| Autor: |
Cordeaux, Y., Briddon, S. J., Alexander, S. P. H., Kellam, B., Hill, S. J. |
| Předmět: |
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| Zdroj: |
FASEB Journal; Mar2008, Vol. 22 Issue 3, p850-860, 11p, 4 Diagrams, 5 Graphs |
| Abstrakt: |
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 (pKB=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 (~0.2 µm²) revealed two agonist-occupied A3-AR components with differing diffusion characteristics (diffusion coefficients= 2.65 x 10-8 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/µm², 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. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
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