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. Homologous desensitization of G protein-coupled receptors (GPCRs) is mediated by G protein receptor kinases (GRKs). Upon agonist binding and activation, the GPCR becomes a substrate for GRK phosphorylation at serine and/or threonine residues. In, FY2003 we discovered evidence that the GRK4 isoform exhibits constitutive or agonist-independent phosphorylation of the D1 dopamine receptor. Co-expression of GRK4 and the rat D1 receptor in HEK293T cells results in increased basal phosphorylation of the receptor that is similar to the level of phosphorylation induced by dopamine stimulation in the absence of GRK4. The addition of dopamine to GRK4 co-transfected cells produces only a modest increase in phosphorylation over basal levels. Radioligand binding assays and confocal fluorescence microscopy using a D1 receptor-GFP chimera reveals that GRK4 co-expression induces a shift in the number of receptors from the plasma membrane into intracellular locations independent of agonist stimulation. GRK4 co-expression also results in a ~50% decline in the accumulation of cAMP in response to a maximally-effective concentration of dopamine. However, the population of receptors that remain at the plasma membrane in the presence of GRK4 desensitize and internalize similarly as D1 receptors co-expressed without GRK4. Mutation or truncation of serine and threonine residues in the carboxyl terminus of the D1 receptor reduces the amount of phosphorylation when the receptor is co-expressed with GRK4, but the reduction in phosphorylation is independent of agonist stimulation. These data suggest that the D1 dopamine receptor can be constitutively phosphorylated by a GRK4 in an agonist-independent manner and that this phosphorylation results in constitutive desensitization and internalization of the receptor. GRK4 might thus play a critical role in regulating the D1 receptor within cells or tissues that co-express these two proteins. Previously, D2 dopamine receptors (D2DARs) have been shown to undergo G protein-coupled receptor kinase (GRK) phosphorylation in an agonist-specific fashion. In FY2003 have investigated the ability of the second-messenger activated protein kinases, protein kinase A (PKA) and protein kinase C (PKC) to mediate phosphorylation and desensitization of the D2DAR. HEK293T cells were transiently transfected with the rat D2DAR and then treated with various intracellular activators and inhibitors of PKA or PKC. Treatment with agents that increase cAMP, and activate PKA, had no effect on the phosphorylation state of the D2DAR suggesting that PKA does not phosphorylate the D2DAR, at least in HEK293T cells. In contrast, cellular treatment with 1 uM phorbol 12-myristate 13-acetate (PMA), a PKC activator, resulted in ~3-fold increase in D2DAR phosphorylation within 10 min of treatment. The phosphorylation was specific for PKC as the PMA effect was mimicked by PDBu, but not by 4-alpha-PDD, active and inactive, phorbol diesters, respectively. PMA treatment produced only a small change in the cell surface expression of the D2DAR (5% decrease). The PMA-mediated D2DAR phosphorylation was completely blocked by co-treatment with the PKC inhibitor, bisindolylmaleimide II. In contrast, PKC inhibition had no effect on agonist-promoted phosphorylation suggesting that PKC is not involved in this response. To assess D2DAR desensitization, we examined the ability of the receptor to attenuate cAMP accumulation. PMA pretreatment of the cells resulted in a ~30-40% desensitization in the maximum response for dopamine inhibition of forskolin-stimulated cAMP accumulation. We also examined agonist-stimulated [35S]-GTP-gamma-S binding. PMA treatment diminished dopamine-stimulated [35S]-GTP-gamma-S binding by ~ 50% and also produced >5 fold shift (to the lower affinity) in the EC50. These results suggest that PKC mediates phosphoryation of the D2DAR and may be involved in its heterologous desensitization. We have previously shown that D3 dopamine receptors (D3DARs) and AMPA receptor subunits can be co-immunoprecipitated from HEK293T cells. These effects were specific and suggested that D3 and AMPA receptors may form hetero-oligomers when expressed in the same cell. In FY2003, we have investigated the functional consequences of these receptor-receptor interactions using intact cells. Using radioligand binding assays, we found that co-expression of the GluR2 or GluR3 subunits resulted in a 25-50% decrement in D3DAR expression levels. To examine D3DAR function, it was necessary to co-transfect adenylyl cyclase type V in the cells and then examine D3DAR-mediated inhibition of forskolin-stimulated cAMP accumulation. Both dopamine and quinpirole inhibited the adenylyl cyclase activity in a dose-dependent fashion. Co-expression of GluR2 with the D3DAR resulted in a 4-6 fold rightward shift in the agonist dose-response curves (increase in EC50) without a major effect on the maximum response. Interestingly, the addition of 30 uM AMPA to the cAMP assay abolished the shift in agonist potency such that the GluR2 subunit expression was without effect. These results suggest that D3DAR and GluR subunits may constitutively hetero-oligomerize in HEK293T cells and that this attenuates D3DAR-G protein coupling. Further, the state of D3DAR/GluR hetero-oligomerization may be dependent on agonist occupancy/activation of the GluR receptor. In terms of AMPA receptor function, co-expression of D3DARs with GluR1+2 in HEK293T cells did not alter the functional expression of GluR-mediated responses as measured by the density of current activated by 100 uM AMPA + 100 uM cyclothiazide. The expression of GluR2 also did not appear to be altered, as current/voltage relationships were linear. We are currently further investigating the effects of D3DAR expression on GluR activity. The D2 dopamine receptor exists in two protein isoforms that are generated by alternative mRNA splicing. In order to identify proteinsthat may differentially interact with these isofomrs, we have performed yeast two-hybrid screens of rat brain cDNA libraries using the 3rd cytoplasmic loops of the receptors as baits. Our strategy was to first identify positive interacting clones using the D2S and D2L 3rd loops and then test for cross-interaction using the opposite 3rd loop. Using the D2S 3rd loop, we identified 282 positive clones whereas 193 clones were isolated using the D2L 3rd loop. Following sib elimination, insert identification and sizing, as well as DNA sequencing, there remained 55 and 81 unique clones positive for D2S and D2L, respectively. Cross transformations revealed that, of the 55 clones identified with the D2S isoform, 16 were found to be highly positive with the D2L receptor whereas 21 of the 81 clones originally identified with the D2L isoform, where found to be highly positive with the D2S receptor. The identification and characterization of the unique as well as the cross-reacting clones are currently in progress.
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