that an anti-alpha 3 antibody (P1B5) inhibited the attachment of PC-3 cells to entactin whereas this antibody did not inhibit the attachment of these cells to laminin. Attachment to laminin was, however, blocked by anti-alpha 6 antibody (G0H3). These data demonstrate that the cell surface receptor for entactin on these prostate carcinoma cells is the integrin alpha 3 beta 1 and that these cells utilize alpha 6 beta 1 as the receptor for laminin.     

The alpha 2 beta 1 integrin cell surface collagen receptor binds to the alpha 1 (I)-CB3 peptide of collagen

We have previously shown that platelets adhere to collagen substrates via a Mg2(+)-dependent mechanism mediated by the surface glycoprotein Ia-IIa (human leukocyte very late activation protein 2, alpha 2 beta 1 integrin) complex. The adhesion is specific for collagen and is supported by collagen types I, II, III, IV, and VI. Several other members of the integrin family of adhesive protein receptors recognize discrete linear amino acid sequences within their adhesive glycoprotein ligands. Experiments with both intact platelets and with liposomes containing the purified receptor complex indicated that the alpha 2 beta 1 receptor recognized denatured type I collagen in a Mg2(+)-dependent manner. To further localize the binding site, the alpha 1 and alpha 2 chains of type I collagen were purified by gel filtration and ion exchange chromatography and tested as adhesive substrates. Both the alpha 1(I) and alpha 2(I) chains effectively supported Mg2(+)-dependent platelet adhesion. The purified alpha 1(I) collagen chain was then subjected to cleavage with cyanogen bromide, and the resultant peptides were separated by chromatography on carboxymethylcellulose. Only the alpha 1(I)-CB3 fragment supported Mg2(+)-dependent platelet adhesion. The monoclonal antibody P1H5 which recognizes an epitope on the alpha 2 subunit of the integrin receptor and which inhibits the adhesion of both intact platelets and liposomes bearing the purified receptor to collagen also inhibited platelet adhesion to the alpha 1(I)-CB3 fragment. These results indicate that the alpha 2 beta 1 receptor recognizes a sequence of amino acids present in the alpha 1(I)-CB3 fragment of type I collagen. An identical or similar sequence likely mediates binding of the receptor to other collagen polypeptides.     

Complete primary structure of the alpha 1-chain of human basement membrane (type IV) collagen

We have determined the primary structure of the alpha 1(IV)-chain of human type IV collagen by nucleotide sequencing of overlapping cDNA clones that were isolated from a human placental cDNA library. The present data provide the sequence of 295 amino acids not previously determined. Altogether, the alpha 1(IV)-chain contains 1642 amino acids and has a molecular mass of 157625 Da. There are 1413 residues in the collagenous domain and 229 amino acids in the carboxy-terminal globular domain. The human alpha 1(IV)-chain contains a total of 21 interruptions in the collagenous Gly-X-Y repeat sequence. These interruptions vary in length between two and eleven residues. The alpha 1(IV)-chain contains four cysteine residues in the triple-helical domain, four cysteines in the 15-residue long noncollagenous sequence at the amino-terminus and 12 cysteines in the carboxy-terminal NC-domain.     

Characterization of a type IV collagen major cell binding site with affinity to the alpha 1 beta 1 and the alpha 2 beta 1 integrins

The aim of this investigation was to identify the domains of type IV collagen participating in cell binding and the cell surface receptor involved. A major cell binding site was found in the trimeric cyanogen bromide-derived fragment CB3, located 100 nm away from the NH2 terminus of the molecule, in which the triple-helical conformation is stabilized by interchain disulfide bridges. Cell attachment assays with type IV collagen and CB3 revealed comparable cell binding activities. Antibodies against CB3 inhibited attachment on fragment CB3 completely and on type IV collagen to 80%. The ability to bind cells was strictly conformation dependent. Four trypsin derived fragments of CB3 allowed a closer investigation of the binding site. The smallest, fully active triple-helical fragment was (150)3-amino acid residues long. It contained segments of 27 and 37 residues, respectively, at the NH2 and COOH terminus, which proved to be essential for cell binding. By affinity chromatography on Sepharose-immobilized CB3, two receptor molecules of the integrin family, alpha 1 beta 1 and alpha 2 beta 1, were isolated. Their subunits were identified by sequencing the NH2 termini or by immunoblotting. The availability of fragment CB3 will allow for a more in-depth study of the molecular interaction of a short, well defined triple-helical ligand with collagen receptors alpha 1 beta 1 and alpha 2 beta 1.     

Affinity of integrin alpha 1 beta 1 from liver sinusoidal membranes for type IV collagen.

Hepatic sinusoidal membranes isolated from adult rats were extracted with detergent and fractionated on a wheat germ agglutinin affinity column. Bound glycoproteins were eluted with N-acetyl glucosamine and chromatographed on a type IV collagen affinity column. Recovery of the bound fraction by EDTA and analysis by SDS-PAGE revealed two glycoproteins with apparent molecular weights of 180,000 and 117,000. These were identified immunologically by Western blotting as the alpha and beta subunits of integrin alpha 1 beta 1.     

Role of ADMIDAS cation-binding site in ligand recognition by integrin alpha 5 beta 1

Integrin-ligand interactions are regulated in a complex manner by divalent cations, and multiple cation-binding sites are found in both alpha and beta integrin subunits. A key cation-binding site that lies in the beta subunit A-domain is known as the metal-ion dependent adhesion site (MIDAS). Recent x-ray crystal structures of integrin alpha V beta 3 have identified a novel cation binding site in this domain, known as the ADMIDAS (adjacent to MIDAS). The role of this novel site in ligand recognition has yet to be elucidated. Using the interaction between alpha 5 beta 1 and fibronectin as a model system, we show that mutation of residues that form the ADMIDAS site inhibits ligand binding but this effect can be partially rescued by the use of activating monoclonal antibodies. The ADMIDAS mutants had decreased expression of activation epitopes recognized by 12G10, 15/7, and HUTS-4, suggesting that the ADMIDAS is important for stabilizing the active conformation of the integrin. Consistent with this suggestion, the ADMIDAS mutations markedly increased the dissociation rate of the integrin-fibronectin complex. Mutation of the ADMIDAS residues also reduced the allosteric inhibition of Mn2+-supported ligand binding by Ca2+, suggesting that the ADMIDAS is a Ca2+-binding site involved in the inhibition of Mn2+-supported ligand binding. Mutations of the ADMIDAS site also perturbed transduction of a conformational change from the MIDAS through the C-terminal helix region of the beta A domain to the underlying hybrid domain, implying an important role for this site in receptor signaling.     

Collagen XVI harbors an integrin alpha1 beta1 recognition site in its C-terminal domains

Collagen XVI is integrated tissue-dependently into distinct fibrillar aggregates, such as D-banded cartilage fibrils and fibrillin-1-containing microfibrils. In skin, the distribution of collagen XVI overlaps that of the collagen-binding integrins alpha1 beta1 and alpha2 beta1. Basal layer keratinocytes express integrin alpha2 beta1, whereas integrin alpha1 beta1 occurs in smooth muscle cells surrounding blood vessels, in hair follicles, and on adipocytes. Cells bearing the integrins alpha1 beta1 and alpha2 beta1 attach and spread on recombinant collagen XVI. Furthermore, collagen XVI induces the recruitment of these integrins into focal adhesion plaques, a principal step in integrin signaling. Of potential physiological relevance, these integrin-collagen XVI interactions may connect cells with specialized fibrils, thus contributing to the organization of fibrillar and cellular components within connective tissues. In cell-free binding assays, collagen XVI is more avidly bound by alpha1 beta1 integrin than by alpha2 beta1 integrin. Both integrins interact with collagen XVI via the A domain of their alpha subunits. A tryptic collagen XVI fragment comprising the collagenous domains 1-3 is recognized by alpha1 beta1 integrin. Electron microscopy of complexes of alpha1 beta1 integrin with this tryptic collagen XVI fragment or with full-length collagen XVI revealed a unique alpha1 beta1 integrin-binding site within collagen XVI located close to its C-terminal end.     

Rapid synthesis of a register-specific heterotrimeric type I collagen helix encompassing the integrin alpha2beta1 binding site

We report a rapid method to synthesize cystine cross-linked heterotrimeric collagenous peptides. They can be engineered to favour one particular axial alignment of the strands, called the register of the helix. Here, the sequence of the constituent peptides contains 18 residues of "guest" collagen type I sequence flanked by N and C-terminal (Gly-Pro-Pro)5 "host" modules which ensure helicity. Further C-terminal residues include appropriately spaced cysteine residues and alanine to provide the necessary flexibility for helix formation. The cross-linking reaction and subsequent separation protocols have been designed for any inserted collagen sequence that does not contain a cysteine residue. Mass spectrometry and ion-exchange chromatography allow us to distinguish between different disulphide-bonded species and to monitor the formation of side-products. Starting peptide can be recovered simply from the reaction mixture by reduction and separation. Yields are typically 30%, working on a 10 mg scale. 15N-1H NMR and platelet adhesion studies show that the peptide heterotrimers presented here can reshuffle to cover all three axial registers. Less flexible spacers between the disulphide linkages and the helix will restrict each heterotrimer to one register only.     

The spatial orientation of the essential amino acid residues arginine and aspartate within the alpha1beta1 integrin recognition site of collagen IV has been resolved using fluorescence resonance energy transfer

The interaction of collagen IV with cells is mediated mainly by the integrin alpha1beta1. The recognition site has been located to a segment of the triple-helical domain 100 nm away from the N terminus of the collagen molecule. The three essential amino acid residues of the alpha1beta1 binding site, arginine alpha2(IV)461 and the two aspartate residues alpha1(IV)461, are all located on different chains. Since the spatial array of the three residues depends on the stagger of the chains within the triple helix, the stagger has been elucidated using fluorescence resonance energy transfer with phenylalanine alpha1(IV)473 and tryptophan alpha2(IV)479 as the fluorescent donor/acceptor pair. The distance R between phenylalanine and tryptophan was determined by analysis of the energy transfer efficiency, E, and the orientation factor, kappa(2). In parallel, distance R and orientation factor, kappa(2 )were also calculated from the coordinates of the triple helix. Comparison of the calculated and empirically determined values unequivocally showed the stagger to be alpha1'alpha1alpha2. This arrangement of the three alpha chains describes the conformation of the alpha1beta1 integrin recognition site, that is the distinct orientation of the side-chains of the essential residues aspartate and arginine in respect to the helix axis.     

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

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

Polarized expression of integrin receptors (alpha 6 beta 4, alpha 2 beta 1, alpha 3 beta 1, and alpha v beta 5) and their relationship with the cytoskeleton and basement membrane matrix in cultured human keratinocytes

In human keratinocytes cultured in conditions which allow differentiation and stratification and are suitable to reconstitute a fully functional epidermis, alpha 6 beta 4 and two members of the beta 1 integrin family (alpha 2 beta 1 and alpha 3 beta 1) were respectively polarized to the basal and lateral domains of the plasmamembrane both in growing colonies and in the reconstituted epidermis. Conversely, the alpha v integrin subunit, presumably in association with beta 5, was expressed at the basal surface in growing and migrating but not in stationary keratinocytes. The integrin alpha 6 beta 4: (a) was organized in typical patches which often showed a "leopard skin" pattern where spots corresponded to microfilament-free areas; (b) was not associated with focal contacts containing vinculin and talin but rather corresponded to relatively removed contact areas of the basal membrane as shown by interference reflection microscopy; and (c) was coherent to patches of laminin secreted and deposited underneath the ventral membrane of individual cells. The two beta 1 integrins (alpha 2 beta 1 and alpha 3 beta 1), both endowed with laminin receptor properties, were not associated with focal adhesions under experimental conditions allowing full epidermal maturation but matched the lateral position of vinculin (but not talin), cingulin, and desmoplakin, all makers of intercellular junctions. Often thin strips of laminin were observed in between the lateral aspects of individual basal keratinocytes. The integrin complex alpha v beta 5 had a topography similar to that of talin- and vinculin-containing focal adhesions mostly in the peripheral cells of expanding keratinocyte colonies and in coincidence with fibronectin strands. The discrete topography of beta 1 and beta 4 integrins has a functional role in the maintenance of the state of aggregation of cultured keratinocytes since lateral aggregation was impaired by antibodies to beta 1 whereas antibodies to beta 4 prevented cell-matrix adhesion (De Luca, M., R. N. Tamura, S. Kajiji, S. Bondanza, P. Rossino, R. Cancedda, P. C. Marchisio, and V. Quaranta. Proc. Natl. Acad. Sci. USA. 87:6888-6892). Moreover, the surface polarization of integrins followed attachment and depended both on the presence of Ca2+ in the medium and on the integrity of the cytoskeleton. We conclude that our in vitro functional tests and structural data suggest a correlation between the pattern of integrin expression on defined plasmamembrane domains and the mechanism of epidermal assembly.     

Collagen receptors integrin alpha2beta1 and discoidin domain receptor 1 regulate maturation of the glomerular basement membrane and loss of integrin alpha2beta1 delays kidney fibrosis in COL4A3 knockout mice

Maturation of the glomerular basement membrane (GBM) is essential for maintaining the integrity of the renal filtration barrier. Impaired maturation causes proteinuria and renal fibrosis in the type IV collagen disease Alport syndrome. This study evaluates the role of collagen receptors in maturation of the GBM, matrix accumulation and renal fibrosis by using mice deficient for discoidin domain receptor 1 (DDR1), integrin subunit α2 (ITGA2), and type IV collagen α3 (COL4A3). Loss of both collagen receptors DDR1 and integrin α2β1 delays maturation of the GBM: due to a porous GBM filtration barrier high molecular weight proteinuria that more than doubles between day 60 and day 100. Thereafter, maturation of the GBM causes proteinuria to drop down to one tenth until day 200. Proteinuria and the porous GBM cause accumulation of glomerular and tubulointerstitial matrix, which both decrease significantly after GBM-maturation until day 250. In parallel, in a disease with impaired GBM-maturation such as Alport syndrome, loss of integrin α2β1 positively delays renal fibrosis: COL4A3^−/−/ITGA2^−/− double knockouts exhibited reduced proteinuria and urea nitrogen compared to COL4A3^−/−/ITGA2^+/− and COL4A3^−/−/ITGA2^+/+ mice. The double knockouts lived 20% longer and showed less glomerular and tubulointerstitial extracellular matrix deposition than the COL4A3^−/− Alport mice with normal integrin α2β1 expression. Electron microscopy illustrated improvements in the glomerular basement membrane structure. MMP2, MMP9, MMP12 and TIMP1 were expressed at significantly higher levels (compared to wild-type mice) in COL4A3^−/−/ITGA2^+/+ Alport mice, but not in COL4A3^+/+/ITGA2^−/− mice. In conclusion, the collagen receptors DDR1 and integrin α2β1 contribute to regulate GBM-maturation and to control matrix accumulation. As demonstrated in the type IV collagen disease Alport syndrome, glomerular cell–matrix interactions via collagen receptors play an important role in the progression of renal fibrosis.     

CD44/chondroitin sulfate proteoglycan and alpha 2 beta 1 integrin mediate human melanoma cell migration on type IV collagen and invasion of basement membranes.

Tumor cell invasion of basement membranes (BM) represents one of the critical steps in the metastatic process. Tumor cell recognition of individual BM matrix components may involve individual cell adhesion receptors, such as integrins or cell surface proteoglycans, or may involve a coordinate action of both types of receptors. In this study, we have focused on the identification of a cell surface CD44/chondroitin sulfate proteoglycan (CSPG) and alpha 2 beta 1 integrin on human melanoma cells that are both directly involved in the in vitro invasion of reconstituted BM via a type IV collagen-dependent mechanism. Interfering with cell surface expression of human melanoma CSPG with either p-nitro-phenyl-beta-D-xylopyranoside treatment or anti-CD44 monoclonal antibody (mAb) preincubation (mAb) preincubation inhibits melanoma cell invasion through reconstituted BM. These treatments also strongly inhibit melanoma cell migration on type IV collagen, however, they are ineffective at inhibiting cell adhesion to type IV collagen. Purified melanoma cell surface CD44/CSPG, or purified chondroitin sulfate, bind to type IV collagen affinity columns, consistent with a role for CD44/CSPG-type IV collagen interactions in mediating tumor cell invasion. In contrast, melanoma cell migration on laminin (LM) does not involve CD44/CSPG, nor does CD44/CSPG bind to LM, suggesting that CD44/CSPG-type IV collagen interactions are specific in nature. Additionally, anti-alpha 2 and anti-beta 1 integrin mAbs are capable of blocking melanoma cell invasion of reconstituted BM. Both of these anti-integrin mAbs inhibit melanoma cell adhesion and migration on type IV collagen, whereas only anti-beta 1 mAb inhibits cell adhesion to LM. Collectively, these results indicate that melanoma cell adhesion to type IV collagen is an important consideration in invasion of reconstituted BM in vitro, and suggest that CD44/CSPG and alpha 2 beta 1 integrin may collaborate to promote human melanoma cell adhesion, migration, and invasion in vivo.     

A novel binding site in collagen type III for integrins alpha1beta1 and alpha2beta1

Previously identified high affinity integrin-binding motifs in collagens, GFOGER and GLOGER, are not present in type III collagen. Here, we first characterized the binding of recombinant I domains from integrins alpha(1) and alpha(2) (alpha(1)I and alpha(2)I) to fibrillar collagen types I-III and showed that each I domain bound to the three types of collagens with similar affinities. Using rotary shadowing followed by electron microscopy, we identified a high affinity binding region in human type III collagen recognized by alpha(1)I and alpha(2)I. Examination of the region revealed the presence of two sequences that contain the critical GER motif, GROGER and GAOGER. Collagen-like peptides containing these two motifs were synthesized, and their triple helical nature was confirmed by circular dichroism spectroscopy. Experiments show that the GROGER-containing peptide was able to bind both alpha(1)I and alpha(2)I with high affinity and effectively inhibit the binding of alpha(1)I and alpha(2)I to type III and I collagens, whereas the GAOGER-containing peptide was considerably less effective. Furthermore, the GROGER-containing peptide supported adhesion of human lung fibroblast cells when coated on a culture dish. Thus, we have identified a novel high affinity binding sequence for the collagen-binding integrin I domains.