Abstract
Molecular evolution and chemical genetics have been applied to generate functional pairings of mutated G protein-coupled receptors (GPCRs) and nonendogenous ligands. These mutant receptors, referred to as receptors activated solely by synthetic ligands (RASSLs) or designer receptors exclusively activated by designer drugs (DREADDs), have huge potential to define physiological roles of GPCRs and to validate receptors in animal models as therapeutic targets to treat human disease. However, appreciation of ligand bias and functional selectivity of different ligands at the same receptor suggests that RASSLs may signal differently than wild-type receptors activated by endogenous agonists. We assessed this by generating forms of wild-type human M3 muscarinic receptor and a RASSL variant that responds selectively to clozapine N-oxide. Although the RASSL receptor had reduced affinity for muscarinic antagonists, including atropine, stimulation with clozapine N-oxide produced effects very similar to those generated by acetylcholine at the wild-type M3-receptor. Such effects included the relative movement of the third intracellular loop and C-terminal tail of intramolecular fluorescence resonance energy transfer sensors and the ability of the wild type and evolved mutant to regulate extracellular signal-regulated kinase 1/2 phosphorylation. Each form interacted similarly with β-arrestin 2 and was internalized from the cell surface in response to the appropriate ligand. Furthermore, the pattern of phosphorylation of specific serine residues within the evolved receptor in response to clozapine N-oxide was very similar to that produced by acetylcholine at the wild type. Such results provide confidence that, at least for the M3 muscarinic receptor, results obtained after transgenic expression of this RASSL are likely to mirror the actions of acetylcholine at the wild type receptor.
Footnotes
These studies were supported by the Biotechnology and Biosciences Research Council [Grant BB/E006302/1]; the Medical Research Council [Grant G0900050]; the Wellcome Trust [Grant 047600]; and a Biotechnology and Biosciences Research Council-Heptares Inc. studentship [Grant RM36G0146].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.111.074674.
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ABBREVIATIONS:
- GPCR
- G protein-coupled receptor
- RASSL
- receptor activated solely by synthetic ligand
- DREADD
- designer receptor exclusively activated by designer drug
- hM3
- human muscarinic M3
- VSV
- vesicular stomatitis virus
- hM3-R
- human muscarinic M3 receptor
- FlAsH
- fluorescein arsenical hairpin
- IL3
- third intracellular loop
- FRET
- fluorescence resonance energy transfer
- DMEM
- Dulbecco's modified Eagle's medium
- PAGE
- polyacrylamide gel electrophoresis
- MOPS
- 3-(N-morpholino)propanesulfonic acid
- ERK
- extracellular signal-regulated kinase
- FBS
- fetal bovine serum
- BRET
- bioluminescence resonance energy transfer
- HEK
- human embryonic kidney
- Rluc
- Renilla reniformis luciferase 8
- HBSS
- Hanks' balanced saline solution
- EDT
- ethanedithiol
- DMSO
- dimethyl sulfoxide
- eCFP
- enhanced cyan fluorescent protein
- QNB
- quinuclidinyl benzilate.
- Received July 8, 2011.
- Accepted August 30, 2011.
- Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
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