(3,4), and two SH2-containing adapter proteins, Grb2 and Shc, have been implicated in its activation. Specifically, these proteins have been shown to bind directly to tyrosine-phosphorylated receptors (5-7) or SH2 docking proteins (such as the insulin receptor substrate 1) (8). Grb2, a 25-kDa protein with two SH3 domains flanking one SH2 domain, shuttles the Ras guanine nucleotide exchange factor, Sosl, to activated receptors (or to insulin receptor substrate 1) (5, 7-11) so that Sosl can activate Ras by catalyzing the exchange of GDP for GTP (5, 9-11). Shc, another widely expressed protein that contains an N-terminal phosphotyrosine binding (PTB) domain (12-16) and a C-terminal SH2 domain (17), can associate, in its tyrosine-phosphorylated form, with Grb2-Sosl complexes and may increase Grb2-Sosl interactions after growth factor stimulation (9,18,19 MATERIALS AND METHODS Reagents. COS-cell-derived murine IL-3 and GM-CSF were produced as described (29). Rabbit antiserum to the Shcassociated p145 protein was generated by immunizing rabbits with a 15-mer obtained from amino acid sequencing (VPAE-GVSSLNEMINP) and crosslinked to keyhole limpet hemocyanin with glutaraldehyde (30). The glutathione S-transferase (GST) fusion proteins, consisting of the 27-kDa N-terminal and of GST linked to the Grb2 N-terminal (amino acid residues 5-56) or C-terminal (residues 163-215) SH3 domains of Grb2, were expressed in Escherichia coli in pGEX-2T plasmids (Pharmacia/LKB) and the fusion proteins were recovered from clarified lysates with glutathione (GSH)-agarose beads as described (31 Abbreviations: GM-CSF, granulocyte-macrophage colony-stimulating factor; GSH, glutathione; GST, glutathione S-transferase; IL-3, interleukin 3; NRS, normal rabbit serum; 5-ptase, inositol polyphosphate 5-phosphatase; Ptd1ns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; Ins(1,3,4,5)P4, inositol 1,3,4,5-tetrakisphosphate; SH2, src homology 2 domain; PTB, phosphotyrosine binding; p145, the Shc-associated 145-kDa protein.