Allosteric interactions across native adenosine-A3 receptor homodimers: quantification using single-cell ligand-binding kinetics

FASEB J. 2011 Oct;25(10):3465-76. doi: 10.1096/fj.11-186296. Epub 2011 Jun 29.


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 A(1) 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine / pharmacology
  • Adenosine-5'-(N-ethylcarboxamide) / pharmacology
  • Allosteric Regulation / drug effects*
  • Animals
  • CHO Cells
  • Cricetinae
  • Cricetulus
  • Gene Expression Regulation
  • Humans
  • Kinetics
  • Protein Binding
  • Receptor, Adenosine A3 / chemistry
  • Receptor, Adenosine A3 / metabolism*
  • Xanthines / pharmacology


  • Receptor, Adenosine A3
  • Xanthines
  • Adenosine-5'-(N-ethylcarboxamide)
  • 8-(4-((2-aminoethyl)aminocarbonylmethyloxy)phenyl)-1,3-dipropylxanthine
  • Adenosine