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. The role of cAMP-mediated phosphorylation in regulating D1 receptor function was further examined using site-directed mutagenesis techniques. We had previously found that when all four protein kinase A (PKA) phosphorylation sites in the receptor were eliminated, the mutant receptor exhibited slower kinetics of desensitization subsequent to dopamine exposure. Further analyses of single mutated receptors, in which only one of the four sites is modified, reveals that Thr-268 in the 3rd cytoplasmic loop of the receptor is primarily responsible for the effects of PKA. Evidence was also obtained suggesting that G protein-coupled receptor kinase (GRK) phosphorylation sites reside within the 3rd cytoplasmic loop of the D1 receptor. We are currently testing this further by mutating each potential GRK site and assaying for functional effects. We have also made epitope (FLAG)-tagged D1 and D2 receptors for use in cellular trafficking (receptor internalization) studies and for the direct examination of receptor phosphorylation in situ. The epitope-tagged D 1 receptor was visualized in transiently transfected COS cells using both fluorescence and confocal microscopy. Progress was also achieved in the creation of an expression vector which will allow for the large scale expression and purification of the D2 receptor from 51 insect cells for use in biochemical studies. In other work, zinc ion was found to allosterically modulate both agonist and antagonist binding to all D2-like receptors (D2, D3 and D4), although the mechanisms of inhibition appear to differ among the receptor subtypes. Zinc was not found to inhibit the functional coupling of any of the receptors per se. Work also continued on cloning a third """"""""D1-like"""""""" receptor which stimulates phosphatidylinositol (PI) turnover and calcium mobilization. Proof that this receptor exists was obtained by showing that dopamine stimulation of PI turnover is unchanged in transgenic """"""""knock-out"""""""" mice lacking D1 receptors. Finally, homologous recombination was used to inactivate the D5 dopamine receptor gene in embryonic stem (ES) cells. These cells were used to create chimeric mice, several of which were found to carry the mutation in their germ line. Heterozygous animals have now been produced and are currently being mated to produce homozygous D5 knock-out mice.
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