1. health and fitness
2. disease

How to build a glycinergic postsynaptic membrane
HEINRICH BETZt, JOCHEN KUHSE, VOLKER SCHMIEDEN, MARIA-LUISA MALOSIO*,
DIETER LANGOSCH, PETER PRIOR, BERTRAM SCHMITT and JOACHIM KIRSCH
Department o f Neurochemistry, Max-Planck-Institute for Brain Research, Deutschordenstrasse 46, D-6000 Frankfurt/M. 71,
Federal Republic o f Germany
Present address: D epartm ent o f Pharm acology, U niversity o f M ilano, 1-20129 M ilano, Italy
t Author for correspondence
*
Summary
The inhibitory glycine receptor (GlyR) is a ligandgated chloride channel protein found at many
synapses of the mammalian central nervous system.
During development, distinct isoforms of the GlyR
are generated by the sequential expression of differ­
ent a subunit variants. The appearance of adult-type
GlyRs in spinal cord is accompanied by the accumu­
lation of a 93xlOs Mr receptor-associated peripheral
membrane protein. The latter has been localized at
the cytoplasmic face of glycinergic postsynaptic
membranes and is thought to anchor GlyRs beneath
glycinergic nerve terminals. The 9 3 x l0 3Mr protein
binds with high affinity to polymerized tubulin,
suggesting that it functions as a receptor-microtubule linking component. Our data suggest that the
interaction of developmentally regulated receptor
isoforms with specialized microtubule-associated
proteins represents a crucial step in the assembly of
postsynaptic receptor matrices.
K ey words: postsynaptic inhibition, glycine receptor, receptor
isoforms, receptor-associated proteins.
Introduction
Each neuron in the mammalian brain carries up to
thousands of postsynaptic membrane specializations.
These postsynaptic sites are characterized by receptor
proteins, which mediate signal transduction upon binding
of neurotransmitter released from the apposed nerve
terminal. At present, little is known about the mechan­
isms involved in the selective localization of neurotrans­
mitter receptors in postsynaptic membrane areas. This
report describes some characteristics of receptor regu­
lation at glycinergic synapses in the developing mam­
malian central nervous system (CNS).
The amino acid glycine is a major inhibitory neurotrans­
mitter in the vertebrate CNS (Aprison and Daly, 1978).
Glycine-mediated inhibition of neuronal activity results
from activation of the inhibitory glycine receptor (GlyR), a
ligand-gated chloride channel in spinal cord and certain
brain regions (reviewed by Langosch et al. 1990; Betz,
1991). The GlyR has been purified from mammalian spinal
cord and represents a pentameric protein composed of
ligand-binding subunits of 4 8 x l0 3Mr (a) and homologous
polypeptides of 58 x 10s Mr (/3) (Pfeiffer et al. 1982; Graham
et al. 1985; Langosch et al. 1988). The primary structures of
these GlyR subunits have been determined by cDNA
Journal o f Cell Science, Supplement 15, 23 -25 (1991)
Printed in Great Britain © The Company o f Biologists Limited 1991
sequencing and shown to share a common transmembrane
topology and significant sequence homology with nicotinic
acetylcholine and GABAa receptor proteins (Grenningloh
et al. 1987, 1990a). All these receptors, therefore, are
thought to constitute members of a superfamily of ligandgated ion-channel proteins that evolved from a common
ancestral polypeptide (Betz, 1990).
Biochemical and molecular cloning techniques indicate
considerable heterogeneity of GlyRs during development
(Betz, 1991). Moreover, a 93x10 Mr protein co-purifying
with GlyR subunits upon affinity chromatography has
been implicated in the synaptic topology of GlyRs (Triller
et al. 1985; Schmitt et al. 1987). Here we summarize data
which suggest mechanistic models for GlyR localization
during development.
Developmental heterogeneity of the GlyR
Evidence for subtype heterogeneity of the GlyR was first
detected in studies of rodent spinal cord development, in
which a neonatal isoform prevalent at birth was shown to
differ from the adult GlyR in pharmacological, immuno­
logical and biochemical properties (Becker et al. 1988).
This neonatal receptor shows only low affinity for the
glycinergic antagonist strychnine and contains an a
subunit of 49xlOsMr. Within 2-3 weeks after birth, the
neonatal GlyR in spinal cord is completely replaced by the
adult-type receptor.
Further evidence for developmental heterogeneity of the
GlyR came from expression studies (Akagi and Miledi,
1988). Injection of poly(A)+ RNA isolated from rat brain or
spinal cord into Xenopus oocytes caused expression of
glycine-gated chloride channels in the oocyte membrane.
By sedimentation on sucrose density gradients, two
classes of GlyR mRNAs could be separated, both of which
gave rise to functional channels when expressed in
oocytes. A rapidly sedimenting heavy mRNA species was
abundant in spinal cord of adult rats, whereas fractions
from neonatal spinal cord and adult cerebral cortex
contained a low molecular weight GlyR mRNA.
Molecular cloning studies in our laboratory now have
revealed that the developmental heterogeneity of GlyRs
observed at both the protein and mRNA expression level
reflects the existence of several GlyR a subunit genes. By
low-stringency screening of cDNA and genomic libraries,
clones encoding different variants of the originally
described GlyR a subunit (now termed al) have been
isolated. Two different oil cDNAs, oil (Grenningloh et al.
19906; Kuhse et al. 1991) and a2* (Kuhse et al. 1990a), as
well as an a3 cDNA (Kuhse et al. 19906) were found to be
23
differentially expressed in rats and humans. Moreover,
genomic sequences encoding a fourth variant, a4, have
been isolated from mouse (Y. Maulet, B. Matzenbach, and
H. Betz, unpublished observations). All these a sequences
display a high degree of amino acid identity and
correspond to GlyR proteins, whose expression is under
distinct temporal and regional control. The a2 polypep­
tides represent ligand-binding subunits of neonatal GlyRs
in spinal cord and forebrain, whereas o3 receptors appear
mainly to be expressed in the postnatal cerebellum
(Malosio et al. 1991). The location of a4 GlyRs is presently
unknown; in adult rodent brain, a4 mRNA is found only at
very low levels.
Northern blot analysis, amplification by polymerase
chain reaction (PCR) and in situ hybridization have been
used to monitor the accumulation of GlyR subunit
transcripts during CNS development. All presently avail­
able data indicate high levels of a2 and [5 transcripts in
embryos and neonatal animals, whereas al and a3
sequences become significantly expressed only at about 2
weeks after birth (reviewed by Betz, 1991). This is
consistent with the biochemically demonstrated exchange
of GlyR isoforms in spinal cord during the first two
postnatal weeks and suggests that adult glycinergic
synapses contain GlyRs built of al or, to a much lesser
extent, a3 polypeptides. Interestingly, the different a
subunit variants show high sequence divergence in their
predicted intracellular domains. This may relate to a
differential regulation of their cell surface distribution by
intracellular receptor-associated proteins.
Gephyrin, a 93x103 Mr glycine receptorassociated protein, is a putative
receptor-microtubule linker
GlyR solubilized from rat, pig or mouse spinal cord
membranes co-purifies with a 93xlOsAfr protein upon
affinity chromatography (Pfeiffer et al. 1982; Graham et al.
1985). Immuno-electron microscopy using selective mono­
clonal antibodies (Pfeiffer et al. 1984) indicates that this
polypeptide decorates the cytoplasmic face of the glyciner­
gic postsynaptic membrane (Triller et al. 1985; Altschuler
et al. 1986). Moreover, in biochemical studies the
93 x 103Mr protein behaves as a typical peripheral mem­
brane component, that can be extracted by alkaline pH or
acylation (Schmitt et al. 1987). It therefore was proposed
that this polypeptide may anchor the GlyR in the
postsynaptic membrane by interaction with cytoskeletal
proteins or structural components of postsynaptic den­
sities. Consistent with this view, the increase o f93 x 103Mr
protein immunoreactivity in developing spinal cord paral­
lels the ontogenetic appearance of the adult GlyR isoform
(Becker et al. 1988).
Sequencing of 93x103Mr protein cDNAs (P. Prior, B.
Schmitt, G. Grenningloh, I. Pribilla, G. Multhamp, K.
Beyreuther, Y. Maulet, P. Werner, D. Langosch, J. Kirsch
and H. Betz, unpublished observations) revealed some
similarities in the amino acid composition of the deduced
protein sequence to that of microtubule-associated protein
5 (MAP5). We therefore investigated whether the
93 x 10s Mr protein may bind to tubulin (Kirsch et al. 1991).
Indeed, significant amounts of tubulin were found to copurify with the 93xl0s Mr protein upon fractionation of
GlyR polypeptides. Also, tubulin binds to the isolated and
immobilized 9 3x l0 3Mr protein in an overlay procedure.
Finally, the 93 X103Mr protein co-assembles with polym­
24
H. Betz et al.
erized tubulin or microtubules through repeated cycles of
polymerization and depolymerization and mediates sedi­
mentation of GlyR a and ß subunits with polymerized
tubulin. This interaction displays high affinity CKd5“
2.5 nM), significant cooperativity (Hill coefficients. 1) and
approaches a stoichiometry of about 1:4 under saturating
conditions (Kirsch et al. 1991). Similar cooperative
characteristics and stoichiometries of tubulin binding
have been reported for the interaction of MAP2 and MAP5
with polymerized tubulin. The 93xl03Mr protein there­
fore may be tentatively classified as a novel type of
tubulin-associated protein, which serves as a receptor-cytoskeleton linking protein. Based on these considerations,
we propose the name gephyrin (from greek gephyra,
bridge) for this polypeptide to indicate its presumed
function (P. Prior et al., unpublished work).
Isolation of gephyrin cDNAs has unraveled considerable
heterogeneity resulting from alternative splicing (P. Prior
et al., unpublished data). The functional significance of
this variability is not known. We speculate however, that a
set of gephyrin variants generated from a common premRNA may be implicated in different membrane protein-microtubule interactions. Moreover, Northern analysis
and PCR amplification indicate that gephyrin transcripts
are present in many rat tissues. Thus, gephyrin may be
important in determining the topography of different cell
surface specializations.
Conclusions and perspectives
The data summarized here suggest that the localization of
GlyRs to postsynaptic membrane areas is a multistep
process during synaptogenesis. First, a neonatal (or
embryonic) GlyR isoform is replaced by adult-type recep­
tors, which characterize postsynaptic membrane areas in
the mature CNS (Triller et al. 1985). This involves both
induction of al (and o3), and repression of a2, gene
transcription. Second, the increased synthesis of gephyrin,
a 93x103Mr GlyR-associated protein, is proposed to be
crucial for anchoring GlyRs at postsynaptic membranes by
interaction with subsynaptic tubulin. Consistent with this
view, the presence of ‘membrane-bound’ tubulin in brain
synaptosomes and postsynaptic densities has been
reported by several investigators (see review by Stephens,
1986). Moreover, a recent immunohistochemical study
indicates the existence of cold-stable acetylated micro­
tubules in the subsynaptic region of the sarcoplasm
underlying the neuromuscular junction (Jasmin et al.
1990). Although the functional and structural relevance of
tubulin as a component of the postsynaptic complex is still
debated (Stephens, 1986), submembraneous tubulin ar­
rays may be a general feature of postsynaptic specializ­
ations and provide a scaffold for anchoring receptors under
nerve terminals. If so, induction of ordered microtubular
structures under ingrowing nerve terminals may consti­
tute a primary event in postsynaptic membrane forma­
tion.
This work was supported by Deutsche Forschungsgem einschaft
(Leibniz-Program m and Schwerpunkt ‘Funktionelle Dom änen’),
Fonds der Chem ischen Industrie and the M inerva Program m o f
the M ax-Planck-G esellschaft. We thank S. W artha for expert
secretarial assistance.
References
A
H. a n d M i l e d i , R. (1988). Heterogeneity o f glycine receptors and
their RNAs in rat brain and spinal cord. Science 242, 270-273.
k a g i,
H., P a r a k k a l , M. H., R e e k s , K. A . a n d
J. (1986). Identification o f glycinergic synapses in the
cochlear nucleus through immunocytochemical localization o f the
postsynaptic receptor. Brain Res. 369, 316-320.
A p r i s o n , M. H. a n d D a l y , E. C. (1978). Biochemical aspects of
transmitter at inhibitory synapses: the role o f glycine. Adv.
Neurochem. 3, 203-294.
B ecker, C.-M., H och , W. a n d Betz , H. (1988). Glycine receptor
heterogeneity in rat spinal cord during postnatal development. EMBO
J. 7, 3717-3726.
B e t z , H. (1990). Ligand-gated ion channels in the brain: the amino acid
receptor family. Neuron 5, 383-392.
B e t z , H. (1991). Glycine receptors: heterogeneous and wide-spread in
the mammalian brain. Trends Neurosci. 14, 458-461.
G r a h a m , D., P f e i f f e r , F., S i m l e r , R. a n d B e t z , H. (1985). Purification
and characterization o f the glycine receptor o f pig spinal cord.
Biochemistry 24, 990-994.
G r e n n i n g l o h , G ., P r i b i l l a , I., P r i o r , P ., M u l t h a u p , G ., B e y r e u t h e r ,
K., T a l e b , 0 . a n d B e t z , H. (1990a). Cloning and expression o f the 58
kd ß subunit o f the inhibitory glycine receptor. Neuron 4, 963-970.
G r e n n i n g l o h , G ., R i e n i t z , A., S c h m i t t , B ., M e t h f e s s e l , C., Z e n s e n ,
M . , B e y r e u t h e r , K., G u n d e l f i n g e r , E. D . a n d B e t z , H. (1 9 8 7 ) . The
strychnine-binding subunit o f the glycine receptor shows homology
with nicotinic acetylcholine receptors. Nature 328, 2 1 5 - 2 2 0 .
G r e n n i n g l o h , G ., S c h m i e d e n , V., S c h o f i e l d , P. R., S e e b u r g , P. H.,
S i d d i q u e , T., M o h a n d a s , T. K., B e c k e r , C . - M . a n d B e t z , H. (19906).
Alpha subunit variants o f the human glycine receptor: primary
structures, functional expression and chromosomal localization of the
corresponding genes. EMBO J. 9, 771-776.
A
ltsch u ler,
W
enthold,
Jasm
in
R . A ., B e t z ,
R.
, B . J ., C h a n g e u x , J .- P .
and
C a r t a u d , J . (1 9 9 0 ).
Compartmentalization o f cold-stable and acetylated microtubules in
the subsynaptic domain o f chick skeletal muscle fibre. Nature 344,
673-675.
K i r s c h , J., L a n g o s c h , D., P r i o r , P ., L i t t a u e r , U. Z., S c h m i t t , B. a n d
H. (1991). The 93 kDa glycine receptor-associated protein binds
to tubulin. J. biol. Chem. (in press).
K u h s e , J., K u r y a t o v , A., M a u l e t , Y., M a l o s i o , M.-L., S c h m i e d e n , V.
a n d B e t z , H. (1991). Alternative splicing generates two isoforms of
the a2 subunit of the inhibitory glycine receptor. FEBS Lett. 283,
73-77.
K u h s e , J., S c h m i e d e n , V. a n d B e t z , H. (1990a). A single amino acid
exchange alters the pharmacology of neonatal rat glycine receptor
subunit. Neuron 5, 867-873.
K u h s e , J., S c h m i e d e n , V. a n d B e t z , H. (19906). Identification and
functional expression of a novel ligand binding subunit of the
inhibitory glycine receptor. J. biol. Chem. 265, 22 317-22320.
L a n g o s c h , D., B e c k e r , C.-M. a n d B e t z , H. (1990). The inhibitory
glycine receptor: A ligand-gated chloride channel of the central
nervous system. Eur. J. Biochem. 194, 1-8.
L a n g o s c h , D., T h o m a s , L . a n d B e t z , H. (1988). Conserved quarternary
structure o f ligand-gated ion channels: the postsynaptic glycine
receptor is a pentamer. Proc. natn. Acad. Sei. U.S.A. 85, 7394-7398.
M a l o s i o , M . - L . , M a r q u ü z e - P o u e y , B . , K u h s e , J. a n d B e t z , H. (1991).
Wide-spread expression o f glycine receptor subunit mRNAs in the
adult and developing rat brain. EMBO J. 10, 2401-2409.
P f e i f f e r , F., G r a h a m , D. a n d B e t z , H. (1982). Purification by affinity
chromatography of the glycine receptor o f rat spinal cord. J. biol.
Chem. 257, 9389-9393.
P f e i f f e r , F., S i m l e r , R., G r e n n i n g l o h , G . a n d B e t z , H. (1984).
Monoclonal antibodies and peptide mapping reveal structural
similarities between the subunits of the glycine receptor of rat spinal
cord. Proc. natn. Acad. Sei. U.S.A. 81, 7224-7227.
S c h m i t t , B ., K n a u s , P., B e c k e r , C.-M. a n d B e t z , H. (1 9 8 7 ) . The M r
93,000 polypeptide of the postsynaptic glycine receptor is a peripheral
membrane protein. Biochemistry 26, 805—811.
S t e p h e n s , R. E. (1986). Membrane tubulin. Biol. Cell 57, 95-100.
T r i l l e r , A., C l u z e a u d , F., P f e i f f e r , F., B e t z , H. a n d K o r n , H. (1985).
Distribution of glycine receptors at central synapses: an
immunoelectron microscopy study. J. Cell Biol. 101, 947-956.
B etz,
Glycinergic synapse
25