5-HT2A receptors are implicated in the mechanism of action of a number of therapeutic agents including antidepressants and atypical antipsychotic drugs. The therapeutic responses to these drugs are delayed, implicating adaptive regulation at the level of receptors or second messenger signaling cascades. This grant proposes a comprehensive analysis integrating molecular, neurochemical and behavioral studies of the 5-HT2A receptor and its close relative, 5-HT2A receptors. Such a multilevel strategy is crucial to an evaluation of the significance of cellular alterations in receptor density or function. We have recently found evidence in cultured cells that the native 5-HT2A and 5-HT2C receptors exhibit spontaneous activity in the absence of agonist (constitutive activity) and that some but not all 5-HT2A antagonists decrease constitutive activity. Behavioral studies indicate that these functionally distinct properties are physiologically significant. This discovery may lead to novel approaches in the development of therapeutic agents.
Five specific aims are proposed (1) to identify the G protein(s) that couples to 5-HT2A and 5-HT2C receptors and the phospholipase C isoforms present in brain regions rich in these receptors. Receptor and G protein alpha subunit cDNAs will be cotransfected into cells to determine which G proteins have the potential for coupling to 5-HT2A and 5-HT2C receptors. These results will guide the design of experiments to determine which G protein actually couples to the 5-HT2A and 5-HT2C receptor in rat brain. The expression of specific phospholipase Cbeta mRNAs in brain will be analyzed by RNase protection. (2) to determine the mechanism of the constitutive activity of 5-HT2A and 5-HT2C receptors and the negative intrinsic activity of receptor antagonists. Receptor-G protein coupling and changes in the phosphorylation state of the receptors will be evaluated. Using a mutagenesis strategy, structural features that confer constitutive activity will be explored. (3) to evaluate the importance of constitutively active 5-HT2A and 5-HT2C receptors. Two models will be used: the elevated plus-maze and transforming properties in cell lines. (4) to investigate the mechanisms of postreceptor regulation of the 5-HT2A receptor signal cascade by selective serotonin uptake inhibitors. Components of the 5-HT2A receptor second messenger pathway will be evaluated using RNase protection. (5) to determine the behavioral relevance of the neurochemical adaptations of the 5-HT2A receptors, using a drug- discrimination paradigm. These studies will provide a better understanding of the action of currently available drugs and perhaps contribute to the development of new drugs with a novel mechanism of action.
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