title: A simple method to detect allostery in GPCR dimers.
abstract: G protein-coupled receptors (GPCRs) represent one of the largest families of cell surface receptors as key targets for pharmacological manipulation. G proteins have long been recognized as allosteric modulators of GPCR ligand binding. More recently, small molecule allosteric modulators have now been widely characterized for a number of GPCRs, and some are now used clinically. Many studies have also underscored the importance of GPCR dimerization or higher-order oligomerization in the control of the physiological responses they modulate. Thus, allosterism can also, between monomer equivalents in the context of a dimer, oligomer, or receptor mosaic, influence signaling pathways downstream. It therefore becomes essential to characterize both small molecule allosteric ligands and allosteric interactions between receptors modulated by canonical orthosteric ligands, in a pathway-specific manner. Here, we describe a simple, radioligand-binding method, which is designed to probe for allosteric modulation mediated by any GPCR interactor, from small molecules to interacting proteins. It can also detect allosteric asymmetries within a GPCR heterodimer, via orthosteric or allosteric ligands. This assay measures time-dependent ligand occupancy of radiolabeled orthosteric or (with adaptations) allosteric ligands as modulated by either small molecules or receptor dimer partners bound or unbound with their own ligands.
authors.affiliation: Department of Pharmacology, McGill University, Montréal, Québec, Canada.
authors.forename: Eugénie
authors.initials: E
authors.lastname: Goupil
authors.forename: Stéphane A
authors.initials: SA
authors.lastname: Laporte
authors.forename: Terence E
authors.initials: TE
authors.lastname: Hébert
mesh_terms: D000494:Allosteric Regulation; D015237:Dinoprost / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000737:chemistry* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008024:Ligands; D055503:Protein Multimerization; D011869:Radioligand Assay; D044140:Receptor, Angiotensin, Type 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011982:Receptors, Prostaglandin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D014316:Tritium
chemical_list: D008024:Ligands; D044140:Receptor, Angiotensin, Type 1; D011982:Receptors, Prostaglandin; C034671:prostaglandin F2alpha receptor; D014316:Tritium; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D015237:Dinoprost
keywords: Allosterism; Dimer; GPCR; Ligand dissociation kinetics