Skip to main content

Research Repository

Advanced Search

Kinetic analysis of antagonist-occupied adenosine-A3 receptors within membrane microdomains of individual cells provides evidence of receptor dimerization and allosterism

Corriden, Ross; Kilpatrick, Laura E.; Kellam, Barrie; Briddon, Stephen J.; Hill, Stephen J.

Kinetic analysis of antagonist-occupied adenosine-A3 receptors within membrane microdomains of individual cells provides evidence of receptor dimerization and allosterism Thumbnail


Authors

Ross Corriden

Profile Image

BARRIE KELLAM BARRIE.KELLAM@NOTTINGHAM.AC.UK
Professor of Medicinal Chemistry

STEPHEN HILL STEVE.HILL@NOTTINGHAM.AC.UK
Professor of Molecular Pharmacology



Abstract

© The Author(s). In our previous work, using a fluorescent adenosine-A3 receptor (A3AR) agonist and fluorescence correlation spectroscopy (FCS), we demonstrated highaffinity 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 μm2/s, respectively. FCS analysis of a green fluorescent protein (GFP)-tagged A3AR exhibited a single diffusing species (0.105 μm2/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.

Citation

Corriden, R., Kilpatrick, L. E., Kellam, B., Briddon, S. J., & Hill, S. J. (2014). Kinetic analysis of antagonist-occupied adenosine-A3 receptors within membrane microdomains of individual cells provides evidence of receptor dimerization and allosterism. FASEB Journal, 28(10), 4211-4222. https://doi.org/10.1096/fj.13-247270

Journal Article Type Article
Acceptance Date Jun 9, 2014
Online Publication Date Jun 26, 2014
Publication Date Oct 1, 2014
Deposit Date Jul 18, 2016
Publicly Available Date Mar 29, 2024
Journal FASEB Journal
Print ISSN 0892-6638
Electronic ISSN 1530-6860
Publisher Federation of American Society of Experimental Biology
Peer Reviewed Peer Reviewed
Volume 28
Issue 10
Pages 4211-4222
DOI https://doi.org/10.1096/fj.13-247270
Keywords GPCR; dissociation; fluorescence correlation spectroscopy; fluorescent ligand
Public URL https://nottingham-repository.worktribe.com/output/994216
Publisher URL http://www.fasebj.org/content/28/10/4211

Files





You might also like



Downloadable Citations