CHRONIC EXPOSURE OF RAT GLIOMA C6 CELLS TO CHOLERA-TOXIN INDUCES LOSS OF THE ALPHA-SUBUNIT OF THE STIMULATORY GUANINE NUCLEOTIDE-BINDING PROTEIN (GS)

Rat glioma C6 BU1 cells were treated in tissue culture with cholera toxin. Incubation of membranes derived from these cells with fresh cholera toxin and [P-32]NAD+ failed to promote incorporation of radioactivity into polypeptides corresponding to forms of G(s)-alpha. This is generally assumed to reflect prior ADP ribosylation of these polypeptides in vivo using endogenous NAD+ as substrate. However, immunological studies with anti-peptide antisera which identify all forms of G(s)-alpha demonstrated that concentrations of this polypeptide were now substantially reduced in the membranes. This effect was specific for G(s)-alpha as neither the alpha-subunits of the pertussis toxin-sensitive G-proteins G(i)2 and G(i)3, nor the beta-subunit common to the various G-proteins were lost in parallel. Pertussis toxin-catalysed ADP ribosylation did not cause the downregulation of G(s)-alpha nor the alpha-subunits of G(i)2 or G(i)3 although it did cause ADP ribosylation of the entire complement of both G(i)2 and G(i)3 in the membranes. Despite the reduction in levels of immunoreactive G(s)-alpha from the membranes of cholera toxin-treated cells, no alterations in levels of mRNA corresponding to this G-protein were noted.