The long term goal of this project is to characterize neurotransmitter receptor-mediated information transduction, and its regulation, across neuronal membranes. The primary receptor systems under investigation are those for dopamine. In order to characterize these receptors at the biochemical and molecular levels, and study their regulation, there are two interrelated lines of research being performed: 1) investigation of the cell biology, function and regulation of the receptors at the protein level; and 2) the molecular cloning of the receptor cDNAs/genes and investigation of receptor structure, pharmacology and regulation in cultured cell lines and transgenic mice. In FY99, the role of protein phosphorylation in regulating D1 receptor function was examined using metabolic labeling techniques. We created an epitope-tagged D1 receptor construct by adding the FLAG peptide sequence to the NH2 terminus of the rat receptor. This receptor construct was stably expressed to high levels in C6 glioma cells. Prior investigations by this laboratory, and others, have shown the D1 receptor to undergo rapid (t1/2 = 10-15 min) agonist-induced desensitization in C6 cells which is characterized by a reduction in agonist potency and maximum cAMP response. In our present studies, we used metabolic labeling to perform whole cell phosphorylation assays in which the receptor is immunopurified using antisera directed against the FLAG epitope. We find that the D1 receptor is phosphorylated under basal conditions and that its phosphorylation state is increased by about 3-fold upon pre-exposure of the cells to dopamine. The rate of agonist-induced phosphorylation is rapid, t1/2 < 1 min, and is much faster than the rate of agonist- induced desensitization. This suggests that receptor phosphorylation is not the rate limiting step for receptor desensitization. The dopamine- induced receptor phosphorylation is blocked by D1-selective antagonists and is mimicked by D1-selective agonists. The recovery of receptor phosphorylation to basal levels upon agonist removal is also rapid (t1/2 = 6-7 min) and is not blocked by agents (i.e., Con A) which inhibit receptor internalization. This suggests that receptor internalization may not be required for receptor de-phosphorylation. - dopamine, receptor, desensitization, mutation, transgenic
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