A fluorescent live imaging screening assay based on translocation criteria identifies novel cytoplasmic proteins implicated in GPCR signalling pathways.
Fiche publication
Date publication
mars 2015
Auteurs
Membres identifiés du Cancéropôle Est :
Dr GALZI Jean-Luc
Tous les auteurs :
Lecat S, Matthes HW, Pepperkok R, Simpson JC, Galzi JL
Lien Pubmed
Résumé
Several cytoplasmic proteins that are involved in G protein-coupled receptor signalling 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 8 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 towards 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 signalling pathways downstream of G protein-coupled receptors and could be scaled-up for high-content screening.
Référence
Mol Cell Proteomics. 2015 Mar 10. pii: mcp.M114.046698.