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: 5.98). 05/2008; 19(4):1540-7. DOI: 10.1091/mbc.E07-09-0942
Source: PubMed

ABSTRACT 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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    • "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.
    Cell calcium 03/2015; DOI:10.1016/j.ceca.2015.03.005 · 4.21 Impact Factor
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    • "We narrowed the region important to NPY2R ciliary localization to the intracellular loops in the IC3- IC4 region (Figure S2). The previously characterized Ax[A/S] xxQ CTS within intracellular loop 3 (IC3) of SSTR3 is not present in either NPY2R or the closely related GPR83 (Figure 3A) (Berbari et al., 2008a). Instead, we determined that the [R/K][I/L]W sequence in IC3 is required for ciliary localization by mutating amino acid residues that were similar among Pgr15l, GPR83, and NPY2R but dissimilar in nonciliary NPY1R (Figures 3A, 3B, and S3). "
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    ABSTRACT: Human monogenic obesity syndromes, including Bardet-Biedl syndrome (BBS), implicate neuronal primary cilia in regulation of energy homeostasis. Cilia in hypothalamic neurons have been hypothesized to sense and regulate systemic energy status, but the molecular mechanism of this signaling remains unknown. Here, we report a comprehensive localization screen of 42 G-protein-coupled receptors (GPCR) revealing seven ciliary GPCRs, including the neuropeptide Y (NPY) receptors NPY2R and NPY5R. We show that mice modeling BBS disease or obese tubby mice fail to localize NPY2R to cilia in the hypothalamus and that BBS mutant mice fail to activate c-fos or decrease food intake in response to the NPY2R ligand PYY3-36. We find that cells with ciliary NPY2R show augmented PYY3-36-dependent cAMP signaling. Our data demonstrate that ciliary targeting of NPY receptors is important for controlling energy balance in mammals, revealing a physiologically defined ligand-receptor pathway signaling within neuronal cilia.
    Cell Reports 12/2013; 5. DOI:10.1016/j.celrep.2013.11.011 · 7.21 Impact Factor
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    • "/ mice (Berbari et al., 2008b). The BBSome interacts with the ciliary targeting motif in the IP3 loop of SSTR3, and IP3 SSTR3 -GFP fusion proteins translocate into cilia in a BBSome–dependent manner (Berbari et al., 2008a; Jin et al., 2010; Domire et al., 2011). The BBSome could facilitate the transport of proteins from the plasma membrane through the barrier of the ciliary transition zone into the ciliary membrane proper (Nachury et al., 2010). "
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    ABSTRACT: The BBSome is a complex of seven proteins, including BBS4, that is cycled through cilia by intraflagellar transport (IFT). Previous work has shown that the membrane-associated signaling protein phospholipase D (PLD) accumulates abnormally in cilia of Chlamydomonas reinhardtii bbs mutants. Here we show that PLD is a component of wild-type cilia but is enriched ∼150-fold in bbs4 cilia; this accumulation occurs progressively over time and results in altered ciliary lipid composition. When wild-type BBSomes were introduced into bbs cells, PLD was rapidly removed from the mutant cilia, indicating the presence of an efficient BBSome-dependent mechanism for exporting ciliary PLD. This export requires retrograde IFT. Importantly, entry of PLD into cilia is BBSome and IFT independent. Therefore, the BBSome is required only for the export phase of a process that continuously cycles PLD through cilia. Another protein, carbonic anhydrase 6, is initially imported normally into bbs4 cilia but lost with time, suggesti
    The Journal of Cell Biology 04/2013; 201(2-2):249-261. DOI:10.1083/jcb.201207139 · 9.69 Impact Factor
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