ESTABLISHMENT OF A CELLULAR-ASSAY SYSTEM FOR G-PROTEIN-LINKED RECEPTORS - COUPLING OF HUMAN NK2 AND 5-HT(2) RECEPTORS TO PHOSPHOLIPASE-C ACTIVATES A LUCIFERASE REPORTER GENE

A functional cellular assay system was developed for the detection of substances modulating the activity of G protein-coupled receptors, lin ked to the phospholipase C second messenger system. The human adenocar cinoma cell line A549 was transformed with the Photinus pyralis lucife rase gene under the control of the ICAM-1 gene 5' regulatory region an d, subsequently, stably transfected with the human neurokinin 2 (NK2) receptor gene. The ICAM-1 promoter is known to be inducible via the ph ospholipase C signal transduction pathway. In this NK2 receptor test c ell line, expression of luciferase was inducible by neurokinin A and o ther NK2-specific agonists. The order of potency of the three neurokin ins substance P, neurokinin A and neuromedin K was consistent with pub lished data and results from ligand binding studies performed with the same NK2 test cell line. The agonistic effect of neurokinin A could b e inhibited in a dose-dependent manner by simultaneous addition of NK2 -specific antagonists or protein kinase C-inhibitors. Similarly, a sta ble test cell line expressing the human serotonin 2 receptor was estab lished. Agonist-induced luciferase expression in this cell line was ab olished in the presence of 5-HT2-specific antagonists. These cellular assay systems can be employed for the identification of competitive, n on-competitive and allosteric modulators of the NK2 and the 5-HT, rece ptor, and they represent prototypes for analogous test cell lines for other phospholipase C-coupled receptors.