D-1 DOPAMINE RECEPTORS IN DIFFERENT CELLULAR SYSTEMS

描述：（改编自申请人的摘要） (DA)神经传递与神经退行性疾病有关 例如帕金森病（PD）和其它神经病理学疾病 诸如高泌乳素血症、高血压、精神分裂症和 毒瘾该实验室的长期目标是 确定DA及其受体在 神经传递，在正常和患病的人脑中。的 申请人假设不同的细胞系统含有DA 受体与不同的效应物有差异的相互作用， 不同的信号转导系统，导致激活不同的 信号响应。为此，他们研究了刺激性 D-1DA受体在药理学、生理学和分子生物学上的研究进展 水平，在不同的细胞系统中：用人D-1转染的GH4 C1 cDNA，人SK-N-MC神经母细胞瘤细胞和大鼠纹状体。进展 事实证明了这一假设的正确性。 在这项资助计划中，申请人将把他们的研究扩展到 包括新的和遗传上不同的DA受体，D-5， 与D-1受体相似但又不同。使用转染的GH4 C1 细胞，他们将分析和比较D-1和 D-5受体通过生化和生理研究。D-5 受体将被溶解，并使用开发的方法进行研究 D-1受体。D-5受体氧化还原状态的重要性， 将分析其对氧化/还原剂的敏感性，因为 申请人已经描述了D-1受体。使用特异性抗G抗体 蛋白抗体，纯化的G蛋白，并通过开发“敲除” 细胞，其中G蛋白表达被选择性地抑制， 使用反义寡核苷酸，他们将识别不同的G 与D-1（G 0？）和D-5（G2？）受体。小说 D-5受体抑制磷酸肌醇代谢的机制将 详细描述。他们将识别G蛋白和 使用特定的蛋白激酶，可能参与这种抑制作用 可能的候选成分的抗体和抑制剂。的能力 D-1受体下调Gs-alpha的蛋白质和mRNA 将进一步详细调查，因为这直接 在PD的基于激动剂的治疗中的治疗意义。从 分子研究，他们将评估DA在改变 SK-N-MC细胞中Gs-α mRNA稳定性和合成速率。 还将进行研究，分析可以 逆转DA对Gs-alpha的下调。这些系统性研究 可能有助于阐明D-1和D-5 DA受体的作用机制 大脑的激活和功能。

DESCRIPTION: (adapted from Applicant's Abstract) Defective dopamine (DA) neurotransmission has been implicated in neurodegenerative diseases such as Parkinson's disease (PD) and in other neuropathological conditions such as hyperprolactemia, hypertension, schizophrenia and drug addiction. The long term objective of this laboratory is to identify specific functional roles of DA and its receptors in neurotransmission, in the normal and diseased human brain. The applicants hypothesize that different cellular systems contain DA receptors which interact differentially with different effectors of different signal transducing systems, resulting in activation of diverse signalling responses. To this end, they have examined the stimulatory D-1 DA receptors at the pharmacological, physiological and molecular levels, in different cellular systems: GH4C1 transfected with human D-1 cDNA, human SK-N-MC neuroblastoma cells and rat striata. Progress to date has verified the validity of this hypothesis. In this grant proposal, the applicants will extend their studies to include the novel and genetically distinct DA receptor, D-5, which is similar to and yet different form D-1 receptors. Using transfected GH4C1 cells, they will analyze and compare the molecular properties of D-1 and D-5 receptors through biochemical and physiological studies. D-5 receptors will be solubilized and studied using methods developed for D-1 receptors. The importance of the redox state of D-5 receptors and its sensitivity to oxidizing/reducing agents will be analyzed, as the applicants have described for D-1 receptors. Using specific anti-G protein antibodies, purified G proteins and by developing "knock-out" cells in which G protein expression is selectively suppressed with the use of antisense oligonucleotides, they will identify the different G proteins which couple to D-1 (G0?) and D-5 (G2?) receptors. The novel mechanism by which D-5 receptors inhibit phosphoinositide metabolism will be characterized in detail. They will identify the G proteins and the protein kinases which may be involved in this inhibition, using specific antibodies and inhibitors of possible candidate components. The ability of D-1 receptors to down-regulate Gs-alpha at both the protein and mRNA levels will be further investigated in detail, since this has direct therapeutic significance in the agonist-based therapies of PD. From molecular studies, they will assess the effect of DA in altering the stability and rate of synthesis of Gs-alpha mRNA in SK-N-MC cells. Studies will also be conducted to analyze the processes which can reverse the down-regulation of Gs-alpha by DA. These systematic studies may enable an elucidation of the mechanisms of D-1 and D-5 DA receptor activation and function in the brain.