Assays of RGS Box Activity and Allosteric Control
Fluorescence-Based Assays for RGS Box Function

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Abstract

Ligand-activated, seven transmembrane-spanning receptors interact with inactive G-protein heterotrimers (Gαβγ) to catalyze GTP loading and, consequently, activation of Gα subunits and the liberation of Gβγ. Gα·GTP and Gβγ are then competent to regulate independent effector pathways. The duration of heterotrimeric G-protein signaling is determined by the lifetime of the Gα subunit in the GTP-bound state. Signal termination is facilitated by the intrinsic guanosine triphosphatase (GTPase) activity of Gα and subsequent reformation of the inactive heterotrimer. Regulators of G-protein signaling (RGS) proteins act enzymatically, via their hallmark “RGS box,” as GTPase-accelerating proteins (GAPs) for Gα subunits and thus function as negative regulators of G-protein signaling in vitro and in vivo. This article describes the use of fluorescence resonance energy transfer (FRET) to monitor the interaction between a Gα subunit and an RGS box protein. Furthermore, this article describes optimization of this assay for high-throughput screening and the evaluation of mutant RGS box and Gα proteins. Finally, this article describes the novel application of this FRET technique to measure the activity of RGS protein-derived GoLoco peptides that modulate Gα activation by aluminum tetrafluoride.

Introduction

Seven transmembrane-spanning receptors couple to second messenger pathways via heterotrimeric, guanine nucleotide-binding proteins (G proteins), which are composed of three subunits termed Gα, Gβ, and Gγ (Gilman 1987, Pierce 2002). The Gα subunit is a molecular switch that is conformationally sensitive to guanine nucleotides. GDP-bound Gα is inactive and tightly associated with Gβγ. Activated receptors catalyze guanine nucleotide exchange on the GDP-bound heterotrimer. GTP binding to Gα is thought to cause subunit dissociation and the activation of second messenger pathways by both Gα·GTP and Gβγ. Signal termination is facilitated by the intrinsic guanosine triphosphatase (GTPase) activity of Gα. Thus, the duration of heterotrimeric G-protein signaling is determined by the lifetime of the Gα subunit in the GTP-bound state.

Regulator of G-protein signaling (RGS) proteins, characterized by their hallmark, ∼120 amino acid “RGS box,” are GTPase-accelerating proteins (GAPs) for Gα subunits and thus act as negative regulators of G-protein signaling in vitro and in vivo (Neubig and Siderovski, 2002). Given their dynamic spatiotemporal regulation and receptor selectivity, RGS proteins represent promising targets for developing new therapeutic interventions (Neubig and Siderovski, 2002). In vitro measurements of RGS box GAP activity generally encompass radiolabeled guanine nucleotide hydrolysis assays (Berman et al., 1996a), coprecipitation assays (Druey and Kehrl, 1997), or surface plasmon resonance (Kimple 2001, Popov 1997; see also this volume, Willard and Siderovski, 2004). Measurement of the RGS box-catalyzed nucleotide cycle has also been performed using both Gα-subunit intrinsic fluorescence (Lan et al., 2000) and quench flow kinetic methods (Mukhopadhyay and Ross, 1999). This article describes methods for the use of fluorescence resonance energy transfer (FRET) to monitor the archetypal Gα⧸RGS box pair, namely the Gαi1⧸RGS4 interaction (Tesmer et al., 1997), both kinetically and at end point using a high-throughput methodology. Finally, we examine the application of this FRET technique to assay GoLoco peptides derived from RGS proteins that inhibit Gαi1 activation.

Section snippets

Materials

Unless otherwise specified, all chemicals are of the highest purity obtainable from Sigma (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA).

Fluorescence Resonance Energy Transfer Assay Design Considerations

Fluorescence resonance energy transfer is the nonradiative transfer of energy from a donor fluorophore to an acceptor fluorophore. For a comprehensive discussion of the physical principles and practicalities of FRET, the reader is directed to the excellent resources of dos Remedios and Lakowicz (1999) and Moens (1995). Essentially, according to the Förster equation, FRET will be determined by (1) donor fluorophore quantum yield, (2) donor⧸acceptor spectral overlap, (3) physical distance between

Cloning and Purification of Gαi1-CFP and YFP-RGS4

To create an expression construct for the YFP-RGS4 fusion protein, the citrine mutant of YFP [amino acids 1–237 of the pEYFP-C1 open reading frame (ORF); BD Biosciences Clontech, Palo Alto, CA] with a Q69M mutation (Heikal et al., 2000) and the entire ORF of mouse RGS4 (amino acids 1–205; SwissProt RGS4_MOUSE) were cloned in-frame with the N-terminal hexahistidine tag (His6) and tobacco-etch virus (TEV) protease cleavage site of the prokaryotic expression vector pProEXHTb (Invitrogen, Carlsbad,

FRET-Based Assay of RGS4⧸Gαi1 Interaction

Measurements of CFP and YFP fluorescence are made in a LS55 luminescence spectrofluorimeter (Perkin Elmer, Boston, MA). The LS55 cuvette holder is water jacketed and thus can be connected to a circulating water bath to provide controlled temperature between 4 and 40°. Proteins are incubated in 2-ml quartz cuvettes (Fisher) with magnetic stirring bars (Fisher) containing 1 ml of buffer F (10 mM Tris–HCl, pH 8.0, 1 mM EDTA, 50 mM NaCl, 10 mM MgCl2, 2 μM GDP). The water bath temperature is set to

Development of a High-Throughput FRET Assay for Gαi1⧸RGS4 Interaction

Aluminum tetrafluoride-dependent FRET between Gαi1-CFP and YFP-RGS4 can be measured in a 96-well plate format using a SpectraMax Gemini fluorescence plate reader (Molecular Devices, Sunnyvale, CA). Samples are placed in Costar 96-well, flat-bottom, “special optics” black plates (Corning, Acton, MA). The SpectraMax Gemini is first optimized for CFP⧸YFP FRET using the SPECTRUM (FLUORESCENCE) function under the INSTRUMENT SETTINGS dialog box in the SOFTmax PRO v3.11 control software (Molecular

Use of RGS Box Proteins as Biosensors for G-Protein Activation

The FRET assay for the Gα⧸RGS box interaction can also be utilized to assay the effect of G-protein modulatory peptides on Gα function. We have explored this approach using a synthetic peptide derived from the GoLoco motif of RGS12. GoLoco motifs are guanine nucleotide dissociation inhibitors (GDIs) for the adenylyl cyclase inhibitory class of G-protein α subunits. GoLoco motif-containing proteins have essential roles in metazoan cell division processes via their regulation of the

Concluding Remarks

We have described protocols for the accurate measurement of the interaction between Gαi1 and RGS4 using FRET. This assay is effective in measuring the activities of both mutant Gαi1 (i.e., G183S) and mutant RGS4 (i.e., R167M) proteins and should be amenable to any Gα⧸RGS box pair that can be purified as soluble recombinant proteins. There remains the potential for this assay to be modified and utilized in vivo to detect receptor-mediated heterotrimer activation and we are currently pursuing

Acknowledgements

We thank Dr. Catherine Berlot for the provision of ECFP and EYFP cDNAs (Weis Centre for Research, Danville, PA). FSW is an American Heart Association Postdoctoral Fellow. RJK is supported by a predoctoral fellowship from the National Institutes of Mental Health (F30 MH64319). This work was funded by NIH Grants R01 GM062338 and P01 GM065533. Thanks to Dr. Benjamin Yerxa and the rest of Inspire Pharmaceuticals Inc. for support in the initial phase of this project. DPS is a recipient of the

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