title: Allosteric interactions across native adenosine-A3 receptor homodimers: quantification using single-cell ligand-binding kinetics.
abstract: 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.
authors.affiliation: Institute of Cell Signalling, School of Biomedical Sciences, The University of Nottingham, Nottingham, UK, NG7 2UH.
authors.forename: Lauren T
authors.initials: LT
authors.lastname: May
authors.forename: Lloyd J
authors.initials: LJ
authors.lastname: Bridge
authors.forename: Leigh A
authors.initials: LA
authors.lastname: Stoddart
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Briddon
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Hill
mesh_terms: D000241:Adenosine / Q000494:pharmacology; D019830:Adenosine-5'-(N-ethylcarboxamide) / Q000494:pharmacology; D000494:Allosteric Regulation / Q000187:drug effects*; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005786:Gene Expression Regulation; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D043684:Receptor, Adenosine A3 / Q000737:chemistry / Q000378:metabolism*; D014970:Xanthines / Q000494:pharmacology
chemical_list: D043684:Receptor, Adenosine A3; D014970:Xanthines; D019830:Adenosine-5'-(N-ethylcarboxamide); C051869:8-(4-((2-aminoethyl)aminocarbonylmethyloxy)phenyl)-1,3-dipropylxanthine; D000241:Adenosine