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-related cDNAs/genes and investigation of receptor structure, pharmacology and regulation in cultured cell lines and transgenic miceIn FY-2000, the mechanisms of agonist-induced regulation of D1 and D2 receptors were further investigated. We attempted to identify the sites of regulatory phosphorylation (PHOS) in the D1 dopamine receptor (DAR) using mutagenesis techniques and expression in HEK-293 cells. We created two 3rd cytoplasmic loop mutants, one in which all serine and threonine residues were mutated (3rdTOT) and another in which just serines 256, 258 & 259 were mutated (3rd234). We also prepared a series of mutants where the D1 DAR was truncated subsequent to the indicated positions in the carboxyl terminus: T347, T369, T394 & T404. Expression of the mutant and wild-type (WT) constructs revealed similar levels of receptor binding except for the T347 mutant which was 50% lower. Dopamine (DA) stimulated cAMP accumulation in a similar fashion for all constructs except for the 3rdTOT mutant which showed a lower potency for DA. DA-induced desensitization (DES) was investigated for the 3rdTOT and T347 mutants. Interestingly, DES of the 3rdTOT and T347 mutants was impaired and enhanced, respectively, compared to WT. DA-induced PHOS of all constructs was investigated. Relative to WT, the 3rdTOT mutant exhibited a 50% decrease which was mimicked by the 3rd234 mutant. This suggests that serines 256, 258 & 259 account for the majority of 3rd loop PHOS. Mutant T404 also exhibited a 50% decrease in PHOS with further reductions observed for each of the truncation mutants. Surprisingly, PHOS of the T347 mutant was barely detectable. These results suggest that PHOS of carboxyl terminal residues is necessary for PHOS of 3rd loop residues. The mechanism of DES in the T347 mutant is currently under investigation. To investigate the intracellular trafficking of the D2 receptor in response to DA treatment, we constructed D2-YFP chimeras in which YFP was fused to the carboxyl termini of the human D2L and D2S receptors. Expression of these constructs in HEK293 cells revealed receptor binding characteristics identical to those of wild-type receptors. Fluorescent visualization of the expressed D2-YFP constructs was performed via confocal microscopy and a live-cell, real-time format. Under basal conditions, the D2L-YFP receptor is expressed in the plasma membrane (PM), however, significant levels of cytoplasmic (CP) fluorescence (FL) is also observed. After DA treatment, the PM FL is decreased and more diffuse whereas the CP FL is increased and more punctate. Similar results are observed with the D2S-YFP construct. We also co-expressed the D2L-YFP receptor with GRKs 2, 3, 5 or 6, or arrestin 2 or 3. Co-expression with any of the GRKs, or either of the arrestins, results in a decrease in PM FL and an increase in CP FL such that the D2L-YFP receptor is mostly intracellular. In GRK-2 and/or arrestin-2 co-transfected cells, DA exposure results in a slight further decrease in PM FL and increase in CP FL. In other experiments, we found that DA treatment results in a doubling of the D2L receptor binding activity. These results suggest that the DA-induced D2 receptor up-regulation is not due to recruitment of CP receptors to the PM.
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