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(Received for publication, June 13, 1996, and in revised form, August 23, 1996)
From the ¶ Program in Molecular Biology and Cancer, Samuel
Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario
M5G 1X5, Canada, the Epidermal growth factor (EGF) stimulation leads
to autophosphorylation of the epidermal growth factor receptor (EGFR)
and tyrosine phosphorylation of Shc. The Grb2 SH2 domain binds to Tyr1068 of EGFR and Tyr317 of Shc while its SH3
domains bind to mSos1. Therefore, EGF treatment potentially results in
the formation of several multimeric signaling complexes, including
EGFR-Grb2-mSos1, EGFR-Shc-Grb2-mSos1, and Shc-Grb2-mSos1, linking the
receptor to activation of the Ras GTPase. We have purified Grb2, mSos1,
and the Grb2-mSos1 complex to high homogeneity, and used these isolated
proteins to obtain binding affinities of mSos1 for Grb2 and of either
Grb2 or Grb2-mSos1 for phosphotyrosine-containing peptides. mSos1 bound
Grb2 with a KD of 0.4 µM; the
stoichiometry of the Grb2-mSos1 complex was 1:1. An EGFR-derived
phosphopeptide bound Grb2 with a KD of 0.7 µM, whereas the Shc-derived phosphopeptide bound Grb2
with a KD of 0.2 µM. Since Grb2
exists in a stable complex with mSos1, and both proteins can exist in a
constitutive complex in unstimulated cells, we performed phosphopeptide
binding studies 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-derived phosphopeptides with higher affinities
(KD of 0.3 µM and 31 nM,
respectively) than Grb2 alone. These findings suggest that the
proximity of mSos1 to Grb2 in the complex can influence the
interactions of the Grb2 SH2 domain with phosphopeptides and raise the
possibility that in the Grb2-mSos1 complex the SH2 and SH3 domains of
Grb2 are not independent of each other but may be indirectly linked by
mSos1.
Volume 271, Number 48,
Issue of November 29, 1996
pp. 30472-30478
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
¶
,
,
and
Department of Biochemistry,
University of Toronto, Toronto, Ontario M5S 1A8, Canada, the 
MRC
Group in Protein Structure and Function, Department of Biochemistry,
University of Alberta and the Protein Engineering Network of Centres of
Excellence, Edmonton, Alberta T6G 2H7, Canada
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