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

doi:10.1074/jbc.272.9.5413

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

From the Department of Pharmacology, University of Tennessee, Memphis, Memphis, Tennessee 38163 and
^‡ Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706-1532

^§ To whom correspondence should be addressed:
Dept. of Pharmacology, Health Science Center, University of Tennessee, 874 Union Ave., Memphis, TN 38163.
Tel.: 901-448-6006; Fax: 901-448-7300.

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_sα 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_sα we have investigated G_sα 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_sα. 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 G_sα. However, interactions of these regions of the EGF receptor with constitutively active G_sα were diminished in the two-hybrid assay. Employing purified proteins, our studies demonstrate that the EGF receptor, directly and stoichiometrically, associates with G_sα (1 mol of G_sα/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_sα. However, neither the C-terminally truncated EGF receptor (Δ1022-1186) nor a peptide corresponding to residues 985-996 of the receptor altered association with G_sα, thus indicating the selectivity of the G protein interaction with the juxtamembrane region. Interestingly, peptides corresponding to N and C termini of G_sα did not alter the association of G_sα with the EGF receptor. Consistent with the findings from the two-hybrid assay where constitutively active G_sα poorly associated with the EGF receptor, in vitro experiments with purified proteins also demonstrated that activation of G_sα 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_sα and that upon activation of G_sα this association is decreased.

Footnotes

↵* This work was supported in part by National Institutes of Health Grant HL 48308, a Grant-in-Aid from the American Heart Association, National Center (to T. B. P.), and National Institutes of Health Grant GM 53271 (to P. J. B.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

EGF

epidermal growth factor

EGFR

epidermal growth factor receptor

EGFR_C

cytosolic region of the EGFR (amino acids 644-1186)

EGFR_CJM

cytosolic juxtamembrane region of the EGFR (amino acids 644-707)

EGFR_CΔJM

cytosolic region of the EGFR encompassing residues 695-1186 in which juxtamembrane 50 amino acids (residues 645-694) are deleted

EGFR-13

peptide corresponding to amino acids 645-657 of the EGFR

P-EGFR-13

EGFR-13 in which residue corresponding to Thr⁶⁵⁴ in EGFR is phosphorylated

EGFR-14

peptide corresponding to amino acids 679-692 of the EGF receptor

G-protein

GTP-binding regulatory protein

G_s

stimulatory GTP binding regulatory protein of adenylyl cyclase

G_sα

α-subunit of G_s

G_sα*

constitutively active Q213L mutant of G_sα

G_sα 15-29

G_sα 371-380, and G_sα 354-372, peptides corresponding to amino acids indicated by numbers in G_sα

GTPγS

guanosine 5′-3-O-(thio)triphosphate

mAb

monoclonal antibody

PAGE

polyacrylamide gel electrophoresis

APP

amyloid precursor protein.
- Received September 5, 1996.
- Revision received December 16, 1996.