Regulation of Thyroid Adenylate Cyclase: Guanyl Nucleotide Modulation of Thyrotropin Receptor-Adenylate Cyclase Function*

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Abstract
The role of guanyl nucleotides in regulating the hormonal responsiveness of adenylate cyclase was studied in thyroid plasma membranes. Guanyl-5′-yl-imidodiphosphate [Gpp(NH)p] alone stimulated the enzyme. At a low ATP concentration (0.2 mM), TSH alone had little or no effect, but when added with Gpp(NH)p, it resulted in a dose-dependent increase of enzyme activity. Kinetic studies revealed that Gpp(NH)p alone stimulated adenylate cyclase activity only after a 10-min lag. TSH abolished the lag, resulting in an apparent increase in activity and a lowering of the activation constant for Gpp(NH)p. GTP caused an initial increase in activity at 2 min, followed by a gradual decline below basal levels. This inhibition was not prevented by TSH. Further examination revealed that GDP caused inhibition of Gpp(NH)p-stimulated activity in a competitive manner, suggesting that conversion of GTP to GDP may be responsible for the time-dependent decay seen with GTP. To study the effects of guanyl nucleotides on coupling of the TSH receptor to adenylate cyclase, plasma membranes were preactivated with saturating amounts of Gpp(NH)p and washed extensively to remove unbound Gpp(NH)p. Incubation of preactivated membranes with either Gpp(NH)p or TSH gave no further stimulation of adenylate cyclase. However, Gpp(NH)p plus TSH produced 30% more stimulation. In contrast, the addition of TSH plus GDP to preactivated membranes led to a dose-dependent decrease in activity. Furthermore, promotion of further stimulation by TSH plus Gpp(NH)p was competitively inhibited by GDP, in the same manner as untreated membranes. This suggested that the dual action of TSH, i.e. stimulation and inhibition, is governed and mediated by specific guanyl nucleotides. Analysis of guanyl nucleotides effects on the binding of [l25I]iodo-TSH to membranes revealed that although some inhibition of binding exists, this effect is 1) unrelated to the concentration effect of nucleotides on adenylate cyclase, and 2) not specific for guanosine phosphates. Scatchard analysis of TSH binding in the presence of guanyl nucleotides demonstrated that even at high concentrations, GTP had no effect on the high affinity, low capacity receptor for TSH. These results suggest that GTP or Gpp(NH)p plays no role in modulating TSHreceptor interaction. Thus, guanyl nucleotide regulation of TSH action appears to be limited to adenylate cyclase.