Radioligand Binding Analysis as a Tool for Quality Control of GPCR Production for Structural Characterization: Adenosine A2aR as a Template for Study

Shweta Singh1, Minghao Zhang1, Nicolas Bertheleme1, Elodie Kara2, Philip G. Strange2, Bernadette Byrne1

1 Membrane Protein Crystallography Group, Division of Molecular Biosciences, Imperial College London, London, United Kingdom, 2 School of Pharmacy, University of Reading, Whiteknights, Reading, United Kingdom
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 29.3
DOI:  10.1002/0471140864.ps2903s67
Online Posting Date:  February, 2012
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Abstract

Functional characterization of G protein–coupled receptors is essential to ascertain the suitability of a protein target for downstream studies and to help develop optimal expression and isolation procedures. Radioligand binding analysis is a well‐established technique, which allows direct measurement of the amount of functional receptor in a sample. It can be readily applied to both membrane‐bound and soluble receptor samples and is an ideal method for monitoring the amount of functional protein at each stage in the expression and isolation process. This unit presents protocols for the radioligand binding analysis of the human adenosine A2a receptor and provides examples of how these assays can be used at several stages to help optimize expression, solubilization, and isolation procedures.

Keywords: G protein–coupled receptor; adenosine A2aR; saturation radioligand binding assay; competitive radioligand binding assay; dissociation constant; specific activity

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Small‐Scale Membrane Preparation for Radioligand Binding Assay
  • Alternate Protocol 1: Large‐Scale Membrane Preparation
  • Support Protocol 1: Protein Concentration Determination: Modified Lowry Assay
  • Support Protocol 2: Modified Protein Assay for Samples Containing High Concentrations of Detergent Using TCA Precipitation and SDS
  • Basic Protocol 2: Radioligand Binding Analysis for Membrane‐Bound Receptors: Saturation Assay
  • Alternate Protocol 2: Single‐Point Radioligand Binding Assay
  • Basic Protocol 3: Competition Radioligand Binding Assay
  • Basic Protocol 4: Preparation of Soluble Receptor for Radioligand Binding Analysis
  • Basic Protocol 5: Estimating Recovery of Soluble Receptor by Radioligand Binding Assay
  • Basic Protocol 6: Tm Determination to Assess Stability of Soluble Receptors
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Small‐Scale Membrane Preparation for Radioligand Binding Assay

  Materials
  • P. pastoris culture containing recombinantly expressed protein of interest (see protocol introduction for details)
  • Cell breaking buffer (see recipe), ice‐cold
  • Membrane buffer (see recipe)
  • Benchtop centrifuge
  • −80°C freezer
  • 2‐ml safe‐lock tubes
  • Glass beads (Biospec)
  • Tissue lyser (Qiagen)
  • Thick‐walled 1.5‐ml tubes (Beckman)
  • Benchtop ultracentrifuge, Optima L‐XP series (Beckman Coulter)
  • 1‐ml syringe and a 21‐G needle

Alternate Protocol 1: Large‐Scale Membrane Preparation

  Materials
  • P. pastoris culture containing recombinantly expressed protein of interest growing in a 5‐liter bioreactor
  • Cell breaking buffer (see recipe)
  • Membrane buffer (see recipe)
  • Constant cell disruptor (Constant Systems)
  • Benchtop centrifuge
  • 50‐ml polycarbonate tubes (Beckman Coulter)
  • 45Ti rotor in an ultra XP centrifuge (Beckman Coulter)
  • Glass or Teflon homogenizer (tissue grinder; Sigma‐Aldrich)

Support Protocol 1: Protein Concentration Determination: Modified Lowry Assay

  Materials
  • Bovine serum albumin (BSA) stock solution at 1 mg/ml (see recipe)
  • Test protein
  • 10% (w/v) trichloroacetic acid (TCA; Sigma‐Aldrich)
  • Lowry solutions (Lowry Solution A, Solution B, and Solution C; see recipes)
  • Folin‐Ciocalteu reagent (Sigma‐Aldrich)
  • Vortex mixer
  • 1.5‐ml microcentrifuge tubes
  • Spectrophotometer
  • Software (e.g., GraphPad Prism, MS Excel)

Support Protocol 2: Modified Protein Assay for Samples Containing High Concentrations of Detergent Using TCA Precipitation and SDS

  Materials
  • Membrane protein sample
  • 25% (w/v) trichloroacetic acid (TCA; Sigma‐Aldrich)
  • Lowry solution A:B:C at 100:1:1 (v/v) (see recipes)
  • 10% (w/v) SDS
  • Folin‐Ciocalteu reagent (Sigma‐Aldrich)
  • Vortex mixer
  • Spectrophotometer

Basic Protocol 2: Radioligand Binding Analysis for Membrane‐Bound Receptors: Saturation Assay

  Materials
  • Membranes containing the expressed adenosine A 2aR (see introduction to protocol 1 for details)
  • Radioactive ligand (radioligands; e.g., [3H] ZM241385)
  • Cold ligand (nonradioactive; e.g., Theophylline; see recipe)
  • Binding buffer (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Scintillation cocktail (e.g., Ultima Gold MV; Perkin Elmer)
  • 5‐ml round‐bottom polystyrene tubes (Fisher)
  • 25°C incubator
  • Brandel harvester
  • Glass micro‐fiber GF/C filter (Whatman)
  • Scintillation vials
  • Scintillation counter (Beckman)
  • Prism software (GraphPad)

Alternate Protocol 2: Single‐Point Radioligand Binding Assay

  Materials
  • Membrane buffer (see recipe)
  • Solubilization buffer (see recipe)
  • Dodecyl β‐D maltoside (DDM; see recipe for detergent stock solution)
  • Decyl β‐D maltoside (DM; see recipe for detergent stock solutions)
  • Cholestryl hemisuccinate (Sigma‐Aldrich)
  • 4°C incubator
  • 45Ti rotor in an XP ultracentrifuge (Beckman)
  • Additional reagents and equipment for preparing the membrane proteins ( protocol 1)

Basic Protocol 3: Competition Radioligand Binding Assay

  Materials
  • Binding buffer (see recipe)
  • Dodecyl β‐D maltoside (DDM; see recipe for detergent stock solution)
  • Decyl β‐D maltoside (DM; see recipe for detergent stock solution)
  • Cholestryl hemisuccinate (Sigma‐Aldrich)
  • NaCl
  • Theophylline (see recipe)
  • [3H] ZM241385 (see recipe)
  • 4°C incubator
  • Illustra microspin G‐50 columns (GE LifeSciences)
  • Centrifuge
  • Scintillation vials
  • Scintillation cocktail (Ultima Gold MV; Perkin Elmer)
  • Scintillation counter (Beckman)

Basic Protocol 4: Preparation of Soluble Receptor for Radioligand Binding Analysis

  Materials
  • Purified adenosine A 2aR (see unit 29.4 for purification protocols)
  • Decyl β‐D maltoside (DM; see recipe for detergent stock solution)
  • Cholestryl hemisuccinate (CH; Sigma‐Aldrich)
  • Binding buffer (see recipe)
  • Ice
  • ZM241385 (see recipe)
  • [3H] ZM241385 (see recipe)
  • Theophylline (see recipe)
  • Heating blocks or water baths
  • Illustra microspin G‐50 columns (GE LifeSciences)
  • 4°C incubator
  • Additional reagents and equipment for solubilizing adenosine ( protocol 8) and calculating the amount of active receptor in the sample using a single‐point binding assay ( protocol 6)
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Figures

Videos

Literature Cited

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