BINDING OF THE GRB2 SH2 DOMAIN TO PHOSPHOTYROSINE MOTIFS DOES NOT CHANGE THE AFFINITY OF ITS SH3 DOMAINS FOR SOS PROLINE-RICH MOTIFS

Phosphotyrosine peptide binding to Grb2 induces tryptophan fluorescenc e changes in the Src homology 2 (SH2) domain. Affinities are in the na nomolar range, the She peptide having the highest affinity, followed b y peptides mimicking Grb2 binding sites on EGF and HGF receptors, the putative sites on insulin and IGF-1 receptors having much lower affini ties. Proline-rich peptide binding to the SH3 domains induces fluoresc ence changes mainly in the C-terminal SH3. Affinities are in the micro molar range, the highest affinity peptides mimicking the first proline -rich motif of the Sos C-terminus. Additional residues before this PVP PPVPP motif provide a minor contribution to the binding, but the two r esidues after this motif are important and may contribute to specifici ty. The affinity of each SH3 for each proline-rich motif is too low to account for the high stability of the Grb2-Sos complex, suggesting th at Grb2 recognizes other structural features in the Sos C-terminus. Bi nding of a phosphotyrosine peptide to the SH2 has no effect on the SH3 s. Thus the binding of Grb2 to a receptor or to an associated protein phosphorylated on tyrosines is unlikely to activate the exchange facto r activity of Sos through a conformational change transmitted from the SH2 to the SH3 domains.