Quantitative analysis of Grb2-Sos1 interaction: the N-terminal SH3 domain of Grb2 mediates affinity.
Grb2 is an adaptor protein that links receptor and cytoplasmic tyrosine kinases to the Ras signalling pathway by binding the Ras-specific guanine nucleotide exchange factor, Sos1, through its SH3 domains. The Grb2-SH3 domain binding has been localized to the carboxy-terminal two hundred amino acids of Sos1 (Sos1-c). By using real time biospecific interaction analysis (BIAcore), we studied the kinetic parameters and binding affinity of the Grb2-Sos1-c interaction. The binding of Grb2 to Sos1-c is a high affinity interaction with a moderate association rate (9.45 x 10(4) per M per s), a slow dissociation rate (13.8 x 10(-5) s), and an affinity constant of 1.48 nM. BIAcore measurements on isolated N-terminal and C-terminal SH3 domains (NSH3 and CSH3) further indicate that the high affinity Grb2-Sos1-c interaction is primarily mediated through the NSH3 domain (Kd = 1.68 nM). The CSH3 domain shows substantially reduced binding to Sos1-c in these measurements. Inhibition studies with BIAcore using proline rich peptides derived from the C-terminus of Sos1 show that there is a single major binding site for Grb2 in Sos1. This binding site is contained within the peptide N20, which corresponds to amino acids 1143-1162 of Sos1. This peptide completely blocks the Grb2-Sos1-c and NSH3-Sos1-c interactions with IC50 values of 8 microM and 4 microM respectively. The discrete interaction between the NSH3 domain and the N20 peptide may be amenable for drug discovery through screening or peptidomimetic approaches.
Sastry
L
L
Oncologix Inc., Gaithersburg, Maryland 20878, USA.
Lin
W
W
Wong
W T
WT
Di Fiore
P P
PP
Scoppa
C A
CA
King
C R
CR
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D005656:Fungal Proteins / Q000378:metabolism*; D051380:GRB2 Adaptor Protein; D051379:Mice; D008969:Molecular Sequence Data; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits; D012097:Repressor Proteins / Q000378:metabolism*; D020837:SOS1 Protein; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D005656:Fungal Proteins; D051380:GRB2 Adaptor Protein; C495949:Grb2 protein, mouse; D011506:Proteins; D012097:Repressor Proteins; D020837:SOS1 Protein