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Detection of receptor ligands by monitoring selective stabilization of a Renilla luciferase‐tagged, constitutively active mutant, G‐protein‐coupled receptor
Author(s) -
Ramsay Douglas,
Bevan Nicola,
Rees Stephen,
Milligan Graeme
Publication year - 2001
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1038/sj.bjp.0704077
Subject(s) - luciferase , mutant , receptor , agonist , ligand (biochemistry) , downregulation and upregulation , microbiology and biotechnology , chemistry , biology , g protein coupled receptor , biochemistry , transfection , gene
The wild‐type β 2 ‐adrenoceptor and a constitutively active mutant of this receptor were C‐terminally tagged with luciferase from the sea pansy Renilla reniformis . C‐terminal addition of Renilla luciferase did not substantially alter the levels of expression of either form of the receptor, the elevated constitutive activity of the mutant β 2 ‐adrenoceptor nor the capacity of isoprenaline to elevate cyclic AMP levels in intact cells expressing these constructs. Treatment of cells expressing constitutively active mutant β 2 ‐adrenoceptor‐ Renilla luciferase with antagonist/inverse agonist ligands resulted in upregulation of levels of this polypeptide which could be monitored by the elevated luciferase activity. The pEC 50 for ligand‐induced luciferase upregulation and ligand affinity to bind the receptor were highly correlated. Similar upregulation could be observed following sustained treatment with agonist ligands. These effects were only observed at a constitutively active mutant of the β 2 ‐adrenoceptor. Co‐expression of the wild‐type β 2 ‐adrenoceptor C‐terminally tagged with the luciferase from Photinus pyralis did not result in ligand‐induced upregulation of the levels of activity of this luciferase. Co‐expression of the constitutively active mutant β 2 ‐adrenoceptor‐ Renilla luciferase and an equivalent mutant of the α 1b ‐adrenoceptor C‐terminally tagged with green fluorescent protein allowed pharmacological selectivity of adrenoceptor antagonists to be demonstrated. This approach offers a sensitive and convenient means, which is amenable to high throughput analysis, to monitor ligand binding to a constitutively active mutant receptor. As no prior knowledge of receptor ligands is required this approach may be suitable to identify ligands at orphan G protein‐coupled receptors.British Journal of Pharmacology (2001) 133 , 315–323; doi: 10.1038/sj.bjp.0704077