Expression of orphan receptors GPR22 and GPR162 in streptozotocin-induced diabetic rats

Abstract

Aims/Introduction: Diabetes mellitus is a chronic degenerative disease characterized by high blood glucose levels as a result of problems in the action or insulin secretion. Although there are many treatments for this pathology, it has been associated with a high mortality rate. For this reason, it is important to try to identify new pathways that could be involved in diabetic complications. Recently, a new class of receptors has been reported, called orphan receptors because the associated ligand and signaling pathways are unknown, these receptors have been associated with certain pathologies. Therefore, the aim of this work was to study the expression of the orphan receptors GPR22 and GPR162 in heart, aorta, brain and kidney of diabetic rats. Materials and methods: We used Wistar male rats with 10–12 weeks of age. Diabetes was induced by a single dose of streptozotocin (60?mg/kg i.p.). After four weeks, the tissue was obtained and the expression of the mRNA was measured by RT-PCR. Results: Our results showed that the orphan receptors are expressed in a different way in heart, kidney, brain and aorta of diabetic and non-diabetic rats. Conclusions: We conclude that orphan receptors could be involved in the development of diabetes complications.     

Evidence of alterations in the expression of orphan receptors GPR26 and GPR39 due to the etiology of the metabolic syndrome

Aims: Metabolic syndrome (MS) is composed of several metabolic abnormalities that increase the risk of cardiovascular diseases and diabetes. Although there are treatments for the components of MS, this pathology maintains a high mortality, suggesting that there are other mechanisms in which orphan receptors such as GPR26 and GPR39 may be involved. For this reason, the aim of this work was to evaluate the expression of GPR26 and GPR39 orphan receptors in two models of MS (diet and genetics).

Materials and methods: We used male Wistar rats, which received 70% fructose in drinking water for 9 weeks, and obese Zucker rats. We measured weight, blood pressure, glucose, triglycerides, total cholesterol, HDL cholesterol, LDL cholesterol to determine the MS and the expression of the orphan receptors GPR26 and GPR39 in brain, heart, aorta, liver, and kidney by RT-PCR.

Results: The analysis of the expression of the orphan receptors GPR26 and GPR39 showed that the receptors are expressed in some tissues, but the expression of the GPR26 tends to decrease in the heart and aorta, whereas in the brain, no changes were observed, this receptor is not expressed in the liver and kidney of both strains. The expression of GPR39 isoforms depends on the tissue and MS model.

Conclusions: We conclude that the orphan receptors GPR26, GPR39v1, and GPR39v2 are expressed in different tissues and their profile expression is dependent on the etiology of the MS.     

Seven evolutionarily conserved human rhodopsin G protein-coupled receptors lacking close relatives

We report seven new members of the superfamily of human G protein-coupled receptors (GPCRs) found by searches in the human genome databases, termed GPR100, GPR119, GPR120, GPR135, GPR136, GPR141, and GPR142. We also report 16 orthologues of these receptors in mouse, rat, fugu (pufferfish) and zebrafish. Phylogenetic analysis shows that these are additional members of the family of rhodopsin-type GPCRs. GPR100 shows similarity with the orphan receptor SALPR. Remarkably, the other receptors do not have any close relative among other known human rhodopsin-like GPCRs. Most of these orphan receptors are highly conserved through several vertebrate species and are present in single copies. Analysis of expressed sequence tag (EST) sequences indicated individual expression patterns, such as for GPR135, which was found in a wide variety of tissues including eye, brain, cervix, stomach and testis. Several ESTs for GPR141 were found in marrow and cancer cells, while the other receptors seem to have more restricted expression patterns.     

Cutting edge: identification of the orphan receptor G-protein-coupled receptor 2 as CCR10, a specific receptor for the chemokine ESkine

A number of orphan G-protein coupled receptors (GPR) have been reported as putative chemokine receptors. One previously reported orphan receptor is an incomplete PCR clone, called GPR2. Here we report the cloning of full-length human (h)GPR2 and mouse (m)GPR2 cDNAs, and the identification of GPR2 as a receptor for a novel CC chemokine called ESkine. hGPR2 is expressed at high levels in testis and small intestine, and at lower levels in other tissues. mGPR2 was expressed at high levels in small intestine, colon, lymph nodes, and Peyer's patches and at lower levels in thymus and spleen. Stimulation of L1.2/hGPR2 transfectants with hESkine induced their migration and resulted in intracellular calcium mobilization. These results provide evidence that GPR2 is a specific receptor for ESkine. We propose that GPR2 be renamed as CCR10. The expression pattern of mGPR2/CCR10 suggests that it may play a role in the homing/trafficking of leukocytes within intestinal and lymphoid environments.     

An expressed sequence tag (EST) data mining strategy succeeding in the discovery of new G-protein coupled receptors

We have developed a comprehensive expressed sequence tag database search method and used it for the identification of new members of the G-protein coupled receptor superfamily. Our approach proved to be especially useful for the detection of expressed sequence tag sequences that do not encode conserved parts of a protein, making it an ideal tool for the identification of members of divergent protein families or of protein parts without conserved domain structures in the expressed sequence tag database. At least 14 of the expressed sequence tags found with this strategy are promising candidates for new putative G-protein coupled receptors. Here, we describe the sequence and expression analysis of five new members of this receptor superfamily, namely GPR84, GPR86, GPR87, GPR90 and GPR91. We also studied the genomic structure and chromosomal localization of the respective genes applying in silico methods. A cluster of six closely related G-protein coupled receptors was found on the human chromosome 3q24-3q25. It consists of four orphan receptors (GPR86, GPR87, GPR91, and H963), the purinergic receptor P2Y1, and the uridine 5'-diphosphoglucose receptor KIAA0001. It seems likely that these receptors evolved from a common ancestor and therefore might have related ligands. In conclusion, we describe a data mining procedure that proved to be useful for the identification and first characterization of new genes and is well applicable for other gene families.     

Constitutive Activity among Orphan Class-A G Protein Coupled Receptors

The purpose of this study was to evaluate the extent of constitutive activity among orphan class-A G protein coupled receptors within the cAMP signaling pathway. Constitutive signaling was revealed by changes in gene expression under control of the cAMP response element. Gene expression was measured in Chinese hamster ovary cells transiently co-transfected with plasmids containing a luciferase reporter and orphan receptor. Criteria adopted for defining constitutive activation were: 1) 200% elevation over baseline reporter gene expression; 2) 40% inhibition of baseline expression; and 3) 40% inhibition of expression stimulated by 3 μM forskolin. Five patterns of activity were noted: 1) inhibition under both baseline and forskolin stimulated expression (GPR15, GPR17, GPR18, GPR20, GPR25, GPR27, GPR31, GPR32, GPR45, GPR57, GPR68, GPR83, GPR84, GPR132, GPR150, GPR176); 2) no effect on baseline expression, but inhibition of forskolin stimulated expression (GPR4, GPR26, GPR61, GPR62, GPR78, GPR101, GPR119); 3) elevation of baseline signaling coupled with inhibition of forskolin stimulated expression (GPR6, GPR12); 4) elevation of baseline signaling without inhibition of forskolin stimulated expression (GPR3, GPR21, GPR52, GPR65); and 5) no effect on expression (GPR1, GPR19, GPR22, GPR34, GPR35, GPR39, GPR63, GPR82, GPR85, GPR87). Constitutive activity was observed in 75% of the orphan class-A receptors examined (30 of 40). This constitutive signaling cannot be explained by simple overexpression of the receptor. Inhibition of cAMP mediated expression was far more common (65%) than stimulation of expression (15%). Orphan receptors that were closely related based on amino acid homology tended to have similar effects on gene expression. These results suggest that identification of inverse agonists may be a fruitful approach for categorizing these orphan receptors and targeting them for pharmacological intervention.     

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.     

Quantitative expression profiling of G-protein-coupled receptors (GPCRs) in metastatic melanoma: the constitutively active orphan GPCR GPR18 as novel drug target

G-protein-coupled receptors (GPCRs) have been implicated in the tumorigenesis and metastasis of human cancers and are considered amongst the most desirable targets for drug development. Utilizing a robust quantitative PCR array, we quantified expression of 94 human GPCRs, including 75 orphan GPCRs and 19 chemokine receptors, and 36 chemokine ligands, in 40 melanoma metastases from different individuals and benign nevi. Inter-metastatic site comparison revealed that orphan GPR174 and CCL28 are statistically significantly overexpressed in subcutaneous metastases, while P2RY5 is overexpressed in brain metastases. Comparison between metastases (all three metastatic sites) and benign nevi revealed that 16 genes, including six orphan receptors (GPR18, GPR34, GPR119, GPR160, GPR183 and P2RY10) and chemokine receptors CCR5, CXCR4, and CXCR6, were statistically significantly differentially expressed. Subsequent functional experiments in yeast and melanoma cells indicate that GPR18, the most abundantly overexpressed orphan GPCR in all melanoma metastases, is constitutively active and inhibits apoptosis, indicating an important role for GPR18 in tumor cell survival. GPR18 and five other orphan GPCRs with yet unknown biological function may be considered potential novel anticancer targets in metastatic melanoma.     

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.     

Tissue-specific expression of PPAR mRNAs in diabetic rats and divergent effects of cilostazol

Cilostazol and ligands of peroxisome proliferator-activated receptors (PPARs) have been effectively used to alleviate diabetic complications, but the common and tissue-specific expression patterns of PPARs in different tissues in diabetic patients and those treated with cilostazol have not been reported. Here, we aimed to assess the effects of diabetes and cilostazol on mRNA expression of PPARalpha and PPARgamma in the aorta, renal cortex, and retina of diabetic rats treated with cilostazol for 8 weeks. PPARalpha mRNA expression showed uniform downregulation in all these tissues in diabetic rats, and this effect was reversed by cilostazol treatment. Surprisingly, PPARgamma mRNA expression was reduced in the renal cortex and retina, yet increased in the aorta of diabetic rats, although cilostazol still reversed these changes. Interestingly, cilostazol, a well-known phosphodiesterase 3 inhibitor and cAMP elevator, augmented cAMP content only in the aorta, but showed no significant effects in the renal cortex of diabetic rats. In conclusion, mRNA expression of PPARs is tissue-specific in diabetes and may be differently affected by cilostazol, possibly because of its tissue-specific effects on cAMP content.     

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.     

Involvement of the chemokine-like receptor GPR33 in innate immunity

Chemokine receptors control leukocyte chemotaxis and cell-cell communication but have also been associated with pathogen entry. GPR33, an orphan member of the chemokine-like receptor family, is a pseudogene in most humans. After the appearance of GPR33 in first mammalian genomes, this receptor underwent independent pseudogenization in humans, other hominoids and some rodent species. It was speculated that a likely cause of GPR33 inactivation was its interplay with a rodent-hominoid-specific pathogen. Simultaneous pseudogenization in several unrelated species within the last 1 million years (myr) caused by neutral drift appears to be very unlikely suggesting selection on the GPR33 null-allele. Although there are no signatures of recent selection on human GPR33 we found a significant increase in the pseudogene allele frequency in European populations when compared with African and Asian populations. Because its role in the immune system was still hypothetical expression analysis revealed that GPR33 is highly expressed in dendritic cells (DC). Murine GPR33 expression is regulated by the activity of toll-like receptors (TLR) and AP-1/NF-κB signaling pathways in cell culture and in vivo. Our data indicate an important role of GPR33 function in innate immunity which became dispensable during human evolution most likely due to past or balancing selection.     

Functional characterization of human receptors for short chain fatty acids and their role in polymorphonuclear cell activation

Short chain fatty acids (SCFAs), including acetate, propionate, and butyrate, are produced at high concentration by bacteria in the gut and subsequently released in the bloodstream. Basal acetate concentrations in the blood (about 100 microm) can further increase to millimolar concentrations following alcohol intake. It was known previously that SCFAs can activate leukocytes, particularly neutrophils. In the present work, we have identified two previously orphan G protein-coupled receptors, GPR41 and GPR43, as receptors for SCFAs. Propionate was the most potent agonist for both GPR41 and GPR43. Acetate was more selective for GPR43, whereas butyrate and isobutyrate were more active on GPR41. The two receptors were coupled to inositol 1,4,5-trisphosphate formation, intracellular Ca2+ release, ERK1/2 activation, and inhibition of cAMP accumulation. They exhibited, however, a differential coupling to G proteins; GPR41 coupled exclusively though the Pertussis toxin-sensitive Gi/o family, whereas GPR43 displayed a dual coupling through Gi/o and Pertussis toxin-insensitive Gq protein families. The broad expression profile of GPR41 in a number of tissues does not allow us to infer clear hypotheses regarding its biological functions. In contrast, the highly selective expression of GPR43 in leukocytes, particularly polymorphonuclear cells, suggests a role in the recruitment of these cell populations toward sites of bacterial infection. The pharmacology of GPR43 matches indeed the effects of SCFAs on neutrophils, in terms of intracellular Ca2+ release and chemotaxis. Such a neutrophil-specific SCFA receptor is potentially involved in the development of a variety of diseases characterized by either excessive or inefficient neutrophil recruitment and activation, such as inflammatory bowel diseases or alcoholism-associated immune depression. GPR43 might therefore constitute a target allowing us to modulate immune responses in these pathological situations.     

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。     

Tissue specific expression and immunohistochemical localization of glutathione S-transferase in streptozotocin induced diabetic rats: modulation by Momordica charantia (karela) extract

In streptozotocin (STZ)-induced diabetes, destruction of pancreatic beta-cell causes an acute shortage of insulin. Increased oxidative stress is believed to be one of the main factors in the etiology and complications of diabetes. In this study we have reported hyperglycemia and glutathione-associated oxidative stress in rats one week after treatment with STZ. In our previous studies, we have reported oxidative stress-related changes in xenobiotic metabolism in tissues from STZ-induced chronic diabetic rats. Here, we demonstrate by immunohistochemistry, that glutathione S-transferase (GST) isoenzymes are differentially expressed in the liver, kidney and testis of diabetic rats. The distribution of GST isoenzymes was found to be tissue- and regio-specific. In addition, we have also shown that treatment with an extract of Momordica charantia (karela), an antidiabetic herb, modulates GST expression in diabetic rats and reverts them to the normal distribution as seen in the tissues of control rats. These results suggest that glutathione metabolism and GST distribution in the tissues of diabetic rats may play an important role in the etiology, pathology and prevention of diabetes.     

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.     

The Gs-Linked Receptor GPR3 Inhibits the Proliferation of Cerebellar Granule Cells during Postnatal Development

Background

During postnatal murine and rodent cerebellar development, cerebellar granule precursors (CGP) gradually stop proliferating as they differentiate after migration to the internal granule layer (IGL). Molecular events that govern this program remain to be fully elucidated. GPR3 belongs to a family of Gs-linked receptors that activate cyclic AMP and are abundantly expressed in the adult brain.

Methodology/Principal Findings

To investigate the role of this orphan receptor in CGP differentiation, we determined that exogenous GPR3 expression in rat cerebellar granule neurons partially antagonized the proliferative effect of Sonic hedgehog (Shh), while endogenous GPR3 inhibition by siRNA stimulated Shh-induced CGP proliferation. In addition, exogenous GPR3 expression in CGPs correlated with increased p27/kip expression, while GPR3 knock-down led to a decrease in p27/kip expression. In wild-type mice, GPR3 expression increased postnatally and its expression was concentrated in the internal granular layer (IGL). In GPR3 −/− mice, the IGL was widened with increased proliferation of CGPs, as measured by bromodeoxyuridine incorporation. Cell cycle kinetics of GPR3-transfected medulloblastoma cells revealed a G0/G1 block, consistent with cell cycle exit.

Conclusions/Significance

These results thus indicate that GPR3 is a novel antiproliferative mediator of CGPs in the postnatal development of murine cerebellum.     

GPR35 is a functional receptor in rat dorsal root ganglion neurons

GPR35, previously an orphan G-protein coupled receptor, is a receptor for kynurenic acid. Here we examine the distribution of GPR35 in the rat dorsal root ganglion (DRG) and the effects of its selective activation. GPR35 was expressed predominantly by small- to medium-diameter neurons of the DRG. Many of these same neurons also expressed the transient receptor potential vanilloid 1 channel, a nociceptive neuronal marker. The GPR35 agonists kynurenic acid and zaprinast inhibited forskolin-stimulated cAMP production by cultured rat DRG neurons. Inhibition required G_i/o proteins as the effect was completely abolished by pretreatment with pertussis toxin. This is the first study to report the expression and function of GPR35 in rat nociceptive DRG neurons. We propose that GPR35 modulates nociception and that continued study of this receptor will provide additional insight into the role of kynurenic acid in pain perception.     

Differential effects of selective endothelin type a receptor antagonist on the gene expression of vascular endothelial growth factor and its receptors in streptozotocin-induced diabetic heart

Cardiovascular complications are an important feature of diabetes mellitus (DM). Abnormal and decreased coronary collateral development has been implicated in the pathogenesis of cardiac complications in DM. More recently, decreased expression of vascular endothelial growth factor (VEGF) and its receptors has been found in diabetic heart. To our knowledge, no study has focused on the therapeutic improvement associated with VEGF in diabetic heart. DM was induced by intraperitoneal injection of streptozotocin (65 mg/kg) in Sprague-Dawley rats, while control rats received only citrate buffer. After 1 week, the streptozotocin-treated rats were randomly divided into two groups: one group received the selective endothelin (ET) type A receptor antagonist TA-0201 at a dose of 1 mg/kg/day for 2 weeks by osmotic mini-pump, and the vehicle group received saline only. The plasma glucose level was 504 +/- 75 mg/dl in the diabetic rats and was unchanged by treatment with ET antagonist. The body weight was decreased in the diabetic rats compared with the control rats, but the left ventricular (LV)-body weight ratio was increased in the diabetic group and was unaffected by treatment with ET antagonist. mRNA expression of VEGF and its receptors (Flt-1 and Flk-1) in the LV tissues was assessed using real-time polymerase chain reaction. VEGF expression was significantly decreased in diabetic heart and was greatly improved by treatment with ET antagonist. The expression of VEGF receptors was down-regulated in early diabetic heart but was not recovered by treatment with ET antagonist. ET and its receptor A might have differential regulation on the gene expressions of VEGF and its receptors in early diabetic heart.