Adaptive changes in the responsiveness of adrenergic receptors are thought to be involved in the pathology and/or therapeutic treatment of a variety of disease states, ranging from cardiovascular and pulmonary to neuropsychiatric. Similar changes have been shown to occur in isolated cells in culture, where the cellular and molecular mechanisms involved can be more easily investigated. Exposure of mammalian cells in culture to catecholamines or other adrenergic receptor agonists leads to desensitization of beta-adrenergic receptors and their coupling to adenylate cyclase and to desensitization of alpha1-adrenergic receptors and their coupling to polyphosphoinositide turnover. Recent studies indicate that regulatory interactions also occur between receptors coupled to different signal transduction mechanisms. The proposed studies are aimed at understanding the mechanisms involved in these changes in adrenergic receptor responsiveness, with an emphasis on the likely involvement of protein phosphorylation. Studies of beta-adrenergic receptor regulation will focus on 1321N1 human astrocytoma cells. Studies of alpha1-adrenergic receptor regulation will focus on DDT1 MF-2 hamster smooth muscle cells. Regulatory interactions will be studied in both cell lines. 1) The apparent desensitization of alpha1-adrenergic receptors will be further characterized using approaches similar to those applied previously to beta-adrenergic receptor desensitization, including studies of specificity, guanine nucleotide-binding protein involvement, and receptor internalization. 2) The ability of drugs activating different receptors to cause translocation of beta-adrenergic receptor kinase and protein kinase C will be determined. 3) Inhibitors of protein kinases will be used to directly determine whether phosphorylation is required for desensitization and elucidate the site(s) of involvement of phosphorylation in response regulation. 4) The mechanisms of agonist-induced changes in receptor binding properties measured on intact cells and their relationship to receptor internalization and phosphorylation will be further investigated. 5) The occurrence, significance, and molecular mechanism(s) of sensitization of adenylate cyclase activity by activators of polyphosphoinositide turnover and by activators of protein kinase C will be further investigated.
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