title: Establishment of a cellular assay system for G protein-linked receptors: coupling of human NK2 and 5-HT2 receptors to phospholipase C activates a luciferase reporter gene.
abstract: A functional cellular assay system was developed for the detection of substances modulating the activity of G protein-coupled receptors, linked to the phospholipase C second messenger system. The human adenocarcinoma cell line A549 was transformed with the Photinus pyralis luciferase gene under the control of the ICAM-1 gene 5'regulatory region and, subsequently, stably transfected with the human neurokinin 2 (NK2) receptor gene. The ICAM-1 promoter is known to be inducible via the phospholipase C signal transduction pathway. In this NK2 receptor test cell line, expression of luciferase was inducible by neurokinin A and other NK2-specific agonists. The order of potency of the three neurokinins substance P, neurokinin A and neuromedin K was consistent with published data and results from ligand binding studies performed with the same NK2 test cell line. The agonistic effect of neurokinin A could be inhibited in a dose-dependent manner by simultaneous addition of NK2-specific antagonists or protein kinase C-inhibitors. Similarly, a stable test cell line expressing the human serotonin 2 receptor was established. Agonist-induced luciferase expression in this cell line was abolished in the presence of 5-HT2-specific antagonists. These cellular assay systems can be employed for the identification of competitive, non-competitive and allosteric modulators of the NK2 and the 5-HT2 receptor, and they represent prototypes for analogous test cell lines for other phospholipase C-coupled receptors.
authors.affiliation: Ernst Boehringer Institut, Bender+Co., Vienna, Austria.
authors.forename: U
authors.initials: U
authors.lastname: Weyer
authors.forename: R
authors.initials: R
authors.lastname: Schäfer
authors.forename: A
authors.initials: A
authors.lastname: Himmler
authors.forename: S K
authors.initials: SK
authors.lastname: Mayer
authors.forename: E
authors.initials: E
authors.lastname: Bürger
authors.forename: A P
authors.initials: AP
authors.lastname: Czernilofsky
authors.forename: C
authors.initials: C
authors.lastname: Stratowa
mesh_terms: D001681:Biological Assay / Q000379:methods; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics; D008156:Luciferases / Q000235:genetics; D015288:Neurokinin A / Q000494:pharmacology; D015287:Neurokinin B / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism; D018041:Receptors, Neurokinin-2 / Q000032:analysis*; D011985:Receptors, Serotonin / Q000032:analysis*; D015290:Second Messenger Systems; D013373:Substance P / Q000494:pharmacology; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D010716:Phosphatidylinositols; D018041:Receptors, Neurokinin-2; D011985:Receptors, Serotonin; D018799:Intercellular Adhesion Molecule-1; D013373:Substance P; D015288:Neurokinin A; D015287:Neurokinin B; D008156:Luciferases; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins