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.

GPCR ontology: development and application of a G protein-coupled receptor pharmacology knowledge framework.

MOTIVATION: Novel tools need to be developed to help scientists analyze large amounts of available screening data with the goal to identify entry points for the development of novel chemical probes and drugs. As the largest class of drug targets, G protein-coupled receptors (GPCRs) remain of particular interest and are pursued by numerous academic and industrial research projects. RESULTS: We report the first GPCR ontology to facilitate integration and aggregation of GPCR-targeting drugs and demonstrate its application to classify and analyze a large subset of the PubChem database. The GPCR ontology, based on previously reported BioAssay Ontology, depicts available pharmacological, biochemical and physiological profiles of GPCRs and their ligands. The novelty of the GPCR ontology lies in the use of diverse experimental datasets linked by a model to formally define these concepts. Using a reasoning system, GPCR ontology offers potential for knowledge-based classification of individuals (such as small molecules) as a function of the data. AVAILABILITY: The GPCR ontology is available at http://www.bioassayontology.org/bao_gpcr and the National Center for Biomedical Ontologies Web site.

Naïve Bayes classification using 2D pharmacophore feature triplet vectors.

A naïve Bayes classifier, employed in conjunction with 2D pharmacophore feature triplet vectors describing the molecules, is presented and validated. Molecules are described using a vector where each element in the vector contains the number of times a particular triplet of atom-based features separated by a set of topological distances occurs. Using the feature triplet vectors it is possible to generate naïve Bayes classifiers that predict whether molecules are likely to be active against a given target (or family of targets). Two retrospective validation experiments were performed using a range of actives from WOMBAT, the Prous Integrity database, and the Arena screening library. The performance of the classifiers was evaluated using enrichment curves, enrichment factors, and the BEDROC metric. The classifiers were found to give significant enrichments for the various test sets.

Development of a BRET2 screening assay using beta-arrestin 2 mutants.

This study has focused on enhancing the signal generated from the interaction between a G-protein-coupled receptor (GPCR) and beta-arrestin 2 (beta-arr2), measured by the bioluminescence resonance energy transfer (BRET(2)) technology. Both class A (beta(2)-adrenergic receptor [beta(2)-AR]) and class B (neurokinin-type 1 receptor [NK1-R]) GPCRs, classified based on their internalization characteristics, have been analyzed. It was evaluated whether the BRET(2) signal can be enhanced by using (1) beta-arr2 phosphorylation-independent mutant (beta-arr2 R169E) and (2) beta-arr2 mutants deficient in their ability to interact with the components of the clathrin-coated vesicles (beta-arr2 R393E, R395E and beta-arr2 373 stop). For the class B receptor, there was no major difference in the agonist-promoted BRET(2) signal when comparing results obtained with wild-type (wt) and mutant beta-arr2. However, with the class A receptor, a more than 2-fold increase in the BRET(2) signal was observed with beta-arr2 mutants lacking the AP-2 or both AP-2 and clathrin binding sites. This set of data suggests that the inability of these beta-arr2 mutants to interact with the components of the clathrin-coated vesicle probably prevents their rapid dissociation from the receptor, thus yielding an increased and more stable BRET(2) signal. The beta-arr2 R393E, R395E mutant also enhanced the signal window with other members of the GPCR family (neuropeptide Y type 2 receptor [NPY2-R] and TG1019 receptor) and was successfully applied in full-plate BRET(2)-based agonist and antagonist screening assays.

Distribution and regulation of chicken growth hormone secretagogue receptor isoforms.

Chicken ghrelin has recently been isolated as a hormone which stimulates growth hormone and corticosterone secretion in chicken. Ghrelin mediates these actions in mammals by binding to the growth hormone secretagogue receptor (GHS-R). In this study, we describe the partial cloning of two chicken GHS-R (cGHS-R) isoforms: cGHS-R1a and cGHS-R1c. cGHS-R1a and cGHS-R1c cDNA show, respectively, 81 and 78% homology with the corresponding parts of the human GHS-R1a cDNA. In contrast to the human GHS-R1b isoform, which is truncated after transmembrane domain 5 (TM-5), the chicken GHS-R1c isoform lacks 16 amino acids in TM-6 suggesting that this isoform is not active in ghrelin signal transduction. The cystein residues, N-linked glycosylation sites and potential phosphorylation sites, found in the human GHS-R1a, were also conserved in both chicken isoforms. RT-PCR analysis demonstrated cGHS-R1a and cGHS-R1c mRNA expression in all tissues tested, except liver and pancreas, with highest levels in the pituitary and the hypothalamus. Intermediate levels of expression were detected, in descending order, in the ovary, telencephalon, heart, adrenal gland, cerebellum, and optic lobes whereas low expression was detected in the brainstem, lung, kidney, proventriculus, duodenum, and colon. Very low expression was found in skin, stomach, and muscle. cGHS-R1c was expressed in lower amounts than cGHS-R1a in all analysed tissues. Administration of 1 microM chicken ghrelin to pituitaries in vitro resulted in a down-regulation of both cGHS-R isoforms within 15 min, whereas after 1h levels returned to control values. Growth hormone and corticosterone down-regulated cGHS-R1a and cGHS-R1c mRNA expression within 60 min of exposure, whereas growth hormone-releasing factor 1-29 (1 microM) only reduced cGHS-R1a mRNA expression after 60min. Thyrotropin-releasing hormone (1 microM) did not alter cGHS-R expression.