This first aim of this competing application will be to continue to elucidate the molecular basis of ligand recognition by DA (especially the D1-like) receptors, and the functional consequences of such interactions. We shall continue to utilize several strategies of ligand-based molecular modeling (including traditional and novel Comparative Molecular Field Analysis [CoMFA] techniques) to refine existing models, and test the hypothesis of alternate binding models for these receptors. This will be complemented by structure-based receptor modeling and drug design. Site-directed receptor mutagenesis, coupled with the synthesis and use of novel rigid ligands that can probe the mutated space of the receptor, are hypothesized to provide definitive structural information about several key aspects of receptor topography. This in turn can then be used to test and refine models of the D1-like receptors. In an iterative fashion, we shall continue to develop new ligands with novel mechanisms of receptor binding or activation, and with selectivity for D1 versus D5 receptors. These ligands will be powerful tools for continued research on receptor structure and function. The second major direction of this work is to understand the consequence of drug-receptor interaction. Although our past work has provided the first full D1 agonists and an understanding of the molecular characteristics of drugs that affect their intrinsic efficacy, it is unclear what molecular mechanisms engender full versus partial agonism. We shall determine the role of activation of various isoforms of G-proteins and transduction systems to determine if partial agonism is quantal or graded in terms of activation of: (1) individual populations of G-proteins; and 2) various transduction systems (especially adenylate cyclases). Finally, we shall study the molecular mechanisms involved in desensitization and down-regulation. Despite extensive understanding of these phenomena in other G-protein systems, our work has shown that drugs of apparently similar characteristics (e.g., high affinity full D1 agonists) can cause markedly different changes over time, in both in vitro cellular systems and the intact brain. Using site directed mutagenesis, a series of in vitro and in vivo system, and a powerful armamentarium of pharmacological probes, we shall seek to determine what aspects of receptor structure are important for regulatory events. In addition, we shall determine how co-activation of one or more populations of DA D2-like receptors affects long-term adaptive changes in vivo. These studies will not only be useful for heuristic reasons, but may affect the pending use of D1 agonists in several clinical conditions.
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