Agonist regulation of cellular G(s) α-subunit levels in neuroblastoma x glioma hybrid NG108-15 cells transfected to express different levels of the human β2 adrenoceptor

Abstract

Neuroblastoma x glioma hybrid NG108-15 cells endogenously express at least three receptors which activate adenylate cyclase via the intermediacy of the stimulatory G-protein, G(s). Sustained exposure of the cells to agonists at the IP prostanoid receptor results in a substantial decrease in cellular levels of the α-subunit of G(s) (G(s)α). By contrast, equivalent treatments of the cells with agonists at either the A2 adenosine receptor or the secretin receptor have no measurable effect on cellular amounts of G(s)α. To examine whether this is a feature specific to the IP prostanoid receptor or is related to the level of expression of the individual receptors, NG108-15 cells were transfected with a construct containing a human β2-adrenoceptor cDNA under the control of the β-actin promoter. Two clones of these cells were examined in detail, βN22, which expressed some 4000 fmol/mg of membrane protein, and clone βN17, which expressed approx. 300 fmol/mg of membrane protein of the receptor. Exposure of βN22 cells to the β-adrenergic agonist isoprenaline resulted maximally in some 55% decrease in membrane-associated levels of G(s)α without effect on membrane levels of G(i)2α, G(i)3α, G(o)α or G(q)α/G11α. Dose-response curves to isoprenaline in βN22 cells indicated that half-maximal down-regulation of G(s)α was produced by approx. 1 nM agonist. Equivalent exposure of βN17 cells to isoprenaline did not significantly modify levels of any of the G-protein α subunits, including G(s)α. In βN22 cells the IP prostanoid 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)α levels. Iloprost was also effective in causing down-regulation of G(s)α levels in clone βN17. Concurrent addition of both isoprenaline and iloprost to clone βN22 resulted in less than additive down-regulation of G(s)α. These results demonstrate that the phenomenon of agonist-induced specific G-protein down-regulation is determined by the levels of expression of the receptor.