Ligand stimulation of the platelet-derived growth factor (PDGF) P-receptor leads to activation of its intrinsic tyrosine kinase and autophosphorylation of the intracellular part of the receptor. The autophosphorylated tyrosine residues mediate interactions with downstream signal transduction molecules and thereby initiate different signalling pathways. A pathway leading to activation of the GTP-binding protein Ras involves the adaptor molecule GRB2. Here we show that Tyr-716, a novel autophosphorylation site in the PDGF ,8-receptor kinase insert, mediates direct binding of GRB2 in vitro and in vivo. In a panel of mutant PDGF ,-receptors, in which Tyr-716 and the previously known autophosphorylation sites were individually mutated, only PDGFRjY716F failed to bind GRB2. Furthermore, a synthetic phosphorylated peptide containing Tyr-716 bound GRB2, and this peptide specifically interrupted the interaction between GRB2 and the wild-type receptor. In addition, the Y716(P) peptide significantly decreased the amount of GTP bound to Ras in response to PDGF in permeabilized fibroblasts as well as in porcine aortic endothelial cells expressing transfected PDGF ,8-receptors. The mutant PDGFRjIY716F still mediated activation of mitogen-activated protein kinases and an increased DNA synthesis in response to PDGF, indicating that multiple signal transduction pathways transduce mitogenic signals from the activated PDGF j-receptor.Platelet-derived growth factor (PDGF) is a connective tissue cell mitogen, consisting of dimers of disulfide-bonded A and B polypeptide chains, which combine to form the three isoforms, PDGF-AA, -AB, and -BB (for a review, see reference 21). Two types of PDGF tyrosine kinase receptors have been identified and cloned. The ox-receptor binds both A and B chains with high affinity, whereas the p-receptor binds only the B chain.Signal transduction through tyrosine kinase receptors follows a general scheme whereby ligand binding induces dimerization or oligomerization of receptor molecules, activation of the receptor tyrosine kinase, and autophosphorylation in trans on multiple tyrosine residues in the intracellular region (reviewed in reference 44). Thereby, binding sites are created for intracellular signal transduction molecules, containing one or two copies of Src homology 2 (SH2) domains. There are two principal groups of signal transduction molecules: those which are equipped with a catalytic domain, and those which lack such domains but which serve as adaptors and associate with catalytically active molecules (50). For the PDGF P-receptor, considerable information has accumulated regarding the positions of autophosphorylation sites as well as which signal transduction molecules interact with individual sites. Thus, members of the Src family, i.e., phospholipase C--y, the Ras GTPase-activating protein (GAP), phosphatidylinositol 3'-kinase (P13-kinase), the phosphotyrosine phosphatase PTP1D/ Syp, and the adaptor molecules Shc in the PDGF P-receptor (reviewed in reference 9). The specificity of these in...