MOLECULAR REGULATION OF D1 DOPAMINE RECEPTOR FUNCTION

D1 多巴胺受体功能的分子调控

• 批准号: 6803198
• 负责人: Richard B Mailman
• 金额: $ 25.26万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-30 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6803198
• 关键词: G protein coupled receptor kinase; Parkinson' s disease; arrestins; cell line; dopamine agonists; dopamine receptor; enzyme activity; mass spectrometry; matrix assisted laser desorption ionization; protein binding; protein kinase A; protein localization; protein protein interaction; receptor binding; receptor coupling; receptor sensitivity

DESCRIPTION(Adapted from applicant's abstract): This is a revised application based on data showing that the first full D1 dopamine agonist dihydrexidine that we developed caused profound acute antiparkinsonian effects in primates. Recent data indicate that some, but not all, full D1 agonists produce marked tolerance when administered repeatedly. We also have shown that D1 dopamine receptor agonists of similar efficacy can differ dramatically in desensitization liability. Using a series of rigid Dl agonists, we shall determine if selective conformations are promoted by different full Dl agonists, and whether these conformations represent different, if overlapping, receptor states compared to those that promote G protein coupling. First, we shall study differences in how novel D1 agonists functionally desensitize hemagglutinin (HA)-tagged human D1 receptors (HA-D1R) in C-6 glioma cells as affected by the extent and/or pattern of GRK activity. The rate and extent of D1 receptor phosphorylation by different agonists, and the effects of dominant negative GRK mutants, will be determined. Molecular and pharmacological tools will be used to assess the relative roles of endogenous GRKs and PKA in such desensitization. We also shall determine the cellular response of GRK to agonist exposure by assessing its translocation and interaction with G-beta-gamma complements. Second, we shall map the phosphorylated residues of the hD1R receptor. Patterns of agonist-induced receptor phosphorylation induced by GRKs and second-messenger-kinases (e.g., PKA) will be assessed by mutating subsets of serines and threonines in HA-hDl-C-6 cells. We also shall overexpress HA-D1R with and without various GRKs in HEK293 cells. The phosphorylated HA-hD1 receptor will be isolated by immunoprecipitation, cleaved with protease, and sequenced by MALDVToF, Ion Trap, and/or nano-ESI-mass spectrometry. Finally, we shall examine the relationship between GRK activity and arrestin binding in the initiation of G protein uncoupling and functional desensitization in the HA-hD1 C-6 system. The complement of Q-arrestins will be determined, and alterations in high affinity binding of GRKs and arrestins to D1 receptors measured following exposure to select D1 agonists. The loss of receptor-G-protein coupling from binding of labeled GTP-analogs will be assessed to correlate receptor uncoupling with levels of )-arrestin binding. The necessity of 5-arrestin(s) in functional desensitization of the HA-hD1 receptor in C-6 cells will be studied using dominant-negative mutants, and the relation between GRK and p-arrestins assessed using tools developed in Aim 1 studies. These studies of beta-arrestin binding and recruitment will provide another functional endpoint in the delineation of the molecular mechanisms of D1 receptor desensitization.

描述（改编自申请人摘要）： 这是一份修改后的申请，数据显示，第一个完整的D1 我们开发的多巴胺激动剂dihydrexidine引起了严重的急性 对灵长类动物的抗帕金森作用。最近的数据表明，一些，但不是 当重复给药时，所有的完全D1激动剂都产生显著的耐受性。我们 也已经表明，具有类似功效的D1多巴胺受体激动剂可以 在脱敏责任方面有很大的不同。使用一系列刚性Dl 激动剂，我们将确定是否促进选择性构象 不同的完整Dl激动剂，以及这些构象是否代表 不同的，如果重叠，受体状态相比，促进G 蛋白质偶联首先，我们将研究新型D1激动剂 功能性脱敏血凝素（HA）标记的人D1受体（HA-D1 R） C-6胶质瘤细胞中GRK活性的程度和/或模式的影响。 不同激动剂引起的D1受体磷酸化的速率和程度， 将确定显性负性GRK突变体的影响。分子和 药理学工具将被用来评估内源性的相对作用， GRKs和PKA在这种脱敏中的作用。我们还将确定 通过评估GRK的易位， 与G-β-γ补体的相互作用。第二，我们将绘制 hD 1 R受体的磷酸化残基。 激动剂诱导的GRKs受体磷酸化模式和 第二信使激酶（例如，PKA）将通过突变的 丝氨酸和苏氨酸。我们还将过度表达HA-D1 R 在HEK 293细胞中有和没有各种GRK。磷酸化的HA-hD 1 受体将通过免疫沉淀分离，用蛋白酶切割， 通过MALDVToF、离子阱和/或纳米-ESI-质谱法测序。最后我们 将检查GRK活性和抑制蛋白结合之间的关系， HA-hD 1中G蛋白解偶联和功能脱敏的启动 C-6系统将确定Q-抑制蛋白的补体，并将改变 在GRKs和抑制蛋白与D1受体的高亲和力结合中 在暴露于选择的D1激动剂之后。受体G蛋白的丢失 将评估来自标记的GTP-类似物结合的偶联， 受体解偶联与抑制蛋白结合水平。的必要性 5-arrestin对C-6细胞HA-hD 1受体功能性脱敏的作用 将使用显性-阴性突变体进行研究，GRK 和使用Aim 1研究中开发的工具评估的β-抑制蛋白。这些研究 β-抑制蛋白的结合和募集将提供另一种功能， 描述D1受体分子机制的终点 脱敏