Investigating the trafficking mechanisms and signaling consequences of dopamine receptor primary cilia localization

研究多巴胺受体初级纤毛定位的运输机制和信号传导后果

• 批准号: 10595554
• 负责人: Rita Reale Fagan
• 金额: $ 6.95万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10595554
• 关键词: Address; Binding; Binding Proteins; Biochemical; Biological Assay; Biosensor; Brain; CRISPR/Cas technology; Cell Culture Techniques; Cell Line; Cell membrane; Cell model; Cell surface; Cells; Cilia; Clustered Regularly Interspaced Short Palindromic Repeats; Co-Immunoprecipitations; Cognition; Complex; Corpus striatum structure; Cultured Cells; Cyclic AMP; Defect; Development; Dopamine; Dopamine Receptor; Exclusion; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; Genes; Genetic Diseases; Goals; Institution; Learning; Mediating; Mediator; Modeling; Molecular; Morphology; Movement; Mus; Neurologic Symptoms; Neuromodulator; Neurons; Neurotransmitters; Pathway interactions; Physiological; Population; Proteins; Proteomics; Publishing; Rattus; Receptor Activation; Receptor Signaling; Regulation; Reporting; Rewards; Scientist; Sensory; Signal Pathway; Signal Transduction; Slice; Structural Protein; Surface; Techniques; Testing; Time; Training; Transfection; Validation; Work; career; ciliopathy; experimental study; monoamine; neuroregulation; novel; protein transport; prototype; receptor; structural determinants; tool; trafficking

Project Summary/Abstract Ciliopathies are genetic disorders that arise from cilia loss or dysfunctional signaling, characterized by a multitude of defects, including neurological symptoms. A subset of G protein-coupled receptors (GPCRs) are enriched in primary cilia, including receptors for the modulatory neurotransmitter, dopamine. Dopamine receptor (DR) cilia localization was described a decade ago, yet the underlying cellular trafficking machinery remains largely undefined. Interestingly, published work from our lab and others supports the hypothesis that the DR cilia targeting mechanism is selective and distinct from other ciliary GPCRs. Moreover, the impact of cilia-specific DR signaling on neuronal function and activity is largely unexplored. This proposal aims to test two central hypotheses: (1) DR cilia trafficking is mechanistically distinct from the prevailing understanding of GPCR cilia localization, and (2) DR signaling from the primary cilium impacts neuronal cAMP downstream of dopaminergic stimulation. We will take a multifaceted approach in order to tackle these questions using IMCD3 cells, an established ciliated cell culture model ideal for investigating the molecular mechanisms underpinning GPCR cilia trafficking. First, we will elucidate and refine the DR sequence motifs necessary and sufficient for cilia localization. We will then employ unbiased, quantitative proteomics to uncover novel factors that bind directly to DRs, and are required for cilia localization. Next, we will test whether DR cilia targeting requires a distinct subset of cilia transport machinery from other cilia-localized GPCRs using standard biochemical and CRISPR/Cas9 gene editing techniques. Furthermore, this proposal will test the hypothesis that the structural determinants and cilia trafficking proteins required for DR cilia trafficking in heterologous cells are conserved in striatal medium spiny neurons, the endogenous DR context. Finally, we will examine the downstream consequences of cilia-specific DR activation, focusing on cAMP, in striatal neurons. The proposed experiments will greatly expand our understanding of selective ciliary trafficking mechanisms, and be the first to examine the impact of localized DR signaling from primary cilia in neurons. In addition, the training plan and institutional support detailed in this proposal provide exceptional tools for advancing the applicant towards a career as an independent scientist.

项目摘要/摘要 纤毛病是由纤毛丢失或信号功能障碍引起的遗传性疾病，其特征是 包括神经症状在内的缺陷。G蛋白偶联受体(GPCRs)的一个子集富含 初级纤毛，包括调节性神经递质多巴胺的受体。多巴胺受体(DR)纤毛 本地化是十年前描述的，然而潜在的蜂窝贩运机制在很大程度上仍然存在 未定义。有趣的是，我们实验室和其他实验室发表的工作支持了cilia博士的假设 靶向机制是选择性的，不同于其他睫状GPCR。此外，纤毛特异性DR的影响 神经元功能和活动的信号转导在很大程度上是未知的。 这项提议旨在检验两个核心假设：(1)cilia traffing博士在机制上与 对纤毛定位的普遍理解，以及(2)来自初级纤毛撞击的DR信号 多巴胺能刺激下游的神经元cAMP。我们将采取多方面的方法来解决 这些问题利用IMCD3细胞，建立了纤毛细胞培养模型，理想地用于研究分子 支持GPCR纤毛贩运的机制。首先，我们将阐明和提炼DR序列基序 纤毛定位的必要性和充分性。然后，我们将使用无偏见的定量蛋白质组学来揭示 直接与DRS结合的新因子，是纤毛定位所必需的。接下来，我们将测试西莉亚博士 目标定位需要纤毛运输机械的不同子集，不同于其他纤毛定位的GPCR，使用标准 生物化学和CRISPR/Cas9基因编辑技术。此外，这一提议将检验这样一个假设： 异种细胞中DR纤毛运输所需的结构决定因素和纤毛运输蛋白是 在纹状体中保守的棘神经元，内源性DR的背景。最后，我们将研究 纤毛特异性DR激活的下游后果，集中在cAMP，在纹状体神经元。建议数 实验将极大地扩展我们对选择性纤毛运输机制的理解，并成为第一个 研究初级纤毛对神经元局部DR信号的影响。此外，培训计划和 本提案中详细说明的机构支持提供了特殊工具，以推动申请者实现 独立科学家的职业生涯。