Ins and outs of GPCR signaling in primary cilia
2015; Springer Nature; Volume: 16; Issue: 9 Linguagem: Inglês
10.15252/embr.201540530
ISSN1469-3178
AutoresKenneth Bødtker Schou, Lotte B. Pedersen, Søren T. Christensen,
Tópico(s)Retinal Development and Disorders
ResumoReview21 August 2015free access Ins and outs of GPCR signaling in primary cilia Kenneth Bødtker Schou Kenneth Bødtker Schou Department of Biology, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Lotte Bang Pedersen Lotte Bang Pedersen Department of Biology, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Søren Tvorup Christensen Corresponding Author Søren Tvorup Christensen Department of Biology, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Kenneth Bødtker Schou Kenneth Bødtker Schou Department of Biology, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Lotte Bang Pedersen Lotte Bang Pedersen Department of Biology, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Søren Tvorup Christensen Corresponding Author Søren Tvorup Christensen Department of Biology, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Author Information Kenneth Bødtker Schou1, Lotte Bang Pedersen1 and Søren Tvorup Christensen 1 1Department of Biology, University of Copenhagen, Copenhagen, Denmark *Corresponding author. Tel: +45 51322997; E-mail: [email protected] EMBO Reports (2015)16:1099-1113https://doi.org/10.15252/embr.201540530 See the Glossary for abbreviations used in this article. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Primary cilia are specialized microtubule-based signaling organelles that convey extracellular signals into a cellular response in most vertebrate cell types. The physiological significance of primary cilia is underscored by the fact that defects in assembly or function of these organelles lead to a range of severe diseases and developmental disorders. In most cell types of the human body, signaling by primary cilia involves different G protein-coupled receptors (GPCRs), which transmit specific signals to the cell through G proteins to regulate diverse cellular and physiological events. Here, we provide an overview of GPCR signaling in primary cilia, with main focus on the rhodopsin-like (class A) and the smoothened/frizzled (class F) GPCRs. We describe how such receptors dynamically traffic into and out of the ciliary compartment and how they interact with other classes of ciliary GPCRs, such as class B receptors, to control ciliary function and various physiological and behavioral processes. Finally, we discuss future avenues for developing GPCR-targeted drug strategies for the treatment of ciliopathies. Glossary 5-HT6 5-hydroxytryptamine (serotonin) receptor 6 7TM Seven transmembrane ABCC4 ATP-binding cassette sub-family C member 4 ACIII Adenylate cyclase type III ARF ADP-ribosylation factor ARL Arf-related protein ASAP1 Arf-GAP with SH3 domain, ANK repeat and PH domain-containing protein 1 BBS Bardet–Biedl Syndrome cAMP 3′-5′-cyclic adenosine monophosphate CDE Clathrin-dependent endocytosis CiPo Ciliary pocket CNS Central nervous system CTS Ciliary targeting sequence DR Dopamine receptor DRD2S Short isoform of dopamine receptor D2 EP4 Prostaglandin E receptor 4 ERK Extracellular signal-regulated kinase/Mitogen activated protein kinase EVC Ellis–van Creveld syndrome protein FZD Frizzled G protein Guanosine nucleotide-binding protein GAS Growth arrest-specific protein GALR Galanin receptor GEF Guanine nucleotide exchange factor GFP Green fluorescent protein GIPR Gastric inhibitory polypeptide receptor GLI Glioma-associated oncogene homolog GLI-A Activator form of GLI GLI-R Repressor form of GLI GNP Granule neuron progenitor GnRH Gonadotropin-releasing hormone GPCR/GPR G protein-coupled receptor GRK G protein-coupled receptor kinase GTP Guanosine triphosphate HH Hedgehog HTR6 5-hydroxytryptamine (serotonin) receptor 6 IFT Intraflagellar transport ILK Integrin-linked kinase IMCD3 Inner medullary collecting duct cells 3 KIF3A Kinesin-like protein 3A KIF7 Kinesin family member 7 KISS1R Kisspeptin receptor 1 MB Medulloblastoma MCHR1 Melanin-concentrating hormone receptor 1 NMUR1 Neuromedin U receptor 1 NPFFR1 Neuropeptide FF receptor 1 NPYR Neuropeptide Y receptor PAC1 PACAP receptor 1 PACAP Pituitary adenylate cyclase-activating polypeptide PGE2 Prostaglandin E2 PKA cAMP-dependent protein kinase PRLHR Prolactin-releasing hormone receptor PTC Patched QRFPR Pyroglutamylated RF amide peptide receptor RAB Ras-related protein in brain RABIN RAB-interacting protein RAN Ras-related nuclear protein RPE Retinal pigment epithelium SCN Suprachiasmatic nucleus SHH Sonic HH SMO Smoothened SNARE NSF attachment protein receptor SSTR3 Somatostatin receptor 3 T2R Bitter taste receptor 2 TGN Trans-Golgi network TGR G protein-coupled bile acid receptor TRAPP Transport protein particle TUB Tubby TULP Tubby-like protein TZ Transition zone VNO Vomeronasal organ VPAC Vasoactive intestinal peptide receptor WNT Wingless/Int Introduction Signaling through G protein-coupled receptors (GPCRs) regulates a vast array of cellular and physiological processes throughout the eukaryotic kingdom. GPCRs constitute a substantial and highly diverse family of seven transmembrane (7TM) receptors that transmit assorted signals from the extracellular environment to the cell through both G protein-dependent and G protein-independent pathways, which regulate the activity of various cellular signaling networks. GPCRs are encoded by about 800 different genes in humans 12. This large number of GPCRs enables cells to respond to sensory inputs as diverse as odorants, light, lipids, ions, amines, and nucleotides as well as signaling peptides and proteins, such as hormones, morphogens, and neurotransmitters 3. Hence, GPCRs are well-established targets for almost half of all therapeutic drugs. Yet, many GPCRs have been denominated “orphan receptors,” because their natural ligands have escaped identification so far 4. GPCRs are grouped into six classes based on sequence homology and functional similarity. These include the rhodopsin-like receptors (class A), the secretin receptor family (class B), metabotropic glutamate/pheromone (class C), fungal mating pheromone receptors (class D), cyclic AMP (cAMP) receptors (class E) and frizzled/smoothened (class F) 5. All GPCRs share a common topology consisting of an extracellular N-terminus (e1) followed by 7TM-spanning alpha-helices (H1–H7) that are separated by three intracellular (i1–i3) and three extracellular loops (e2–e4), respectively, and a C-terminus (i4) projecting into the cytosol (Fig 1A) 6. Most GPCRs exert their function through pathways involving interaction and activation of heterotrimeric G proteins, although G protein-independent signaling mechanisms occur, for example via receptor-interacting proteins that regulate both agonist-promoted and agonist-inhibited GPCR signaling 7 as well as G protein-interacting protein cross talk with non-GPCR signaling 89. In the absence of an agonist, GPCRs bind to the heterotrimeric G protein complex: a GDP-bound Gα protein (Gαs, Gαq, Gαi/o, and/or Gα12/13) and the Gβγ heterodimer (Fig 1A). Once the GPCR encounters its agonist, the receptor transmutes into its active conformation, which allows GTP exchange with GDP on the Gα protein that in turn dissociates from the Gβγ subunits and prompts downstream signaling through secondary messenger pathways 1011. Similarly, Gβγ activates a variety of signaling events as outlined in Figure 1A. Figure 1. Overview on GPCR signaling and function of ciliary GPCRs in vision and olfaction(A) Examples of sensory inputs for GPCR-mediated signaling pathways through activation of G proteins. (B) List of known ciliary GPCRs in photoreceptor and olfactory receptor neurons. (C) A cartoon of a rod photoreceptor neuron and localization of light-sensitive GPCRs (rhodopsin) in the disk array of the outer segment, which comprises a modified primary cilium. (D) Cartoon of olfactory receptor cells and localization of ciliary GPCRs that detect odorants. Please see text for details and references. Download figure Download PowerPoint In many cases, GPCRs localize to specific subcellular domains for optimization of detection and transduction of both external and cytoplasmic cues to ensure proper regulation of cell-specific functions. As an example, synaptic processes are modulated through the spatiotemporal localization of GPCRs in the highly polarized neuronal membrane environment such as for the type-1 cannabinoid receptor (CB1R), which is a major brain GPCR that predominantly localizes and functions in axons and specific presynaptic nerve terminals 12. Growing evidence further points to the function of GPCR-mediated signaling from the nuclear membrane to activate intranuclear signaling events that regulate physiological function in cardiac myocytes 13. The subcellular domains include defined membrane parts at the cell surface, including lipid rafts/caveolae as well as β-arrestin-dependent endocytosis via clathrin-coated pits that serve as signaling platforms to control compartmentalization of GPCR-mediated signaling 1415. In this regard, GPCRs may function as scaffolds for the recruitment of GPCR-interacting proteins, which modulate the localization of GPCRs to specific intracellular compartments known as signalosomes 15. In this review, we focus on the cilium as a unique domain for GPCR-mediated signaling. Seminal work dating back to the early 1980s and 1990s established how specialized non-motile cilia of photoreceptor cells and olfactory receptor neurons mediate sensory signaling by displaying light and odorant stimulated GPCRs in close physical vicinity to their cognate sensory stimuli (reviewed in 1617) (Fig 1B and C). Such vital sensory roles of cilia in the visual and olfactory systems naturally led to the question whether non-motile primary cilia, displayed on the surface of most non-dividing cells in our body, could have evolved an analogous disposition for GPCR signaling, that is, could the primary cilium afford functional benefits to GPCR signaling stimulated by a diffusible agonist? Indeed, through the last decade, a number of GPCRs, once thought to be activated by freely diffusible ligands on the plasma membrane, have been shown to exhibit a pronounced functional and subcellular preference for the ciliary membrane compartment. The ciliary GPCRs identified so far belong to three major classes of the GPCR superfamily: A, B, and F. In the following, we present an overview of ciliary GPCR signaling and describe how the dynamic localization and trafficking of these receptors into and out the cilium is regulated, as well as how such receptors cross-talk with other classes of GPCRs for the spatiotemporal regulation of cellular and physiological processes. Ciliary structure and assembly Cilia are microtubule-based, membrane-enclosed projections on the surface of most eukaryotic cells 18. They generally fall into two classes, defined by their axonemal arrangement of microtubules and capacity to function as motile and/or signaling units. Classical motile (9 + 2) cilia have axonemes with nine outer doublets and two inner singlets of microtubules as well as radial spokes and axonemal dynein arms that promote motility. Non-motile (9 + 0) cilia typically lack the central pair of microtubules and structures associated with motility, and emanate as solitary organelles from the centrosomal mother centriole (basal body) in most non-dividing vertebrate cell types (Fig 2A). They function as mechano-, osmo-, and chemosensory units that control cellular and physiological processes during development and in tissue homeostasis. Ciliary axonemes of both motile and non-motile cilia are assembled onto the basal body by intraflagellar transport (IFT), which is characterized by kinesin-2- and cytoplasmic dynein-2-mediated bidirectional transport of IFT particles, with associated ciliary cargo, from the ciliary base toward the tip and back 19 (Fig 2B). The membrane surrounding the ciliary axoneme is continuous with the plasma membrane of the cell, but is enriched for specific membrane proteins and lipids that confer the cilium with unique sensory properties. Structural and functional barriers comprising a transition zone (TZ) at the ciliary base ensure the selective passage of proteins into and out of the ciliary compartment, and transition fibers basal to the functional barriers connect the ciliary base to the plasma membrane 202122. The region between the primary ciliary membrane and the plasma membrane, referred to as the periciliary membrane, is frequently infolded to produce a ciliary pocket (CiPo) that comprises an active site for exocytosis and clathrin-dependent endocytosis (CDE) of ciliary receptors 2324. Figure 2. Primary cilia are sensory organelles that coordinate GPCR signaling during development and in tissue homeostasis(A) Electron micrographs (EMs) of the primary cilium: (i) transmission EM of a longitudinal section of a neuronal primary cilium emerging from the centrosomal mother centriole, which functions as a basal body (BB). The region between the ciliary membrane and plasma membrane, referred to as the periciliary membrane, is often infolded to produce a ciliary pocket (CiPo) that comprises an active site for exocytosis and clathrin-dependent endocytosis of ciliary receptors (courtesy of Joseph Gleeson). (ii) Scanning EM of a fibroblast primary cilium with a ciliary pocket; asterisks mark microvilli (courtesy of Peter Satir). (iii) Transmission EM of a cross section of a fibroblast primary cilium at the ciliary transition zone showing the 9 + 0 microtubules arrangement of the axoneme (with permission 144). (B) Cartoon illustrating IFT and trafficking processes at the primary cilium to control ciliary assembly and targeting of GPCRs to the ciliary membrane. Receptor transport from the TGN to the ciliary pocket is mediated by ARF4, IFT20, and TCTEX-1 together with a complex consisting of FIP3, ASAP1, and RAB11/RAB8/Rabin8 subcomplex. The docking of vesicles near the periciliary membrane switches to IFT in a process involving the IFT-B components IFT57 and IFT20 as well as ARL6 and the RAB8-binding protein, Rabaptin5. Please see text for further details. (C) List of GPCRs known to localize to the primary cilium. (D) Immunofluorescence micrographs with examples of GPCRs localizing along the axis of the primary cilium in various cell types: (i) localization of melanin-concentrating hormone receptor 1 (MCHR1) in cultured mouse hypothalamic neurons (courtesy of Nicholas Berbari and Kirk Mykytyn); (ii) localization of somatostatin receptor 3 (SSTR3) in neuronal primary cilia of the mouse hindbrain (with permission 145); (iii) localization of dopamine 5 receptor (DR5) to primary cilia in porcine kidney proximal tubule (LLC-PK1) cells (with permission 146); (iv) localization of kisspeptin receptor 1 (KISSR1) to primary cilia the medial hypothalamus in adult CiliaGFP mice (with permission 77); and (v) localization of smoothened (SMO) to primary cilia in SMO agonist (SAG)-stimulated human embryonic stem cells (hESC) (with permission 147). Keys: β-tubulin III (neuronal marker), ACIII (adenylate cyclase III, neuronal primary cilium marker), Ac-tub (acetylated α-tubulin, primary cilium marker), Glu-tub (glutamylated α-tubulin, primary cilium marker), and DAPI/DRAQ5 (stains DNA, nuclear marker). Please see text for references and further details Download figure Download PowerPoint The sensory capacity of primary cilia is maintained through the spatiotemporal localization of specific receptors and downstream signaling components along the cilium–centrosome axis, including receptor tyrosine kinases (RTK) 25, transforming growth factor beta receptors (TGFβRs) 24, Notch receptors 26, receptors for extracellular matrix (ECM) proteins 27, and ion channels 28 as well as class A, B, and F GPCRs, which are the focus of this review. The medical significance of primary cilia is becoming increasingly evident, since defects in assembly, structure, and sensory function of these organelles are associated with a plethora of diseases and syndromic disorders (ciliopathies), including nephronophthisis and polycystic kidney disease as well as Bardet–Biedl (BBS), Alström (AS), Joubert (JS), and Meckel–Gruber (MKS) syndromes 29, manifested by congenital heart disease, craniofacial and skeletal patterning defects, neurodevelopmental disorders, and cognitive impairment as well as obesity 303132. Sorting and targeting of receptors to the cilium rely on multiple pathways. They include the polarized trafficking of vesicles from the trans-Golgi network (TGN) and recycling endosomes directly to the ciliary pocket followed by selective conveyance of the proteins across the ciliary barriers. Alternatively, receptors may move through a lateral transport pathway from the plasma membrane to the ciliary membrane 333435 (Fig 2B), a scenario recently proposed for the dopamine receptor, D1R, as discussed below. Targeting of receptors from the TGN to the cilium is thought to be guided by discrete ciliary targeting sequences (CTSs; Table 1) that interact with specific trafficking modules, which regulate the budding, transport, docking, and fusion of post-Golgi carriers or recycling endosomes at the ciliary base (for recent reviews, see 2133). This process has been particularly well studied in the outer segment of vertebrate rod and cone photoreceptors in the retina, which are modified cilia with vision class A GPCRs (rhodopsin and photopsins) that absorb photons to activate the G protein transducin, causing hyperpolarization of the cell thus inhibiting synaptic release 36. In rod cells, rhodopsin localizes to flattened disks of discrete self-contained vesicles within the compartment of the outer segment, whereas photopsins localize to disks, which are contiguous with the outer segment plasma membrane of the cone cells, although mammalian cones may contain disks, which are separated from the plasma membrane 37. For example, the C-terminal CTS of rhodopsin (see below) was shown to directly bind to the small GTPase ARF4, which mediates budding of rhodopsin carrier vesicles at the TGN followed by their translocation to photoreceptor connecting cilia by a complex mechanism involving the RAB11/ARF effector FIP3, the ARF GTPase-activating protein ASAP1 (Arf-GAP with SH3 domain, ANK repeat, and PH domain-containing protein 1), and the RAB11/RAB8/Rabin8 complex 38394041. These post-Golgi carriers are likely transported by the cytoplasmic dynein-1 motor to the ciliary base via direct interaction between rhodopsin's C-terminal tail and the dynein light chain Tctex-1 42. Additional regulators of ciliary trafficking include the transport protein particle (TRAPP)II complex and TRAPPC8, which are required for the recruitment of Rabin8 to the centrosome 4344, as well as the IFT-B complex protein IFT20 4546. Table 1. Examples of proposed ciliary targeting sequences (CTSs) for various GPCRs in mammalian cells GPCR Examples on proposed ciliary targeting sequences (CTSs) VXPX motif in C-tail AXXXQ motif in i3 Other motifs in i3 Rhodopsin 40149 [SQVAPA] Not necessary SSTR3 71 [APSCQ] HTR6 71 [ATAGQ] MCHR1 71150 Not necessary [APASQ] NPY2R, GPR88 80 Not present [R/K][I/L]W We note that some studies have suggested the presence of phenylalanine-arginine (FR) CTSs in the C-terminus of several ciliary GPCRs, but structural evidence suggests that these FR motifs are inaccessible for the ciliary targeting machinery and rather mediate proper protein folding (discussed in 148). Additional studies have implicated Bardet–Biedl syndrome (BBS) proteins in ciliary membrane biogenesis/homeostasis, for example, by promoting ciliary trafficking of specific GPCRs 474849. The i3-CTSs of these receptors (see Table 1 and below) appear to interact directly with components of the BBSome 4849, a stable complex of seven BBS proteins that cooperates and interacts with the RAB11/RAB8/Rabin8 complex to promote cilia membrane biogenesis 5051. At the ciliary base, the BBS4 component of the BBSome helps the vesicle–motor complexes to dock near the periciliary membrane in order to switch the vesicle receptor trafficking to IFT, a process involving the IFT-B components IFT57 and IFT20 as well as ARL6 and the RAB8-binding protein, Rabaptin5 52. This leads to stimulation of the GEF activity of Rabin8 toward RAB8, which then interacts with the exocyst complex to mediate receptor fusion with the membrane. RAB8 itself is targeted to cilia via the transition zone-associated complex of RPGR and CEP290, and likely BBS4 52. Of note, the BBSome also interacts with the IFT machinery to regulate ciliary export of signaling proteins 53. As a testament to the importance of the BBSome in proficient targeting of GPCRs to cilia, knockout mice defective in the ciliary protein Tubby or BBSome components display aberrant ciliary localization and signaling of the melanin-concentrating hormone receptor 1 (MCHR1) and somatostatin receptor 3 (SSTR3), which causes blindness and obesity 475455. For a recent review on the mechanisms in sorting, targeting, and trafficking of rhodopsin to the outer segment, and how mistrafficking is associated with degeneration of photoreceptors, please see 56. In addition to the GPCRs mentioned above, a growing number of GPCRs are specifically targeted to cilia, including both motile and non-motile cilia. For example, in the airway epithelium, motile 9 + 2 cilia harbor bitter taste class A GPCRs (T2Rs) that signal through the G protein Gustducin when encountering a bitter substance. This causes airways to relax and protects the respiratory system from noxious compounds 57. Similarly, olfactory transduction is regulated through the combination of hundreds of class A GPCRs that rely on activation of the G protein Gαolf in cilia of the main olfactory epithelium (Fig 1B and C) This activates an adenylate cyclase that leads to cAMP production and depolarization of the cell through the opening of several ion channels 58 (Fig 3A). GPCR signaling is also well documented in invertebrate sensilla, such as in chemosensory cilia on dendritic endings of sensory neurons in Caenorhabditis elegans (C. elegans). These cilia play a unique role in regulating behavioral responses, including social feeding and dauer formation as well as avoidance and mating responses 59. Systematic characterization of these cilia has contributed significantly to the understanding of ciliary targeting of GPCRs. As an example, a recent study identified novel cis- and trans-acting mechanisms, that is, amino acid motifs and trafficking pathways, required for ciliary localization of GPCRs 60. The components shown to be required for localization of GFP-tagged GPCRs to sensilla in C. elegans include a number of vesicular and adaptor proteins such as the bbs-1, bbs-8, rab-8, arl-3, rl-13, odr-4, unc-101, and daf-25 as well as TZ and IFT subunits. These GPCRs use different CTSs for ciliary targeting within a given cell type, and CTSs within individual GPCRs mediate ciliary localization via diverse trafficking mechanisms across cell types 60. In most amphibia, reptiles, and non-primate mammals, the vomeronasal organ (VNO) at the nasal septum also bears GPCRs that in mice are associated with a extensive array of instinctive behaviors, such as aggression, predator avoidance, and sexual attraction 61. However, VNO cells are generally microvillar rather than ciliary. Similarly, gustatory hair cells in the taste buds of the tongue use microvilli as cellular extensions for sweet, umami, and bitter tasting through the activation of a series of class A and C GPCRs 62. Figure 3. Examples of ciliary GPCR signaling(A) GPCR signaling in olfactory cilia relies on the cAMP-dependent opening of ion channels, leading to an influx of Na+ and Ca2+ ions into the ciliary compartment, which in turn activates chloride channels, causing efflux of Cl−, which results in a further depolarization of the cell. Abbreviations: Olf: ligands for olfactory receptors. (B) Outline of trafficking and signaling processes associated with MCHR1 and SSTR3 signaling in neuronal primary cilia. The BBSome and TULP3, IFT-A, and KIF3A control the localization of the receptors to the ciliary base and further into the ciliary membrane. Abbreviations: M: melanin-concentrating hormone; S: somatostatin. (C) Outline of signaling processes associated with D1R and GPR88 signaling in primary cilia. D1R is activated by catecholamines both in the cilium and at the plasma membrane, but receptor activation is specifically inhibited within the cilium by GPR88. (D) Outline of trafficking and signaling processes associated with HH signaling in primary cilia. Please see text for references and further details. Download figure Download PowerPoint Recently, signaling molecules were shown to be released into the extracellular environment from the ciliary membrane by the shedding of ectosomes 63646566. This adds an additional layer of complexity to the trafficking mechanisms of receptors to and from the cilium, although it is currently unknown whether receptors in extracellular vesicles fuse with the ciliary membrane to control signaling processes within the ciliary compartment. Rhodopsin-like (class A) GPCRs in neuronal primary cilia A small, but growing number of rhodopsin-like (class A) GPCRs have been demonstrated to localize to primary cilia. In neuronal cells, ciliary GPCRs act as extra synaptic or “unwired” receptors believed to regulate neuronal function by sensing neuromodulators in the local environment. The first class A GPCRs found to be enriched in neuronal primary cilia were SSTR3 6768 and serotonin receptor 6 (5-HT6 or HTR6) 6970, which were detected by immunofluorescence confocal microscopy of cilia on, for example, neurons from the coronal rat brain section, island of Calleja and the olfactory tubercle. Employing C-terminal chimeras and sequence analysis, the discrete GPCR-specific AxxxQ CTS was deduced in the third intracellular loop (i3) of SSTR3 and HTR6 (Table 1), leading to the identification of a third ciliary class A GPCR, MCHR1. As with SSTR3 and HTR6, this CTS is sufficient to localize MCHR1 to cilia in neurons 71. Interestingly, while most neurons in the brain possess a primary cilium 72, it has been demonstrated that only a subset of ciliated neurons display MCHR1 and HTR6 in the ciliary membrane 47676970. MCHR1, which is critical for proficient feeding behavior, was shown to concentrate in neuronal cilia in the hypothalamus in mice 73. The hypothalamus, a brain region controlling appetite behavior and energy metabolism, relies on ciliary signaling to sense satiety signals from the surroundings. Disruption of cilia by conditional depletion of Kif3A or Tg737/Ift88 specifically on pro-opiomelanocortin (POMC)-expressing neurons in the hypothalamus causes hyperphagia-induced obesity in mice 74, thus raising the possibility that MCHR1 in neuronal cilia might regulate energy homeostasis. In line with this, as discussed above, the ciliary localization of SSTR3 and MCHR1 was demonstrated to rely on the BBSome in mouse brain sections and in cultured hippocampal neurons 474950). Mouse models of BBS support a role for the BBSome in targeting GPCRs to cilia, as neurons from mice lacking either the BBS2 or BBS4 protein retain structurally normal primary cilia but fail to accumulate MCHR1 and SSTR3 in the ciliary membrane 47. These mouse models of BBS provided some of the first mechanistic clues linking BBS phenotypes, for example, obesity, to a defective molecular mis-targeting of GPCRs to the neuronal cilium. Members of the Tubby family, namely Tubby and Tubby-like protein (TULP3), have been demonstrated to mediate IFT complex A-dependent trafficking of ectopically expressed, GFP-tagged, SSTR3 and MCHR1 to cilia 75. In the neurons of Tubby-deficient (Tub) mutant mice, SSTR3 and MCRH1 as well as HTR6 are diminished or excluded from the neuronal primary cilia. As with the Bbs2 and Bbs4 mutant mice, Tub mice are obese 76, lending further credence to the linkage between GPCR targeting to cilia and energy homeostasis. However, Tubby is not essential for all GPCR trafficking to cilia since another receptor, the odorant receptor mOR28, remains correctly localized to the distal cilia of olfactory epithelial cells of Tub mutant mice 54, thus emphasizing the specificity of action exerted by Tubby. More recently, the kisspeptin receptor (KISS1R) was identified as a novel ciliary class A GPCR in gonadotropin-releasing hormone (GnRH) neurons in the mouse hypothalamus, and it was suggested that primary cilia are required for normal KISS1R signaling in these neurons 77. KISS1R regulates the onset of puberty and adult reproductive function 7778, but conditional ablation of primary cilia in GnRH neurons in transgenic mice did not affect their sexual maturation, so further analysis will be needed to understand the function of ciliary KISS1R signaling in the brain 77. Additional subtypes of rhodopsin-like GPCRs, including dopamine D1, D2, and D5 79 and neuropeptide Y NPY2R and NPY5R 80 receptors, as discussed below, were found to be specifically localized to primary cilia in different cell types. These findings have been highly instructive for understanding the complex machinery of how GPCRs are ta
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