PT - JOURNAL ARTICLE AU - Fu-Yue Zeng AU - Alison J. McLean AU - Graeme Milligan AU - Michael Lerner AU - Derek T. Chalmers AU - Dominic P. Behan TI - Ligand Specific Up-Regulation of a <em>Renilla reniformis</em> Luciferase-Tagged, Structurally Unstable Muscarinic M<sub>3</sub> Chimeric G Protein-Coupled Receptor AID - 10.1124/mol.64.6.1474 DP - 2003 Dec 01 TA - Molecular Pharmacology PG - 1474--1484 VI - 64 IP - 6 4099 - http://molpharm.aspetjournals.org/content/64/6/1474.short 4100 - http://molpharm.aspetjournals.org/content/64/6/1474.full SO - Mol Pharmacol2003 Dec 01; 64 AB - The rat muscarinic acetylcholine receptor subtype 3 was modified by swapping the third intracellular loop with the corresponding region of a constitutively active mutant human β2-adrenergic receptor and attaching Renilla reniformis luciferase to its C terminus. The chimeric fusion receptor displayed constitutive Gq- and Gs-coupled activity as demonstrated in nuclear factor of activated T cell and cAMP response element reporter gene assays. The chimeric receptor displayed a pharmacological binding profile comparable with that of the wild-type receptor for agonists, antagonists, and inverse agonists but showed a large decrease in expression in both human embryonic kidney 293 and COS-7 cells. Long-term treatment of cells expressing the chimeric receptor with agonists, antagonists, and inverse agonists resulted in a concentration-dependent up-regulation in the steady-state levels that was not observed for the wild-type receptor. The EC50 of neutral antagonists and inverse agonists was significantly correlated to their binding affinities at the wild-type receptor, whereas agonists demonstrated greater EC50 values for the chimeric receptor. To validate the approach as a means of discovering novel receptor modulators, a cell-based, high-throughput screening assay was developed and used to screen a small molecule compound collection against the chimeric fusion receptor. Several novel hits were identified and confirmed by ligand binding assay and functional assays using the wild-type rat muscarinic acetylcholine receptor subtype 3.