Independent binding of peptide ligands to the SH2 and SH3 domains of Grb2.
Grb2, composed entirely of SH2 and SH3 domains, serves as an adaptor protein in signaling from growth factor-activated tyrosine kinase receptors. It interacts via its SH2 domain with the autophosphorylated carboxyl-terminal tail of activated epidermal growth factor (EGF) receptor and via its SH3 domains with proline-rich sequences in the Ras guanine nucleotide releasing factor, Son of sevenless (Sos). Recruitment of the Grb2-Sos complex to the receptor upon its stimulation leads to Ras activation. A major question remains as to whether SH2-mediated binding of Grb2 to the activated receptor results in conformational changes that influence its SH3-mediated association with Sos, thereby affecting Sos activity. This question is addressed through studies of the binding to intact Grb2 of an EGF receptor-derived phosphotyrosine-containing peptide and a Sos-derived proline-rich peptide using isothermal titration calorimetry and surface plasmon resonance measurements. The phosphopeptide binds to Grb2 in a 1:1 complex, with a KD of 0.4 microns. The Sos proline-rich peptide binds to Grb2 in a 2:1 complex, with a KD of 22 microns. Saturation of the SH2 domain of Grb2 with the EGFR phosphopeptide was found not to affect its subsequent binding to the Sos peptide. Thus we detected no influence of SH2 binding upon SH3-mediated interactions, suggesting that the domains do not communicate, and that recruitment itself of Sos to the cell surface is sufficient for Ras signaling.
Lemmon
M A
MA
Department of Pharmacology, New York University Medical Center, New York 10016.
Ladbury
J E
JE
Mandiyan
V
V
Zhou
M
M
Schlessinger
J
J
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D002151:Calorimetry; D066246:ErbB Receptors / Q000737:chemistry / Q000378:metabolism*; D051380:GRB2 Adaptor Protein; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism*; D019000:Phosphotyrosine; D011506:Proteins / Q000378:metabolism*; D015398:Signal Transduction; D020664:Son of Sevenless Proteins; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D008024:Ligands; D008565:Membrane Proteins; D010748:Phosphopeptides; D011506:Proteins; D020664:Son of Sevenless Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D066246:ErbB Receptors