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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：
Rita Reale Fagan
金额：
$ 6.72万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
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：
https://reporter.nih.gov/project-details/10464628
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 G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors Genes Genetic Diseases Goals Learning Light Mediating Mediator of activation protein 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 Validation Work career ciliopathy experimental study monoamine neuroregulation novel protein transport receptor tool trafficking

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.

项目总结/文摘