MOLECULAR REGULATION OF D1 DOPAMINE RECEPTOR FUNCTION

D1 多巴胺受体功能的分子调控

• 批准号: 6394198
• 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/6394198
• 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（改编自申请人的摘要）： 这是根据数据修改后的申请，显示第一个完整的 D1 我们开发的多巴胺激动剂二羟西定引起了严重的急性 对灵长类动物具有抗帕金森病作用。最近的数据表明，有一些，但不是 所有 D1 完全激动剂在重复给药时都会产生显着的耐受性。我们 还表明，具有类似功效的 D1 多巴胺受体激动剂可以 脱敏责任差异很大。采用一系列刚性Dl 激动剂，我们将确定选择性构象是否被促进 不同的全Dl激动剂，以及这些构象是否代表 与促进 G 的受体状态相比，如果重叠的话，受体状态也不同 蛋白质偶联。首先，我们将研究新型 D1 激动剂的差异 功能性使血凝素 (HA) 标记的人类 D1 受体 (HA-D1R) 脱敏 C-6 神经胶质瘤细胞中的 GRK 活性程度和/或模式的影响。 不同激动剂对 D1 受体磷酸化的速率和程度，以及 显性失活GRK突变体的影响将被确定。分子和 药理学工具将用于评估内源性的相对作用 GRKs和PKA在这种脱敏中的作用。我们还应确定蜂窝 通过评估其易位和GRK对激动剂暴露的反应 与 G-β-γ 补体的相互作用。其次，我们要绘制地图 hD1R 受体的磷酸化残基。 GRK 和 GRK 诱导的激动剂诱导受体磷酸化模式 第二信使激酶（例如 PKA）将通过突变子集进行评估 HA-hD1-C-6细胞中的丝氨酸和苏氨酸。我们还将过度表达 HA-D1R HEK293 细胞中有或没有各种 GRK。磷酸化HA-hD1 受体将通过免疫沉淀分离，用蛋白酶切割，并且 通过 MALDVToF、离子阱和/或纳米 ESI 质谱法进行测序。最后，我们 应检查 GRK 活性与抑制蛋白结合之间的关系 HA-hD1 中 G 蛋白解偶联和功能脱敏的启动 C-6系统。 Q-arrestins 的补体将被确定，并且改变 测量 GRK 和视紫红质抑制蛋白与 D1 受体的高亲和力结合 暴露于选择的 D1 激动剂后。受体-G-蛋白的丧失 将评估标记 GTP 类似物结合的耦合以进行关联 受体与 )-arrestin 结合水平解偶联。的必要性 5-抑制蛋白在 C-6 细胞中 HA-hD1 受体功能脱敏中的作用 将使用显性失活突变体进行研究，并且GRK之间的关系 和 p-arrestins 使用 Aim 1 研究中开发的工具进行评估。这些研究 β-抑制蛋白的结合和招募将提供另一种功能 D1 受体分子机制描述的终点 脱敏。