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Coupling of the Canine Renal Parathyroid Hormone Receptor to Adenylate Cyclase: Modulation by Guanyl Nucleotides and iV-Ethylmaleimide^*

ANNE P. TEITELBAUM, ROBERT A. NISSENSON and CLAUDE D. ARNAUD

Endocrine Unit, Medical Service, Veterans Administration Medical Center, San Francisco, California 94121, and the Departments of Medicine and Physiology, University of California, San Francisco, California 94143

Address requests for reprints to: Dr. Anne Teitelbaum, Endocrine Unit, Veterans Administration Medical Center, 4150 Clement Street (111N), San Francisco, California 94121.

Abstract

Guanyl nucleotides greatly enhance PTH stimulation of renal adenylate cyclase activity. As part of an investigation of the mechanism of this potentiation, we evaluated the effect of guanosine triphosphate (GTP) and a GTP analog, guanylimidodiphosphate [Gpp(NH)p] on the specific binding of ^l25I-labeled bovine (b) PTH-(l–34) to canine renal cortical plasma membranes. Dissociation of [^l25I]bPTH-(l–34) from receptors was slow (t 200 min) in the absence of added guanyl nucleotide, whereas in the presence of GTP or Gpp(NH)p (100 µM), dissociation was rapid (t 1 min). The increase in dissociation rate prompted by Gpp(NH)p was reflected in a 3- to 4-fold decrease in PTH receptor affinity, from 5 nM to 17 nM. In contrast to the slow dissociation rate of the ^l25I-labeled agonists [Nle⁸,Nle¹⁸,Tyr³⁴]bPTH-(l–34)amide and bPTH(l–34), the dissociation rate of the ^l25I-labeled antagonist analog of PTH [Nle⁸,Nle¹⁸,Tyr³⁴]bPTH-(3–34)amide was rapid (t 2 min). Moreover, binding of the labeled PTH antagonist analog was not affected by 100 ;µM Gpp(NH)p, whereas specific binding of the labeled PTH agonist analog to membranes was decreased by 40% within 2 min. These data suggest that guanyl nucleotide regulation of PTH receptors is agonist specific.

N-Ethylmaleimide (NEM), a sulfhydryl alkylating reagent that blocks the coupling of hormone receptors to adenylate cyclase was used to obtain supportive evidence for the notion that formation of the guanyl nucleotide-sensitive state of the PTH receptor is required for coupling of the receptor to adenylate cyclase. NEM (100 µM) almost completely blocked the Gpp(NH)p enhancement of the dissociation rate of labeled PTH from receptors and reduced PTH-stimulated adenylate cyclase activity, whereas 10 JUM NEM was ineffective, and the effect of 30 µM on PTH binding and enzyme activity was intermediate. The slow dissociation rate of [^l25I]bPTH-(l–34) measured in the absence of exogenous guanyl nucleotides was unaffected by concentrations of NEM up to 10 mM. Thus, NEM reduced the Gpp(NH)p effect on PTH binding and decreased PTH-stimulated adenylate cyclase activity over the same concentration range. These data suggest that hormonal activation of the enzyme is associated with the ability of guanyl nucleotides to regulate PTH receptors and support the notion that the formation of a guanyl nucleotide-sensitive state of the PTH receptor is necessary for PTH activation of adenylate cyclase.

Footnotes

^* This work was supported in part by the Veterans Administration, by NIH Grants AM-21614 and AM-27755, and by the Academic Senate Committee on Research of the University of California, San Francisco and was presented in part a.t the National Meeting of the American Federation for Clinical Research, Washington, D.C., 1980.

Recipient of a USPHS Postdoctoral Fellowship

Received February 22, 1982.

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