Intracellular association of glycine receptor with gephyrin increases its plasma membrane accumulation rate.
Gephyrin, a tubulin-binding protein, is the core of inhibitory postsynaptic scaffolds stabilizing glycine receptors (GlyRs) and/or GABA(A) receptors. Previous ultrastructural studies in vivo and in vitro have reported a localization of gephyrin to intracellular cisternas during development or after glycinergic denervation (Seitanidou et al., 1992; Colin et al., 1996, 1998). These data were compatible with a traffic of this cytoplasmic, but membrane-associated, protein together with membrane proteins such as GlyR after exocytosis and/or endocytosis pathways. We have now investigated the consequences of a GlyR-gephyrin interaction on the localization and the dynamics of these two molecules in African green monkey kidney cells (COS-7) cells and in neurons transfected with green fluorescent protein-tagged-gephyrin and myc-tagged GlyR alpha1 subunits. In these experiments, myc-tagged GlyR alpha1 contained, or did not contain, the gephyrin-binding sequence (betagb) of the GlyR beta subunit. We report here that GlyR-gephyrin interaction localizes gephyrin to GlyR-containing organelles. Videomicroscopy and nocodazole treatment indicate that the movements of these vesicles are microtubule dependent. Expressing GlyR alpha1 with a thrombin cleavage site between the myc-tag and the N terminal of the GlyR alpha1 subunit (Rosenberg et al., 2001) allowed monitoring of newly inserted receptors in the cell surface. Using temperature changes to block GlyR in, and then release it from, the trans-Golgi network, we show that gephyrin accelerates the accumulation of GlyR at the cell surface. Therefore, our data strongly suggest that some GlyR clusters are associated with gephyrin on their way to the cell surface and that this association increases the accumulation of GlyR at the plasma membrane.
Hanus
Cyril
C
Laboratoire de Biologie Cellulaire de la Synapse Normale et Pathologique, Institut National de la Santé et de la Recherche Médicale, Ecole Normale Supérieure, 75005 Paris, France.
Vannier
Christian
C
Triller
Antoine
A
D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D049452:Green Fluorescent Proteins; D007424:Intracellular Fluid / Q000378:metabolism*; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D018715:Microscopy, Video; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015739:Nocodazole / Q000494:pharmacology; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000502:physiology; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D018009:Receptors, Glycine / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D002352:Carrier Proteins; D008164:Luminescent Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D016271:Proto-Oncogene Proteins c-myc; D018009:Receptors, Glycine; D011993:Recombinant Fusion Proteins; C073441:gephyrin; D049452:Green Fluorescent Proteins; D015739:Nocodazole