Determinants of Ligand Binding Specificity of the α1β1 and α2β1Integrins

The integrin ␣9␤1 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 ␣9 subunit is structurally similar to the integrin ␣4 subunit, and ␣9␤1 and ␣4␤1 both recognize VCAM-1 as a ligand. We therefore examined whether the disintegrin EC3, which we have recently shown specifically inhibits the binding of ␣4 integrins to ligands, would also be a functional inhibitor of ␣9␤1. EC3 and a novel heterodimeric disintegrin that we identified, EC6, both were potent inhibitors of ␣9␤1-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 ␣9␤1-and ␣4␤1-mediated adhesion to VCAM-1. Surprisingly though, concentrations of EC3 that completely inhibited adhesion of ␣9-transfected cells to VCAM-1 had little or no effect on adhesion to either of the other ␣9␤1 ligands, osteopontin and tenascin-C. Furthermore, peptides AEIDGIEL and SV-VYGLR, which we have previously shown inhibit binding of ␣9␤1-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 ␣9␤1 integrin with VCAM-1 and the extracellular matrix ligands osteopontin and tenascin-C.The integrin ␣9 subunit forms a single known heterodimer, ␣9␤1, that is widely expressed in epithelia and smooth and skeletal muscle and on neutrophils (1, 2). We and others have identified three distinct ligands for ␣9␤1: the extracellular matrix proteins tenascin-C (3) and osteopontin (4 -6) and the inducible endothelial immunoglobulin family member, VCAM-1 1 (1). We have mapped the ligand-binding site in tenascin-C to an exposed peptide loop in the third fibronectin type III repeat containing the sequence AEIDGIEL (7). We have also mapped the ␣9␤1 ligand-binding site in osteopontin (6). Although an initial report suggested that ␣9␤1 might bind to an RGD-containing sequence in osteopontin (5), we were able to demonstrate by extensive mutagenesis that the binding site is within the linear peptide sequence SVVYGLR immediately adjacent to the RGD site (6).Structurally, the ␣9 subunit is closely related to the ␣4 subunit, and on the basis of sequence homology ␣4 and ␣9 appear to be the only known members of a subfamily of integrin ␣ subunits that lack both an insertional domain and an extracellular disulfide-linked cleavage site (2). Furthermore, both subunits are expressed on leukocytes and mediate leukocyte migration (1, 8). Finally, both integrins recognize VCAM-1 as a ligand (1, 9). We have shown that both ␣4␤1 and ␣9␤1 contribute to the chemotactic migration of human neutrophils across endothelial cell monolayers activated by tumor necrosis factor-␣ (TNF␣), an effect that is due at least in part to interaction of these integrins with VCAM-1 induced in resp...

Section: Fig 11mentioning

confidence: 78%

Inhibitory Effects of MLDG-containing Heterodimeric Disintegrins Reveal Distinct Structural Requirements for Interaction of the Integrin α9β1 with VCAM-1, Tenascin-C, and Osteopontin

Marcinkiewicz

Taooka

Yokosaki³

et al. 2000

“…Binding of DMP1 to Immobilized Type I Collagen-A solidphase binding assay was used as the method of choice for characterization of interactions between collagen and noncollagenous proteins (8,23). The binding curves shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The collagen was then dissolved in 0.01 M HCl at a 1 mg/ml concentration. Collagen binding property (23) was determined by coating microtiter plates (Immulon 4, Dynex Technologies) with 5 g of type I collagen in 100 l of 0.1 M acetic acid/well overnight at 4°C. Wells were washed three times with PBS and blocked with 0.5% (w/v) bovine serum albumin in PBS at 37°C for 2 h before the addition of varying concentrations of the GST-fused recombinant DMP1 (24).…”

Section: Solid-phase Binding Assay Of Dmp1 With Monomeric Collagen-mentioning

confidence: 99%

Dentin Matrix Protein 1 Immobilized on Type I Collagen Fibrils Facilitates Apatite Deposition in Vitro

George

2004

212

208

During bone and dentin mineralization, the crystal nucleation and growth processes are considered to be matrix regulated. Osteoblasts and odontoblasts synthesize a polymeric collagenous matrix, which forms a template for apatite initiation and elongation. Coordinated and controlled reaction between type I collagen and bone/dentin-specific noncollagenous proteins are necessary for well defined biogenic crystal formation. However, the process by which collagen surfaces become mineralized is not understood. Dentin matrix protein 1 (DMP1) is an acidic noncollagenous protein expressed during the initial stages of mineralized matrix formation in bone and dentin. Here we show that DMP1 bound specifically to type I collagen, with the binding region located at the N-telopeptide region of type I collagen. Peptide mapping identified two acidic clusters in DMP1 responsible for interacting with type I collagen. The collagen binding property of these domains was further confirmed by site-directed mutagenesis. Transmission electron microscopy analyses have localized DMP1 in the gap region of the collagen fibrils. Fibrillogenesis assays further demonstrated that DMP1 accelerated the assembly of the collagen fibrils in vitro and also increased the diameter of the reconstituted collagen fibrils. In vitro mineralization studies in the presence of calcium and phosphate ions demonstrated apatite deposition only at the collagenbound DMP1 sites. Thus specific binding of DMP1 and possibly other noncollagenous proteins on the collagen fibril might be a key step in collagen matrix organization and mineralization.Mineralized tissues such as bone and dentin are hierarchically organized biocomposites possessing unique mechanical properties (1). Understanding the biomineralization process is of great value for synthesis of bioengineered bone and dentinlike materials with optimum structural properties. Type I collagen accounts for 90% of the total protein in the organic matrix of bone and dentin (1). It not only provides the structural framework with viscoelastic properties but also defines compartments for ordered mineral deposition. Studies have demonstrated that the apatite crystals are first nucleated in the gap region, and the growing mineral platelets are highly organized in a staggered manner within the collagen fibrils (2, 3).It is well established that type I collagen matrix does not have the capacity to induce matrix-specific mineral formation from metastable calcium phosphate solutions that do not spontaneously precipitate. Several in vitro studies have demonstrated that ordered mineralization of apatite on collagen fibril is impossible without additives (4, 5). As a result, much attention has been drawn to the noncollagenous proteins (NCPs) 1 that are tightly bound to the collagen fibers in mineralized tissues. Bone and/or dentin-specific NCPs are mostly acidic in nature and are rich in glutamic acid, aspartic acid, and phosphoserines (6, 7). They possess high calcium binding capacity and hydroxyapatite affinity (8 -10). In vitro ...

“…In contrast, the VWF-A3 domain appears to function as an independent structural unit, and there is no evidence for modulation of its collagen binding affinity, nor does binding of A3 to collagen appear to affect the affinity of the VWF-A1 domain for platelet receptor GpIb␣. Thus, the collagen-binding site of A3 merely performs an adhesive function, whereas binding sites of I domains are more sophisticated and also play a regulatory role (31).…”

Section: Discussionmentioning

confidence: 99%

Mapping the Collagen-binding Site in the von Willebrand Factor-A3 Domain

Romijn

Westein

Bouma

et al. 2003

The multimeric glycoprotein von Willebrand factor (VWF) mediates platelet adhesion to collagen at sites of vascular damage. The binding site for collagen types I and III is located in the VWF-A3 domain. Recently, we showed that His 1023 , located near the edge between the "front" and "bottom" faces of A3, is critical for collagen binding (Romijn, R. A., Bouma, B., Wuyster, W., Gros, P., Kroon, J., Sixma, J. J., and Huizinga, E. G. (2001) reduced binding affinity about 10-fold. Together, these residues define a flat and rather hydrophobic collagenbinding site located at the front face of the A3 domain. The collagen-binding site of VWF-A3 is distinctly different from that of the homologous integrin ␣ 2 I domain, which has a hydrophilic binding site located at the top face of the domain. Based on the surface characteristics of the collagen-binding site of A3, we propose that it interacts with collagen sequences containing positively charged and hydrophobic residues. Docking of a collagen triple helix on the binding site suggests a range of possible engagements and predicts that at most eight consecutive residues in a collagen triple helix interact with A3.Under conditions of high shear stress, platelet adhesion to collagen at sites of vascular injury is initiated by the interaction of platelet receptor glycoprotein (Gp) 1 Ib-IX-V with collagen-bound von Willebrand factor (VWF) (1). Transient interactions between VWF and GpIb-IX-V mediate platelet rolling, which slows down the platelet and allows other platelet receptors such as integrin ␣ 2 ␤ 1 (2) and GpVI to bind to collagen (2-4). These interactions result in firm adhesion and activation of platelets at the site of vascular injury.VWF is a multimeric glycoprotein consisting of ϳ270-kDa monomers that are linked by disulfide bonds (5). The affinity of VWF for collagen depends on multimer size (6). The binding site for fibrillar collagens type I and III is located in the VWF-A3 domain (7), whereas collagen type VI has been shown to bind to the VWF-A1 domain (8, 9). The latter domain also contains the binding site for GpIb␣ of the GpIb-IX-V complex (10, 11).VWF A-type domains and homologous integrin I domains adopt a so-called dinucleotide-binding fold, or Rossman fold, composed of a central ␤-sheet flanked on both sides by amphipathic ␣-helices (12-15). Binding of the I domains of integrins ␣ 1 ␤ 1 , ␣ 2 ␤ 1 , ␣ 10 ␤ 1 , and ␣ 11 ␤ 1 to collagen involves a divalent cation (16, 17) located in the metal ion-dependent adhesion site (MIDAS) motif, and amino acid residues at the top face of the domain (18,19). Binding of the I domain of integrin ␣ 2 ␤ 1 to collagen induces a major displacement of its carboxyl-terminal ␣-helix that is thought to be critical for integrin signaling (18). The A3 domain of VWF does not contain a functional MIDAS motif, and binding of A3 to collagen is cation-independent (20, 21). The involvement of the top face of A3 in collagen binding has been excluded by mutagenesis studies (13,22). Recently, we showed that His 1023 , located close to th...