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, structure, function and regulation of the receptors at the protein level; and 2) the molecular cloning and identification of proteins that directly interact with the receptors to modify their function, expression, regulation and trafficking. Projects involing mice that are deficient in specific dopamine receptor subtypes are also in progress. In FY2005, we have continued investigating the role of G protein-coupled receptor kinase (GRK) and protein kinase C (PKC)-mediated phosphorylation in regulating both D1 and D2 receptor function. D1 dopamine receptors (D1 DARs) are known to be phosphorylated by GRKs and we have recently reported that the D1 DAR is also phosphorylated by PKC. Our current efforts have been directed at identifying specific GRK and PKC phosphorylation sites within the D1 DAR and investigating potential interactions between GRK- and PKC-mediated regulation of the receptor. We previously reported that DA-induced desensitization of the D1 DAR is correlated with phosphorylation of residues within its 3rd cytoplasmic loop as well as the carboxy terminus and that phosphorylation of these sites may take place in a hierarchical fashion (Kim et al., JBC 279: 7999-8010, 2004). As a test of this hypothesis, we simultaneously mutated all potential phosphorylation sites (20 Ser/Thr residues) within the carboxy terminus of the receptor. This resulted in the complete abolishment of DA-induced phosphorylation of the D1 DAR suggesting that, indeed, phosphorylation of the carboxy termimus is required for GRK-mediated phosphorylation of 3rd loop residues. In a separate set of experiments we have attempted to identify the PKC phosphorylation sites within the D1 DAR by simultaneously mutating 7 Ser/Thr residues, representing potential PKC recognition sites. This mutant D1 receptor (PKC-) displayed reduced basal phosphorylation that was minimally increased upon phorbol ester treatment suggesting that the majority of the PKC sites are eliminated in this receptor. Interestingly, DA stimulation of the PKC- receptor also exhibited reduced phosphorylation even though we have previously shown that DA-induced receptor phosphorylation is not mediated by PKC. Further, the PKC- mutant exhibited increased DA-stimulated cAMP accumulation as well as decreased desensitization when treated with DA. Thus, the D1 DAR lacking PKC phosphorylation sites exhibited reduced basal phosphorylation that corresponded to an increase in cAMP accumulation and reduced receptor desensitization. These data suggest that inhibition of PKC phosphorylation of the D1 DAR sensitizes the receptor to agonist treatment and concomitantly diminishes GRK modulation of receptor. In FY2005, we have identified a novel mechanism of GRK regulation of the D2 receptor. Previously, we and others have shown that the D2 dopamine receptor (D2DAR) is phosphorylated by GRK and PKC. In HEK293T cells, dopamine (DA) stimulates D2DAR phosphorylation by 2-3 fold. Co-expression of GRK2/3 with D2DAR potentiates basal and DA-stimulated D2DAR phosphorylation. Simultaneous mutation of 6 serines and 2 threonines within the 3rd cytoplasmic loop decreased basal and completely abolished DA- and GRK2-stimulated D2DAR phosphorylation. However, this mutant (GRK-) receptor is phosphorylated to same extent as the wild-type (WT) receptor via PKC activation with PMA. Treatment with PKC inhibitors completely eliminates basal phosphorylation of the GRK- mutant. Further mutation of the PKC phosphorylation sites within the GRK- receptor completely eliminates basal as well as DA-, GRK- and PMA-stimulated receptor phosphorylation. Taken together, these data suggest that the GRK and PKC phosphorylation sites are distinct and only GRK and PKC phosphorylate the D2DAR in HEK293T cells. We next investigated agonist-stimulated D2DAR internalization using the GRK- and WT receptors. Surprisingly, DA induced internalization of the GRK- mutant receptor to the same extent as WT receptor. Co-expression of GRK2 with the WT D2DAR enhances receptor internalization in the absence and presence of DA. This GRK2-enhanced D2DAR internalization was unaffected in the GRK- mutant receptor. Over-expression of arrestin2 also increased DA-induced D2DAR internalization similarly in both the WT and GRK- mutant receptors. This effect was enhanced by GRK2 co-transfection with the WT receptor, but was not with the GRK- receptor. Using arrestin-GFP translocation assays, we found that DA induced translocation of arrestin to same extent via the WT and GRK- receptors. These data suggest that the phosphorylation of the D2DAR is not a prerequisite for agonist or GRK/arrestin mediated internalization, although receptor phosphorylation may enhance arrestin association with the receptor. The mechanism by which GRK mediates D2DAR internalization in the absence of receptor phosphorylation is under investigation. In FY2005, we initiated new proteomics projects involving co-immunoprecipitation (co-IP) assays for D1 and D2 DARs coupled with mass spectrometry (MS) sequencing to identify interacting partners. Following IP of the D1 DAR from HEK293 cells, proteins were separated using 1D PAGE. Independent bands were excised, trypsinized, and subjected to MS-based peptide sequencing. Approximately 40 proteins were identified by searching against a non-redundant protein database. Approximately, 14 of these proteins were also found in immunoprecipitates from mock-transfected controls and therefore identified as non-specific. One specific interacting protein is the molecular chaperone calnexin (CNX). CNX normally acts as an ER retention protein for glycosylated proteins thus facilitating their correct folding and delivery to the golgi. Selective interaction was verified in a number of different systems using co-IP followed by both western blot and MS (three individual peptides with 100% match to CNX were identified by MS). To determine the influence of CNX on D1 DAR expression we conducted biological assays using a variety of CNX modifying conditions. Over-expression of CNX in HEK293T cells results in a marked decrease in D1 DAR receptor expression as determined by radiolabeled ligand binding assays. Treatment of cells with the glycosylation inhibitor tunicamycin, or the glucosidase inhibitor castanospermine, both of which can inhibit the interaction of CNX with the D1 DAR, results in little effect on total receptor expression, as determined by binding assays, but confocal fluorescence microscopy reveals the accumulation of the D1-GFP receptor in internal stores. To further investigate the D1-CNX interaction, we employed the use of a D1 DAR mutant lacking most of the carboxy terminus. This mutant is known to have retarded trafficking to the cell surface versus wild-type DAR. When the D1-CNX interaction was examined with IP and western blot, the mutant receptor co-precipitates significantly more CNX indicating that more of the mutant receptor is bound to CNX. Taken together these data suggest that CNX acts as an ER retention protein capable of influencing the amount of D1 receptor protein trafficked to the cell surface. Similar experiments are currently being conducted for the D2 receptor using both transfected cell systems and mouse brains.