Use of Flow Cytometric Methods to Quantify Protein‐Protein Interactions

Levi L. Blazer1, David L. Roman2, Molly R. Muxlow1, Richard R. Neubig3,1,4

1 Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, Michigan, 2 Division of Medicinal and Natural Products Chemistry, The University of Iowa, Iowa City, Iowa, 3 Department of Internal Medicine, The University of Michigan Medical School, Ann Arbor, Michigan, 4 Center for Chemical Genomics, The University of Michigan Medical School, Ann Arbor, Michigan
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 13.11
DOI:  10.1002/0471142956.cy1311s51
Online Posting Date:  January, 2010
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Abstract

A method is described for the quantitative analysis of protein-protein interactions using the flow cytometry protein interaction assay (FCPIA). This method is based upon immobilizing protein on a polystyrene bead, incubating these beads with a fluorescently labeled binding partner, and assessing the sample for bead-associated fluorescence in a flow cytometer. This method can be used to calculate protein-protein interaction affinities or to perform competition experiments with unlabeled binding partners or small molecules. Examples described in this protocol highlight the use of this assay in the quantification of the affinity of binding partners of the regulator of G-protein signaling protein, RGS19, in either a saturation or a competition format. An adaptation of this method that is compatible for high-throughput screening is also provided. Curr. Protoc. Cytom. 51:13.11.1-13.11.15. © 2010 by John Wiley & Sons, Inc.

Keywords: RGS; G protein; protein-protein interaction; FCPIA; high-throughput screening; multiplexing

     
 
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Table of Contents

  • Introduction
  • Characterizing Protein-Protein Binding Using the Flow Cytometry Protein-Protein Interaction Assay
  • Basic Protocol 1: Saturation Analysis of Biotin-RGS with Alexa Fluor532-Go
  • Basic Protocol 2: FCPIA Competition Assay
  • Basic Protocol 3: High-Throughput Screening with FCPIA
  • Support Protocol 1: Biotinylation of RGS Protein
  • Support Protocol 2: Alexa Fluor532 Labeling of Go
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Saturation Analysis of Biotin-RGS with Alexa Fluor532-Go

 Materials
  • LumAvidin polystyrene beads (L100-Lxxx-01, where the “xxx” represents the bead region)
  • Bead coupling buffer (BCB; see recipe)
  • Biotinylated RGS protein (see Support Protocol 1)
  • Flow buffer (FB; see recipe)
  • Alexa Fluor532-conjugated Go (see Support Protocol 2)
  • 50 µM aluminum chloride, aqueous
  • 50 mM magnesium chloride, aqueous
  • 50 mM sodium fluoride, aqueous
  • 1.5-ml microcentrifuge tubes
  • Microcentrifuge, preferably fixed-angle rotor
  • Vortex
  • Full-skirted 96-well polypropylene PCR plate (ISC Bioexpress, cat. no. T-3082-1)
  • Multichannel pipet
  • Aluminum foil
  • Luminex 200 flow cytometer (Luminex)

Basic Protocol 2: FCPIA Competition Assay

 Materials
  • LumAvidin polystyrene beads (L100-Lxxx-01, where the “xxx” represents the bead region)
  • Bead coupling buffer (BCB; see recipe)
  • Biotinylated RGS protein (see Support Protocol 1)
  • Flow buffer (FB; see recipe)
  • Compound or competing protein (e.g., unlabeled RGS protein)
  • Alexa Fluor532-conjugated Go (see Support Protocol 2)
  • 50 µM aluminum chloride, aqueous
  • 50 mM magnesium chloride, aqueous
  • 50 mM sodium fluoride, aqueous
  • 1.5-ml microcentrifuge tubes
  • Microcentrifuge, preferably fixed-angle rotor
  • Vortex
  • Multichannel pipette or Multidrop micro reagent dispenser (Thermo-Fisher)
  • Luminex 200 flow cytometer (Luminex)

Basic Protocol 3: High-Throughput Screening with FCPIA

 Materials
  • SPHERO streptavidin-coated particles, 5- to 5.9-µm diameter (Spherotech, SVP-50-5)
  • Bead coupling buffer (BCB; see recipe)
  • Biotinylated RGS protein (see Support Protocol 1)
  • Flow buffer (FB; see recipe)
  • Test or library compounds
  • Alexa Fluor-532-conjugated Go (see Support Protocol 2)
  • 50 µM aluminum chloride, aqueous
  • 50 mM magnesium chloride, aqueous
  • 50 mM sodium fluoride, aqueous
  • 1.5-ml microcentrifuge tubes
  • Microcentrifuge, preferably with a fixed-angle rotor
  • Vortex mixer
  • Multidrop micro reagent dispenser (Thermo-Fisher)
  • Low volume, nonstick, black 384-well plate (Corning, cat. no. 3676)
  • HyperCyt liquid sampling unit (IntelliCyt)
  • Accuri C6 cytometer (Accuri Cytometers)
  • HyperView software

Support Protocol 1: Biotinylation of RGS Protein

 Materials
  • 1 to 10 mg of purified protein, preferably >90% homogeneous
  • Reaction buffer: 50 mM HEPES, pH 9.0, at 4°C, 250 mM NaCl, 5% glycerol supplemented with 1 mM tris(2-carboxyethyl)phosphine (TCEP)
  • N-hydroxysuccidimyl ester-biotin (NHS-biotin; Sigma-Aldrich, cat. no. B2643)
  • Dimethyl sulfoxide (DMSO)
  • 1 M glycine (MP Biomedicals, cat. no. 808831) in 1 M HEPES, pH 9.0, at 4°C
  • Storage buffer: 50 mM HEPES, pH 7.4, at 4°C, 250 mM NaCl, 5% glycerol supplemented with 1 mM tris(2-carboxyethyl)phosphine (TCEP)
  • 10-ml Sephadex G25 gel filtration column (essentially any standard desalting column can be used)
  • Amicon Ultra centrifugal concentrators (MWCO, 10,000; Millipore, cat. no. UFC901096)

Support Protocol 2: Alexa Fluor532 Labeling of Go

 Materials
  • 1 to 10 mg of purified Go, preferably >90% homogeneous
  • Tris(2-carboxyethyl)phosphine (TCEP)
  • Reaction buffer: 50 mM HEPES, pH 7.4, at 4°C, 250 mM NaCl, 5% glycerol, supplemented with 10 µM guanosine diphosphate (GDP)
  • AlexaFluor532 maleimide, 1 mg (Invitrogen, cat. no. A10255)
  • Dimethyl sulfoxide (DMSO)
  • 1 M dithiothreitol (powdered DTT), aqueous
  • Storage buffer: 50 mM HEPES, pH 7.4, at 4°C, 250 mM NaCl, 5% glycerol supplemented with 1 mM TCEP and 10 µM GDP
  • Amicon ultra centrifugal concentrators (MWCO, 10,000; Millipore, cat. no. UFC901096)
  • 10 ml Sephadex G25 gel filtration column (essentially any standard desalting column can be used)
  • Spectrophotometer
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Figures

  •  FigureFigure 13.11.1 Schematic of FCPIA approach. Avidin-coated microspheres are labeled with biotinylated RGS proteins. The immobilized RGS proteins are incubated with Alexa Fluor-532-labeled Go. The ability of small molecules or competing unlabeled RGS proteins to inhibit this interaction can be characterized by their ability to diminish bead-associated Alexa Fluor-532 fluorescence. The assay can be expanded by labeling distinctly identifiable beads (e.g., Luminex bead regions) with different RGS proteins and performing the experiment in a multiplex format.
    View Image
  •  FigureFigure 13.11.2 Saturation of RGS19 binding to Go in the presence or absence of GDP, aluminum fluoride, and magnesium. Nonspecific binding is defined by residual affinity of RGS19 for Go-GDP in the absence of aluminum fluoride (–AMF). As can be observed, the interaction between RGS19 and Go binding is dependent on aluminum fluoride and is of high affinity (Kd ~ 12 nM).
    View Image
  •  FigureFigure 13.11.3 Characterization of the RGS19-GIPC protein-protein interaction. Saturation of RGS19 binding to GIPC is independent of which protein is immobilized on the bead. (A) Saturation of biotinylated RGS19 by Alexa Fluor-532-labeled GIPC. (B) Saturation of biotinylated GIPC by Alexa Fluor-532-labeled RGS19. Notice that there is no difference in Kd or Bmax. (C) Immobilized wild-type RGS19 is competed by unlabeled wild-type but not PDZ RGS19 (dc11RGS19) for binding to Alexa Fluor-532-labeled GIPC. Deletion of the PDZ domain abolishes the affinity of the wild-type RGS19.
    View Image
  •  FigureFigure 13.11.4 Multiplexed flow cytometry analysis during monoclonal antibody development. (A) Multiplexed titer analysis of sera from a representative mouse challenged with RGS19. (B) Representative single-point multiplexed hybridoma screening on 74 clones derived from the mouse in panel A. Notice the high specificity for the target antigen (RGS19) over MBP or RGS4-MBP fusion protein. This method allows a large number of hybridomas (>200) to be screened in a single day for antigenic specificity.
    View Image

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Literature Cited

Literature Cited
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    Blazer, L.L. and Neubig, R.R. 2008. Small molecule protein-protein interaction inhibitors as CNS therapeutic agents: Current progress and future hurdles. Neuropsychopharmacology 34:126-141.
    Buranda, T., Wu, Y., and Sklar, L.A. 2009. Chapter 11. Subsecond analyses of G-protein coupled-receptor ternary complex dynamics by rapid mix flow cytometry. Methods Enzymol. 461:227-247.
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    Jeanneteau, F., Diaz, J., Sokoloff, P., and Griffon, N. 2004a. Interactions of GIPC with dopamine D2, D3 but not D4 receptors define a novel mode of regulation of G protein-coupled receptors. Mol. Biol. Cell 15:696-705.
    Jeanneteau, F., Guillin, O., Diaz, J., Griffon, N., and Sokoloff, P. 2004b. GIPC recruits GAIP (RGS19) to attenuate dopamine D2 receptor signaling. Mol. Biol. Cell 15:4926-4937.
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    Roman, D.L., Ota, S., and Neubig, R.R. 2009. Polyplexed Flow Cytometry Protein Interaction Assay: A Novel High-Throughput Screening Paradigm for RGS Protein Inhibitors. J. Biomol. Screen. 14:610-619.
    Roof, R.A., Jin, Y., Roman, D.L., Sunahara, R.K., Ishii, M., Mosberg, H.I., and Neubig, R.R. 2006. Mechanism of action and structural requirements of constrained peptide inhibitors of RGS proteins. Chem. Biol. Drug Des. 67:266-274.
    Roof, R.A., Sobczyk-Kojiro, K., Turbiak, A.J., Roman, D.L., Pogozheva, I.D., Blazer, L.L., Neubig, R.R., and Mosberg, H.I. 2008. Novel peptide ligands of RGS4 from a focused one-bead, one-compound library. Chem. Biol. Drug Des. 72:111-119.
    Sarvazyan, N.A., Remmers, A.E., and Neubig, R.R. 1998. Determinants of gi1alpha and beta gamma binding. Measuring high affinity interactions in a lipid environment using flow cytometry. J. Biol. Chem. 273:7934-7940.
    Shankaranarayanan, A., Thal, D.M., Tesmer, V.M., Roman, D.L., Neubig, R.R., Kozasa, T., and Tesmer, J.J. 2008. Assembly of high order G alpha q-effector complexes with RGS proteins. J. Biol. Chem. 283:34923-34934.
    Shoichet, B.K. 2006. Screening in a spirit haunted world. Drug Discov. Today 11:607-615.
    Simons, P.C., Shi, M., Foutz, T., Cimino, D.F., Lewis, J., Buranda, T., Lim, W.K., Neubig, R.R., McIntire, W.E., Garrison, J., Prossnitz, E., and Sklar, L.A. 2003. Ligand-receptor-G-protein molecular assemblies on beads for mechanistic studies and screening by flow cytometry. Mol. Pharmacol. 64:1227-1238.
    Sklar, L.A., Edwards, B.S., Graves, S.W., Nolan, J.P., and Prossnitz, E.R. 2002. Flow cytometric analysis of ligand-receptor interactions and molecular assemblies. Annu. Rev. Biophys. Biomol. Struct. 31:97-119.
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