REGULATION OF HORMONE-RESPONSIVE ADENYLATE CYCLASE

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1. Exposure of mammalian cells to isoproterenol resulted in a rapid loss of agonist-stimulated adenylate cyclase activity. Desensitization was accompanied by a sequestration of Beta-adrenergic receptors into a lighter density membrane fraction inaccessible to hydrophilic antagonists. When the cells were washed free of agonist, resensitization and reappearance of the sequestered receptors occurred. Pretreatment of the cells with concanavalin A blocked sequestration but not desensitization. Using a membrane fusion technique to transfer the receptors to a foreign adenylate cyclase, we were able to show that a reduction in receptor function occurred during desensitization and in the absence of sequestration. Upon resensitization, receptor function was recovered. Thus, the key event in agonist-mediated desensitization is a reduction in receptor function. 2. Exposure of rat striatal membranes to N-ethylmaleimide (NEM) caused a loss of D-1 dopamine receptors. D-1 specific agonists or antagonists, respectively, fully and partially protected the receptors from inactivation by NEM. Upon transfer of the receptors to a foreign adenylate cyclase by membrane fusion, agonist- but not antagonist-protected D-1 receptors remained functional as measured by agonist stimulation of the cyclase. We have now succeeded in solubilizing the D-1 receptor and are using the selective protection by agonist as a means to purify the receptor. 3. Deglycosylated human chorionic gonadotropin (DG-hCG) is a potent antagonist of hCG. When DG-hCG is bound to hCG-receptors on MLTC-1 cells, addition of anti-hCG reverses the antagonism and results in stimulation of adenylate cyclase. Rabbit antibodies raised to DG-hCG cross-reacted with hCG. When the antiserum was purified on an hCG-agarose affinity column, the nonabsorbed fraction only reacted with DG-hCG and did not reverse its antagonism. In contrast, the absorbed antibodies reacted preferentially with the Beta-subunit of hCG and DG-hCG, reversed the antagonism of DG-hCG and this reversal was blocked by prior incubation of the antibodies with the Beta- but not the Alpha-subunits. Thus, epitopes on the Beta-subunit may be important for determining whether the hormone is an agonist or antagonist.