Hyperactivity of dopamine receptor systems has been implicated in a variety of neuropsychiatric disorders and may result in alterations in the function of signaling proteins such as adenylate cyclase. Acute activation of D2-like dopamine receptors inhibits cyclic AMP accumulation; however, short- (2 hr) and long- (18 hr) term activation induces heterologous sensitization of certain isoforms of adenylate cyclase. This heterologous sensitization is blocked by pertussis toxin treatment, indicating the involvement of Galphai/o proteins. It is hypothesized that persistent activation of Galphai/o-linked receptors leads to enhanced Galphas-adenylate cyclase coupling and that there are mechanistic differences between short- and long-term sensitization that involve decreased Galphai protein subunits. These hypotheses will be addressed in the proposed experiments that will manipulate the expression of recombinant Galphai/o, adenylate cyclase isoforms, and mutant Galphas in characterized cell lines expressing D2 receptors to elucidate the molecular mechanisms involved in both short- and long-term sensitization by D2 receptors. The first objective is to define the Galphai/o subunits (Galphai1, Galphai2, Galphai3 or Galphao) specificity for D2 receptor-induced short- and long-term sensitization in neuronal cells. This objective will be accomplished by expressing individual pertussis-toxin -insensitive Galpha subunits in cells in which endogenous Galphai/o proteins are inactivated. The effects of short- and long-term agonist exposure on G protein subunit levels will also be determined by quantitative immunoblotting. The second objective is to examine the adenylate cyclase isoform specificity and potential mechanisms for short- and long-term sensitization of wild type and mutant recombinant isoforms of adenylate cyclase. These studies will use cell lines expressing D2 receptors and individual neuronal isoforms of adenylate cyclase, and take advantage of the observation that each adenylate cyclase isoform has a distinct pattern of regulation by Ca2+, PKC, Galphas, and beta/gamma subunits. Finally the hypothesis that sensitization of adenylate cyclase requires Galphas activation and Galphas-adenylate cyclase interactions will be addressed. For these studies the effectors of several Galphas mutants on heterologous sensitization in cell lines expressing recombinant D2 receptors and adenylate cyclase will be examined. D2 receptors and mutant Galphas proteins will also be expressed in S49 cells and cyc- mutants of these cells (lacking Galphas) to examine the requirement for Galphas in heterologous sensitization. The goals of the present studies are to examine and elucidate the biochemical pathways and mechanism(s) responsible for both short- and long-term sensitization of adenylate cyclase by D2 receptors. The data that we obtain are likely to increase out understanding of the pathophysiology of central nervous system disorders and may also lead to improved treatment strategies.
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