Detection of receptor ligands by monitoring selective stabilization of a Renilla luciferase-tagged, constitutively active mutant, G-protein-coupled receptor.
The wild-type beta2-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 beta2-adrenoceptor nor the capacity of isoprenaline to elevate cyclic AMP levels in intact cells expressing these constructs. Treatment of cells expressing constitutively active mutant beta2-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 pEC50 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 beta2-adrenoceptor. Co-expression of the wild-type beta2-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 beta2-adrenoceptor-Renilla luciferase and an equivalent mutant of the alpha1b-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.
Ramsay
D
D
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK.
Bevan
N
N
Rees
S
S
Milligan
G
G
D000262:Adenylyl Cyclases / Q000378:metabolism; D000319:Adrenergic beta-Antagonists / Q000494:pharmacology; D000818:Animals; D015784:Betaxolol / Q000494:pharmacology; D002460:Cell Line; D003063:Cnidaria / Q000235:genetics*; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D008024:Ligands; D008156:Luciferases / Q000235:genetics*; D008164:Luminescent Proteins; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects
D000319:Adrenergic beta-Antagonists; D018076:DNA, Complementary; D008024:Ligands; D008164:Luminescent Proteins; D018343:Receptors, Adrenergic, beta-2; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D008156:Luciferases; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D015784:Betaxolol