Real-time visualization of heterotrimeric G protein Gq activation in living cells
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METHODOLOGY ARTICLE
Open Access
Real-time visualization of heterotrimeric G protein Gq activation in living cells Merel JW Adjobo-Hermans1,2, Joachim Goedhart1, Laura van Weeren1, Saskia Nijmeijer3, Erik MM Manders1, Stefan Offermanns4 and Theodorus WJ Gadella Jr1*
Abstract Background: Gq is a heterotrimeric G protein that plays an important role in numerous physiological processes. To delineate the molecular mechanisms and kinetics of signalling through this protein, its activation should be measurable in single living cells. Recently, fluorescence resonance energy transfer (FRET) sensors have been developed for this purpose. Results: In this paper, we describe the development of an improved FRET-based Gq activity sensor that consists of a yellow fluorescent protein (YFP)-tagged Gg2 subunit and a Gaq subunit with an inserted monomeric Turquoise (mTurquoise), the best cyan fluorescent protein variant currently available. This sensor enabled us to determine, for the first time, the kon (2/s) of Gq activation. In addition, we found that the guanine nucleotide exchange factor p63RhoGEF has a profound effect on the number of Gq proteins that become active upon stimulation of endogenous histamine H1 receptors. The sensor was also used to measure ligand-independent activation of the histamine H1 receptor (H1R) upon addition of a hypotonic stimulus. Conclusions: Our observations reveal that the application of a truncated mTurquoise as donor and a YFP-tagged Gg2 as acceptor in FRET-based Gq activity sensors substantially improves their dynamic range. This optimization enables the real-time single cell quantification of Gq signalling dynamics, the influence of accessory proteins and allows future drug screening applications by virtue of its sensitivity.
Background Heterotrimeric G proteins are composed of Ga subunits and Gbg dimers, and can be activated by G-proteincoupled receptors (GPCRs). Upon binding of an agonist, the receptor changes its conformation, and acts as a guanine nucleotide exchange factor (GEF). By inducing the exchange of guanine diphosphate (GDP) for guanine triphosphate (GTP) in the Ga subunit, the G protein becomes active [1]. Gbg interacts with Ga through two interfaces: the switch region and the region formed by the N terminus of the Ga subunit. Binding of GTP upon receptor activation disrupts the switch interface, which is thought to trigger the dissociation of Gbg from Ga [2]. The family of Ga subunits consists of four classes; the subunits Gaq, Ga11, Ga14 and Ga16 * Correspondence: [email protected] 1 Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, The Netherlands Full list of author information is available at the end of the article
belong to the Gq class. The principal target of the Gq class is phospholipase (PL)Cb [3]. Recently, RhoGEF proteins such as leukemia-associated Rho-guanine nucleotide exchange factor (LARG) and p63RhoGEF have been shown to direc
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