TIGHT ASSOCIATION OF GRB2 WITH RECEPTOR PROTEIN-TYROSINE-PHOSPHATASE-ALPHA IS MEDIATED BY THE SH2 AND C-TERMINAL SH3 DOMAINS

Receptor protein-tyrosine phosphatase alpha (RPTP alpha), a transmembr ane member of the extensive family of protein-tyrosine phosphatases (P TPs), is constitutively phosphorylated on Tyr789, a consensus binding site for the SH2 domain of the SH3-SH2-SH3 adaptor protein GRB2, We ha ve previously shown that GRB2 binds to P.Tyr789 in vivo and in vitro v ia its SH2 domain, Here, we report that not only the GRB2 SH2 domain, but also the C-terminal SH3 domain is involved in binding to RPTP alph a in vitro and in vivo, Although the N-terminal SH3 domain of GRB2 is essential for binding to the Ras guanine nucleotide exchange factor So n of Sevenless (Sos), an RPTP alpha-GRB2-Sos complex could not be dete cted, The inclusion of peptides encompassing an hSos1 proline-rich mot h in cell lysates resulted in enhanced binding of RPTP alpha to GRB2 i n vitro, suggesting that steric hindrance prohibits formation of the R PTP alpha-GRB2 Sos complex, In vitro binding experiments indicated tha t the binding of GRB2 to Sos/dynamin and RPTP alpha was mutually exclu sive. Analysis of in vitro binding kinetics coupled with results from transient co-transfections demonstrated that RPTP alpha is tightly bou nd to GRB2 The site of interaction of the C-terminal SH3 domain of GRB 2 with RPTP alpha was mapped using deletion mutants to an 18-residue r egion in the N-terminal PTP domain, Arg469, within this region, was id entified as one of the residues that is involved in the interaction wi th the C-terminal SH3 domain of GRB2, RPTP alpha residues 469-486 are localized dose to the catalytic site cleft in the structure of the N-t erminal PTP-domain, suggesting that interaction with the C-terminal SH 3 domain may block access to the catalytic site, thus inhibiting RPTP alpha activity.