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

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

?Regulator of G?protein Signaling? (RGS) proteins constitute a class of intracellular signaling regulators that accelerate GTP hydrolysis by heterotrimeric G? 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/G? interactions (most avid in vitro when G? 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 ? subunits; Förster resonance energy transfer (FRET); single?turnover GTP hydrolysis; fluorescence polarization (FP)

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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 MgCl₂ , 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 MgCl₂ , 400 µM GTPγS
GTP[γ‐³² P] (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)
Gα_i1 N1 buffer (see recipe )
Gα_i1 N2 buffer (see recipe )
Gα_i1 QA buffer (see recipe )
Gα_i1 QB buffer (see recipe )
Gα_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

Figure 2.8.1 Example of compound screen setup for Gα_i1 and RGS4. (A ) Add 2 µl test compounds to rows A‐P/columns 3‐22 and add 2 µl DMSO control to rows A‐P/columns 1, 2, 23, and 24. (B ) Add 18 µl tracer control (T) and buffer control (B) to rows A‐C/column 1 and rows D‐F/column 1, respectively. Add 18 µl of Gα_i1 alone control to all rows in column 23. Add 18 µl of Gα_i1 + RGS4 mix to rows A‐P/columns 2‐22.

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Figure 2.8.2 Gα_i1 ‐CFP and YFP‐RGS4 emission spectra in GDP and AMF buffers. 500 nM Gα_i1 ‐CFP and 900 nM YFP‐RGS4 were excited at 433 nm and emission spectra measured with a cuvette‐based PerkinElmer LS‐55 spectrometer. In the presence of AMF, RGS4 and Gα_i1 bind with high affinity, leading to FRET between their fluorescent tags and an increased FRET ratio [(525‐nm emission)/(474‐nm emission)] compared to GDP buffer.

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Figure 2.8.3 Example RGS protein/Gα FRET experimental setup. (A ) Each reaction mixture consists of 150 µl 3× RGS protein solution, 150 µl 3× Gα_i1 , and compound or DMSO to the desired test concentration. The total reaction volume is 450 µl. (B ) The reaction mixtures, in both AMF and GDP buffers, are distributed in triplicate across a 96‐well plate. The shading gradient represents varying concentrations of test compound.

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Figure 2.8.4 Inhibition of Gα_i1 /RGS4 interaction by the cysteine‐reactive compound CCG‐4986. A previously identified compound, CCG‐4986, (Roman et al., ) covalently modifies surface cysteines on RGS4, preventing association with Gα_i1 (Kimple et al., ). The FRET method allows determination of an IC₅₀ for RGS protein inhibition.

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Figure 2.8.5 RGS4 is allosterically modulated by phospholipids in single‐turnover hydrolysis assays. Phosphatidyl inositol triphosphate (PIP₃ ) modulates RGS4 activity though an allosteric “B” site, described previously (Ishii and Kurachi, ; Tu and Wilkie, ). In single‐turnover assays, inclusion of phospholipid vesicles containing PIP₃ , but not phosphatidyl choline (PC) only, inhibits RGS4 GAP activity. Similar reversal of RGS4‐mediated GTP hydrolysis acceleration would be expected for an inhibitory compound.

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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.