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Chapter 9). As with the mammalian family members,
the DLG protein has a PDZ binding domain, and the
membrane proteins that it anchors have the conserved
C-terminal motif. The developmental importance of
MAGUK family proteins is underlined by studies
showing moderate to severe mental retardation when
one of the genes is truncated (Tarpey et al., 2004).
Even while receptor clustering is underway, the
synthesis of new synaptic proteins increases dramati-
cally. The majority of AChRs within a cluster are newly
inserted a short time after innervation (Salpeter and
Harris, 1983; Ziskind-Conhaim et al., 1984; Role et al.,
1985; Dubinsky et al., 1989). The contribution of exist-
ing and newly inserted AChRs was examined at newly
formed synapses in chick nerve-muscle cultures by
labeling the receptors before and after innervation
(Figure 8.24). Before innervation, all of the AChRs
present on the muscle membrane surface were labeled
with a-Btx (“old” AChRs). Following the addition of
neurons, a monoclonal antibody directed against an
extracellular AChR epitope was applied to label all
AChRs (“old” and “new” AChRs). In this way, it was
possible to determine the contribution of both “old”
receptors (i.e., a-Btx labeled) and “new” receptors (i.e.,
antibody-labeled minus a-Btx labeled). Within eight
hours of neuron addition, more than 60% of the AChRs
are newly inserted into the muscle membrane, indi-
cating that synthesis is rapidly upregulated (Role et al.,
1985). These results suggest that synthesis is regulated
by the presynaptic terminal. Similarly, when innerva-
tion of the Drosophila neuromuscular junction is
delayed or prevented in prospero mutants, the normal
increase in functional glutamate receptors fails to
occur (Broadie and Bate, 1993b).
Further evidence that motor neuron terminals selec-
tively regulate AChR synthesis comes from the local-
ized expression of the specific mRNAs. Muscle cells
are polynuclear, and the nuclei that lay directly below
the synaptic cleft are distinctive from those found
extrasynaptically in that they preferentially transcribe
AChR mRNA; this is controlled by the presence of
motoneuron terminals (Klarsfeld et al., 1991; Sanes
et al., 1991; Simon et al., 1992; Merlie and Sanes, 1985).
As new receptors are added, they also become more
stable. This has been shown by measuring how long
the receptors remain in the membrane before being
replaced. In the chick, the rate of AChR turnover gradu-
ally increases from from a half-life of ª30 hours at the
FIGURE 8.23 Gephyrin is required for glycine receptor cluster-
ing. When spinal neurons are grown in culture, the peripheral mem-
brane protein, Gephyrin, co-localizes with glycine receptor clusters
(top). When translation of the Gephyrin protein is blocked with an
antisense oligonucleotide (bottom), the glycine receptors do not
form clusters in the neuronal membrane. (Adapted from Kirsch
et al., 1993)
vated, and this is directly correlated with an increase in
AMPA-type glutamate receptors at these spines
(Mauceri et al., 2004). This may provide a mechanism by
which activity does lead to receptor clustering.
There is evidence that GABA A receptor clustering
involves a novel internal membrane protein, called
GABARAP, which was isolated on the basis of its inter-
action with the g2 subunit of GABA A receptors. An N-
terminal subdomain of GABARAP binds microtubules,
suggesting that it may stabilize receptors at the
membrane (Wang et al., 1999; Wang and Olsen, 2000).
Although GABARAP has been shown to aggregate
GABA A receptors and enhance their conductance in
tissue culture (Chen et al., 2000; Everitt et al., 2004), its
role in normal development has yet to be established.
Clustering proteins are conserved across species. A
MAGUK family member, called discs-large (DLG), co-
localizes with glutamate receptors at the Drosophila
nerve-muscle junction. When the dlg gene is inacti-
vated, synaptic structure and function are profoundly
altered (Woods and Bryant, 1991; Lahey et al., 1994;
Budnick et al., 1996). The synaptic localization of DLG
is regulated by CaMKII activity (Koh et al., 1999). DLG
is necessary for the localization of FASII, a cell adhe-
sion molecule that regulates synapse formation (see
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