Biochemical, Biophysical and Cellular Techniques to Study the Guanine Nucleotide Exchange Factor, GIV/Girdin

Pradipta Ghosh1, Nicolas Aznar1, Lee Swanson1, I‐Chung Lo2, Inmaculada Lopez‐Sanchez1, Jason Ear1, Cristina Rohena1, Nicholas Kalogriopoulos1, Linda Joosen1, Ying Dunkel1, Nina Sun1, Peter Nguyen3, Deepali Bhandari3

1 Department of Medicine, University of California at San Diego, La Jolla, 2 Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, 3 Department of Chemistry and Biochemistry, California State University Long Beach, Long Beach
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/cpch.13
Online Posting Date:  December, 2016
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Abstract

Canonical signal transduction via heterotrimeric G proteins is spatiotemporally restricted, i.e., triggered exclusively at the plasma membrane, only by agonist activation of G protein‐coupled receptors via a finite process that is terminated within a few hundred milliseconds. Recently, a rapidly emerging paradigm has revealed a noncanonical pathway for activation of heterotrimeric G proteins via the nonreceptor guanidine‐nucleotide exchange factor, GIV/Girdin. Biochemical, biophysical, and functional studies evaluating this pathway have unraveled its unique properties and distinctive spatiotemporal features. As in the case of any new pathway/paradigm, these studies first required an in‐depth optimization of tools/techniques and protocols, governed by rationale and fundamentals unique to the pathway, and more specifically to the large multimodular GIV protein. Here we provide the most up‐to‐date overview of protocols that have generated most of what we know today about noncanonical G protein activation by GIV and its relevance in health and disease. © 2016 by John Wiley & Sons, Inc.

Keywords: GIV/Girdin; trimeric G proteins; immunoblotting; immunoprecipitation; in cellulo GST‐pull down; immunofluorescence; FRET

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Detection of Full‐Length GIV by Immunoblotting
  • Basic Protocol 2: Detection of Full‐Length GIV mRNA by Quantitative Real‐Time Polymerase Chain Reaction (QRT‐PCR)
  • Basic Protocol 3: Immunoprecipitation of Endogenous or Exogenously Expressed GIV
  • Basic Protocol 4: Transfection of GIV Constructs into Mammalian Cells
  • Basic Protocol 5: In Cellulo GST Pull‐Down Assay Using GST‐Tagged GIV from Mammalian Cells
  • Basic Protocol 6: Whole‐Cell Immunofluorescence
  • Basic Protocol 7: Förster Resonance Energy Transfer (FRET) Studies to Assess the Spatiotemporal Dynamics of GIV‐Associated Protein Complexes and GIV‐Dependent G Protein Signaling
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Detection of Full‐Length GIV by Immunoblotting

  Materials
  • Samples
  • 8% SDS‐PAGE gel
  • Running buffer (see recipe)
  • Transfer buffer (see recipe)
  • Primary antibodies (see Table 13.0.2):
    • Girdin‐T13 (Santa Cruz Biotechnology, cat. no. sc‐133371; 1:500 in 5% milk/PBS‐T)
    • Girdin coiled‐coil antibodies (Millipore, cat. nos. ABT80 and ABT168; 1:500 in 5% milk/PBS‐T)
  • Anti‐pY1764 GIV (Spring Bio)
  • Bovine serum albumin (BSA; Sigma) or dry milk powder (Store bought brand)
  • Phosphate buffered saline with 0.05% Tween 20 (PBS‐T)
  • IRDye Infrared dye‐conjugated secondary antibodies (Li‐COR)
  • PVDF membrane: 0.45‐μm pore size (Millipore)
  • Minigel system (Bio‐Rad)
  • 2000 volt power supply (Bio‐Rad)
  • LiCOR Odyssey CLx Imaging system (Li‐COR Odyssey)
Table h.3.2   MaterialsAntibodies Tested to Successfully Immunoprecipitate and/or Immunoblot Endogenous or Exogenously Expressed GIV a

Antibody/cat. no. Source Type Application
GIV‐CT (Girdin T‐13)/sc‐133371 Santa Cruz Biotechnology (SCBT) Rabbit Polyclonal IB, IP, IF, PLA
GIV CC/ABT80, ABT168 Millipore Rabbit Polyclonal IB, IP, IF, PLA
GIV CC/MABT100 Millipore Mouse Monoclonal IB
GIV pY1765/P158 Spring Bioscience Rabbit Monoclonal IB, IP, IF, PLA

 aAbbreviations: CT, Carboxyl terminus; CC, coiled‐coil; IB, immunoblotting; IF, immunofluorescence; IP, immunoprecipitation; PLA, proximity ligation assay.

Basic Protocol 2: Detection of Full‐Length GIV mRNA by Quantitative Real‐Time Polymerase Chain Reaction (QRT‐PCR)

  Materials
  • Cultured cells or tissues
  • RNeasy kit (QIAGEN)
  • SuperScript II Reverse Transcriptase (Invitrogen)
  • RNase H (Invitrogen)
  • TaqMan Fast Universal PCR Master Mix (2×) (Applied Biosystems) or Fast SYBR Green Master Mix (2×)
  • GIV primers or GADPH primers designed by Primer Express Software v3.0.1 (Applied Biosystems) (see Table 13.0.3) for SYBR green detection
  • CCDC88A (GIV) Assay Mix: Hs01554973_m1 (see Table 13.0.4) for Taqman assay detection and GAPDH Assay mix: Hs99999905_m1(see Table 13.0.4) for Taqman assay detection
  • RNase‐free water
  • Sterile filter pipet tips
  • 0.2‐ml PCR microtubes with attached caps size
  • MicroAmp Optical 96‐Well Reaction Plate (Applied Biosystems)
  • MicroAmp Optical 96‐Well Optical Adhesive Film (Applied Biosystems)
  • ABI StepOnePlus (Applied Biosystems)
  • Microcentrifuge
Table h.3.3   MaterialsSequence of Probes for Taqman Gene Expression Assay for Detection of GIVPrimers for Detection of GIV mRNA

Gene name Assay number NCBI Ref Seq(s) Amplicon length
GAPDH Hs99999905_m1 NM_002046.3 122
CCDC88A Hs01554973_m1 NM_001135597.1 130 bp
NM_001254943.1
NM_018084.4
Gene name Primer sequence NCBI Ref Seq(s) Amplicon length PCR efficiency
CCDC88A (Mouse) Fwd: 5′‐GTGATCTCTACTGCT GAAGG‐3′ Rev: 5′‐TGTTGCT CCCTAGACCTGCT‐3′ NC_000077.6 185 bp 96.6%
CCDC88A (Human) Fwd: 5′‐ ATCTCAACTGCCG AAGGAACT‐3′ Rev: 5′‐TGT TGCTCCCTAGACCTGCT‐3′ NG_031944.1 182 bp 97.7%
GAPDH (Human) Fwd: 5′‐TCAGTTGTAGGCAA GCTGCGACGT‐3′ Rev: 5′‐AA GCCAGAGGCTGGTACCTAG AAC‐3′ NG_007073.2 185 bp 98.2%
GAPDH (Mouse) Fwd: 5′‐ CTGCAGCCTCGTCCC GTAGAC A ‐ 3′ Rev: 5′‐TGCCG TGAGTGGAGTCATA CTGGA‐ 3″ NC_000072.6 181 bp 97.1%

Table h.3.4   MaterialsSequence of Probes for Taqman Gene Expression Assay for Detection of GIVPrimers for Detection of GIV mRNA

Gene name Assay number NCBI Ref Seq(s) Amplicon length
GAPDH Hs99999905_m1 NM_002046.3 122
CCDC88A Hs01554973_m1 NM_001135597.1 130 bp
NM_001254943.1
NM_018084.4
Gene name Primer sequence NCBI Ref Seq(s) Amplicon length PCR efficiency
CCDC88A (Mouse) Fwd: 5′‐GTGATCTCTACTGCT GAAGG‐3′ Rev: 5′‐TGTTGCT CCCTAGACCTGCT‐3′ NC_000077.6 185 bp 96.6%
CCDC88A (Human) Fwd: 5′‐ ATCTCAACTGCCG AAGGAACT‐3′ Rev: 5′‐TGT TGCTCCCTAGACCTGCT‐3′ NG_031944.1 182 bp 97.7%
GAPDH (Human) Fwd: 5′‐TCAGTTGTAGGCAA GCTGCGACGT‐3′ Rev: 5′‐AA GCCAGAGGCTGGTACCTAG AAC‐3′ NG_007073.2 185 bp 98.2%
GAPDH (Mouse) Fwd: 5′‐ CTGCAGCCTCGTCCC GTAGAC A ‐ 3′ Rev: 5′‐TGCCG TGAGTGGAGTCATA CTGGA‐ 3″ NC_000072.6 181 bp 97.1%

Basic Protocol 3: Immunoprecipitation of Endogenous or Exogenously Expressed GIV

  Materials
  • IP lysis buffer (see recipe)
  • Ice
  • Cos7 cells (ATCC)
  • 1× phosphate‐buffered saline (PBS; see recipe)
  • Antibodies (see Table 13.0.2)
  • Protein A/G Sepharose beads (GE Healthcare)
  • 1× PBS‐T wash buffer (see recipe)
  • 5× Laemmli sample buffer (see recipe)
  • Cell scraper
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge
  • Vortex mixer
  • 30‐G needles
  • 4°C rotator
  • Additional reagents and equipment for determining the protein concentration (Bradford, )

Basic Protocol 4: Transfection of GIV Constructs into Mammalian Cells

  Materials
  • Polyethylenimine (PEI) (Polysciences, cat. no. 23966)
  • Deionized water
  • Cells (e.g., Cos‐7 cells; ATCC)
  • Complete medium (e.g., for Cos‐7 cells use DMEM supplemented with 10% FBS and 1× penicillin‐streptomycin‐glutamine)
  • Transfection quality pDNA constructs of GIV (see Table 7)
  • 1× phosphate‐buffered saline (PBS; see recipe)
  • Serum‐free medium
  • 0.22‐μm syringe filter
  • Vortex mixer
  • 1.5‐ml microcentrifuge tubes

Basic Protocol 5: In Cellulo GST Pull‐Down Assay Using GST‐Tagged GIV from Mammalian Cells

  Materials
  • Glutathione Sepharose beads (GE Healthcare Life Sciences)
  • GST pull‐down lysis buffer (see recipe)
  • 1× phosphate‐buffered saline (PBS; see recipe)
  • 5× Laemmli sample buffer (see recipe)
  • 1× PBS‐T wash buffer (see recipe)
  • Rotator
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge
  • Additional reagents and equipment for lysing the cells (see protocol 3) and immunoblotting (see protocol 1)

Basic Protocol 6: Whole‐Cell Immunofluorescence

  Materials
  • Cells (either HeLa, HEK, Cos‐7 or others; obtained from the ATCC)
  • 3% paraformaldehyde (PFA) in 1× phosphate‐buffered saline (PBS)
  • 0.1 M glycine in 1× PBS (Quenching solution)
  • 0.2% Triton X‐100 (TX‐100) in 1× PBS (Permeabilizing solution)
  • 1% BSA and 0.1% TX‐100 in 1× PBS (Blocking buffer)
  • Primary antibodies (against target antigen whose localization one seeks to study, e.g., GIV in this case)
  • 1× phosphate‐buffered saline (PBS; see recipe)
  • Secondary antibodies, e.g., Alexa Fluor conjugated (Life Technologies)
  • DAPI (4′,6‐diamino‐2‐phenylindole) (Life Technologies), optional
  • ProLong (Life Technologies)
  • Store‐bought clear nail polish (sealant)
  • Multi‐well plates containing sterile coverslips
  • 37°C, 5% CO 2 incubator
  • Confocal or fluorescence microscope

Basic Protocol 7: Förster Resonance Energy Transfer (FRET) Studies to Assess the Spatiotemporal Dynamics of GIV‐Associated Protein Complexes and GIV‐Dependent G Protein Signaling

  Materials
  • Mammalian cells (e.g., HeLa, Cos7)
  • DMEM (Corning, cat. no. 10‐013‐CV) supplemented with 10% fetal bovine serum (FBS; HyClone, cat. no. SH30071.03) and 1× penicillin‐streptomycin‐glutamine (Gibco, cat. no. 10378‐016)
  • Trans‐IT‐LT1 tansfection reagent (Mirus Bio)
  • Plasmid(s) of interest for transient expression
  • Imaging medium: DMEM without Phenol Red (Corning, cat. no. 17‐205‐CV)
  • 35‐mm FluoroDish (World Precision Instruments, cat. no. FD35‐100)
  • Microscope: Inverted confocal laser scanning microscope (Olympus FV1000/3000 or equivalent) equipped with:
    • Oil‐immersed objective (60×, 1.49 N.A)
    • Excitation lasers for donor (CFP) in the range of 405 to 440 nm (HeNe) and for direct excitation of acceptor (YFP) a 514‐ to 515‐nm laser (Argon‐ion)
    • Detection via gated spectral detection or suitable bandpass filter
    • Software for imaging and analysis purposes:
    • In‐built software (Olympus Fluoview or equivalent)
  • ImageJ software with RiFRET plugin installed [free download platform: https://imagej.nih.gov/ij/ (Roszik et al., ; Schneider et al., )]
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Figures

Videos

Literature Cited

Literature Cited
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