A widespread distribution of hormone receptors which inhibit rather than stimulate adenylate cyclase activity has been noted in recent years. Modulators which may utilize this inhibitory pathway include opiates, adenosine, dopamine, prostaglandins, muscarinic cholinergic and alpha-adrenergic effectors. Functional requirements for the expression of inhibition are distinct from those of stimulation. However, it is unclear whether distinct sets of hormone receptors and GTP regulatory proteins are associated with this inhibitory regulation. Primary goals of the present proposal are: i) to characterize the interaction of the alpha2-adrenergic and adenosine-Ri receptors with GTP regulatory proteins (Ni) in promoting inhibition of adenylate cyclase in platelets and adipocytes, respectively; ii) to solubilize inhibitory receptors and GTP regulatory proteins; and iii) to compare the properties of the inhibitory proteins with what is known of their counterparts which stimulate adenylate cyclase. Achievement of these goals is dependent on the development of functional assays for the regulatory components either when present in membranes or following solubilization. The role of GTP hydrolysis in inhibitory regulation will also be assessed. Hydrolysis of GTP plays a central role in stimulation of the enzyme; preliminary results indicate a separate role in inhibition. Kinetic studies of enzyme activity will be performed on the interaction of GTP and the nonhydrolyzable analog, GPPNHP. These studies are intended to expand our insights into the structural and kinetic properties of dually regulated adenylate cyclase systems. The long term outcome of these studies will be to provide an understanding, which is presently not available, of the basic mechanism of action of a large group of hormones and neurotransmitters.
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