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.

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.

We have used the chemotactic ability of Dictyostelium cells to examine the roles of Rho family members, known regulators of the assembly of F-actin, in cell movement. Wild-type cells polarize with a leading edge enriched in F-actin toward a chemoattractant. Overexpression of constitutively active Dictyostelium Rac1B 61L or disruption of DdRacGAP1, which encodes a Dictyostelium Rac1 GAP, induces membrane ruffles enriched with actin filaments around the perimeter of the cell and increased levels of F-actin in resting cells. Whereas wild-type cells move linearly toward the cAMP source, Rac1B 61L and Ddracgap1 null cells make many wrong turns and chemotaxis is inefficient, which presumably results from the unregulated activation of F-actin assembly and pseudopod extension. Cells expressing dominant-negative DdRac1B 17N do not have a well-defined F-actin-rich leading edge and do not protrude pseudopodia, resulting in very poor cell motility. From these studies and assays examining chemoattractant-mediated F-actin assembly, we suggest DdRac1 regulates the basal levels of F-actin assembly, its dynamic reorganization in response to chemoattractants, and cellular polarity during chemotaxis.C hemotaxis, directed cell movement toward a chemoattractant agent, is involved in diverse biological responses, including wound healing in vertebrates, migration of tumor cells, metastasis of cancer cells, and aggregation leading to the formation of the multicellular organism in Dictyostelium (1-5). This process is activated by a large and diverse number of extracellular ligands that bind to cell surface receptors and leads to the directed reorganization of the actin and myosin cytoskeletons, pseudopod extension in the direction of the chemoattractant source, and cell movement via pathways that are thought to be highly conserved between mammals and Dictyostelium (3, 6-8).In polymorphonuclear leukocytes, macrophage, and Dictyostelium cells, chemotaxis can be mediated through G proteincoupled cell surface receptors, and in mammalian cells, the response is thought to function through G i via release of G␤␥ subunits (9-11). In Dictyostelium, the chemoattractants folic acid and cAMP (the chemoattractant that mediates aggregation) function through the folate receptor and cAMP receptor cAR1 via heterotrimeric G proteins containing the coupled G␣ subunits G␣4 and G␣2, respectively (3, 12, 13). In Dictyostelium, the second messenger cGMP, produced by receptor activation of guanylyl cyclase, plays an essential role in regulating changes in the actin and myosin cytoskeletons (14). Other components such as the Dictyostelium Akt͞PKB, Ras protein RasG, the Ras exchange factor AleA, a MAP kinase cascade, a novel Rasinteracting protein RIP3, phosphatidyl inositol-3 kinase, and PAKa, a homologue of mammalian PAK1, are involved in different aspects of the signaling pathway that leads to cell movement and chemotaxis (2,(15)(16)(17)(18)(19)(20). Transduction of the chemotactic signal to second messengers induces rearrangement of cytoskeletal components, ...

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.