The process of agonist-induced desensitization of hormone-sensitive adenylate cyclase systems in cultured human and animal cells will be investigated. Previous studies have established that cells regulate the level of their response to hormones and neurohormones in a manner that compensates for the effects of prolonged exposure to excessively high or low concentrations of the effector. Our previous work has shown that catecholamine-induced desensitization of the B-adrenergic receptor/adenylate cyclase system of human astrocytoma and mouse lymphoma cells involves a multistep process which includes an initial uncoupling of the receptor from adenylate cyclase followed by an actual loss of the receptor from the cell membrane. In this proposal, we outline a series of experiments to define the chemical modification of the B-receptor that results in uncoupling. This reaction exhibits characteristics consistent with an enzyme catalyzed covalent modification of the B-receptor. Therefore, we will initially determine if the B-receptor is phosphorylated, or alternatively methylated, during the early stages of the desensitization process. We also have shown that concomitant with the uncoupling process the B-receptor is converted to a membrane-bound form with sedimentation properties that allow its separation from native receptors upon sucrose gradient centrifugation. We propose to determine the characteristics of B-receptors in this membrane fraction in terms of their binding properties, susceptibility to detergent extraction and functional capacity. The desensitization process will be studied not only utilizing intact cells but will involve the use of isolated plasma membrane preparations. The development of an isolated membrane system that exhibits agonist-induced desensitization similar to that observed in whole cells should greatly facilitate the determination of the molecular mechanisms involved.
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