Therefore, studies were performed to investigate the hypothesis that the EGFR protein tyrosine kinase phosphorylates G s␣ and activates this protein. Employing purified EGFR and G s␣ , we have demonstrated that the EGFR kinase phosphorylates G s␣ in a time-dependent manner with a stoichiometry of 2 mol of phosphate incorporated/mol of G s␣ . As determined by phosphoamino acid analysis, the phosphorylation of G s␣ by the EGFR kinase was exclusively on tyrosine residues. Interestingly, GDP and guanosine 5-3-O-(thio)triphosphate (GTP␥S) inhibited the phosphorylation of G s␣ without altering EGFR autophosphorylation. However, G protein ␥ subunits protected against GDP-and GTP␥S-mediated inhibition of phosphorylation of G s␣ . In functional studies, phospho-G s␣ demonstrated a greater GTPase activity and also a greater capacity to bind GTP␥S as compared to the nonphosphorylated G s␣ . Moreover, the phospho-G s␣ augmented adenylyl cyclase activity in S49 cyc ؊ cell membranes to a greater extent than its nonphosphorylated counterpart. Therefore, we conclude that phosphorylation of G s␣ on tyrosine residues by the EGFR kinase activates this G protein and increases its ability to stimulate adenylyl cyclase. Epidermal growth factor (EGF)1 exerts a variety of biological actions ranging from increased DNA synthesis, hyperplasia, and increased glucose and fatty acid metabolism, to alterations in muscular function (see Ref. 1 for review). These pleiotropic actions of EGF are mediated via the activation of several second messenger systems. For instance, following binding of EGF to its receptors, the intrinsic protein tyrosine kinase activity of the EGF receptor is increased, resulting in autophosphorylation of the EGF receptor as well as of other cellular proteins (reviewed in Refs. 1 and 2). The autophosphorylation of the EGF receptor serves to recruit proteins containing the Src homology 2 (SH2) domains such as phospholipase C␥ (3), the subsequent phosphorylation of which increases phosphatidylinositol metabolism, and the generation of the second messengers inositol 1,4,5-trisphosphate and diacylglycerol (3). Likewise, recruitment of the SH2-containing proteins such as Grb2 and other adaptor proteins to the phosphotyrosine-containing domains on the EGF receptor also leads to the activation of serine/threonine phosphorylation cascades such as the mitogen-activated protein kinase cascade (4). In addition, EGF has also been documented to modulate the cAMP second messenger system. Studies from our laboratory have shown that EGF increases contractility, beating rate, and cAMP accumulation in the heart (5) by stimulating adenylyl cyclase via a process involving G s␣ (6, 7). Moreover, the protein tyrosine kinase activity of the EGF receptor is important for EGF-mediated stimulation of cardiac adenylyl cyclase (8). One implication of this latter finding is that one, or more, of the signaling elements involved in stimulation of adenylyl cyclase by the activated EGF receptor is(are) phosphorylated. Therefore, we have proposed the hyp...
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␣ 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␣ we have investigated G s␣ 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␣ . 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␣ . However, interactions of these regions of the EGF receptor with constitutively active G s␣ were diminished in the two-hybrid assay. Employing purified proteins, our studies demonstrate that the EGF receptor, directly and stoichiometrically, associates with G s␣ (1 mol of G s␣ /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␣ . However, neither the C-terminally truncated EGF receptor (⌬1022-1186) nor a peptide corresponding to residues 985-996 of the receptor altered association with G s␣ , thus indicating the selectivity of the G protein interaction with the juxtamembrane region. Interestingly, peptides corresponding to N and C termini of G s␣ did not alter the association of G s␣ with the EGF receptor. Consistent with the findings from the two-hybrid assay where constitutively active G s␣ poorly associated with the EGF receptor, in vitro experiments with purified proteins also demonstrated that activation of G s␣ 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␣ and that upon activation of G s␣ this association is decreased. The pleiotropic actions of epidermal growth factor (EGF)1 are elicited by stimulation of a number of second messenger systems by the ligand-activated EGF receptor (1, 2). In addition to its well documented effects on the mitogen-activated protein kinase cascade (3) and phospholipase C␥ (4), EGF has also been demonstrated to regulate the cAMP second messenger system (5). We have previously demonstrated that in cardiac myocytes EGF elevates cAMP accumulation (6) by augmenting the activity of adenylyl cyclase (7,8) and that this increase in cAMP accumulation also augments the beating rate and contractility in intact hearts (9). EGF stimulates cardiac adenylyl cyclase by activation of the ␣-subunit of the stimu...
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