Molecular And Pharmacological Studies Of Dopamine Recept

Neurological Disorders And Stroke

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Abstract

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 the neurotransmitter dopamine. To characterize these receptors at the biochemical and molecular levels and study their regulation, two interrelated lines of research are underway: 1) investigation of the cell biology, function and regulation of the receptors at the protein level; and 2) the molecular cloning of receptor-interacting cDNAs/genes and investigation of receptor structure, pharmacology and regulation in cultured cell lines and transgenic mice. In FY-2002, the mechanisms of agonist-induced regulation of D1 receptors were further investigated. D1 dopamine receptor (DAR) is stoichiometrically phosphorylated in C6 glioma cells and this phosphorylation is increased 2-3 fold upon agonist activation and desensitization. Potential G protein-coupled receptor kinase (GRK) phosphorylation sites exist in both the COOH terminus and the 3rd intracellular loop (3rdICL) of the receptor. To investigate the role of these two regions in desensitization, we created mutant receptors where either the cytoplasmic tail is mostly deleted (T347) or where three serines in the 3rdICL were mutated (3rdICL-234). The T347 mutant desensitized normally, while the 3rdICL-234 mutant exhibited impaired desensitization. Since potential GRK phosphorylation sites have been mutated or deleted in these two mutants, we investigated how over-expression of different GRKs would affect their desensitization. Desensitization was documented as a decrease in the maximal cAMP response as well as an EC50 shift in the dopamine dose-response curve. When co-transfected with the 3rdICL-234 receptor, GRK2, GRK3, and GRK6 each corrected the impairment of desensitization seen previously with this mutant. When co-transfected with the T347 receptor, GRK3 and GRK6 had no effect on desensitization whereas GRK2 induced a larger EC50 shift. Transfection of GRK5 to either the 3rdICL-234 or T347 receptors did not enhance their desensitization; however, GRK5 expression induced a larger agonist-induced EC50 shift with the wild-type receptor. These results suggest that GRK2/3/5/6 phosphorylation sites exist in the receptor's COOH terminus whereas only GRK2 phosphorylates residues in the receptor's 3rdICL. We have begun to investigate the role that protein phosphorylation plays in D2 receptor regulation. In the present year, we used HEK293T cells to investigate D2 dopamine receptor phosphorylation and sequestration. For receptor sequestration, we used [3H]sulpiride, a hydrophilic ligand that is cell surface-restricted and intact cell radioligand binding assays. Treatment with dopamine for 1 hr resulted in a 25% loss of cell surface receptor binding. This effect was not blocked by pertussis toxin indicating that G-protein coupling was not required. Agonist-induced receptor sequestration was blocked by co-expression with a dominant-negative mutant of dynamin which itself resulted in increased receptor expression at the cell surface. Co-expression of GRKs 2&3 decreased cell surface D2 receptor expression and enhanced agonist-induced sequestration, whereas GRKs 5&6 were without effect. Expression of either arrestin2 or arrestin3 increased agonist-induced receptor sequestration and this effect was enhanced by GRK2. These results suggest that the D2 receptor exhibits constitutive sequestration that is blocked by the dynamin mutant and enhanced by GRK2/3 phosphorylation. The D2 receptor is also phosphorylated under basal conditions and phosphorylation is increased 2-3 fold upon exposure to dopamine. GRKs 2&3 enhanced both basal and dopamine-stimulated receptor phosphorylation whereas GRKs 5&6 had no effect. Simultaneous mutation of serines 285,286,288 and threonines 287,293 within the 3rd cytoplasmic loop attenuated, but did not completely block, agonist- and GRK2- enhanced receptor phosphorylation and sequestration. These results suggest that GRK2/3 phosphorylation of Ser/Thr residues in the 3rd loop modulates intracellular trafficking of the D2 receptor. D2/D3 dopamine receptors directly interact with GRIP, a PDZ domain-containing protein that also interacts with GluR2/3 AMPA receptor subunits. GRIP functions as a scaffolding protein linking AMPA receptors and other signaling proteins into macromolecular complexes within postsynaptic membranes. Given this, and the fact that D2-like and AMPA receptors show cellular co-localization in the CNS we wondered if D2 or D3 receptors might form heterodimers with AMPA receptors, perhaps in a GRIP-facilitated fashion. As an initial test of this hypothesis, we co-expressed a FLAG-tagged D2 receptor or a c-myc-tagged D3 receptor with either GluR1, GluR2 or GluR4 AMPA receptor subunits in HEK293T cells. Radioligand binding assays confirmed the expression of the dopamine receptors while immunoblots confirmed the expression of the AMPA receptors. When the cells were detergent-solubilized and the D2 or D3 receptors were immunoprecipitated with either anti-FLAG or anti-c-myc antisera, respectively, D2 and D3 receptors were co-immunoprecipitated with AMPA receptor subunits GluR1, GluR2 and GluR4. However, D3 receptors were only co-immunoprecipitated with the GluR2 subunit when co-expressed with GRIP. These results suggest that GRIP facilitates interactions between D3 and GluR2. GluR3 subunit interactions are currently under investigation. In contrast, no immunoprecipitation of any AMPA receptor subunit was observed from cells expressing the GluR subunits alone or with the GluR subunit co-expressed with either the FLAG or c-myc peptides only. Further, co-expression with AMPA receptors altered D2 or D3 radioligand binding activity in HEK293T cells. Thus, D2 and D3 dopamine receptors directly interact with AMPA receptors in a heterologous cellular expression system.