Visualization of G Protein−Coupled Receptor (GPCR) Interactions in Living Cells Using Bimolecular Fluorescence Complementation (BiFC)

Pierre‐Alexandre Vidi1, Julie A. Przybyla1, Cheng‐Deng Hu1, Val J. Watts1

1 Purdue University, West Lafayette, Indiana
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 5.29
DOI:  10.1002/0471142301.ns0529s51
Online Posting Date:  April, 2010
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Abstract

Members of the G protein−coupled receptor (GPCR) superfamily have been shown to homo‐ and hetero‐oligomerize both in vitro and in vivo. Although the functional and pharmacological significance of GPCR oligomerization is far from being completely understood, evidence suggests that, depending on the receptor, oligomerization may influence ligand binding, G protein coupling, and receptor targeting. Bimolecular fluorescence complementation (BiFC) is a technique based on the complementation of fragments from fluorescent proteins that allows the measurement and visualization of protein interactions in living cells. It can be extended to the simultaneous detection of distinct protein‐protein interactions using a multicolor setup. This unit describes the application of BiFC and multicolor BiFC to the visualization of GPCR oligomerization in a neuronal cell model. Oligomerization of GPCR fusions to BiFC tags is visualized and measured using fluorescence microscopy and fluorometry. The effect of ligands on the relative formation of distinct oligomeric species is monitored with a ratiometric multicolor BiFC approach. Curr. Protoc. Neurosci. 51:5.29.1‐5.29.15. © 2010 by John Wiley & Sons, Inc.

Keywords: G protein−coupled receptor; oligomerization; bimolecular fluorescence complementation; multiple protein‐protein interactions; neuronal cells; ligand effects

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Microscopic Detection of GPCR Interactions in Living Cells Using Bimolecular Fluorescence Complementation (BiFC)
  • Support Protocol 1: Quantification of Microscopic Fluorescent Signals
  • Alternate Protocol 1: Microscopic Detection of Multiple GPCR Interactions in Living Cells
  • Support Protocol 2: Ratiometric Analysis of Multiple GPCR Interactions Using Fluorescence Microscopy
  • Alternate Protocol 2: BiFC and Multicolor BiFC Measurements Using Fluorometry
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Microscopic Detection of GPCR Interactions in Living Cells Using Bimolecular Fluorescence Complementation (BiFC)

  Materials
  • Plasmid vectors containing BiFC fragment sequences of Venus (e.g., pBiFC vectors; Shyu et al., ; Fig. ; pBiFC vectors and their sequences may be requested from Dr. C.‐D. Hu at hu1@purdue.edu)
  • DNA encoding receptor coding sequences
  • Plasmid Midi Kit (Qiagen) or equivalent
  • Cell system for expressing BiFC constructs (e.g., HEK293, COS‐1, HeLa, or the neuronal CAD cell line; also see Critical Parameters)
  • Appropriate growth medium, with and without pH indicator
  • Lipofectamine 2000 (Invitrogen) or other transfection reagent
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Cell culture vessels appropriate for microscopy (e.g., chambered coverglass [Nunc] or cluster plates with round glass coverslips at the bottom of the wells)
  • Inverted fluorescence microscope (e.g., Nikon TE2000‐U) equipped with:
    • 20× and 60× objectives
    • Excitation source (e.g., mercury lamp)
    • Filter set for Venus/YFP (excitation 500/20 nm; emission 535/30 nm)
  • Sensitive digital monochrome CCD camera (e.g., Photometrics CoolSNAP‐ES)
  • Image acquisition software (e.g., Metamorph, Molecular Devices)
  • Additional reagents and equipment for PCR and DNA sequencing ( appendix 1A), receptor binding experiments (unit 1.4), cAMP assays (e.g., unit 7.12) or other downstream assays for receptor function, protein immunodetection (unit 5.19), and radioreceptor binding assays (units 1.4& 7.5)

Support Protocol 1: Quantification of Microscopic Fluorescent Signals

  • Plasmid encoding Cerulean or mCherry to be used as normalizing FP (assuming VN/VC was used in the protocol 1)
  • Filter set for Cerulean/CFP (excitation 430/25 nm; emission 470/30 nm) or mCherry/DsRed (excitation 572/23 nm; emission 640/50 nm)
  • ImageJ image processing and analysis software (http://rsbweb.nih.gov/ij/) or equivalent

Alternate Protocol 1: Microscopic Detection of Multiple GPCR Interactions in Living Cells

  • Plasmid vectors containing the following BiFC fragments: VN (Venus N‐terminal), CN (Cerulean N‐terminal), CC (Cerulean C‐terminal); pBiFC vectors may be used (Shyu et al., ; available from C.‐D. Hu at hu1@purdue.edu)
  • Filter set for Cerulean/CFP (excitation 430/25 nm; emission 470/30 nm)

Support Protocol 2: Ratiometric Analysis of Multiple GPCR Interactions Using Fluorescence Microscopy

  • Reagents for protein concentration determination (e.g., BCA from Pierce)
  • 12‐well cluster plates
  • Black 96‐well plates
  • Multiwell plate reader that efficiently separates Venus and Cerulean fluorescent signals (e.g., Fusion, Packard‐Perkin Elmer)
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Figures

Videos

Literature Cited

Literature Cited
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