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

Arrestin 介导的多巴胺 D3R 对 T 型 Ca2 通道 CaV3.2 调节的分子决定因素

• 批准号: 10585908
• 负责人: Caroline Marie Keeshen
• 金额: $ 4万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10585908
• 关键词: Acute; Affect; Affinity; Agonist; Antipsychotic Agents; Arrestins; Axon; Behavior; Behavioral; Behavioral Model; Binding; Biological Assay; Biological Models; Bipolar Disorder; Cell model; Cells; Chronic; Clinical; Clozapine; Clozaril; Cognitive; Coupled; Couples; Detection; Development; Dopamine; Dopamine D2 Receptor; Dopamine Receptor; Electrophysiology (science); Endocytosis; Event; Exhibits; Family; G-Protein-Coupled Receptors; GTP-Binding Proteins; Generations; Goals; Impairment; In Vitro; Ligand Binding; Ligands; LoxP-flanked allele; MAPK3 gene; Mediating; Mental Depression; Midbrain structure; Molecular; Motor Activity; Mus; Mutation; Names; Outcome; Patients; Pharmaceutical Preparations; Phosphorylation; Phosphorylation Site; Phosphotransferases; Population; Prefrontal Cortex; Property; Protein Kinase C; Protein Sortings; Quinpirole; Receptor Down-Regulation; Receptor Signaling; Receptor Up-Regulation; Research; Resistance; Schizophrenia; Second Messenger Systems; Serine; Seroquel; Signal Pathway; Signal Transduction; Signaling Protein; Site-Directed Mutagenesis; Startle Reaction; System; Testing; Therapeutic; Threonine; Time; Work; antagonist G; behavior measurement; desensitization; dopamine D3 receptor; effective therapy; experimental study; flexibility; improved; in vivo; insight; mimetics; mutant; neural; neuropsychiatry; neuroregulation; next generation; novel therapeutics; patch clamp; patient population; pre-clinical; prepulse inhibition; prevent; profiles in patients; protein activation; protein kinase C kinase; quetiapine; radioligand; receptor; receptor downregulation; receptor upregulation; recruit; response; scaffold; severe mental illness; side effect; stable cell line; trafficking

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.

项目总结 多巴胺(DA)信号的失调被认为是严重精神疾病(SMI)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 抗精神病药物(SGAs)，它与抑制性Gi/o三聚体G蛋白的DA D2样家族具有高亲和力 偶联受体，从而削弱DA受体向G蛋白发送信号以响应DA的能力。而当 SGAs的主要靶点被认为是D2DA受体(D2R)，体外结合试验有 证明这些药物对D3DA受体(D3R)的亲和力相等或更高，已知D3DA受体 定位于与情感相关的神经部位。最近，我们发现D3Rs在大脑中的轴突起始节段(AIS)。 中脑和前额叶皮质通过arrestin介导的ERK1/2依赖性调节细胞兴奋性 与T-型钙通道(CaV3.2)的相互作用，而不是典型的GI介导的钙通道抑制。 这特别耐人寻味，因为DA与D3R的结合不会促进Arrestin的招募，除非我们已经 发现，伴随着蛋白激酶C(PKC)通过第二信使和/或去极化而激活。 此外，我们还发现，一些SGAs甚至在 缺乏G蛋白激活。我假设配体结合和PKC激活促进独立 D3R上的磷酸化事件，这两个事件都是arrestin参与和通道调节所必需的。 在这里，我将研究D3R和CaV3.2之间神经调节相互作用的分子决定因素。 我将确定D3R上的配体依赖和PKC依赖的激酶磷酸化位点，它们是 这是使用定点突变的这种功能相互作用的组成部分。我将稳定地建立异源细胞系 利用体外全细胞表达这些突变型受体并检测它们对D3R钙调节的影响 膜片钳在这个异种系统中的电生理学。我还将审查SGA小组是否有能力 补充Arrestin和调制通道功能。最后，我将检查哪些SGA配体，作为结果 Arrestin参与，促进D3R内吞和长期给药后的降解，以及 这种不同的贩卖行为是否可以解释通常在 患者群体，使用活体小鼠行为模型。我假设虽然所有的SGA都与G对抗 蛋白信号转导，只有一些与arrestin结合，支持AIS中CaV3.2的急性抑制，而另一些 不会的。推而广之，我还假设与arrestin结合的部分配体将促进D3R 在重复SGA管理期间下调，而不这样做的将通过以下方式促进D3R上调 防止DA介导的内吞和降解。我的目标是更好地描述SGA的D3R Arrestin介导的信号和转运，从而提供对SGAs的效果/副作用概况的洞察 也许还可以指导下一代治疗SMI的方法的开发。