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Ligand Specific Up-Regulation of a Renilla reniformis Luciferase-Tagged, Structurally Unstable Muscarinic M₃ Chimeric G Protein-Coupled Receptor

Arena Pharmaceuticals Inc., San Diego, California (F.-Y.Z., M.L., D.C., D.B.); and Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom (A.M, G.M.)

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 ₂-adrenergic receptor and attaching Renilla reniformis luciferase to its C terminus. The chimeric fusion receptor displayed constitutive G_q- and G_s-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.

Address correspondence to: Dr. Fu-Yue Zeng, Arena Pharmaceuticals Inc. 6166 Nancy Ridge Drive, San Diego, CA 92121. E-mail: fzeng{at}arenapharm.com

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