IDENTIFICATION OF TYR-762 IN THE PLATELET-DERIVED GROWTH-FACTOR ALPHA-RECEPTOR AS THE BINDING-SITE FOR CRK PROTEINS

Tyr-762 is an autophosphorylation site in the human platelet-derived g rowth factor (PDGF) alpha-receptor. In order to investigate whether ph osphorylated Tyr-762 serves as a docking site for downstream signal tr ansduction molecules, affinity purification using an immobilized synth etic peptide containing phosphorylated Tyr-762 and its surrounding ami no acid residues was performed. Proteins in HeLa cell lysate of molecu lar sizes 27, 38 and 40 kDa bound to the phosphorylated, but not to th e unphosphorylated peptide, Analyses of partial amino acid sequences o f the purified proteins indicated that they were identical to CrkI, Cr kII and CrkL respectively. The wild-type PDGF alpha-receptor, when exp ressed in porcine aortic endothelial cells, formed complexes with CrkI I and CrkL upon ligand stimulation, which was specifically inhibited b y a synthetic peptide containing phosphorylated Tyr-762, Replacement o f Tyr-762 with a phenylalanine residue in the PDGF alpha-receptor abro gated ligand-induced binding of Crk proteins, Tyrosine phosphorylation of CrkII and CrkL increased by 1.8- and 1.3-fold, respectively, upon ligand stimulation of the wild-type alpha-receptor, In contrast, the Y 762F mutant PDGF alpha-receptor failed to induce tyrosine phosphorylat ion of Crk proteins, CrkII and CrkL constitutively formed complex with the guanine nucleotide exchange factor C3G, in unstimulated as well a s PDGF-stimulated cells, Moreover, the activated wild-type PDGF alpha- receptor but not the Y762F mutant receptor was found in a C3G immunopr ecipitate, suggesting that a ternary complex between the activated PDG F alpha-receptor, Crk and C3G was formed. DNA synthesis stimulated by PDGF-BB as well as PDGF-induced MAP kinase activation was similar in c ells expressing wild-type and mutant receptors. Interestingly, the act ivated PDGF beta-receptor was found not to bind Crk proteins. Instead, Tyr-771 of the beta-receptor, which is localized at an analogous posi tion to Tyr-762 in the alpha-receptor, binds RasGAP, RasGAP is not bou nd to the alpha-receptor. Thus, this region in the kinase inserts of t he two receptors may be important for the divergency in signaling from the two PDGF receptors.