Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Sep;78(3):511-23.
doi: 10.1124/mol.110.064493. Epub 2010 Jun 22.

The effect of allosteric modulators on the kinetics of agonist-G protein-coupled receptor interactions in single living cells

Lauren T May  1 , Tim J Self, Stephen J Briddon, Stephen J Hill
Affiliations

The effect of allosteric modulators on the kinetics of agonist-G protein-coupled receptor interactions in single living cells

Lauren T May et al. Mol Pharmacol. 2010 Sep.

Abstract

Allosteric binding sites on adenosine -A(1) and -A(3) receptors represent attractive therapeutic targets for amplifying, in a spatially and temporally selective manner, the tissue protective actions of endogenous adenosine. This study has directly quantified the kinetics of agonist/G protein-coupled receptor interactions at the single-cell level, reflecting the physiological situation in which intracellular signaling proteins can exert major allosteric effects on agonist-receptor interactions. The association and dissociation rate constants at both A(1) and A(3) receptors, and therefore the affinity of the fluorescent adenosine derivative ABA-X-BY630 (structure appears in J Med Chem 50:782-793, 2007), were concentration-independent. The equilibrium dissociation constants of ABA-X-BY630 at A(1) and A(3) receptors were approximately 50 and 10 nM, respectively, suggesting that, even in live cells, low agonist concentrations predominantly detect high-affinity receptor states. At A(1) receptors, the dissociation of ABA-X-BY630 (30 nM) was significantly faster in the absence (k(off) = 1.95 +/- 0.09 min(-1)) compared with the presence of the allosteric enhancer (2-amino-4,5-dimethyl-3-thienyl)(3-(trifluoromethyl)phenyl)-methanone (PD81,723; 10 microM; k(off) = 0.80 +/- 0.03 min(-1)) and allosteric inhibitor 4-methoxy-N-(7-methyl-3-(2-pyridinyl)-1-isoquinolinyl)benzamide (VUF5455; 1 microM; k(off) = 1.48 +/- 0.16 min(-1)). In contrast, ABA-X-BY630 dissociation from A(3) receptors was significantly slower in the absence (k(off) = 0.78 +/- 0.18 min(-1)) than in the presence of the allosteric inhibitors VUF5455 (1 microM; k(off) = 3.15 +/- 0.12 min(-1)) and PD81,723 (10 microM; k(off) = 2.46 +/- 0.18 min(-1)). An allosteric mechanism of action has previously not been identified for PD81,723 at the A(3) receptor or VUF5455 at the A(1) receptor. Furthermore, the marked enhancement in fluorescent agonist dissociation by VUF5455 in living cells contrasts previous observations from broken cell preparations and emphasizes the need to study the allosteric regulation of agonist binding in living cells.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
The nature of fluid transfer upon reservoir exchange within the perfusion system. a, a schematic of the perfusion system setup. b, the addition of 50 μM Brilliant Black BN within the viewing cell caused a rapid decrease in the phase image intensity, which was readily reversed upon perfusion of HBSS only. c, the influence of different flow rates [3–5 (●), 6–10 (○), 11–15 (closed gray circle), and 16–20 ml/min (open gray circle)] on the association (0–2 min) and dissociation (2–5 min) kinetics of 100 nM ABA-X-BY630 at CHO-A3 cells. Data represent the mean ± the S.E.M. from three to five separate experiments in which each replicate reflects the fluorescence intensity from the plasma membrane of 10 cells.
Fig. 2.
Fig. 2.
Characterization of the binding and functional properties of ABA-X-BY630 at the human adenosine A1 receptor. a, ABA-X-BY630 mediated a robust, concentration-dependent increase in intracellular calcium mobilization (○) and ERK1/2 phosphorylation (●) in CHO-A1 cells. Data are expressed as a percentage of the response mediated by 1 μM NECA and represent the mean ± S.E.M. from four experiments. b, a confocal image showing discrete membrane binding of 30 nM ABA-X-BY630 at CHO-A1 cells. Association and dissociation kinetics of 10 (closed gray circle), 30 (○), and 100 (●) nM ABA-X-BY630 at CHO-A1 (c) and CHO-NT (d) cells. Normalized association (e) and dissociation (f) kinetics of 10 (closed gray circle), 30 (○), and 100 (●) nM ABA-X-BY630 at CHO-A1 cells. For the purposes of direct comparison, confocal configurations remained constant for both total and nonspecific binding. Confocal fluorescence and phase images were obtained at 2-s intervals for the duration of the experiment. Data represent the mean ± S.E.M. from three to six separate experiments in which each replicate reflects the fluorescence intensity from the plasma membrane of 10 cells.
Fig. 3.
Fig. 3.
Characterization of the binding and functional properties of ABA-X-BY630 at the human adenosine A3 receptor. a, ABA-X-BY630 mediated a robust, concentration-dependent increase in intracellular calcium mobilization (○) and ERK1/2 phosphorylation (●) in CHO-A3 cells. Data are expressed as a percentage of the response mediated by 1 μM NECA and represent the mean ± S.E.M. from four experiments. b, a confocal image showing discrete membrane binding of 30 nM ABA-X-BY630 at CHO-A3 cells. Association and dissociation kinetics of 3 (open gray circle), 10 (closed gray circle), 30 (○), and 100 (●) nM ABA-X-BY630 at CHO-A3 (c) and CHO-NT (d) cells. Normalized association (e) and dissociation (f) kinetics of 3 (open gray circle), 10 (closed gray circle), 30 (○), and 100 (●) nM ABA-X-BY630 at CHO-A3 cells. For the purpose of direct comparison, confocal configurations remained constant for both total and nonspecific binding. Confocal fluorescence and phase images were obtained at 2-s intervals for the duration of the experiment. Data represent the mean ± S.E.M. from four to eight separate experiments in which each replicate reflects the fluorescence intensity from the plasma membrane of 10 cells.
Fig. 4.
Fig. 4.
Single-cell analysis of the kinetic and equilibrium binding parameters of ABA-X-BY630 at the human adenosine A3 receptor. The dominant influence of ABA-X-BY630 concentration was on the observed association rate (a) as opposed to the dissociation rate constant (b), association rate constant (c), or equilibrium dissociation constant (d). The mean ± S.E.M. is shown for 60 to 80 replicates, which are plotted as individual data points and represent the parameter estimates from single cells from six to eight experiments.
Fig. 5.
Fig. 5.
Allosteric modulation of agonist dissociation in live cells. a, a schematic illustrating the topographically distinct nature of allosteric and orthosteric receptor sites. b, the dissociation of 100 nM ABA-X-BY630 in the absence (●; n = 15) and presence of 100 nM DPCPX (closed gray circle; n = 2; data represent the mean only) from CHO-A1 cells. c, the dissociation of 30 nM ABA-X-BY630 in the absence (●; n = 14) and presence of 10 μM PD81,723 (closed gray circle; n = 6) from CHO-A1 cells. d, single-cell analysis of the influence of 10 μM PD81,723 on the dissociation rate of 30 nM ABA-X-BY630 from CHO-A1 cells. The mean ± S.E.M. is shown for 120 replicates in the absence of 10 μM PD81,723 and 60 replicates in the presence of 10 μM PD81,723 that are plotted as individual data points and represent the rate of ABA-X-BY630 dissociation from single cells. e, the dissociation of 30 nM ABA-X-BY630 in the absence (●; n = 4) and presence of 1 μM VUF5455 (closed gray circle; n = 4) from CHO-A3 cells. Unless otherwise stated, data represent the mean ± S.E.M. and each replicate reflects the fluorescence intensity from the plasma membrane of 10 cells. f, single-cell analysis of the influence of 1 μM VUF5455 on the dissociation rate of 30 nM ABA-X-BY630 from CHO-A3 cells. The mean ± S.E.M. is shown for 30 replicates in the absence of 1 μM VUF5455 and 40 replicates in the presence of 1 μM VUF5455 that are plotted as individual data points and represent the rate of ABA-X-BY630 dissociation from single cells.
Fig. 6.
Fig. 6.
The influence of PD81,723 and VUF5455 on the binding and function of 30 nM ABA-X-BY630 at the human adenosine A1 receptor. a, the association and dissociation kinetics of 30 nM ABA-X-BY630 in the absence (●; n = 12) and presence of 1 (○; n = 10) and 10 μM (closed gray circle; n = 6) PD81,723 at CHO-A1 cells. b, the intracellular calcium mobilization mediated by 30 nM ABA-X-BY630 in the absence (●; n = 12) and presence of 1 (○; n = 10) and 10 μM (closed gray circle; n = 6) PD81,723 at CHO-A1 cells. c, normalized association kinetics of 30 nM ABA-X-BY630 in the absence (●; n = 12) and presence of 1 (○; n = 10) and 10 μM (closed gray circle; n = 6) PD81,723 at CHO-A1 cells. d, normalized dissociation kinetics of 30 nM ABA-X-BY630 in the absence (●; n = 12) and presence of 1 (○; n = 10) and 10 μM (closed gray circle; n = 6) PD81,723 at CHO-A1 cells. e, the association and dissociation kinetics of 30 nM ABA-X-BY630 in the absence (●; n = 12) and presence of 0.1 (○; n = 3) and 1 μM (closed gray circle; n = 5) VUF5455 at CHO-A1 cells. f, the intracellular calcium mobilization mediated by 30 nM ABA-X-BY630 in the absence (●; n = 12) and presence of 0.1 (○; n = 3) and 1 μM (closed gray circle; n = 5) VUF5455 at CHO-A1 cells. g, normalized association kinetics of 30 nM ABA-X-BY630 in the absence (●; n = 12) and presence of 0.1 (○; n = 3) and 1 μM (closed gray circle; n = 5) VUF5455 at CHO-A1 cells. h, normalized dissociation kinetics of 30 nM ABA-X-BY630 in the absence (●; n = 12) and presence of 0.1 (○; n = 3) and 1 μM (closed gray circle; n = 5) VUF5455 at CHO-A1 cells.
Fig. 7.
Fig. 7.
The influence of PD81,723 and VUF5455 on the binding and function of 30 nM ABA-X-BY630 at the human adenosine A3 receptor. a, the association and dissociation kinetics of 30 nM ABA-X-BY630 in the absence (●; n = 14) and presence of 1 (○; n = 9) and 10 μM (closed gray circle; n = 6) PD81,723 at CHO-A3 cells. b, the intracellular calcium mobilization mediated by 30 nM ABA-X-BY630 in the absence (●; n = 14) and presence of 1 (○; n = 9) and 10 μM (closed gray circle; n = 6) PD81,723 at CHO-A3 cells. c, normalized association kinetics of 30 nM ABA-X-BY630 in the absence (●; n = 14) and presence of 1 (○; n = 9) and 10 μM (closed gray circle; n = 6) PD81,723 at CHO-A3 cells. d, normalized dissociation kinetics of 30 nM ABA-X-BY630 in the absence (●; n = 14) and presence of 1 (○; n = 9) and 10 μM (closed gray circle; n = 6) PD81,723 at CHO-A3 cells. e, the association and dissociation kinetics of 30 nM ABA-X-BY630 in the absence (●; n = 14) and presence of 0.1 (○; n = 7) and 1 μM (closed gray circle; n = 6) VUF5455 at CHO-A3 cells. f, the intracellular calcium mobilization mediated by 30 nM ABA-X-BY630 in the absence (●; n = 14) and presence of 0.1 (○; n = 7) and 1 μM (closed gray circle; n = 6) VUF5455 at CHO-A3 cells. g, normalized association kinetics of 30 nM ABA-X-BY630 in the absence (●; n = 14) and presence of 0.1 (○; n = 7) and 1 μM (closed gray circle; n = 6) VUF5455 at CHO-A3 cells. h, normalized dissociation kinetics of 30 nM ABA-X-BY630 in the absence (●; n = 14) and presence of 0.1 (○; n = 7) and 1 μM (closed gray circle; n = 6) VUF5455 at CHO-A3 cells.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Avlani V, May LT, Sexton PM, Christopoulos A. (2004) Application of a kinetic model to the apparently complex behavior of negative and positive allosteric modulators of muscarinic acetylcholine receptors. J Pharmacol Exp Ther 308:1062–1072 - PubMed
    1. Birdsall NJ, Lazareno S, Matsui H. (1996) Allosteric regulation of muscarinic receptors. Prog Brain Res 109:147–151 - PubMed
    1. Bruns RF, Fergus JH. (1990) Allosteric enhancement of adenosine A1 receptor binding and function by 2-amino-3-benzoylthiophenes. Mol Pharmacol 38:939–949 - PubMed
    1. Christopoulos A, Kenakin T. (2002) G protein-coupled receptor allosterism and complexing. Pharmacol Rev 54:323–374 - PubMed
    1. Christopoulos A, Lanzafame A, Ziegler A, Mitchelson F. (1997) Kinetic studies of co-operativity at atrial muscarinic M2 receptors with an “infinite dilution” procedure. Biochem Pharmacol 53:795–800 - PubMed

Publication types