NOVEL MOLECULAR SITE FOR ANTIDOPAMINERGIC EFFECTS

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These neuropharmacological studies application will study the biochemical and molecular mechanisms by which the D1 subclass of dopamine receptors cause their psychopharmacological effects. Several hypotheses will be tested: that there is biochemical heterogeneity of D1 receptors; that only a subset of these receptors are linked biochemically to cAMP synthesis; that a subset of these D1 dopamine receptors interact functionally with D2 receptors as part of the same multimolecular complex; and that it is possible to model this receptor class at the molecular level and to use such models to design drugs that have selectivity for certain subpopulations of D1 receptors. These hypotheses will be tested by studying the basis for apparent multiplicity of D1-like receptors, e.g., by comparing the occurrence of [3H]-SCH23390 binding sites versus dopamine-sensitive adenylate cyclase activity (DA-ACase) or the potency of selected drugs to cause dopaminergically-mediated behaviors. Lesioning and pharmacological studies will be used to compare limbic areas to striatum on the basis of these functional and receptor characteristics. Several series of rigid and semi-rigid dopamine agonists and antagonists will be compared using both in vivo and in vitro pharmacological methods. These data will be used in computer-assisted molecular modeling studies to model the active site of D1 receptors. Although initially these studies will assume a single receptor, it is assumed that the biological studies ultimately will provide data permitting at least two types of sites (e.g., one linked to stimulation of adenylate cyclase, and one not) to be modeled and defined. The molecular modeling studies will be modified continually to incorporate the data from receptor solubilization, purification, and characterization studies which will be a major emphasis of this research. As a consequence of our demonstration that SCH23390 binds tenaciously to its physiologically important receptor(s), the purification experiments will rely heavily on affinity chromatography using a 4-alkylphenyl-substituted analog of SCH23390 that we will synthesize. The availability of purified or partially purified receptor(s) will lead to raising of antibodies against these proteins, and to the initiation of molecular biological study of these proteins. We will perform immunohistochemical localization of these receptors, and also conduct immunoneutralization studies. The molecular biological studies will be aimed at the physiological mechanisms involved in expression and regulation of D1 receptors (e.g., synthesis, posttranslational modifications, sites of expression, etc.).