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Tyrosine Phosphorylation of the ß3-Subunit of the Vß3 Integrin Is Required for Membrane Association of the Tyrosine Phosphatase SHP-2 and Its Further Recruitment to the Insulin-Like Growth Factor I Receptor

University of North Carolina, School of Medicine, Chapel Hill, North Carolina 27599

Address all correspondence and requests for reprints to: David R. Clemmons, M.D., CB-7170, 6111 Thurston-Bowles, Division of Endocrinology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7170. E-mail: endo{at}med.unc.edu.

Ligand occupancy of the Vß3 integrin is required for IGF-I receptor (IGF-IR) phosphorylation of an appropriate duration and for stimulation of IGF-I actions. In vascular smooth muscle cells (SMCs), the tyrosine phosphatase SHP-2 regulates the duration of IGF-IR phosphorylation and biological actions. We determined the role of ligand occupancy of the Vß3 integrin on ß3 phosphorylation and studied the role of ß3 phosphorylation in regulating both SHP-2 recruitment to the cell membrane and IGF-I-dependent biological responses. Vitronectin binding to Vß3 induced tyrosine phosphorylation of the ß3-subunit in subconfluent SMCs and was accompanied by increased association of SHP-2 with ß3. In confluent SMCs, the ß3-subunit was constitutively phosphorylated leading to basal binding of SHP-2. The Src kinase inhibitor PP2 caused a concentration-dependent decrease in ß3 phosphorylation and resulted in decreased SHP-2 association with ß3 and with the cell membrane. In contrast to control cells, SMCs expressing a mutant ß3 that had two tyrosines changed to phenylalanines showed a 89.9 ± 1.2% decrease in ß3 phosphorylation. This decrease was associated with reduced SHP-2 binding to nonphosphorylated ß3 and a corresponding decrease in the membrane association of SHP-2. When IGF-I was added to cells expressing mutant ß3, SHP-2 was not recruited to the Src homology 2 domain-containing tyrosine phosphatase substrate-1 or to IGF-IR. This was associated with prolonged IGF-IR phosphorylation and an impaired cellular DNA synthesis response to IGF-I. These results define a mechanism by which ligand occupancy of Vß3 regulates the SMC response to IGF-I.

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