RESUMO
Previously, we have demonstrated that epidermal growth factor (EGF) can stimulate adenylyl cyclase activity via activation of Gs in the heart. Moreover, we have recently shown that Gsalpha is phosphorylated by the EGF receptor protein tyrosine kinase and that the juxtamembrane region of the EGF receptor can stimulate Gs directly. Therefore, employing isolated cardiac membranes, the two-hybrid assay, and in vitro association studies with purified EGF receptor and Gsalpha we have investigated Gsalpha complex formation with the EGF receptor and elucidated the region in the receptor involved in this interaction. In isolated cardiac membranes, immunoprecipitation of EGF receptor was accompanied by co-immunoprecipitation of Gsalpha. In the yeast two-hybrid assay, the cytosolic domain of the EGF receptor and the N-terminal 64 amino acids of this region (Met644-Trp707) associated with Gsalpha. However, interactions of these regions of the EGF receptor with constitutively active Gsalpha were diminished in the two-hybrid assay. Employing purified proteins, our studies demonstrate that the EGF receptor, directly and stoichiometrically, associates with Gsalpha (1 mol of Gsalpha/mol of EGF receptor). This association was not altered in the presence or absence of ATP and therefore, was independent of tyrosine phosphorylation of either of the proteins. Peptides corresponding to the juxtamembrane region of the receptor decreased association of the EGF receptor with Gsalpha. However, neither the C-terminally truncated EGF receptor (Delta1022-1186) nor a peptide corresponding to residues 985-996 of the receptor altered association with Gsalpha, thus indicating the selectivity of the G protein interaction with the juxtamembrane region. Interestingly, peptides corresponding to N and C termini of Gsalpha did not alter the association of Gsalpha with the EGF receptor. Consistent with the findings from the two-hybrid assay where constitutively active Gsalpha poorly associated with the EGF receptor, in vitro experiments with purified proteins also demonstrated that activation of Gsalpha by guanosine 5'-3-O-(thio)triphosphate decreased the association of G protein with the EGF receptor. Thus we conclude that the juxtamembrane region of the EGF receptor, directly and stoichiometrically, associates with Gsalpha and that upon activation of Gsalpha this association is decreased.
