Molecular determinants of Arrestin-mediated dopamine D3R modulation of the T-type Ca2+ channel CaV3.2

项目摘要 多巴胺（DA）信号的失调被认为是严重精神疾病（SMI）的神经缺陷的基础， 包括精神分裂症、躁郁症和抑郁症。SMI的治疗依赖于第二代 抗精神病药（SGAs），其以高亲和力结合抑制性Gi/o三聚体G蛋白的DA D2样家族 偶联受体，从而损害DA受体响应DA向G蛋白发出信号的能力。 SGAs的主要靶点被认为是D2 DA受体（D2 R），体外结合试验 证明了这些药物对D3 DA受体（D3 R）的亲和力相等或甚至更高，已知D3 DA受体 定位于与情感相关的神经位点。最近，我们发现D3受体在轴突起始段（AIS）中表达， 中脑和前额叶皮层通过Arrestin介导的ERK 1/2依赖性调节细胞兴奋性 与T型Ca 2+通道（CaV3.2）相互作用，而不是典型的Gi介导的Ca 2+通道抑制。 这是特别有趣的，因为DA与D3 R的结合并不促进Arrestin募集，除了，正如我们所做的， 发现，伴随着蛋白激酶C（PKC）通过第二信使和/或去极化的激活。 此外，我们发现一些SGAs甚至在细胞中也参与这种Arrestin特异性信号传导机制。 没有G蛋白激活。我假设配体结合和PKC激活促进独立的 D3 R上的磷酸化事件，这两者都是抑制蛋白接合和通道调节所需的。 在这里，我将研究D3 R和CaV3.2之间的神经调节相互作用的分子决定因素。 我将确定D3 R上的配体依赖性和PKC依赖性激酶磷酸化位点， 使用定点诱变将其整合到这种功能性相互作用中。我会稳定地创造异源细胞系 表达这些突变体受体，并使用体外全细胞检测它们对D3 R Ca 2+调节的影响 膜片钳电生理学在这个异源系统。我还将研究一个SGAs小组的能力， 募集抑制蛋白并调节通道功能。最后，我将检查哪些SGA配体，作为一个结果， 抑制蛋白接合，促进D3 R内吞作用和在延长的药物施用后降解，以及 这种差异性的贩运是否可以解释在人类中常见的各种认知副作用， 患者群体，使用体内小鼠行为模型。我假设，虽然所有的SGAs拮抗G 蛋白质信号传导，只有一些将参与Arrestin支持AIS中CaV3.2的急性抑制，而其他 不会的推而广之，我还假设，选择配体，从事抑制蛋白将促进D3 R 在重复SGA给药期间下调，而那些不下调的药物将促进D3 R上调， 防止DA介导的内吞作用和降解。我的目标是更好地描述SGA的D3 R特性 抑制蛋白介导的信号传导和运输，从而提供对SGAs的作用/副作用特征的了解 或许还能指导下一代SMI治疗方法的发展。

PROJECT SUMMARY Dysregulation of dopamine (DA) signaling is thought to underlie the neural deficits of serious mental illness (SMI), including schizophrenia, bipolar disorder, and depression. Treatment for SMI relies on second generation antipsychotics (SGAs), which bind with high affinity to the DA D2-like family of inhibitory Gi/o trimeric G protein coupled receptors, thereby impairing the ability of DA receptors to signal to G protein in response to DA. While the primary target of SGAs was thought to be the D2 DA receptor (D2R), in-vitro binding assays have demonstrated equal or even higher affinity of these drugs for the D3 DA receptor (D3R), which is known to be localized in neural loci associated with affect. Recently, we found that D3Rs in the axon initial segment (AIS) of midbrain and prefrontal cortex modulate cellular excitability via an Arrestin-mediated, ERK1/2 dependent interaction with T-type Ca2+ channels (CaV3.2), as opposed to canonical Gi-mediated Ca2+ channel inhibition. This is particularly intriguing as DA binding to D3R does not promote Arrestin recruitment except, as we have found, with concomitant activation of protein kinase C (PKC) via second messengers and/or depolarization. Furthermore, we have found that some SGAs engage this Arrestin-specific signaling mechanism even in the absence of G protein activation. I hypothesize that ligand binding and PKC activation promote independent phosphorylation events on the D3R, both of which are required for Arrestin engagement and channel modulation. Here, I will examine the molecular determinants of the neuromodulatory interaction between the D3R and CaV3.2. I will determine the ligand-dependent and PKC-dependent kinase phosphorylation sites on the D3R that are integral to this functional interaction using site-directed mutagenesis. I will create heterologous cell lines stably expressing these mutant receptors and examine their impact on D3R Ca2+ modulation using in vitro whole cell patch clamp electrophysiology in this heterologous system. I will also examine the ability of a panel of SGAs to recruit Arrestin and modulate channel function. Lastly, I will examine which SGA ligands, as a consequence of Arrestin engagement, promote D3R endocytosis and degradation upon prolonged drug administration, and whether this differential trafficking can account for the variable cognitive side-effects commonly observed in patient populations, using in vivo mouse behavioral models. I hypothesize that while all SGAs antagonize G protein signaling, only some will engage Arrestin supporting acute inhibition of CaV3.2 in the AIS while others will not. By extension, I also hypothesize that the select ligands that engage Arrestin will promote D3R downregulation during repeated SGA administration while those that do not will promote D3R upregulation by preventing DA-mediated endocytosis and degradation. My goal is to better characterize SGAs for their D3R Arrestin-mediated signaling and trafficking, and thereby provide insight into the effect/side-effect profiles of SGAs and perhaps guide the development of next generation treatments for SMI.