Variations in receptor number at a given synapse are known to contribute to synaptic plasticity, but methods used to establish this idea usually do not allow for the determination of the dynamics of these phenomena. We used single-particle tracking to follow in real time, on the cell surface, movements of the glycine receptor (GlyR) with or without the GlyR stabilizing protein gephyrin. GlyR alternated within seconds between diffusive and confined states. In the absence of gephyrin, GlyR were mostly freely diffusing. Gephyrin induced long confinement periods spatially associated with submembranous clusters of gephyrin. However, even when most receptors were stabilized, they still frequently made transitions through the diffusive state. These data show that receptor number in a cluster results from a dynamic equilibrium between the pools of stabilized and freely mobile receptors. Modification of this equilibrium could be involved in regulation of the number of receptors at synapses.

Glycine is the major inhibitory neurotransmitter in the spinal cord and brain stem. Gephyrin is required to achieve a high concentration of glycine receptors (GlyRs) in the postsynaptic membrane, which is crucial for efficient glycinergic signal transduction. The interaction between gephyrin and the GlyR involves the E-domain of gephyrin and a cytoplasmic loop located between transmembrane segments three and four of the GlyR b subunit. Here, we present crystal structures of the gephyrin E-domain with and without the GlyR b-loop at 2.4 and 2.7 Å resolutions, respectively. The GlyR b-loop is bound in a symmetric 'key and lock' fashion to each E-domain monomer in a pocket adjacent to the dimer interface. Structure-guided mutagenesis followed by in vitro binding and in vivo colocalization assays demonstrate that a hydrophobic interaction formed by Phe 330 of gephyrin and Phe 398 and Ile 400 of the GlyR b-loop is crucial for binding.

High local concentrations of glycine receptors (GlyRs) at inhibitory postsynaptic sites are achieved through their binding to the scaffold protein gephyrin. The N-and C-terminal domains of gephyrin are believed to trimerize and dimerize, respectively, thus contributing to the formation of submembranous gephyrin clusters at synapses. GlyRs are associated with gephyrin also at extrasynaptic locations. We have investigated how gephyrin oligomerization influences GlyR dynamics and clustering in COS-7 cells and in cultured spinal cord neurons. To this aim, we have expressed isolated N-and C-terminal domains of gephyrin that interfere with the oligomerization of the full-length protein. We also studied the effect of an endogenous splice variant, ge(2,4,5), with a decreased propensity to trimerize. A reduction of the size and number of gephyrin-GlyR clusters was found in cells expressing the various interfering gephyrin constructs. Using fluorescence recovery after photobleaching, we studied the exchange kinetics of synaptic gephyrin clusters. Real-time singleparticle tracking was used to analyze the mobility of GlyRs. We found that all the tested constructs displayed faster rates of recovery than wild-type gephyrin and increased the mobility of extrasynaptic receptors, showing that gephyrin-gephyrin interactions modulate the lateral diffusion of GlyRs. Furthermore, we observed an inverse correlation between GlyR diffusion properties and gephyrin cluster size that depended on the number of binding sites blocked by the different constructs. Since alterations in the oligomerization properties of gephyrin are related to the dynamics of GlyRs, the gephyrin splice variant ge(2,4,5) may be implicated in the modulation of synaptic strength.

The scaffolding protein gephyrin is known to anchor glycine receptors (GlyR) at synapses and to participate in the dynamic equilibrium between synaptic and extrasynaptic GlyR in the neuronal membrane. Here we investigated the properties of this interaction in cells cotransfected with YFP-tagged gephyrin and GlyR subunits possessing an extracellular myc-tag. In HeLa cells and young neurons, single particle tracking was used to follow in real time individual GlyR, labeled with quantum dots, traveling into and out of gephyrin clusters. Analysis of the diffusion properties of two GlyR subunit types--able or unable to bind gephyrin--gave access to the association states of GlyR with its scaffolding protein. Our results indicated that an important portion of GlyR could be linked to a few molecules of gephyrin outside gephyrin clusters. This emphasizes the role of scaffolding proteins in the extrasynaptic membrane and supports the implication of gephyrin-gephyrin interactions in the stabilization of GlyR at synapses. The kinetic parameters controlling the equilibrium between GlyR inside and outside clusters were also characterized. Within clusters, we identified two subpopulations of GlyR with distinct degrees of stabilization between receptors and scaffolding proteins.

Tyrosylprotein sulfotransferase (TPST) is a 54-to 50-kDa integral membrane glycoprotein of the transGolgi network found in essentially all tissues investigated, catalyzing the tyrosine O-sulfation of soluble and membrane proteins passing through this compartment. Here we describe (i) an approach to identify the TPST protein, referred to as MSC (modification after substrate crosslinking) labeling, which is based on the crosslinking of a substrate peptide to TPST followed by intramolecular Protein tyrosine sulfation is a widespread posttranslational modification found in all metazoan species and tissues examined (1, 2). It is catalyzed by tyrosylprotein sulfotransferase (TPST) (3), an integral membrane glycoprotein residing in the trans-Golgi network (TGN) whose catalytic site is oriented toward the TGN lumen (2). Accordingly, proteins trafficking through the TGN have been found to become tyrosinesulfated, including several identified plasma membrane and secretory proteins (2). As for its physiological role, tyrosine sulfation has been shown to promote protein-protein interaction (2, 4), be it between (i) two secretory proteins (4, 5), (ii) a secretory protein and its cell surface receptor (6, 7), or (iii) two plasma membrane proteins (8-10).Recognition by TPST requires the occurrence of certain structural features in the substrate protein; a hallmark of these is the presence of acidic amino acid residues in the vicinity of the tyrosine (11-15). The comparison of the sequence motifs of various tyrosine sulfation sites (12), the characterization of the enzymatic properties of TPST from various tissues (16), and the observation that upon SDS͞PAGE purified TPST appears as a 54-to 50-kDa doublet of two, albeit highly related, polypeptides (17), have led to the suggestion (2) that for a given organism, isoenzymes of TPST may exist. Here we report the cDNA cloning and molecular characterization of a human TPST, referred to as TPST-2, which is distinct from the human TPST (referred to as TPST-1), whose molecular cloning was reported (18) while this study was in progress. MATERIALS AND METHODS Synthesis of [35 S]PAPS. ''Carrier-free'' [ 35 S]PAPS was synthesized as described (19), purified by thin-layer electrophoresis on cellulose sheets at pH 3.5, eluted, and stored at Ϫ20°C. The [ 35 S]PAPS was adjusted to the indicated concentration by addition of unlabeled PAPS. except for the presence of one rather than two N-terminal lysine residues] was dissolved in 50% acetonitrile͞50 mM sodium borate, pH 8.5, to a final concentration of 3 mM. NHS-LC-biotin (Fluka) in acetonitrile was added from a 30-mM stock solution to a final concentration of 12 mM, and the mixture was incubated for 90 min at room temperature. The pH was maintained at a value of 8.5 by repeated addition of appropriate amounts of 1 M NaOH. After incubation, the mixture was acidified to pH Ͻ 3 by addition of trifluoroacetic acid, and ␣,-bis-(biotinyl-Ahx)-KE(EPEYGE) 3 -OH (referred to as biotinyl-SgI 3 ) was isolated by using reverse-phase chromatograph...

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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.

Gephyrin is required for the formation of clusters of the glycine receptor (GlyR) in the neuronal postsynaptic membrane. It can make trimers and dimers through its N-and C-terminal G and E domains, respectively. Gephyrin oligomerization could thus create a submembrane lattice providing GlyR-binding sites. We investigated the relationships between the stability of cell surface GlyR and the ability of gephyrin splice variants to form oligomers. Using truncated and full-length gephyrins we found that the 13-amino acid sequence (cassette 5) prevents G domain trimerization. Moreover, E domain dimerization is inhibited by the gephyrin central L domain. All of the gephyrin variants bind GlyR ␤ subunit cytoplasmic loop with high affinity regardless of their cassette composition. Coexpression experiments in COS-7 cells demonstrated that GlyR bound to gephyrin harboring cassette 5 cannot be stabilized at the cell surface. This gephyrin variant was found to deplete synapses from both GlyR and gephyrin in transfected neurons. These data suggest that the relative expression level of cellular variants influence the overall oligomerization pattern of gephyrin and thus the turnover of synaptic GlyR.Fast neurotransmission at synapses depends on the enrichment of ionotropic receptors in the postsynaptic membrane. Scaffolding proteins present in the postsynaptic densities participate in the local and selective accumulation of most excitatory and inhibitory receptors in front of the corresponding transmitter release sites (1, 2). Synaptic localization of clusters of inhibitory ␥-aminobutyric acid, type A and glycine receptors relies on gephyrin, initially discovered as a GlyR-associated extrinsic membrane protein (3, 4). The pivotal role of gephyrin has been largely demonstrated by antisense experiments (5, 6) and the use of knock-out mice (7,8). Gephyrin binds both GlyR via an 18-amino acid amphipathic helix within the M3-M4 cytoplasmic loop of the ␤ subunit (9, 10) and tubulin via a motif similar to Tau/MAP2 tubulin-binding domain (11, 12). Therefore, gephyrin is functionally adapted to anchor GlyR at synapses via the cytoskeleton (8), but its physical link with ␥-aminobutyric acid, type A receptors is not yet understood.Gephyrin has a modular structure resulting from the fusion of two genes of bacterial origin, encoding the enzymes MogA and MoeA that catalyze the biosynthesis of the molybdenum cofactor in Escherichia coli (13,14). These enzymes are homologous to the gephyrin G and E domains (N-and C-terminal domains), respectively, which flank a 170-residue highly variable region (linker domain or L domain). The recent determination of the tertiary and quaternary structures of the MogA, MoeA, and gephyrin G domains (15-18) provides clues for delineating structure-function relationships for gephyrin. The binding site of the GlyR ␤ subunit has been mapped on a gephyrin-specific structure in the E domain crystal (19 -21). Isolated G and E domains form stable trimers and dimers, respectively, implying that gephyrin has two distinct ...