Kinetic analysis of antagonist-occupied adenosine-A3 receptors within membrane microdomains of individual cells provides evidence of receptor dimerization and allosterism.
Bottom Line: 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
Affiliation: Institute of Cell Signalling, School of Life Sciences, Medical School, and.Show MeSH
Mentions: To determine the relative occupancy levels of CA200645 and the previously described A3AR agonist, ABEA-X-BY630, we performed autocorrelation analysis to determine the number of receptor–ligand complexes (τD3 component) for both compounds relative to the total number of A3AR-GFP receptors in transfected CHO-K1 cells (103±14/μm2; n=31). Our analysis revealed that the receptor occupancy levels for the two compounds at the concentration used in the two studies were not significantly different, at 24.5 ± 2.7/μm2 (23.7%) and 21.8 ± 1.6/μm2 (21.0%) for ABEA-X-BY630 and CA200645, respectively (n=31–34; Fig. 7A); however, the diffusion coefficients of ABEA-X-BY630-A3AR complexes (0.24±0.02 μm2/s) were significantly faster than those observed for the CA200645-A3AR complexes in CHO-A3AR-GFP cells (0.16±0.04 μm2/s) (Fig. 7B). The influence of agonists, antagonists, and VUF 5455 on diffusional characteristics of GFP-tagged A3ARs was also investigated. However, the diffusion coefficient of A3AR-GFP was not significantly affected by treatment with NECA, MRS 1220, or VUF 5455 (Fig. 8B). It was notable, however, that there was a small but significant (P<0.05) increase in the number of particles with MRS 1220 and a significant (P<0.01) decrease in the number with VUF 5455 (Fig. 8A).
Affiliation: Institute of Cell Signalling, School of Life Sciences, Medical School, and.