title: Stimulus bias provides evidence for conformational constraints in the structure of a G protein-coupled receptor.
abstract: A key characteristic of G protein-coupled receptors (GPCRs) is that they activate a plethora of signaling pathways. It is now clear that a GPCR coupling to these pathways can be regulated selectively by ligands that differentially drive signaling down one pathway in preference to another. This concept, termed stimulus bias, is revolutionizing receptor biology and drug discovery by providing a means of selectively targeting receptor signaling pathways that have therapeutic impact. Herein, we utilized a novel quantitative method that determines stimulus bias of synthetic GPCR ligands in a manner that nullifies the impact of both the cellular background and the "natural bias" of the endogenous ligand. By applying this method to the M(2) muscarinic acetylcholine receptor, a prototypical GPCR, we found that mutation of key residues (Tyr-80(2.61) and Trp-99(3.28)) in an allosteric binding pocket introduces stimulus bias in response to the atypical ligands AC-42 (4-n-butyl-1-(4-(2-methylphenyl)-4-oxo-1-butyl)piperidine HCl) and 77-LH-28-1 (1-(3-(4-butyl-1-piperidinyl)propyl)- 3,3-dihydro-2(1H)-quinolinone). By comparing stimulus bias factors among receptor internalization, G protein activation, extracellular-regulated protein kinase 1/2 (ERK1/2) signaling, and receptor phosphorylation, we provide evidence that Tyr-80(2.61) and Trp-99(3.28) act either as molecular switches or as gatekeeper residues that introduce constraints limiting the active conformation of the M(2) muscarinic acetylcholine receptor and thereby regulate stimulus bias. Furthermore, we provide evidence that downstream signaling pathways previously considered to be related to each other (i.e. receptor phosphorylation, internalization, and activation of ERK1/2) can act independently.
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, and Department of Pharmacology, Monash University, Victoria 3800, Australia.
authors.forename: Karen J
authors.initials: KJ
authors.lastname: Gregory
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Sexton
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Tobin
authors.forename: Arthur
authors.initials: A
authors.lastname: Christopoulos
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D000495:Allosteric Site; D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D002217:Carbachol / Q000494:pharmacology; D006224:Cricetinae; D006207:Half-Life; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D018721:Muscarinic Agonists / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010862:Pilocarpine / Q000494:pharmacology; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D021381:Protein Transport / Q000187:drug effects; D043585:Receptor, Muscarinic M2 / Q000819:agonists / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D018721:Muscarinic Agonists; D043585:Receptor, Muscarinic M2; D010862:Pilocarpine; D002217:Carbachol; D000109:Acetylcholine