The juxtamembrane, cytosolic region of the epidermal growth factor receptor is involved in association with alpha-subunit of G(s)

Previously, we have demonstrated that epidermal growth factor (EGF) can stimulate adenylyl cyclase activity via activation of G(s) in the heart. Moreover, we have recently shown that G(s alpha) is phosphorylated by the EGF receptor protein tyrosine kinase and that the juxtamembrane region of the EGF receptor can stimulate G(s) directly. Therefore, employing isolated cardiac membranes, the two-hybrid assay, and in vitro association studies with purified EGF receptor and G(s alpha) we have investigated G(s alpha) 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 G(s alpha). In the yeast two-hybrid assay, the cytosolic domain of the EGF receptor and the N-terminal 64 amino acids of this region (Met(644)-Trp(707)) associated with G(s alpha). However, interactions of these regions of the EGF receptor with constitutively active G(s alpha) were diminished in the two-hybrid assay. Employing purified proteins, our studies demonstrate that the EGF receptor, directly and stoichiometrically, associates with G(s alpha) (1 mol of G(s alpha)/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 G(s alpha). However, neither the C-terminally truncated EGF receptor (Delta 1022-1186) nor a peptide corresponding to residues 985-996 of the receptor altered association with G(s alpha), thus indicating the selectivity of the G protein interaction with the juxtamembrane region. Interestingly, peptides corresponding to N and C termini of G(s alpha) did not alter the association of G(s alpha) with the EGF receptor. Consistent with the findings from the two-hybrid assay where constitutively active G(s alpha) poorly associated with the EGF receptor, in vitro experiments with purified proteins also demonstrated that activation of G(s alpha) 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 G(s alpha) and that upon activation of G(s alpha) this association is decreased.