FIG. 4.

Binding of Grb2 to the receptor and recruitment of p85 PI 3-kinase are dependent on Y867. (A) Binding of Grb2 to the receptors requires phosphorylation of Y867 in the Grb-2 consensus motif. Filters containing immunoprecipitates (IP), obtained with the indicated antibodies, of 3-mg portions of protein from lysates of IL-3-starved cells were Western blotted (WB) with the indicated antibodies. (B) Grb2 complex formation. Protein (3 mg) from lysates of IL-3-starved cells transfected with pLXSN vector or with a plasmid encoding CDMer or the F867 mutant receptor were incubated with GST-Grb2 fusion proteins. GST was fused to full-length (FL) Grb2 or to either of two Grb2 domains: the SH2 domain and the C-terminal SH3 domain (C-SH3). GST alone was used as a control. The filter was probed with antiphosphotyrosine antibody (pY), stripped, and reprobed with the indicated antibodies (WB). Immunoprecipitation of a 95-kDa phosphorylated protein (p95) with anti-p85 PI 3-kinase antibody (C) or with GST-p85 fusion proteins (prepared as for the GST-Grb2 fusion proteins) (D). This phosphorylated protein associated with p85 only in cells expressing receptors with Y867 in the Grb2 binding site.