Purification and In Vitro Functional Analysis of the Arabidopsis thaliana Regulator of G-Protein Signaling-1

doi:10.1016/S0076-6879(04)89019-0

Methods in Enzymology

Volume 389, 2004, Pages 320-338

https://doi.org/10.1016/S0076-6879(04)89019-0 Get rights and content

Abstract

The model organism Arabidopsis thaliana contains a restricted set of heterotrimeric G-protein subunits, with only one canonical Gα subunit (AtGPA1), one Gβ subunit (AtAGB1), and two Gγ subunits (AtAGG1 and AtGG2) identified. We have identified a novel additional component of heterotrimeric G-protein signaling in the A. thaliana genome, regulator of G-protein signaling-1 (AtRGS1). This protein has the predicted topology and structure of a G-protein-coupled receptor in that it contains seven transmembrane domains, but AtRGS1 also contains a unique C-terminal extension, namely a regulator of G-protein signaling domain (RGS box). This article describes methods for the purification and in vitro functional analysis of the RGS box of AtRGS1.

Introduction

Yeast, plants, and metazoan organisms all use heterotrimeric G-protein signaling for signal processing and homeostasis. However, the model organism Arabidopsis thaliana (thale cress) contains an unusually restricted complement of G-protein signaling pathway components. Arabidopsis contains only one canonical Gα subunit (AtGPA1), one Gβ subunit (AtAGB1), and two Gγ subunits (AtAGG1 and AtGG2) (Jones, 2002). Enigmatically, neither a G-protein-coupled receptor (GPCR) nor a direct Gα or Gβγ effector has been described for Arabidopsis (reviewed in Jones, 2002). We have completed in vitro and in vivo functional analyses of the Arabidopsis regulator of G-protein signaling-1 [AtRGS1 (Chen et al., 2003)]. AtRGS1 has predicted topological and structural similarity to GPCRs in that it contains seven transmembrane domains, but uniquely it contains a C-terminal regulator of G-protein signaling domain (or “RGS box”). This article describes methods for the identification, purification, and in vitro functional analysis of the RGS box of AtRGS1. AtGPA1 and the RGS box of AtRGS1 can be purified following overexpression in Escherichia coli. Complex formation between AtGPA1 and the AtRGS1 RGS box can be quantified using coprecipitation and surface plasmon resonance approaches. AtRGS1 RGS box-catalyzed GTPase-accelerating protein (GAP) activity on AtGPA1 can be measured in single turnover GAP assays using a fluorescent biosensor for inorganic phosphate. This article also describes fluorescence-based methods for using 2′-(or-3′)-O-(N-methylanthraniloyl)guanosine 5′-triphosphate (MANT-GTP) to measure AtRGS1-catalyzed GAP activity on AtGPA1.

Section snippets

Materials

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

Bioinformatic Identification and Analysis of AtRGS1

A search for A. thaliana open reading frames (ORF) with similarity to an RGS box-hidden Markov model was performed using the Simple Modular Architecture Research Tool (SMART) web server (http:⧸⧸smart.embl-heidelberg.de) (Letunic et al., 2002). This search revealed a ORF of 459 amino acids, hereafter called AtRGS1, that encodes an extended N-terminal region with seven predicted transmembrane domains and a C-terminal RGS box (Fig. 1A). A database search of Arabidopsis ORFs using the N- and

Purification of AtGPA1 and AtRGS1

AtGPA1 and AtRGS1 are purified following overexpression in E. coli as hexahistidine (His₆) and glutathione-S-transferase (GST) fusion proteins, respectively. Using conventional recombinant DNA methods, the ORF of AtGPA1 is force directionally cloned into the EcoRI and XhoI sites of pPROEXHTb (Invitrogen, Carlsbad, CA). pPROEXHT vectors provide fusion proteins with a N-terminal tobacco etch virus (TEV)-protease cleavable His₆ tag. Amino acids 249–459 encompassing the RGS box of AtRGS1 are cloned

Coprecipitation Assay

The nucleotide-dependent interaction between GST-AtRGS1(249–459) and His₆-AtGPA1 can be determined using coprecipitation with glutathione agarose (known idiomatically as the “GST-pulldown” assay). RGS proteins have a characteristic high affinity toward Gα · GDP · AlF₄⁻ but low affinity for either the GDP- or the GTPγS-bound forms of Gα. RGS proteins act catalytically by stabilizing the transition state for nucleotide hydrolysis (Berman et al., 1996). The Gα · GDP · AlF₄⁻ · Mg²⁺ complex mimics

Single Turnover GAP Assays using E. coli Phosphate-Binding Protein

RGS boxes accelerate the GTPase activity of their cognate Gα partners. Standard assays of RGS protein-mediated GAP activity rely on the release of ³²P-labeled inorganic phosphate (P_i) from [γ-³²P]GTP-bound G-protein α subunits (Krumins 2002, Ross 2002). Generally, this assay of GAP activity is conducted in a single turnover format, as nucleotide release from Gα, rather than GTP hydrolysis, is the rate-limiting step in the G-protein cycle in the absence of guanine nucleotide exchange factors

Acknowledgements

We thank Dr. Martin R. Webb (National Institute of Medical Research, Mill Hill, UK) for provision of the PBP expression construct and Drs. Adam Shutes (UNC) and Krister Wennerburg (UNC) for assistance with the production of MDCC-PBP. Thanks also to Dr. Alan Jones (UNC) for the construct pDEST15-AtRGS1(249–459) and the UNC Department of Pharmacology Protein Core for provision of instrumentation. FSW is an American Heart Association Postdoctoral Fellow. This work was funded by NIH Grants R01

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Purification and In Vitro Functional Analysis of the Arabidopsis thaliana Regulator of G-Protein Signaling-1

Abstract

Introduction

Section snippets

Materials

Bioinformatic Identification and Analysis of AtRGS1

Purification of AtGPA1 and AtRGS1

Coprecipitation Assay

Single Turnover GAP Assays using E. coli Phosphate-Binding Protein

Acknowledgements

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