Factors determining specificity of signal transduction by G-protein-coupled receptors. Regulation of signal transfer from receptor to G-protein.
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 alpha 2A/D 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 alpha 2-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 35S-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.
Sato
M
M
Department of Pharmacology, Medical University of South Carolina, Charleston 29425, USA.
Kataoka
R
R
Dingus
J
J
Wilcox
M
M
Hildebrandt
J D
JD
Lanier
S M
SM
D016475:3T3 Cells; D000316:Adrenergic alpha-Agonists / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000378:metabolism; D000068438:Brimonidine Tartrate; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D016716:PC12 Cells; D037342:Pertussis Toxin; D011810:Quinoxalines / Q000378:metabolism; D051381:Rats; D018341:Receptors, Adrenergic, alpha-2 / Q000096:biosynthesis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000316:Adrenergic alpha-Agonists; D011810:Quinoxalines; D018341:Receptors, Adrenergic, alpha-2; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D006165:Guanylyl Imidodiphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000068438:Brimonidine Tartrate; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins