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

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. INTRODUCTIONPrimary cilia are appendages that project from almost all human cell types (Wheatley et al., 1996). It is generally accepted that primary cilia serve important specialized signaling functions (Pazour and Witman, 2003;Marshall and Nonaka, 2006;Singla and Reiter, 2006). In the eye, photoreceptors, which are modified primary cilia, sense and respond to light. In the nose, specialized olfactory cilia detect odors and initiate signaling cascades in olfactory neurons. In the kidney, it is proposed that bending of cilia on epithelial cells by fluid flow triggers an increase in intracellular calcium mediated by an ion channel located on the cilium (Praetorius and Spring, 2001;Nauli et al., 2003). In each case, the function of the cilium is defined by the signaling proteins that are enriched in the ciliary membrane (i.e., light receptors, odorant receptors, and mechanoreceptors). Importantly, disruption of the signaling mediated by these receptors can cause disease and altered development (Davenport and Yoder, 2005;Hildebrandt and Otto, 2005;Pan et al., 2005;Bisgrove and Yost, 2006). Yet, the specific signaling proteins that localize to the vast majority of cilia in the mammalian body are unknown. Thus, the functions of primary cilia on most cell types in the body are unknown.Neuronal primary cilia are abundant throughout the rodent brain (Bishop et al., 2007). The functional importance of these cilia is suggested by the fact that several human ciliary disorders, including Bardet-Biedl syndrome (BBS), Joubert syndrome (JS), and Meckel syndrome (MKS), have prominent functional and structural CNS phenotypes (Badano et al., 2006). Although the specific functions of neuron...

Bardet–Biedl syndrome proteins are required for the localization of G protein-coupled receptors to primary cilia

“…Therefore, we asked whether other GPCRs, and specifically GPCRs whose functions are consistent with the BBS phenotypes, fail to localize to cilia in BBS animals. We recently found that Mchr1, which is involved in the regulation of feeding and energy balance (21), localizes to cilia in regions of the brain involved in feeding and reward pathways, including the olfactory bulb, olfactory tubercle, nucleus accumbens, and hypothalamus (43). To test whether the BBS proteins are required for Mchr1 ciliary localization, we colabeled WT, Bbs2 Ϫ/Ϫ , and Bbs4 Ϫ/Ϫ brain sections with anti-ACIII and antiMchr1.…”

Far from solved: A perspective on what we know about early mechanisms of left–right asymmetry

“…Cilia do indeed possess ion channels (Teilmann et al, 2005) and even neurotransmitter receptors (Brailov et al, 2000;Berbari et al, 2008), so perhaps gradients other than calcium (McGrath et al, 2003;Schneider et al, 2008) also function during ciliary flow at the node. The very early presence of the relevant ion transporter proteins does not rule out this model, because, like ciliary proteins themselves which can also be expressed long before ciliogenesis, they could be present but not functioning in LR asymmetry until cilia appear.…”

Primary cilia enhance kisspeptin receptor signaling on gonadotropin-releasing hormone neurons

“…To test whether Kiss1r localizes to cilia, a construct encoding mouse Kiss1r fused at the carboxy-terminus to EGFP was expressed in inner medullary collecting duct (IMCD) cells. We have previously shown that ciliary GPCRs selectively localize to cilia when expressed in IMCD cells (12,14). Notably, Kiss1r localized to cilia on IMCD cells (Fig.…”

“…Specific signaling proteins are selectively targeted to and retained within neuronal cilia, which are restricted compartments and regulate entry and exit of proteins through multiple mechanisms (6,7). These signaling proteins include type 3 adenylyl cyclase (AC3) (8), which converts ATP to cAMP, and the GPCRs, somatostatin receptor 3 (Sstr3) (9), serotonin receptor 6 (10,11), melanin-concentrating hormone receptor 1 (Mchr1) (12,13), dopamine receptor 1 (14), and neuropeptide Y receptors 2 and 5 (15). The functions of primary cilia are determined by the proteins that are enriched within them, thus, it is likely that neuronal cilia sense neuromodulators in the extracellular milieu and initiate signaling cascades.…”

“…We previously identified ciliary localization sequences in the third intracellular loop and carboxy tail of ciliary GPCRs and used these sequences to predict novel ciliary GPCRs (12,14). One of these, the kisspeptin receptor (Kiss1r, also known as GPR54), was considered a strong candidate ciliary GPCR given that loss of Kiss1r leads to hypogonadotropic hypogonadism in humans and mice (16,17) and hypogonadotropic hypogonadism is a feature of the ciliopathy Bardet-Biedl syndrome (18).…”