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 [32P]NAD+ and then cultured for 18 h. Resolution of the cell proteins on polyacrylamide gels revealed [32P]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 [32P]NAD+ and hydrolysis of the product revealed mono(ADP-ribosyl)ation. Immunoprecipitation of these products with anti-Gs alpha antiserum revealed a single band identical to cholera toxin substrate. Culture of [32P]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 Gs alpha, whether measured by Western blotting with anti-Gs alpha antibody (two separate antisera) or by cholera toxin-dependent [32P]ADP-ribosylation. There was no accompanying change in the abundance of G beta. The increase in Gs 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 [3H]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 Gs alpha by eukaryotic ADP-ribosyltransferase modifies the abundance and activity of Gs alpha in NG108-15 cells, and hence may play a role in the hormonal regulation of cell function.
D036002:ADP Ribose Transferases / Q000378:metabolism*; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D015153:Blotting, Western; D002772:Cholera Toxin / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005910:Glioma; D006822:Hybrid Cells; D016285:Iloprost / Q000378:metabolism / Q000494:pharmacology; D007163:Immunosorbent Techniques; D009243:NAD / Q000378:metabolism; D009447:Neuroblastoma; D009536:Niacinamide / Q000494:pharmacology; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D009243:NAD; D000246:Adenosine Diphosphate Ribose; D009536:Niacinamide; D002772:Cholera Toxin; D036002:ADP Ribose Transferases; D019204:GTP-Binding Proteins; D016285:Iloprost