The epidermal growth factor receptor is coupled to a pertussis toxin-sensitive guanine nucleotide regulatory protein in rat hepatocytes

M.N Liang,J.C Garrison

doi:10.1016/s0021-9258(18)98845-9

Abstract

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.

Highlights

Activation of epidermal growth factor(EGF) recep- Activation of EGF’ receptors elicits multiple intracellular tors stimulates inositolphosphateproduction in rat signals that lead to DNA synthesis and cell divihepatocytes viaa pertussis toxin-sensitive mechanism, sion
EGF-stimulated [36S]GTPrS binding tyrosine kinase have been discovered, including microtubulewas completelyabolished in hepatocytemembranes associated protein kinase and phospholipase C-y
Microtuprepared from pertussis toxin-treated rats and was bule-associated protein kinase is a serine protein inhibited in hepatocyte membranes that were treated kinase whose phosphorylation is increased upon EGF receptor directly with threesolved A-subunitof pertussis toxin. activation [5]

Summary

RESULTS

Binding was calculated as thedifference between the GTPyS bound in the presence or absence of agonist or agonist and. The EGFeffect on [35S]GTPySbinding was completely abolished by including 100 PM GDPPS in the binding assay (Fig. 2B).To directly testthe hypothesis thattheEGF receptor in rat hepatocytes interacts with a pertussis toxinsensitive G proteint,he effect of EGF on [35S]GTPySbinding was measured in hepatic membranes prepared from pertussis toxin-treatedrats. W, controltime course; 0, time course inthe presence of 10 pM perimental Procedures." The average densities for the EGF receptor bands on the filmswere 8.04 & 1.72 and 7.08 & 1.64 OD x mm' for the normal and pertussis toxin-treated rat hepatic membranes, respectively (Fig. 4) These results suggested that there is no difference in the ability of EGF to isoproterenol; 0,time course in the presence of 10 pM isoproterenol and 1 mM I-propranolol.

GDP dissociation from the inactive protein and stimulating with G proteins?

Findings

Go and Gi reconstituted into a phospholipid vesicle system