Regulation of TNF-alpha secretion by a specific melanocortin-1 receptor peptide agonist.

The lack of specific pharmacological tools has impeded the evaluation of the role of each melanocortin receptor (MCR) subtype in the myriad physiological effects of melanocortins. 154N-5 is an octapeptide (MFRdWFKPV-NH(2)) that was first identified as an MC1R antagonist in Xenopus melanophores [J. Biol. Chem. 269 (1994) 29846]. In this manuscript, we show that 154N-5 is a specific agonist for human and murine MC1R. The peptide has negligible activity at MC3R and MC4R and is 25-fold less potent and a weak agonist at MC5R. 154N-5 was tested in both a cellular and an animal model of tumor necrosis factor-alpha (TNF-alpha) secretion. The inhibitory efficacy of 154N-5 on TNF-alpha secretion in both models was similar to the nonselective agonist NDP-alpha-melanocyte stimulating hormone (NDP-alphaMSH), thus, we conclude that inhibition of TNF-alpha secretion by melanocortin peptides is mediated by MC1R. 154N-5 is a valuable new tool for the evaluation of specific contribution of MC1R agonism to physiological and pathological processes.

The Orphan G protein-coupled receptors GPR41 and GPR43 are activated by propionate and other short chain carboxylic acids.

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.

The orphan G protein-coupled receptor GPR40 is activated by medium and long chain fatty acids.

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 alpha(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 ligand-mediated luciferase induction was inhibited by pertussis toxin treatment, suggesting that GPR40 was coupled to G alpha(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 beta-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.