Cholera toxin differentially decreases membrane levels of α and β subunits of G proteins in NG108‐15 cells

Abstract

Treatment of NG108-15 neuroblastoma .times. glioma cells (24 h) with cholera toxin (0.1-10 .mu.g/ml) resulted in a concentration-dependent reduction of the membrane levels of subunits of GTP-binding regulatory proteins (G-proteins), as determined by quantitative immunoblot procedures. The extent of reduction differed for different types of subunits: the levels of Go.alpha. and G.beta.1 were reduced by 40-50%, whereas those of G.alpha.common immunoreactivity and Gi2.alpha. were only reduced by 10-20% following treatment with 10 .mu.g/ml cholera toxin. This effect of the toxin could not be mimicked by incubation with the resolved B oligomer of cholera toxin, nor by exposure of cells to agents able to raise the intracellular levels of cAMP. Basal adenylate cyclase was stimulated in a biphasic manner by cholera toxin, being stimulated at low concentrations (0.1 - 10 ng/ml) and then decreased at high (0.1 - 10 .mu.g/ml) concentrations. Thus, the down regultion of G-protein subunits produced by cholera toxin requires its (ADP-ribosyl)transferase activity but does not result from a cAMP-mediated mechanism. The toxin-mediated decrease of GO.alpha. in the membrane was correlated with a diminution of opioid-receptor-mediated stimulation of high-affinity GTPase activity, suggesting that opioid receptors interact with Go in native membranes of NG108-15 cells. Northern-blot analysis of cytoplasmic RNA prepared from cells treated with cholera toxin showed that the levels of mRNA coding for G.beta.1 did not change. Thus, the cholera-toxin-induced decrease of G-protein subunits may not result from an alteration in mRNA levels, but may involve a direct effect of the toxin on the process of insertion and/or clearance of G proteins into and/or from the membrane. These data indicate that cholera toxin, besides catalyzing the ADP-ribosylation of Gs and Gi/Go types of G proteins, can also reduce the steady state levels of Go.alpha. and G.beta.1 subunits in the membrane and thus alter by an additonal mechanism the function of inhibitory receptor systems.