The overall goal of this research program is to investigate the regulation of receptors for neurotransmitters and hormones. These experiments will emphasize studies of Beta-adrenergic receptors. Activation of these receptors results in an increase in adenylate cyclase activity and ultimately, in an increase in intracellular concentrations of cyclic AMP. The effects of agonists appear to involve formation of a ternary complex composed of agonist, receptor (R), and guanine nucleotide binding protein (G/F). The principal aim of this program is to increase our understanding, at a molecular level, of the factors that regulate the coupling of agonist occupied receptors to the guanine nucleotide binding regulatory protein. An appreciation of these interactions and the factors that modulate them is central to an understanding of information transfer across cell membranes. In this proposal experiments are described that utilize radiolabelled agonists and antagonists. The properties of the binding of these radioligands will be assessed both in membrane preparations and in intact cells. L6 myoblasts will be used for some of these experiments to take advantage of the high density of receptors on these cells. Other experiments will utilize S49 lymphoma cells and the UNC and AC- variants of these cells which are missing or have a defective G/F protein. The mechanism underlying the marked decrease in the affinity of receptors on intact cells for some full agonists that occurs during a binding assay will be explored. Determination of the properties of receptors will involve both kinetic and equilibrium determinations. Studies of the temperature dependence of the interactions of agonists and antagonists with Beta-adrenergic receptors will be carried out. The equilibrium and transition state thermodynamic parameters of these interactions will then be determined. The molecular basis for the anomalous kinetics sometimes observed in studies of the binding of radiolabelled antagonists will be investigated. The possibility that R and G/F are precoupled will be explored and the stoichiometry of R and G/F will be investigated. The effects of experimentally varying this stoichiometry will be determined. The effects of ions, nucleotides, alkylating agents, and reducing agents will also be explored. Effects of these agents on the interactions of agonists and antagonists will be compared to try to increase our understanding, at a molecular level, of the way in which the interactions of receptors with the guanine nucleotide binding protein are regulated.
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