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Endocrinology, Vol 119, 1805-1809, Copyright © 1986 by Endocrine Society


ARTICLES

Guanyl nucleotide regulation of human chorionic gonadotropin binding to rat luteal tissue

PJ McIlroy and RP Stewart

Guanyl nucleotides are known to have a dual effect on most hormone- sensitive adenylate cyclase systems, regulating activation of the adenylate cyclase enzyme and binding of hormone to receptor. In the ovary, guanyl nucleotides have been shown to be required for hCG stimulation of luteal adenylate cyclase, but no effect on binding has been observed. Evidence has been obtained which suggests that guanyl nucleotide regulation of hCG binding is masked in most luteal membrane preparations by the presence of a large excess of unregulated receptor. A highly purified urea-washed membrane fraction has been prepared. Binding of hCG to this preparation was decreased (approximately 45%) in the presence of 5'-guanylylimidodiphosphate (GMPPnP). The maximal effect of GMPPnP occurred at 10 microM, with the half-maximal effect at 0.2 microM. These levels compare favorably with the GTP concentrations required for hCG stimulation of luteal adenylate cyclase. Analysis of equilibrium binding experiments showed that GMPPnP acted by reducing the number of binding sites by 45%. However, kinetic experiments suggested that this effect was due to a significant decrease in the affinity of a fraction of the hCG-binding sites. Association of hCG and its receptor was unaffected by the presence of GMPPnP (100 microM), whereas the dissociation of 40-50% of bound hormone was significantly accelerated (30-fold) by its presence. The guanyl nucleotide effect required the presence of MG+2; other divalent cations (Ca+2, Mn+2, and Co+2 could not be substituted. The ratio of beta-adrenergic to hCG- binding sites in the urea-washed heavy membrane preparation was elevated, suggesting that a sizable fraction of uncoupled hCG receptor had been removed. The results show that hCG binding to its luteal receptor is modulated by guanyl nucleotide and suggest that the modulation only occurs in those receptors that are directly coupled to the adenylate cyclase enzyme.





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