(FAK) is a pathway shared by several integrins. It seems USA to be particularly important in anchorage dependence 1 Corresponding author (Frisch et al., 1996;Xu et al., 1996). Moreover, several growth factor receptors have been shown to be present in Integrin-mediated cell attachment modulates growth isolated focal adhesion complexes and in other membrane responses and growth factors regulate cell attachment.structures containing aggregated integrins (Plopper et al., Moreover, both cell attachment to extracellular matrix 1995; Miyamoto et al., 1996). and mitogenic signaling by growth factors are necessaryThe pathways that are known to be restricted to indifor the proliferation of most types of normal cells, vidual integrins include up-regulation of the anti-apoptosis suggesting that integrin and growth factor receptor protein Bcl-2 by the α5β1 integrin (Zhang et al., 1995), signaling pathways meet at some downstream point.activation of the adaptor protein Shc by α6β4 (Mainiero We report here that a small, highly tyrosine-phos et al., 1995) and, presumably by an unrelated process, by phorylated fraction of PDGFβ and insulin receptors certain other integrins (Wary et al., 1996). Moreover, the co-immunoprecipitates with the αvβ3 integrin from αvβ3 integrin interacts with the PDGF and insulin receptor cells. The integrin association requires growth factor signaling pathways (Bartfeld et al., 1993; Vuori and stimulation of the receptors. Several signaling mole- Ruoslahti, 1994). cules that are known to be associated with activatedThat the αvβ3 integrin is somehow linked to PDGF growth factor receptors were present in the αvβ3 signaling was first suggested by the finding of Bartfeld integrin complexes. Mitogenicity and chemotaxis et al. (1993) that a 190 kDa tyrosine-phosphorylated induced by PDGF-BB were enhanced in cells plated protein could be immunoprecipitated together with αvβ3 on the αvβ3 ligand vitronectin compared with cells from PDGF-BB-stimulated cells. The identity of the 190 plated on the β1 integrin ligand collagen. Thus, the kDa protein is not known. An involvement of αvβ3 in engagement of the αvβ3 integrin in cell-matrix interinsulin signaling was discovered when IRS-1, a cytoactions appears to coordinate an intense response to growth factors, helping to explain the importance of plasmic signal transduction mediator of the insulin (and this integrin for tissue regeneration, angiogenesis and insulin-like growth factor, IGF) receptors, was found to tumor metastasis.co-immunoprecipitate with αvβ3 (Vuori and Ruoslahti, Keywords: αvβ3 integrin/cell attachment/cell-matrix 1994). IRS-1 is tyrosine-phosphorylated by the activated interaction/growth factors/PDGFβ insulin (and IGF) receptor tyrosine kinases (RTK) and as a result binds a number of downstream signaling molecules (White and Kahn, 1994). The mitogenic activity of insulin is enhanced in cells that have adhered to a substrate (such

Eph receptor tyrosine kinases play key roles in pattern formation during embryonic development, but little is known about the mechanisms by which they elicit specific biological responses in cells. Here, we investigate the role of tyrosines 605 and 611 in the juxtamembrane region of EphB2, because they are conserved Eph receptor autophosphorylation sites and demonstrated binding sites for the SH2 domains of multiple signaling proteins. Mutation of tyrosines 605 and 611 to phenylalanine impaired EphB2 kinase activity, complicating analysis of their function as SH2 domain binding sites and their contribution to EphB2-mediated signaling. In contrast, mutation to the negatively charged glutamic acid disrupted SH2 domain binding without reducing EphB2 kinase activity. By using a panel of EphB2 mutants, we found that kinase activity is required for the changes in cell-matrix and cell - cell adhesion, cytoskeletal organization, and activation of mitogen-activated protein (MAP) kinases elicited by EphB2 in transiently transfected cells. Instead, the two juxtamembrane SH2 domain binding sites were dispensable for these effects. These results suggest that phosphorylation of tyrosines 605 and 611 is critical for EphB2-mediated cellular responses because it regulates EphB2 kinase activity.

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