Allosteric interactions across native adenosine-A 3 receptor homodimers: Quantification using single-cell ligand-binding kinetics

Lauren Therese May, Lloyd J Bridge, Leigh A Stoddart, Stephen J Briddon, Stephen J Hill

Research output: Contribution to journalArticleResearchpeer-review

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 +, respectively. At the A3 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.
Original languageEnglish
Pages (from-to)3465 - 3476
Number of pages12
JournalFASEB Journal
Volume25
Issue number10
DOIs
Publication statusPublished - 2011
Externally publishedYes

Cite this

May, Lauren Therese ; Bridge, Lloyd J ; Stoddart, Leigh A ; Briddon, Stephen J ; Hill, Stephen J. / Allosteric interactions across native adenosine-A 3 receptor homodimers: Quantification using single-cell ligand-binding kinetics. In: FASEB Journal. 2011 ; Vol. 25, No. 10. pp. 3465 - 3476.
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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 +, respectively. At the A3 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.",
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Allosteric interactions across native adenosine-A 3 receptor homodimers: Quantification using single-cell ligand-binding kinetics. / May, Lauren Therese; Bridge, Lloyd J; Stoddart, Leigh A; Briddon, Stephen J; Hill, Stephen J.

In: FASEB Journal, Vol. 25, No. 10, 2011, p. 3465 - 3476.

Research output: Contribution to journalArticleResearchpeer-review

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T1 - Allosteric interactions across native adenosine-A 3 receptor homodimers: Quantification using single-cell ligand-binding kinetics

AU - May, Lauren Therese

AU - Bridge, Lloyd J

AU - Stoddart, Leigh A

AU - Briddon, Stephen J

AU - Hill, Stephen J

PY - 2011

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N2 - 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 +, respectively. At the A3 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.

AB - 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 +, respectively. At the A3 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.

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DO - 10.1096/fj.11-186296

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JO - FASEB Journal

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SN - 0892-6638

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ER -