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.

项目概要/摘要 纤毛病是由纤毛缺失或信号传导功能障碍引起的遗传性疾病，其特征是多种 缺陷，包括神经系统症状。 G 蛋白偶联受体 (GPCR) 的一个子集富含 初级纤毛，包括调节神经递质多巴胺的受体。多巴胺受体 (DR) 纤毛 十年前就描述了定位，但潜在的细胞贩运机制在很大程度上仍然存在 不明确的。有趣的是，我们实验室和其他实验室发表的工作支持了这样的假设：DR 纤毛 靶向机制具有选择性，与其他睫状 GPCR 不同。此外，纤毛特异性 DR 的影响 神经元功能和活动的信号传导在很大程度上尚未被探索。 该提案旨在检验两个中心假设：(1) DR 纤毛运输在机制上不同于 对 GPCR 纤毛定位的普遍理解，以及 (2) 来自初级纤毛影响的 DR 信号传导 多巴胺能刺激的神经元 cAMP 下游。我们将采取多方面的措施来解决 这些问题使用 IMCD3 细胞，这是一种已建立的纤毛细胞培养模型，非常适合研究分子生物学 支持 GPCR 纤毛贩运的机制。首先，我们将阐明和完善 DR 序列基序 对于纤毛定位来说是必要且充分的。然后，我们将采用公正的定量蛋白质组学来揭示 直接与 DR 结合的新因子，是纤毛定位所必需的。接下来我们来测试一下DR cilia是否 靶向需要使用标准的与其他纤毛定位 GPCR 不同的纤毛运输机制子集 生化和 CRISPR/Cas9 基因编辑技术。此外，该提案将检验以下假设： 异源细胞中 DR 纤毛运输所需的结构决定因素和纤毛运输蛋白是 保守于纹状体中型多棘神经元，即内源性 DR 环境。最后，我们将检查 纤毛特异性 DR 激活的下游后果，重点是纹状体神经元中的 cAMP。拟议的 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 检查来自神经元初级纤毛的局部 DR 信号传导的影响。此外，培训计划和 该提案中详细说明的机构支持为推动申请人走向成功提供了特殊的工具 作为一名独立科学家的职业生涯。