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 FY2006, we have continued investigating the role of protein kinase C (PKC)-mediated phosphorylation in regulating D1 receptor function and how this might be modulated by ethanol. Ethanol consumption is well known to potentiate dopaminergic signaling and this is partially mediated by the D1 receptor. The mechanism responsible for ethanol modulation of D1 receptor signaling is unclear and has been the focus of our study. We found that ethanol pretreatment of D1 receptor-transfected HEK293 cells potentiates dopamine-stimulated cAMP accumulation and decreases D1 receptor phosphorylation without altering receptor expression. We hypothesized that ethanol may decrease D1 receptor phosphorylation and enhance signaling by either activating a protein phosphatase or inhibiting a protein kinase. To examine the potential involvement of phosphatases or kinases on D1 receptor phosphorylation and signaling, HEK293 cells expressing the D1 receptor were pretreated with several phosphatase or protein kinase C (PKC) inhibitors prior to dopamine-stimulation. D1 receptor-mediated signaling was evaluated using cAMP accumulation assays and D1 receptor phosphorylation was assessed via in situ phosphorylation assays. Pretreatment with phosphatase inhibitors did not abolish the ethanol potentiation of dopamine-stimulated cAMP levels or the decrease in D1 receptor phosphorylation. Furthermore, co-expression of the D1 receptor with a constitutively active subunit of calcineurin (protein phosphatase 2B) did not potentiate dopamine-stimulated cAMP levels or reduce basal D1 receptor phosphorylation levels when compared to control cells expressing the D1 receptor alone. However, cellular pretreatment with PKC inhibitors mimicked the effects of ethanol on both dopamine-stimulated cAMP levels and D1 receptor phosphorylation, suggesting that ethanol functions to inhibit basal PKC phosphorylation of the receptor. This idea is supported by the observation that treatment of the cells with both ethanol and PKC inhibitors promote non-additive effects on D1 receptor phosphorylation and activity. In cells cotransfected with the D1 receptor and the PKC isozymes gamma or delta, the ethanol-dependent decrease of D1 receptor phosphorylation appears to be augmented suggesting that the effects of ethanol may be mediated by these PKC isozymes. The ability of ethanol to modulate PKC activity in the cells was directly assessed using vitro kinase assays following selective immunoprecipitation of specific PKC isozymes. We found that ethanol pretreatment of the cells indeed attenuated the membrane kinase activities of gamma and delta whereas those of beta and epsilon were unaffected. Taken together, these results suggest that PKC gamma and delta constitutively phosphorylate the D1 dopamine receptor under basal conditions and that this dampens receptor-G protein coupling. Exposure to ethanol specifically inhibits the activity of these PKC isozymes resulting in decreased basal receptor phosphorylation and enhanced D1 receptor-mediated signaling.? ? In FY2006, 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 protein partners. One protein identified this way was sorting nexin-25 (SNX25). Sorting nexins are a diverse group of cellular trafficking proteins that are defined by the presence of a phospholipid-binding motif, the phox (PX) domain, a sequence of 100-130 residues that bind phosphatidylinositol phosphates, thereby targeting these proteins to membranes enriched in these lipids. Mammalian SNXs have been suggested to be involved in intracellular trafficking, internalization, and endosomal recycling or sorting. Thus far, 27 SNXs have been identified in humans, all defined by the presence of the PX domain. SNX25 also contains an RGS (regulator of G-protein signaling) domain, a sequence of ~120 residues that functions as a GTPase activator thus stimulating the inactivation of heterotrimeric G proteins. The physiological role of SNX25 is unknown. Using radioligand binding assays, we found that increasing the expression levels of SNX25 in HEK293T cells dose-dependently increased the amount of D1 receptor expressed in the plasma membrane. This increase in receptor number was accompanied by an increase in D1 receptor-mediated cAMP accumulation. We also evaluated the effects of SNX25 on D2 receptor expression and found that, as with the D1 receptor, increasing cellular SNX25 expression increased total D2 receptor binding activity. This increase in receptor expression was correlated with increased D2 receptor-mediated signaling as determined via inhibition of forskolin-stimulated cAMP accumulation. There were no effects of increasing SNX25 levels on basal or forskolin-stimulated cAMP levels in the cells. These results suggest that SNX25 plays an important role in the expression of both D1 and D2 dopamine receptor expression. The mechanism(s) by which this occurs is currently under investigation. ? ? A second protein identified in our proteomics projects was the alpha1 subunit of the Na+/K+-ATPase (NKA alpha1). The NKA is a transmembrane protein consisting of both alpha and beta subunits, with alpha1 being the predominant alpha isoform. Studies indicate that the alpha subunit is primarily responsible for the transport of Na+ and K+ across the plasma membrane. Western analysis and reverse co-immunoprecipitation experiments have confirmed the MS results, revealing specific D1 and D2 DAR interactions with NKA alpha1. To determine the impact of NKA on DAR function, biological assays were conducted in the presence of enhanced levels of NKA alpha1 in HEK293T cells. In this system, over-expression of NKA alpha1 yields a dramatic decrease in total D1 and D2 DAR number. A concomitant functional decrease in both D1 and D2 DAR-mediated regulation of cAMP production was also observed with NKA alpha1 over-expression. Interestingly, pharmacological inhibition of either over-expressed or endogenous NKA with ouabain produced an increase in D2 DAR activity, as measured by cAMP production. Furthermore, over-expression of the D2 DAR also impacts NKA function, causing a decrease in endogenous NKA activity as measured by 86Rb uptake. These preliminary data indicate that the D2 DAR and NKA can reciprocally regulate one another. Current studies are underway to determine both the impact of NKA pharmacological blockade on D1 DAR function, and the consequence of receptor stimulation or desensitization on DAR-NKA interactions and NKA function. Whether this reciprocal regulation is mediated by direct or indirect interactions will also be investigated.
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