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Real-time visualization of heterotrimeric G protein Gq activation in living cells

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      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 Gγ2 subunit and a Gαq 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 k on (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 Gγ2 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.

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      Most cited references 69

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      Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells.

      Many proteins associated with the plasma membrane are known to partition into submicroscopic sphingolipid- and cholesterol-rich domains called lipid rafts, but the determinants dictating this segregation of proteins in the membrane are poorly understood. We suppressed the tendency of Aequorea fluorescent proteins to dimerize and targeted these variants to the plasma membrane using several different types of lipid anchors. Fluorescence resonance energy transfer measurements in living cells revealed that acyl but not prenyl modifications promote clustering in lipid rafts. Thus the nature of the lipid anchor on a protein is sufficient to determine submicroscopic localization within the plasma membrane.
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        Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins.

        Fluorescence resonance energy transfer (FRET) technology has been used to develop genetically encoded fluorescent indicators for various cellular functions. Although most indicators have cyan- and yellow-emitting fluorescent proteins (CFP and YFP) as FRET donor and acceptor, their poor dynamic range often prevents detection of subtle but significant signals. Here, we optimized the relative orientation of the two chromophores in the Ca(2+) indicator, yellow cameleon (YC), by fusing YFP at different angles. We generated circularly permuted YFPs (cpYFPs) that showed efficient maturation and acid stability. One of the cpYFPs incorporated in YC absorbs a great amount of excited energy from CFP in its Ca(2+)-saturated form, thereby increasing the Ca(2+)-dependent change in the ratio of YFP/CFP by nearly 600%. Both in cultured cells and in the nervous system of transgenic mice, the new YC enables visualization of subcellular Ca(2+) dynamics with better spatial and temporal resolution than before. Our study provides an important guide for the development and improvement of indicators using GFP-based FRET.
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          The 2.0 A crystal structure of a heterotrimeric G protein.

          The structure of a heterotrimeric G protein reveals the mechanism of the nucleotide-dependent engagement of the alpha and beta gamma subunits that regulates their interaction with receptor and effector molecules. The interaction involves two distinct interfaces and dramatically alters the conformation of the alpha but not of the beta gamma subunits. The location of the known sites for post-translational modification and receptor coupling suggest a plausible orientation with respect to the membrane surface and an activated heptahelical receptor.
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            Author and article information

            Affiliations
            [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
            [2 ]Nijmegen Centre for Molecular Life Sciences, Department of Biochemistry, Radboud University Nijmegen Medical Centre, Geert Grooteplein 28, 6525 GA Nijmegen, The Netherlands
            [3 ]Department of Chemistry and Pharmaceutical Sciences, Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
            [4 ]Department of Pharmacology, Max-Planck-Institute for Heart and Lung Research, Ludwigstrasse 43, 61231 Bad Nauheim, Germany
            Contributors
            Journal
            BMC Biol
            BMC Biology
            BioMed Central
            1741-7007
            2011
            27 May 2011
            : 9
            : 32
            3129320
            1741-7007-9-32
            21619590
            10.1186/1741-7007-9-32
            Copyright ©2011 Adjobo-Hermans et al; licensee BioMed Central Ltd.

            This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

            Categories
            Methodology Article

            Life sciences

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