Developing chemical genetic approaches to explore G protein-coupled receptor function: validation of the use of a Receptor Activated Solely by Synthetic Ligand (RASSL)

Alvarez-Curto, E. , Prihandoko, R., Tautermann, C. S., Zwier, J. M., Pediani, J. D. , Lohse, M. J., Hoffmann, C., Tobin, A. B. and Milligan, G. (2011) Developing chemical genetic approaches to explore G protein-coupled receptor function: validation of the use of a Receptor Activated Solely by Synthetic Ligand (RASSL). Molecular Pharmacology, 80(6), pp. 1033-1046. (doi:10.1124/mol.111.074674)

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Publisher's URL: http://dx.doi.org/10.1124/mol.111.074674

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 M(3) 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 M(3)-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 <i>N</i>-oxide was very similar to that produced by acetylcholine at the wild type. Such results provide confidence that, at least for the M<sub>3</sub> muscarinic receptor, results obtained after transgenic expression of this RASSL are likely to mirror the actions of acetylcholine at the wild type receptor

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Milligan, Professor Graeme and Pediani, Dr John and Alvarez-Curto, Dr Elisa
Authors: Alvarez-Curto, E., Prihandoko, R., Tautermann, C. S., Zwier, J. M., Pediani, J. D., Lohse, M. J., Hoffmann, C., Tobin, A. B., and Milligan, G.
College/School:College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Molecular Pharmacology
Journal Abbr.:Mol. Pharmacol.
Publisher:American Society for Pharmacology and Experimental Therapeutics
ISSN:0026-895X
ISSN (Online):1521-0111
Published Online:31 August 2011
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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
437051Exploring the selectivity and consequences of GPCR homo and hetero dimerisation/oligomerisation using receptors activated solely by ......Graeme MilliganBiotechnology and Biological Sciences Research Council (BBSRC)BB/E006302/1Institute of Neuroscience and Psychology
510631The organisational structure of class A GPCRs: implications for function and drug designGraeme MilliganMedical Research Council (MRC)G0900050RI NEUROSCIENCE & PSYCHOLOGY