SOLUBLE LIGANDS OF THE ALPHA(V)BETA(3) INTEGRIN MEDIATE ENHANCED TYROSINE PHOSPHORYLATION OF MULTIPLE PROTEINS IN ADHERENT BOVINE PULMONARY-ARTERY ENDOTHELIAL-CELLS

Binding of substrate-bound extracellular matrix proteins to cell surfa ce integrins results in a variety of cellular responses including adhe sion, cytoskeletal reorganization, and gene expression. We have previo usly shown that addition of soluble SC5b-9, the complement-vitronectin complex, resulted in an RGD-dependent increase in lung venular hydrau lic conductivity (Ishikawa, S., Tsukada, H., and Bhattacharya, J. (199 3) J. Clin. Invest. 91, 103-109). To identify specific integrin(s) and signal transduction pathways that are responsive to soluble vitronect in-containing ligands, we exposed confluent bovine pulmonary artery ce lls to purified soluble human mono- or multimeric vitronectin, or SC5b -9, and determined the extent of endothelial cell protein tyrosine pho sphorylation. Monomeric vitronectin (Vn) did not induce enhanced prote in tyrosine phosphorylation. However, multimeric Vn and SC5b-9 elicite d time and concentration-dependent increases in tyrosine phosphorylati on of numerous proteins. Antiserum against vitronectin, RGD peptides, and monoclonal and polyclonal antibodies against the alpha(v) beta(3) integrin blocked the vitronectin- or SC5b-9-induced enhanced accumulat ion of tyrosine phosphoproteins, while antibodies against beta(1) inte grins and the alpha(v) beta(5) integrin did not. Clustering of the alp ha(v) beta(3) integrin using monoclonal antibody LM609 caused a patter n of enhanced tyrosine phosphorylation similar to that caused by multi meric Vn and SC5b-9, suggesting that aggregation of alpha(v) beta(3), was critical for signaling. Among the proteins that underwent enhanced tyrosine phosphorylation in response to vitronectin were the cytoskel etal proteins paxillin, cortactin, and ezrin, as well as the SH2 domai n containing protein She, and p125(FAK). We conclude that ligation of the alpha(v) beta(3) integrin by soluble ligands promotes enhanced pho sphorylation of several proteins implicated in tyrosine kinase signali ng and suggest that this pathway may be important in inflammatory stat es which are accompanied by accumulation of SC5b-9.