RT Journal Article
SR Electronic
T1 Developing Chemical Genetic Approaches to Explore G Protein-Coupled Receptor Function: Validation of the Use of a Receptor Activated Solely by Synthetic Ligand (RASSL)
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 1033
OP 1046
DO 10.1124/mol.111.074674
VO 80
IS 6
A1 Elisa Alvarez-Curto
A1 Rudi Prihandoko
A1 Christofer S. Tautermann
A1 Jurriaan M. Zwier
A1 John D. Pediani
A1 Martin J. Lohse
A1 Carsten Hoffmann
A1 Andrew B. Tobin
A1 Graeme Milligan
YR 2011
UL http://molpharm.aspetjournals.org/content/80/6/1033.abstract
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.