Detection of receptor ligands by monitoring selective stabilization of a Renilla luciferase-tagged, constitutively active mutant, G-protein-coupled receptor

1 The wild-type beta (2)-adrenoceptor and a constitutively active mutant of this receptor were C-terminally tagged with luciferase from the sea pansy Renilla reniformis. 2 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 beta (2)-adrenoceptor nor the capacity of isoprenaline to elevate cyclic AMP levels in intact cells expressing these constructs. 3 Treatment of cells expressing constitutively active mutant beta (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. 4 The pEC(50) for ligand-induced luciferase upregulation and ligand affinity to bind the receptor were highly correlated. 5 Similar upregulation could be observed following sustained treatment with agonist ligands. 6 These effects were only observed at a constitutively active mutant of the beta (2)-adrenoceptor. Coexpression of the wild-type beta (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. 7 Co-expression of the constitutively active mutant beta (2)-adrenoceptor-Renilla luciferase and an equivalent mutant of the alpha (1b)-adrenoceptor C-terminally tagged with green fluorescent protein allowed pharmacological selectivity of adrenoceptor antagonists to be demonstrated. 8 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. 9 As no prior knowledge of receptor ligands is required this approach may be suitable to identify ligands at orphan G protein-coupled receptors.