G-Protein-Coupled Receptor Signaling Through Gpr176, Gz, and RGS16 Tunes Time in the Center of the Circadian Clock

Home , Hitoshi Okamura

2017, 64 (1), 1-10 Advance Publication doi: 10.1507/endocrj.EJ17-0130 REVIEW G-protein-coupled receptor signaling through Gpr176, Gz, and RGS16 tunes time in the center of the circadian clock

Kaoru Goto*, Masao Doi*, Tianyu Wang, Sumihiro Kunisue, Iori Murai and Hitoshi Okamura

Department of Systems Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan

Abstract. G-protein-coupled receptors (GPCRs) constitute an immensely important class of drug targets with diverse clinical applications. There are still more than 120 orphan GPCRs whose cognate ligands and physiological functions are not known. A set of circadian pacemaker neurons that governs daily rhythms in behavior and physiology resides in the suprachiasmatic nucleus (SCN) in the brain. Malfunction of the circadian clock has been linked to a multitude of diseases, such as sleeping disorders, obesity, diabetes, cardiovascular diseases, and cancer, which makes the clock an attractive target for drug development. Here, we review a recently identified role of Gpr176 in the SCN. Gpr176 is an SCN-enriched orphan GPCR that sets the pace of the circadian clock in the SCN. Even without known ligand, this orphan receptor has an agonist-independent basal activity to reduce cAMP signaling. A unique cAMP-repressing G-protein subclass Gz is required for the activity of Gpr176. We also provide an overview on the circadian regulation of G-protein signaling, with an emphasis on a role for the regulator of G-protein signaling 16 (RGS16). RGS16 is indispensable for the circadian regulation of cAMP in the SCN. Developing drugs that target the SCN remains an unfulfilled opportunity for the circadian pharmacology. This review argues for the potential impact of focusing on GPCRs in the SCN for the purpose of tuning the body clock.

Key words: Circadian clock, Orphan GPCR, Gpr176, Gz, RGS16

Introduction the enormous clinical relevance of this class of receptors. Astonishingly, of the 356 non-olfactory GPCRs G-protein-coupled receptors (GPCRs), also known encoded in the human genome, about 38% are still as 7 transmembrane receptors, constitute the largest considered ‘orphans’ whose physiological roles and family of cell surface molecules involved in signal endogenous ligands are not known. Deciphering their transmission. They are activated by a wide range of functions therefore remains of hot interest from both stimulants, including light, odorant molecules, peptide basic sciences and drug development perspectives. and non-peptide neurotransmitters, hormones, growth A set of the central pacemaker neurons in the supra- factors and lipids, and control their corresponding chiasmatic nucleus (SCN) in the brain serves as a downstream processes, including sensory transduction, guardian of our daily life: it co-ordinates the daily cell–cell communication, neuronal transmission, and rhythms of sleep and wakefulness, as well as behav- hormonal signaling. Given the central role of GPCRs ior and physiology. Through its ability to set the pace in diverse physiological processes and diseases, it may and phase of physiological rhythms in anticipation of not come as a surprise that more than 20% of drugs in regular day/night cycles, the clock forms a highly effi- current clinical use act on specific GPCRs, attesting to cient adaptation system to the predictive changes in environment. Disturbance of this endogenous clock, Submitted Mar. 26, 2017; Accepted Mar. 29, 2017 as EJ17-0130 with, e.g., jet-lag, rotational shiftwork, and irregular Released online in J-STAGE as advance publication May 13, 2017 night-owl lifestyles in current society, therefore causes Correspondence to: Masao Doi, Ph.D., Department of Systems a range of healthcare problems. One of the most sig- Biology, Graduate School of Pharmaceutical Sciences, Kyoto nificant conceptual changes brought about by the anal- University, 46-29 Yoshida-Shimo-Adachi-cho, Sakyo-ku, Kyoto 6068501, Japan. ysis of circadian clock-deficient mice is that -abnor E-mail: [email protected] malities in the circadian clock are linked not only to * These authors contributed equally to this work. sleep arousal disorder but also to a wide variety of ©The Japan Endocrine Society 2 Goto et al. common diseases, including hypertension, diabetes, The SCN is the center of obesity, and cancer [1-5]. It is worth noting that drug the body clock efficacy and toxicity also change with time in a manner depending on the endogenous clock [6]. These The SCN is the chief orchestrator for the lines of growing evidence support the potential value rhythms generated in the body. Almost all cells in of developing drugs that target the circadian clock. the body express a set of the essential clock genes. Here, we review a recently identified role of Gpr176 Correspondingly, all major organs have their own in the SCN. Gpr176 is an SCN-enriched orphan GPCR circadian clocks and they constitute a hierarchical that sets the pace of the central clock in the SCN [7]. multi-oscillator network, in which the SCN serves as Gpr176 couples to Gz, and even in the absence of known the chief orchestrator dominating the rhythms in the ligand, it possesses an agonist-independent basal activ- peripheral clocks (Fig. 1). ity to repress cAMP signaling [7, 8]. Besides Gpr176 In searching for the anatomical structure of the and Gz, this review also emphasizes a role for the regu- master clock, lesion studies led to the identification lator of G-protein signaling 16 (RGS16) in the tempo- of a part of the brain called the “suprachiasmatic ral regulation of G-protein-cAMP signaling in the SCN nucleus” (SCN). As its name suggests, the SCN is [9, 10]. Developing drugs that specifically target the located just above (supra to) the optic chiasm, the SCN remains an unfulfilled opportunity for the circa- place where the optic nerves cross-pass beneath the dian clock therapy. In this context, this review argues brain hypothalamus. Physical lesion of this structure for the potential merit of focusing on GPCRs in the abolishes circadian behavioral rhythms, which can be SCN for the purpose of tuning the body clock. restored by transplantation of fetal SCN tissue [11].

Central clock SCN Pineal

Light (retina) Melatonin secretion 024 Time (hr)

SCN governs periphery via signals involving neuronal, endocrine, feeding, behavior rhythms

Autonomic Adrenal Heart Liver nervous system Heart rate Cortisol secretion Detoxication activity Body temperature 024 024 024 024 Time (hr) Time (hr) Time (hr) Time (hr)

Fig. 1 The SCN is the center of the body clock The SCN oscillator synchronizes peripheral clocks in organs such as liver, heart, and adrenal via direct and indirect routes. SCN neurons receive light information directly from retina. This photic entrainment corrects the phase of the endogenous SCN clock to ultimately adjust the organismal physiological rhythms to the regular geographical light/dark cycle.

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In addition, SCN transplants from Tau mutant ham- The SCN-GENE PROJECT identifies sters displaying a short period phenotype restore Gpr176: Gpr176 is an SCN-enriched rhythms in SCN-lesioned wild-type hamsters with a orphan GPCR that sets the pace of period length characteristic of the mutant donor [12], circadian behavior demonstrating that the period is encoded by the SCN. Moreover, SCN transplants from wild-type mice In order to identify a new GPCR that tunes the cen- restore circadian rhythmicity in global clock-gene tral clock, we launched the SCN-orphan GPCR project knockout mice, where no functional clocks operate [7, 9], in which we (i) searched for the orphan GPCRs throughout the body [13], providing evidence that the whose expression is enriched in the SCN, (ii) generated transplanted SCN alone is sufficient to produce organ- knockout mice of candidate GPCR genes of interest, ismal rhythmicity. Recent cell-type-specific condi- and (iii) asked whether there is a defect in their circa- tional clock-gene knockout studies also support the dian rhythms in behavior. Based on this screening strat- assertion that the SCN is the central clock [14-16]. egy, Gpr176 was identified as an SCN-enriched orphan The SCN is the site of the circadian center; it is GPCR that sets the pace of circadian behavior (Fig. 2). hence logical that the SCN expresses all of known Gene ablation of Gpr176 shortens circadian period of genes that determine the circadian period of locomotor behavior. A similar short-period phenotype was also activity rhythms. Taking advantage of this nature, we observed for the clock gene Per1 transcription rhythms initiated the SCN-GENE PROJECT [17] that focuses in ex vivo organotypic SCN slice cultures. Thus, the on the genes highly expressed in the SCN. defect in the SCN mirrors the behavioral rhythm output.

A B 11 PACAP DNA microarray 11 Gpr176 SCN-GPCR Project 10 receptor SCN-GPCR 10 9 Top100 9 SCN microarray Prokineticin receptor 8 V1a 8 7 Vipr2 (VPAC2) 7 In situ hybridization 6 6 5 5 Creation of KO mice 4 4 3 3 Behavioral test 2 2 Log2 GPCR mRNA levels 1 1 Identication of a new 0 0 relevant SCN-GPCR

C Gpr176

SCN In situ Behavioral microarray hybridization test Orphan GPCRs Candidates Candidates Gpr176 > 120 26 3

Fig. 2 The SCN-GENE PROJECT leading to the identification of Gpr176 (A) Experimental design of the SCN orphan GPCR project. (B) The top 100 GPCRs in SCN microarray. Note that Gpr176 was estimated to be abundant, relative to prokineticin receptor, vasopressin V1a receptor, and Vip receptor, Vipr2; all of them are known to be important in the SCN function. (C) The genes highlighted in blue in (B), which include all listed orphan GPCRs, were assessed through in situ hybridization. Arrows indicate robust positive SCN signals for Gpr176, Calcr, and Gpr19. (D) The results form behavioral tests on respective knockout mice for Gpr176, Calcr, and Gpr19 led us to prioritize Gpr176 as a target for further analysis [Adapted from Ref. 7].

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Gpr176 cycles in abundance and intestinal peptide (Vip) receptor Vipr2, a receptor colocalizes with Vipr2 known to play a key role in circadian timing [19, 20]. Temporal expression profiles of Gpr176 and Vipr2 Gpr176 is an evolutionally conserved, verte- reveal that while these two receptors exist in the same brate class A orphan GPCR (Fig. 3). It was cloned SCN neurons, they are opposite in peak expression in 1995 [18]. Notably, Gpr176 is expressed mainly phase. Namely, Gpr176 peaks at night, whereas Vipr2 in the brain, with most being found in the SCN, and during the day [7, 21]. These spatiotemporal expres- its protein abundance within the SCN fluctuates in sion profiles of Gpr176 and Vipr2 are congruent with a circadian fashion, peaking during the night. More their antiphasic, opposite actions on cAMP signaling specifically, Gpr176 colocalizes with the vasoactive in the SCN (see below).

L N A G A S A E A G S T N R P H S T N P S V W S S N H G M 1 F G V W S E T E N L W W C S S C I A Y I E T L L C S M D V N A Y G 200 V Extracellular H A T Q P T N V P S T H Y Q L S L M V V T N R Y 100 F V L S S A Y T L V 300 Q I L K C V F V A V T F I L V I L L F T P Y P Y L D K S V V V T P F L F A N T L Q T V C S A C V K H V V I L I V V V V F V A V T L W F I V I F P S C A H T V I C A V S I W P V K S G A V M V S G I T M Y L S L L C S S L V V V V L L N G L F L N A A F E L A T F N L A P S R F V P V M I L L H L I K I K E L F L A A L A L F S W R D D I E T N S R Q R K C V T Y R R T K V V R Y I S S Intracellular S S A A V C G Q T V K N V T K L L Y L T L V F R S P I S R I T N L H 340 Y E A Q P H R Y S R R N L S P V L S S S Q T P E A V G G T V K V I R E K K I A A L P S M R S G S Q L L E M F E S P K F I Q Q Q G I H D E E E S E A K Y L G S A D E P C S T L V E K A Q F 411 G E Q E P P Q L A P S V P P P S V R P E S D V T G P V A P M E S G I F P D K Y S P P L E F P G F G F Q L Q W L S E T R N S K K R L L Q I L E E P T N G L P P T K V P R V N R V E R K M S D V K P F I S V K N N R 515 S

Fig. 3 Snake-plot representation of Gpr176 The full-length amino acid sequence of the mouse Gpr176. The conserved motifs of the class A GPCR family (DRYxxV at TM3, NPxxY at TM7) are highlighted in yellow. As indicated, Gpr176 contains four putative glycosylation sites at the N-terminal part. A relatively large, C-terminal domain also characterizes Gpr176.

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Gpr176 bears an agonist-independent results in reduced suppression of cAMP signal during activity to counteract with the night. In vivo data suggest that Gpr176 serves as Vipr2-cAMP signaling a nighttime cAMP suppressor in the SCN. Circadian fluctuation of cAMP signal has been Molecular characterization of Gpr176 revealed that demonstrated to be crucial for the maintenance of this orphan receptor has an agonist-independent con- circadian clock function in the SCN [22]. A sim- stitutive activity to repress cAMP production [7]. In ple explanation for the possible downstream mech- general, all GPCRs display two distinct structural con- anisms of action of Gpr176 and Vipr2 may involve formations, active and inactive. Agonists lock the cAMP signal-mediated temporal regulation of Per1 receptor structure in its active form, whereas inverse transcription through a cAMP-responsive element agonists stabilize it in its inactive form. Even in the (CRE) on its promoter [23]. Yet, the molecular absence of ligands, GPCRs spontaneously interchange frameworks through which the circadian cAMP sig- between the two states, generating agonist-independent nal is integrated to the core clock mechanism are not baseline activity. The magnitude of this basal activ- fully understood. ity differs considerably between GPCRs. Comparative studies examining basal activities of class-A GPCRs Gpr176 couples to Gz [8] revealed that Gpr176 is among those possessing high-level constitutive activities. Notably, the unique G-protein subclass Gz is The basal activity of Gpr176 antagonizes with required for the basal activity of Gpr176 [7]. Gz is an Vipr2-Gs mediated cAMP signaling. Thus, Gpr176 evolutionally conserved Gi/o subfamily member. It has appears to constitute a previously unknown “nega- been biochemically shown that GTP-bound Gz inhibits tive” limb that could counteract with the “positive” the activity of adenylyl cyclases [24]. Gz resembles Gi cAMP signaling via Vipr2 (Fig. 4). Consistent with in terms of the repressing activity toward cAMP signal- this “yin-yang” model, the lack of Gpr176 in the SCN ing [25]. However, Gz is not just an ersatz Gi.

Vip

Vipr2 Gpr176

CELL MEMBRANE

CYTOPLASM Gs AC Gz Day Night Gi

cAMP

RGS16

Circadian clock Day

Night

Fig. 4 A model of circadian Gpr176/Gz/RGS16/cAMP axis in the SCN The orphan G-protein-coupled receptor Gpr176 couples to Gz and thereby antagonizes with Vip-Vipr2-Gs-cAMP signaling that controls the intracellular clock cycling. The clock enhances expression of Gpr176 at night, thereby defining repression time of day. In the beginning of the day the clock in turn induce expression of RGS16, which deactivates cAMP-repressing signal through Gi, perhaps also Gz. This temporarily permits the daytime enhancement of cAMP production in the SCN. AC, adenylate cyclase. The photograph behind the schematic model is a representative mouse coronal brain section immunolabeled for Gpr176. Gpr176 is mainly located in the SCN.

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Echoing its unique name, Gz displays a range of The long lived Gz signaling might partly account unique properties that distinguish it from the other Gi for the robust basal activity of Gpr176. Among the family members. Firstly, differently from Gi, Gz is other known GPCRs expressed in the SCN, the recep- mainly expressed in the brain plus platelets [26-28]. tors for serotonin (5-HT1A) and melatonin (MT1 and Secondly, unlike Gi, pertussis toxin (PTX) does not MT2) are also able to couple to Gz [33-36], extending a inhibit Gz. As shown in Fig. 5, PTX mediates ADP potential contribution of Gz to the circadian clock reg- ribosylation at the conserved consensus cysteine resi- ulatory mechanisms. due in the fourth position from the C terminus of Gi/o proteins. The lack of this cysteine residue renders Gz The SCN-GENE PROJECT identifies refractory to PTX (Fig. 5). Lastly, compared to Gi RGS16 as a GAP required for and other G-protein families Gs and Gq, Gz is unique circadian timing in that it bears a relatively low intrinsic GTPase activity. The kcat values for GTP hydrolysis by Gs, Gi, and Regulator of G-protein signaling (RGS) proteins Gq are in the range of 10 min-1, whereas Gz shows an constitute a family of over 20 proteins that nega- approximately 100 times slower rate of activity (0.05 tively regulate GPCR signaling pathways by enhanc- min-1) (Table 1) [24, 28-32]. Thus, once GTP binds ing endogenous GTPase activities of G-protein α to Gz, the activated Gz appears reluctant to go back subunits. The SCN-GENE PROJECT focusing on to the inactive state, which results in a signal with a this family [9] revealed a role of RGS16 in determi- long life-time. A limited time resolution might be a ning the pace of the SCN clock. Gene knockout and trade-off of the long-life signal, while the latter would knockdown of RGS16 both compromise the periodi- rather fit to the hourly control of the clock. city of behavioral rhythm [9, 10]. Moreover, RGS16

Phylogeny of Gi/o family ADP ribosylation site

Gi1 NNLKDCGLF Gi2 NNLKDCGLF Gi3 NNLKECGLY GoA NNLRGCGLY GoB KNLRGCGLY Gt1 ENLKDCGLF Gt2 ENLKDCGLF Gz NNLKYIGLC Fig. 5 Comparison of the C-terminal sequences of the Gi/o family members Note that Gz is a unique Gi/o subfamily member that inhibits adenylyl cyclase activity in a PTX-insensitive manner.

Table 1 kcat for GTP hydrolysis by Gα subtypes –1 Subtype kcat (min ) Source Reference Gαz 0.05 Human, recombinant, purified from E. coli [28] Gαi1 2.4 Rat, recombinant, purified from E. coli [29] Gαi2 2.7 Rat, recombinant, purified from E. coli [29] Gαi3 1.8 Rat, recombinant, purified from E. coli [29] Gαo 2.2 Rat, recombinant, purified from E. coli [29] Gαs-long 3.2 Bovine, recombinant, purified from E. coli [30] Gαs-short 4.5 Bovine, recombinant, purified from E. coli [30] Gαq/11 0.8 Bovine, native, purified from cerebrum [31] Gα12 0.2 Murine, recombinant, purified from Sf9 cell [24] Gα13 0.2 Murine, recombinant, purified from Sf9 cell [32]

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knockout leads to the loss of circadian production for example, the molecular clockwork of the SCN of cAMP in the SCN [9], implying an intimate link phase-leads that of peripheral clocks by 7 to 11 hours between RGS16 and Gpr176. [46]. Any drug targeting common clock machinery Biochemically, RGS16 functions as a GTPase- would reset all the clocks simultaneously and equally, accelerating protein (GAP) for Gi [37-39]. By promot- and this might be deleterious for keeping the adap- ing GTP hydrolysis, RGS16 terminates GTP-bound Gi tive phasic order between the tissues (unless such signaling. A critical feature of RGS16 is that it func- drugs were to be delivered to the target tissue selec- tions in the SCN in a time-restricted manner [9, 10, tively- a very demanding regimen). On the other 40-42]. Via a direct transcriptional control by the cir- hand, the SCN-specific components would make it cadian clock, protein accumulation of RGS16 drasti- possible to design compounds that shift the SCN but cally changes with time, peaking during the day [9]. It have no direct action on peripheral clocks, but leav- is therefore likely that RGS16 acts as a daytime deac- ing them still subject to SCN-dependent cues and tivator for Gi singling in the SCN. Systematically, this thereby retaining relative circadian phases and ampli- may help to gate SCN cAMP signal in a timely fash- tudes. We reason therefore that, in conjunction with ion, the gate of cAMP signal open during the day and the other methods, the SCN-GENE PROJECT would closed during the night (Fig. 4). RGS16 acts on Gi provide an alternative valuable way to finding poten- as well as Gq [37]. It remains to be shown whether tial drug targets for the body clock. RGS16 regulates Gz signal in the SCN. A new twist also came from three independent Perspectives research teams, one of which is a Google-backed pri- vate company (23andMe) offering personal genome While Gpr176 and other orphan GPCRs have no DNA test service in US. The teams utilized human known natural ligands, small molecules that act as genome DNA information from about 100,000 individ- ligands could be developed as potential drug candi- uals whom they queried about their propensity to get dates. A key impediment to GPCR deorphanization up early or sleep in the morning. Interestingly, all three is uncertainty about the coupling partner via which teams led to the same conclusion that genetic variants they signal, making functional assays problem- of RGS16 are closely associated with being a morning atic. However, we already know that Gpr176 cou- person [43-45]. These findings provide reason to sus- ples to Gz. In the presence of Gz, Gpr176 is able to pect that RGS16-dependent similar circadian mecha- display a relatively strong constitutive activity. This nisms operate in humans as well. would provide a merkmal activity for the purpose of assaying inverse agonists. Another important fea- The rationales behind ture of Gpr176 is the site specificity of expression. the SCN-GENE PROJECT In an optimistic view, a drug targeting the SCN may aid in normalization of circadian sleep and associated Finally, we elaborate on what we believe to be a body temperature rhythms with fewer side effects on potential advantage of the SCN-GENE PROJECT. peripheral clocks. We initiated this tissue-specific approach [17] with the longer term goal of identifying novel targets for Acknowledgements circadian therapeutics. So far, the identification of the components of the circadian clock has advanced This work was supported in part by the Core Research through the use of a number of approaches including for Evolutional Science and Technology, Japan Science phylogenetic methods, mutagenesis-based forward and Technology Agency (to H.O.), the Grant-in-Aid for genetics, in silico analysis, and cell reporter based Scientific Research on Innovative Areas (JP15H05933), screening. In contrast to these approaches, the genes the Translational Research Network Program, the identified by the project with its focus on SCN-GENE Platform for Drug Discovery, Informatics, and may not be important for the peripheral clocks. In Structural Life Science, from the Ministry of Education, the longer term this should be an advantage for ther- Culture, Sports, Science and Technology of Japan, and apeutic development. The clocks distributed across grants from the Sumitomo Foundation and the Suzuken the body are not functionally uniform; In rodents, Memorial Foundation (to M.D.).

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