Intracellular Association of Glycine Receptor with Gephyrin Increases Its Plasma Membrane Accumulation Rate

Abstract

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-taggedgephyrin and myc-tagged GlyR ␣ 1 subunits. In these experiments, myc-tagged GlyR ␣ 1 contained, or did not contain, the gephyrinbinding sequence (␤gb) of the GlyR ␤ 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 ␣ 1 with a thrombin cleavage site between the myc-tag and the N terminal of the GlyR ␣ 1 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.

Molecular and functional heterogeneity of GABAergic synapses

“…However, there are a number of important differences between the two types of synapses, which suggest that their formation and regulation might depend on distinct mechanisms (Table 1) Second, as discussed in the next section, postsynaptic clustering of some GABA A R subtypes is possible in the absence of gephyrin. In contrast, GlyR postsynaptic clustering appears to be dependent on the presence of gephyrin clusters [52], mirroring the high affinity of their interaction and confirming observations that GlyR trafficking in neurons is facilitated by association with gephyrin [53].…”

Activity-Dependent Movements of Postsynaptic Scaffolds at Inhibitory Synapses

“…S2, available at www.jneurosci.org as supplemental material). This contrasted with gephyrin chimera tagged with GFP at their C terminus, which formed intracellular aggregates (Meier et al, 2000a;Hanus et al, 2004). The distribution patterns of N terminus tagged and untagged gephyrin (in the absence or presence of GlyR) were independent of the variant used (supplemental Fig.…”

“…The ability of VeGe to interact with the GlyR was tested in cotransfection experiments in COS-7 cells (Meier et al, 2000a(Meier et al, , 2001Hanus et al, 2004) (supplemental methods, available at www.jneurosci.org as supplemental material). VeGe redistributed to GlyR-containing intracellular membranes and formed submembranous clusters coaggregated with cell-surface receptors when coexpressed with GlyR ␣1 subunit containing the gephyrin-binding sequence of the ␤ subunit (␣1␤gb) (supplemental Fig.…”

“…These results led to the notion that the interactions of receptors with scaffolding proteins are more dynamic than previously expected. Recent evidence indicates that some postsynaptic scaffold proteins at excitatory (Inoue and Okabe, 2003;Huang and El-Husseini, 2005) and inhibitory (Hanus et al, 2004) synapses can be exchanged between the cell surface and intracellular compartments. Using fluorescence recovery after photobleaching assays, the dynamics of scaffolding proteins have been documented on the time scale of a few seconds at excitatory synapses (Okabe et al, 2001).…”

“…Immunofluorescence (IF) staining was performed essentially as described previously (Hanus et al, 2004). Briefly, cells were fixed for 15 min at room temperature (RT) in 4% (w/v) paraformaldehyde (Serva Feinbiochemica, Heidelberg, Germany) in PBS, quenched for 20 min at RT with 50 mM NH 4 Cl, and permeabilized for 7 min with 0.1% (w/v) Triton X-100 at 4°C.…”