Evaluating Modulators of “Regulator of G‐protein Signaling” (RGS) Proteins

Dustin E. Bosch1, Thomas Zielinski2, Robert G. Lowery2, David P. Siderovski1

1 UNC Neuroscience Center and Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 2 BellBrook Labs, Madison, Wisconsin
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 2.8
DOI:  10.1002/0471141755.ph0208s56
Online Posting Date:  March, 2012
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Abstract

“Regulator of G‐protein Signaling” (RGS) proteins constitute a class of intracellular signaling regulators that accelerate GTP hydrolysis by heterotrimeric Gα subunits. In recent years, RGS proteins have emerged as potential drug targets for modulation by small molecules. Described in this unit are high‐throughput screening procedures for identifying modulators of RGS protein‐mediated GTPase acceleration (GAP activity), for assessment of RGS domain/Gα interactions (most avid in vitro when Gα is bound by aluminum tetrafluoride), and for validation of candidate GAP‐modulatory molecules with the single‐turnover GTP hydrolysis assay. Curr. Protoc. Pharmacol. 56:2.8.1‐2.8.15. © 2012 by John Wiley & Sons, Inc.

Keywords: regulator of G‐protein signaling (RGS) proteins; heterotrimeric G‐protein α subunits; Förster resonance energy transfer (FRET); single‐turnover GTP hydrolysis; fluorescence polarization (FP)

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Identifying Candidate RGS Protein Modulators with the Transcreener GDP Assay and a Rate‐Modified Gα Subunit
  • Basic Protocol 2: Measuring Disruption of the RGS Domain/Gα Interaction by Förster Resonance Energy Transfer (FRET)
  • Basic Protocol 3: Measuring Modulation of GAP Activity by Single‐Turnover GTP Hydrolysis
  • Support Protocol 1: Purification of Gαi1, Gαi1(R178M/A326S), and Gαi1‐CFP
  • Support Protocol 2: Purification of RGS4 and YFP‐RGS4
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Identifying Candidate RGS Protein Modulators with the Transcreener GDP Assay and a Rate‐Modified Gα Subunit

  Materials
  • Compound library (e.g., Sigma LOPAC or ChemBridge DIVERSet)
  • Dimethyl sulfoxide (DMSO)
  • Assay buffer, final concentration in 20‐µl reaction: 10 mM Tris⋅Cl (pH 7.5), 1 mM EDTA, 10 mM MgCl 2, 10 µM GTP, 10 µg/ml GDP antibody, and 2 nM fluorescent nucleotide tracer; for the protocol below, this will be prepared as a 1.1× solution (18 µl added to a final 20 µl volume)
  • Transcreener GDP FP Assay kit (Bellbrook Labs, cat. no. 3009‐1K) containing:
    • GDP antibody
    • Tracer
  • Recombinant Gα i1(R178M/A326S) subunit (purified as in Zielinski et al., ; also see protocol 4)
  • Recombinant RGS4 protein (purified as in Zielinski et al., ; also see protocol 5)
  • Black, 384‐well assay plates with a nonbinding surface (Corning, cat. no. 3676)
  • Fluorescence polarization‐capable plate reader (e.g., Tecan Safire2, BMG PHERAstar Plus)

Basic Protocol 2: Measuring Disruption of the RGS Domain/Gα Interaction by Förster Resonance Energy Transfer (FRET)

  Materials
  • GDP buffer (see recipe)
  • AMF (aluminum, magnesium, and fluoride) buffer (see recipe)
  • Recombinant YFP‐RGS4 fusion protein (purified as in Willard et al., ; see protocol 5)
  • Recombinant Gα i1‐CFP fusion protein (purified as in Willard et al., ; see protocol 4]
  • Candidate modulatory compounds in solution [assay is robust to ∼5% (v/v) DMSO]
  • 1.5‐ml microcentrifuge tubes
  • Black polystyrene 96‐well plates (e.g., Costar, Corning)
  • Fluorescence dual emission plate reader [e.g., POLARstar Omega (BMG Labtech), EnVision (PerkinElmer)]

Basic Protocol 3: Measuring Modulation of GAP Activity by Single‐Turnover GTP Hydrolysis

  Materials
  • Recombinant Gα i1 protein (purified as in Kimple et al., ; see protocol 4)
  • Buffer C (see recipe)
  • Recombinant RGS4 protein (purified as in Kimple et al., ; see protocol 5)
  • Candidate modulatory compounds (assay is robust to at least 10% DMSO; Blazer et al., )
  • Buffer D: 40 mM MgCl 2, 400 µM GTPγS
  • GTP[γ‐32P] (PerkinElmer product no. BLU004Z250UC, specific activity of 6000 Ci/mmol)
  • Ice
  • Charcoal slurry: 5% (w/v) activated charcoal (e.g., Sigma, cat. no. C4386), 50 mM phosphoric acid, pH 3.0; stored stably long‐term at 4°C
  • Scintillation fluid (e.g., Fisher Scintisafe gel)
  • Safety equipment and designated space for radioactive materials
  • Benchtop vortexer
  • 30°C water bath
  • 5‐ml polypropylene test tubes
  • Refrigerated centrifuge adapted for 5‐ml test tubes, suitable for radioactive materials (e.g., Hermle Z400K)
  • Scintillation vials
  • Scintillation counter

Support Protocol 1: Purification of Gαi1, Gαi1(R178M/A326S), and Gαi1‐CFP

  Materials
  • 100 ml of a BL21(DE3) E. coli (Novagen) overnight stock transformed with a vector encoding hexahistidine‐tagged Gα i1, Gα i1(R178M/A326S), or hexahistidine‐Gα i1 in frame with a C‐terminal cyan fluorescent protein
  • 4 liters Luria broth prepared and autoclaved in baffled 2.8‐liter flasks, supplemented with selection antibiotic (e.g., 50 µg/ml ampicillin)
  • 2 ml of 1 M IPTG (isopropyl‐beta‐D‐thiogalactopyranoside) stock solution (Acros Organics)
  • i1 N1 buffer (see recipe)
  • i1 N2 buffer (see recipe)
  • i1 QA buffer (see recipe)
  • i1 QB buffer (see recipe)
  • i1 S200 buffer (see recipe)
  • Liquid nitrogen or dry ice and ethanol bath
  • 37°C shaking incubator
  • Beckman J‐6 centrifuge
  • High‐pressure homogenizer (Emulsiflex, Avestin)
  • Beckman LM‐8 ultracentrifuge
  • HisTrap Fast Flow nickel nitriloacetic acid (NTA) column (GE Healthcare)
  • Akta FPLC (GE Healthcare)
  • Superdex 200 gel filtration column (GE Healthcare)
  • Source 15Q anion‐exchange column (GE Healthcare)
  • Vivaspin 20 ultrafiltration spin column (Sartorius Stedim Biotech) with 10‐kDa molecular weight (MW) cutoff

Support Protocol 2: Purification of RGS4 and YFP‐RGS4

  Materials
  • 100 ml of a BL21(DE3) E. coli (Novagen) overnight stock transformed with a vector encoding hexahistidine‐tagged RGS4, or hexahistidine‐RGS4 in‐frame with an N‐terminal yellow fluorescent protein
  • Four liters Luria broth prepared and autoclaved in baffled 2.8‐liter flasks, supplemented with selection antibiotic (e.g., 50 µg/ml ampicillin)
  • 2 ml of 1 M IPTG (isopropyl‐beta‐D‐thiogalactopyranoside) stock solution (Acros Organics)
  • RGS4 N1 buffer (see recipe)
  • RGS4 N2 buffer (see recipe)
  • RGS4 QA buffer (see recipe)
  • RGS4 QB buffer (see recipe)
  • RGS4 S200 buffer (see recipe)
  • Liquid nitrogen or dry ice and ethanol bath
  • 37°C shaking incubator
  • Beckman J‐6 centrifuge
  • High‐pressure homogenizer (Emulsiflex, Avestin)
  • Beckman LM‐8 ultracentrifuge
  • HisTrap Fast Flow nickel nitriloacetic acid (NTA) column (GE Healthcare)
  • Akta FPLC (GE Healthcare)
  • Superdex 200 gel filtration column (GE Healthcare)
  • Source 15Q anion‐exchange column (GE Healthcare)
  • Vivaspin 20 ultrafiltration spin column (Sartorius Stedim Biotech) with 10 kDa molecular weight (MV) cutoff
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Figures

Videos

Literature Cited

   Berman, D.M., Wilkie, T.M., and Gilman, A.G. 1996. GAIP and RGS4 are GTPase‐activating proteins for the Gi subfamily of G protein alpha subunits. Cell 86:445‐452.
   Blazer, L.L., Roman, D.L., Chung, A., Larsen, M.J., Greedy, B.M., Husbands, S.M., and Neubig, R.R. 2010. Reversible, allosteric small‐molecule inhibitors of regulator of G protein signaling proteins. Mol. Pharmacol. 78:524‐533.
   Ishii, M. and Kurachi, Y. 2004. Assays of RGS protein modulation by phosphatidylinositides and calmodulin. Methods Enzymol. 389:105‐118.
   Kimple, A.J., Willard, F.S., Giguère, P.M., Johnston, C.A., Mocanu, V., and Siderovski, D.P. 2007. The RGS protein inhibitor CCG‐4986 is a covalent modifier of the RGS4 Gα‐interaction face. Biochim. Biophys. Acta 1774:1213‐1220.
   Kimple, R.J., De Vries, L., Tronchere, H., Behe, C.I., Morris, R. A., Gist Farquhar, M., and Siderovski, D.P. 2001. RGS12 and RGS14 GoLoco motifs are G alpha(i) interaction sites with guanine nucleotide dissociation inhibitor activity. J. Biol. Chem. 276:29275‐29281.
   Oldham, W.M. and Hamm, H.E. 2008. Heterotrimeric G protein activation by G‐protein‐coupled receptors. Nat. Rev. Mol. Cell. Biol. 9:60‐71.
   Popov, S., Yu, K., Kozasa, T., and Wilkie, T.M. 1997. The regulators of G protein signaling (RGS) domains of RGS4, RGS10, and GAIP retain GTPase activating protein activity in vitro. Proc. Natl. Acad. Sci. U.S.A. 94:7216‐7220.
   Roman, D.L., Talbot, J.N., Roof, R.A., Sunahara, R.K., Traynor, J.R., and Neubig, R.R. 2007. Identification of small‐molecule inhibitors of RGS4 using a high‐throughput flow cytometry protein interaction assay. Mol. Pharmacol. 71:169‐175.
   Tu, Y. and Wilkie, T.M. 2004. Allosteric regulation of GAP activity by phospholipids in regulators of G‐protein signaling. Methods Enzymol. 389:89‐105.
   Willard, F.S., Kimple, R.J., Kimple, A.J., Johnston, C.A., and Siderovski, D.P. 2004. Fluorescence‐based assays for RGS box function. Methods Enzymol. 389:56‐71.
   Zhang, J.H., Chung, T.D., and Oldenburg, K.R. 1999. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen. 4:67‐73.
   Zielinski, T., Kimple, A.J., Hutsell, S.Q., Koeff, M.D., Siderovski, D.P., and Lowery, R.G. 2009. Two Gα(i1) rate‐modifying mutations act in concert to allow receptor‐independent, steady‐state measurements of RGS protein activity. J. Biomol. Screen. 14:1195‐1206.
Key Reference
   Kimple, A.J., Bosch, D.E., Giguère, P.M., and Siderovski, D.P. 2011. Regulators of G‐protein signaling and their Gα substrates: Promises and challenges in their use as drug discovery targets. Pharmacol. Rev. 63:728‐749.
  A recent review of the RGS proteins focused on the rationale for their consideration as valuable drug discovery targets and current efforts to identify chemical probes that modify RGS protein GAP activity.
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