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
| Autor: | U, Weyer, R, Schäfer, A, Himmler, S K, Mayer, E, Bürger, A P, Czernilofsky, C, Stratowa |
|---|---|
| Rok vydání: | 1993 |
| Předmět: |
Neurokinin B
Neurokinin A Receptors Neurokinin-2 Substance P Intercellular Adhesion Molecule-1 Phosphatidylinositols Transfection Second Messenger Systems Cell Line Gene Expression Regulation GTP-Binding Proteins Genes Reporter Receptors Serotonin Type C Phospholipases Humans Biological Assay Luciferases |
| Zdroj: | Receptorschannels. 1(3) |
| ISSN: | 1060-6823 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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