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

项目总结/摘要 纤毛病是由纤毛损失或功能失调的信号传导引起的遗传性疾病，其特征在于多种 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，包括调节性神经递质多巴胺的受体。多巴胺受体（DR）纤毛 十年前就有人描述了局部化，但基本的细胞贩运机制仍然主要是 未定义。有趣的是，我们实验室和其他人发表的工作支持DR纤毛 靶向机制是选择性的并且不同于其他睫状GPCR。此外，纤毛特异性DR的影响 对神经元功能和活性的信号传导在很大程度上未被探索。 该提议旨在测试两个中心假设：（1）DR纤毛运输在机制上不同于 GPCR纤毛定位的普遍理解，和（2）来自初级纤毛的DR信号影响 多巴胺能刺激下游的神经元cAMP。我们将采取多方面的办法， 这些问题使用IMCD 3细胞，一个建立的纤毛细胞培养模型，理想的研究分子 支持GPCR纤毛运输的机制。首先，我们将阐明和完善DR序列基序 这是纤毛定位的必要条件和充分条件。然后，我们将采用无偏见的定量蛋白质组学来揭示 新的因子，直接结合DR，纤毛定位所需的。接下来，我们将测试DR纤毛是否 靶向需要纤毛运输机制的不同子集，从其他纤毛定位的GPCR使用标准 生物化学和CRISPR/Cas9基因编辑技术。此外，本提案将检验以下假设： 在异源细胞中DR纤毛运输所需的结构决定簇和纤毛运输蛋白是 在纹状体中等多刺神经元中保守，内源性DR背景。最后，我们将研究 在纹状体神经元中，纤毛特异性DR激活的下游后果，集中于cAMP。拟议 实验将大大扩展我们对选择性纤毛运输机制的理解，并成为第一个 检查神经元中来自初级纤毛的局部DR信号的影响。此外，培训计划和 本建议书中详述的机构支持为推动申请人实现 作为一名独立的科学家。

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.