D-1 DOPAMINE RECEPTORS IN DIFFERENT CELLULAR SYSTEMS

描述：(改编自申请者摘要)有缺陷的多巴胺 (DA)神经传递与神经退行性疾病有关 例如帕金森氏病(PD)和其他神经病理学 高催乳素血症、高血压、精神分裂症和 吸毒成瘾。这个实验室的长期目标是 识别多巴胺及其受体在脑内的特定功能作用 神经传递，在正常和疾病的人脑中。这个 申请者假设不同的蜂窝系统含有DA 与不同的效应器相互作用的受体 不同的信号转导系统，导致激活不同的 发信号回应。为此，他们研究了刺激性 D-1DA受体在药理学、生理学和分子水平上的研究进展 不同细胞系统中的水平：转染人D-1的GH4C1 C DNA、人SK-N-MC神经母细胞瘤细胞和大鼠纹状体。进展至 数据验证了这一假设的正确性。 在这项助学金计划中，申请者将把他们的学习扩展到 包括新的和遗传上不同的DA受体D-5，它是 与D-1受体相似但又不同。利用转基因的GH4C1 细胞，他们将分析和比较D-1和 D-5受体通过生化和生理学研究。D-5 将使用开发的方法对受体进行增溶和研究 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 申请者已经描述了D-1受体。使用特定的抗荷抗体 蛋白抗体，纯化的G蛋白和发展的“敲除” G蛋白表达被选择性抑制的细胞 使用反义寡核苷酸，他们将识别不同的G D-1偶联蛋白(G0？)和D-5(G2？)感受器。这部小说 D-5受体抑制肌醇磷脂代谢的机制 被详细地描述出来。他们将确定G蛋白和 可能参与这种抑制的蛋白激酶，使用特定的 可能的候选成分的抗体和抑制剂。一种能力 D-1受体在蛋白和mRNA上下调Gs-α 级别将进一步详细调查，因为这直接 激动剂为主的帕金森病治疗意义。从… 分子研究，他们将评估DA在改变 SK-N-MC细胞Gs-αmRNA合成的稳定性和速率。 还将进行研究，以分析可以 逆转DA对Gs-α的下调作用。这些系统的研究 可能有助于阐明D-1和D-5多巴胺受体的作用机制 大脑中的活动和功能。

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.