Structural requirements for alpha 9 beta 1-mediated adhesion and migration to thrombin-cleaved osteopontin.
Osteopontin is an RGD-containing glycoprotein that mediates adhesion and migration of a variety of different cell types. We recently showed that a recombinant osteopontin fragment that was expected to be formed following thrombin cleavage was not only biologically active, but also could support alpha 9 beta 1-mediated adhesion, an activity not found in the full-length molecule. In this study we defined binding sites on the N-terminal osteopontin fragment important for alpha 9 beta 1-mediated cell interactions. In addition to adhesion, we showed that alpha 9 beta 1 could mediate cell migration, a function not previously identified for this integrin. Using site-directed mutagenesis, we made specific mutations in the RGD region of the N-terminal osteopontin fragment. Mutation of RGD to RGE resulted in a 50% decrease in cell adhesion. The residual binding to the RGE mutant could be blocked by alpha 9 and beta 1 antibodies. Adhesion to the RGE mutant was due to residual recognition of the RGE site by alpha 9 beta 1 since mutants containing more drastic mutations in the RGD domain achieved by mutating RGD to RAA or by eliminating the RGD completely failed to support cell adhesion and migration. In contrast, alpha 9 beta 1-mediated adhesion to tenascin was RGD independent. These data demonstrate that alpha 9 beta 1 is one of the few integrin receptors that can interact with two distinct RGD-containing ligands through different adhesive domains.
Department of Pathology, University of Washington, Seattle 98195, USA.
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Giachelli
D001665:Binding Sites; D002448:Cell Adhesion / Q000502:physiology*; D002465:Cell Movement / Q000502:physiology*; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D008545:Melanoma; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000235:genetics; D053495:Osteopontin; D011993:Recombinant Fusion Proteins; D012795:Sialoglycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019063:Tenascin; D013917:Thrombin / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D016023:Integrins; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; D019063:Tenascin; C113256:integrin alpha 9 beta 1; D053495:Osteopontin; C047981:arginyl-glycyl-aspartic acid; D013917:Thrombin