[18]

[18]. material == The online version of this article (doi:10.1007/s00018-010-0603-4) contains supplementary I-CBP112 material, which is available to authorized users. Keywords:Ciliopathy, Neuronal cilia, Dopamine receptor 1, Bardet-Biedl syndrome == Introduction == Nonmotile main cilia are typically solitary microtubule-based organelles present on nearly every cell type in the mammalian body. Main cilia play crucial functions in coordinating numerous cellular I-CBP112 and developmental signaling pathways [13]. Neurons throughout the mammalian brain possess a main cilium projecting from your cell body. The fact that several ciliopathies have prominent neurological phenotypes indicates that neuronal cilia provide important functions in the brain [4]. Subsets of neuronal cilia contain certain G protein-coupled receptors (GPCRs), including somatostatin receptor 3 (Sstr3) [5], serotonin receptor 6 (Htr6) [6,7], and melanin-concentrating hormone receptor 1 (Mchr1) [8,9]. This suggests that some neuronal cilia sense neuromodulators in the brain. Yet, the signaling pathways mediated by the vast majority of neuronal cilia in the mammalian brain are unknown. The ciliopathy Bardet-Biedl syndrome (BBS) is characterized by obesity, retinal dystrophy, renal anomalies, hypogenitalism, polydactyly, and cognitive deficits [10]. There are at least 14 causative BBS genes [11], and seven of the BBS proteins (BBS1, 2, 4, 5, 7, 8, and 9) form a complex, called the BBSome, that constitutes a HMMR coat complex and mediates vesicular trafficking to the cilium [12,13]. We previously reported that Sstr3 and Mchr1 fail to localize to neuronal cilia in mouse models of BBS [9], suggesting BBS proteins are required for trafficking of GPCRs to neuronal cilia. This is supported by a recent study showing that this BBSome interacts with a fragment of human SSTR3 and this interaction is required for ciliary targeting [12]. Furthermore, as Mchr1 is an important regulator of feeding behavior [14] and BBS mice develop hyperphagia-induced obesity [15,16], we hypothesize that mislocalization of cell-type-specific ciliary signaling proteins together with disruption of signaling is the fundamental mechanism underlying the pathophysiology of the pleiotropic BBS phenotypes. A critical step in determining the signaling pathways mediated by neuronal cilia is usually to identify the signaling proteins that specifically localize to cilia. Even though ciliary membrane is usually contiguous with the plasma membrane, ciliary localization of proteins is usually tightly regulated and only certain proteins localize to the ciliary membrane. We recently reported that ciliary GPCRs selectively localize to cilia when expressed in inner medullary collecting duct (IMCD) cells whereas nonciliary GPCRs do not localize to cilia [8]. Using expression of chimeric GPCRs in IMCD cells, we found that the third intracellular (i3) loops I-CBP112 of Sstr3 and Htr6 are sufficient to mediate ciliary localization of the nonciliary receptors Sstr5 and Htr7, respectively [8]. Comparison of the i3 loops from Sstr3 and Htr6 recognized a loose consensus sequence that we used to identify Mchr1 as a novel ciliary GPCR. Interestingly, the i3 loops of Sstr3 and Htr6 are sufficient but not necessary for ciliary localization [8], suggesting you will find additional ciliary localization sequences within Sstr3 and Htr6. Here, we use the presence of putative ciliary targeting sequences in the i3 loop and carboxy terminal tail to predict novel ciliary GPCRs. We show, for the first time, that dopamine receptor 1 (D1) localizes to mouse neuronal main cilia. Notably, this localization is usually dynamic and D1 is usually recruited to cilia in response to an increase in cAMP levels and treatment with receptor ligand causes a rapid decrease in D1 ciliary localization. Interestingly, D1 ciliary localization is usually increased in neurons from mice lacking BBS proteins and does not decrease in response to ligand treatment. Furthermore, we find that a subunit of the BBSome, Bbs5, interacts with D1. Together, these results suggest that D1 dynamically localizes to neuronal cilia and that translocation of D1 out of cilia in response to agonist requires the BBSome and entails conversation with Bbs5. == Materials and methods == == Plasmid construction == Coding sequences of the D1 receptor, BBSome subunits, and Gapdh were amplified from reverse-transcribed mouse whole brain RNA using the.