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.