The long-term goal of this project is the biochemical and molecular characterization of dopaminergic receptor mediated information transduction, and its regulation, across neuronal membranes. Two major interrelated areas of research on Dl and D2 dopamine receptors are currently under investigation: 1. Cell Biology and Regulation of Dopamine Receptors. The functional and regulatory properties of Dl ana D2 dopamine receptors on various neuroblastoma and retinoblastoma cell lines were characterized. The Dl receptors were shown to undergo homologous desensitization in response to agonist stimulation while a heterologous desensitization was observed in response to cAMP. D2 receptors were also shown to undergo desensitization and down-regulation in response to agonist treatment. A variety of fluorescent, affinity, and antibody probes for dopamine receptors were developed. Utilizing these probes, it was shown that DI receptors were localized to most of the cells in the striatum as well as numerous fibers while the D2 receptors appeared to be confined to a smaller population of neurons. 2. Molecular Cloning of Dopamine Receptors. Investigations concerning the RNA splice variants of the D-2 dopamine receptor were continued in FY 90. A series of stably transfected cell lines were constructed which express either the short (D-2S) or long (D-2L) Isoforms of the D2 receptor. Both isoforms were shown to exhibit identical pharmacologic and functional properties with respect to adenylyl cyclase inhibition. Both isoforms were also found to augment arachidonic acid release. A cDNA encoding the Dl dopamine receptor linked to the stimulation of adenylyl cyclase was cloned and expressed. A novel subtype of the D-1 receptor was discovered by expressing rat striatal mRNA in Xenopus oocytes. This new D-1 receptor subtype is linked to the stimulation of phosphatidylinositol turnover and calcium mobilization.
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