title: The Grb2-mSos1 complex binds phosphopeptides with higher affinity than Grb2.
abstract: 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 microM; the stoichiometry of the Grb2-mSos1 complex was 1:1. An EGFR-derived phosphopeptide bound Grb2 with a KD of 0.7 microM, whereas the Shc-derived phosphopeptide bound Grb2 with a KD of 0.2 microM. 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 microM 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.
authors.affiliation: Department of Biochemistry, University of Toronto, Ontario, Canada.
authors.forename: Y M
authors.initials: YM
authors.lastname: Chook
authors.forename: G D
authors.initials: GD
authors.lastname: Gish
authors.forename: C M
authors.initials: CM
authors.lastname: Kay
authors.forename: E F
authors.initials: EF
authors.lastname: Pai
authors.forename: T
authors.initials: T
authors.lastname: Pawson
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D051380:GRB2 Adaptor Protein; D020662:Guanine Nucleotide Exchange Factors; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008970:Molecular Weight; D010748:Phosphopeptides / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins; D015398:Signal Transduction; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship; D020663:ras Guanine Nucleotide Exchange Factors; D018909:src Homology Domains
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495949:Grb2 protein, mouse; D020662:Guanine Nucleotide Exchange Factors; D046911:Macromolecular Substances; D010748:Phosphopeptides; D011506:Proteins; D011994:Recombinant Proteins; D020663:ras Guanine Nucleotide Exchange Factors