Hormones-sensitive effector systems regulate the levels of intracellular second messengers such as cyclic nucleotides and inositol phosphates in response to stimulation by hormones, neurotransmitters, and autacoids. Hormones bind to specific cell-surface receptors and modulate these effector systems via a family of heterotrimeric, GTP-binding regulatory proteins (""""""""N-proteins"""""""") that share common beta-/gamma- subunits. The N-proteins include Ns and Ni, that regulate stimulation and inhibition of adenylate cyclase, respectively, and No. The No-protein, like Ni, is a substrate for specific ADP-ribosylation by pertussis toxin. Pertussis toxin treatment results in ADP-ribosylation of Ni and No and abolishes receptor-mediated inhibition of adenylate cyclase and regulation of phosphatidylinositol (PI) metabolism in rat fat cells and 3T2-L1 cells. A major focus of this application is to define (i) to what extent N-protein-mediated pathways interact through common subunits, and (ii) how alterations at the level of N-proteins that are induced by thyroid hormones and cell differentiation express their effects on control of effector systems, adenylate cyclase and PI metabolism. The status of N-proteins will be probed by use of assays of subunit function, bacterial toxin-catalyzed modification, and antibodies against the alpha-subunits of Ni and No, and the beta-subunit. The second major goal focuses on the study of specific alterations modifying the responses of the effector systems that are expressed at the level of hormone receptors, specifically beta-adrenergic receptors. First, beta-receptors possess sulfhydryl groups and disulfide bridges that are essential for ligand binding. By using chemical analyses of pure receptor in tandem with an immunological approach that employs anti-receptor antisera to probe the structure of the beta-adrenergic receptor, we propose to investigate the role of sulfhydryl groups and disulfide bridges in receptor activation by agonist. Second, mechanisms of the long-term regulation of beta-adrenergic receptors that involve receptor function and metabolism are poorly understood. Differentiation of 3T3-L1 cells from the fibroblast to adipocyte phenotype is accompanied both by a marked increase in the number of beta-receptors and by a switch in the subtype specificity of the receptors, from beta-1 to beta-2. The nature and metabolism of these receptors will be probed using anti-receptor antisera in addition to other techniques. Third, the structural basis for the loss of function of the beta-receptor of fat cells from hypothyroid rats will be probed using purified receptor and techniques described above. These studies will aid in elucidating the biochemical basis of hormone action in normal and pathophysiological states.
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