The evolutionary history and tissue mapping of GPR123: specific CNS expression pattern predominantly in thalamic nuclei and regions containing large pyramidal cells

The Adhesion family of G protein-coupled receptors (GPCRs) includes 33 receptors and is the second largest GPCR family. Most of these proteins are still orphans and fairly little is known of their tissue distribution and evolutionary context. We report the evolutionary history of the Adhesion family protein GPR123 as well as mapping of GPR123 mRNA expression in mouse and rat using in situ hybridization and real-time PCR, respectively. GPR123 was found to be well conserved within the vertebrate lineage, especially within the transmembrane regions and in the distal part of the cytoplasmic tail, containing a potential PDZ binding domain. The real-time PCR data indicates that GPR123 is predominantly expressed in CNS. The in situ data show high expression in thalamic nuclei and regions containing large pyramidal cells like cortex layers 5 and 6 and subiculum. Moreover, we found distinct expression in amygdala, hypothalamus, inferior olive and spinal cord. The CNS specific expression, together with the high sequence conservation between the vertebrate sequences investigated, indicate that GPR123 may have an important role in the regulation of neuronal signal transduction.     

Cloning, Pharmacology, and Tissue Distribution of G-Protein-Coupled Receptor GPR105 (KIAA0001) Rodent Orthologs

It has recently been shown that UDP-glucose is a potent agonist of the orphan G-protein-coupled receptor (GPCR) KIAA0001. Here we report cloning and analysis of the rat and mouse orthologs of this receptor. In accordance with GPCR nomenclature, we have renamed the cDNA clone, KIAA0001, and its orthologs GPR105 to reflect their functionality as G-protein-coupled receptors. The rat and mouse orthologs show 80% and 83% amino acid identity, respectively, to the human GPR105 protein. We demonstrate by genomic Southern blot analysis that there are no genes in the mouse or rat genomes with higher sequence similarity. Chromosomal mapping shows that the mouse and human genes are located on syntenic regions of chromosome 3. Further analyses of the rat and mouse GPR105 proteins show that they are activated by the same agonists as the human receptor, responding to UDP-glucose and closely related molecules with similar affinities. The mouse and rat receptors are widely expressed, as is the human receptor. Thus we conclude that we have identified the rat and mouse orthologs of the human gene GPR105.     

Chromosomal localization of GPR48, a novel glycoprotein hormone receptor like GPCR, in human and mouse with radiation hybrid and interspecific backcross mapping

We report the chromosomal localization in both mouse and human of a novel G-protein-coupled receptor, GPR48, which resembles glycoprotein hormone receptors, that may be implicated in Wilms tumor deletion syndromes such as WAGR. This receptor forms a novel sub-family of glycoprotein hormone-like GPCRs. We have mapped this receptor to human chromosome 11p14-->p13 by several approaches, including radiation hybrid and interspecific backcross mapping, and show that GPR48 is close to BDNF. This data differs from the recently published mapping of LGR4 (5q34-->q35.1) (Hsu et al., 1998). Additionally, we show that Gpr48 and Bdnf are tightly linked on mouse chromosome 2, in a region with conserved synteny to human 11p14-->p13.     

The G-protein-coupled receptors GPR3 and GPR12 are involved in cAMP signaling and maintenance of meiotic arrest in rodent oocytes

In mammalian and amphibian oocytes, the meiotic arrest at the G2/M transition is dependent on cAMP regulation. Because genetic inactivation of a phosphodiesterase expressed in oocytes prevents reentry into the cell cycle, suggesting autonomous cAMP synthesis, we investigated the presence and properties of the G-protein-coupled receptors (GPCRs) in rodent oocytes. The pattern of expression was defined using three independent strategies, including microarray analysis of GV oocyte mRNAs, EST database scanning, and RT-PCR amplification with degenerated primers against transmembrane regions conserved in the GPCR superfamily. Clustering of the GPCR mRNAs from rat and mouse oocytes indicated the expression of the closely related Gpr3, Gpr12, and Edg3, which recognize sphingosine and its metabolites as ligands. Expression of these mRNAs was confirmed by RT-PCR with specific primers as well as by in situ hybridization. That these receptors are involved in the control of cAMP levels in oocytes was indicated by the finding that expression of the mRNA for Gpr3 and Gpr12 is downregulated in Pde3a-deficient oocytes, which have a chronic elevation of cAMP levels. Expression of GPR3 or GPR12 in Xenopus laevis oocytes prevented progesterone-induced meiotic maturation, whereas expression of FSHR had no effect. A block in spontaneous oocyte maturation was also induced when Gpr3 or Gpr12 mRNA was injected into mouse oocytes. Downregulation of GPR3 and GPR12 caused meiotic resumption in mouse and rat oocytes, respectively. However, ablation of the Gpr12 gene in the mouse did not cause a leaky meiotic arrest, suggesting compensation by Gpr3. Incubation of mouse oocytes with the GPR3/12 ligands SPC and S1P delayed spontaneous oocyte maturation. We propose that the cAMP levels required for maintaining meiotic arrest in mouse and rat oocytes are dependent on the expression of Gpr3 and/or Gpr12.     

Nine new human Rhodopsin family G-protein coupled receptors: identification, sequence characterisation and evolutionary relationship

We report nine new members of the Rhodopsin family of human G protein-coupled receptors (GPCRs) found by searches in the genome databases. BLAST searches and phylogenetic analyses showed that only four of the receptors are closely related to previously characterised GPCRs, GPR150 and GPR154 to oxytocin/vasopressin receptors, GPR152 to CRTH2/FPRs and GPR165 to GPR72/NPYR. Four of the receptors, GPR139, GPR146, GPR153 and GPR162, have one other orphan GPCRs as close relative while GPR148 lacks close relatives. We have identified in total 37 orthologues for the new receptors, primarily from rat, mouse, chicken, fugu and zebrafish. GPR162 and GPR139 are remarkably well conserved while GPR148 seems to be evolving rapidly. Analyses using expressed sequence tags (ESTs) indicate that all the new receptors except GPR153 have the CNS as a major site of expression.     

The regulation of adipogenesis through GPR120

Recently, it has been found that long-chain fatty acids activate the G protein-coupled receptors (GPRs), GPR120 and GPR40. However, there have been no reports to date on the possible physiological roles of these GPRs in adipose tissue development and adipocyte differentiation. GPR120 mRNA was highly expressed in the four different adipose tissues, and the amount of mRNA was elevated in adipose tissues of mice fed a high fat diet. However, GPR40 mRNA was not detected in any of the adipose tissues. The expression of GPR120 mRNA was higher in adipocytes compared to stromal-vascular (S-V) cells. The level of GPR120 mRNA increased during adipocyte differentiation in 3T3-L1 cells. Similar results were observed in human adipose tissue, human preadipocytes, and cultured adipocytes. Moreover, use of a small interference RNA (siRNA) to down-regulate GPR120 expression resulted in inhibition of adipocyte differentiation. Our results suggest that GPR120 regulates adipogenic processes such as adipocyte development and differentiation.     

N-arachidonoyl glycine induces macrophage apoptosis via GPR18

N-arachidonoyl glycine (NAGly), a member of lipoamino acids, was reported to exhibit anti-inflammatory effects in experimental ear edema or peritonitis. However the underlying mechanisms have not been clarified so far. In this study, we attempt to investigate the effects of NAGly on macrophages, including the relevant signaling pathways. NAGly potently induced apoptosis in mouse macrophage-derived cell line, RAW264.7. Pretreatment with inhibitors for MEK and p38 MAPK prevented the apoptosis induced by NAGly, although NAGly activated ERK1/2, p38 MAPK and JNK. Further, we focused on implication of GPR18, one of the orphan G protein-coupled receptors, because NAGly has been reported as a candidate ligand for GPR18. Pretreatment with pertussis toxin or siRNA to knock down the expression of GPR18 significantly attenuated the apoptosis induced by NAGly. In mouse peritoneal macrophages, the expression of GPR18 mRNA was elevated in proinflammatory stimulated macrophages but not in anti-inflammatory stimulated macrophages; consistently, NAGly remarkably reduced cell viability of the former, as compared to the latter. These results suggest that NAGly might be involved in function of macrophages through GPR18.     

Differential changes in GPR55 during microglial cell activation

We examined how lipopolysaccharide (LPS) and interferon gamma (IFN-γ), known to differentially activate microglia, affect the expression of G protein-coupled receptor 55 (GPR55), a novel cannabinoid receptor. We found that GPR55 mRNA is significantly expressed in both primary mouse microglia and the BV-2 mouse microglial cell line, and that LPS down-regulates this message. Conversely, IFN-γ slightly decreases GPR55 mRNA in primary microglia, while it upregulates this message in BV-2 cells. Moreover, the GPR55 agonist, lysophosphatidylinositol, increases ERK phosphorylation in BV-2 stimulated with IFN-γ, in correlation with the increased amount of GPR55 mRNA. Remarkably, these stimuli-induced changes in GPR55 expression are similar to those observed with CB₂-R, suggesting that both receptors might be involved in neuroinflammation and that their expression is concomitantly controlled by the state of microglial activation.     

Cloning,molecular characterization,and spatial and developmental expression analysis of GPR41 and GPR43 genes in New Zealand rabbits

Short-chain fatty acids (SCFAs) play a regulatory role in various physiological processes in mammals and act as endogenous ligands for the G protein-coupled receptors (GPR) 41 and 43. The role of GPR41 and GPR43 in mediating SCFA signaling in the rabbit remains unclear. The present study was to investigate the sequence of the GPR41 and GPR43 messenger RNA (mRNA) and their expression pattern in different tissues and developmental stages in New Zealand rabbit. Comparison of genomic sequences in GenBank using the Basic Local Alignment Search Tool program suggested that the New Zealand rabbit GPR41 mRNA has high similarities with the human (84%), bovine (84%) and Capra hircus (84%) genes. Similarly, GPR43 mRNA has high similarity with the rat (84%) and mouse (84%) genes. Real-time PCR results indicated that GPR41 and GPR43 mRNA were expressed throughout rabbit’s whole development and were expressed in several tissues. G protein-coupled receptor 41 and GPR43 mRNA were most highly expressed in pancreas (P<0.05) and s.c. adipose tissue (P<0.05), respectively. The expression levels of GPR41 mRNA was down-regulated in duodenum, cecum (P<0.05) and pancreas and up-regulated in jejunum, ileum, adipose tissue and spleen during growth. G protein-coupled receptor 43GPR43 mRNA was highly expressed in the duodenum, jejunum, ileum, colon, cecum and lung at 15^th day (P<0.05), whereas the expression levels in the pancreas and spleen increased later after birth, with the highest expression at 60^th day (P<0.05).     

GPR155: Gene organization, multiple mRNA splice variants and expression in mouse central nervous system

Emerging evidence suggests that GPR155, an integral membrane protein related to G-protein coupled receptors, has specific roles in Huntington disease and autism spectrum disorders. This study reports the structural organization of mouse GPR155 gene and the generation of five variants (Variants 1-5) of GPR155 mRNA, including so far unknown four variants. Further, it presents the level of expression of GPR155 mRNA in different mouse tissues. The mRNAs for GPR155 are widely expressed in adult mouse tissues and during development. In situ hybridization was used to determine the distribution of GPR155 in mouse brain. The GPR155 mRNAs are widely distributed in forebrain regions and have more restricted distribution in the midbrain and hindbrain regions. The highest level of expression was in the lateral part of striatum and hippocampus. The expression pattern of GPR155 mRNAs in mouse striatum was very similar to that of cannabinoid receptor type 1. The predicted protein secondary structure indicated that GPR155 is a 17-TM protein, and Variant 1 and Variant 5 proteins have an intracellular, conserved DEP domain near the C-terminal.     

猪脂肪组织和原代脂肪细胞中GPR43基因的转录表达规律

根据GenBank已发表的人、小鼠及大鼠GPR43(G protein-coupled receptor 43)基因序列, 设计并合成一对引物, RT-PCR扩增获得猪GPR43基因cDNA, 并利用PCR技术检测该基因在不同猪种、不同发育阶段、不同部位脂肪组织及原代脂肪细胞中的转录表达规律。结果显示, 成功克隆猪GPR43 cDNA片段, 长度为486 bp (GenBank登陆号为EU122439); 同源性分析发现, 猪GPR43与人、小鼠和大鼠同源性达83%以上; GPR43 mRNA表达量在脂肪型猪种上显著高于瘦肉型猪种, 随月龄增长表达量逐渐上升, 且皮下脂肪表达量较内脏脂肪高; 在猪前体脂肪细胞诱导分化过程中, GPR43 mRNA表达量呈时间依赖性升高。揭示GPR43 mRNA表达与猪肥胖程度、年龄、脂肪沉积部位以及脂肪细胞分化程度密切相关。     

Minireview: recent developments in the physiology and pathology of the lysophosphatidylinositol-sensitive receptor GPR55

Emerging data suggest that off-target cannabinoid effects may be mediated via novel seven-transmembrane spanning/G protein-coupled receptors. Due to its cannabinoid sensitivity, the G protein-coupled receptor 55 (GPR55) was recently proposed as a candidate; however, GPR55 is phylogenetically distinct from the traditional cannabinoid receptors, and the conflicting pharmacology, signaling, and functional data have prevented its classification as a novel cannabinoid receptor. Indeed, the most consistent and potent agonist to date is the noncannabinoid lysophospholipid, lysophosphatidylinositol. Here we present new human GPR55 mRNA expression data, providing supportive evidence of GPR55 expression in a vast array of tissues and cell types. Moreover, we summarize major recent developments in GPR55 research and aim to update the reader in the rapidly expanding fields of GPR55 pharmacology, physiology, and pathology.     

Cloning, expression, and pharmacological characterization of the GPR120 free fatty acid receptor from cynomolgus monkey: Comparison with human GPR120 splice variants

Activation of the GPCR GPR120 by free fatty acids has been reported to cause GLP-1 release in rodent intestine. One genetic sequence was reported for rodents, while two sequences were reported for human GPR120, BC101175 and NM_181745. A 1086 base pair sequence cloned from cynomolgus monkey colon cDNA has 85.1% and 83.4% homology with the mouse and rat GPR120 sequences, and 97.5% homology with the human BC101175 sequence. No splice variants of the cynomolgus monkey GPR120 receptor were found. Eight non-synonymous cSNPs were discovered with frequencies less than 4% in monkey samples tested. Real-time PCR demonstrated that, like the human, the highest GPR120 expression in cynomolgus monkey is in lung and colon. Studies measuring intracellular calcium release produced by free fatty acids and the small molecule GPR120 agonist GW9508 in cells expressing the cynomolgus monkey GPR120 receptor were compared to those expressing the human BC101175 splice variant. Long-chain free fatty acids produced the greatest response in cynomolgus monkey GPR120-expressing cells. GW9508 had similar efficacy at the cynomolgus monkey and at the BC101175 human GPR120 receptors. The cynomolgus monkey and the human GPR120 (BC101175) receptors have similar sequences and pharmacology. The possible significance of the alternate splice variant in human is discussed.     

Conformational restriction in a series of GPR119 agonists: Differences in pharmacology between mouse and human

A series of conformationally restricted GPR119 agonists were prepared based around a 3,8-diazabicyclo[3.2.1]octane scaffold. Examples were found to have markedly different pharmacology in mouse and human despite similar levels of binding to the receptor. This highlights the large effects on GPCR phamacology that can result from small structural changes in the ligand, together with inter-species differences between receptors.     

Identification and characterization of mouse SSX genes: a multigene family on the X chromosome with restricted cancer/testis expression

Human SSX was first identified as the gene involved in the t(X;18) translocation in synovial sarcoma. SSX is a multigene family, with 9 complete genes on chromosome Xp11. Normally expressed almost exclusively in testis, SSX mRNA is expressed in various human tumors, defining SSX as a cancer/testis antigen. We have now cloned the mouse ortholog of SSX. Mouse SSX genes can be divided into Ssxa and Ssxb subfamilies based on sequence homology. Ssxa has only one member, whereas 12 Ssxb genes, Ssxb1 to Ssxb12, were identified by cDNA cloning from mouse testis and mouse tumors. Both Ssxa and Ssxb are located on chromosome X and show tissue-restricted mRNA expression to testis among normal tissues. All putative human and mouse SSX proteins share conserved KRAB and SSX-RD domains. Mouse tumors were found to express some, but not all, Ssxb genes, similar to the SSX activation in human tumors.     

Molecular Cloning and Chromosomal Localization of the MouseGpr37Gene Encoding an Orphan G-Protein-Coupled Peptide Receptor Expressed in Brain and Testis

We report the cloning of the mouse ortholog of the humanGPR37gene, which encodes an orphan G-protein-coupled receptor highly expressed in brain tissues and homologous to neuropeptide-specific receptors ([20],Genomics 45:68–77;[45],Biochem. Biophys. Res. Commun. 233:559–567). The genomic organization of theGPR37gene is conserved in both mouse and human species with a single intron interrupting the receptor-coding sequence within the presumed third transmembrane domain. Comparative genetic mapping of theGPR37gene showed that it maps to a conserved chromosomal segment on proximal mouse chromosome 6 and human chromosome 7q31. The mouseGpr37gene contains an open reading frame coding for a 600-amino-acid protein 83% identical to the humanGPR37gene product. The predicted mouse GPR37 protein contains seven putative hydrophobic transmembrane domains, as well as a long (249 amino acid residues), arginine- and proline-rich amino-terminal extracellular domain, which is also a distinctive feature of the human GPR37 receptor. Northern blot analysis of mouse tissues withGpr37-specific probes revealed a main 3.8-kb mRNA and a much less abundant 8-kb mRNA, both expressed in the brain. A 3-kb mRNA is also expressed in the testis. Both the mouse and the humanGPR37genes may belong to a class of highly conserved mammalian genes encoding a novel type of G-protein-coupled receptor predominantly expressed in the brain.     

GPR40 gene expression in human pancreas and insulinoma

To assess gene expression of a membrane-bound G-protein-coupled fatty acid receptor, GPR40, in the human pancreas and islet cell tumors obtained at surgery were analyzed. The mRNA level of the GPR40 gene in isolated pancreatic islets was approximately 20-fold higher than that in the pancreas, and the level was comparable to or rather higher than that of the sulfonylurea receptor 1 gene, which is known to be expressed abundantly in human pancreatic beta cells. A large amount of GPR40 mRNA was detected in tissue extracts from two cases of insulinoma, whereas the expression was undetectable in glucagonoma or gastrinoma. The present study demonstrates that GPR40 mRNA is expressed predominantly in pancreatic islets in humans and that GPR40 mRNA is expressed solely in human insulinoma among islet cell tumors. These results indicate that GPR40 is probably expressed in pancreatic beta cells in the human pancreas.