Quantification of Allosteric Interactions at G Protein–Coupled Receptors Using Radioligand Binding Assays
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- Abstract
- Table of Contents
- Materials
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- Literature Cited
Abstract
Allosteric interactions involve the simultaneous binding of two ligands to the same receptor. An allosteric modulator causes a conformational change in the receptor protein that yields a change in the binding or signaling of an orthosteric agent, i.e., an agonist or competitive antagonist that binds to the endogenous agonist binding site. Because of the complex nature of allosteric phenomena, the detection and quantification of their effects on orthosteric ligand binding relies on the use of both equilibrium and non?equilibrium assays to ensure proper interpretation of the findings. Outlined in this unit are the most common experimental approaches for measuring allosteric effects on orthosteric ligand affinity at G protein?coupled receptors. There is also a discussion of the analysis of experimental data derived from such assays. Curr. Protoc. Pharmacol. 52:1.22.1?1.22.41. © 2011 by John Wiley & Sons, Inc.
Keywords: allosterism; allosteric interaction; cooperativity; radioligand binding; G protein coupled?receptors; dissociation kinetics; non?equilibrium
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PDF or HTML at Wiley Online LibraryTable of Contents
- Introduction
- Basic Protocol 1: Measurement of Allosteric Modulation of Radioligand Binding: Saturation Experiments
- Alternate Protocol 1: Determination of the Affinity Ratio
- Basic Protocol 2: Measurement of Allosteric Modulation of Radioligand Binding: Inhibition (or Potentiation) Experiments
- Alternate Protocol 2: Measurement of Allosteric Modulation of Radioligand Binding: Inhibition (or Potentiation) Experiments Under Non‐Equilibrium Conditions
- Basic Protocol 3: Measurement of Allosteric Modulation of Radioligand Binding to Cloned Receptors in Membranes: Dissociation Kinetic Studies Using Isotopic Dilution
- Alternate Protocol 3: “Two‐Point” Kinetic Experiments
- Alternate Protocol 4: Measurement of Allosteric Modulation of Radioligand Binding: Dissociation Kinetic Studies Using “Infinite Dilution” in Buffer
- Support Protocol 1: Data Analysis
- Commentary
- Literature Cited
- Figures
- Tables
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Basic Protocol 1: Measurement of Allosteric Modulation of Radioligand Binding: Saturation Experiments
Materials
Alternate Protocol 1: Determination of the Affinity Ratio
Materials
Basic Protocol 2: Measurement of Allosteric Modulation of Radioligand Binding: Inhibition (or Potentiation) Experiments
Materials
Alternate Protocol 2: Measurement of Allosteric Modulation of Radioligand Binding: Inhibition (or Potentiation) Experiments Under Non‐Equilibrium Conditions
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Figure 1.22.1 Schematic representation of the non‐equilibrium binding assay methodology. Tubes in “Set A” are exposed to prelabeled receptors, whereas tubes in “Set B” are exposed to the receptor and radioligand at the same time. View Image -
Figure 1.22.2 Analysis of the saturation binding data obtained for the antagonist, [3 H]N‐methylscopolamine ([3 H]NMS), at the cloned human muscarinic M2 receptor in the absence or presence of the indicated concentrations of the allosteric modulator, gallamine. Experimental data are taken from Table . (A ) Constrained, simultaneous nonlinear regression analysis, using the program GraphPad PRISM, according to Equation . (B ) Scatchard (Rosenthal) transformation of the same data. Note the reduction in slope (1/ K App ) with no significant change in B max . (C ) Nonlinear regression analysis of the data using GraphPad PRISM, according to Equation followed by Equation . (D ) Nonlinear regression analysis of the data using GraphPad PRISM, according to Equation followed by Equation . In each instance, the relevant parameter estimates derived from the curve‐fitting procedure are shown. View Image -
Figure 1.22.3 Example of the affinity‐ratio assay for the interaction of the allosteric modulator N‐benzyl brucine at the human M2 and M3 muscarinic acetylcholine receptors expressed in CHO cells. (A ) Effects of N‐benzyl brucine on the specific binding of [3 H]N‐methylscopolamine ([3 H]NMS (0.4 nM at the M2 receptor and 0.2 nM at the M3 receptor) in the absence or presence of unlabeled acetylcholine (ACh; 2 µM at the M2 receptor and 20 µM at the M3 receptor) in the presence of 0.2 mM GTP, and on [3 H]ACh binding to the M2 receptor measured in the absence of GTP. Incubation was at 30°C. (B ) Conversion of the data shown in A to affinity ratios. The fractional effect of N‐benzyl brucine on the binding of [3 H]ACh at M2 receptors is also shown. Note the ability of the affinity ratio plot to display the direction of the cooperativity, as well as a semi‐quantitative estimate of the magnitude of the cooperativity. Taken from Lazareno et al. (). View Image -
Figure 1.22.4 Equations that may be utilized to directly fit the ternary complex model to experimental binding data derived from equilibrium binding studies. View Image -
Figure 1.22.5 Analysis of the potentiation of [3 H]N‐methylscopolamine (0.1 nM) binding (LogKA = –9.52) by the allosteric modulator, alcuronium, at the cloned M2 muscarinic acetylcholine receptor. Incubation was for 5 hr at 37°C. The curve was analyzed using GraphPad PRISM according to Equation . View Image -
Figure 1.22.6 Analysis of the inhibition of binding of a low (0.12 nM, solid circles) and a high (1 nM, open circles) concentration of [3 H]N‐methylscopolamine (LogKA = –9.52) by the allosteric modulator, gallamine, at the cloned M2 muscarinic acetylcholine receptor. Data are taken from Table . Incubation was for 90 min at 37°C. Analysis was performed using a constrained, simultaneous nonlinear regression of both experimental datasets to Equation using the program GraphPad PRISM. Inset: the same data are shown normalized as percentage of respective control radioligand binding. This better illustrates the limited ability of weak negative allosteric modulators to fully inhibit radioligand binding when the concentration of the latter is increased. View Image -
Figure 1.22.7 Nonequilibrium assay of the effects of the allosteric modulator, McN‐A‐343, at the human M2 muscarinic acetylcholine receptor expressed in CHO cells. Receptors were either unlabeled or prelabeled with [3 H]N‐methylscopoloamine, as indicated, before the incubation with McN‐A‐343. The final radioligand concentration after dilution was 0.3 nM, and the incubation time was 1 hr and 20 min. The incubation temperature was 37°C. The lines represent the best fit, via constrained, simultaneous nonlinear regression, to Equation . View Image -
Figure 1.22.8 Effect of the allosteric modulator, heptane‐1,7‐bis‐(dimethyl‐3‐phthalimidopropyl)‐ammonium bromide (C7 /3‐phth), on the dissociation rate of [3 H]N‐methylscopolamine in CHO cell membranes expressing the cloned M2 muscarinic receptor. Membranes were incubated with 0.2 nM [3 H]N‐methylscopolamine at 37°C for 60 min before dissociation was monitored after the addition of 1 µM atropine alone or in combination with C7 /3‐phth at the indicated concentrations. Main figure: normalized curves represent the best fit via constrained, simultaneous nonlinear regression analysis, using GraphPad PRISM, according to Equations and . Inset: nonlinear regression analysis of the same data set using GraphPad PRISM according to Equations and . View Image -
Figure 1.22.9 Ternary complex model of allosteric interaction. R denotes the receptor, A and B denote the orthosteric and allosteric ligands, respectively; K A and K B denote the equilibrium dissociation constants of AR and BR, respectively. The symbol α denotes the cooperativity factor, and is a quantitative measure of the maximal, reciprocal alteration of the equilibrium dissociation constant of A and B for their respective binding sites, when both ligands bind concomitantly to form the ternary complex ARB. View Image -
Figure 1.22.10 Flow chart strategy for dealing with compounds identified as potential allosteric modulators. View Image
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| Key References | |
| Ehlert, F.J. 1988. See above. | |
| These citations cover the detection and quantification of allosteric interactions at GPCRs in great detail. | |
| Lazareno, S. and Birdsall, N.J.M. 1995. See above. |
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