Molecular Pharmacology, Vol 16, 709-718, Copyright © 1979 by the American Society for Pharmacology and Experimental Therapeutics

Multiple Reactive Sulfhydryl Groups Modulate the Function of Adenylate Cyclase Coupled Beta-Adrenergic Receptors

¹ From the Howard Hughes Medical Institute Laboratory, Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

The beta-adrenergic receptor adenylate cyclase complex in the frog erythrocyte contains at least two reactive sulfhydryl groups, which have been identified by their different sensitivities to N-ethylmaleimide (NEM). Adenylate cyclase catalytic activity is completely inhibited by [NEM] = 1 mM. In contrast, ability of beta-adrenergic agonists to form a high affinity guanine nucleotide sensitive state is reduced by NEM only at concentrations 1 mM. The inhibition of cyclase activity did not affect high affinity agonist binding nor perturb the ability of guanine nucleotides to reduce agonist affinity as determined in direct radioligand binding assays utilizing the beta-adrenergic agonist [³H]hydroxybenzylisoproterenol and (-)isoproterenol competition for antagonist [³H]dihydroalprenolol binding. Preincubation of frog erythrocyte membranes with antagonists or guanine nucleotide did not alter the effect of NEM on agonist affinity. However, once formed by preincubation of membranes with agonist, the high affinity state is resistant to the effects of NEM. The data suggest that the sulfhydryl group modulating agonist affinity 1) is located on the receptor complex but distal to the hormonal binding site and 2) may be associated with guanine nucleotide regulation of agonist affinity.

The widely different sensitivities of adenylate cyclase activity and agonist binding affinity toward NEM have been exploited to investigate the mechanism of desensitization in frog erythrocytes. Incubation of intact erythrocytes with NEM under appropriate conditions inactivates the cyclase enzyme without altering the ability of membranes prepared from these cells to bind agonist with high affinity. This NEM treatment prior to prolonged exposure to (-)isoproterenol prevents receptor desensitization in frog erythrocytes, suggesting that agonist occupancy of a nucleotide-sensitive receptor is insufficient, by itself, to initiate the receptor regulation process.

Note:
ACKNOWLEDGMENTS The authors wish to thank Dr. Lee Limbird for critically reading the manuscript and Ms. D. Addison for helping to prepare the manuscript.