Agonist-occupied A3 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 A3 adenosine receptor (A3-AR) agonist, ABEA-X-BY630, and the technique of fluorescence correlation spectroscopy (FCS) to investigate the diffusional characteristics of functional agonist-occupied A3-AR complexes in single living cells. In Chinese hamster ovary cells expressing the human A3-AR, the fluorescent A3-AR agonist was able to inhibit forskolin-stimulated [3H]cAMP production (pEC50=8.57), and this was antagonized by the A3-selective antagonist MRS1220 (pK(B)=9.32). The fluorescent ligand also stimulated phosphoinositide hydrolysis (pEC50=7.34). Ligand binding to the A3-AR on the membranes of single cells and subsequent increases in single cell [Ca2+]i were monitored simultaneously in real time using confocal microscopy. FCS measurements in small-membrane microdomains (approximately 0.2 microm2) revealed two agonist-occupied A3-AR components with differing diffusion characteristics (diffusion coefficients=2.65x10(-8) and 1.19x10(-9) cm2/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/microm2, respectively, by MRS1220 (100 nM). These data provide direct evidence for at least two populations of agonist-occupied A3-receptor complexes, showing different motilities within the membrane of single living cells.
Cordeaux
Y
Y
Institute of Cell Signalling, School of Biomedical Sciences, Medical School, University of Nottingham, Nottingham, NG7 2UH, UK.
Briddon
S J
SJ
Alexander
S P H
SP
Kellam
B
B
Hill
S J
SJ
D000241:Adenosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D058909:Adenosine A3 Receptor Agonists; D000818:Animals; D001667:Binding, Competitive; D001896:Boron Compounds / Q000737:chemistry* / Q000494:pharmacology*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000494:pharmacology*; D006801:Humans; D008024:Ligands; D046911:Macromolecular Substances / Q000737:chemistry / Q000378:metabolism; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods; D015394:Molecular Structure; D043684:Receptor, Adenosine A3 / Q000032:analysis / Q000378:metabolism*
C527805:ABEA-X-BY630; D058909:Adenosine A3 Receptor Agonists; D001896:Boron Compounds; D005456:Fluorescent Dyes; D008024:Ligands; D046911:Macromolecular Substances; D043684:Receptor, Adenosine A3; D000241:Adenosine