G Protein-coupled Receptors Genes Research Articles

Diversity and impact of rare variants in genes encoding the platelet G protein-coupled receptors.

SummaryPlatelet responses to activating agonists are influenced by common population variants within or near G protein-coupled receptor (GPCR) genes that affect receptor activity. However, the impact of rare GPCR gene variants is unknown. We describe the rare single nucleotide variants (SNVs) in the coding and splice regions of 18 GPCR genes in 7,595 exomes from the 1,000-genomes and Exome Sequencing Project databases and in 31 cases with inherited platelet function disorders (IPFDs). In the population databases, the GPCR gene target regions contained 740 SNVs (318 synonymous, 410 missense, 7 stop gain and 6 splice region) of which 70% had global minor allele frequency (MAF) < 0.05%. Functional annotation using six computational algorithms, experimental evidence and structural data identified 156/740 (21%) SNVs as potentially damaging to GPCR function, most commonly in regions encoding the transmembrane and C-terminal intracellular receptor domains. In 31 index cases with IPFDs (Gi-pathway defect n=15; secretion defect n=11; thromboxane pathway defect n=3 and complex defect n=2) there were 256 SNVs in the target regions of 15 stimulatory platelet GPCRs (34 unique; 12 with MAF<1% and 22 with MAF ≥ 1%). These included rare variants predicting R122H, P258T and V207A substitutions in the P2Y12 receptor that were annotated as potentially damaging, but only partially explained the platelet function defects in each case. Our data highlight that potentially damaging variants in platelet GPCR genes have low individual frequencies, but are collectively abundant in the population. Potentially damaging variants are also present in pedigrees with IPFDs and may contribute to complex laboratory phenotypes.

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.

Identification of G protein coupled receptors for opsines and neurohormones in Rhodnius prolixus. Genomic and transcriptomic analysis

The importance of Chagas disease motivated the scientific effort to obtain the complete genomic sequence of the vector species Rhodnius prolixus, this information is also relevant to the understanding of triatomine biology in general. The central nervous system is the key regulator of insect physiology and behavior. Neurohormones (neuropeptides and biogenic amines) are the chemical messengers involved in the regulation and integration of neuroendocrine signals. In insects, this signaling is mainly mediated by the interaction of neurohormone ligands with G protein coupled receptors (GPCRs). The recently sequenced R. prolixus genome provides us with the opportunity to analyze this important family of genes in triatomines, supplying relevant information for further functional studies. Next-generation sequencing methods offer an excellent opportunity for transcriptomic exploration in key organs and tissues in the presence of a reference genome as well as when a reference genome is not available. We undertook a genomic analysis to obtain a genome-wide inventory of opsines and the GPCRs for neurohormones in R. prolixus. Furthermore, we performed a transcriptomic analysis of R. prolixus central nervous system, focusing on neuropeptide precursor genes and neurohormone and opsines GPCRs. In addition, we mined the whole transcriptomes of Triatoma dimidiata, Triatoma infestans and Triatoma pallidipennis – three sanitary relevant triatomine species – to identify neuropeptide precursors and GPCRs genes. Our study reveals a high degree of sequence conservation in the molecular components of the neuroendocrine system of triatomines.

A Novel F2RL3 Variant Predicting a Tyr157Cys Substitution in Protease-Activated Receptor 4 Is Associated with Reduced Receptor Function in Platelets

IntroductionProtease-activated receptor 4 (PAR4) is expressed widely in haemopoietic and vascular tissues and mediates thrombin-induced platelet activation primarily through Gq, protein kinase C and Ca2+ dependent pathways. Platelet PAR4 function may be modified by common non-coding variants near F2RL3 which encodes PAR4, and by epigenetic regulators which affect F2RL3 expression. However, coding sequence variants which affect PAR4 expression or function have not been reported previously. Here we characterise a novel missense variant in F2RL3associated with loss of PAR4 function in platelets from heterozygous subjects and reduced surface expression in platelets and a cell model.MethodsThe index case (P1) was identified by re-sequencing platelet G-protein coupled receptor genes in sub-groups of a collection of 2,400 adult cardiac surgery patients (GRAPHICS study; UK REC 12/SW/0286). Platelets were studied by light transmission aggregation (LTA) and by measuring P-selectin exposure, PAC-1 binding and intracellular Ca2+mobilisation in response to activating agonists. Platelet PAR4 expression was measured in platelets by flow cytometry and immunoblot and in transiently transfected HEK293 cells by flow cytometry and fluorescence microscopy.ResultsP1 was a 66 year old male with lifelong mild mucocutaneous bleeding and two gastrointestinal bleeds during aspirin treatment who harboured a heterozygous c.471A>G transition in F2RL3predicting a p.Tyr157Cys (Y157C) substitution in PAR4.Platelet phenotypeCompared to healthy controls, platelets from P1 showed reduced aggregation responses to the PAR4 agonist AYPGKF (60-600 μM; EC50 P1 266 μM vs controls 153 μM (n=11)) and to α-thrombin (5-100 mU.ml-1; EC50 P1 51 mU.ml-1 vs controls 15 mU.ml-1 (n=8)) with the greatest reduction in responses at 150 μM AYPGKF (maximum amplitude (MA) P1 1% vs controls 56%; p<0.0001) and 10 mU.ml-1 α-thrombin (MA P1 3% vs controls 67%; p<0.0001). Platelets from P1 also showed reduced P-selectin exposure, PAC-1 binding and intracellular Ca2+mobilisation in response to AYPGKF (25-1000 μM) compared to healthy controls. Functional responses of platelets from P1 to the PAR1 agonist SFLLRN (0.25-100μM) were similar to controls suggesting a selective defect in PAR4 function. Platelets from P1 showed a mean number of PAR4 receptors/platelet of 13 by flow cytometry, compared to 295 in controls (SEM 98; n=9) indicating markedly reduced surface expression. However, PAR1 surface expression in platelets from P1 was similar to controls. Immunoblots of platelet protein extracts for PAR4 from P1 and controls were similar, suggesting comparable total PAR4 expression.The c.471A>G F2RL3 variant was identified in two sons of P1, who also showed reduced platelet functional responses to AYPGKF and PAR4 surface expression that were similar to P1, indicating co-segregation of platelet phenotype and the variant F2LR3 genotype. However, these subjects did not display abnormal bleeding.Y157C PAR4 in HEK293 cellsVariant PAR4 with a C-terminal CFP tag (Y157C PAR4-CFP) expressed in HEK293 cells showed reduced surface expression by flow cytometry (mean of median fluorescence intensities 28% that of wild-type (WT) PAR4-CFP with SEM 5%; n=3). Consistent with this, fluorescence microscopy revealed predominantly intracellular expression of Y157C PAR4-CFP in the HEK293 cells with limited cell surface expression when compared to WT PAR4-CFP.ConclusionThe Y157 residue is highly conserved within transmembrane domain 3 of PAR4 that, in other G protein coupled receptors, is crucial for maintaining normal receptor structure. Therefore, the Y157C substitution identified in the index case in PAR4 is likely to adversely affect receptor function. Consistent with this, our data from multiple functional assays show reduced platelet responses to α-thrombin and to a PAR4 specific agonist, but not to a PAR1 specific agonist. This platelet phenotype co-segregated with the heterozygous Y157C substitution in the study kindred suggesting a causal association. However, this was not associated with bleeding in all kindred members. In both platelets and HEK293 cells we demonstrated the Y157C substitution was associated with loss of PAR4 function and reduced surface receptor expression.This is the first description of a missense PAR4 variant causing loss of receptor function as a heritable trait. DisclosuresNo relevant conflicts of interest to declare.

Somatostatin receptor type 2 contributes to the self-renewal of murine embryonic stem cells.

The roles of G-protein coupled receptors (GPCRs) in stem cell biology remain unclear. In this study, we aimed to identify GPCRs that might contribute to the self-renewal of mouse embryonic stem cells (mESCs). The expression levels of pluripotent genes and GPCR gene were detected in E14 mESCs using PCR array and RT-PCR. Immunofluorescent staining was used to examine the expression of pluripotent markers and the receptor translocation. Western blot analysis was used to detect phosphorylation of signal proteins. Knock-down of receptor was conducted to confirm its role in pluripotency maintenance. In leukemia inhibitory factor (LIF)-free medium, mESCs lost the typical morphology of pluripotency, accompanied by markedly decreases in expression of somatostatin receptor type 2 (SSTR2), as well as the pluripotency biomarkers Oct4, Sox2, Rex1 and Nanog. Addition of the SSTR2 agonist octreotide or seglitide (0.1-30 μmol/L) in LIF-free medium dose-dependently promoted the self-renewal of mESCs, whereas the SSTR2 antagonist S4 (0.03-3 μmol/L) dose-dependently blocked octreotide-induced self-renewal. Knock-down of SSTR2 significantly decreased the self-renewal of mESCs even in the presence of LIF. Addition of LIF (1000 U/mL) or octreotide (1 μmol/L) in LIF-free medium significantly increased both phosphorylation and nuclear ocalization of STAT3. The activation of SSTR2 contributes to the self-renewal of mESCs via activation of the STAT3 pathway.

Research resource: Gene profiling of G protein-coupled receptors in the arcuate nucleus of the female.

The hypothalamic arcuate nucleus controls many critical homeostatic functions including energy homeostasis, reproduction, and motivated behavior. Although G protein-coupled receptors (GPCRs) are involved in the regulation of these functions, relatively few of the GPCRs have been identified specifically within the arcuate nucleus. Here, using TaqMan low-density arrays we quantified the mRNA expression of nonolfactory GPCRs in mouse arcuate nucleus. An unprecedented number of GPCRs (total of 292) were found to be expressed, of which 183 were known and 109 were orphan GPCRs. The known GPCR genes expressed were classified into several functional clusters including hormone/neurotransmitter, growth factor, angiogenesis and vasoactivity, inflammation and immune system, and lipid messenger receptors. The plethora of orphan genes expressed in the arcuate nucleus were classified into 5 structure-related classes including class A (rhodopsin-like), class B (adhesion), class C (other GPCRs), nonsignaling 7-transmembrane chemokine-binding proteins, and other 7-transmembrane proteins. Therefore, for the first time, we provide a quantitative estimate of the numerous GPCRs expressed in the hypothalamic arcuate nucleus. Finally, as proof of principle, we documented the expression and function of one of these receptor genes, the glucagon-like peptide 1 receptor (Glp1r), which was highly expressed in the arcuate nucleus. Single-cell RT-PCR revealed that Glp1r mRNA was localized in proopiomelanocortin neurons, and using whole-cell recording we found that the glucagon-like peptide 1-selective agonist exendin-4 robustly excited proopiomelanocortin neurons. Thus, the quantitative GPCR data emphasize the complexity of the hypothalamic arcuate nucleus and furthermore provide a valuable resource for future neuroendocrine/endocrine-related experiments.

Chaperoning G Protein-Coupled Receptors: From Cell Biology to Therapeutics

G protein-coupled receptors (GPCRs) are membrane proteins that traverse the plasma membrane seven times (hence, are also called 7TM receptors). The polytopic structure of GPCRs makes the folding of GPCRs difficult and complex. Indeed, many wild-type GPCRs are not folded optimally, and defects in folding are the most common cause of genetic diseases due to GPCR mutations. Both general and receptor-specific molecular chaperones aid the folding of GPCRs. Chemical chaperones have been shown to be able to correct the misfolding in mutant GPCRs, proving to be important tools for studying the structure-function relationship of GPCRs. However, their potential therapeutic value is very limited. Pharmacological chaperones (pharmacoperones) are potentially important novel therapeutics for treating genetic diseases caused by mutations in GPCR genes that resulted in misfolded mutant proteins. Pharmacoperones also increase cell surface expression of wild-type GPCRs; therefore, they could be used to treat diseases that do not harbor mutations in GPCRs. Recent studies have shown that indeed pharmacoperones work in both experimental animals and patients. High-throughput assays have been developed to identify new pharmacoperones that could be used as therapeutics for a number of endocrine and other genetic diseases.

Transcriptome analysis of neuropeptides and G-protein coupled receptors (GPCRs) for neuropeptides in the brown planthopper Nilaparvata lugens

The genes encoding neuropeptides, neurohormones and their putative G-protein coupled receptors were identified in the brown planthopper (BPH), Nilaparvata lugens (Stål) by transcriptome analysis (RNA-seq). Forty-eight candidate genes were found to encode neuropeptides or peptide hormones. These include all known insect neuropeptides and neurohormones, with the exception of neuropeptide-like precursor 2 (NPLP2) and trissin. The gene coding for prothoracicotropic hormone (PTTH) was first identified from hemimetabolous insect. A total of 57 putative neuropeptide GPCR genes were identified and phylogenetic analysis showed most of them to be closely related to insect GPCRs. A notable finding was the occurrence of vertebrate hormone receptors, thyrotropin-releasing hormone receptor (TRHR)-like GPCR and parathyroid hormone receptor (PTHR)-like GPCRs. These results suggest that N. lugens possesses the most comprehensive neuropeptide system yet found in insects. Moreover, our findings demonstrate the power of RNA-seq as a tool for analyzing the neuropeptide-related genes in the absence of whole genome sequence information.

An mRNA atlas of G protein-coupled receptor expression during primary human monocyte/macrophage differentiation and lipopolysaccharide-mediated activation identifies targetable candidate regulators of inflammation

G protein-coupled receptors (GPCRs) are among the most important targets in drug discovery. In this study, we used TaqMan Low Density Arrays to profile the full GPCR repertoire of primary human macrophages differentiated from monocytes using either colony stimulating factor-1 (CSF-1/M-CSF) (CSF-1 Mϕ) or granulocyte macrophage colony stimulating factor (GM-CSF) (GM-CSF Mϕ). The overall trend was a downregulation of GPCRs during monocyte to macrophage differentiation, but a core set of 10 genes (e.g. LGR4, MRGPRF and GPR143) encoding seven transmembrane proteins were upregulated, irrespective of the differentiating agent used. Several of these upregulated GPCRs have not previously been studied in the context of macrophage biology and/or inflammation. As expected, CSF-1 Mϕ and GM-CSF Mϕ exhibited differential inflammatory cytokine profiles in response to the Toll-like Receptor (TLR)4 agonist lipopolysaccharide (LPS). Moreover, 15 GPCRs were differentially expressed between these cell populations in the basal state. For example, EDG1 was expressed at elevated levels in CSF-1 Mϕ versus GM-CSF Mϕ, whereas the reverse was true for EDG6. 101 GPCRs showed differential regulation over an LPS time course, with 65 of these profiles being impacted by the basal differentiation state (e.g. GPRC5A, GPRC5B). Only 14 LPS-regulated GPCRs showed asynchronous behavior (divergent LPS regulation) with respect to differentiation status. Thus, the differentiation state primarily affects the magnitude of LPS-regulated expression, rather than causing major reprogramming of GPCR gene expression profiles. Several GPCRs showing differential profiles between CSF-1 Mϕ and GM-CSF Mϕ (e.g. P2RY8, GPR92, EMR3) have not been widely investigated in macrophage biology and inflammation. Strikingly, several closely related GPCRs displayed completely opposing patterns of regulation during differentiation and/or activation (e.g. EDG1 versus EDG6, LGR4 versus LGR7, GPRC5A versus GPRC5B). We propose that selective regulation of GPCR5A and GPCR5B in CSF-1 Mϕ contributes to skewing toward the M2 macrophage phenotype. Our analysis of the GPCR repertoire expressed during primary human monocyte to macrophage differentiation and TLR4-mediated activation provides a valuable new platform for conducting future functional analyses of individual GPCRs in human macrophage inflammatory pathways.

The effect of clozapine on mRNA expression for genes encoding G protein-coupled receptors and the protein components of clathrin-mediated endocytosis

ObjectivesClathrin-mediated endocytosis (CME) is an intracellular trafficking mechanism for packaging cargo, including G protein-coupled receptors (GPCRs), into clathrin-coated vesicles (CCVs). The antipsychotic chlorpromazine inhibits CCV assembly of adaptor protein AP2 whereas clozapine increases serotonin2A receptor internalization. We hypothesized that clozapine alters the expression of CME genes modulating vesicle turnover and GPCR internalization.Materials and methodsSH-SY5Y human neuroblastoma cells were incubated with clozapine (1–20 µmol/l) for 24–72 h. GPCR and CME-related gene mRNA expression was measured using RT-PCR. We quantified changes in the same genes using expression data from a microarray study of mice brains after 12 weeks of treatment with 12 mg/kg/day clozapine.ResultsThe expression of genes encoding adaptor and clathrin assembly proteins, AP2A2, AP2B1, AP180, CLINT1, HIP1, ITSN2, and PICALM, increased relative to the control in SH-SY5Y cells incubated with 5–10 µmol/l clozapine for 24–72 h. The microarray study showed significantly altered expression of the above CME-related genes, with a marked 641-fold and 17-fold increase in AP180 and the serotonin1A GPCR, respectively. The expression of three serotonergic receptor and lysophosphatidic acid receptor 2 (EDG4) GPCR genes was upregulated in SH-SY5Y cells incubated with 5 µmol/l clozapine for 24 h. EDG4 expression was also increased with 10–20 µmol/l clozapine treatment at 48–72 h. Clozapine significantly decreased the expression of β-arrestin, involved in GPCR desensitization, both in vitro and vivo.ConclusionThe changes we report in CME and GPCR mRNAs implicate CCV-mediated internalization of GPCRs and the serotonergic system in clozapine’s mechanism of action, which may be useful in the design of more effective and less toxic antipsychotic therapies.

Identification of novel arthropod vector G protein-coupled receptors.

BackgroundThe control of vector-borne diseases, such as malaria, dengue fever, and typhus fever is often achieved with the use of insecticides. Unfortunately, insecticide resistance is becoming common among different vector species. There are currently no chemical alternatives to these insecticides because new human-safe classes of molecules have yet to be brought to the vector-control market. The identification of novel targets offer opportunities for rational design of new chemistries to control vector populations. One target family, G protein-coupled receptors (GPCRs), has remained relatively under explored in terms of insecticide development.MethodsA novel classifier, Ensemble*, for vector GPCRs was developed. Ensemble* was validated and compared to existing classifiers using a set of all known GPCRs from Aedes aegypti, Anopheles gambiae, Apis Mellifera, Drosophila melanogaster, Homo sapiens, and Pediculus humanus. Predictions for unidentified sequences from Ae. aegypti, An. gambiae, and Pe. humanus were validated. Quantitative RT-PCR expression analysis was performed on previously-known and newly discovered Ae. aegypti GPCR genes.ResultsWe present a new analysis of GPCRs in the genomes of Ae, aegypti, a vector of dengue fever, An. gambiae, a primary vector of Plasmodium falciparum that causes malaria, and Pe. humanus, a vector of epidemic typhus fever, using a novel GPCR classifier, Ensemble*, designed for insect vector species. We identified 30 additional putative GPCRs, 19 of which we validated. Expression of the newly discovered Ae. aegypti GPCR genes was confirmed via quantitative RT-PCR.ConclusionA novel GPCR classifier for insect vectors, Ensemble*, was developed and GPCR predictions were validated. Ensemble* and the validation pipeline were applied to the genomes of three insect vectors (Ae. aegypti, An. gambiae, and Pe. humanus), resulting in the identification of 52 GPCRs not previously identified, of which 11 are predicted GPCRs, and 19 are predicted and confirmed GPCRs.

Chemotaxis and oospore formation in Phytophthora sojae are controlled by G‐protein‐coupled receptors with a phosphatidylinositol phosphate kinase domain

G-protein-coupled receptors (GPCRs) are key cellular components that mediate extracellular signals into intracellular responses. Genome mining revealed that Phytophthora spp. have over 60 GPCR genes among which a prominent class of 12 encoding novel proteins with an N-terminal GPCR domain fused to a C-terminal phosphatidylinositol phosphate kinase (PIPK) domain. This study focuses on two GPCR-PIPKs (GKs) in Phytophthora sojae. PsGK4 and PsGK5 are differentially expressed during the life cycle with the highest expression in cysts and during cyst germination, and at late infection stages. In P. sojae transformants that constitutively express RFP-tagged PsGK4 and PsGK5, the fusion proteins in hyphae reside in small, rapidly moving vesicular-like structures. Functional analysis using gene silencing showed that PsGK4-silenced transformants displayed higher levels of encystment and a reduced cyst germination rate when compared with the recipient strain. Moreover, GK4 deficiency (or reduction) resulted in severe defects in zoospore chemotaxis towards isoflavones and soybean roots. In contrast, PsGK5-silenced transformants exhibited no obvious defects in asexual development but oospore production was severely impaired. Both, PsGK4- and PsGK5-silenced transformants showed reduced pathogenicity. These results point to involvement of GKs in zoospore behaviour, chemotaxis and oospore development, and suggest that PsGK4 and PsGK5 each head independent signalling pathways.

The Discovery Road of G-protein-coupled Receptors: Introduction of the Nobel Prize Winners in Chemistry 2012, Robert J. Lefkowitz and Brian K. Kobilka

The Nobel Prize in Chemistry 2012 was awarded jointly to Robert J. Lefkowitz and Brian K. Kobilka based on their remarkable contributions to the studies of G-protein-coupled receptors (GPCRs). Robert J. Lefkowitz is the Godfather of the modern GPCRs studies. He developed the radioligand binding assay to study the receptors, verified the existence of the receptors, cloned the first GPCR gene, found G protein-coupled receptor kinases (GRKs) and β-arrestins which mediate the desensitization and endocytosis of the receptors, and found the siganling pathways mediated by β-arrestins. Brian K. Kobilka developed several breakthrough methods in GPCRs crystallography. His team first solved the high-resolution structure of β2-adrenergic receptor. His findings led rapid development of GPCRs crystallography. Amazingly, he and his research team got a snapshot of the β-adrenergic receptor at the exact moment when it is activated. The present review summarizes the GPCRs discovery experiences of these two outstanding scientists.

Inflammatory Mediators Alter the Astrocyte Transcriptome and Calcium Signaling Elicited by Multiple G-Protein-Coupled Receptors

Inflammation features in CNS disorders such as stroke, trauma, neurodegeneration, infection, and autoimmunity in which astrocytes play critical roles. To elucidate how inflammatory mediators alter astrocyte functions, we examined effects of transforming growth factor-β1 (TGF-β1), lipopolysaccharide (LPS), and interferon-gamma (IFNγ), alone and in combination, on purified, mouse primary cortical astrocyte cultures. We used microarrays to conduct whole-genome expression profiling, and measured calcium signaling, which is implicated in mediating dynamic astrocyte functions. Combinatorial exposure to TGF-β1, LPS, and IFNγ significantly modulated astrocyte expression of >6800 gene probes, including >380 synergistic changes not predicted by summing individual treatment effects. Bioinformatic analyses revealed significantly and markedly upregulated molecular networks and pathways associated in particular with immune signaling and regulation of cell injury, death, growth, and proliferation. Highly regulated genes included chemokines, growth factors, enzymes, channels, transporters, and intercellular and intracellular signal transducers. Notably, numerous genes for G-protein-coupled receptors (GPCRs) and G-protein effectors involved in calcium signaling were significantly regulated, mostly down (for example, Cxcr4, Adra2a, Ednra, P2ry1, Gnao1, Gng7), but some up (for example, P2ry14, P2ry6, Ccrl2, Gnb4). We tested selected cases and found that changes in GPCR gene expression were accompanied by significant, parallel changes in astrocyte calcium signaling evoked by corresponding GPCR-specific ligands. These findings identify pronounced changes in the astrocyte transcriptome induced by TGF-β1, LPS, and IFNγ, and show that these inflammatory stimuli upregulate astrocyte molecular networks associated with immune- and injury-related functions and significantly alter astrocyte calcium signaling stimulated by multiple GPCRs.

Abstract 4860: Exon capture analysis of G-protein coupled receptors reveals activating mutations in GRM3 in melanoma

Abstract An important feature of cellular signaling involves the activation of plasma membrane-bound receptor complexes via endogenous ligands. G protein-coupled receptors (GPCRs), the largest human gene family, are known to be activated by ligands such as, thrombin, chemokines or glutamate, and have been shown to be important regulators of signaling pathways. Dysregulation via overexpression of diverse GPCRs has been well documented in primary and metastatic tumors cells such as breast, lung, prostate and gastric tumors, head and neck squamous cell carcinoma, and melanoma (1), however, knowledge of their genetic alterations is limited especially in malignant melanoma. Thus, further analysis of this gene family is warranted to determine if GPCR proteins play a role in melanomagenesis. In our current study, we used exon capture and illumina sequencing methods to analyze the mutational status of all the GPCRs in melanoma. This investigation revealed that one family member, GRM3 was frequently mutated in melanoma and that one of its mutations clustered within one position (Glu870Lys). Biochemical analysis of GRM3 alterations revealed that mutant GRM3 selectively regulated the phosphorylation of MEK leading to increased anchorage-independent growth and migration. In vivo studies revealed that overexpression of mutant forms of GRM3 result in increased lung macrometastases; whereas depletion of GRM3 in cells expressing mutant forms of GRM3 resulted in reduced xenograft tumor formation compared to equivalently treated wild type GRM3 cells. Furthermore, melanoma cells expressing mutant forms of GRM3 had reduced cell growth and reduced cellular migration after shRNA-mediated knockdown of GRM3 or treatment with a selective MEK inhibitor, AZD-6244, which is currently being used in phase II clinical trials. Our study yields a comprehensive map of genetic alterations in the GPCR gene family and suggests a potential novel therapeutic target for melanoma patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4860. doi:1538-7445.AM2012-4860

Select Neuropeptides and their G-Protein Coupled Receptors in Caenorhabditis Elegans and Drosophila Melanogaster

The G-protein coupled receptor (GPCR) family is comprised of seven transmembrane domain proteins and play important roles in nerve transmission, locomotion, proliferation and development, sensory perception, metabolism, and neuromodulation. GPCR research has been targeted by drug developers as a consequence of the wide variety of critical physiological functions regulated by this protein family. Neuropeptide GPCRs are the least characterized of the GPCR family as genetic systems to characterize their functions have lagged behind GPCR gene discovery. Drosophila melanogaster and Caenorhabditis elegans are genetic model organisms that have proved useful in characterizing neuropeptide GPCRs. The strength of a genetic approach leads to an appreciation of the behavioral plasticity that can result from subtle alterations in GPCRs or regulatory proteins in the pathways that GPCRs control. Many of these invertebrate neuropeptides, GPCRs, and signaling pathway components serve as models for mammalian counterparts as they have conserved sequences and function. This review provides an overview of the methods to match neuropeptides to their cognate receptor and a state of the art account of neuropeptide GPCRs that have been characterized in D. melanogaster and C. elegans and the behaviors that have been uncovered through genetic manipulation.

Reduced Food Intake and Body Weight in Mice Deficient for the G Protein-Coupled Receptor GPR82

G protein-coupled receptors (GPCR) are involved in the regulation of numerous physiological functions. Therefore, GPCR variants may have conferred important selective advantages during periods of human evolution. Indeed, several genomic loci with signatures of recent selection in humans contain GPCR genes among them the X-chromosomally located gene for GPR82. This gene encodes a so-called orphan GPCR with unknown function. To address the functional relevance of GPR82 gene-deficient mice were characterized. GPR82-deficient mice were viable, reproduced normally, and showed no gross anatomical abnormalities. However, GPR82-deficient mice have a reduced body weight and body fat content associated with a lower food intake. Moreover, GPR82-deficient mice showed decreased serum triacylglyceride levels, increased insulin sensitivity and glucose tolerance, most pronounced under Western diet. Because there were no differences in respiratory and metabolic rates between wild-type and GPR82-deficient mice our data suggest that GPR82 function influences food intake and, therefore, energy and body weight balance. GPR82 may represent a thrifty gene most probably representing an advantage during human expansion into new environments.

Exon capture analysis of G protein-coupled receptors identifies activating mutations in GRM3 in melanoma.

G protein-coupled receptors (GPCRs), the largest human gene family, are important regulators of signaling pathways. However, knowledge of their genetic alterations is limited. In this study, we used exon capture and massively parallel sequencing methods to analyze the mutational status of 734 GPCRs in melanoma. This investigation revealed that one family member, GRM3, was frequently mutated and that one of its mutations clustered within one position. Biochemical analysis of GRM3 alterations revealed that mutant GRM3 selectively regulated the phosphorylation of MEK, leading to increased anchorage-independent growth and migration. Melanoma cells expressing mutant GRM3 had reduced cell growth and cellular migration after short hairpin RNA-mediated knockdown of GRM3 or treatment with a selective MEK inhibitor, AZD-6244, which is currently being used in phase 2 clinical trials. Our study yields the most comprehensive map of genetic alterations in the GPCR gene family.

Functional and Structural Overview of G-Protein-Coupled Receptors Comprehensively Obtained from Genome Sequences

An understanding of the functional mechanisms of G-protein-coupled receptors (GPCRs) is very important for GPCR-related drug design. We have developed an integrated GPCR database (SEVENS http://sevens.cbrc.jp/) that includes 64,090 reliable GPCR genes comprehensively identified from 56 eukaryote genome sequences, and overviewed the sequences and structure spaces of the GPCRs. In vertebrates, the number of receptors for biological amines, peptides, etc. is conserved in most species, whereas the number of chemosensory receptors for odorant, pheromone, etc. significantly differs among species. The latter receptors tend to be single exon type or a few exon type and show a high ratio in the numbers of GPCRs, whereas some families, such as Class B and Class C receptors, have long lengths due to the presence of many exons. Statistical analyses of amino acid residues reveal that most of the conserved residues in Class A GPCRs are found in the cytoplasmic half regions of transmembrane (TM) helices, while residues characteristic to each subfamily found on the extracellular half regions. The 69 of Protein Data Bank (PDB) entries of complete or fragmentary structures could be mapped on the TM/loop regions of Class A GPCRs covering 14 subfamilies.

Pharmacological tools for lysophospholipid GPCRs: development of agonists and antagonists for LPA and S1P receptors

Previous studies on lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) using various approaches have shown that both the molecules can act as intercellular signaling molecules. The discovery of the Edg subfamily of G-protein-coupled receptors (GPCRs) (later renamed LPA(1-3) and S1P(1-5)) for these molecules has opened up a new avenue for pathophysiological research on lysophospholipids. Genetic and molecular studies on lysophospholipid GPCRs have elucidated pathophysiological impacts and roles in cellular signaling pathways. Recently, lysophospholipid GPCR genes have been used to develop receptor subtype-selective agonists and antagonists. The discovery of FTY720, a novel immune modulator, along with other chemical tools, has provided a means of elucidating the functions of each lysophospholipid GPCR on an organ and the whole body level. This communication attempts to retrospectively review the development of agonists and antagonists for lysophospholipid GPCRs, provide integrated information on pharmacological tools for lysophospholipid GPCR signaling, and speculate on future drug development.