Tissue distribution and functional analyses of the constitutively active orphan G protein coupled receptors, GPR26 and GPR78

GPR26 and GPR78 are orphan GPCRs (oGPCRs) that share 51% amino acid sequence identity and are widely expressed in selected tissues of the human brain as well as the developing and adult mouse brain. Investigation of the functional activity of GPR26 and GPR78 via expression in HEK293 cells showed that both proteins are constitutively active and coupled to elevated cAMP production. Accordingly, in yeast, GPR26 demonstrated apparent agonist-independent coupling to a chimeric Gpa1 protein in which the 5 C-terminal amino acids were from Galphas. A comparison of the proteins revealed an atypical glutamine residue in GPR78 in place of the conserved arginine residue (R3.50) in the so-called DRY box. Site-directed mutants R3.50 in GPR26 were constructed and retained their constitutive activity suggesting that these 2 receptors activate G proteins in a manner that is distinct from other group 1 GPCRs.     

家鸡G蛋白偶联受体85基因的结构、序列与组织表达分析

G蛋白偶联受体(GPCR)超家族是细胞膜上广泛存在的一类受体,是细胞跨膜信号转导的一类重要受体分子,参与许多生理过程调节。它们中仍有很多至今尚未找到内源性配体,这类受体被称为孤儿型受体。G蛋白偶联受体85(GPR85)是GPCR超家族中孤儿型受体的一员。目前,在非哺乳类脊椎动物中,针对GPR85的研究极少。本研究以家鸡Gallus gallus domesticus为模型,通过反转录PCR和RACE-PCR等方法从脑中克隆到GPR85基因的cDNA全长序列,揭示其基因结构,并用实时荧光定量PCR(qPCR)方法探究了该基因在家鸡各组织中的表达情况。结果显示:家鸡GPR85基因位于1号染色体上,由2个外显子组成,其编码区位于第2个外显子上,长为1 113 bp,可编码1个370个氨基酸的7次跨膜受体蛋白。家鸡GPR85与其他脊椎动物(人Homo sapiens、小鼠Mus musculus、大鼠Rattus norvegicus、热带爪蟾Xenopus tropicalis和斑马鱼Danio rerio)的GPR85具有高度的氨基酸序列一致性(>93%)。qPCR分析发现,GPR85基因mRNA在家鸡全脑、垂体、肾上腺、精巢中有较高表达,而在所检测的其他外周组织中表达极低。本研究首次揭示了家鸡GPR85基因的结构与表达特征,为后续探究GPR85基因在家鸡等非哺乳类中的生理功能奠定基础。     

Oxytocin and oxytocin-receptor mRNA expression in the human gastrointestinal tract: a polymerase chain reaction study

BACKGROUND/AIM: Oxytocin (OT) has a wide range of effects throughout the body. However, the role of OT on the gastrointestinal (GI) tract has to be settled. So far, the few studies performed reveal no conclusive results. The aim of this study was to examine the expression of OT and OT-receptor mRNA in the human GI tract. MATERIAL AND METHODS: Full-thickness biopsies from all segments of the GI tract and the gallbladder were collected during operations at the Department of Surgery, Malm? University Hospital. Biopsies were taken and put immediately into fluid nitrogen and stored at -70 degrees C until total RNA was extracted after mechanical tissue homogenization. Subsequently, poly A(+) mRNA was isolated from the total RNA extract using an automated nucleic acid extractor and converted into single-stranded cDNA. PCR amplifications were carried out using gene-specific OT and OT-receptor primers. The specificity of the PCR amplicons was further confirmed by Southern blot analyses using gene specific OT and OT-receptor hybridization probes. RESULTS: Expression of OT and OT-receptor mRNA was detected in nearly all segments of the GI tract analyzed. In most of the biopsy specimens analyzed, co-expression of both OT and OT-receptor mRNA appeared to take place. CONCLUSION: The present study demonstrates that OT and OT-receptor mRNAs are expressed throughout the GI tract. A possible physiological and/or pathophysiological role of OT and OT-receptor expression in the human GI tract and the cellular location of its expression remain to be shown.     

Urocortin 2 expression in the rat gastrointestinal tract under basal conditions and in chemical colitis

It is becoming increasingly evident that the urocortins (Ucns) and their receptors are involved in the initiation and development of inflammation in the gastrointestinal (GI) tract. There has not been a systematic study of the basal expression of Ucns or their receptors in the GI tract. Here, we examined basal expression of Ucn 2 and its high-affinity receptor, CRF-R2 in the rat GI tract. Ucn 2 mRNA was expressed throughout the small and large intestine. Surprisingly, CRF-R2 mRNA expression was detected in only a subset of GI regions that expressed Ucn 2. Immunohistochemical study showed that both Ucn 2 immuno-reactivity (Ucn 2-IR) and CRF-R2-IR were consistently seen in the neurons of the myenteric plexus and the nerve fibers innervating the circular muscle. By and large, Ucn 2-IR was detected in all layers, including the mucosal and the submucosal layers throughout the GI regions. In contrast, CRF-R2-IR was very low or undetectable in the mucosal layers of all regions examined. The role of Ucn 2 and CRF-R2 was then examined in a rat model of chemically-induced colitis. In the early phase of colitis, Ucn 2 mRNA levels peaked, whereas, in striking contrast, CRF-R2 mRNA expression decreased approximately 2.5-fold below control levels. At the peptide level, Ucn 2-IR was specifically induced in a large population of immune cells that infiltrated the lamina propria and submucosa of the distal colon, whereas CRFR2-IR was detected in only a small fraction of infiltrated immune cells. CRF-R2-IR was dramatically reduced in the neurons of the myenteric plexus. Thus, we show, for the first time, that in the acute phase of inflammation, Ucn 2 levels are increased whereas expression levels of its only identified receptor, CRF-R2, are decreased. This suggests that Ucn 2 exerts its effects only in part via CRF-R2.     

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.     

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.     

A family of fatty acid binding receptors

The family of G protein-coupled receptors (GPCRs) serves as the target for almost a third of currently marketed drugs, and provides the predominant mechanism through which extracellular factors transmit signals to the cell. The discovery of GPCRs with no known ligand has initiated a frenzy of research, with the aim of elucidating the physiological ligands for these "orphan" receptors and revealing new drug targets. The GPR40 family of receptors, tandemly located on chromosome 19q13.1, exhibit 30-40% homology to one another and diverse tissue distribution, yet all are activated by fatty acids. Since agonists of GPR40 are medium to longchain fatty acids and those for GPR41 and 43 are short-chain fatty acids, the family clearly provides an intriguing example of how the ligand specificity, patterns of expression, and function of GPCRs can diverge through evolution. Here we summarize the identification, structure, and pharmacology of the receptors and speculate on the respective physiological roles that the GPR40 family members may play.     

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.     

GPRC5B a putative glutamate-receptor candidate is negative modulator of insulin secretion

GPRC5B is an orphan receptor belonging to the group C family of G protein-coupled receptors (GPCRs). GPRC5B is abundantly expressed in both human and mouse pancreatic islets, and both GPRC5B mRNA and protein are up-regulated 2.5-fold in islets from organ donors with type 2 diabetes. Expression of Gprc5b is 50% lower in islets isolated from newborn (<3 weeks) than in adult (>36 weeks) mice. Lentiviral shRNA-mediated down-regulation of Gprc5b in intact islets from 12 to 16 week-old mice strongly (2.5-fold) increased basal (1 mmol/l) and moderately (40%) potentiated glucose (20 mmol/l) stimulated insulin secretion and also enhanced the potentiating effect of glutamate on insulin secretion. Down-regulation of Gprc5b protected murine insulin-secreting clonal MIN6 cells against cytokine-induced apoptosis. We propose that increased expression of GPRC5B contributes to the reduced insulin secretion and β-cell viability observed in type-2 diabetes. Thus, pharmacological targeting of GPRC5B might provide a novel means therapy for the treatment and prevention of type-2 diabetes.     

Identification of novel splice variants of Adhesion G protein-coupled receptors

Alternative splicing is an important mechanism to generate proteome diversity in higher eukaryotic organisms. We searched for splice variants of the human Adhesion family of G protein-coupled receptors (GPCRs) using mRNA sequences and expressed sequence tags. The results presented here describe 53 human splice variants among the 33 Adhesion GPCRs. Many of these variants appear to be coding for "functional" proteins (29) while the others are seemingly "non-functional" (24). Novel functional splice variants were found for: CD97, CELR3, EMR2, EMR3, GPR56, GPR110, GPR112-GPR114, GPR116, GPR123-GPR126, GPR133, HE6, and LEC1-LEC3. Splice variants for GPR116, GPR125, GPR126, and HE6 were found conserved in other species. Several of the functional splice variants lack one or more of the functional domains that are found in the N-termini of these receptors. These functional domains are likely to affect ligand binding or interaction with other proteins and these novel splice variants may have important roles for the specificity of interactions between these receptors and extracellular molecules. Another type of splice variants found here lacks a GPCR proteolytic site (GPS). The GPS domain has been shown to be essential for the proteolytic cleavage of the receptors N-termini and for cellular surface expression. We suggest that these alternative splice variants may be crucial for the function of the receptors while the seemingly non-functional splice variants may be a part of a regulative mechanism.     

Cloning and characterization of a human orphan family C G-protein coupled receptor GPRC5D

Recently three orphan G-protein coupled receptors, RAIG1, GPRC5B and GPRC5C, with homology to members of family C (metabotropic glutamate receptor-like) have been identified. Using the protein sequences of these receptors as queries we identified overlapping expressed sequence tags which were predicted to encode an additional subtype. The full length coding regions of mouse mGprc5d and human GPRC5D were cloned and shown to contain predicted open reading frames of 300 and 345 amino acids, respectively. GPRC5D has seven putative transmembrane segments and is expressed in the cell membrane. The four human receptor subtypes, which we assign to group 5 of family C GPCRs, show 31-42% amino acid sequence identity to each other and 20-25% sequence identity to the transmembrane domains of metabotropic glutamate receptor subtypes 2 and 3 and other family C members. In contrast to the remaining family C members, the group 5 receptors have short amino terminal domains of some 30-50 amino acids. GPRC5D was shown to be clustered with RAIG1 on chromosome 12p13.3 and like RAIG1 and GPRC5B to consist of three exons, the first exon being the largest containing all seven transmembrane segments. GPRC5D mRNA is widely expressed in the peripheral system but all four receptors show distinct expression patterns. Interestingly, mRNA levels of all four group 5 receptors were found in medium to high levels in the kidney, pancreas and prostate and in low to medium levels in the colon and the small intestine, whereas other organs only express a subset of the genes. In an attempt to delineate the signal transduction pathway(s) of the orphan receptors, a series of chimeric receptors containing the amino terminal domain of the calcium sensing receptor or metabotropic glutamate receptor subtype 1, and the seven transmembrane domain of the orphan receptors were constructed and tested in binding and functional assays.     

The roles of GRP81 as a metabolic sensor and inflammatory mediator

GPR81 (also named as HCA1) is a member of a subfamily of orphan G-protein coupled receptors (GPCRs), coupled to G_i-type G proteins. GPR81 was discovered in 2001 and identified as the only known endogenous receptor of lactate under physiological conditions in 2008, which opened a new field of research on how lactate may act as a signal molecule along with the GPR81 expression in the roles of metabolic process and inflammatory response. Recent studies showed that the physiological functions of GPR81 include lipid metabolism in adipose tissues, metabolic excitability in the brain, cellular development, and inflammatory response modulation. These findings may reveal a novel therapeutic strategy to treat clinical, metabolic, and inflammatory diseases. This article will summarize past research on GPR81, including its characteristics of distribution and expression, functional residues, pharmacological, and physiological agonists, involvement in signal transduction, and pharmacological applications.     

Role of free fatty acid receptors in the regulation of energy metabolism

Free fatty acids (FFAs) are energy-generating nutrients that act as signaling molecules in various cellular processes. Several orphan G protein-coupled receptors (GPCRs) that act as FFA receptors (FFARs) have been identified and play important physiological roles in various diseases. FFA ligands are obtained from food sources and metabolites produced during digestion and lipase degradation of triglyceride stores. FFARs can be grouped according to ligand profiles, depending on the length of carbon chains of the FFAs. Medium- and long-chain FFAs activate FFA1/GPR40 and FFA4/GPR120. Short-chain FFAs activate FFA2/GPR43 and FFA3/GPR41. However, only medium-chain FFAs, and not long-chain FFAs, activate GPR84 receptor. A number of pharmacological and physiological studies have shown that these receptors are expressed in various tissues and are primarily involved in energy metabolism. Because an impairment of these processes is a part of the pathology of obesity and type 2 diabetes, FFARs are considered as key therapeutic targets. Here, we reviewed recently published studies on the physiological functions of these receptors, primarily focusing on energy homeostasis.     

Neuronal Orphan G-Protein Coupled Receptor Proteins Mediate Plasmalogens-Induced Activation of ERK and Akt Signaling

The special glycerophospholipids plasmalogens (Pls) are enriched in the brain and reported to prevent neuronal cell death by enhancing phosphorylation of Akt and ERK signaling in neuronal cells. Though the activation of Akt and ERK was found to be necessary for the neuronal cells survival, it was not known how Pls enhanced cellular signaling. To answer this question, we searched for neuronal specific orphan GPCR (G-protein coupled receptor) proteins, since these proteins were believed to play a role in cellular signal transduction through the lipid rafts, where both Pls and some GPCRs were found to be enriched. In the present study, pan GPCR inhibitor significantly reduced Pls-induced ERK signaling in neuronal cells, suggesting that Pls could activate GPCRs to induce signaling. We then checked mRNA expression of 19 orphan GPCRs and 10 of them were found to be highly expressed in neuronal cells. The knockdown of these 10 neuronal specific GPCRs by short hairpin (sh)-RNA lentiviral particles revealed that the Pls-mediated phosphorylation of ERK was inhibited in GPR1, GPR19, GPR21, GPR27 and GPR61 knockdown cells. We further found that the overexpression of these GPCRs enhanced Pls-mediated phosphorylation of ERK and Akt in cells. Most interestingly, the GPCRs-mediated cellular signaling was reduced significantly when the endogenous Pls were reduced. Our cumulative data, for the first time, suggest a possible mechanism for Pls-induced cellular signaling in the nervous system.