title: Native and recombinant human Edg4 receptor-mediated Ca(2+) signalling.
abstract: We have developed an assay system suitable for assessment of compound action on the Edg4 subtype of the widely expressed lysophosphatidic acid (LPA)-responsive Edg receptor family. Edg4 was stably overexpressed in the rat hepatoma cell line Rh 7777, and a Ca(2+)-based FLIPR assay developed for measurement of functional responses. In order to investigate the mechanisms linking Edg4 activation to cytosolic Ca(2+) elevation, we have also studied LPA signalling in a human neuroblastoma cell line that endogenously expresses Edg4. LPA responses displayed similar kinetics and potency in the two cell lines. The Ca(2+) signal generated by activation of LPA-sensitive receptors in these cells is mediated primarily by endoplasmic reticulum. However, there is a substantial inhibition of the LPA response by FCCP, indicating that mitochondria also play a key role in the LPA response. Partial inhibition of the response by cyclosporin A could indicate an active Ca(2+) release role for mitochondria in the LPA response. The inositol 1,4,5-triphosphate receptor antagonist 2-aminoethyl diphenyl borate markedly inhibits, but does not abolish, the Ca(2+) response to LPA, suggesting further complexity to the signalling pathways activated by Edg receptors. In comparing Edg signalling in recombinant and native cells, there is a striking overall similarity in receptor expression pattern, agonist potency, and the effect of modulators on the Ca(2+) response. This indicates that the Edg4-overexpressing Rh7777 cell line is a very useful model system for studying receptor pharmacology and signalling mechanisms, and for investigating the Edg4 receptor's downstream effects.
authors.affiliation: Merck Sharp & Dohme Research Laboratories, Neuroscience Research Centre, Harlow, Essex, UK. peter_simpson@merck.com
authors.forename: Peter B
authors.initials: PB
authors.lastname: Simpson
authors.forename: Israel Ramos
authors.initials: IR
authors.lastname: Villullas
authors.forename: Irina
authors.initials: I
authors.lastname: Schurov
authors.forename: Julie
authors.initials: J
authors.lastname: Kerby
authors.forename: Rachel
authors.initials: R
authors.lastname: Millard
authors.forename: Christine
authors.initials: C
authors.lastname: Haldon
authors.forename: Margaret S
authors.initials: MS
authors.lastname: Beer
authors.forename: George
authors.initials: G
authors.lastname: McAllister
mesh_terms: D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D045744:Cell Line, Tumor; D004396:Coloring Agents; D004721:Endoplasmic Reticulum / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D008113:Liver Neoplasms / Q000378:metabolism; D008246:Lysophospholipids / Q000494:pharmacology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000187:drug effects / Q000502:physiology*; D049368:Receptors, Lysophosphatidic Acid; D011994:Recombinant Proteins; D015398:Signal Transduction / Q000187:drug effects; D019284:Thapsigargin / Q000494:pharmacology; D000099157:Sphingosine Kinase
chemical_list: D002118:Calcium; D004396:Coloring Agents; D004791:Enzyme Inhibitors; D015544:Inositol 1,4,5-Trisphosphate; D008246:Lysophospholipids; D017853:Phosphotransferases (Alcohol Group Acceptor); D043562:Receptors, G-Protein-Coupled; D049368:Receptors, Lysophosphatidic Acid; D011994:Recombinant Proteins; D019284:Thapsigargin; D000099157:Sphingosine Kinase