Molecular and Behavioral Analysis of Dopamine Receptor Function

描述（由申请人提供）： 多巴胺受体是多种神经和精神疾病（包括精神分裂症和帕金森病）的主要治疗靶点。多巴胺受体的五种亚型（D1- D5）是G蛋白偶联受体超家族的成员。D1和D2亚型是最丰富的，介导最经典的多巴胺依赖性行为。该研究计划的总体长期目标是描述和区分D1和D2受体机制，从配体与受体结合的初始步骤到多巴胺依赖性行为的最终表现。第二个目的是评估D2受体的可变剪接形式D2 S和D2 L的不同功能作用。第一个具体目标是由D1受体激动剂而不是D2激动剂必须具有儿茶酚环的假设驱动的，因为在跨膜螺旋6和7中的两个位置处具有不同的残基。我们将描述相互突变体，其中一种受体亚型中的一个或两个残基被改变为另一种亚型的相应残基。我们将确定突变对高亲和力和低亲和力结合的影响，并通过儿茶酚胺和非儿茶酚胺激动剂调节环AMP积累。第二个具体目标是基于这样的假设，即D1和D2受体的激活调节其在脂筏中的定位，并差异调节其他筏富集蛋白的丰度。我们建议检查非筏组分中受体的分布以及急性或重复激动剂处理对受体分布的影响。此外，筏组分的蛋白质组学分析将用于鉴定药物诱导的其他筏蛋白的变化。第三个具体的目标将测试的假设，D2 L是突触后D2受体介导增加的运动活动和D2 S是突触前自身受体，调节多巴胺的释放和酪氨酸羟化酶活性在小鼠基底前脑。我们将评估HSV介导的D2 L或D2 S在D2受体缺失突变小鼠的突触后核（突触后）或腹侧被盖区（突触前）表达的行为和神经化学效应。第四个具体目标是由以下假设驱动的：S100 B与D2受体的结合是最有效的功能所必需的，并且仍有D1和D2受体相互作用蛋白有待鉴定，其调节多巴胺受体信号传导和定位。我们将进行额外的轮的cDNA文库筛选与D1和D2受体的细胞内循环，以确定新的多巴胺受体相互作用的蛋白质，并调查这些蛋白质和S100 B多巴胺受体的亚细胞定位和受体的信号传导特性的结合的后果。我们希望，通过鉴定以亚型选择性方式与配体相互作用的D1和D2受体的结构特征，以确定信号通路的结合和激活，解剖多巴胺受体亚型对多巴胺和合成激动剂的生化和行为反应的贡献，并表征调节多巴胺受体信号传导的新型蛋白质，这些研究将有助于开发涉及多巴胺系统的神经精神障碍的更有选择性的药物治疗。 公共卫生相关性： 每年，VA医疗中心住院治疗约45，000名退伍军人治疗精神分裂症，并治疗至少40，000名帕金森病患者。这些疾病的原因尚不清楚，但多巴胺D2样受体密度的变化与精神分裂症的病理生理学有关，这些疾病的主要治疗方法是阻断或刺激多巴胺受体。分析神经元和哺乳动物细胞系中表达的内源性和重组多巴胺受体，以及行为啮齿动物中多巴胺受体的功能，是进行基础研究的强大模型系统，应有助于发展改善精神分裂症和运动障碍治疗的合理策略。

DESCRIPTION (provided by applicant): Project Summary/Abstract Dopamine receptors are the primary therapeutic targets for a variety of neurological and psychiatric disorders, including schizophrenia and Parkinson's disease. The five subtypes of dopamine receptors (D1- D5) are members of the superfamily of G protein-coupled receptors. The D1 and D2 subtypes are the most abundant and mediate most classic dopamine-dependent behaviors. The overall long-term goal of this research program is to delineate and distinguish between D1 and D2 receptor mechanisms, from the initial step of ligand binding to the receptor to the final manifestation of dopamine-dependent behaviors. A secondary aim is to assess the differential functional roles of the alternatively spliced forms of the D2 receptor, D2S and D2L. The first specific aim is driven by the hypothesis that D1 receptor agonists, but not D2 agonists, must have a catechol ring because of distinct residues at two positions in transmembrane helices 6 and 7. We will characterize reciprocal mutants in which one or both of these residues in one receptor subtype is changed to the corresponding residue(s) of the other subtype. We will determine the effect of the mutations on high- and low-affinity binding of and regulation of cyclic AMP accumulation by catecholamine and non-catecholamine agonists. The second specific aim is based on the hypothesis that activation of D1 and D2 receptors regulates their localization in lipid rafts and differentially regulates the abundance of other raft-enriched proteins. We propose to examine the distribution of the receptors in non- raft fractions and the effect of acute or repeated agonist treatment on receptor distribution. In addition, proteomic analyses of raft fractions will be used to identify drug-induced changes in other raft proteins. The third specific aim will test the hypotheses that D2L is the postsynaptic D2 receptor that mediates increased locomotor activity and D2S is the presynaptic autoreceptor that regulates dopamine release and tyrosine hydroxylase activity in the mouse basal forebrain. We will evaluate the behavioral and neurochemical effects of HSV-mediated expression of D2L or D2S in the nucleus accumbens (postsynaptic) or ventral tegmental area (presynaptic) of D2 receptor null-mutant mice. The fourth specific aim is driven by the hypotheses that S100B binding to the D2 receptor is necessary for most efficient function, and that there are D1 and D2 receptor-interacting proteins still to be identified that regulate dopamine receptor signaling and localization. We will carry out additional rounds of cDNA library screening with D1 and D2 receptor intracellular loops to identify new dopamine receptor-interacting proteins and investigate the consequences of the binding of these proteins and S100B to dopamine receptors for the subcellular localization and signaling properties of the receptors. We hope that, by identifying structural features of D1 and D2 receptors that interact with ligands in a subtype-selective manner to determine binding and activation of signaling pathways, dissecting the contributions of dopamine receptor subtypes to biochemical and behavioral responses to dopamine and synthetic agonists, and characterizing novel proteins that regulate dopamine receptor signaling, these studies will contribute to the development of more selective pharmaceutical treatments of neuropsychiatric disorders that involve the dopamine system. PUBLIC HEALTH RELEVANCE: Project Narrative Each year, VA Medical Centers hospitalize about 45,000 veterans for treatment of schizophrenia and treat at least 40,000 Parkinson's disease patients. The causes of the diseases are unknown, but changes in the density of dopamine D2-like receptors have been implicated in the pathophysiology of the schizophrenia, and the primary treatments for the diseases are blockade or stimulation of dopamine receptors. Analysis of endogenous and recombinant dopamine receptors expressed in neurons and in mammalian cell lines, and of the function of dopamine receptors in the behaving rodent, are powerful model systems with which to perform basic research that should contribute to the development of rational strategies for improved treatment of schizophrenia and movement disorders.