Functional coupling of endogenous serotonin (5-HT1B) and calcitonin (C1a) receptors in CHO cells to a cyclic AMP-responsive luciferase reporter gene.
A cyclic AMP-responsive reporter cell line has been established through the stable expression of a luciferase reporter plasmid in Chinese hamster ovary (CHO) cells. Reporter cells showed a dose-dependent expression of luciferase in response to incubation with forskolin. These CHO cells were screened for endogenous G protein-coupled receptors capable of stimulating or inhibiting adenylyl cyclase, by monitoring changes in luciferase expression. Serotonin (5-HT) receptor agonist ligands caused an inhibition of forskolin-stimulated luciferase expression in the rank order 5-carboxamidotryptamine > 5-HT > sumatriptan > 8-hydroxy-2-(di-n-propylamino)tetralin. The response to 5-HT was reversed by the 5-HT1 receptor antagonists cyanopindolol and pindolol, but not the 5-HT2 receptor antagonist ketanserin. Calcitonin was more potent than calcitonin gene-related peptide (CGRP) at stimulating luciferase expression in this cell line, and these responses were insensitive to the CGRP receptor antagonist, CGRP (8-37). These results were consistent with the presence of 5-HT(1B-like) and calcitonin (C1a-like) receptors in CHO cells, with the responses to 5-HT and CGRP being pertussis and cholera toxin-sensitive, respectively. This reporter gene assay gave the expected pharmacological profile for these receptors when compared with cyclic AMP accumulation assays, confirming its value as a functional assay for G protein-coupled receptors linked to adenylyl cyclase.
George
S E
SE
Department of Biosciences, The University of Kent at Canterbury, England.
Bungay
P J
PJ
Naylor
L H
LH
D000818:Animals; D016466:CHO Cells; D002116:Calcitonin / Q000378:metabolism / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D004298:Dopamine / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter; D007700:Kinetics; D008156:Luciferases / Q000096:biosynthesis; D010869:Pindolol / Q000494:pharmacology; D044302:Receptor, Serotonin, 5-HT1B; D018003:Receptors, Calcitonin / Q000819:agonists / Q000096:biosynthesis / Q000502:physiology*; D011985:Receptors, Serotonin / Q000096:biosynthesis / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012701:Serotonin / Q000494:pharmacology*; D017366:Serotonin Receptor Agonists / Q000494:pharmacology*; D013004:Somatostatin / Q000494:pharmacology; D014162:Transfection
D044302:Receptor, Serotonin, 5-HT1B; D018003:Receptors, Calcitonin; D011985:Receptors, Serotonin; D011993:Recombinant Fusion Proteins; D017366:Serotonin Receptor Agonists; D005576:Colforsin; D012701:Serotonin; D013004:Somatostatin; D002116:Calcitonin; D010869:Pindolol; D000242:Cyclic AMP; D008156:Luciferases; D004298:Dopamine