Identification of Ciliary Localization Sequences within the Third Intracellular Loop of G Protein-coupled Receptors

Department of Pharmacology, Division of Human Genetics, and College of Medicine, The Ohio State University, Columbus, OH 43210, USA.
Molecular biology of the cell (Impact Factor: 4.47). 05/2008; 19(4):1540-7. DOI: 10.1091/mbc.E07-09-0942
Source: PubMed


Primary cilia are sensory organelles present on most mammalian cells. The functions of cilia are defined by the signaling proteins localized to the ciliary membrane. Certain G protein-coupled receptors (GPCRs), including somatostatin receptor 3 (Sstr3) and serotonin receptor 6 (Htr6), localize to cilia. As Sstr3 and Htr6 are the only somatostatin and serotonin receptor subtypes that localize to cilia, we hypothesized they contain ciliary localization sequences. To test this hypothesis we expressed chimeric receptors containing fragments of Sstr3 and Htr6 in the nonciliary receptors Sstr5 and Htr7, respectively, in ciliated cells. We found the third intracellular loop of Sstr3 or Htr6 is sufficient for ciliary localization. Comparison of these loops revealed a loose consensus sequence. To determine whether this consensus sequence predicts ciliary localization of other GPCRs, we compared it with the third intracellular loop of all human GPCRs. We identified the consensus sequence in melanin-concentrating hormone receptor 1 (Mchr1) and confirmed Mchr1 localizes to primary cilia in vitro and in vivo. Thus, we have identified a putative GPCR ciliary localization sequence and used this sequence to identify a novel ciliary GPCR. As Mchr1 mediates feeding behavior and metabolism, our results implicate ciliary signaling in the regulation of body weight.

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Available from: Kirk Mykytyn, Jan 13, 2014
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    • "To begin to explore the biochemical mechanism of D1R ciliary targeting, we searched for structural determinants within the receptor that are required for ciliary localization. Previous studies of other cilia-localized receptors have identified targeting determinants located either in a cytoplasmic loop (Berbari et al., 2008a) or the cytoplasmic tail (C-tail; Deretic et al., 1998; Corbit et al., 2005; Geng et al., 2006; Jenkins et al., 2006). Progressive truncation of the D1R C-tail (Figure 3A) strongly reduced ciliary localization of receptors. "
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    ABSTRACT: Appropriate physiological signaling by primary cilia depends on the specific targeting of particular receptors to the ciliary membrane, but how this occurs remains poorly understood. In this study, we show that D1-type dopaminergic receptors are delivered to cilia from the extra-ciliary plasma membrane by a mechanism requiring the receptor cytoplasmic tail, the intraflagellar transport complex-B (IFT-B), and ciliary kinesin KIF17. This targeting mechanism critically depends on Rab23, a small guanine nucleotide binding protein that has important effects on physiological signaling from cilia but was not known previously to be essential for ciliary delivery of any cargo. Depleting Rab23 prevents dopamine receptors from accessing the ciliary membrane. Conversely, fusion of Rab23 to a non-ciliary receptor is sufficient to drive robust, nucleotide-dependent mis-localization to the ciliary membrane. Dopamine receptors thus reveal a previously unrecognized mechanism of ciliary receptor targeting and functional role of Rab23 in promoting this process.
    Full-text · Article · Jul 2015 · eLife Sciences
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    • "In addition to housing ion channels, the primary cilium is enriched with molecular components from a variety of signaling pathways. These molecules include GPCRs such as serotonin receptor isoform 6 (5HT6) and somatostatin receptor 3 (SSTR3) [33], RTKs such as PDGFR-␣ and EGFR [34], as well as class III adenylate cyclases (ACs) such as AC 3, 5 and 6 [35] [36] [37]. The enrichment of these molecules inside primary cilia suggests that the local signal strength of the downstream pathways could be more robust in the cilium than the surrounding cell membrane. "
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    ABSTRACT: The primary cilium is a solitary hair-like organelle on the cell surface that serves as an antenna sensing ever-changing environmental conditions. In this review, we will first recapitulate the molecular basis of the polymodal sensory function of the primary cilia, specifically focusing on transient receptor potential (TRP) channels that accumulate inside the organelle and conduct calcium ions (Ca(2+)). Each subfamily member, namely TRPP2 TRPP3, TRPC1 and TRPV4, is gated by multiple environmental factors, including chemical (receptor ligands, intracellular second messengers such as Ca(2+)), mechanical (fluid shear stress, hypo-osmotic swelling), or physical (temperature, voltage) stimuli. Both activity and heterodimer compositions of the TRP channels may be dynamically regulated for precise tuning to the varying dynamic ranges of the individual input stimuli. We will thus discuss the potential regulation of TRP channels by local second messengers. Despite its reported importance in embryonic patterning and tissue morphogenesis, the precise functional significance of the downstream Ca(2+) signals of the TRP channels remains unknown. We will close our review by featuring recent technological advances in visualizing and analyzing signal transduction inside the primary cilia, together with current perspectives illuminating the functional significance of intraciliary Ca(2+) signals. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Full-text · Article · Mar 2015 · Cell calcium
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    • "Primary cilia have been implicated in the proper transduction of multiple signaling pathways. These include pathways necessary for development and differentiation, such as Sonic hedgehog (Shh) and Wnt, [10], [11], as well as those necessary for regulation of response to physiological cues, such as insulin [12] and melanin-concentrating hormone [13]. Additionally, neuronal signaling receptors, including several neuropeptide receptors have been localized to cilia and require interaction with ciliary proteins. "
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    ABSTRACT: Primary cilia regulate an expanding list of signaling pathways in many different cell types. It is likely that identification of the full catalog of pathways associated with cilia will be necessary to fully understand their role in regulation of signaling and the implications for diseases associated with their dysfunction, ciliopathies. Bardet-Biedl Syndrome (BBS) is one such ciliopathy which is characterized by a spectrum of phenotypes. These include neural defects such as impaired cognitive development, centrally mediated hyperphagia and peripheral sensory defects. Here we investigate potential defects in a signaling pathway associated with neuronal function, brain derived neurotrophic factor (BDNF) signaling. Upon loss of BBS4 expression in cultured cells, we observed decreased phosphorylation and activation by BDNF of its target receptor, TrkB. Assessment of ciliary localization revealed that, TrkB localized to the axonemes or basal bodies of cilia only in the presence of BDNF. Axonemal localization, specifically, was abrogated with loss of BBS4. Finally, we present evidence that loss of the ciliary axoneme through depletion of KIF3A impedes activation of TrkB. Taken together, these data suggest the possibility of a previously uninvestigated pathway associated with perturbation of ciliary proteins.
    Full-text · Article · May 2014 · PLoS ONE
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