Ligand-stimulated autophosphorylation of the platelet-derived growth factor receptor (PDGFR) beta subunit creates a number of binding sites for SH2-containing proteins. One of the PDGFR-associated proteins is a 64-kDa protein of unknown identity and function. We present data indicating that the 64-kDa protein that associates with the activated PDGFR is Syp (also called SH-PTP2, PTP-1D, or SH-PTP3), the ubiquitously expressed 64-kDa SH2-containing protein-tyrosine phosphatase. Phosphorylation of Tyr-1009 in the C terminus of the PDGFR is required for the stable association of Syp, suggesting that phosphorylation of this residue creates a binding site for the Syp SH2 domains. Although Syp stably associates with the PDGFR, this event is not required for PDGF-stimulated tyrosine phosphorylation of Syp. These data raise the interesting possibility that protein-tyrosine phosphatases contribute to the intracellular relay of biological signals originating from receptor tyrosine kinases such as the PDGFR.

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In response to binding of platelet-derived growth factor (PDGF), the PDGF receptor (PDGFR) beta subunit is phosphorylated on tyrosine residues and associates with numerous signal transduction enzymes, including the GTPase-activating protein of ras (GAP) and phosphatidylinositol 3-kinase (PI3K). Previous studies have shown that association of PI3K requires phosphorylation of tyrosine 751 (Y751) in the kinase insert and that this region of receptor forms at least a portion of the binding site for PI3K. In this study, the in vitro binding of GAP to the PDGFR was investigated. Like PI3K, GAP associates only with receptors that have been permitted to autophosphorylate, and GAP itself does not require tyrosine phosphate in order to stably associate with the phosphorylated PDGFR. To define which tyrosine residues are required for GAP binding, a panel of PDGFR phosphorylation site mutants was tested. Mutation of Y771 reduced the amount of GAP that associates to an undetectable level. In contrast, the F771 (phenylalanine at 771) mutant bound wild-type levels of PI3K, whereas the F740 and F751 mutants bound 3 and 23%, respectively, of the wild-type levels of PI3K but wild-type levels of GAP. The F740/F751 double mutant associated with wild-type levels of GAP, but no detectable PI3K activity, while the F740/F751/F771 triple mutant could not bind either GAP or PI3K. The in vitro and in vivo associations of GAP and PI3K activity to these PDGFR mutants were indistinguishable. The distinct tyrosine residue requirements suggest that GAP and PI3K bind different regions of the PDGFR. This possibility was also supported by the observation that the antibody to the PDGFR kinase insert Y751 region that blocks association of PI3K had only a minor effect on the in vitro binding of GAP. In addition, highly purified PI3K and GAP associated in the absence of other cellular proteins and neither cooperated nor competed with each other's binding to the PDGFR. Taken together, these studies indicate that GAP and PI3K bind directly to the PDGFR and have discrete binding sites that include portions of the kinase insert domain.