Agonist‐occupied A 3 adenosine receptors exist within heterogeneous complexes in membrane microdomains of individual living cells

G protein‐coupled receptors are known to be organized within different membrane compartments or microdomains of individual cells. Here, we have used a fluorescent A 3 adenosine receptor (A 3 ‐AR) agonist, ABEA‐X‐BY630, and the technique of fluorescence correlation spectroscopy (FCS) to investigate the diffusional characteristics of functional agonist‐occupied A 3 ‐AR complexes in single living cells. In Chinese hamster ovary cells expressing the human A 3 ‐AR, the fluorescent A 3 ‐AR agonist was able to inhibit forskolin‐stimulated [ 3 H]cAMP production (pEC 50 =8.57), and this was antagonized by the A 3 ‐selective antagonist MRS1220 (pK B =9.32). The fluorescent ligand also stimulated phosphoinositide hydrolysis (pEC 50 = 7.34). Ligand binding to the A 3 ‐AR on the membranes of single cells and subsequent increases in single cell [Ca 2+ ] i were monitored simultaneously in real time using confocal microscopy. FCS measurements in small‐membrane microdomains (~0.2 μm 2 ) revealed two agonist‐occupied A 3 ‐AR components with differing diffusion characteristics (diffusion coefficients=2.65×10 −8 and 1.19×10 −9 cm 2 /s, respectively). The binding of ligand to these two components was reduced from 5.1 and 14.9 to 2.6 and 3.3 receptors/μm 2 , respectively, by MRS1220 (100 nM). These data provide direct evidence for at least two populations of agonist‐occupied A 3 ‐receptor complexes, showing different motilities within the membrane of single living cells.—Cordeaux, Y., Briddon, S. J., Alexander, S. P. H., Kellam, B., Hill, S. J. Agonist‐occupied A3 adenosine receptors exist within heterogeneous complexes in membrane microdomains of individual living cells. FASEB J . 22, 850–860 (2008)