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

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Abstract
Treatment of NG108‐15 neuroblastoma × glioma cells (24 h) with cholera toxin (0.1 – 10 μg/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 Goα and Gβ1 were reduced by 40 – 50%, whereas those of Gαcommon immunoreactivity and Gi2α were only reduced by 10 – 20% following treatment with 10 μg/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 (0.01 – 10 ng/ml) and then decreased at high (0.1 – 10 μg/ml) concentrations. Thus, the down regulation of G‐proteins 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 Goα 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 Gβ1 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 Goα and Gβ1 subunits in the membrane and thus alter by an additional mechanism the function of inhibitory receptor systems.
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