Differential roles of the Src homology 2 domains of phospholipase C-gamma l (PLC-gamma l) in platelet-derived growth factor-induced activation of PLC-gamma l in intact cells

Upon stimulation of cells with platelet-derived growth factor (PDGF), phosp holipase C-gamma 1 (PLC-gamma 1) binds to the tyrosine-phosphorylated PDGF receptor through one or both of its Src homology 2 (SH2) domains, is phosph orylated by the receptor kinase, and is thereby activated to hydrolyze phos phatidylinositol 4,5-bisphosphate, Association of PLC-gamma 1 with the inso luble subcellular fraction is also enhanced in PDGF-stimulated cells. The i ndividual roles of the two SH2 domains of PLC-gamma 1 in mediating the inte raction between the enzyme and the PDGF receptor have now been investigated by functionally disabling each domain. A critical Arg residue in each SH2 domain was mutated to Ala, Both wild-type and mutant PLC-gamma 1 proteins w ere transiently expressed in a PLC-gamma 1-deficient fibroblast cell line, and these transfected cells were stimulated with PDGF, The mutant protein i n which the COOH-terminal SH2 domain was disabled bound to the PDGF recepto r. Accordingly, it was phosphorylated by the receptor, catalyzed the produc tion of inositol phosphates, and mobilized intracellular calcium to extents similar to (but slightly less than) those observed with the wild-type enzy me. In contrast, the mutant in which the NH2-terminal SH2 domain was impair ed did not bind to the PDGF receptor and consequently was neither phosphory lated nor activated. These results suggest that the NH2-terminal SH2 domain , but not the COOH-terminal SH2 domain, of PLC-gamma 1 is required for PDGF -induced activation of PLC-gamma 1, Functional impairment of the SH2 domain s did not affect the PDGF-induced redistribution of PLC-gamma 1, suggesting that recruitment of PLC-gamma 1 to the particulate fraction does not invol ve the SH2 domains.