The epidermal growth factor receptor is coupled to a pertussis toxin-sensitive guanine nucleotide regulatory protein in rat hepatocytes.
Activation of epidermal growth factor (EGF) receptors stimulates inositol phosphate production in rat hepatocytes via a pertussis toxin-sensitive mechanism, suggesting the involvement of a G protein in the process. Since the first event after receptor-G protein interaction is exchange of GTP for GDP on the G protein, the effect of EGF was measured on the initial rates of guanosine 5'-O-(3-[35S]thiotriphosphate) [( 35S]GTP gamma S) association and [alpha-32P]GDP dissociation in rat hepatocyte membranes. The initial rate of [35S]GTP gamma S binding was stimulated by EGF, with a maximal effect observed at 8 nM EGF. EGF also increased the initial rate of [alpha-32P]GDP dissociation. The effect of EGF on [35S]GTP gamma S association was blocked by boiling the peptide for 5 min in 5 mM dithiothreitol or by incubation of the membranes with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S). EGF-stimulated [35S]GTP gamma S binding was completely abolished in hepatocyte membranes prepared from pertussis toxin-treated rats and was inhibited in hepatocyte membranes that were treated directly with the resolved A-subunit of pertussis toxin. The amount of guanine nucleotide binding affected by occupation of the EGF receptor was approximately 6 pmol/mg of membrane protein. Occupation of angiotensin II receptors, which are known to couple to G proteins in hepatic membranes, also stimulated [35S]GTP gamma S association with and [alpha-32P]GDP dissociation from the membranes. The effect of angiotensin II on [alpha-32P]GDP dissociation was blocked by the angiotensin II receptor antagonist [Sar1,Ile8]angiotensin II, demonstrating that the guanine nucleotide binding was receptor-mediated. In A431 human epidermoid carcinoma cells, EGF stimulates inositol lipid breakdown, but the effect is not blocked by treatment of the cells with pertussis toxin. In these cells, EGF had no effect on [35S]GTP gamma S binding. Occupation of the beta-adrenergic receptor in A431 cell membranes with isoproterenol did stimulate [35S] GTP gamma S binding, and the effect could be completely blocked by l-propranolol. These results support the concept that in hepatocyte membranes, EGF receptors interact with a pertussis toxin-sensitive G protein via a mechanism similar to other hormone receptor-G protein interactions, but that in A431 human epidermoid carcinoma cells, EGF may activate phospholipase C via different mechanisms.
Liang
M N
MN
Department of Pharmacology, University of Virginia School of Medicine, Charlottesville 22908.
Garrison
J C
JC
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002294:Carcinoma, Squamous Cell; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000302:isolation & purification / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008297:Male; D037342:Pertussis Toxin; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D010566:Virulence Factors, Bordetella; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004815:Epidermal Growth Factor; D037342:Pertussis Toxin; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D019204:GTP-Binding Proteins; D007545:Isoproterenol; D002118:Calcium