Grb2 monomer–dimer equilibrium determines normal versus oncogenic function

The SH2 and SH3 domain structural integrity and functionality were confirmed by peptide binding assays. Binding studies using two phosphotyrosine peptides derived from EGFR (FLPVPE{pY}IN-QSVPKR) and Shc (EEPPDHQ{pY}YNDFPGK) for the Grb2 SH2 domain and a SOS1-derived proline-rich peptide (PVPPPVPPRRRPEY) for the SH3 domains were performed using MST 1. Grb2 was fluorescently labeled using Atto488 dye. A solution of unlabeled peptide was serially diluted from about 100 μM to 55 nM in the presence of 200 nM labeled domain. (a, b and c) binding of phospho-Shc peptide to WT, Y160 and N188/214D with comparable binding affinities. (d, e and f) binding of SOS1 peptide to WT, Y160 and N188/214D with comparable binding affinities. g) Wild type Grb2 binding to phospho-EGFR peptide.