Abstract
Several cytoplasmic proteins that are involved in G protein-coupled receptor signaling cascades are known to translocate to the plasma membrane upon receptor activation, such as beta-arrestin2. Based on this example and in order to identify new cytoplasmic proteins implicated in the ON-and-OFF cycle of G protein-coupled receptor, a live-imaging screen of fluorescently labeled cytoplasmic proteins was performed using translocation criteria. The screening of 193 fluorescently tagged human proteins identified eight proteins that responded to activation of the tachykinin NK2 receptor by a change in their intracellular localization. Previously we have presented the functional characterization of one of these proteins, REDD1, that translocates to the plasma membrane. Here we report the results of the entire screening. The process of cell activation was recorded on videos at different time points and all the videos can be visualized on a dedicated website. The proteins BAIAP3 and BIN1, partially translocated to the plasma membrane upon activation of NK2 receptors. Proteins ARHGAP12 and PKM2 translocated toward membrane blebs. Three proteins that associate with the cytoskeleton were of particular interest : PLEKHH2 rearranged from individual dots located near the cell-substrate adhesion surface into lines of dots. The speriolin-like protein, SPATC1L, redistributed to cell-cell junctions. The Chloride intracellular Channel protein, CLIC2, translocated from actin-enriched plasma membrane bundles to cell-cell junctions upon activation of NK2 receptors. CLIC2, and one of its close paralogs, CLIC4, were further shown to respond with the same translocation pattern to muscarinic M3 and lysophosphatidic LPA receptors. This screen allowed us to identify potential actors in signaling pathways downstream of G protein-coupled receptors and could be scaled-up for high-content screening.
Highlights
From the ‡G protein-coupled receptor (GPCR), Pain and Inflammation Team, UMR7242, CNRS-University of Strasbourg, LabEx Medalis, 300 Bvd Sebastien Brant, 67412 Illkirch, France; §European Molecular Biology Laboratory, Advanced Light Microscopy Facility, Meyerhofstr 1, 69117 Heidelberg, Germany; ¶¶School of Biology and Environmental Science and UCD Conway Institute, University College Dublin, Dublin 4, Ireland
Setting-up the Translocation Screen of the Cytoplasmic Protein Collection—The aim of the screen was to find cytoplasmic proteins that would respond to activation of the cells with the agonist neurokinin A (NKA) by changing their intracellular localization as an indication that the hit protein might be a downstream target of the NK2 receptor signaling cascades
The process of cell activation with the NK2 receptor agonist was recorded on videos at different time points and positive fluorescent clones were selected based upon a change in their intracellular localization
Summary
From the ‡GPCRs, Pain and Inflammation Team, UMR7242, CNRS-University of Strasbourg, LabEx Medalis, 300 Bvd Sebastien Brant, 67412 Illkirch, France; §European Molecular Biology Laboratory, Advanced Light Microscopy Facility, Meyerhofstr 1, 69117 Heidelberg, Germany; ¶¶School of Biology and Environmental Science and UCD Conway Institute, University College Dublin, Dublin 4, Ireland. Localization per se, without additional information, is not sufficient to indicate the potential function of cytoplasmic, or cytoplasmic and nuclear proteins that account for almost half of the proteome diversity, when compared with proteins located in dedicated organelles, such as the Golgi apparatus or lysosomes. To tackle this need for additional information, we have implemented a live-imaging screening strategy that is based on changes in localization of fluorescently labeled cytoplasmic proteins studied upon the activation of cells. The cDNA library of this GFP-cDNA localization project was selected for our live-imaging screen: from this library, we screened the proteins that had been identified residing in the cytoplasm or cytoplasm and nucleus
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