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.     

Insights into unbound–bound states of GPR142 receptor in a membrane-aqueous system using molecular dynamics simulations

G protein coupled receptors (GPCRs) are source machinery in signal transduction pathways and being one of the major therapeutic targets play a significant in drug discovery. GPR142, an orphan GPCR, has been implicated in the regulation of insulin, thereby having a crucial role in Type II diabetes management. Deciphering of the structures of orphan, GPCRs (O-GPCRs) offer better prospects for advancements in research in ion translocation and transduction of extracellular signals. As the crystallographic structure of GPR142 is not available in PDB, therefore, threading and ab initio-based approaches were used for 3D modeling of GPR142. Molecular dynamic simulations (900 ns) were performed on the 3D model of GPR142 and complexes of GPR142 with top five hits, obtained through virtual screening, embedded in lipid bilayer with aqueous system using OPLS force field. Compound 1, 3, and 4 may act as scaffolds for designing potential lead agonists for GPR142. The finding of GPR142 MD simulation study provides more comprehensive representation of the functional properties. The concern for Type II diabetes is increasing worldwide and successful treatment of this disease demands novel drugs with better efficacy.     

GPR158/179 regulate G protein signaling by controlling localization and activity of the RGS7 complexes

The extent and temporal characteristics of G protein-coupled receptor (GPCR) signaling are shaped by the regulator of G protein signaling (RGS) proteins, which promote G protein deactivation. With hundreds of GPCRs and dozens of RGS proteins, compartmentalization plays a key role in establishing signaling specificity. However, the molecular details and mechanisms of this process are poorly understood. In this paper, we report that the R7 group of RGS regulators is controlled by interaction with two previously uncharacterized orphan GPCRs: GPR158 and GPR179. We show that GPR158/179 recruited RGS complexes to the plasma membrane and augmented their ability to regulate GPCR signaling. The loss of GPR179 in a mouse model of night blindness prevented targeting of RGS to the postsynaptic compartment of bipolar neurons in the retina, illuminating the role of GPR179 in night vision. We propose that the interaction of RGS proteins with orphan GPCRs promotes signaling selectivity in G protein pathways.     

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.     

Ligand screening system using fusion proteins of G protein-coupled receptors with G protein alpha subunits

G protein-coupled receptors (GPCRs) constitute one of the largest families of genes in the human genome, and are the largest targets for drug development. Although a large number of GPCR genes have recently been identified, ligands have not yet been identified for many of them. Various assay systems have been employed to identify ligands for orphan GPCRs, but there is still no simple and general method to screen for ligands of such GPCRs, particularly of G(i)-coupled receptors. We have examined whether fusion proteins of GPCRs with G protein alpha subunit (Galpha) could be utilized for ligand screening and showed that the fusion proteins provide an effective method for the purpose. This article focuses on the followings: (1) characterization of GPCR genes and GPCRs, (2) identification of ligands for orphan GPCRs, (3) characterization of GPCR-Galpha fusion proteins, and (4) identification of ligands for orphan GPCRs using GPCR-Galpha fusion proteins.     

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。     

The orphan GPR50 receptor specifically inhibits MT1 melatonin receptor function through heterodimerization

One-third of the approximately 400 nonodorant G protein-coupled receptors (GPCRs) are still orphans. Although a considerable number of these receptors are likely to transduce cellular signals in response to ligands that remain to be identified, they may also have ligand-independent functions. Several members of the GPCR family have been shown to modulate the function of other receptors through heterodimerization. We show that GPR50, an orphan GPCR, heterodimerizes constitutively and specifically with MT(1) and MT(2) melatonin receptors, using biochemical and biophysical approaches in intact cells. Whereas the association between GPR50 and MT(2) did not modify MT(2) function, GPR50 abolished high-affinity agonist binding and G protein coupling to the MT(1) protomer engaged in the heterodimer. Deletion of the large C-terminal tail of GPR50 suppressed the inhibitory effect of GPR50 on MT(1) without affecting heterodimerization, indicating that this domain regulates the interaction of regulatory proteins to MT(1). Pairing orphan GPCRs to potential heterodimerization partners might be of clinical importance and may become a general strategy to better understand the function of orphan GPCRs.     

The Orphan Adhesion G Protein-coupled Receptor GPR97 Regulates Migration of Lymphatic Endothelial Cells via the Small GTPases RhoA and Cdc42

The important role of the lymphatic vascular system in pathological conditions such as inflammation and cancer has been increasingly recognized, but its potential as a pharmacological target is poorly exploited. Our study aimed at the identification and molecular characterization of lymphatic-specific G protein-coupled receptors (GPCRs) to assess new targets for pharmacological manipulation of the lymphatic vascular system. We used a TaqMan quantitative RT-PCR-based low density array to determine the GPCR expression profiles of ex vivo isolated intestinal mouse lymphatic (LECs) and blood vascular endothelial cells (BECs). GPR97, an orphan adhesion GPCR of unknown function, was the most highly and specifically expressed GPCR in mouse lymphatic endothelium. Using siRNA silencing, we found that GPR97-deficient primary human LECs displayed increased adhesion and collective cell migration, whereas single cell migration was decreased as compared with nontargeting siRNA-transfected control LECs. Loss of GPR97 shifted the ratio of active Cdc42 and RhoA and initiated cytoskeletal rearrangements, including F-actin redistribution, paxillin and PAK4 phosphorylation, and β1-integrin activation. Our data suggest a possible role of GPR97 in lymphatic remodeling and furthermore provide the first insights into the biological functions of GPR97.     

Discovery and validation of novel peptide agonists for G-protein-coupled receptors

G-protein-coupled receptors (GPCRs) represent an important group of targets for pharmaceutical therapeutics. The completion of the human genome revealed a large number of putative GPCRs. However, the identification of their natural ligands, and especially peptides, suffers from low discovery rates, thus impeding development of therapeutics based on these potential drug targets. We describe the discovery of novel GPCR ligands encrypted in the human proteome. Hundreds of potential peptide ligands were predicted by machine learning algorithms. In vitro screening of selected 33 peptides on a set of 152 GPCRs, including a group of designated orphan receptors, was conducted by intracellular calcium measurements and cAMP assays. The screening revealed eight novel peptides as potential agonists that specifically activated six different receptors in a dose-dependent manner. Most of the peptides showed distinct stimulatory patterns targeted at designated and orphan GPCRs. Further analysis demonstrated a significant in vivo effect for one of the peptides in a mouse inflammation model.     

An overview of the diverse roles of G-protein coupled receptors (GPCRs) in the pathophysiology of various human diseases

G-protein coupled receptors (GPCRs) modulate diverse cellular responses to the majority of neurotransmitters and hormones within the human body. They exhibit much structural and functional diversity, and are responsive to a plethora of endogenous (biogenic amines, cations, lipids, peptides, and glycoproteins) and exogenous (therapeutic drugs, photons, tastants, and odorants) ligands and stimuli. Due to the key roles of GPCRs in tissue/cell physiology and homeostasis, signaling pathways associated with GPCRs are implicated in the pathophysiology of various diseases, ranging from metabolic, immunological, and neurodegenerative disorders, to cancer and infectious diseases. Approximately 40% of clinically approved drugs mediate their effects by modulating GPCR signaling pathways, which makes them attractive targets for drug screening and discovery. The pace of discovery of new GPCR-based drugs has recently accelerated due to rapid advancements in high-resolution structure determination, high-throughput screening technology and in silico computational modeling of GPCR binding interaction with potential drug molecules. This review aims to provide an overview of the diverse roles of GPCRs in the pathophysiology of various diseases that are the major focus of biopharmaceutical research as potential drug targets.     

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.     

Multiplex GPCR assay in reverse transfection cell microarrays

G protein-coupled receptors (GPCRs) are a superfamily of proteins that include some of the most important drug targets in the pharmaceutical industry. Despite the success of this group of drugs, there remains a need to identify GPCR-targeted drugs with greater selectivity, to develop screening assays for validated targets, and to identify ligands for orphan receptors. To address these challenges, the authors have created a multiplexed GPCR assay that measures greater than 3000 receptor: ligand interactions in a single microplate. The multiplexed assay is generated by combining reverse transfection in a 96-well plate format with a calcium flux readout. This assay quantitatively measures receptor activation and inhibition and permits the determination of compound potency and selectivity for entire families of GPCRs in parallel. To expand the number of GPCR targets that may be screened in this system, receptors are cotransfected with plasmids encoding a promiscuous G protein, permitting the analysis of receptors that do not normally mobilize intracellular calcium upon activation. The authors demonstrate the utility of reverse transfection cell microarrays to GPCR-targeted drug discovery with examples of ligand selectivity screening against a panel of GPCRs as well as dose-dependent titrations of selected agonists and antagonists.     

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.     

Orphan endogenous lipids and orphan GPCRs: A good match

A large and growing family of over 70 endogenous lipids of the basic structure N-acyl amide has been identified during the last 10 years. Only a few of these lipids have been characterized for biological activity, however, those that have shown a wide-range of activity may act at G-protein coupled receptors (GPCRs). Like orphan GPCRs that are identified as being in the genome and expressed in tissue, the majority of these endogenous lipids many produced throughout the body, some predominately in nervous tissue, remain orphaned. Here, we give a brief history of these orphan lipids and highlight the activity of N-arachidonoyl glycine, and farnesyl pyrophosphate at the orphan receptors GPR18 and GPR92, respectively, as well as summarizing the biological and pharmacological data for the recently identified N-palmitoyl glycine that suggests activity at a novel GPCR. Working to deorphanize both lipids and GPCRs together provides a unique opportunity for a greater understanding of cellular signaling and a challenge to find them all a home.     

Trends in application of advancing computational approaches in GPCR ligand discovery

G protein-coupled receptors (GPCRs) comprise the most important superfamily of protein targets in current ligand discovery and drug development. GPCRs are integral membrane proteins that play key roles in various cellular signaling processes. Therefore, GPCR signaling pathways are closely associated with numerous diseases, including cancer and several neurological, immunological, and hematological disorders. Computer-aided drug design (CADD) can expedite the process of GPCR drug discovery and potentially reduce the actual cost of research and development. Increasing knowledge of biological structures, as well as improvements on computer power and algorithms, have led to unprecedented use of CADD for the discovery of novel GPCR modulators. Similarly, machine learning approaches are now widely applied in various fields of drug target research. This review briefly summarizes the application of rising CADD methodologies, as well as novel machine learning techniques, in GPCR structural studies and bioligand discovery in the past few years. Recent novel computational strategies and feasible workflows are updated, and representative cases addressing challenging issues on olfactory receptors, biased agonism, and drug-induced cardiotoxic effects are highlighted to provide insights into future GPCR drug discovery.     

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.     

GPCRs heterodimerization: a new way towards the discovery of function for the orphan receptors?

G protein-coupled receptors (GPCRs), also called seven transmembrane domain (7TM) proteins, represent the largest family of cell surface receptors. GPCRs control a variety of physiological processes, are involved in multiple diseases and are major drug targets. Despite a vast effort of academic and industrial research, more than one hundred receptors remain orphans. These orphan GPCRs offer a great potential for drug discovery, as almost 60% of currently prescribed drugs target GPCRs. Deorphenization strategies have concentrated mainly on the identification of the natural ligands of these proteins. Recent advances have shown that orphan GPCRs, similar to orphan nuclear receptors, can regulate the function of non-orphan receptors by heterodimerization. These findings not only help to better understand the extraordinary diversity of GPCRs, but also open new perspectives for the identification of the function of these orphan receptors that hold great therapeutic potential.