AGONIST REGULATION OF CELLULAR G(S) ALPHA-SUBUNIT LEVELS IN NEUROBLASTOMA X GLIOMA HYBRID NG108-15 CELLS TRANSFECTED TO EXPRESS DIFFERENT LEVELS OF THE HUMAN BETA-2 ADRENOCEPTOR

Neuroblastoma x glioma hybrid NG108-15 cells endogenously express at l east three receptors which activate adenylate cyclase via the intermed iacy of the stimulatory G-protein, G(s). Sustained exposure of the cel ls to agonists at the IP prostanoid receptor results in a substantial decrease in cellular levels of the alpha-subunit of G(s) (G(s) alpha) [McKenzie and Milligan (1990) J. Biol. Chem. 265, 17084-17093; Adie, M ullaney, McKenzie and Milligan (1992) Biochem J. 285, 529-536]. By con trast, equivalent treatments of the cells with agonists at either the A2 adenosine receptor or the secretin receptor have no measurable effe ct on cellular amounts of G(s) alpha. To examine whether this is a fea ture specific to the IP prostanoid receptor or is related to the level of expression of the individual receptors, NG108-15 cells were transf ected with a construct containing a human beta 2-adrenoceptor cDNA und er the control of the beta-actin promoter. Two clones of these cells w ere examined in detail, beta N22, which expressed some 4000 fmol/mg of membrane protein, and clone beta N17, which expressed approx. 300 fmo l/mg of membrane protein of the receptor. Exposure of beta N22 cells t o the beta-adrenergic agonist isoprenaline resulted maximally in some 55% decrease in membrane-associated levels of G(s) alpha, without effe ct on membrane levels of G(i)2 alpha, G(i)3 alpha, G(D) alpha or G(q) alpha/G(11)alpha. Dose-response curves to isoprenaline in beta N22 cel ls indicated that half-maximal down-regulation of G(s) alpha was produ ced by approx. 1 nM agonist. Equivalent exposure of beta N17 cells to isoprenaline did not significantly modify levels of any of the G-prote in alpha subunits, including G(s) alpha. In beta N22 cells the IP pros tanoid receptor was expressed at similar levels to those in wild-type NG108-15 cells, and treatment with iloprost resulted in a similar down -regulation of cellular G(s) alpha levels. Iloprost was also effective in causing down-regulation of G(s) alpha. levels in clone beta N17. C oncurrent addition of both isoprenaline and iloprost to clone beta N22 resulted in less than additive down-regulation of G(s) alpha. These r esults demonstrate that the phenomenon of agonist-induced specific G-p rotein downregulation is determined by the levels of expression of the receptor.