ADP ribosylation and G protein regulation in the thyroid.
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Adenosine Diphosphate Ribose↗
Biomedical subjects
F Ribeiro-Neto
Publications and source records attributed to F Ribeiro-Neto.
Prevention of alpha 2-adrenergic inhibition on ADH action by pertussis toxin in rabbit CCT.
The present studies were performed to investigate the mechanism whereby alpha 2-adrenergic receptor occupancy inhibits the hydrosmotic action of antidiuretic hormone (ADH) in isolated cortical collecting tubules (CCT). The ADH-ribosyltransferase activity of pertussis toxin (PT) was used to promote covalent modification in CCT Ni, the inhibitory regulatory protein of adenylate cyclase, which presumably mediates the alpha 2-adrenergic inhibition of water flow. Tubules preincubated with PT were studied after the addition of ADH and then after the superimposition of clonidine. In these studies, the inhibition of Jv (water absorption, nl X mm-1 X min-1) and Pf (water permeability coefficient, cm/s), by the addition of 10(-4) M clonidine to the bath, was attenuated by PT in a concentration-dependent manner. Reversal of the inhibitory action of clonidine was accomplished with a concentration of 1.0 micrograms/ml PT. To further elucidate the molecular basis of Ni-mediated transduction of the alpha 2-adrenergic signal, ADP-ribosylation studies were undertaken in membrane preparations of dissected CCT segments. PT ADP ribosylated a 40,000 Mr peptide which was proportional to the amount of membrane protein added. Furthermore, pretreatment of CCT during dissection with 0.5 micrograms/ml PT dramatically decreased the susceptibility of the subunit of Ni (alpha i) to be subsequently ADP ribosylated by PT, when compared with CCT preparations not previously treated with PT. Cholera toxin ADP ribosylated a 42,000 Mr peptide from CCT membranes and PT pretreatment did not interfere with the reaction. We conclude that CCT segments have both the pertussis and cholera toxin substrates and the effect of clonidine to attenuate ADH action is mediated through Ni.
Adenosine Diphosphate↗
Adenosine diphosphate ribosylation of G proteins by pertussis and cholera toxin in isolated membranes. Different requirements for and effects of guanine nucleotides and Mg2+.
ADP ribosylation of membranes by pertussis toxin (PT) and cholera toxin (CT) was studied as a function of addition of ATP, various guanine nucleotides, Mg2+, and inorganic phosphate (Pi). ADP ribosylation of a 40 kilodalton (kDa) band by PT is markedly enhanced by ATP and GTP and is strongly inhibited by Pi or Mg2+. GTP analogs (GTP gamma S and GMP-adenyl-5'-yl imidodiphosphate) were less effective. In contrast, ADP ribosylation of two substrates for CT (of 42 and 50 kDa) is stimulated by Pi, Mg2+, and GTP or GTP analogs such as GTP gamma S, but is unaffected by ATP. These stimulatory conditions correlate well with GTP-mediated activation of stimulated nucleotide-binding regulatory component of adenyl cyclase. Optimal conditions for ADP ribosylation by PT do not correlate simply with conditions thought to lead to stabilization of an inactive form of inhibitory nucleotide-binding regulatory component of adenyl cyclase (Gi) or Gi-like protein; rather, the data suggest the involvement of both a stimulatory nucleotide site on PT (positively affected by either ATP or GTP) and a stabilizing site on the PT substrate (affected by GDP, GDP beta S, or GTP). Treatment of membranes with Lubrol PX increased ADP ribosylation by PT by as much as 25- to 30-fold, but inhibited the action of CT. Using defined conditions for ADP ribosylation by PT and CT, distinct labeling patterns were observed in thyroid, brain, corpus luteum, liver, heart, and erythrocytes membranes. All membranes were more intensely labeled by PT rather than CT.
Adenosine Diphosphate Ribose↗
Incubation of bovine thyroid slices with thyrotropin is associated with a decrease in the ability of pertussis toxin to adenosine diphosphate-ribosylate guanine nucleotide regulatory component(s).
Pretreatment of bovine thyroid slices with TSH resulted in desensitization of TSH-sensitive adenylyl cyclase activity but no change in stimulatory nucleotide binding regulatory component of adenylyl cyclase (Gs) activity assessed by reconstitution of the Gs-defective cyc-S49 adenylyl cyclase system. Possible changes in substrates for pertussis toxin (PT)-induced ADP ribosylation due to TSH treatment and/or in endogenous ADP ribosylation of membrane proteins were explored. Using 10 microM [32P]NAD+ as substrate, endogenous ADP ribosylation was not observed in membranes from control or TSH-treated slices. ADP ribosylation of alpha-subunits of Gs by cholera toxin was also unaffected by incubation of thyroid slices with TSH. In contrast, ADP ribosylation of 40 kilodalton (kDa) substrates for PT was decreased between 40% and 60% by TSH treatment. This effect of TSH was dependent on its concentration and the time of incubation of the slices and was specific for labeling of the 40 kDa PT substrate. Prostaglandin E1 treatment of thyroid slices, which results in a much smaller homologous desensitizing effect, did not result in changes in ADP ribosylation by PT. The effect of incubation of slices with TSH was abolished by pretreatment of the membranes with 0.3-1.0% Lubrol PX, which increased the labeling of the 40 kDa polypeptides. The data suggests that TSH induces in thyroid tissue a redistribution of 40 kDa polypeptides changing their availability to PT.
Adenosine Diphosphate Ribose↗