Factors Determining Specificity of Signal Transduction by G-protein-coupled Receptors

doi:10.1074/jbc.270.25.15269

Factors Determining Specificity of Signal Transduction by G-protein-coupled Receptors

REGULATION OF SIGNAL TRANSFER FROM RECEPTOR TO G-PROTEIN (*)

From the (1)Department of Pharmacology, Medical University of South Carolina, Charleston, South Carolina 29425

^¶ To whom correspondence should be addressed:
Dept. of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425.
Tel.: 803-792-2574; Fax: 803-792-2475.

Abstract

Among subfamilies of G-protein-coupled receptors, agonists initiate several cell signaling events depending on the receptor subtype (R) and the type of G-protein (G) or effector molecule (E) expressed in a particular cell. Determinants of signaling specificity/efficiency may operate at the R-G interface, where events are influenced by cell architecture or accessory proteins found in the receptor's microenvironment. This issue was addressed by characterizing signal transfer from R to G following stable expression of the α adrenergic receptor in two different membrane environments (NIH-3T3 fibroblasts and the pheochromocytoma cell line, PC-12). Receptor coupling to endogenous G-proteins in both cell types was eliminated by pertussis toxin pretreatment and R-G signal transfer restored by reconstitution of cell membranes with purified brain G-protein. Thus, the receptor has access to the same population of G-proteins in the two different environments. In this signal restoration assay, agonist-induced activation of G was 3-9-fold greater in PC-12 as compared with NIH-3T3 α²-adrenergic receptor transfectants. The cell-specific differences in signal transfer were observed over a range of receptor densities or G-protein concentration. The augmented signal transfer in PC-12 versus NIH-3T3 transfectants occurred despite a 2-3-fold lower level of receptors existing in the R-G-coupled state (high affinity, guanyl-5′-yl imidodiphosphate-sensitive agonist binding), suggesting the existence of other membrane factors that influence the nucleotide binding behavior of G-protein in the two cell types. Detergent extraction of PC-12 but not NIH-3T3 membranes yielded a heat-sensitive, macromolecular entity that increased S-labeled guanosine 5′-O-(thiotriphosphate) binding to brain G-protein in a concentration-dependent manner. These data indicate that the transfer of signal from R to G is regulated by a cell type-specific, membrane-associated protein that enhances the agonist-induced activation of G.

Footnotes

↵^§ Contributed equally to this work and are visiting scientists from Asahikawa Medical College, Asahikawa, Japan.
↵* This work was supported by National Institutes of Health Grants NS24821 (to S. M. L.) and DK37219 (to J. D. H.) and the Council for Tobacco Research CTR 2235 (to S. M. L.). This paper is the fourth in the series “Factors Determining Specificity of Signal Transduction by G-protein-coupled Receptors.” The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

G

G-protein

R

receptor

E

effector molecule

α²-AR

α² adrenergic receptor

PT

pertussis toxin

GTPS

guanosine 5′-3-O-(thio)triphosphate

Gpp(NH)p

guanosine 5′-(β,-imido)triphosphate.
↵²M. Sato, E. Duzic, and S. M. Lanier, unpublished observations.
↵³A portion of these studies was presented in preliminary form (Tian, W. N., Deth, R., Lanier, S. M., and Duzic, E. (1992) FASEB J.6, 2423).
↵⁴E. Duzic and S. M. Lanier, unpublished observations.
↵⁵M. Sato, R. Kataoka, J. Hildebrandt, and S. M. Lanier, unpublished observations.
↵⁶M. Sato, J. Hildebrandt, and S. M. Lanier, unpublished observations.