4-4-difluoro-4-bora-3a-4a-diaza-s-indacene and Insulinoma

GPR40 is a G protein-coupled receptor (GPCR) whose endogenous ligands have recently been identified as medium- and long-chain free fatty acids (FFAs), and it is thought to play an important role in insulin release. Despite recent research efforts, much still remains unclear in our understanding of its pharmacology, mainly because the receptor-ligand interaction has not been analyzed directly. To study the pharmacology of GPR40 in a more direct fashion, we developed a flow cytometry-based binding assay. FLAG-tagged GPR40 protein was expressed in Sf9 cells, solubilized, immobilized on immunomagnetic beads, and labeled with the fluorescent probe C1-BODIPY-C12. Flow cytometry analysis showed that C1-BODIPY-C12 specifically labels a single class of binding site in a saturable and reversible manner with an apparent dissociation constant of approximately 3 microM. The FFAs that activate GPR40 competed with C1-BODIPY-C12 binding; thus, medium- to long-chain FFAs could compete, whereas short-chain FFAs and methyl linoleate had no inhibitory effect. Furthermore, ligands that are known to activate GPR40 competed for binding in a concentration-dependent manner. All the ligands that inhibited the binding promoted phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 in human embryonic kidney (HEK) 293 cells that expressed GPR40 and [Ca(2+)](i) responses in mouse insulinoma (MIN6) cells that natively express GPR40; however, pioglitazone, a thiazolidinedione that failed to compete for the binding, did not activate ERK or [Ca(2+)](i) response. This study showed that a flow cytometry-based binding assay can successfully identify direct interactions between GPR40 and its ligands. This approach would be of value in studying the pharmacology of GPCRs.

Topics: Animals; Baculoviridae; Binding Sites; Binding, Competitive; Biological Assay; Boron Compounds; Calcium; Cell Line; Cell Line, Tumor; Flow Cytometry; Fluorescent Dyes; Humans; Inhibitory Concentration 50; Insulinoma; Kidney; Ligands; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Models, Biological; Phosphorylation; Protein Binding; Receptors, G-Protein-Coupled; Spodoptera

The endoplasmic reticulum (ER) plays a pivotal role in the regulation of cytosolic Ca(2+) concentrations ([Ca(2+)](cyt)) and hence in insulin secretion from pancreatic beta-cells. However, the molecular mechanisms involved in both the uptake and release of Ca(2+) from the ER are only partially defined in these cells, and the presence and regulation of ER ryanodine receptors are a matter of particular controversy. To monitor Ca(2+) fluxes across the ER membrane in single live MIN6 beta-cells, we have imaged changes in the ER intralumenal free Ca(2+) concentration ([Ca(2+)](ER)) using ER-targeted cameleons. Resting [Ca(2+)](ER) (approximately 250 micromol/l) was markedly reduced after suppression (by approximately 40%) of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)-2b but not the SERCA3 isoform by microinjection of antisense oligonucleotides, implicating SERCA2b as the principle ER Ca(2+)-ATPase in this cell type. Nutrient secretagogues that elevated [Ca(2+)](cyt) also increased [Ca(2+)](ER), an effect most marked at the cell periphery, whereas inositol 1,4,5-trisphosphate-generating agents caused a marked and homogenous lowering of [Ca(2+)](ER). Demonstrating the likely presence of ryanodine receptors (RyRs), caffeine and 4-chloro-3-ethylphenol both caused an almost complete emptying of ER Ca(2+) and marked increases in [Ca(2+)](cyt). Furthermore, photolysis of caged cyclic ADP ribose increased [Ca(2+)](cyt), and this effect was largely abolished by emptying ER/Golgi stores with thapsigargin. Expression of RyR protein in living MIN6, INS-1, and primary mouse beta-cells was also confirmed by the specific binding of cell-permeate BODIPY TR-X ryanodine. RyR channels are likely to play an important part in the regulation of intracellular free Ca(2+) changes in the beta-cell and thus in the regulation of insulin secretion.

Topics: Boron Compounds; Caffeine; Calcium; Calcium Channels; Calcium-Transporting ATPases; Carbachol; Chlorophenols; Cholinergic Agonists; Cytoplasm; Endoplasmic Reticulum; Fluorescent Dyes; Humans; Inositol 1,4,5-Trisphosphate Receptors; Insulin; Insulin Secretion; Insulinoma; Islets of Langerhans; Phosphodiesterase Inhibitors; Photochemistry; Receptors, Cytoplasmic and Nuclear; Recombinant Proteins; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Staining and Labeling; Transfection; Tumor Cells, Cultured