Methods of Measuring Internalization of G Protein–Coupled Receptors

Nicholas Evans1

1 GlaxoSmithKline Research, Harlow
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 12.6
DOI:  10.1002/0471141755.ph1206s24
Online Posting Date:  May, 2004
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Abstract

This unit provides detailed protocols for measuring receptor internalization. The techniques are sufficiently generalized to be applicable to most receptors in a wide variety of cell types. Both radioactive and non‐radioactive techniques are described that may be used to quantify receptor internalization, and the differences between the two are highlighted. This unit discusses how quantification of internalization may be achieved, and the advantages and drawbacks of each technique. Low‐ and higher‐throughput methods are compared, and the technologies required to conduct the analyses are discussed.

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of Receptor Internalization Using Radiolabeled Ligands
  • Alternate Protocol 1: Direct Measurement of Receptor Internalization after Radioligand Binding
  • Alternate Protocol 2: Separation of External and Internalized ligand Using Sucrose Density Centrifugation
  • Basic Protocol 2: Colorimetric Technologies to Measure Receptor Internalization Using Elisa
  • Alternate Protocol 3: Fluorescence Technologies to Measure Receptor Internalization Using Flow Cytometry
  • Basic Protocol 3: Analysis of Receptor Internalization in Fixed Cells Using Fluorescence Microscopy
  • Alternate Protocol 4: Monitoring Real‐Time Receptor Internalization Using Time Lapse Confocal Microscopy
  • Alternate Protocol 5: Measurement of Internalization of Receptor–Green Fluorescent Protein Chimeras by Confocal Microscopy
  • Basic Protocol 4: Measuring Receptor Internalization Using High‐Throughput Technologies
  • Support Protocol 1: Preparation of Cells for Receptor Internalization Studies
  • Support Protocol 2: Preparation of Ligand and Compound Plates for Receptor Internalization Studies
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Measurement of Receptor Internalization Using Radiolabeled Ligands

  Materials
  • Cells of interest seeded into poly‐L‐lysine‐coated 24‐well plates at 2 × 105 cells/well or 96‐well plates at 1.5–2.5 × 104 cells/well (see protocol 10)
  • Ligand of interest
  • HBSS/Ca2+ (see recipe), prewarmed to 37°C
  • PBS (see recipe), ice cold
  • Low‐pH stripping buffer 1 (see recipe) or high‐pH stripping buffer (see recipe), ice cold
  • Radioligand of interest
  • 0.1 M NaOH

Alternate Protocol 1: Direct Measurement of Receptor Internalization after Radioligand Binding

  • Binding buffer (see recipe), 37°C and ice‐cold
  • Low‐pH stripping buffer 2 (see recipe), ice‐cold
  • 0.5 M NaOH

Alternate Protocol 2: Separation of External and Internalized ligand Using Sucrose Density Centrifugation

  • Serum‐free EMEM (see recipe), prewarmed to 37°C
  • 1% (w/v) BSA in PBS (see recipe), ice‐cold
  • Lysis buffer (see recipe)

Basic Protocol 2: Colorimetric Technologies to Measure Receptor Internalization Using Elisa

  Materials
  • Agonist for receptor of interest in HBSS/Ca2+ (see recipe)
  • Cells of interest in 24‐well plates (∼2 × 105 cells/well) coated with 0.1 mg/ml poly‐L‐lysine (see protocol 10)
  • TBS (see recipe)
  • 4% (w/v) paraformaldehyde in TBS
  • 1% (w/v) BSA in TBS
  • Primary antibody for receptor of interest diluted in 1% (w/v) BSA in TBS
  • Alkaline phosphatase–conjugated secondary antibody, diluted according to manufacturer's instructions in 1% (w/v) BSA in TBS
  • Alkaline phosphatase substrate (e.g., Bio‐Rad)
  • 96‐well plate with 100 µl of 0.4 M NaOH in each well
  • ELISA plate reader (e.g., Spectramax; Molecular Devices)

Alternate Protocol 3: Fluorescence Technologies to Measure Receptor Internalization Using Flow Cytometry

  • PBS (see recipe), ice cold and room temperature
  • Appropriate anti‐epitope primary antibody diluted in 1% (w/v) BSA in PBS
  • Fluorescently tagged secondary antibody (e.g., 1:200 goat anti‐mouse; Sigma) diluted in 1% (w/v) BSA in PBS
  • 10 mM Tris·Cl/5 mM EDTA, pH 7.4
  • 3.6% (w/v) paraformaldehyde in PBS
  • Additional reagents and equipment for flow cytometry

Basic Protocol 3: Analysis of Receptor Internalization in Fixed Cells Using Fluorescence Microscopy

  Materials
  • Cells of interest plated on glass coverslips in 6‐well plates (see protocol 10)
  • Appropriate growth medium (e.g., DMEM/30 mM HEPES, pH 7.4), 4°C
  • Fluorescently tagged transferrin (Molecular Probes) diluted in recommended buffer according to manufacturer's instructions
  • Appropriate anti‐epitope primary antibody diluted in 1% (w/v) BSA in PBS (see recipe)
  • Appropriate agonist diluted in HBSS/Ca2+ (see recipe)
  • 3.7% (w/v) formaldehyde in PBS, pH 7.4 (see recipe)
  • 1 mM CaCl 2 in TBS (see recipe)
  • Permeabilizing buffer (see recipe)
  • Appropriate FITC‐labeled secondary antibody, diluted in 1% (w/v) BSA in PBS according to manufacturer's instructions
  • Additional reagents and equipment for fluorescence microscopy

Alternate Protocol 4: Monitoring Real‐Time Receptor Internalization Using Time Lapse Confocal Microscopy

  • Cells plated onto chambered coverglass slides for confocal microscopy (see protocol 10)
  • PBS (see recipe), ice‐cold
  • Additional reagents and equipment for confocal microscopy, including temperature‐controlled stage

Alternate Protocol 5: Measurement of Internalization of Receptor–Green Fluorescent Protein Chimeras by Confocal Microscopy

  • Cells expressing receptor‐GFP chimera of interest, grown on microscope slides in 6‐well plates (see protocol 10)
  • PBS (see recipe), optional
  • 10 µg/ml red fluorescent lectin (e.g., rhodamine‐concanavalin A; Molecular Probes) diluted in recommended buffer according to manufacturer's instructions, optional
  • Appropriate ligand
  • Software for image analysis (e.g., Zeiss LSM; Zeiss) and data analysis (e.g., GraFit, Erithacus Software or Prism, GraphPad Software)
  • Additional reagents and equipment for confocal or fluorescence microscopy, including temperature‐controlled stage (for time course studies)

Basic Protocol 4: Measuring Receptor Internalization Using High‐Throughput Technologies

  Materials
  • Cells of interest grown in 96‐well plates (see protocol 10)
  • Assay buffer (see recipe)
  • 4% (w/v) paraformaldehyde containing 15 µg/ml Hoechst 3334
  • Plates containing agonist or antagonist (see protocol 11)
  • Multichannel pipettors (e.g., Biomek Fx, Multimek Multi‐Channel Pipettor, and Biomek 2000 laboratory automated workstation; Beckman Coulter) and appropriate pipet tips
  • Plate washer (Elx405; Bio‐Tek Instruments) with 96‐well pipetting head
  • Arrayscan II imaging system (Cellomics)
  • Analysis software, such as Microsoft Excel (Microsoft) or GraFit version 4.0.13 (Erithacus Software)

Support Protocol 1: Preparation of Cells for Receptor Internalization Studies

  Materials
  • Cells grown to confluence in 175‐cm2 (T175) flask
  • PBS Dulbecco's without Ca2+ and Mg2+ (Life Technologies)
  • Versene (Life Technologies)
  • Standard growth medium for cells being seeded
  • Assay buffer (see recipe)
  • Biocoat poly‐L‐lysine‐coated black‐walled plates (Becton Dickinson)
  • 50‐ml centrifuge tubes (Nunc)
  • Benchtop centrifuge (e.g., CR4.22, Jouan)
  • Multi‐well plates with or without appropriate coverslips or chambered coverglass slides for confocal microscopy
  • Additional reagents and equipment for counting cells

Support Protocol 2: Preparation of Ligand and Compound Plates for Receptor Internalization Studies

  Materials
  • Agonist of interest
  • Assay buffer (see recipe)
  • Agonist or antagonist to be tested
  • 96‐well polypropylene round‐bottom plates (Becton Dickinson)
  • Biomek 2000 laboratory automated workstation (Beckman Coulter) and appropriate pipet tips
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Figures

Videos

Literature Cited

Literature Cited
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   Bunemann, M. and Hosey, M.M. 1999. G‐protein coupled receptor kinases as modulators of G‐protein signalling. J. Physiol. 517:5‐23.
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