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

摘要: 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 (R*) conformation. current study, used A3AR antagonist (CA200645) to study binding characteristics antagonist-occupied inactive (R) conformations in membrane microdomains individual cells. FCS analysis CA200645-occupied A3ARs revealed 2 species, τD2 τD3, that diffused at 2.29 ± 0.35 0.09 0.03 μm(2)/s, respectively. green protein (GFP)-tagged exhibited single diffusing species (0.105 μm(2)/s). The CA200645 τD3 was antagonized by nanomolar concentrations A3 MRS 1220, but not NECA (up 300 nM), consistent with R. normally dissociated slowly from A3AR, inclusion xanthine amine congener (XAC) or VUF 5455 during washout markedly accelerated reduction number particles exhibiting characteristics. It is notable this effect accompanied significant increase diffusion. These data show ligand-occupied receptors provides unique means monitoring ligand residence times are significantly reduced as consequence allosteric interaction across dimer interface