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 surface integrins results in a variety of cellular responses including adhesion, cytoskeletal reorganization, and gene expression. We have previously shown that addition of soluble SC5b-9, the complement-vitronectin complex, resulted in an RGD-dependent increase in lung venular hydraulic conductivity (Ishikawa, S., Tsukada, H., and Bhattacharya, J. (1993) J. Clin. Invest. 91, 103-109). To identify specific integrin(s) and signal transduction pathways that are responsive to soluble vitronectin-containing ligands, we exposed confluent bovine pulmonary artery cells to purified soluble human mono- or multimeric vitronectin, or SC5b-9, and determined the extent of endothelial cell protein tyrosine phosphorylation. Monomeric vitronectin (Vn) did not induce enhanced protein tyrosine phosphorylation. However, multimeric Vn and SC5b-9 elicited time- and concentration-dependent increases in tyrosine phosphorylation 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 accumulation of tyrosine phosphoproteins, while antibodies against beta 1 integrins and the alpha v beta 5 integrin did not. Clustering of the alpha v beta 3 integrin using monoclonal antibody LM609 caused a pattern of enhanced tyrosine phosphorylation similar to that caused by multimeric 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 cytoskeletal proteins paxillin, cortactin, and ezrin, as well as the SH2 domain-containing protein Shc, and p125FAK. We conclude that ligation of the alpha v beta 3 integrin by soluble ligands promotes enhanced phosphorylation of several proteins implicated in tyrosine kinase signaling and suggest that this pathway may be important in inflammatory states which are accompanied by accumulation of SC5b-9.
Bhattacharya
S
S
St. Luke's-Roosevelt Hospital Center, New York, New York 10019, USA.
Fu
C
C
Bhattacharya
J
J
Greenberg
S
S
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D015938:Complement Membrane Attack Complex / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism*; D006023:Glycoproteins / Q000494:pharmacology*; D006493:Heparin / Q000378:metabolism; D016023:Integrins / Q000502:physiology*; D009842:Oligopeptides / Q000378:metabolism; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011651:Pulmonary Artery / Q000378:metabolism; D016024:Receptors, Cytoadhesin / Q000502:physiology*; D019030:Receptors, Vitronectin; D014443:Tyrosine / Q000378:metabolism*; D019096:Vitronectin
D015938:Complement Membrane Attack Complex; D003598:Cytoskeletal Proteins; D006023:Glycoproteins; D016023:Integrins; D009842:Oligopeptides; D051419:Paxillin; D010750:Phosphoproteins; D016024:Receptors, Cytoadhesin; D019030:Receptors, Vitronectin; D019096:Vitronectin; D014443:Tyrosine; C047981:arginyl-glycyl-aspartic acid; D006493:Heparin