The role of individual SH2 domains in mediating association of phospholipase C-gamma 1 with the activated EGF receptor

The two SH2 (Src homology domain 2) domains present in phospholipase C-gamm a 1 (PLC-gamma 1) were assayed for their capacities to recognize the five a utophosphorylation sites in the epidermal growth factor receptor. Plasmon r esonance and immunological techniques were employed to measure interactions between SH2 fusion proteins and phosphotyrosine-containing peptides. The N -SH2 domain recognized peptides in the order of pY1173 > pY992 > pY1068 > p Y1148 much greater than pY1086, while the C-SH2 domain recognized peptides in the order of pY992 > pY1068 > pY1148 much greater than pY1086 and pY1173 . The major autophosphorylation site, pY1173, was recognized only by the N- SH2 domain. Contributions of the N-SH2 and C-SH2 domains to the association of the intact PLC-gamma 1 molecule with the activated epidermal growth fac tor (EGF) receptor were assessed in vivo. Loss of function mutants of each SH2 domain were produced in a full-length epitope-tagged PLC-gamma 1. After expression of the mutants, cells were treated with EGF and association of exogenous PLC-gamma 1 with EGF receptors was measured. In this context the N-SH2 is the primary contributor to PLC-gamma 1 association with the EGF re ceptor. The combined results suggest an association mechanism involving the N-SH2 domain and the pY1173 autophosphorylation site as a primary event an d the C-SH2 domain and the pY992 autophosphorylation site as a secondary ev ent.