MOLECULAR REGULATION OF D1 DOPAMINE RECEPTOR FUNCTION

D1 多巴胺受体功能的分子调控

• 批准号: 6655078
• 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 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6655078
• 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（根据申请人的摘要改编）： 这是一个基于数据的修订应用程序，显示了第一个完整的D1 我们开发的多巴胺激动剂二羟化二羟化二羟化引起了深刻的急性 抗帕金森氏症对灵长类动物的影响。最近的数据表明有些但没有 全部，全D1激动剂反复给药时产生明显的公差。我们 还表明，具有相似疗效的D1多巴胺受体激动剂可以 脱敏责任差异很大。使用一系列刚性DL 激动剂，我们将确定是否促进了选择性构象 不同的完整DL激动剂，以及这些构象是否代表 与促进g的受体相比，受体状态不同，如果重叠，则 蛋白质耦合。首先，我们将研究新型D1激动剂的差异 在功能上脱敏血凝素（HA）标记的人D1受体（HA-D1R） 在C-6神经胶质瘤细胞中受GRK活性的程度和/或模式的影响。 不同激动剂的D1受体磷酸化的速率和程度， 将确定主要的负GRK突变体的影响。分子和 药理学工具将用于评估内源性的相对作用 在这种脱敏中GRK和PKA。我们还将确定细胞 通过评估其易位和 与G-Beta-Gamma的相互作用补充。其次，我们将绘制 HD1R受体的磷酸化残基。 激动剂诱导的受体磷酸化的模式由GRK诱导和 第二届摄影酶（例如，PKA）将通过突变的子集进行评估 HA-HDL-C-6细胞中的丝氨酸和苏氨酸。我们还将过表达HA-D1R HEK293细胞中有和没有各种GRK。磷酸化的HA-HD1 受体将通过免疫沉淀分离，用蛋白酶切割，然后 由MALDVTOF，离子陷阱和/或纳米质量质量光谱测序。最后，我们 应检查GRK活性与抑制蛋白结合之间的关系 h-hd1中G蛋白解偶联和功能脱敏的启动 C-6系统。将确定Q-arrestins的补充，并进行更改 在GRK的高亲和力结合中，与D1受体的逮捕蛋白相结合 暴露于选择D1激动剂之后。受体-G蛋白的丧失 将评估标记为GTP-Analogs的结合耦合以相关 受体与） - arrestin结合水平解开。必要 在C-6细胞中HA-HD1受体的功能脱敏的5-次素脱敏 将使用显性阴性突变体和GRK之间的关系研究 使用AIM 1研究中开发的工具评估的P- arrestins。这些研究 Beta-arrestin的绑定和招聘将提供另一个功能 D1受体分子机制的描述中的终点 脱敏。