G protein-coupled receptor Gpr4 senses amino acids and activates the cAMP-PKA pathway in Cryptococcus neoformans

The Galpha protein Gpa1 governs the cAMP-PKA signaling pathway and plays a central role in virulence and differentiation in the human fungal pathogen Cryptococcus neoformans, but the signals and receptors that trigger this pathway were unknown. We identified seven putative proteins that share identity with known G protein-coupled receptors (GPCRs). One protein, Gpr4, shares limited sequence identity with the Dictyostelium discoideum cAMP receptor cAR1 and the Aspergillus nidulans GPCR protein GprH and also shares structural similarity with the Saccharomyces cerevisiae receptor Gpr1. gpr4 mutants exhibited reduced capsule production and mating defects, similar to gpa1 mutants, and exogenous cAMP suppressed both gpr4 mutant phenotypes. Epistasis analysis provides further evidence that Gpr4 functions upstream of the Galpha subunit Gpa1. Gpr4-Gpr4 homomeric interactions were observed in the yeast two-hybrid assay, and Gpr4 was shown to physically interact with Gpa1 in the split-ubiquitin system. A Gpr4::DsRED fusion protein was localized to the plasma membrane and methionine was found to trigger receptor internalization. The analysis of intracellular cAMP levels showed that gpr4 mutants still respond to glucose but not to certain amino acids, such as methionine. Amino acids might serve as ligands for Gpr4 and could contribute to engage the cAMP-PKA pathway. Activation of the cAMP-PKA pathway by glucose and amino acids represents a nutrient coincidence detection system shared in other pathogenic fungi.     

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.     

Glucose and sucrose act as agonist and mannose as antagonist ligands of the G protein-coupled receptor Gpr1 in the yeast Saccharomyces cerevisiae

Several examples of G protein-coupled receptors have recently been suggested to respond to common sugars in millimolar concentrations. This low affinity has made it difficult to demonstrate direct receptor-ligand interaction. In the yeast Saccharomyces cerevisiae, rapid activation of the cAMP pathway by glucose and sucrose requires the GPCR Gpr1. Our results obtained by cysteine scanning mutagenesis and SCAM (substituted cysteine accessibility method) of residues in TMD VI provide strong evidence that glucose and sucrose directly interact as ligands with Gpr1. The affinity for sucrose is much higher. Structurally similar sugars such as galactose, mannose, and fructose do not act as agonists, but mannose acts as an antagonist for both sucrose and glucose. These results support the idea that Gpr1 directly senses sugars and that sugars can effectively bind GPCRs with a low affinity in a binding pocket formed by the transmembrane domains. The ligand repertoire of GPCRs can thus be extended to common sugars in millimolar concentrations.     

Gpr116 Receptor Regulates Distinctive Functions in Pneumocytes and Vascular Endothelium

Despite its known expression in both the vascular endothelium and the lung epithelium, until recently the physiological role of the adhesion receptor Gpr116/ADGRF5 has remained elusive. We generated a new mouse model of constitutive Gpr116 inactivation, with a large genetic deletion encompassing exon 4 to exon 21 of the Gpr116 gene. This model allowed us to confirm recent results defining Gpr116 as necessary regulator of surfactant homeostasis. The loss of Gpr116 provokes an early accumulation of surfactant in the lungs, followed by a massive infiltration of macrophages, and eventually progresses into an emphysema-like pathology. Further analysis of this knockout model revealed cerebral vascular leakage, beginning at around 1.5 months of age. Additionally, endothelial-specific deletion of Gpr116 resulted in a significant increase of the brain vascular leakage. Mice devoid of Gpr116 developed an anatomically normal and largely functional vascular network, surprisingly exhibited an attenuated pathological retinal vascular response in a model of oxygen-induced retinopathy. These data suggest that Gpr116 modulates endothelial properties, a previously unappreciated function despite the pan-vascular expression of this receptor. Our results support the key pulmonary function of Gpr116 and describe a new role in the central nervous system vasculature.     

An siRNA screen in pancreatic beta cells reveals a role for Gpr27 in insulin production

The prevalence of type 2 diabetes in the United States is projected to double or triple by 2050. We reasoned that the genes that modulate insulin production might be new targets for diabetes therapeutics. Therefore, we developed an siRNA screening system to identify genes important for the activity of the insulin promoter in beta cells. We created a subclone of the MIN6 mouse pancreatic beta cell line that expresses destabilized GFP under the control of a 362 base pair fragment of the human insulin promoter and the mCherry red fluorescent protein under the control of the constitutively active rous sarcoma virus promoter. The ratio of the GFP to mCherry fluorescence of a cell indicates its insulin promoter activity. As G protein coupled receptors (GPCRs) have emerged as novel targets for diabetes therapies, we used this cell line to screen an siRNA library targeting all known mouse GPCRs. We identified several known GPCR regulators of insulin secretion as regulators of the insulin promoter. One of the top positive regulators was Gpr27, an orphan GPCR with no known role in beta cell function. We show that knockdown of Gpr27 reduces endogenous mouse insulin promoter activity and glucose stimulated insulin secretion. Furthermore, we show that Pdx1 is important for Gpr27's effect on the insulin promoter and insulin secretion. Finally, the over-expression of Gpr27 in 293T cells increases inositol phosphate levels, while knockdown of Gpr27 in MIN6 cells reduces inositol phosphate levels, suggesting this orphan GPCR might couple to Gq/11. In summary, we demonstrate a MIN6-based siRNA screening system that allows rapid identification of novel positive and negative regulators of the insulin promoter. Using this system, we identify Gpr27 as a positive regulator of insulin production.     

GPR125 positively regulates osteoclastogenesis potentially through AKT-NF-κB and MAPK signaling pathways

G-protein-coupled receptors (GPCRs) signaling is critical to cell differentiation and activation. However, the function of GPCRs in osteoclast differentiation and activation remains unclear. We found that the G-protein coupled receptor 125 (GPCR 125) gene (Gpr125) gene was highly expressed in osteoclasts through RNA-sequencing technology, qRT-PCR, and Western blot analysis. We characterized the role of GPCR125 in osteoclast differentiation and activation by loss-of-function and gain-of-function methods in osteoclasts. Osteoclasts with lentivirus-mediated GPR125 silencing demonstrated a dramatic reduction in differentiation and impaired bone resorption function. In contrast, overexpression of Gpr125 in osteoclasts increased NFATC1 expression and enhanced osteoclast differentiation and enhanced osteoclast-mediated bone resorption. These results indicated that GPCR125 positively regulates osteoclast formation and function. Following receptor activator of nuclear factor kappa-Β ligand (RANKL) stimulation, the expression levels of MAPK signaling pathway proteins phosphorylated-ERK (p-ERK) and phosphorylated-p38 (p-p38) were significantly decreased in the Gpr125 knockdown (sh-GPR125) group compared to its control group. We also found that phosphorylated AKT (p-AKT) expression was downregulated, as well as nuclear factor kappa-B (NF-κB) signaling pathway protein phosphorylated-IKB alpha (p-IKBα). Our results demonstrated that GPCR125 positively regulates osteoclasts via RANKL-stimulated MAPK and AKT-NF-κB signaling pathways, and GPCR125 could potentially be utilized as a novel therapeutic target in bone related diseases including osteoporosis.     

Human GPR107 and murine Gpr108 are members of the LUSTR family of proteins found in both plants and animals, having similar topology to G-protein coupled receptors.

Two new cDNAs, human GPR107 and murine GPR108, were cloned from mammalian lung that are members of a novel gene family encoding proteins that are predicted to have an amino-terminal hydrophobic signal peptide sequence, a long extracellular domain and a carboxy-terminal seven transmembrane domain (LUSTR domain) similar to GPCRs. The 18-exon human GPR107 gene is located at 9q34.2-3 and spans 86.4 kb and the cDNA encodes a 552 residue protein. The closely related, but not homologous, 17-exon murine Gpr108 gene is located at 17C-D and spans 12.8 kb. The murine Gpr108 cDNA encodes a 562 residue protein that has 49% identity to human GPR107. They are distantly related to two other genes, transmembrane protein 87A and 87B that encode LUSTR domain-containing proteins in the human genome. LUSTR proteins are also found in Drosophila, Saccharomyces and Arabidopsis, but are absent from bacteria, archaea and viruses. This suggests that GPCRs are present in higher plants.     

Gpr97 is essential for the follicular versus marginal zone B-lymphocyte fate decision

Gpr97 is an orphan adhesion GPCR and is highly conserved among species. Up to now, its physiological function remains largely unknown. Here, we show that Gpr97 deficiency results in an extensive reduction in B220⁺ lymphocytes in mice. More intensive analyses reveal an expanded marginal zone but a decreased follicular B-cell population in Gpr97^−/−spleen, which displays disorganized architecture characterized by diffuse, irregular B-cell areas and the absence of discrete perifollicular marginal and mantle zones. In vivo functional studies reveal that the mutant mice could generate antibody responses to T cell-dependent and independent antigens, albeit enhanced response to the former and weakened response to the latter. By screening for the molecular events involved in the observed phenotypes, we found that lambda 5 expression is downregulated and its upstream inhibitor Aiolos is increased in the spleen of mutant mice, accompanied by significantly enhanced phosphorylation and nuclear translocation of cAMP response element-binding protein. Interestingly, increased constitutive Nf-κb p50/p65 expression and activity were observed in Gpr97^−/− spleen, implicating a crucial role of Gpr97 in regulating Nf-κb activity. These findings uncover a novel biological function of Gpr97 in regulating B-cell development, implying Gpr97 as a potential therapeutic target for treatment of immunological disorders.     

The Orphan G Protein-Coupled Receptor Gene GPR178 Is Evolutionary Conserved and Altered in Response to Acute Changes in Food Intake

G protein-coupled receptors (GPCRs) are a class of integral membrane proteins mediating physiological functions fundamental for survival, including energy homeostasis. A few years ago, an amino acid sequence of a novel GPCR gene was identified and named GPR178. In this study, we provide new insights regarding the biological significance of Gpr178 protein, investigating its evolutionary history and tissue distribution as well as examining the relationship between its expression level and feeding status. Our phylogenetic analysis indicated that GPR178 is highly conserved among all animal species investigated, and that GPR178 is not a member of a protein family. Real-time PCR and in situ hybridization revealed wide expression of Gpr178 mRNA in both the brain and periphery, with high expression density in the hypothalamus and brainstem, areas involved in the regulation of food intake. Hence, changes in receptor expression were assessed following several feeding paradigms including starvation and overfeeding. Short-term starvation (12–48h) or food restriction resulted in upregulation of Gpr178 mRNA expression in the brainstem, hypothalamus and prefrontal cortex. Conversely, short-term (48h) exposure to sucrose or Intralipid solutions downregulated Gpr178 mRNA in the brainstem; long-term exposure (10 days) to a palatable high-fat and high-sugar diet resulted in a downregulation of Gpr178 in the amygdala but not in the hypothalamus. Our results indicate that hypothalamic Gpr178 gene expression is altered during acute exposure to starvation or acute exposure to palatable food. Changes in gene expression following palatable diet consumption suggest a possible involvement of Gpr178 in the complex mechanisms of feeding reward.     

Impaired working memory,cognitive flexibility and reward processing in mice genetically lacking Gpr88: Evidence for a key role for Gpr88 in multiple cortico-striatal-thalamic circuits

The GPR88 orphan G protein-coupled receptor is expressed throughout the striatum, being preferentially localised in medium spiny neurons. It is also present in lower densities in frontal cortex and thalamus. Rare mutations in humans suggest a role in cognition and motor function, while common variants are associated with psychosis. Here we evaluate the influence of genetic deletion of GPR88 upon performance in translational tasks interrogating motivation, reward evaluation and cognitive function. In an automated radial arm maze ‘N-back’ working memory task, Gpr88 KO mice showed impaired correct responding, suggesting a role for GPR88 receptors in working memory circuitry. Associative learning performance was similar to wild-type controls in a touchscreen task but performance was impaired at the reversal learning stage, suggesting cognitive inflexibility. Gpr88 KO mice showed higher breakpoints, reduced latencies and lengthened session time in a progressive ratio task consistent with enhanced motivation. Simultaneously, locomotor hyperactivity was apparent in this task, supporting previous findings of actions of GPR88 in a cortico-striatal-thalamic motor loop. Evidence for a role of GPR88 in reward processing was demonstrated in a touchscreen-based equivalent of the Iowa gambling task. Although both Gpr88 KO and wild-type mice showed a preference for an optimum contingency choice, Gpr88 KO mice selected more risky choices at the expense of more advantageous lower risk options. Together these novel data suggest that striatal GPR88 receptors influence activity in a range of procedures integrated by prefrontal, orbitofrontal and anterior cingulate cortico-striatal-thalamic loops leading to altered cognitive, motivational and reward evaluation processes.     

Comprehensive repertoire and phylogenetic analysis of the G protein-coupled receptors in human and mouse

Understanding differences in the repertoire of orthologous gene pairs is vital for interpretation of pharmacological and physiological experiments if conclusions are conveyed between species. Here we present a comprehensive dataset for G protein-coupled receptors (GPCRs) in both human and mouse with a phylogenetic road map. We performed systematic searches applying several search tools such as BLAST, BLAT, and Hidden Markov models and searches in literature data. We aimed to gather a full-length version of each human or mouse GPCR in only one copy referring to a single chromosomal position. Moreover, we performed detailed phylogenetic analysis of the transmembrane regions of the receptors to establish accurate orthologous pairs. The results show the identity of 495 mouse and 400 human functional nonolfactory GPCRs. Overall, 329 of the receptors are found in one-to-one orthologous pairs, while 119 mouse and 31 human receptors originate from species-specific expansions or deletions. The average percentage similarity of the orthologue pairs is 85%, while it varies between the main GRAFS families from an average of 59 to 94%. The orthologous pairs for the lipid-binding GPCRs had the lowest levels of conservation, while the biogenic amines had highest levels of conservation. Moreover, we searched for expressed sequence tags (ESTs) and identified more than 17,000 ESTs matching GPCRs in mouse and human, providing information about their expression patterns. On the whole, this is the most comprehensive study of the gene repertoire that codes for human and mouse GPCRs. The datasets are available for downloading.     

沉默Gpr3基因表达对猪卵泡颗粒细胞凋亡的影响

G蛋白偶联受体3（G protein-coupled receptor 3,Gpr3）属于G蛋白偶联受体超家族成员,能够维持卵泡卵母细胞减数分裂的前期阻滞,但在卵泡颗粒细胞中的作用不清。该研究利用RNAi技术,以化学合成的siRNA转染体外培养的猪卵泡颗粒细胞,并利用Real-time PCR和Western blot技术检验Gpr3基因的沉默效果;利用MTT（四甲基偶氮唑盐）、流式细胞术和Real-time PCR技术检测沉默Gpr3基因表达对猪卵泡颗粒细胞凋亡以及凋亡相关基因表达的影响。结果显示,Gpr3-siRNA能够有效地抑制猪卵泡颗粒细胞中Gpr3基因mRNA和蛋白的表达（P〈0.01）;在沉默Gpr3基因表达后,猪卵泡颗粒细胞的细胞活性由0.419升高至0.586,同时细胞凋亡率由2.67%下降至0.42%,并在显著上调Bcl-2表达的同时,下调了Bax的表达（P〈0.05）。结果表明,沉默Gpr3基因的表达抑制了猪卵泡颗粒细胞的凋亡,其机制可能与调控Bcl-2和Bax表达有关。     

A Saccharomyces cerevisiae G-protein coupled receptor, Gpr1, is specifically required for glucose activation of the cAMP pathway during the transition to growth on glucose

In the yeast Saccharomyces cerevisiae the accumulation of cAMP is controlled by an elaborate pathway. Only two triggers of the Ras adenylate cyclase pathway are known. Intracellular acidification induces a Ras-mediated long-lasting cAMP increase. Addition of glucose to cells grown on a non-fermentable carbon source or to stationary-phase cells triggers a transient burst in the intracellular cAMP level. This glucose-induced cAMP signal is dependent on the G alpha-protein Gpa2. We show that the G-protein coupled receptor (GPCR) Gpr1 interacts with Gpa2 and is required for stimulation of cAMP synthesis by glucose. Gpr1 displays sequence homology to GPCRs of higher organisms. The absence of Gpr1 is rescued by the constitutively activated Gpa2Val-132 allele. In addition, we isolated a mutant allele of GPR1, named fil2, in a screen for mutants deficient in glucose-induced loss of heat resistance, which is consistent with its lack of glucose-induced cAMP activation. Apparently, Gpr1 together with Gpa2 constitute a glucose-sensing system for activation of the cAMP pathway. Deletion of Gpr1 and/or Gpa2 affected cAPK-controlled features (levels of trehalose, glycogen, heat resistance, expression of STRE-controlled genes and ribosomal protein genes) specifically during the transition to growth on glucose. Hence, an alternative glucose-sensing system must signal glucose availability for the Sch9-dependent pathway during growth on glucose. This appears to be the first example of a GPCR system activated by a nutrient in eukaryotic cells. Hence, a subfamily of GPCRs might be involved in nutrient sensing.     

过表达Gpr3诱导猪颗粒细胞G1阻滞及凋亡

G蛋白偶联受体3（Gpr3）属于G蛋白偶联受体视紫质家族成员. Gpr3通过激活Gs蛋白介导的下游信号通路,维持卵泡卵母细胞减数分裂的前期阻滞,但在卵泡颗粒细胞中的作用不清. 为了明确Gpr3在猪卵泡颗粒细胞中的功能,构建了Gpr3基因的真核表达载体,利用过表达的方式激活其介导的信号通路,并利用MTT、流式细胞术和real-time PCR等方法检测了过表达Gpr3对猪卵泡颗粒细胞增殖及凋亡的影响. 结果显示,过表达Gpr3后,猪颗粒细胞的增殖水平显著下调,G0/G1期细胞的百分比增加,S期细胞减少,Cyclin B1和CDK1 mRNA的表达量也显著降低;同时,显著增加了颗粒细胞的凋亡率,在抑制Bcl-2表达的同时,促进了Bax的表达. 结果表明：过表达Gpr3在猪颗粒细胞中具有抑增殖促凋亡的作用,丰富了其在调节卵泡发育过程中的生物学功能.     

The G protein-coupled receptor GPR162 is widely distributed in the CNS and highly expressed in the hypothalamus and in hedonic feeding areas

The Rhodopsin family is a class of integral membrane proteins belonging to G protein-coupled receptors (GPCRs). To date, several orphan GPCRs are still uncharacterized and in this study we present an anatomical characterization of the GPR162 protein and an attempt to describe its functional role. Our results show that GPR162 is widely expressed in GABAergic as well as other neurons within the mouse hippocampus, whereas extensive expression is observed in areas related to energy homeostasis and hedonic feeding such as hypothalamus, amygdala and ventral tegmental area, regions known to be involved in the regulation of palatable food consumption.     

Two novel genes, Gpr113, which encodes a family 2 G-protein-coupled receptor, and Trcg1, are selectively expressed in taste receptor cells

To identify genes important for taste receptor cell function, we analyzed the sequences and expression patterns of clones isolated from a mouse taste receptor cell-enriched cDNA library. Here, we report the analyses of two novel genes, Gpr113 and Trcg1. Gpr113 encodes a G-protein-coupled receptor belonging to family 2B, members of which are characterized by having long N-terminal, extracellular domains. The predicted N-terminal extracellular domain of GPR113 contains 696 amino acids with two functional domains, a peptide hormone-binding domain and a G-protein-coupled receptor proteolytic site. Expression analyses indicate that Gpr113 expression is highly restricted to a subset of taste receptor cells. TRCG1 is also selectively expressed in a subset of taste receptor cells. Trcg1 is alternatively spliced and encodes Trcg1 isoforms of 209 and 825 amino acids. BLAST searches of genomic sequences indicate that a putative homolog of Trcg1 resides on human chromosome 15q22.