Quantification of Allosteric Interactions at G Protein–Coupled Receptors Using Radioligand Binding Assays

Katie Leach1, Patrick M. Sexton1, Arthur Christopoulos1

1 Drug Discovery Biology, Monash Institute of Pharmaceutical Science and Department of Pharmacology, Monash University, Parkville, Victoria, Australia
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 1.22
DOI:  10.1002/0471141755.ph0122s52
Online Posting Date:  March, 2011
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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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Table 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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Materials

Basic Protocol 1: Measurement of Allosteric Modulation of Radioligand Binding: Saturation Experiments

  Materials
  • Appropriate membrane preparation (see other units of Chapter 1)
  • Assay buffer (e.g., HEPES or Tris‐based buffers)
  • Radioligand
  • Test compound (allosteric modulator)
  • Unlabeled (non‐radioactive) competitive (orthosteric) ligand for determination of nonspecific binding
  • Wash buffer (usually the same as the assay buffer)
  • Scintillation cocktail (e.g., Packard Ultima Gold; Wallac HiSafe)
  • 12 × 75–mm glass or polypropylene culture tubes
  • Shaking water bath
  • Glass fiber filters (e.g., Whatman GF/B)
  • Cell harvester (e.g., Brandell, http://www.brandel.com/, or Skatron)
  • Scintillation counter and vials
  • Additional reagents and equipment for determination of protein concentration ( appendix 3A)

Alternate Protocol 1: Determination of the Affinity Ratio

  Materials
  • Appropriate membrane preparation (see other units of Chapter 1)
  • Assay buffer (e.g., HEPES or Tris‐based buffers)
  • Radioligand
  • Test compound (allosteric modulator)
  • Unlabeled (non‐radioactive) competitive (orthosteric) ligand for determination of nonspecific binding
  • Wash buffer (usually the same as the assay buffer), ice cold
  • Scintillation cocktail (e.g., Packard Ultima Gold; Wallac HiSafe)
  • 12 × 75–mm glass or polypropylene culture tubes
  • Shaking water bath
  • Glass fiber filters (e.g., Whatman GF/B)
  • Cell harvester (e.g., Brandell, http://www.brandel.com/, or Skatron)
  • Scintillation counter and vials
  • Additional reagents and equipment for determination of protein concentration ( appendix 3A)

Basic Protocol 2: Measurement of Allosteric Modulation of Radioligand Binding: Inhibition (or Potentiation) Experiments

  Materials
  • Appropriate membrane preparation (see other units of Chapter 1)
  • Assay buffer (e.g., HEPES or Tris‐based buffers)
  • Radioligand
  • Test compound (allosteric modulator)
  • Unlabeled (non‐radioactive) competitive (orthosteric) ligand for determination of nonspecific binding
  • Wash buffer (usually the same as the assay buffer), ice cold
  • Scintillation cocktail (e.g., Packard Ultima Gold; Wallac HiSafe)
  • 12 × 75–mm glass or polypropylene culture tubes
  • Shaking water bath
  • Glass fiber filters (e.g., Whatman GF/B)
  • Cell harvester (e.g., Brandell, http://www.brandel.com/, or Skatron)
  • Scintillation counter and vials
  • Additional reagents and equipment for determination of protein concentration ( appendix 3A)

Alternate Protocol 2: Measurement of Allosteric Modulation of Radioligand Binding: Inhibition (or Potentiation) Experiments Under Non‐Equilibrium Conditions

  • Sampling manifold (e.g., Millipore 1225) that allows for individual samples to be filtered at different times
  • Flask for holding large volumes of buffer
  • Magnetic stirrer to maintain adequate mixing of the buffer at the desired temperature
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Figures

Videos

Literature Cited

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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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