Molecular And Pharmacological Studies Of Dopamine Recept

多巴胺受体的分子和药理学研究

• 批准号: 7322993
• 负责人: DAVID R. SIBLEY
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7322993
• 关键词: Molecular; Pharmacological; Studies; Dopamine; Recept

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.

该项目的长期目标是表征神经递质受体介导的信息转导及其跨神经元膜的调节。正在研究的主要受体系统是神经递质多巴胺的受体系统。为了在生化和分子水平上表征这些受体并研究它们的调节，两个相互关联的研究正在进行中：1）在蛋白质水平上研究受体的细胞生物学、结构、功能和调节； 2) 直接与受体相互作用以改变其功能、表达、调节和运输的蛋白质的分子克隆和鉴定。涉及缺乏特定多巴胺受体亚型的小鼠的项目也在进行中。 2006 财年，我们继续研究蛋白激酶 C (PKC) 介导的磷酸化在调节 D1 受体功能中的作用以及乙醇如何对其进行调节。众所周知，乙醇消耗会增强多巴胺能信号传导，这部分是由 D1 受体介导的。乙醇调节 D1 受体信号传导的机制尚不清楚，这一直是我们研究的重点。我们发现，用乙醇预处理 D1 受体转染的 HEK293 细胞可增强多巴胺刺激的 cAMP 积累，并降低 D1 受体磷酸化，而不会改变受体表达。我们假设乙醇可以通过激活蛋白磷酸酶或抑制蛋白激酶来降低 D1 受体磷酸化并增强信号传导。为了检查磷酸酶或激酶对 D1 受体磷酸化和信号传导的潜在参与，在多巴胺刺激之前，用几种磷酸酶或蛋白激酶 C (PKC) 抑制剂对表达 D1 受体的 HEK293 细胞进行预处理。使用 cAMP 积累测定评估 D1 受体介导的信号传导，并通过原位磷酸化测定评估 D1 受体磷酸化。用磷酸酶抑制剂进行预处理并不能消除乙醇对多巴胺刺激的 cAMP 水平的增强作用或 D1 受体磷酸化的降低。此外，与单独表达 D1 受体的对照细胞相比，D1 受体与钙调神经磷酸酶（蛋白磷酸酶 2B）的组成型活性亚基（蛋白磷酸酶 2B）的共表达不会增强多巴胺刺激的 cAMP 水平或降低基础 D1 受体磷酸化水平。然而，用 PKC 抑制剂进行细胞预处理模拟了乙醇对多巴胺刺激的 cAMP 水平和 D1 受体磷酸化的影响，表明乙醇具有抑制受体基础 PKC 磷酸化的作用。这一观点得到了以下观察结果的支持：用乙醇和 PKC 抑制剂处理细胞可促进对 D1 受体磷酸化和活性的非加和效应。在用 D1 受体和 PKC 同工酶 γ 或 δ 共转染的细胞中，D1 受体磷酸化的乙醇依赖性降低似乎增强，表明乙醇的作用可能是由这些 PKC 同工酶介导的。在对特定 PKC 同工酶进行选择性免疫沉淀后，使用体外激酶测定直接评估乙醇调节细胞中 PKC 活性的能力。我们发现细胞的乙醇预处理确实减弱了γ和δ的膜激酶活性，而β和ε的膜激酶活性不受影响。总而言之，这些结果表明 PKC γ 和 δ 在基础条件下组成型磷酸化 D1 多巴胺受体，并且这抑制了受体与 G 蛋白的偶联。接触乙醇会特异性抑制这些 PKC 同工酶的活性，导致基础受体磷酸化减少并增强 D1 受体介导的信号传导。 2006 财年，我们启动了新的蛋白质组学项目，涉及 D1 和 D2 DAR 的免疫共沉淀 (co-IP) 测定以及质谱 (MS) 测序，以鉴定相互作用的蛋白质伙伴。通过这种方式鉴定出的一种蛋白质是排序 nexin-25 (SNX25)。分选连接蛋白是一组不同的细胞运输蛋白，其定义是存在磷脂结合基序、phox (PX) 结构域、结合磷脂酰肌醇磷酸的 100-130 个残基序列，从而将这些蛋白靶向富含这些脂质。哺乳动物 SNX 被认为参与细胞内运输、内化和内体回收或分类。迄今为止，已在人类中鉴定出 27 个 SNX，所有这些都是由 PX 结构域的存在来定义的。 SNX25 还包含一个 RGS（G 蛋白信号传导调节器）结构域，这是一段约 120 个残基的序列，可充当 GTP 酶激活剂，从而刺激异源三聚体 G 蛋白的失活。 SNX25 的生理作用尚不清楚。使用放射性配体结合测定，我们发现增加 HEK293T 细胞中 SNX25 的表达水平会剂量依赖性地增加质膜中表达的 D1 受体的量。受体数量的增加伴随着 D1 受体介导的 cAMP 积累的增加。我们还评估了 SNX25 对 D2 受体表达的影响，发现与 D1 受体一样，增加细胞 SNX25 表达会增加总 D2 受体结合活性。受体表达的增加与 D2 受体介导的信号传导增加相关，这是通过抑制毛喉素刺激的 cAMP 积累来确定的。增加 SNX25 水平对细胞中基础或毛喉素刺激的 cAMP 水平没有影响。这些结果表明SNX25在D1和D2多巴胺受体表达中发挥重要作用。目前正在研究发生这种情况的机制。 我们的蛋白质组学项目中发现的第二种蛋白质是 Na+/K+-ATP 酶 (NKA alpha1) 的 alpha1 亚基。 NKA 是一种跨膜蛋白，由 α 和 β 亚基组成，其中 α1 是主要的 α 亚型。研究表明，α 亚基主要负责 Na+ 和 K+ 跨质膜的运输。 Western 分析和反向免疫共沉淀实验证实了 MS 结果，揭示了 D1 和 D2 DAR 与 NKA α1 的特异性相互作用。为了确定 NKA 对 DAR 功能的影响，在 HEK293T 细胞中 NKA α1 水平增强的情况下进行了生物学测定。在该系统中，NKA α1 的过度表达导致 D1 和 D2 DAR 总数急剧减少。 NKA α1 过表达也观察到 D1 和 D2 DAR 介导的 cAMP 产生调节功能随之下降。有趣的是，用哇巴因药理抑制过表达或内源性 NKA 会增加 D2 DAR 活性（通过 cAMP 产生来测量）。此外，D2 DAR 的过度表达也会影响 NKA 功能，导致通过 86Rb 摄取测量的内源性 NKA 活性降低。这些初步数据表明 D2 DAR 和 NKA 可以相互调节。目前正在进行研究以确定NKA药理阻断对D1 DAR功能的影响，以及受体刺激或脱敏对DAR-NKA相互作用和NKA功能的影响。还将研究这种相互调节是否是通过直接或间接相互作用介导的。