Previous studies have shown that Src is required for platelet-derived growth factor (PDGF)-dependent cell cycle progression in fibroblasts. Since fibroblasts usually express both PDGF receptors (PDGFRs), these findings suggested that Src was mandatory for signal relay by both the ␣ and ␤PDGFRs. In this study, we have focused on the role of Src in signal relay by the ␣PDGFR.

Activation of the platelet-derived growth factor (PDGF) ␣ receptor (␣PDGFR) leads to cell migration and DNA synthesis. These events are preceded by the ligandinduced tyrosine phosphorylation of the receptor and its association with SH2-containing signaling enzymes including Src family members (Src), the phosphotyrosine phosphatase SHP-2, phosphatidylinositol 3-kinase (PI3K), and phospholipase C-␥1 (PLC␥). In this study, we sought to systematically evaluate the relative roles of the signaling enzymes that are recruited to the ␣PDGFR for DNA synthesis and cell migration. Our approach was to generate and characterize tyrosine to phenylalanine ␣PDGFR mutants that failed to associate with one or more of the above listed signaling enzymes. In a 3T3-like cell line (Ph cells), PDGF-dependent DNA synthesis was strictly dependent on only one of the receptor-associated proteins, PI3K. In contrast, multiple signaling enzymes were required for maximal chemotaxis, as receptors unable to associate with either Src, PI3K, or PLC␥ initiated chemotaxis to 4, 47, or 56% of the wild-type level, respectively. Furthermore, coexpression of mutant receptors revealed that these signaling enzymes do not need to be on the same receptor for a cell to respond chemotactically to PDGF. We conclude that for the ␣PDGFR, PI3K plays a major role in initiating DNA synthesis, whereas PI3K, PLC␥, and especially Src are required for chemotaxis.Receptor tyrosine kinases elicit responses such as cell proliferation and migration via binding and activation of Src homology 2 (SH2) 1 domain-containing signaling molecules. Upon ligand binding, receptor tyrosine kinases dimerize and autophosphorylate, and the phosphorylated tyrosines serve as a key component of the docking sites for SH2 domain-containing signal relay enzymes. There are at least several different ways in which signaling enzymes associate with a tyrosinephosphorylated receptor tyrosine kinase. For the epidermal growth factor receptor, each of the signaling enzymes appears to bind to any one of the phosphorylation sites. The hepatocyte growth factor receptor contains a pair of tyrosine phosphorylation sites, which are required for stable recruitment of at least four signaling enzymes. In contrast, at least some of the signaling enzymes that associate with the ␤PDGFR and the fibroblast growth factor receptor have specific binding sites (1).Activation of the ␣PDGFR results in its phosphorylation at numerous tyrosine residues, and eight such phosphorylation sites have been identified (reviewed in Ref. 2

Following binding of platelet-derived growth factor (PDGF), the PDGF ␣ receptor (␣PDGFR) becomes tyrosine phosphorylated and associates with a number of signal transduction molecules, including phospholipase C␥-1 (PLC␥-1), phosphatidylinositol 3-kinase (PI3K), the phosphotyrosine phosphatase SHP-2, Grb2, and Src. Here, we present data identifying a novel phosphorylation site in the kinase insert domain of the ␣PDGFR at tyrosine (Y) 720. We replaced this residue with phenylalanine and expressed the mutated receptor (F720) in Patch fibroblasts that do not express the ␣PDGFR. Characterization of the F720 mutant indicated that binding of two proteins, SHP-2 and Grb2, was severely impaired, whereas PLC␥-1 and PI3K associated to wild-type levels. In addition, mutating Y720 to phenylalanine dramatically reduced PDGF-dependent tyrosine phosphorylation of SHP-2. Since Y720 was required for recruitment of two proteins, we investigated the mechanism by which these two proteins associated with the ␣PDGFR. SHP-2 bound the ␣PDGFR directly, whereas Grb2 associated indirectly, most probably via SHP-2, as Grb2 and SHP-2 coimmunoprecipitated when SHP-2 was tyrosine phosphorylated. We also compared the ability of the wild-type and F720 ␣PDGFRs to mediate a number of downstream events. Preventing the ␣PDGFR from recruiting SHP-2 and Grb2 did not compromise PDGF-AA-induced activation of Ras, initiation of DNA synthesis, or growth of cells in soft agar. We conclude that phosphorylation of the ␣PDGFR at Y720 is required for association of SHP-2 and Grb2 and tyrosine phosphorylation of SHP-2; however, these events are not required for the ␣PDGFR to activate Ras or initiate a proliferative response. In addition, these findings reveal that while SHP-2 binds to both of the receptors, it binds in different locations: to the carboxy terminus of the ␤PDGFR but to the kinase insert of the ␣PDGFR.