Dopamine Receptor Proteomics

多巴胺受体蛋白质组学

• 批准号: 7969647
• 负责人: David Sibley
• 金额: $ 135.9万
• 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/7969647
• 关键词: Agonist; Alcohol abuse; Alcoholism; Back; Base Sequence; Binding; Biochemical; Biological Assay; Cell Surface Receptors; Cell membrane; Cell surface; Cells; Cellular biology; Chemosensitization; Clinical; Co-Immunoprecipitations; Complement; Complex; Confocal Microscopy; Cyclic AMP; Data; Disease; Dopamine; Dopamine D1 Receptor; Dopamine D2 Receptor; Dopamine Receptor; Economics; Effectiveness; Endocytosis; Ethanol; Excision; Family; Goals; Investigation; Isoenzymes; Label; Laboratories; Link; Lipids; Lysosomes; Macromolecular Complexes; Mass Spectrum Analysis; Mediating; Membrane; Membrane Microdomains; Membrane Proteins; Molecular; Molecular Cloning; Na(+)-K(+)-Exchanging ATPase; Neurons; Neurotransmitter Receptor; Neurotransmitters; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Play; Process; Protein Kinase C; Protein-Serine-Threonine Kinases; Proteins; Proteomics; Receptor Activation; Recycling; Regulation; Reporting; Research; Role; Scaffolding Protein; Signal Pathway; Signal Transduction; Sorting - Cell Movement; Structure; Sulpiride; Surface; System; TAC1 gene; Techniques; Two-Dimensional Gel Electrophoresis; Western Blotting; addiction; alcoholism pharmacotherapy; hepatocyte growth factor-regulated tyrosine kinase substrate; human RIPK1 protein; interest; member; neuropsychiatry; nexin; novel; receptor; receptor binding; receptor coupling; receptor expression; receptor internalization; receptor recycling; research study; response; scaffold; trafficking; Agonist; Alcohol abuse; Alcoholism; Back; Base Sequence; Binding; Biochemical; Biological Assay; Cell Surface Receptors; Cell membrane; Cell surface; Cells; Cellular biology; Chemosensitization; Clinical; Co-Immunoprecipitations; Complement; Complex; Confocal Microscopy; Cyclic AMP; Data; Disease; Dopamine; Dopamine D1 Receptor; Dopamine D2 Receptor; Dopamine Receptor; Economics; Effectiveness; Endocytosis; Ethanol; Excision; Family; Goals; Investigation; Isoenzymes; Label; Laboratories; Link; Lipids; Lysosomes; Macromolecular Complexes; Mass Spectrum Analysis; Mediating; Membrane; Membrane Microdomains; Membrane Proteins; Molecular; Molecular Cloning; Na(+)-K(+)-Exchanging ATPase; Neurons; Neurotransmitter Receptor; Neurotransmitters; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Play; Process; Protein Kinase C; Protein-Serine-Threonine Kinases; Proteins; Proteomics; Receptor Activation; Recycling; Regulation; Reporting; Research; Role; Scaffolding Protein; Signal Pathway; Signal Transduction; Sorting - Cell Movement; Structure; Sulpiride; Surface; System; TAC1 gene; Techniques; Two-Dimensional Gel Electrophoresis; Western Blotting; addiction; alcoholism pharmacotherapy; hepatocyte growth factor-regulated tyrosine kinase substrate; human RIPK1 protein; interest; member; neuropsychiatry; nexin; novel; receptor; receptor binding; receptor coupling; receptor expression; receptor internalization; receptor recycling; research study; response; scaffold; trafficking

In FY2009, we continued our investigation of the Na+, K+-ATPase (NKA, sodium pump) as an interacting protein for the D1 and D2 receptors. Using a co-immunoprecipitation assay for dopamine receptors (DARs) coupled with mass spectrometry (MS) sequencing, we previously identified the Na+/K+-ATPase (Na+ pump, NKA) as a member of both the D1 and D2 DAR signalplexes. Using fluorescent-labeled D2 DAR and NKAα1 constructs in confocal microscopy experiments, co-localization of the two proteins was observed at the plasma membrane. Dopamine stimulation of RFP-tagged D2 DAR-expressing cells resulted in marked DAR internalization twenty minutes after drug treatment. Interestingly, when GFP-tagged NKAα1 was co-expressed, the D2 DAR internalization twenty minutes after dopamine treatment was diminished. To achieve a more quantitative assessment of receptor expression, cell-surface binding assays were performed in the presence and absence of over-expressed NKA. These assays revealed a 45% decrease in cell-surface D2 DAR number twenty minutes after dopamine treatment, and a 30% decrease in receptor number when the NKA was also present. Longer dopamine incubations enhanced D2 DAR internalization, but co-expression of NKA prohibited complete internalization as compared to D2 DAR alone. Current studies are underway to determine the impact of the DAR-NKA complex on receptor recycling, and the role of NKA in lipid rafts on receptor activation and internalization. Our laboratory and others have previously shown that the D2 dopamine receptor (DAR) is internalized by agonist stimulation and either recycled back to the plasma membrane or sorted to lysosomes for degradation. However, the molecular components involved in these processes are only beginning to be characterized. In FY2009, we continued our investigation of sorting nexin-25 (SNX25) as a novel DAR interacting protein using co-immunoprecipitation-coupled mass spectrometry-based sequencing. Mammalian SNXs have been suggested to be involved in the internalization, intracellular trafficking, and endosomal recycling or sorting of membrane-bound cargo. In addition, hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) has been found to play a distinct role in promoting the rapid recycling of internalized receptors back to the plasma membrane. In FY2009, we investigated the role of SNX25 and Hrs on D2 DAR internalization and recycling in HEK293T cells. Receptor internalization and recycling was initially assessed by quantifying cell surface receptors using intact cell 3H-sulpiride binding assays. Treatment with dopamine for 1 hr resulted in a 25% loss of cell surface receptor binding (internalization), which recovered to 93% of control after 1hr of dopamine removal (recycling). Over-expression of either SNX25 or Hrs had no effect on the total surface receptor expression, but decreased the amount of agonist-induced receptor internalization to 17% and 11%, respectively. Surprisingly, D2 DAR recycling was severely impaired in Hrs over-expressing cells. However, SNX25 over-expression resulted in only a slight decrease in receptor recycling. Taken together, over-expression of either SNX25 or Hrs perturbed both internalization and recycling of the D2 DAR. These data suggest that SNX25 and Hrs may play a role in D2 DAR trafficking through membrane compartments and that common machinery exists linking receptor internalization (endocytosis) and recycling. In FY 2009, we also continued our investigations of how protein kinase C regulation of the D1 receptor is regulated by alchohol. Alcohol abuse and alcoholism are of clinical and economic significance worldwide. The effectiveness of the current pharmacotherapies for alcoholism is limited and is partially due to lack of mechanistic data at the molecular level. Aberrant protein kinase C (PKC) signaling is associated with many diseases that include alcoholism and addiction. The PKC family of serine/threonine kinases is comprised of 12 isozymes that differ with respect to their structure, expression, and mechanisms of regulation. In many instances, only a subset of the PKC isozymes is associated with a specific disease state. We are particularly interested in the interplay between PKC and the D1 dopamine receptor in neuropsychiatric disorders such as alcohol abuse and alcoholism. We've recently found that PKC constitutively phosphorylates the D1 receptor and that this negatively regulates dopaminergic signaling. Moreover, we've shown that ethanol (EtOH) treatment decreases constitutive PKC phosphorylation of the D1 receptor with a concomitant potentiation of dopaminergic signaling. Importantly, EtOH was found to directly inhibit the lipid-activated enzymatic activities of PKCγ and PKCδ, but only when they were isolated from the membrane fraction - a response that was not observed for other PKC isozymes, including α, β1 or ε. The molecular mechanisms underlying the EtOH-mediated inhibition of membrane-associated PKCγ and PKCδ kinase activities are at present unclear. We hypothesize that EtOH may regulate the interaction between the PKC isozyme and a membrane-associated interacting protein(s) and/or target the PKC interacting protein itself. We have mow employed a PKC isozyme-specific coimmunoprecipitation approach followed by 2-D gel electrophoresis and mass spectrometry-based sequencing to identify candidate PKC isozyme-specific interacting proteins. RanBP10, a known scaffolding protein, was identified in the proteomics screen involving PKCγ. Significantly, we confirmed the association of RanBP10 with both PKCγ and PKCδ using coimmunoprecipitation techniques followed by Western Blotting. Since PKCγ and PKCδ both phosphorylate the D1 receptor, we postulate that RanBP10 may function as a scaffolding molecule and may also associate with the D1 receptor. This notion was supported by the coimmunoprecipitation of RanBP10 with the D1 receptor. Interestingly, our preliminary functional data suggest that RanBP10 modulates D1 receptor expression and cAMP accumulation. In summary, we have identified a putative scaffolding molecule, RanBP10 that may be a critical component that regulates crosstalk between D1 receptor and PKC isozyme-specific signaling pathways.

在2009财年，我们继续研究Na+，K+-ATPase（NKA，钠泵）作为D1和D2受体的相互作用蛋白。使用共免疫沉淀测定法对多巴胺受体（DARS）结合质谱（MS）测序，我们先前先前鉴定出Na+/K+-ATPase（Na+Pump，NKA，NKA）是D1和D2 DAR信号质量的成员。在共聚焦显微镜实验中，使用荧光标记的D2 DAR和NKAα1构建体，在质膜上观察到了两种蛋白的共定位。对RFP标记的D2表达DAR的细胞的多巴胺刺激导致药物治疗二十分钟后明显的DAR内在化。有趣的是，当GFP标记的NKAα1被共表达时，多巴胺治疗后二十分钟D2 DAR内化。为了对受体表达进行更定量的评估，在存在和不存在过表达的NKA的情况下，进行了细胞表面结合测定。这些测定法显示，在多巴胺治疗后二十分钟后，细胞表面D2 DAR降低了45％，当NKA也存在时，受体数量下降了30％。较长的多巴胺孵育增强了D2 DAR内在化，但与单独使用D2 DAR相比，NKA的共表达禁止完全内在化。目前的研究正在进行中，以确定DAR-NKA复合物对受体回收的影响，以及NKA在脂质筏中对受体激活和内在化的作用。 我们的实验室和其他实验室以前已经表明，D2多巴胺受体（DAR）是通过激动剂刺激内部化的，并且回收回到质膜或分类为溶酶体以降解。 但是，这些过程中涉及的分子成分才刚刚开始表征。 在2009财年，我们使用基于共免疫耦合质谱测序的新型DAR相互作用蛋白继续研究Nexin-25（SNX25）作为一种新型的DAR相互作用蛋白。 已经建议哺乳动物SNX参与内部化，细胞内贩运以及内体回收或膜结合的货物的排序。 此外，已经发现，肝细胞生长因子调节的酪氨酸激酶底物（HRS）在促进内部化受体的快速回收中起着独特的作用，从而恢复了质膜。 在2009财年，我们研究了SNX25和HRS在HEK293T细胞中D2 DAR内在化和回收中的作用。 最初使用完整的细胞3H-磺化结合测定法对细胞表面受体进行定量来评估受体内在化和回收利用。 多巴胺治疗1小时，导致细胞表面受体结合损失25％（内在化），多巴胺去除1小时后恢复到对照的93％（回收）。 SNX25或HR的过表达对总表面受体表达没有影响，但分别将激动剂诱导的受体内在化量降低到17％和11％。出乎意料的是，D2 DAR回收在过度表达的细胞中严重受损。 但是，SNX25的过表达仅导致受体回收略有下降。 总之，SNX25或HRS的过表达扰动了D2 DAR的内在化和回收利用。这些数据表明，SNX25和HRS可能在通过膜室进行D2 DAR运输中发挥作用，并且存在连接受体内在化（内吞作用）和回收利用的常见机械。 在2009财年，我们还继续研究D1受体的蛋白激酶C调节如何受到藻类的调节。 酒精滥用和酒精中毒具有全球临床和经济意义。 当前的酒精中毒药物治疗的有效性受到限制，部分是由于分子水平缺乏机械数据。 异常的蛋白激酶C（PKC）信号传导与包括酒精中毒和成瘾在内的许多疾病有关。 丝氨酸/苏氨酸激酶的PKC家族由12个同工酶组成，它们在调节的结构，表达和机制方面有所不同。 在许多情况下，只有PKC同工酶的一部分与特定疾病状态有关。 我们对PKC和D1多巴胺受体之间在神经精神疾病（例如酗酒和酒精中毒）中的相互作用特别感兴趣。 我们最近发现，PKC组成型磷酸化D1受体，并且这负责调节多巴胺能信号传导。 此外，我们已经表明，乙醇（ETOH）处理可降低D1受体的组成型PKC磷酸化，并伴有多巴胺能信号的增强。 重要的是，发现ETOH直接抑制PKCγ和PKCδ的脂质激活的酶促活性，但仅当它们从膜分数分离出来时，这种响应未观察到其他PKC同工酶，包括α，β1或ε。 目前尚不清楚ETOH介导的抑制膜相关PKCγ和PKCδ激酶活性的抑制作用的分子机制。 我们假设ETOH可以调节PKC同工酶与膜相关相互作用的蛋白（S）和/或靶向PKC相互作用蛋白本身之间的相互作用。 我们已经采用了PKC同工酶特异性共免疫沉淀方法，然后采用2-D凝胶电泳和基于质谱的测序，以鉴定候选PKC同工酶特异性相互作用蛋白。 RANBP10是一种已知的脚手架蛋白，在涉及PKCγ的蛋白质组学筛选中鉴定出来。 值得注意的是，我们使用共免疫沉积技术，随后是蛋白质印迹，证实了RANBP10与PKCγ和PKCδ的关联。 由于PKCγ和PKCδ都磷酸化D1受体，因此我们假设RANBP10可以用作脚手架分子，并且也可能与D1受体相关。 RANBP10与D1受体的共免疫沉淀支持了这一概念。 有趣的是，我们的初步功能数据表明RANBP10调节D1受体表达和cAMP积累。 总而言之，我们已经确定了推定的脚手架分子RANBP10，该分子可能是调节D1受体和PKC同工酶特异性信号通路之间串扰的关键成分。