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 ␣ 9 subunit forms a single heterodimer, ␣ 9 ␤ 1 that mediates cell adhesion to a site within the third fibronectin type III repeat of tenascin-C (TNfn3). In contrast to at least 3 other integrins that bind to this region of tenascin-C, ␣ 9 ␤ 1 does not recognize the common integrin recognition motif, Arg-Gly-Asp (RGD). In this report, we have used substitution mutagenesis to identify a unique ligand recognition sequence in TNfn3. We introduced mutations substituting alanine for each of the acidic residues in or adjacent to each of the exposed loops predicted from the solved crystal structure. Most of these mutations had little or no effect on adhesion of ␣ 9 -transfected SW480 colon carcinoma cells, but mutations of either of two acidic residues in the B-C loop region markedly reduced attachment of these cells. In contrast, cells expressing the integrin ␣ v ␤ 3 , previously reported to bind to the RGD sequence in the adjacent F-G loop, attached to all mutant fragments except one in which the RGD site was mutated to RAA. The peptide, AEIDGIEL, based on the sequence of human tenascin-C in this region blocked the binding of ␣ 9 -transfected cells, but not ␤ 3 -transfected cells to wild type TNfn3. This sequence contains a tripeptide, IDG, homologous to the sequences LDV, IDA, and LDA in fibronectin and IDS in VCAM-1 recognized by the closely related integrin ␣ 4 ␤ 1 . These findings support the idea that this tripeptide motif serves as a ligand binding site for the ␣ 4 /␣ 9 subfamily of integrins.Integrins are cell surface heterodimers that play roles in essential biological processes including development and tissue remodeling (1-4). Ligand binding specificity depends in large part on the specific ␣ and ␤ subunit present in each heterodimer. To date, extracellular matrix proteins, cell surface immunoglobulin superfamily molecules, and cadherins have been identified as ligands for integrins. In most cases where the crystal structure of integrin ligands have been solved, the ligand binding site includes at least one acidic residue, generally displayed in an exposed peptide loop (5-7). The first short peptide sequence identified as an integrin recognition sequence was the tripeptide, Arg-Gly-Asp (RGD) (8), initially identified as a cell-recognition sequence in the large extracellular matrix protein, fibronectin (9, 10). Subsequently, this RGD sequence was found to be present in several integrin ligands and to serve as a recognition sequence for several different integrins (11). As the sequences of multiple integrin ␣ subunits were solved, it became clear that these sequences could be divided into 3 subfamilies based on sequence homology (1). One family includes ␣ subunits with a characteristic disulfide-linked cleavage site. A subset of these form heterodimers that recognize RGD-containing ligands. Another includes ␣ subunits that contain an inserted domain close to the N terminus but no cleavage site. These integrins generally do not recognize RGD-containing ligands. Finally, a third family, including o...

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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.

Osteopontin (OPN) is an integrin-binding inflammatory cytokine that undergoes polymerization catalyzed by transglutaminase 2. We have previously reported that polymeric OPN (polyOPN), but not unpolymerized OPN (OPN*), attracts neutrophils in vitro by presenting an acquired binding site for integrin ␣9␤1. Among many in vitro substrates for transglutaminase 2, only a few have evidence for in vivo polymerization and concomitant function. Although polyOPN has been identified in bone and aorta, the in vivo functional significance of polyOPN is unknown. To determine whether OPN polymerization contributes to neutrophil recruitment in vivo, we injected OPN* into the peritoneal space of mice. Polymeric OPN was detected by immunoblotting in the peritoneal wash of mice injected with OPN*, and both intraperitoneal and plasma OPN* levels were higher in mice injected with a polymerization-incompetent mutant, confirming that OPN* polymerizes in vivo. OPN* injection induced neutrophil accumulation, which was significantly less following injection of a mutant OPN that was incapable of polymerization. The importance of in vivo polymerization was further confirmed with cystamine, a transglutaminase inhibitor, which blocked the polymerization and attenuated OPN*-mediated neutrophil recruitment. The thrombin-cleaved N-terminal fragment of OPN, another ligand for ␣9␤1, was not responsible for neutrophil accumulation because a thrombin cleavage-incompetent mutant recruited similar numbers of neutrophils as wild type OPN*. Neutrophil accumulation in response to both wild type and thrombin cleavage-incompetent OPN* was reduced in mice lacking the integrin ␣9 subunit in leukocytes, indicating that ␣9␤1 is required for polymerization-induced recruitment. We have illustrated a physiological role of molecular polymerization by demonstrating acquired chemotactic properties for OPN. Osteopontin (OPN),3 an integrin-binding cytokine, plays critical roles in physiological and pathological processes, including inflammation, immunomodulation, tissue remodeling, fibrosis, mineralization (1), stem cell retention (2), and tumor metastasis (3). These functions are exerted through interactions with nine integrins and CD44 (4). OPN undergoes many types of post-translational modification, including phosphorylation (5), glycosylation, transglutamination (6), and proteolytic cleavage. Among these post-translational modifications, transglutamination (7) and thrombin-cleavage (8, 9) enhance interactions with integrins. Thrombin-cleaved N-terminal fragment of OPN (nOPN) has been found to play roles in rheumatoid arthritis (10, 11), autoimmune hepatitis (12), and stem cell retention in the bone marrow niche (2). Although OPN was found as a substrate for transglutaminase 2 (TG2; EC 2.3.2.13) earlier than thrombin, little is known about the functional role of the product of transglutamination, polymeric OPN. TG2 is a cross-linking enzyme that catalyzes isopeptide bonding between Gln and Lys residues (13) with specificity for amino acid sequences containing Gln (...