GPR41 and GPR43 are related members of a homologous family of orphan G protein-coupled receptors that are tandemly encoded at a single chromosomal locus in both humans and mice. We identified the acetate anion as an agonist of human GPR43 during routine ligand bank screening in yeast. This activity was confirmed after transient transfection of GPR43 into mammalian cells using Ca 2؉ mobilization and [ 35 S]guanosine 5-O-(3-thiotriphosphate) binding assays and by coexpression with GIRK G protein-regulated potassium channels in Xenopus laevis oocytes. Other short chain carboxylic acid anions such as formate, propionate, butyrate, and pentanoate also had agonist activity. GPR41 is related to GPR43 (52% similarity; 43% identity) and was activated by similar ligands but with differing specificity for carbon chain length, with pentanoate being the most potent agonist. A third family member, GPR42, is most likely a recent gene duplication of GPR41 and may be a pseudogene. GPR41 was expressed primarily in adipose tissue, whereas the highest levels of GPR43 were found in immune cells. The identity of the cognate physiological ligands for these receptors is not clear, although propionate is known to occur in vivo at high concentrations under certain pathophysiological conditions.Within family A of the G protein-coupled receptor (GPCR) 1 gene superfamily (also classified as family 1), there is a phylogenetically related group of ϳ90 receptors that respond to an unusually wide variety of ligand types, considering the relatively close similarity of their primary sequences (1). The group includes receptors that respond to purinergic or pyrimidinergic nucleotides (P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , and P2Y 13 ), modified nucleotides (UDP-glucose), lipids (plateletactivating factor receptor), leukotrienes (BLT 1 and BLT 2 and CysLT 1 and CysLT 2 ), proteases (protease-activated receptor-1-4), chemoattractants (FPR1), and chemokines. To date, these receptors have no clear homologs in invertebrates, unlike the monoamine or neuropeptide receptors, suggesting a relatively recent evolutionary origin (2, 3). At least 50 GPCRs whose cognate ligands are unknown (orphans) (4) are categorized within this group on the basis of sequence homology. Often, these orphans fall into subsets, being more related to each other than to receptors with known ligands; and this, combined with the ligand diversity noted above, makes it difficult to predict the chemical nature of their ligands. One subset comprises GPR40 -43, which were identified as tandemly encoded genes present on cosmids isolated from human chromosomal locus 19q13.1 (5). GPR42 differs from GPR41 at only six amino acid positions; otherwise, the four members of this subfamily share ϳ30% minimum identity. BLAST searches have identified the next most closely related receptors as the proteaseactivated receptors. However, the long N-terminal extracellular domains that serve as protease substrates and that are characteristic of protease-activated receptors are absent in the GPR...

GPR40 is a member of a subfamily of homologous G protein-coupled receptors that include GPR41 and GPR43 and that have no current function or ligand ascribed. Ligand fishing experiments in HEK293 cells expressing human GPR40 revealed that a range of saturated and unsaturated carboxylic acids with carbon chain lengths greater than six were able to induce an elevation of [Ca 2؉ ] i , measured using a fluorometric imaging plate reader. 5,8,11-Eicosatriynoic acid was the most potent fatty acid tested, with a pEC 50 of 5.7. G protein coupling of GPR40 was examined in Chinese hamster ovary cells expressing the G␣ q/i -responsive Gal4-Elk1 reporter system. Expression of human GPR40 led to a constitutive induction of luciferase activity, which was further increased by exposure of the cells to eicosatriynoic acid. Neither the constitutive nor ligandmediated luciferase induction was inhibited by pertussis toxin treatment, suggesting that GPR40 was coupled to G␣ q/11. Expression analysis by quantitative reverse transcription-PCR showed that GPR40 was specifically expressed in brain and pancreas, with expression in rodent pancreas being localized to insulin-producing ␤-cells. These data suggest that some of the physiological effects of fatty acids in pancreatic islets and brain may be mediated through a cell-surface receptor.

Nicotinic acid has been used clinically for over 40 years in the treatment of dyslipidemia producing a desirable normalization of a range of cardiovascular risk factors, including a marked elevation of high density lipoprotein and a reduction in mortality. The precise mechanism of action of nicotinic acid is unknown, although it is believed that activation of a G i -G proteincoupled receptor may contribute. Utilizing available information on the tissue distribution of nicotinic acid receptors, we identified candidate orphan receptors. The selected orphan receptors were screened for responses to nicotinic acid, in an assay for activation of G i -G proteins. Here we describe the identification of the G protein-coupled receptor HM74 as a low affinity receptor for nicotinic acid. We then describe the subsequent identification of HM74A in follow-up bioinformatics searches and demonstrate that it acts as a high affinity receptor for nicotinic acid and other compounds with related pharmacology. The discovery of HM74A as a molecular target for nicotinic acid may facilitate the discovery of superior drug molecules to treat dyslipidemia.

1 Long chain fatty acids have recently been identified as agonists for the G protein-coupled receptors GPR40 and GPR120. Here, we present the first description of GW9508, a small-molecule agonist of the fatty acid receptors GPR40 and GPR120. In addition, we also describe the pharmacology of GW1100, a selective GPR40 antagonist. These molecules were used to further investigate the role of GPR40 in glucose-stimulated insulin secretion in the MIN6 mouse pancreatic b-cell line. 2 GW9508 and linoleic acid both stimulated intracellular Ca 2 þ mobilization in human embryonic kidney (HEK)293 cells expressing GPR40 (pEC 50 values of 7.3270.03 and 5.6570.06, respectively) or GPR120 (pEC 50 values of 5.4670.09 and 5.8970.04, respectively), but not in the parent HEK-293 cell line. 3 GW1100 dose dependently inhibited GPR40-mediated Ca 2 þ elevations stimulated by GW9508 and linoleic acid (pIC 50 values of 5.9970.03 and 5.9970.06, respectively). GW1100 had no effect on the GPR120-mediated stimulation of intracellular Ca 2 þ release produced by either GW9508 or linoleic acid. 4 GW9508 dose dependently potentiated glucose-stimulated insulin secretion in MIN6 cells, but not in primary rat or mouse islets. Furthermore, GW9508 was able to potentiate the KCl-mediated increase in insulin secretion in MIN6 cells. The effects of GW9508 on insulin secretion were reversed by GW1100, while linoleic acid-stimulated insulin secretion was partially attenuated by GW1100. 5 These results add further evidence to a link between GPR40 and the ability of fatty acids to acutely potentiate insulin secretion and demonstrate that small-molecule GPR40 agonists are glucose-sensitive insulin secretagogues.