Premium
Allosteric interactions across native adenosine‐A 3 receptor homodimers: quantification using single‐cell ligand‐binding kinetics
Author(s) -
May Lauren T.,
Bridge Lloyd J.,
Stoddart Leigh A.,
Briddon Stephen J.,
Hill Stephen J.
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.11-186296
Subject(s) - allosteric regulation , chemistry , g protein coupled receptor , receptor , adenosine , agonist , adenosine receptor , dimer , biophysics , cooperativity , xanthine , adenosine a1 receptor , kinetics , stereochemistry , biochemistry , biology , enzyme , physics , organic chemistry , quantum mechanics
A growing awareness indicates that many G‐protein‐coupled receptors (GPCRs) exist as homodimers, but the extent of the cooperativity across the dimer interface has been largely unexplored. Here, measurement of the dissociation kinetics of a fluorescent agonist (ABA‐X‐BY630) from the human A 1 or A 3 adenosine receptors expressed in CHO‐K1 cells has provided evidence for highly cooperative interactions between protomers of the A 3 ‐receptor dimer in single living cells. In the absence of competitive ligands, the dissociation rate constants of ABA‐X‐BY630 from A1 and A 3 receptors were 1.45 ± 0.05 and 0.57 ± 0.07 min −1 , respectively. At the A 3 receptor, this could be markedly increased by both orthosteric agonists and antagonists [15‐, 9‐, and 19‐fold for xanthine amine congener (XAC), 5'‐( N ‐ethyl carboxamido)adenosine (NECA), and adenosine, respectively] and reduced by coexpression of a nonbinding (N250A) A 3 ‐receptor mutant. The changes in ABA‐X‐BY630 dissociation were much lower at the A 1 receptor (1.5‐, 1.4‐, and 1.5‐fold). Analysis of the pEC 50 values of XAC, NECA, and adenosine for the ABA‐X‐BY630‐occupied A 3 ‐receptor dimer yielded values of 6.0 ± 0.1, 5.9 ± 0.1, and 5.2 ± 0.1, respectively. This study provides new insight into the spatial and temporal specificity of drug action that can be provided by allosteric modulation across a GPCR homodimeric interface.—May, L. T., Bridge, L. J., Stoddart, L. A., Briddon, S. J., Hill, S. J. Allosteric interactions across native adenosine‐A 3 receptor homodimers: quantification using single‐cell ligand‐binding kinetics. FASEB J. 25, 3465–3476 (2011). www.fasebj.org