Abstract Gephyrin is a protein involved in both synaptic anchoring of inhibitory ligand-gated ion channels and molybdenum cofactor synthesis. Substantial progress has been made in understanding its gene and protein structures. Furthermore, numerous binding partners of gephyrin have been identified. The mechanisms by which these interactions occur are unclear at present. Alternative splicing has been proposed to contribute to gephyrin&apos;s functional diversity within single cells as well as in different cell types and tissues

B. Schmitt T Saiyed

H Betz

I Paarmann

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Molecular Determinants of Synaptic Function 45
Gephyrin: does splicing affect its function?
I. Paarmann1, T. Saiyed, B. Schmitt and H. Betz
Department of Neurochemistry, Max-Planck-Institute for Brain Research, Deutschordenstrasse 46, D-60528 Frankfurt, Germany
Abstract
Gephyrin is a protein involved in both synaptic anchoring of inhibitory ligand-gated ion channels and
molybdenum cofactor synthesis. Substantial progress has been made in understanding its gene and protein
structures. Furthermore, numerous binding partners of gephyrin have been identified. The mechanisms by
which these interactions occur are unclear at present. Alternative splicing has been proposed to contribute
to gephyrin’s functional diversity within single cells as well as in different cell types and tissues.
Introduction
Gephyrin is a ubiquitously expressed protein with an ap-
parent molecular mass of approx. 93 kDa. In neurons, it is
involved in clustering of inhibitory ligand-gated ion channels,
GlyRs (glycine receptors) and certain GABAA (γ -amino-
butyric acid A) receptor subtypes [1,2]. Additionally, geph-
yrin is required for molybdoenzyme activities such as sulphite
oxidase and xanthine dehydrogenase [2]. The generation of
knockout mice suggested that gephyrin is dispensable for em-
bryonic development, but absolutely essential for postnatal
survival [2]. Numerous gephyrin variants have been found
and described previously. Alternative splicing of gephyrin is
at least partially tissue-specific. One exon, C5, seems to be
much more abundant in muscle compared with other tissues
[3], whereas a different cassette, C3, has been observed to
be very prominently expressed in liver [4]. The same cassette
is largely repressed in neurons by Nova proteins, neuronal
regulators of pre-mRNA splicing [5]. This tissue-specific ex-
pression of at least some cassettes is a clear indication that
gephyrin’s functions may be modified by alternative splicing.
Hence, a better understanding of gephyrin’s gene and protein
structures is likely to provide clues as to how gephyrin man-
ages to fulfil complex and distinct functions in a whole
organism.
Gephyrin gene structure
The structure of the gephyrin gene has been studied most
extensively in mouse and human [3,6]. In mouse, the gephyrin
gene encompasses 457 kb on chromosome 12 D2 containing
30 exons. Ten of these exons, called cassettes, can be spliced
out, giving rise to numerous splice variants. These cassettes
have been named C1–C7 and C4′–C6′ (see Figure 1A).
In human, the gephyrin gene is located on chromosome
14q23.3, with a size of 668 kb and including 27 exons [6].
All constitutively expressed exons are common to mouse
and human. However, for the cassettes some differences
Key words: collybistin, gephyrin, glycine receptor, oligomerization, splicing, vasodilator-
stimulated phosphoprotein (VASP).
Abbreviations used: GABAA, γ -aminobutyric acid A; GlyR, glycine receptor; VASP, vasodilator-
stimulated phosphoprotein.
1To whom correspondence should be addressed (email paarmann@mpih-
frankfurt.mpg.de).
have been observed. The cassettes C1, C6, C7 and C5′
seem not to be transcribed in the human gene. Additionally,
the ‘C4 cluster’ encompassing C4′, C4 and C5 in mouse
contains one more exon in the human gene. C4A, corres-
ponding to C4′ in mouse, is followed by a cassette (C4B)
specific to human, and two further cassettes, C4C and C4D
(C4 and C5 in mouse respectively).
Gephyrin protein structure
The gephyrin gene encodes a protein consisting of three
domains, an N-terminal domain, a C-terminal domain and a
central linker region (see Figure 1B). Both the N- and C-ter-
minal domains have been crystallized [7,8]. The N-terminal
domain has a high structural similarity to the Escherichia
coli MogA protein and forms trimers, whereas the C-terminal
domain shows similarity to the E. coli MoeA protein and
is dimeric. Full-length gephyrin is thought to be a trimer,
which, triggered by an as yet unknown signal, can undergo a
conformational change that allows dimerization of the C-ter-
minal domains. Both the N-terminal trimerization and the
C-terminal dimerization have been suggested to be involved
in the submembranous gephyrin scaffold formation [8].
Gephyrin-binding proteins
Among several proteins reported to bind gephyrin, it is the
interaction of gephyrin with the GlyR that provides the basis
for synaptic GlyR clustering [8]. Collybistin, a guanylate
exchange factor for cdc42, interacts with the C-terminal
domain of gephyrin. This interaction has been shown to
be important for postsynaptic localization of gephyrin and
inhibitory ligand-gated ion channels [9]. Gephyrin also inter-
acts with tubulin and is believed to anchor the GlyR in the
postsynaptic membrane through this interaction [10]. Based
on amino acid sequence analysis, the tubulin-binding site
has been proposed to be located in the linker region [3].
Mena/VASP (vasodilator-stimulated phosphoprotein) and
profilin are further proteins shown to bind gephyrin [11].
Hence, the interactions with VASP and profilin might serve
as a link between the synaptic gephyrin scaffold and the
microfilament system. Additionally, binding of Dlc (dynein
light chain) 1 and 2, components of motor protein complexes,
has been proposed to be involved in retrograde gephyrin
C©2006 Biochemical Society
46 Biochemical Society Transactions (2006) Volume 34, part 1
Figure 1 Maps of gephyrin exon and domain organization
(A) Exon organization of the murine gephyrin gene. Exons are numbered from 1 to 30. Cassettes subject to alternative splicing
are depicted in grey. Arrows represent alternative start and stop codons. (B) Gephyrin domain structure. (C) Localization
of the binding sites of proteins interacting with gephyrin. Regions similar to bacterial and eukaryotic molybdenum cofactor
synthesis proteins are striped. Positions of the binding sites are indicated with black bars. Both (B) and (C) are aligned with
respect to (A). Dlc, dynein light chain; RAFT1, rapamycin and FK506-binding protein 12 target 1.
transport [12]. On the other hand, the interaction of gephyrin
with RAFT1 (rapamycin and FK506-binding protein 12
target 1) is required for rapamycin-sensitive signalling [13].
A tentative map of the known binding sites of the proteins
interacting with gephyrin is presented in Figure 1(C). Further
binding partners of gephyrin, such as the hypothesized linker
protein that mediates gephyrin association with GABAA
receptors, still remain to be discovered.
Effects of splicing on gephyrin function
Gephyrin splicing occurs in all three major domains. Each
domain contains at least three variable expression cassettes,
making alternative splicing an ideal tool for gephyrin to
adjust its abilities to diverse functional demands. However, at
present, experimental proof showing the possible importance
of this mode of regulation is lacking.
The C7 cassette is expressed in many different organs [3].
It contains a stop codon and, as a consequence, only the first
third of the C-terminal domain is translated properly [8].
Therefore, most, if not all, of the known functions of the
C-terminal domain such as dimerization, molybdenum
cofactor synthesis and clustering of the GlyR would be
abolished upon insertion of C7, whereas the N-terminal
domain and the linker region are likely to remain unaffected.
The repression of the C3 cassette in neurons by Nova [5]
indicates that C3 is important in non-neuronal tissues.
Therefore it is likely that C3 might interfere with gephyrin
functions in neurons as well.
The function of the C5′ cassette has been studied in the
most detail. Although C5′ is located in the N-terminal
gephyrin domain and the GlyR binding site lies in the
C-terminal domain [8], C5′ has been reported to abolish
GlyR binding [14]. Furthermore, evidence has been presented
suggesting that C5′ interferes with GlyR anchoring in hip-
pocampal and spinal cord neurons [15].
Alternative splicing has been found to affect the functions
of gephyrin in mouse, but not yet in human. One cassette,
C3, is a very good candidate to also affect the functions of
gephyrin in human. Further studies on the functions of the
distinct expression cassettes in gephyrin may unravel their
roles in the regulation of molybdenum cofactor synthesis,
binding of gephyrin to its interaction partners and gephyrin
oligomerization.
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Received 20 September 2005
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Molecular Determinants of Synaptic Function Gephyrin: does splicing affect its function?

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Molecular Determinants of Synaptic Function Gephyrin: does splicing affect its function?

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