title: Kinetic analysis of antagonist-occupied adenosine-A3 receptors within membrane microdomains of individual cells provides evidence of receptor dimerization and allosterism.
abstract: In our previous work, using a fluorescent adenosine-A3 receptor (A3AR) agonist and fluorescence correlation spectroscopy (FCS), we demonstrated high-affinity labeling of the active receptor (R*) conformation. In the current study, we used a fluorescent A3AR antagonist (CA200645) to study the binding characteristics of antagonist-occupied inactive receptor (R) conformations in membrane microdomains of individual cells. FCS analysis of CA200645-occupied A3ARs revealed 2 species, τD2 and τD3, that diffused at 2.29 ± 0.35 and 0.09 ± 0.03 μm(2)/s, respectively. FCS analysis of a green fluorescent protein (GFP)-tagged A3AR exhibited a single diffusing species (0.105 μm(2)/s). The binding of CA200645 to τD3 was antagonized by nanomolar concentrations of the A3 antagonist MRS 1220, but not by the agonist NECA (up to 300 nM), consistent with labeling of R. CA200645 normally dissociated slowly from the A3AR, but inclusion of xanthine amine congener (XAC) or VUF 5455 during washout markedly accelerated the reduction in the number of particles exhibiting τD3 characteristics. It is notable that this effect was accompanied by a significant increase in the number of particles with τD2 diffusion. These data show that FCS analysis of ligand-occupied receptors provides a unique means of monitoring ligand A3AR residence times that are significantly reduced as a consequence of allosteric interaction across the dimer interface
authors: Institute of Cell Signalling, School of Life Sciences, Medical School, and.
authors: Ross
authors: R
authors: Corriden
authors: Institute of Cell Signalling, School of Life Sciences, Medical School, and.
authors: Laura E
authors: LE
authors: Kilpatrick
authors: School of Pharmacy, Centre for Biomolecular Sciences, University of Nottingham, Nottingham, UK.
authors: Barrie
authors: B
authors: Kellam
authors: Institute of Cell Signalling, School of Life Sciences, Medical School, and.
authors: Stephen J
authors: SJ
authors: Briddon
authors: Institute of Cell Signalling, School of Life Sciences, Medical School, and stephen.hill@nottingham.ac.uk.
authors: Stephen J
authors: SJ
authors: Hill
mesh_terms: D058918:Adenosine A3 Receptor Antagonists / Q000494:pharmacology; D000494:Allosteric Regulation; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007700:Kinetics; D021962:Membrane Microdomains / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D043684:Receptor, Adenosine A3 / Q000378:metabolism*
chemical_list: D058918:Adenosine A3 Receptor Antagonists; D043684:Receptor, Adenosine A3
keywords: GPCR; dissociation; fluorescence correlation spectroscopy; fluorescent ligand