PT  - JOURNAL ARTICLE
AU  - Elisa Alvarez-Curto
AU  - Rudi Prihandoko
AU  - Christofer S. Tautermann
AU  - Jurriaan M. Zwier
AU  - John D. Pediani
AU  - Martin J. Lohse
AU  - Carsten Hoffmann
AU  - Andrew B. Tobin
AU  - Graeme Milligan
TI  - Developing Chemical Genetic Approaches to Explore G Protein-Coupled Receptor Function: Validation of the Use of a Receptor Activated Solely by Synthetic Ligand (RASSL)
AID  - 10.1124/mol.111.074674
DP  - 2011 Dec 01
TA  - Molecular Pharmacology
PG  - 1033--1046
VI  - 80
IP  - 6
4099  - http://molpharm.aspetjournals.org/content/80/6/1033.short
4100  - http://molpharm.aspetjournals.org/content/80/6/1033.full
SO  - Mol Pharmacol2011 Dec 01; 80
AB  - 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.