The integrin ␣ 9 ␤ 1 mediates cell adhesion to tenascin-C and VCAM-1 by binding to sequences distinct from the common integrin-recognition sequence, arginine-glycine-aspartic acid (RGD). A thrombin-cleaved NH 2 -terminal fragment of osteopontin containing the RGD sequence has recently been shown to also be a ligand for ␣ 9 ␤ 1 . In this report, we used site-directed mutagenesis and synthetic peptides to identify the ␣ 9 ␤ 1 recognition sequence in osteopontin. ␣ 9 -transfected SW480, Chinese hamster ovary, and L-cells adhered to a recombinant NH 2 -terminal osteopontin fragment in which the RGD site was mutated to RAA (nOPN-RAA). Adhesion was completely inhibited by anti-␣ 9 monoclonal antibody Y9A2, indicating the presence of a non-RGD ␣ 9 ␤ 1 recognition sequence within this fragment. Alanine substitution mutagenesis of 13 additional conserved negatively charged amino acid residues in this fragment had no effect on ␣ 9 ␤ 1 -mediated adhesion, but adhesion was dramatically inhibited by either alanine substitution or deletion of tyrosine 165. A synthetic peptide, SVVYGLR, corresponding to the sequence surrounding Tyr 165 , blocked ␣ 9 ␤ 1 -mediated adhesion to nOPN-RAA and exposed a ligand-binding-dependent epitope on the integrin ␤ 1 subunit on ␣ 9 -transfected, but not on mocktransfected cells. These results demonstrate that the linear sequence SVVYGLR directly binds to ␣ 9 ␤ 1 and is responsible for ␣ 9 ␤ 1 -mediated cell adhesion to the NH 2 -terminal fragment of osteopontin.Integrins are cell surface heterodimeric receptors that mediate cell-cell and cell-extracellular matrix adhesion (1, 2). Upon ligation by a wide variety of ligands, integrins can initiate signaling cascades that regulate cell growth, cell death, migration, polarization, and tissue remodeling (3). Integrins recognize a surprisingly large number of functionally diverse proteins as ligands, and the list of known integrin ligands continues to grow. New integrin ligands have been identified, and drugs targeting integrins have been developed as a consequence of the description of short linear amino acid sequences that directly bind to integrins. For example, the integrins, and ␣ v ␤ 8 bind to sequences containing the tri-peptide sequence Arg-Gly-Asp (RGD). Several new and biologically important integrin ligands have been identified based on the presence of this sequence (4, 5). Drugs modeled on the structure of the RGD sequence are being used or tested to inhibit integrin function for treatment of thrombosis, inflammation, atherosclerosis, osteoporosis, and cancer (5). The RGD sequence has also been exploited to target cell surface integrins to enhance gene delivery (6). We have previously identified the recognition sequence for the integrin ␣ 9 ␤ 1 in tenascin-C and found that this sequence did not include RGD, but was homologous to the ␣ 4 ␤ 1 recognition sequence in the inducible endothelial adhesion molecule VCAM-1 (7). This finding led to our identification of ␣ 9 ␤ 1 as a receptor for VCAM-1 (8).Osteopontin is a phosphorylated acidic gly...

The integrin α9β1 has been shown to be widely expressed on smooth muscle and epithelial cells, and to mediate adhesion to the extracellular matrix proteins osteopontin and tenascin-C. We have found that the peptide sequence this integrin recognizes in tenascin-C is highly homologous to the sequence recognized by the closely related integrin α4β1, in the inducible endothelial ligand, vascular cell adhesion mole-cule-1 (VCAM-1). We therefore sought to determine whether α9β1 also recognizes VCAM-1, and whether any such interaction would be biologically significant. In this report, we demonstrate that α9β1 mediates stable cell adhesion to recombinant VCAM-1 and to VCAM-1 induced on human umbilical vein endothelial cells by tumor necrosis factor-α. Furthermore, we show that α9β1 is highly and selectively expressed on neutrophils and is critical for neutrophil migration on VCAM-1 and tenascin-C. Finally, α9β1 and α4 integrins contribute to neutrophil chemotaxis across activated endothelial monolayers. These observations suggest a possible role for α9β1/VCAM-1 interactions in extravasation of neutrophils at sites of acute inflammation.

The integrin ␣9 subunit forms a single heterodimer, ␣9␤1. The ␣9 subunit is most closely related to the ␣4 subunit, and like ␣4 integrins, ␣9␤1 plays an important role in leukocyte migration. The ␣4 cytoplasmic domain preferentially enhances cell migration and inhibits cell spreading, effects that depend on interaction with the adaptor protein, paxillin. To determine whether the ␣9 cytoplasmic domain has similar effects, a series of chimeric and deleted ␣9 constructs were expressed in Chinese hamster ovary cells and tested for their effects on migration and spreading on an ␣9␤1-specific ligand. Like ␣4, the ␣9 cytoplasmic domain enhanced cell migration and inhibited cell spreading. Paxillin also specifically bound the ␣9 cytoplasmic domain and to a similar level as ␣4. In paxillin Ϫ/Ϫ cells, ␣9 failed to inhibit cell spreading as expected but surprisingly still enhanced cell migration. Further, mutations that abolished the ␣9-paxillin interaction prevented ␣9 from inhibiting cell spreading but had no effect on ␣9-dependent cell migration. These findings suggest that the mechanisms by which the cytoplasmic domains of integrin ␣ subunits enhance migration and inhibit cell spreading are distinct and that the ␣9 and ␣4 cytoplasmic domains, despite sequence and functional similarities, enhance cell migration by different intracellular signaling pathways.

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...