THE GRB2-MSOS1 COMPLEX BINDS PHOSPHOPEPTIDES WITH HIGHER AFFINITY THAN GRB2

Epidermal growth factor (EGF) stimulation leads to autophosphorylation of the epidermal growth factor receptor (EGFR) and tyrosine phosphory lation of Shc. The Grb2 SH2 domain binds to Tyr(1068) of EGFR and Tyr( 317) of Shc while its SH3 domains bind to mSos1. Therefore, EGF treatm ent potentially results in the formation of several multimeric signali ng complexes, including EGFR-Grb2-mSos1, EGFR-Shc-Grb2-mSos1, and Shc- Grb2-mSos1, linking the receptor to activation of the Ras GTPase. We h ave purified Grb2, mSos1, and the Grb2-mSos1 complex to high homogenei ty, and used these isolated proteins to obtain binding affinities of m Sos1 for Grb2 and of either Grb2 or Grb2-mSos1 for phosphotyrosine-con taining peptides. mSos1 bound Grb2 with a K-D of 0.4 mu M; the stoichi ometry of the Grb2-mSos1 complex was 1:1. An EGFR-derived phosphopepti de bound Grb2 with a K-D of 0.7 mu M, whereas the Shc-derived phosphop eptide bound Grb2 with a K-D of 0.2 mu M. Since Grb2 exists in a stabl e complex with mSos1, and both proteins can exist in a constitutive co mplex in unstimulated cells, we performed phosphopeptide binding studi es on the Grb2-mSos1 complex to gain a better understanding of binding events in the intact cell. Grb2-mSos1 bound to both EGFR- and Shc-der ived phosphopeptides with higher affinities (K-D of 0.3 mu M and 31 nM , respectively) than Grb2 alone. These findings suggest that the proxi mity of mSos1 to Grb2 in the complex can influence the interactions of the Grb2 SH2 domain with phosphopeptides and raise the possibility th at in the Grb2-mSos1 complex the SH2 and SH3 domains of Grb2 are not i ndependent of each other but may be indirectly linked by mSos1.