Elsevier

Methods in Enzymology

Volume 389, 2004, Pages 229-243
Methods in Enzymology

RGS-Insensitive G-Protein Mutations to Study the Role of Endogenous RGS Proteins

https://doi.org/10.1016/S0076-6879(04)89014-1Get rights and content

Abstract

Regulator of G-protein signaling (RGS) proteins are very active GTPase-accelerating proteins (GAPs) in vitro and are expected to reduce signaling by G-protein coupled receptors in vivo. A novel method is presented to assess the in vivo role of RGS proteins in the function of a G protein in which Gα subunits do not bind to RGS proteins or respond with enhanced GTPase activity. A point mutation in the switch I region of Gα subunits (G184S Gαo and G183S Gαi1) blocks the interaction with RGS proteins but leaves intact the ability of Gα to couple to βγ subunits, receptors, and downstream effectors. Expression of the RGS-insensitive mutant G184S Gαo in C6 glioma cells with the μ-opioid receptor dramatically enhances adenylylcyclase inhibition and activation of extracellular regulated kinase. Introducing the same G184S Gαo protein into embryonic stem (ES) cells by gene targeting allows us to assess the functional importance of the endogenous RGS proteins using in vitro differentiation models and in intact mice. Using ES cell-derived cardiocytes, spontaneous and isoproterenol-stimulated beating rates were not different between wild-type and G184S Gαo mutant cells; however, the bradycardiac response to adenosine A1 receptor agonists was enhanced significantly (seven-fold decrease EC50) in GαoRGSi mutant cells compared to wild-type Gαo, indicating a significant role of endogenous RGS proteins in cardiac automaticity regulation. The approach of using RGS-insensitive Gα subunit knockins will reveal the role of RGS protein-mediated GAP activity in signaling by a given Gi⧸o protein. This will reveal the full extent of RGS regulation and will not be confounded by redundancy in the function of multiple RGS proteins.

Introduction

Regulator of G-protein signaling (RGS) proteins modulate the strength and duration of G-protein-mediated signaling by acting as GTPase-accelerating proteins (GAP) though their conserved RGS domain. In addition to their role as negative regulators, RGS proteins also serve as versatile modulators and integrators of G-protein signaling via their diverse functional domains and structural motifs (Hollinger and Hepler, 2002). Considerable knowledge has been gained regarding the biochemical mechanisms of RGS protein stimulation of the GTPase activity of Gα (Ross and Wilkie, 2000). In addition, studies have begun to reveal the physiological roles of RGS proteins (reviewed by Hollinger 2002, Zhong 2001). Much previous work, however, uses overexpression of the individual RGS protein, which complicates conclusions regarding the functional importance of endogenous RGS proteins in vivo. A few RGS knockout mice (i.e., RGS2, RGS9, and RGS14) have been generated (Chen et al., 2000; Oliveira-dos-Santos et al., 2000; Siderovski, personal communication). RGS2 and RGS9 knockout phenotypes are consistent with a negative regulatory function on the vasoconstrictor (Heximer 2003, Tang 2003) and dopamine (Martemyanov 2003, Rahman 2003) signaling, respectively. However, RGS proteins may be functionally redundant, which may make single knockouts of limited value in assessing the overall role of RGS protein function in vivo. Thus, knowledge of the role of RGS proteins in vivo is still in its infancy.

Section snippets

Characterization of an RGS-Insensitive Mutation

The defining feature of RGS proteins is the ∼120 amino acid-conserved RGS domain, which binds preferentially to the transition state of a Gα subunit and accelerates its intrinsic GTPase activity (Berman 1996, Hepler 1997, Hunt 1996). Yeast also has an RGS-regulated G-protein signaling system that is involved in mating pheromone responses. Disruption of the SST2 (“supersensitivity to pheromone-2”) gene leads to enhanced pheromone signaling in terms of both ligand concentration and duration of

Studying the Function of Endogenous RGS Proteins by Transfecting the RGSi Gαo Mutant into Cells

Assessing the role of transfected G proteins in the face of endogenous G proteins is difficult. This problem has been solved by the use of pertussis toxin-insensitive (PTXi) mutants of the Gi⧸o family G proteins (Milligan, 1988). PTX treatment allows examination of responses mediated through the exogenous, expressed G protein without interference by endogenous Gi⧸o proteins. Using cultured hippocampal neurons, Chen and Lambert (2000) employed doubly mutated RGSi⧸PTXi Gα proteins expressed by

Acknowledgements

Supported by NIH Grants GM39561 (RRN), T32HL007853 (XH), MDRTC Grant (NIH P60 DK20572), and an AHA Predoctoral fellowship (YF). We thank Min Liu, Elizabeth Hughes, and Ginny Zawistowski for their technical support.

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