Primary structure and alternative splice variants of gephyrin, a putative glycine receptor-tubulin linker protein

Abstract

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.

Molecular architecture of glycinergic synapses

“…The 93 kDa protein was demonstrated to bind polymerized tubulin with similar aYnity and stoichiometry as microtubule-associated protein 2 (Kirsch et al 1991). It was hypothesized that this protein could form a bridge between the GlyR and the underlying microtubular cytoskeleton and therefore named gephyrin ( ; Greek: bridge) upon elucidation of its primary structure (Prior et al 1992). Gephyrin can interact with a short sequence motif (18 amino acids) within the large cytoplasmic loop of the GlyR subunit thereby establishing a direct, continuous molecular link between hetero-oligomeric GlyRs and gephyrin Kneussel et al 1999b;Meyer et al 1995).…”

“…Three splice variants of gephyrin cDNAs were identiWed (originally named P1-P3), which diVer by the insertion of polypeptide cassettes due to alternative splicing of the premRNA and additional splice variants were identiWed later on Meier et al 2000;Prior et al 1992;Ramming et al 2000). In the meantime, the nomenclature of gephyrin splice cassettes has been revised at a recent meeting exclusively devoted to the biology of gephyrin .…”

“…Moreover, gephyrin containing the C5 cassette has been shown to be unable to catalyze the Wnal step in molybdenum cofactor (see below) biosynthesis in vitro (Smolinsky et al 2008), indicating that alternative splicing may change diVerent properties of gephyrin in a given cellular context. Unexpectedly, Northern blots revealed, that gephyrin mRNAs are expressed in many organs except those of the lymphatic system (Prior et al 1992). In brain and spinal cord, gephyrin expression is restricted to grey matter (Kirsch et al 1993a).…”

“…Surprisingly, the analysis of gephyrin's primary structure revealed signiWcant sequence homologies of the amino-and carboxy-terminal thirds to proteins involved in the biosynthesis of the molybdenum cofactor (moco) (Prior et al 1992;Stallmeyer et al 1999), a pterin cofactor required for the activity of molybdenum-dependent oxidoreductases, namely sulWte oxidase, xanthine dehydrogenase and aldehyde oxidase all of which are predominantly expressed in liver but also in brain (Mendel and Schwarz 2002;Schwarz and Mendel 2006). The amino terminal part of gephyrin (amino acids 1-181), now called the G-domain, shows homologies to the MogA protein, whereas the carboxy terminal part (amino acids 318-736), now called E-domain, is homologous to the MoeA protein of E. coli.…”

Gephyrin: a central GABAergic synapse organizer

“…The gephyrin transcript is subject to extensive alternative splicing 10,19,20 in all three domains. Each domain contains at least three variable expression cassettes, providing a regulatory mechanism through which the signaling properties of specific synapses may be altered.…”