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

In our previous work, using a fluorescent adenosine-A(3) receptor (A(3)AR) 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 A(3)AR 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 A(3)ARs revealed 2 species, tau(D2) and tau(D3), that diffused at 2.29 +/- 0.35 and 0.09 +/- 0.03 mu m(2)/s, respectively. FCS analysis of a green fluorescent protein (GFP)-tagged A3AR exhibited a single diffusing species (0.105 mu m(2)/s). The binding of CA200645 to tau(D3) was antagonized by nano-molar concentrations of the A(3) antagonist MRS 1220, but not by the agonist NECA (up to 300 nM), consistent with labeling of R. CA200645 normally dissociated slowly from the A(3)AR, but inclusion of xanthine amine congener (XAC) or VUF 5455 during washout markedly accelerated the reduction in the number of particles exhibiting tau(D3) characteristics. It is notable that this effect was accompanied by a significant increase in the number of particles with tau(D2) diffusion. These data show that FCS analysis of ligand-occupied receptors provides a unique means of monitoring ligand A(3)AR residence times that are significantly reduced as a consequence of allosteric interaction across the dimer interface.