Determination of GPCR Phosphorylation Status: Establishing a Phosphorylation Barcode

Rudi Prihandoko1, Sophie J. Bradley1, Andrew B. Tobin1, Adrian J. Butcher1

1 MRC Toxicology Unit, University of Leicester, Leicester
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 2.13
DOI:  10.1002/0471141755.ph0213s69
Online Posting Date:  June, 2015
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Abstract

G protein−coupled receptors (GPCRs) are rapidly phosphorylated following agonist occupation in a process that mediates receptor uncoupling from its cognate G protein, a process referred to as desensitization. In addition, this process provides a mechanism by which receptors can engage with arrestin adaptor molecules and couple to downstream signaling pathways. The importance of this regulatory process has been highlighted recently by the understanding that ligands can direct receptor signaling along one pathway in preference to another, the phenomenon of signaling bias that is partly mediated by the phosphorylation status or phosphorylation barcode of the receptor. Methods to determine the phosphorylation status of a GPCR in vitro and in vivo are necessary to understand not only the physiological mechanisms involved in GPCR signaling, but also to fully examine the signaling properties of GPCR ligands. This unit describes detailed methods for determining the overall phosphorylation pattern on a receptor (the phosphorylation barcode), as well as mass spectrometry approaches that can define the precise sites that become phosphorylated. These techniques, coupled with the generation and characterization of receptor phosphorylation‐specific antibodies, provide a full palate of techniques necessary to determine the phosphorylation status of any given GPCR subtype. © 2015 by John Wiley & Sons, Inc.

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Phosphopeptide Mapping of GPCRs
  • Support Protocol 1: Generation of Stable Cell Line Expressing Epitope‐Tagged GPCR
  • Basic Protocol 2: Identification of GPCR Phosphorylation Sites by Mass Spectrometry
  • Basic Protocol 3: Generation and Characterization of Phosphorylation Site–Specific Antibodies
  • Basic Protocol 4: Determination of Receptor Phosphorylation Status by Western Blotting with Phosphorylation‐Specific Antibodies
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Phosphopeptide Mapping of GPCRs

  Materials
  • Cells expressing GPCR of interest, optionally tagged with HA or FLAG
  • Phosphate‐free KHB (see recipe)
  • Protein A−Sepharose beads (GE Healthcare)
  • TEG buffer (see recipe)
  • Radioimmunoprecipitation assay (RIPA) buffer (see recipe)
  • Antibody recognizing receptor epitope or tag sequence (anti‐HA and anti‐FLAG can be purchased from Roche Applied Science or Sigma)
  • Agonist of interest dissolved in KHB, dimethyl sulfoxide (DMSO), or distilled water
  • [32P]Orthophosphate (10 mCi/ml, 370 MBq/ml; PerkinElmer cat. no. NEX053H005MC)
  • 2× Laemmli buffer (see recipe)
  • Transfer buffer (see recipe)
  • Blocking solution (see recipe)
  • 1 mM ammonium bicarbonate solution (Sigma)
  • Sequencing‐grade modified trypsin (Promega)
  • Electrophoresis buffer, pH 1.9 (see recipe)
  • Ascending chromatography buffer (see recipe)
  • Six‐well cell culture plates (Grainier)
  • Humidified CO 2 incubator (Panasonic)
  • 37°C water bath
  • 1.5‐ml microcentrifuge tubes (Eppendorf)
  • Rotator (e.g., Keison)
  • Fine gel‐loading pipet tips
  • 50° to 60°C heating block
  • Nitrocellulose membrane (e.g., Protran from Fisher Scientific)
  • Extra‐thick blot filter paper (Bio‐Rad)
  • Mini‐Blot transfer apparatus (Bio‐Rad)
  • Cling film (e.g., Saran wrap)
  • X‐ray film (Millipore)
  • SpeedVac evaporator (Eppendorf)
  • 20 × 20‐cm cellulose thin‐layer chromatography (TLC) plates (Millipore)
  • Fan drier (Boots)
  • Whatman chromatography paper (Fisher Scientific)
  • Absorbent paper
  • Scalpel
  • Hunter thin‐layer mapping electrophoresis unit (CBS Scientific)
  • Glass ascending thin‐layer chromatography tank
  • Scraper or spatula
  • Storage phosphor screen
  • STORM phosphoimager (GE Healthcare)
  • ImageQuant (AlphaImager) or ImageJ software
  • Additional reagents and equipment for denaturing SDS‐PAGE ( appendix 3B), electroblotting, and autoradiography

Support Protocol 1: Generation of Stable Cell Line Expressing Epitope‐Tagged GPCR

  Materials
  • Chinese hamster ovary (CHO) cells (ATCC)
  • Growth medium: in this case, Hams F‐12 nutrient mixture with 5% penicillin/streptomycin and 10% fetal bovine serum (Life Technologies)
  • Plasmid DNA containing receptor of interest: in this case, pcDNA3.1+ (Life Technologies)
  • Transfection diluent (OptiMEM reduced serum, Life Technologies)
  • FuGENE HD transfection reagent (Roche Applied Science)
  • Selection medium: Growth medium with 500 μg/ml geneticin
  • PBS with 1 mM EDTA (Life Technologies)
  • Anti‐HA antibody (Roche Applied Science)
  • 6‐ and 24‐well tissue culture plates (e.g., Grainier)
  • 10‐cm2 dishes (e.g., Grainier)
  • Light microscope (e.g., Leica)
NOTE: All incubations are carried out in a 37°C humidified CO 2 incubator.

Basic Protocol 2: Identification of GPCR Phosphorylation Sites by Mass Spectrometry

  Materials
  • Cells expressing GPCR of interest, optionally containing HA or FLAG epitope tag
  • Complete KHB (see recipe), 37°C
  • Agonist of interest dissolved in KHB, dimethyl sulfoxide (DMSO), or distilled water
  • PBS containing 1 mM EDTA (Life Technologies), 4°C
  • TE buffer: 10 mM Tris, 1 mM EDTA, pH 7.4
  • Protease inhibitor tablets (Roche Applied Science)
  • Phosphatase inhibitor tablets (Roche Applied Science)
  • Nonidet P‐40 (Sigma)
  • Agarose beads coupled with anti‐FLAG or anti‐HA antibodies (Roche Applied Science)
  • 2× Laemmli buffer (see recipe)
  • 10 mM triethylammonium bicarbonate buffer (TEAB; Sigma)
  • Dithiothreitol (Sigma)
  • Iodoacetamide (Sigma)
  • Acetonitrile (Sigma)
  • Sequencing‐grade modified trypsin (Promega)
  • Trifluoroacetic acid (TFA; Sigma)
  • IMAC buffers, all freshly prepared
    • IMAC buffer A: 250 mM acetic acid in 30% (v/v) acetonitrile
    • IMAC buffer B: 400 mM ammonium hydroxide in 30% (v/v) acetonitrile
    • IMAC buffer C: 0.5% (v/v) TFA in 30% (v/v) acetonitrile
    • IMAC buffer D: 0.1% (v/v) TFA in 80% (v/v) acetonitrile
    • IMAC buffer E: 15% (v/v) ammonium hydroxide
  • PHOS‐Select Iron Affinity Gel (Sigma)
  • HPLC‐grade water (Sigma)
  • Titansphere titanium dioxide gel (GL Sciences)
  • Dihydroxybenzoic acid (DHB; Sigma)
  • 175‐cm2 tissue culture flasks or Cellmaster cell culture roller bottles (Grainier)
  • Polytron homogenizer (PT1200 CL, Kinematica, Basel, Switzerland)
  • Variable‐speed rolling platform (Cole‐Parmer)
  • 1.5‐ml microcentrifuge tubes
  • SpeedVac evaporator
  • End‐over‐end mixer
  • Fritted 1‐ml spin column (Sigma)
  • Additional reagents and equipment for denaturing SDS‐PAGE ( appendix 3B) and Coomassie blue gel staining
NOTE: All incubations are carried out in a 37°C humidified CO 2 incubator.

Basic Protocol 3: Generation and Characterization of Phosphorylation Site–Specific Antibodies

  Materials
  • Cells expressing GPCR of interest with HA or FLAG tag
  • Complete KHB (see recipe), 37°C
  • Test compounds in KHB or water
  • RIPA buffer (see recipe), 4°C
  • Protease inhibitor tablets (Roche Applied Science)
  • Phosphatase inhibitor tablets (Roche Applied Science)
  • Agarose beads coupled with anti‐FLAG or anti‐HA antibodies (Roche Applied Science)
  • Calf intestinal alkaline phosphatase (CIAP) and CIAP buffer with 0.25% n‐octylglucoside (Roche Applied Science)
  • 2× Laemmli buffer (see recipe)
  • Six‐well tissue culture plates (Grainier)
  • 1.5‐ml microcentrifuge tubes (Eppendorf)
  • Variable‐speed rolling platform (Cole‐Parmer)

Basic Protocol 4: Determination of Receptor Phosphorylation Status by Western Blotting with Phosphorylation‐Specific Antibodies

  Materials
  • Western blot of receptor of interest
  • Western blot blocking solution (see recipe)
  • Phosphorylation‐specific primary antibody (see protocol 4)
  • Western blot washing solution (see recipe)
  • Horseradish peroxidase (HRP)–conjugated secondary antibody (Bio‐Rad)
  • Immobilon HRP substrate (Millipore)
  • Nitrocellulose membranes (e.g., Protran from Fisher Scientific)
  • 10‐cm2 Petri dishes (VWR)
  • Glass plate, at least the same size as the membrane
  • Enhanced Chemiluminescent (ECL) X‐ray film and developer (e.g., Xograph)
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Figures

Videos

Literature Cited

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