Physiological requirement for both SH2 domains for phospholipase C-gamma 1function and inter-action with platelet-derived growth factor receptors

Two approaches have been utilized to investigate the role of individual SH2 domains in growth factor activation of phospholipase C-gamma 1 (PLC-gamma 1). Surface plasmon resonance analysis indicates that the individual N-SH2 and C-SH2 domains are able to specifically recognize a phosphotyrosine-cont aining peptide corresponding to Tyr 1021 of the platelet-derived growth fac tor (PDGF) beta receptor. To assess SH2 function in the context of the full -length PLC-gamma 1 molecule as well as within the intact cell, PLC-gamma 1 SH2 domain mutants, disabled by site-directed mutagenesis of the N-SH2 and /or C-SH2 domain(s), were expressed in Plcg1(-/-) fibroblasts. Under equili brium incubation conditions (4 degrees C, 40 min), the N-SH2 domain, but no t the C-SH2 domain, was sufficient to mediate significant PLC-gamma 1 assoc iation with the activated PDGF receptor and PLC-gamma 1 tyrosine phosphoryl ation. When both SH2 domains in PLC-gamma 1 were disabled, the double mutan t did not associate,vith activated PDGF receptors and was not tyrosine phos phorylated. However, no single SH2 mutant was able to mediate growth factor activation of Ca2+ mobilization or inositol 1,4,5-trisphosphate (IP3) form ation. Subsequent kinetic experiments demonstrated that each single SH2 dom ain mutant was significantly impaired in its capacity to mediate rapid asso ciation with activated PDGF receptors and become tyrosine phosphorylated, H ence, when assayed under physiological conditions necessary to achieve a ra pid biological response (Ca2+ mobilization and IP3 formation), both SH2 dom ains of PLC-gamma 1 are essential to growth factor responsiveness.