title: Ligand specific up-regulation of a Renilla reniformis luciferase-tagged, structurally unstable muscarinic M3 chimeric G protein-coupled receptor.
abstract: 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 beta2-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.
authors.affiliation: Arena Pharmaceuticals Inc. 6166 Nancy Ridge Drive, San Diego, CA 92121, USA. fzeng@arenapharm.com
authors.forename: Fu-Yue
authors.initials: FY
authors.lastname: Zeng
authors.forename: Alison J
authors.initials: AJ
authors.lastname: McLean
authors.forename: Graeme
authors.initials: G
authors.lastname: Milligan
authors.forename: Michael
authors.initials: M
authors.lastname: Lerner
authors.forename: Derek T
authors.initials: DT
authors.lastname: Chalmers
authors.forename: Dominic P
authors.initials: DP
authors.lastname: Behan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D018721:Muscarinic Agonists / Q000378:metabolism / Q000494:pharmacology; D018727:Muscarinic Antagonists / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D043587:Receptor, Muscarinic M3 / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000138:chemical synthesis* / Q000235:genetics; D015238:Sulfotransferases / Q000235:genetics*; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology*
chemical_list: D008024:Ligands; D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; D043587:Receptor, Muscarinic M3; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D015238:Sulfotransferases; C022568:Renilla-luciferin sulfotransferase