G-protein-coupled receptors (GPCRs) participate in a broad range of physiological functions. A priority for fundamental and clinical research, therefore, is to decipher the function of over 140 remaining orphan GPCRs. The suprachiasmatic nucleus (SCN), the brain's circadian pacemaker, governs daily rhythms in behaviour and physiology. Here we launch the SCN orphan GPCR project to (i) search for murine orphan GPCRs with enriched expression in the SCN, (ii) generate mutant animals deficient in candidate GPCRs, and (iii) analyse the impact on circadian rhythms. We thereby identify Gpr176 as an SCN-enriched orphan GPCR that sets the pace of circadian behaviour. Gpr176 is expressed in a circadian manner by SCN neurons, and molecular characterization reveals that it represses cAMP signalling in an agonist-independent manner. Gpr176 acts independently of, and in parallel to, the Vipr2 GPCR, not through the canonical Gi, but via the unique G-protein subclass Gz.

G-protein-coupled receptors (GPCRs) are important drug targets with diverse therapeutic applications. However, there are still more than a hundred orphan GPCRs, whose protein functions and biochemical features remain unidentified. Gpr176 encodes a class-A orphan GPCR that has a role in circadian clock regulation in mouse hypothalamus and is also implicated in human breast cancer transcriptional response. Here we show that Gpr176 is N-glycosylated. Peptide-N-glycosidase treatment of mouse hypothalamus extracts revealed that endogenous Gpr176 undergoes N-glycosylation. Using a heterologous expression system, we show that N-glycosylation occurs at four conserved asparagine residues in the N-terminal region of Gpr176. Deficient N-glycosylation due to mutation of these residues reduced the protein expression of Gpr176. At the molecular function level, Gpr176 has constitutive, agonist-independent activity that leads to reduced cAMP synthesis. Although deficient N-glycosylation did not compromise this intrinsic activity, the resultant reduction in protein expression was accompanied by attenuation of cAMP-repressive activity in the cells. We also demonstrate that human GPR176 is N-glycosylated. Importantly, missense variations in the conserved N-glycosylation sites of human GPR176 (rs1473415441; rs761894953) affected N-glycosylation and thereby attenuated protein expression and cAMP-repressive activity in the cells. We show that N-glycosylation is a prerequisite for the efficient protein expression of functional Gpr176/GPR176. G-protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors and are the therapeutic targets of nearly a third of clinically marketed drugs 1,2. Despite their importance, more than one hundred human GPCRs remain poorly characterised due to the lack of useful information on their ligands 3. Included among these so-called orphan GPCRs is GPR176, which is predicted to be a 56-kDa seven-transmembrane protein of class A GPCR with potential sites for N-glycosylation. GPR176 (also known as HB-954) was initially cloned by Hata et al. from a human brain cDNA library 4. In the mouse brain, Gpr176 mRNA levels are predominantly high in the suprachiasmatic nucleus of the hypothalamus (SCN) 5 , the principal circadian pacemaker in mammals, and knockout studies have shown that Gpr176 is required to set the pace of circadian rhythm in behaviour 5. This gene is also expressed in other tissues than the brain 4 and was reported to be involved in the anacardic acid-induced transcriptional response of human breast cancer cells 6. Gpr176 couples to Gz, a subtype of Gi/o, and even in the absence of a known ligand, Gpr176 possesses an agonist-independent constitutive activity that leads to reduced cAMP synthesis 5,7. At the amino acid sequence level, Gpr176 contains five extracellular potential sites for N-glycosylation (Asn-X-Ser/Thr, where X is any amino acid except for Pro); one is located in the third extracellular loop (ECL3) and all other four are located in the N-terminal region. However, wh...

Day-night changes in the storage capacity of the urinary bladder are indispensable for sound sleep. Connexin 43 (Cx43), a major gap junction protein, forms hemichannels as a pathway of ATP in other cell types, and the urinary bladder utilizes ATP as a mechanotransduction signals to modulate its capacity. Here, we demonstrate that the circadian clock of the urothelium regulates diurnal ATP release through Cx43 hemichannels. Cx43 was expressed in human and mouse urothelium, and clock genes oscillated in the mouse urothelium accompanied by daily cycles in the expression of Cx43 and extracellular ATP release into the bladder lumen. Equivalent chronological changes in Cx43 and ATP were observed in immortalized human urothelial cells, but these diurnal changes were lost in both arrhythmic Bmal1-knockout mice and in BMAL1-knockdown urothelial cells. ATP release was increased by Cx43 overexpression and was decreased in Cx43 knockdown or in the presence of a selective Cx43 hemichannel blocker, which indicated that Cx43 hemichannels are considered part of the components regulating ATP release in the urothelium. Thus, a functional circadian rhythm exists in the urothelium, and coordinates Cx43 expression and function as hemichannels that provide a direct pathway of ATP release for mechanotransduction and signalling in the urothelium.

The 3␤-hydroxysteroid dehydrogenase (3␤-HSD) is an enzyme crucial for steroid synthesis. Two different 3␤-HSD isoforms exist in humans. Classically, HSD3B2 was considered the principal isoform present in the adrenal. However, we recently showed that the alternative isoform, HSD3B1, is expressed specifically within the adrenal zona glomerulosa (ZG), where aldosterone is produced, raising the question of why this isozyme needs to be expressed in this cell type. Here we show that in both human and mouse, expression of the ZG isoform 3␤-HSD is rapidly induced upon angiotensin II (AngII) stimulation. AngII is the key peptide hormone regulating the capacity of aldosterone synthesis. Using the human adrenocortical H295R cells as a model system, we show that the ZG isoform HSD3B1 differs from HSD3B2 in the ability to respond to AngII. Mechanistically, the induction of HSD3B1 involves de novo protein synthesis of the nuclear orphan receptors NGFIB and NURR1. The HSD3B1 promoter contains a functional NGFIB/NURR1-responsive element to which these proteins bind in response to AngII. Knockdown of these proteins and overexpression of a dominant negative NGFIB both reduce the AngII responsiveness of HSD3B1. Thus, the AngII-NGFIB/NURR1 pathway controls HSD3B1. Our work reveals HSD3B1 as a new regulatory target of AngII.T he enzyme 3␤-hydroxysteroid dehydrogenase/⌬ 5 -⌬ 4 -isomerase (3␤-HSD) is essential for the biosynthesis of all active steroid hormones, including those secreted from the adrenal gland (1-4). Whereas two distinct 3␤-HSD isoforms (type I 3␤-HSD, which is encoded by HSD3B1, and type II 3␤-HSD, which is encoded by HSD3B2) exist in humans, it has long been thought that type II 3␤-HSD was the only isoform expressed in the human adrenal glands (1). However, this canonical view was recently revised due to the observation that the alternative isoform, HSD3B1, is expressed within zona glomerulosa (ZG) cells (5, 6), where aldosterone is produced. Interestingly, the mouse also has two isoforms in the adrenal: one (Hsd3b1) is ubiquitous in the cortex, but the other (Hsd3b6) is ZG specific (6-9). Thus, in both species, the adrenals possess a ZG-specific isoform, in addition to the ubiquitous one. However, it remains unknown why these different enzymes are expressed simultaneously in ZG cells. Since these isozymes catalyze the same enzymatic reaction, the question remains open why the newly identified ZG-specific isoform needs to be expressed in ZG cells.Angiotensin II (AngII) is the key peptide hormone in the renin-angiotensin-aldosterone system (RAAS). AngII is a potent secretagogue of the mineralocorticoid aldosterone, which is principally synthesized within the adrenal gland ZG cells through a series of enzymatic reactions that involve multiple enzymes, including 3␤-HSDs. The actions of AngII on ZG cells are often divided into two temporally different phases (10-12): (i) an early (within minutes after stimulation) upregulation of aldosterone synthesis through posttranslational activation of steroidogenic acute regulatory...

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.