GS-ALPHA IS A SUBSTRATE FOR MONO(ADP-RIBOSYL)TRANSFERASE OF NG108-15 CELLS - ADP-RIBOSYLATION REGULATES GS-ALPHA ACTIVITY AND ABUNDANCE

NG108-15 neuroblastoma x glioma somatic hybrid cells were permeabilized in the presence of [P-32]NAD+ and then cultured for 18 h. Resolution of the cell proteins on polyacrylamide gels revealed [P-32]ADP-ribosylation of five major protein species with molecular mass values of 52 kDa, 44 kDa, 35 kDa, 30 kDa and 25 kDa. A similar pattern of labelling was also seen when NG108-15 cell membranes were incubated with [P-32]NAD- and hydrolysis of the product revealed mono(ADP-ribosyl)ation. Immunoprecipitation of these products with anti-G(s)alpha antiserum revealed a single band identical to cholera toxin substrate. Culture of [P-32]NAD+-loaded cells for 18 h in the presence of 50 mm-nicotinamide inhibited the eukaryotic mono(ADP-ribosyl)transferase activity. Inhibition of the eukaryotic enzyme was also accompanied by an increase in the abundance of G(s)alpha, whether measured by Western blotting with anti-G(s)alpha antibody (two separate antisera) or by cholera toxin-dependent [P-32]ADP-ribosylation. There was no accompanying change in the abundance of Gbeta. The increase in G(s)alpha abundance in nicotinamide-treated NG108-15 cells was accompanied by a 2-fold increase in basal adenylate cyclase activity (measured in the presence of GTP), and by a smaller but significant increase in iloprost-dependent activation of adenylate cyclase. Receptor number or affinity was not affected by nicotinamide, since this treatment did not alter the binding parameters of [H-3]iloprost to NG108-15 cell membranes. Short-term exposure of cells to nicotinamide for 1 h revealed no significant difference in either basal or agonist-stimulated adenylate cyclase activity. These results reveal that mono(ADP-ribosyl)ation of G(s)alpha by eukaryotic ADP-ribosyltransferase modifies the abundance and activity of G(s)alpha in NG108-15 cells, and hence may play a role in the hormonal regulation of cell function.