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
基本信息
- 批准号:10464628
- 负责人:
- 金额:$ 6.72万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:AddressBindingBinding ProteinsBiochemicalBiological AssayBiosensorBrainCRISPR/Cas technologyCell Culture TechniquesCell LineCell membraneCell modelCell surfaceCellsCiliaClustered Regularly Interspaced Short Palindromic RepeatsCo-ImmunoprecipitationsCognitionComplexCorpus striatum structureCultured CellsCyclic AMPDefectDevelopmentDopamineDopamine ReceptorG Protein-Coupled Receptor SignalingG-Protein-Coupled ReceptorsGenesGenetic DiseasesGoalsLearningLightMediatingMediator of activation proteinModelingMolecularMorphologyMovementMusNeurologic SymptomsNeuromodulatorNeuronsNeurotransmittersPathway interactionsPhysiologicalPopulationProteinsProteomicsPublishingRattusReceptor ActivationReceptor SignalingRegulationReportingRewardsScientistSensorySignal PathwaySignal TransductionSliceStructural ProteinSurfaceTechniquesTestingTimeTrainingValidationWorkcareerciliopathyexperimental studymonoamineneuroregulationnovelprotein transportreceptortooltrafficking
项目摘要
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
纤毛病是由纤毛丧失或功能障碍信号引起的遗传疾病,其特征是
缺陷,包括神经系统症状。 G蛋白偶联受体(GPCR)的一部分富含
原发性纤毛,包括调节神经递质多巴胺的受体。多巴胺受体(DR)纤毛
十年前描述了本地化,但潜在的蜂窝贩运机制仍在很大程度上仍然存在
不明确的。有趣的是,我们实验室和其他人的出版工作支持了Cilia博士的假设
靶向机制是选择性的,并且与其他睫状GPCR不同。此外,纤毛特异性博士的影响
关于神经元功能和活性的信号在很大程度上没有探索。
该提案旨在检验两个中心假设:(1)纤毛贩运博士在机械上与
对GPCR纤毛定位的普遍了解,以及(2)主要纤毛影响的DR信号传导
多巴胺能刺激下游的神经元营地。我们将采用多方面的方法来解决
这些问题使用IMCD3细胞,IMCD3细胞是一种已建立的固定细胞培养模型,非常适合研究分子
GPCR纤毛贩运的机制。首先,我们将阐明和完善DR序列基序
必要且足够用于纤毛本地化。然后,我们将采用公正的定量蛋白质组学来揭示
直接与DR结合的新因素,并且是纤毛定位所必需的。接下来,我们将测试Cilia博士是否
靶向需要使用标准的其他纤毛核心gpcr的纤毛运输机械的不同子集
生化和CRISPR/CAS9基因编辑技术。此外,该提议将检验以下假设。
Cilia在异源细胞中运输所需的结构决定因素和纤毛运输蛋白是
在纹状体培养基神经元中保守,内源性DR上下文。最后,我们将研究
纤毛特异性的DR激活的下游后果,重点是沿纹状体神经元中的cAMP。提议
实验将大大扩展我们对选择性睫毛贩运机制的理解,并成为第一个
检查神经元原发性纤毛的局部DR信号传导的影响。此外,培训计划和
本提案中详述的机构支持提供了杰出的工具,以推动申请人迈向
作为独立科学家的职业。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Rita Reale Fagan其他文献
Rita Reale Fagan的其他文献
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{{ truncateString('Rita Reale Fagan', 18)}}的其他基金
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 - 财政年份:2022
- 资助金额:
$ 6.72万 - 项目类别:
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