title: Detecting ligand interactions with G protein-coupled receptors in real-time on living cells.
abstract: G protein-coupled receptors (GPCRs) are a large group of receptors of great biological and clinical relevance. Despite this, the tools for a detailed analysis of ligand-GPCR interactions are limited. The aim of this paper was to demonstrate how ligand binding to GPCRs can be followed in real-time on living cells. This was conducted using two model systems, the radiolabeled porcine peptide YY (pPYY) interacting with transfected human Y2 receptor (hY2R) and the bombesin antagonist RM26 binding to the naturally expressed gastrin-releasing peptide receptor (GRPR). By following the interaction over time, the affinity and kinetic properties such as association and dissociation rate were obtained. Additionally, data were analyzed using the Interaction Map method, which can evaluate a real-time binding curve and present the number of parallel interactions contributing to the curve. It was found that pPYY binds very slowly with an estimated time to equilibrium of approximately 12h. This may be problematic in standard end-point assays where equilibrium is required. The RM26 binding showed signs of heterogeneity, observed as two parallel interactions with unique kinetic properties. In conclusion, measuring binding in real-time using living cells opens up for a better understanding of ligand interactions with GPCRs.
authors.affiliation: Department of Neuroscience, Unit of Pharmacology, Uppsala University, Husargatan 2, 751 24 Uppsala, Sweden. Electronic address: bo.xu@neuro.uu.se.
authors.forename: Bo
authors.initials: B
authors.lastname: Xu
authors.forename: Zohreh
authors.initials: Z
authors.lastname: Varasteh
authors.forename: Anna
authors.initials: A
authors.lastname: Orlova
authors.forename: Karl
authors.initials: K
authors.lastname: Andersson
authors.forename: Dan
authors.initials: D
authors.lastname: Larhammar
authors.forename: Hanna
authors.initials: H
authors.lastname: Björkelund
mesh_terms: D000818:Animals; D001839:Bombesin / Q000031:analogs & derivatives; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D019894:Peptide YY / Q000737:chemistry / Q000378:metabolism; D011849:Radioactive Tracers; D011869:Radioligand Assay; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D017476:Receptors, Neuropeptide Y / Q000235:genetics / Q000378:metabolism; D013552:Swine; D013997:Time Factors; D014162:Transfection
chemical_list: D008024:Ligands; D011849:Radioactive Tracers; D043562:Receptors, G-Protein-Coupled; D017476:Receptors, Neuropeptide Y; C095889:neuropeptide Y2 receptor; D019894:Peptide YY; D001839:Bombesin
keywords: (111)In; (125)I; G protein-coupled receptor; GPCR; GRPR; Heterogeneity; Interaction Map; Kinetics; LigandTracer; Real-time; gastrin-releasing peptide receptor; hY2R; human Y2 receptor; indium-111; iodine-125; pPYY; porcine peptide YY