The tubulin-binding protein gephyrin copurifies with the inhibitory glycine receptor (GlyR) and is essential for its postsynaptic localization. Here we have analyzed the interaction between the GlyR and recombinant gephyrin and identified a gephyrin binding site in the cytoplasmic loop between the third and fourth transmembrane segments of the beta subunit. GlyR alpha subunits and GABAA receptor proteins failed to bind recombinant gephyrin. However, insertion of an 18 residue segment of the GlyR beta subunit into the GABAA receptor beta 1 subunit conferred gephyrin binding both in an overlay assay and in transfected mammalian cells. These results indicate that beta subunit expression is essential for the formation of a postsynaptic GlyR matrix.

Glycine receptors are anchored at inhibitory chemical synapses by a cytoplasmic protein, gephyrin. Molecular cloning revealed the similarity of gephyrin to prokaryotic and invertebrate proteins essential for synthesizing a cofactor required for activity of molybdoenzymes. Gene targeting in mice showed that gephyrin is required both for synaptic clustering of glycine receptors in spinal cord and for molybdoenzyme activity in nonneural tissues. The mutant phenotype resembled that of humans with hereditary molybdenum cofactor deficiency and hyperekplexia (a failure of inhibitory neurotransmission), suggesting that gephyrin function may be impaired in both diseases.

The formation of postsynaptic GABAA and glycine receptor clusters requires the receptor-associated peripheral membrane protein gephyrin. Here we describe two splice variants of a novel gephyrin-binding protein, termed collybistin I and II, which belong to the family of dbl-like GDP/GTP exchange factors (GEFs). Co-expression of collybistin II with gephyrin induced the formation of submembrane gephyrin aggregates that accumulate hetero-oligomeric glycine receptors. Our data suggest that collybistin II regulates the membrane deposition of gephyrin by activating a GTPase of the Rho/Rac family. Therefore, this protein may be an important determinant of inhibitory postsynaptic membrane formation and plasticity.

A 93 kd polypeptide associated with the mammalian inhibitory glycine receptor (GlyR) is localized at central synapses and binds with high affinity to polymerized tubulin. This protein, named gephyrin (from the Greek gamma epsilon phi upsilon rho alpha, bridge), is thought to anchor the GlyR to subsynaptic microtubules. Here we report its primary structure deduced from cDNA and show that corresponding transcripts are found in all rat tissues examined. In brain, at least five different gephyrin mRNAs are generated by alternative splicing. Expression of gephyrin cDNAs in 293 kidney cells yields polypeptides reactive with a gephyrin-specific antibody, which coprecipitate with polymerized tubulin. Thus, gephyrin may define a novel type of microtubule-associated protein involved in membrane protein-cytoskeleton interactions.

Each neuron in the mammalian brain carries many postsynaptic membrane specializations containing high densities of receptors that mediate signal transduction upon neurotransmitter release from the apposed nerve terminal. Little is known about the mechanisms by which receptors are transported to and anchored at postsynaptic sites, but extracellular as well as intracellular components may be involved. Ultrastructural studies have shown that the peripheral membrane protein gephyrin, which co-purifies with the postsynaptic inhibitory glycine receptor (GlyR) upon affinity chromatography, is situated on the cytoplasmic face of glycinergic postsynaptic membranes. Moreover, gephyrin binds with high affinity to polymerized tubulin and has been postulated to link the GlyR to the subsynaptic cytoskeleton. Here we report that treatment of rat spinal neurons in culture with gephyrin antisense oligonucleotides prevents the formation of GlyR clusters in the dendritic plasma membrane. Thus, gephyrin is essential for localizing the GlyR to presumptive postsynaptic plasma membrane specializations.

The stratum corneum as the outermost epidermal layer protects against exsiccation and infection. Both the underlying cornified envelope (CE) and the intercellular lipid matrix contribute essentially to these two main protective barriers. Epidermis-unique ceramides with ultra-long-chain acyl moities (ULC-Cers) are key components of extracellular lipid lamellae (ELL) and are bound to CE proteins, thereby contributing to the cornified lipid envelope (CLE). Here, we identified human and mouse ceramide synthase 3 (CerS3), among CerS1-6, to be exclusively required for the ULC-Cer synthesis in vitro and of mouse CerS3 in vivo. Deficiency of CerS3 in mice results in complete loss of ULC-Cers (≥C26), lack of continuous ELL and a non-functional CLE. Consequently, newborn mutant mice die shortly after birth from transepidermal water loss. Mutant skin is prone to Candida albicans infection highlighting ULC-Cers to be pivotal for both barrier functions. Persistent periderm, hyperkeratosis and deficient cornification are hallmarks of mutant skin demonstrating loss of Cers to trigger a keratinocyte maturation arrest at an embryonic pre-barrier stage.

The mechanisms underlying the postsynaptic localization of neurotransmitter receptors are poorly understood. Recently, the peripheral membrane protein gephyrin has been shown to be essential for the formation of inhibitory glycine receptor clusters in cultured rat spinal cord neurons. In vitro gephyrin binds with high affinity to polymerized tubulin. Here, the interaction of gephyrin with different components of the cytoskeleton was investigated in primary cultures of rat spinal neurons. After treatment with alkaloids affecting the cytoskeleton, the morphology of post-synaptic gephyrin clusters was analyzed by confocal immunofluorescence microscopy. Depolymerization of microtubules by demecolcine reduced both the percentage of cells with postsynaptic gephyrin clusters and the number of clusters/cell. The size of the remaining gephyrin clusters was increased whereas their gephyrin density was significantly lower than under control conditions. Depolymerization of microfilaments by cytochalasin D in contrast generated smaller clusters of increased gephyrin density. Demecolcine also dispersed postsynaptic glycine receptor clusters as revealed by immunostaining with a specific monoclonal antibody. These findings support the view that in vivo gephyrin anchors receptor polypeptides to the cytoskeleton by a complex interaction with microtubules and microfilaments.

The ability of nerve cells to receive up to several thousands of synaptic inputs from other neurons provides the anatomical basis for information processing in the vertebrate brain. The formation of functional synapses involves selective clustering of neurotransmitter receptors at presumptive postsynaptic regions of the neuronal plasma membrane. Receptor-associated proteins are believed to be crucial for this process. In spinal neurons, synaptic targeting of the inhibitory glycine receptor (GlyR) depends on the expression of the anchoring protein gephyrin. Here we show that the competitive GlyR antagonist strychnine and L-type Ca2+-channel blockers inhibit the accumulation of GlyR and gephyrin at postsynaptic membrane areas in cultured rat spinal neurons. Our data are consistent with a model in which GlyR activation that results in Ca2+ influx is required for the clustering of gephyrin and GlyR at developing postsynaptic sites. Similar activity-driven mechanisms may be of general importance in synaptogenesis.