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 mice. In FY-2001, the mechanisms of agonist-induced regulation of D1 receptors were further investigated. We previously showed that the rat D1 dopamine receptor (DAR) is stoichiometrically phosphorylated in C6 glioma cells and that this phosphorylation is increased 2-3 fold upon agonist activation and desensitization. In the current year, we attempted to identify the sites of regulatory phosphorylation within the D1 DAR using mutagenesis techniques and transient expression in HEK-293 cells. We created two 3rd cytoplasmic loop mutants, one in which all Ser and Thr residues were mutated to Ala or Val residues (3rdTOT) and another in which Ser residues 256, 258 & 259 were mutated to alanines (3rd234). We also prepared a series of mutants where the D1 DAR was truncated subsequent to the indicated positions in the COOH terminus: T347, T369, T394 & T404. Expression of these mutant and wild-type constructs resulted in similar levels of receptor binding activity (~1 pmol/mg) with the exception of the T347 mutant which was about 50% lower. Dopamine (DA) stimulated cAMP accumulation in a similar fashion for all constructs except for the 3rdTOT mutant which showed a 10-fold reduction in DA potency. DA-induced desensitization was investigated for the 3rdTOT and T347 mutants. Both mutants exhibited desensitization, however, desensitization of the 3rdTOT and T347 mutants were impaired and enhanced, respectively, compared to wild-type. DA-induced phosphorylation of all constructs was investigated. Relative to wild-type, the 3rdTOT mutant exhibited about a 50% decrease which was mimicked by the 3rd234 mutant. This suggests that Ser residues 256, 258 & 259 account for the majority of 3rd loop phosphorylation. Mutant T404 also exhibited a 50% decrease in phosphorylation with further reductions observed for each of the truncation mutants. Surprisingly, phosphorylation of T347 mutant was barely detectable. These results suggest that phosphorylation of COOH terminal residues is necessary for phosphorylation of 3rd loop to take place. The mechanism of desensitization in the T347 mutant is currently under investigation. In order to identify potential proteins that interact with the rat D3 dopamine receptor (DAR), we have started to use the DupLex-A yeast two-hybrid system i FY-2001. The entire third cytoplasmic loop (residues 210-375) of the rat D3 DAR was used as a bait to identify clones encoding interacting proteins from a rat whole brain cDNA library. One of the most highly positive clones appeared to represent a partial-length cDNA of GRIP-1, (Glutamate Receptor-Interacting Protein-1) a scaffolding protein known to interact with AMPA receptors. Our partial-length cDNA contains the fourth and fifth PDZ domains of the GRIP-1 protein, which had been shown previously to be required for AMPA receptor interactions. The specificity of the interaction of the D3 DAR and the partial-length GRIP-1 clone was verified using a corresponding yeast expression vector that lacks inserts and showed no growth on -leu plates and no color change on X-gal plates. The partial-length GRIP-1 clone was further shown not to interact with the third cytoplasmic loops of the rat D1, D2L, D4, or D5 DARs. Co-expression of the partial-length GRIP-1 clone with the D3 DAR in HEK-293T cells resulted in ~50% reduction of receptor binding activity. Co-expression of the GRIP-1 clone had no effect the receptor expression or binding activities of the D1, D4 or D5 DARs, however, a decrease in receptor binding was surprisingly observed with the D2L DAR. These findings represent the first report of GRIP-1 interactions with a G-protein coupled receptor. Further experiments confirming and characterizing the interactions between GRIP-1 and the D3 DAR are in progress.
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