The objective of the proposed research is to elucidate the molecular mechanisms involved in desensitization of adrenergic receptors by catecholamines and other agonists. Previous studies in isolated cells in culture have documented several distinct steps involved in the overall process of desensitization; however, the molecular nature of the changes involved remains unknown. Recent studies indicate the widespread occurrence and clinical significance of desensitization of adrenergic receptors in various human tissues, in particular heart, lung, leukocytes, and lymphocytes. Furthermore, preliminary studies suggest that the cellular mechanisms involved in desensitization in these tissues in intact animals are probably similar to those demonstrated in cultured cells. Thus, further studies of the molecular mechanisms involved in desensitization in cultured cell lines, which are more amenable to study, will likely lead to information that will be of relevance to understanding desensitization in various tissues in man. The proposed studies will use recently developed techniques involving short-time assays with intact cells to study the molecular events involved in desensitization of beta-adrenergic receptors (BAR). These assays will further investigate the involvement of internalization of BAR in desensitization of cultured human astrocytoma cells (1321N1) and will be used to identify inhibitors of the various biochemical reactions involved in desensitization. The possible involvement of the cytoskeletal system, cyclic-AMP-independent phosphorylation, transmethylation reactions, and various ions and ion gradients in desensitization will be investigated. Other studies will attempt to develop broken cell systems in which biochemical studies of receptor modification can be carried out. Finally, the intact cell binding techniques developed for study of BAR will also be applied to a study of the likely involvement of similar changes in alpha-1-adrenergic receptors on intact cells in culture.
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