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, Gs. Sustained exposure of the cells to agonists at the IP
prostanoid receptor results in a substantial decrease in cellular levels of the alpha-subunit of Gs (Gs alpha) [McKenzie and Milligan (1990) J. Biol. Chem. 265, 17084-17093; Adie, Mullaney, McKenzie and Milligan (1992) Biochem J. 285, 529-536]. 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 Gs alpha. 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 beta 2-adrenoceptor
cDNA under the control of the
beta-actin promoter. Two clones of these cells were examined in detail, beta N22, which expressed some 4000 fmol/mg of
membrane protein, and clone beta N17, which expressed approx. 300 fmol/mg of
membrane protein of the receptor. Exposure of beta N22 cells to the
beta-adrenergic agonist isoprenaline resulted maximally in some 55% decrease in membrane-associated levels of Gs alpha, without effect on membrane levels of Gi2 alpha, Gi3 alpha, G(o) alpha or Gq alpha/G11 alpha. Dose-response curves to
isoprenaline in beta N22 cells indicated that half-maximal down-regulation of Gs alpha was produced by approx. 1 nM agonist. Equivalent exposure of beta N17 cells to
isoprenaline did not significantly modify levels of any of the
G-protein alpha subunits, including Gs alpha. In beta N22 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 Gs alpha levels.
Iloprost was also effective in causing down-regulation of Gs alpha levels in clone beta N17. Concurrent addition of both
isoprenaline and
iloprost to clone beta N22 resulted in less than additive down-regulation of Gs alpha. These results demonstrate that the phenomenon of agonist-induced specific
G-protein down-regulation is determined by the levels of expression of the receptor.