The Juxtamembrane, Cytosolic Region of the Epidermal Growth Factor Receptor Is Involved in Association with α-Subunit of Gs

Abstract

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...

Divergent requirement for Gαs and cAMP in the differentiation and inflammatory profile of distinct mouse Th subsets

“…Moreover, the cAMP-induced Ca 2+ influx and subsequent Th17 differentiation were PKA dependent ( Figure 7). Previous data have suggested that Gαs can directly activate Ca 2+ channels (28,29) and non-receptor and receptor tyrosine kinases (30)(31)(32); however, our results indicating that addition of a cAMP analog could reverse the phenotype of the Gnas ΔCD4 CD4 + T cells imply that the effects of Gαs on Th subset differentiation is likely to be mediated, at least for the most part, by decreased cAMP accumulation in these cells.…”

Regulation of Epidermal Growth Factor Receptor Degradation by Heterotrimeric Gαs Protein

“…Conceivably, this phosphorylation event might be related to the change in the subcellular localization of G␣s. Furthermore, G␣s has been shown to interact directly with the juxtamembrane region (50 aa) of the EGF receptor in both yeast two-hybrid and coimmunoprecipitation assays; this interaction was suggested to be responsible for the activation of adenylyl cyclase by EGF stimulation in cardiomyocytes (Nair et al, 1990;Sun et al, 1997). Intriguingly, the juxtamembrane region of the EGF receptor contains a dileucine motif that is required for efficient sorting of receptors to lysosomes (Lin et al, 1986;Kil et al, 1999;Bao et al, 2000).…”

Molecular characterization of neurohybrid cell death induced by Alzheimer's amyloid‐β peptides via p75NTR/PLAIDD

“…However, there are several exceptional cases in which single-transmembrane receptors or receptor-like proteins are directly linked to G proteins (Murayama et al 1990;Nishimoto et al 1993;Gong et al 1995;Sun et al 1995Sun et al , 1997. Most importantly, APP has been shown to directly interact with Go, but not Gi, and furthermore to activate Go in a phospholipid vesicle system (Okamoto and Nishimoto 1992;Nishimoto et al 1993;Brouillet et al 1999).…”