title: A Fluorescent Live Imaging Screening Assay Based on Translocation Criteria Identifies Novel Cytoplasmic Proteins Implicated in G Protein-coupled Receptor Signaling Pathways.
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.
authors.affiliation: From the ‡GPCRs, Pain and Inflammation Team, UMR7242, CNRS-University of Strasbourg, LabEx Medalis, 300 Bvd Sébastien Brant, 67412 Illkirch, France; sandra.lecat@unistra.fr.
authors.forename: Sandra
authors.initials: S
authors.lastname: Lecat
authors.affiliation: From the ‡GPCRs, Pain and Inflammation Team, UMR7242, CNRS-University of Strasbourg, LabEx Medalis, 300 Bvd Sébastien Brant, 67412 Illkirch, France;
authors.forename: Hans W D
authors.initials: HW
authors.lastname: Matthes
authors.affiliation: §European Molecular Biology Laboratory, Advanced Light Microscopy Facility, Meyerhofstr 1, 69117 Heidelberg, Germany;
authors.forename: Rainer
authors.initials: R
authors.lastname: Pepperkok
authors.affiliation: ¶¶School of Biology and Environmental Science and UCD Conway Institute, University College Dublin, Dublin 4, Ireland.
authors.forename: Jeremy C
authors.initials: JC
authors.lastname: Simpson
authors.affiliation: From the ‡GPCRs, Pain and Inflammation Team, UMR7242, CNRS-University of Strasbourg, LabEx Medalis, 300 Bvd Sébastien Brant, 67412 Illkirch, France;
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Galzi
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001681:Biological Assay; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D018118:Chloride Channels / Q000235:genetics / Q000378:metabolism; D003598:Cytoskeletal Proteins; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D057054:Molecular Imaging / Q000379:methods*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D018041:Receptors, Neurokinin-2 / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013963:Thyroid Hormones / Q000235:genetics / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism; D000097810:Thyroid Hormone-Binding Proteins
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D001426:Bacterial Proteins; D002352:Carrier Proteins; D018118:Chloride Channels; D003598:Cytoskeletal Proteins; D020690:GTPase-Activating Proteins; D049452:Green Fluorescent Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D018041:Receptors, Neurokinin-2; D011994:Recombinant Proteins; D013963:Thyroid Hormones; D025521:Tumor Suppressor Proteins; D000097810:Thyroid Hormone-Binding Proteins; C454999:ARHGAP12 protein, human; C585199:BAIAP3 protein, human; C101410:BIN1 protein, human; C108828:CLIC2 protein, human; C464459:CLIC4 protein, human; C485182:Cyan Fluorescent Protein; C553461:SPATC1 protein, human; C580984:plekhh2 protein, human; C054562:yellow fluorescent protein, Bacteria