Hormonal stimulation of eucaryotic cell ADP-ribosylation.
The effect of thyrotropin (TSH) on the ADP-ribosylation of endogenous thyroid cell acceptor proteins was examined. Cells were "permeabilized" at 4 degrees C in hypotonic medium and then exposed to [(32)P]- or [(3)H-adenine]NAD(+). The net incorporation of labeled ADP-ribose was measured by trichloroacetic acid precipitation. TSH (100 mU/ml) enhanced ADP-ribosylation with a maximum effect after 30-60 min in the majority of experiments. TSH stimulation was observed even when the incubation contained 1,000-fold more exogenous NAD(+) than the amount of NAD(+) contributed by the permeabilized cells, indicating an effect on enzymatic activity rather than an alteration in NAD(+) pool size or specific activity. No incorporation of radioactivity from labeled NAD(+) was observed in cells not rendered permeable to NAD(+) by hypotonic shock. TSH did not increase the rate of disappearance of trichloroacetic-precipitable radioactivity and did not contain intrinsic NAD(+) glycohydrolase activity. Alkali and snake venom phosphodiesterase, but not ribonuclease or deoxyribonuclease digestion of trichloroacetic acid precipitable thyroid cell radioactivity, revealed primarily 5'-AMP, consistent with an effect of TSH on mono-ADP ribosylation. Nicotinamide and thymidine (50 mM) inhibited both basal and TSH-stimulated ADP-ribosylation of thyroid cell protein. Dibutyryl cyclic (c)AMP (0.1 mM) inhibited endogenous ADP-ribosylation by approximately 35% but had no effect at lower concentrations. 0.5 mM isobutylmethylxanthine inhibited this reaction by approximately 60%. We suggest that TSH enhances thyroid cell ADP-ribosylation by a mechanism independent of cAMP as a second messenger, and that ADP-ribosylation plays a role in the expression of TSH.
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002478:Cells, Cultured; D004285:Dogs; D009243:NAD / Q000378:metabolism; D009536:Niacinamide / Q000494:pharmacology; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D013936:Thymidine / Q000494:pharmacology; D013961:Thyroid Gland / Q000378:metabolism*; D013972:Thyrotropin / Q000494:pharmacology*
D009702:Nucleoside Diphosphate Sugars; D011506:Proteins; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D009536:Niacinamide; D003994:Bucladesine; D013972:Thyrotropin; D013936:Thymidine