Inhibitory effects of MLDG-containing heterodimeric disintegrins reveal distinct structural requirements for interaction of the integrin alpha 9beta 1 with VCAM-1, tenascin-C, and osteopontin

The integrin alpha9beta1 is expressed on epithelial cells, smooth muscle cells, skeletal muscle, and neutrophils and recognizes at least three distinct ligands: vascular cell adhesion molecule 1 (VCAM-1), tenascin-C, and osteopontin. The alpha9 subunit is structurally similar to the integrin alpha4 subunit, and alpha9beta1 and alpha4beta1 both recognize VCAM-1 as a ligand. We therefore examined whether the disintegrin EC3, which we have recently shown specifically inhibits the binding of alpha4 integrins to ligands, would also be a functional inhibitor of alpha9beta1. EC3 and a novel heterodimeric disintegrin that we identified, EC6, both were potent inhibitors of alpha9beta1-mediated adhesion to VCAM-1 and of neutrophil migration across tumor necrosis factor-activated endothelial cells. A peptide containing a novel MLDG motif shared by both of these disintegrins also inhibited alpha9beta1- and alpha4beta1-mediated adhesion to VCAM-1. Surprisingly though, concentrations of EC3 that completely inhibited adhesion of alpha9-transfected cells to VCAM-1 had little or no effect on adhesion to either of the other alpha9beta1 ligands, osteopontin and tenascin-C. Furthermore, peptides AEIDGIEL and SVVYGLR, which we have previously shown inhibit binding of alpha9beta1-expressing cells to tenascin-C and osteopontin, respectively, had no effect on adhesion to VCAM-1. These data suggest that there are structurally distinct requirements for interactions of the alpha9beta1 integrin with VCAM-1 and the extracellular matrix ligands osteopontin and tenascin-C.     

Comparative assessment of the ligand and metal ion binding properties of integrins alpha9beta1 and alpha4beta1

Integrins alpha9beta1 and alpha4beta1 form a distinct structural class, but while alpha4beta1 has been subjected to extensive study, alpha9beta1 remains poorly characterized. We have used the small molecule N-(benzenesulfonyl)-(L)-prolyl-(L)-O-(1-pyrrolidinylcarbonyl)tyrosine (3) to investigate the biochemical properties of alpha9beta1 and directly compare these properties with those of alpha4beta1. Compound 3 has a high affinity for both integrins with K(D) values of < or =3 and 180 pM for alpha9beta1 in 1 mM Mn2+ (activating) and 1 mM Ca2+ and 1 mM Mg2+ (nonactivating) conditions and < or =5 and 730 pM for alpha4beta1 under the corresponding conditions. Ca2+ treatment promoted the binding of 3 to both integrins (EC50 = 30 microM Ca2+ in both cases). Compound 3 binding to both integrins was also stimulated by the addition of the activating monoclonal antibody TS2/16. These findings indicate that the mechanisms by which metal ions and TS2/16 regulate ligand binding to alpha9beta1 and alpha4beta1 are similar. The binding of 3 to both integrins induced the mAb 9EG7 LIBS epitope, a property consistent with occupancy of the receptor's ligand binding site by 3. But whereas EGTA treatment inhibited the binding of 9EG7 to alpha4beta1, it stimulated the binding of 9EG7 to alpha9beta1. The 9EG7 and TS2/16 effects point to contributions of the beta1-chains on binding. Cross-linking data revealed that the integrin alpha-chains are also involved in binding the small molecule, as stable linkages were observed on both the alpha9 chain of alpha9beta1 and the alpha4 chain of alpha4beta1. Extensive structure-activity analyses with natural and synthetic ligands indicate distinct features of the ligand binding pockets. Most notable was the estimated >1000-fold difference in the affinity of the integrins for VCAM-1, which binds alpha4beta1with an apparent K(D) of 10 nM and alpha9beta1 with an apparent K(D) of >10 microM. Differences were also seen in the binding of alpha9beta1 and alpha4beta1 to osteopontin. Compound 3 competed effectively for the binding of VCAM-1 and osteopontin to both integrins. While these studies show many similarities in the biochemical properties of alpha9beta1 and alpha4beta1, they identify important differences in their structure and function that can be exploited in the design of selective alpha9beta1 and alpha4beta1 inhibitors.     

Vipera lebetina venom contains two disintegrins inhibiting laminin-binding beta1 integrins

To explain the myotoxic effects of snake venoms, we searched for inhibitors of alpha7beta1 integrin, the major laminin-binding integrin in skeletal muscle. We discovered two inhibitors in the venom of Vipera lebetina. One of them, lebein-1 (known as lebein), has already been proposed to be a disintegrin because of its RGD-containing primary sequence. The other, lebein-2, is a novel protein that also interacts firmly with alpha3beta1, alpha6beta1, and alpha7beta1 integrins, but not with the collagen-binding alpha1beta1 and alpha2beta1 integrins. Ligand binding of laminin-recognizing beta1 integrins was efficiently blocked by both lebein-1 and lebein-2. In cell attachment assays, lebein-1 and lebein-2 inhibited myoblast attachment not only to laminin, but also to fibronectin. However, neither lebein-1 nor lebein-2 interacted with alpha7beta1 integrin in an RGD-dependent manner, similar to the interaction of the laminin with alpha7beta1 integrin. Identical divalent cation dependence of integrin binding to laminin and to either of the two inhibitors and their mutually exclusive binding suggest that both lebein-1 and lebein-2 interact with the ligand-binding site of laminin-binding beta1 integrins by mimicking the yet unknown integrin-binding structure of laminins. Like lebein-1, lebein-2 is a soluble heterodimeric disintegrin of low molecular mass. Together with membrane-bound ADAM-2 and ADAM-9, the two inhibitors seem to form a small group of disintegrins that can bind to laminin-binding beta1 integrins. Because of their inhibitory capability both in vitro and in vivo, lebein-1 and lebein-2 may be valuable tools in influencing laminin-induced, integrin-mediated cell functions such as cell anchorage, migration, and mechanical force transduction on laminin-rich basement membranes.     

Structural requirements of echistatin for the recognition of alpha(v)beta(3) and alpha(5)beta(1) integrins

There are key differences between the amino acid residues of the RGD loops and the C termini of echistatin, a potent antagonist of alpha(IIb)beta(3), alpha(v)beta(3) and alpha(5)beta(1), and eristostatin, a similar disintegrin selectively inhibiting alpha(IIb)beta(3). In order to identify echistatin motifs required for selective recognition of alpha(v)beta(3) and alpha(5)beta(1) integrins, we expressed recombinant echistatin, eristostatin, and 15 hybrid molecules. We tested them for their ability to inhibit adhesion of different cell lines to fibronectin and von Willebrand factor and to express ligand-induced binding site epitope. The results showed that Asp(27) and Met(28) support recognition of both alpha(v)beta(3) and alpha(5)beta(1). Replacement of Met(28) with Asn completely abolished echistatin's ability to recognize each of the integrins, while replacement of Met(28) with Leu selectively decreased echistatin's ability to recognize alpha(5)beta(1) only. Eristostatin in which C-terminal WNG sequence was substituted with HKGPAT exhibited new activity with alpha(5)beta(1), which was 10-20-fold higher than that of wild type eristostatin. A hypothesis is proposed that the C terminus of echistatin interacts with separate sites on beta(1) and beta(3) integrin molecules.     

Structural requirements for interaction of sodium channel beta 1 subunits with ankyrin

Sodium channel beta subunits modulate channel kinetic properties and cell surface expression levels and function as cell adhesion molecules. beta 1 and beta 2 participate in homophilic cell adhesion resulting in ankyrin recruitment to cell contact sites. We hypothesized that a tyrosine residue in the cytoplasmic domain of beta 1 may be important for ankyrin recruitment and tested our hypothesis using beta 1 mutants replacing Tyr(181) with alanine (beta 1Y181A), phenylalanine (beta 1Y181F), or glutamate (beta 1Y181E), or a truncated construct deleting all residues beyond Tyr(181) (beta 1L182(STOP)). Ankyrin recruitment was observed in beta 1L182(STOP), showing that residues Ile(166)-Tyr(181) contain the major ankyrin recruiting activity of beta 1. Ankyrin recruitment was abolished in beta 1Y181E, suggesting that tyrosine phosphorylation of beta 1 may inhibit beta 1-ankyrin interactions. Ankyrin(G) and beta 1 associate in rat brain membranes and in transfected cells expressing beta 1 and ankyrin(G) in the absence of sodium channel alpha subunits. beta 1 subunits are recognized by anti-phosphotyrosine antibodies following treatment of these cell lines with fibroblast growth factor. beta 1 and ankryin(G) association is not detectable in cells following treatment with fibroblast growth factor. Ankyrin(G) and beta 1Y181E do not associate even in the absence of fibroblast growth factor treatment. beta 1 subunit-mediated cell adhesion and ankyrin recruitment may contribute to sodium channel placement at nodes of Ranvier. The phosphorylation state of beta 1Y181 may be a critical regulatory step in these developmental processes.     

Distinct structural requirements for binding of the integrins alphavbeta6, alphavbeta3, alphavbeta5, alpha5beta1 and alpha9beta1 to osteopontin.

The extracellular matrix protein, osteopontin, is a ligand for several members of the integrin family, including alpha5beta1, alphavbeta3, alphavbeta5 and alpha9beta1. Osteopontin is a substrate for a number of extracellular proteases, including thrombin and the metalloproteases MMP-3 and MMP-7, which cleave osteopontin at sites close to or within the mapped integrin binding sites. Using affinity chromatography and cell adhesion assays, we now identify the integrin alphavbeta6 as an additional osteopontin receptor. Utilizing a series of recombinant forms of osteopontin, we compared the structural requirements for alphavbeta6 binding with those for the 4 other osteopontin-binding integrins. Like alpha5beta1, alphavbeta3 and alphavbeta5 (but not alpha9beta1), alphavbeta6 binds to the RGD site in osteopontin, since RGD peptide or mutation of this site to RAA completely inhibits alphavbeta6-mediated cell adhesion. For both alpha9beta1 and alpha5beta1, the N-terminal fragment generated by thrombin cleavage is a much better ligand than full length osteopontin, whereas thrombin-cleavage does not appear to be required for optimal adhesion to alphavbeta3, alphavbeta5 or alphavbeta6. A recombinant fragment predicted to be generated by MMP cleavage no longer supported alpha5beta1 or alpha9beta1-mediated adhesion, but adhesion mediated by alphavbeta5 or alphavbeta6 was unaffected. Finally, adhesion of alphavbeta5 or alphavbeta6 was inhibited by mutation of two aspartic acid residues upstream of the RGD site, whereas adhesion mediated by alphavbeta3, alpha5beta1 or alpha9beta1 was unaffected by these mutations. These results suggest that the hierarchy of integrin interactions with osteopontin can undergo complex regulation at least in part through the action of extracellular proteases.     

Circulating integrins: alpha 5 beta 1, alpha 6 beta 4 and Mac-1, but not alpha 3 beta 1, alpha 4 beta 1 or LFA-1.

The alpha 5 beta 1, alpha 6 beta 4 and Mac-1 integrins all participate in the endocytotic cycle. By contrast, alpha 3 beta 1, alpha 4 beta 1 and LFA-1 do so much more slowly, or not at all, in the cell lines examined. This indicates that the alpha-chains appear to determine whether an integrin cycles or not, and that alpha 5 beta 1, alpha 6 beta 4 and Mac-1 can be brought to the leading edge of a moving cell by endocytosis and recycling.     

Ligand-binding specificities of laminin-binding integrins: a comprehensive survey of laminin-integrin interactions using recombinant alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4 integrins.

The interactions of cells with basement membranes are primarily mediated via the engagement of laminins by a group of integrin family proteins, including integrins alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4. To explore the ligand-binding specificities of these laminin-binding integrins, we produced these integrins, including two alpha7beta1 splice variants (alpha7X1beta1 and alpha7X2beta1), as soluble recombinant proteins and determined their binding specificities and affinities toward a panel of purified laminin isoforms containing distinct alpha chains. Among the five laminin-binding integrins investigated, alpha3beta1 and alpha6beta4 exhibited a clear specificity for laminin-332 (alpha3beta3gamma2) and laminin-511 (alpha5beta1gamma1)/521 (alpha5beta2gamma1), while integrin alpha6beta1 showed a broad specificity, binding to all laminin isoforms with a preference for laminin-111 (alpha1beta1gamma1), laminin-332 and laminin-511/521. The two alpha7beta1 variants were distinct from alpha3beta1, alpha6beta1 and alpha6beta4 in that they did not bind to laminin-332. alpha7X1beta1 bound to all laminins, except laminin-332, with a preference for laminin-211 (alpha2beta1gamma1)/221 (alpha2beta2gamma1) and laminin-511/521, while alpha7X2beta1 bound preferentially to laminin-111 and laminin-211/221. Laminin-511/521 was the most preferred ligand for all the laminin-binding integrins, except for alpha7X2beta1, whereas laminin-411 was the poorest ligand, capable of binding to alpha6beta1 and alpha7X1beta1 with only modest binding affinities. These comprehensive analyses of the interactions between laminin-binding integrins and a panel of laminins clearly demonstrate that the isoforms of both integrins and laminins differ in their binding specificities and affinities, and provide a molecular basis for better understanding of the adhesive interactions of cells with basement membranes of defined laminin compositions.     

Contribution of alpha2beta1, alpha3beta1, alpha6beta4 integrins and 67 kDa laminin receptor to the interaction of epidermoid carcinoma A-431 cells with laminin-2/4

Laminin-2/4 is the major laminin isoform of normal muscle and nerve tissues and plays an important role in tumor invasion and metastasis. Despite the fact that laminin-2/4 has been found in the skin basement membrane, insufficient evidence is available on the effect of laminin-2/4 on the behavior of both normal and transformed skin cells. A comparison of the contribution of alpha2beta1, alpha3beta1, alpha6beta4 integrins and 67 kDa laminin receptor on the surface of the human epidermoid carcinoma cell, A-431, to interaction with laminin-2/4 was carried out. The cell interaction with extracellular matrix component is a multistage process. We employed new methods for studying different stages of the interaction of A-431 cells with laminin-2/4. We demonstrated that integrins alpha2beta1, alpha3beta1, alpha6beta4 and 67 kDa laminin receptor are involved in the interaction of A-431 cells with laminin-2/4. We found that contribution of the same receptors to different stages of the interaction with laminin can be different. alpha2beta1 integrins are involved in EGF-induced A-431 cells' migration on laminin-2/4. We demonstrated the cooperation between alpha2beta1 and alpha3beta1 integrins during adhesion and spreading of A-431 cells on laminin-2/4-coated substrate. These results provide information about laminin-2/4 receptors and their contribution to different stages of the interaction with cells.     

Expression and functional importance of collagen-binding integrins,alpha 1 beta 1 and alpha 2 beta 1, on virus-activated T cells

Adhesive interactions are crucial to cell migration into inflammatory sites. Using murine lymphocytic choriomeningitis virus as an Ag model system, we have investigated expression and function of collagen-binding integrins, alpha(1)beta(1) and alpha(2)beta(1), on activated and memory T cells. Using this system and MHC tetramers to define Ag-specific T cells, we demonstrate that contrary to being VLAs, expression of alpha(1)beta(1) and alpha(2)beta(1) can be rapidly induced on acutely activated T cells, that expression of alpha(1)beta(1) remains elevated on memory T cells, and that expression of alpha(1)beta(1) parallels that of viral-specific effector CD8(+) T cells (defined by tetramer and IFN-gamma staining). In an adoptive transfer model, mAb-mediated blockade of these integrins on activated effector and memory T cells inhibited Ag-specific delayed-type hypersensitivity responses; similar decreased responses were seen upon transfer of alpha(1)-deficient activated/memory T cells. Thus, expression of alpha(1)beta(1) and alpha(2)beta(1) integrins on activated T cells is directly functionally important for generation of inflammatory responses within tissues. Finally, the inhibitory effect of alpha(1)beta(1) blockade on the delayed-type hypersensitivity response could be bypassed by direct injection of Ag-specific T cells to inflammatory sites, demonstrating for the first time in vivo that collagen-binding integrins are involved in leukocyte migration into tissues.     

Structural requirements for adhesion of soluble recombinant murine vascular cell adhesion molecule-1 to alpha 4 beta 1

This study describes the identification of seven amino acid residues of the vascular cell adhesion molecule (VCAM-1) that influence binding to the alpha 4 beta 1 receptor. Using recombinant murine VCAM-1-IgG, which is bound by both mouse (WEHI 231) and human (Ramos) lymphoid cells, two approaches demonstrated the crucial role of the first two NH2-terminal Ig-like domains in binding: (a) blocking monoclonal anti-mouse VCAM-1 antibodies bound to only truncation variants that included the first two domains; (b) site-direct mutagenesis of the first NH2-terminal domain showed that alanine substitution of the amino acid residues R36, D40, K46, S54, N65, T72, and E81 partially or completely reduced adherence by human and/or mouse cells. Of these D40, when mutated to A, N, or K (but not E), showed complete abrogation of adherence by mouse and human cells, as well as inability to bind blocking anti-murine VCAM- 1 antibody MVCAM.A429, while not inducing gross structural perturbations in VCAM-1. By molecular modeling, the D40 residue was located on a beta turn connecting two beta strands defined as C and D. The residues R36, K46, S54, N65, T72, and E81, which perturb cell adherence and caused small changes to gross structure, are conformationally near or adjacent to D40. Although these residues, identified as crucial for cell adhesion, are all located in domain 1, it is evident that there is a structural requirement for domains 1 and 2 to be intact so that cell adhesive function can occur.     

Structural elements of the osteopontin SVVYGLR motif important for the interaction with alpha(4) integrins

The osteopontin SVVYGLR motif binds the integrins alpha(4)beta(1) and alpha(9)beta(1). We show that alpha(4)beta(7) also interacts with this motif and that an SVVYGLR-OH peptide antagonises the alpha(4)beta(7) MAdCAM interaction. The important elements of this motif required to bind alpha(4)beta(1) and alpha(4)beta(7) were probed using a series of mutated peptides based around SVVYGLR. Leu167 is important for the interaction with alpha(4) integrins, as is the C-terminal carboxylic acid of Arg168 exposed by thrombin cleavage. The importance of the acidic group means that SVVYGLR has structural elements in common with other alpha(4) integrin-binding motifs and suggests why thrombin cleavage activates this motif.     

Tissue transglutaminase, coagulation factor XIII, and the pro-polypeptide of von Willebrand factor are all ligands for the integrins alpha 9beta 1 and alpha 4beta 1

We previously reported that MOLT-3 human lymphocyte-like leukemia cells adhere to tissue-type transglutaminase (tTG) through the integrin alpha(4)beta(1). We now report that G-361 human melanoma cells also adhere to tTG, although they do not express alpha(4)beta(1). G-361 cells utilize two additional integrins, alpha(9)beta(1) and alpha(5)beta(1) to adhere to tTG. Furthermore, blood coagulation factor XIII (FXIII), another member of the transglutaminase family that is highly homologous to tTG, and propolypeptide of von Willebrand factor (pp-vWF) also promoted cell adhesion through alpha(9)beta(1) or alpha(4)beta(1) in G-361 or MOLT-3 cells, respectively. In the case of pp-vWF, alpha(9)beta(1) and alpha(4)beta(1) both bind to the same site, comprised of 15 amino acid residues and designated T2-15. Moreover, SW480 human colon cancer cells stably transfected to express alpha(9)beta(1), but not mock transfectants, adhered to tTG, FXIII, pp-vWF, and T2-15/bovine serum albumin conjugate. These data identify tTG, FXIII, and pp-vWF as shared ligands for the integrins alpha(9)beta(1) and alpha(4)beta(1). This report is the first to unambiguously show that these two integrins share the same cell adhesion site within one protein and provides strong support for classifying alpha(9)beta(1-) and alpha(4)-integrins as functionally related members of an integrin subfamily.     

Analysis of the roles of RGD-binding integrins, alpha(4)/alpha(9) integrins, alpha(6) integrins, and CD9 in the interaction of the fertilin beta (ADAM2) disintegrin domain with the mouse egg membrane

Fertilin beta (also known as ADAM2), a mammalian sperm protein that mediates gamete cell adhesion during fertilization, is a member of the ADAM protein family whose members have disintegrin domains with homology to integrin ligands found in snake venoms. Fertilin beta utilizes an ECD sequence within its disintegrin domain to interact with the egg plasma membrane; the Asp is especially critical. Based on what is known about different integrin subfamilies and their ligands, we sought to characterize fertilin beta binding sites on mouse eggs, focusing on integrin subfamilies that recognize short peptide sequences that include an Asp residue: the alpha(5)/alpha(8)/alpha(v)/alpha(IIb) or RGD-binding subfamily (alpha(5)beta(1), alpha(8)beta(1), alpha(V)beta(1), alpha(V)beta(3), alpha(V)beta(5), alpha(V)beta(6), alpha(V)beta(8), and alpha(IIb)beta(3)) and the alpha(4)/alpha(9) subfamily (alpha(4)beta(1), alpha(9)beta(1), and alpha(4)beta(7)). We tested peptide sequences known to perturb interactions mediated by these integrins in two different assays for fertilin beta binding. Peptides with the sequence MLDG, which perturb alpha(4)/alpha(9) integrin-mediated interactions, significantly inhibit fertilin beta binding to eggs, which suggests a role for a member of this integrin subfamily as a fertilin beta receptor. RGD peptides, which perturb alpha(5)/alpha(8)/alpha(v)/alpha(IIb) integrin-mediated interactions, have partial inhibitory activity. The anti-alpha(6) antibody GoH3 has little or no inhibitory activity. An antibody to the integrin-associated tetraspanin protein CD9 inhibits the binding of a multivalent presentation of fertilin beta (immobilized on beads) but not soluble fertilin beta, which we speculate has implications for the role of CD9 in the strengthening of fertilin beta-mediated cell adhesion but not in initial ligand binding.     

Cross-talk between integrins alpha1beta1 and alpha2beta1 in renal epithelial cells

The collagen-binding integrins α1β1 and α2β1 have profoundly different functions, yet they are often co-expressed in epithelial cells. When both integrins are expressed in the same cell, it has been suggested that α1β1 negatively regulates integrin α2β1-dependent functions. In this study we utilized murine ureteric bud (UB) epithelial cells, which express no functionally detectable levels of endogenous integrins α1β1 and α2β1, to determine the mechanism whereby this regulation occurs. We demonstrate that UB cells expressing integrin α2β1, but not α1β1 adhere, migrate and proliferate on collagen I as well as form cellular cords in 3D collagen I gels. Substitution of the transmembrane domain of the integrin α2 subunit with that of α1 results in decreased cell adhesion, migration and cord formation. In contrast, substitution of the integrin α2 cytoplasmic tail with that of α1, decreases cell migration and cord formation, but increases proliferation. When integrin α1 and α2 subunits are co-expressed in UB cells, the α1 subunit negatively regulates integrin α2β1-dependent cord formation, adhesion and migration and this inhibition requires expression of both α1 and α2 tails. Thus, we provide evidence that the transmembrane and cytoplasmic domains of the α2 integrin subunit, as well as the α1 integrin subunit, regulate integrin α2β1 cell function.     

Multiple binding sites in collagen type I for the integrins alpha1beta1 and alpha2beta1

Integrins alpha(1)beta(1) and alpha(2)beta(1) are two major collagen receptors on the surface of eukaryotic cells. Binding to collagen is primarily due to an A-domain near the N terminus of the alpha chains. Previously, we reported that recombinant A-domain of alpha(1)beta(1) (alpha(1)A) had at least two affinity classes of binding sites in type I collagen (Rich, R. L., et al. (1999) J. Biol. Chem. 274, 24906-24913). Here, we compared the binding of the recombinant A-domain of alpha(2)beta(1) (alpha(2)A) to type I collagen with that of alpha(1)A using surface plasmon resonance and showed that alpha(2)A exhibited only one detectable class of binding sites in type I collagen, with a K(D) of approximately 10 microm at approximately 3 binding sites per collagen molecule. We further demonstrated that alpha(1)A and alpha(2)A competed with each other for binding to type I collagen in enzyme-linked immunosorbent assay (ELISA), suggesting that the binding sites in collagen for the two A-domains overlap or are adjacent to each other. By using rotary shadowing, the complexes of alpha(1)A- and alpha(2)A-procollagen were visualized. Morphometric analyses indicated three major binding regions (near the N terminus, in the central part, and near the C terminus) along the type I procollagen molecule for both A-domains. The positions of the respective binding regions for alpha(1)A and alpha(2)A were overlapping with or adjacent to each other, consistent with the ELISA results. Analysis of the sequences of type I collagen revealed that GER or GER-like motifs are present at each of the binding regions, and notably, the central region contains the GFOGER sequence, which was previously identified as a high affinity site for both alpha(1)A and alpha(2)A (Knight, C. G., et al. (2000) J. Biol. Chem. 275, 35-40). Peptides containing GLOGERGRO (peptide I, near the N terminus), GFOGERGVQ (peptide II, central), and GASGERGPO (peptide III, near the C terminus) were synthesized. Peptides I and II effectively inhibited the binding of alpha(1)A and alpha(2)A to type I collagen, while peptide III did so moderately. The N-terminal site in type I collagen has the sequence GLOGER in all three chains. Thus, it seems that peptide I represents a newly discovered native high affinity site for alpha(1)A and alpha(2)A.     

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.     

Analysis of the alpha4beta1 integrin-osteopontin interaction

The integrin alpha4beta1 is involved in mediating exfiltration of leukocytes from the vasculature. It interacts with a number of proteins up-regulated during the inflammatory response including VCAM-1 and the CS-1 alternatively spliced region of fibronectin. In addition it binds the multifunctional protein osteopontin (OPN), which can act as both a cytokine and an extracellular matrix molecule. Here we map the region of human OPN that supports cell adhesion via alpha4beta1 using GST fusion proteins. We show that alpha4beta1 expressed in J6 cells interacts with intact OPN when the integrin is in a high activation state, and by deletion mapping that the alpha4beta1 binding region in OPN lies between amino acid residues 125 and 168 (aa125-168). This region contains the central RGD motif of OPN, which also interacts with integrins alphavbeta3, alphavbeta5, alphavbeta1, alpha8beta1, and alpha5beta1. Mutating the RGD motif to RAD had no effect on the interaction with alpha4beta1. To define the binding site the region incorporating aa125-168 was divided into 5 overlapping peptides expressed as GST fusion proteins. Two peptides supported adhesion via alpha4beta1, aa132-146, and aa153-168; of these only a synthetic peptide, SVVYGLR (aa162-168), derived from aa153-168 was able to inhibit alpha4beta1 binding to CS-1. These data identify the motif SVVYGLR as a novel peptide inhibitor of alpha4beta1, and the primary alpha4beta1 binding site within OPN.