MOLECULAR REGULATION OF D1 DOPAMINE RECEPTOR FUNCTION

描述(改编自申请人的摘要)： 这是一个基于数据的修订申请，数据显示第一个完整的D1 我们开发的多巴胺激动剂二氢喜定引起严重急性 灵长类动物的抗帕金森效应。最近的数据表明，有一些，但不是 当重复给药时，所有完整的D1激动剂都会产生明显的耐受性。我们 还表明，具有类似疗效的D1多巴胺受体激动剂可以 脱敏能力差异很大。使用一系列刚性数字减震器 激动剂，我们将确定选择性构象是否被 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，不同的受体状态(如果重叠) 蛋白质偶联。首先，我们将研究新型d1激动剂的差异。 功能脱敏血凝素标记的人D1受体(HA-D1R) 在C-6胶质瘤细胞中，受GRK活性的程度和/或模式的影响。 不同激动剂对D1受体磷酸化的速度和程度 显性负性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激素的相对作用。 GRKs和PKA在这种脱敏中的作用。我们还将确定细胞 GRK对激动剂暴露的反应 与G-β-伽马补体相互作用。第二，我们将绘制 HD1R受体的磷酸化残基。 激动剂诱导GRKs和GRKs诱导的受体磷酸化模式 第二信使-激酶(例如，PKA)将通过突变的 HA-HDLC-6细胞中的丝氨酸和苏氨酸。我们还将过度表达HA-D1R HEK293细胞中存在和不存在各种GRK。磷酸化HA-HD1 受体将通过免疫沉淀分离，用蛋白酶切割，并 用MALDVToF、离子陷阱和/或纳米ESI-MS进行测序。最后，我们 应研究GRK活性与Arrestin结合之间的关系 HA-HD1中G蛋白解偶联的启动和功能脱敏 C-6系统。将确定Q-拦截器的补充量，并进行更改 GRKs和抑制素与D1R高亲和力结合的研究 在暴露于选定的D1激动剂后。受体G蛋白的丢失 来自标记的GTP类似物结合的偶联将被评估为相关 受体解偶联与)-arrestin结合水平。……的必要性 5-arrestin(S)对C-6细胞HA-HD1受体的功能性脱敏作用 将利用显性-负性突变体进行研究，以及GRK与 以及使用在Aim 1研究中开发的工具评估的p-拦截剂。这些研究 对β-arrestin的结合和招募将提供另一个功能 D_1受体分子机制研究的终点 脱敏。

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.