Structure-Function Analysis of the D3 Dopamine Receptor

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DESCRIPTION(Provided by applicant): The dopaminergic system and in particular the D3 dopamine receptor have been implicated in the etiology ofneuropsychiatric disorders such as schizophrenia and drug addiction. Studies using genetic, molecul biological and pharmacological techniques suggest that the D3 dopamine receptor plays an important role locomotor activity and behavioral effects involving reinforcement and reward. However the role of L receptor in neuronal signaling and the molecular mechanisms by which it modulates the physiological and behavioral effects are not well understood. It has been proposed that the D3 receptor exerts its phenotyp effects by modulating ion channel function and consequently neuronal signaling. We have recent demonstrated that the human D3 receptor expressed in the AtT-20 neuroendocrine cell line couples endogenous G-protein activated inward rectifier potassium (GIRK) channels and P/Q-type calcium channel thereby modulating spontaneous secretory activity. In this project, we plan to characterize the properties oft] D3 dopamine receptor and identify the structural features of the receptor that are involved in coupling to o about channels. Given the limited expression profile of the D3 receptor in vivo, the lack of selective pharmacologic ligands, and the need to evaluate properties of mutated D3 receptors, this study will primarily use the AtT-2 heterologous expression system. In addition, to assess physiological relevance, we will also study the I receptor-ion channel coupling in primary cultures of rat nucleus accumbens neurons using a combination electrophysiology and single cell reverse transcriptase-PCR. To define the properties of the D3 dopamine receptor, we will employ methods such as single cell electrophysiology, mutagenesis and expression recombinant and tagged receptors. We will address three specific aims: (1) Investigate the coupling of I receptor to ion channels in primary neuronal cultures from rat nucleus accumbens; (2) Identify the G-proteins and other regulatory proteins that couple the D3 receptor to ion channels and characterize the domains of t D3 receptor that interact with these proteins; and (3) Determine the molecular mechanisms underlying I receptor desensitization. These experiments will provide a better understanding of the properties of the I dopamine receptor and the molecular mechanisms by which it modulates neuronal signaling. In doing so, ti study will make significant contributions toward ongoing efforts to define the role of D3 receptor neurological disorders such as schizophrenia.