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example, a phosphatase inhibitor that leads to
increased b-subunit phosphorylation can actually
prevent Agrin-induced receptor clustering (Wallace,
1995). Although a number of cytoplasmic enzymes,
such as Src or PKC, have been implicated in phospho-
rylating AChR subunits, the key events are poorly
understood (Huganir and Greengard, 1983; Huganir et
al., 1984). At the onset of synaptogenesis, the AChR
clusters are labile, and will disperse if the muscle is
denervated. When a-Btx labeled muscles from embry-
onic mice are denervated and placed in calcium-
depleted culture media, the AChR clusters are lost
(Bloch and Steinbach, 1981). By birth, the labeled AChR
clusters have become resistant to this treatment.
During a widespread search for the postsynaptic
Agrin receptor, one candidate has emerged as the
most important transducer. A muscle-specific kinase
(MuSK), identified during a search for novel receptor
tyrosine kinases in denervated muscle, drew attention
because it is localized to the synaptic junction (Figure
8.20A) (Valenzuela et al., 1995). MuSK is expressed
very early in development, beginning at around E13 in
rats, when motor axons are first growing into the
muscle. Agrin induces the tyrosine phosphorylation of
MuSK within minutes (Figure 8.19), and this leads
rapidly to MuSK clustering (Moore et al., 2001). The
importance of MuSK was verified in a targeted disrup-
tion of the gene in mice (Figure 8.20B). These animals
display a dramatic loss of postsynaptic maturation,
including the loss of AChR clustering (DeChiara et al.,
1996). Although Agrin and MuSK display strong
binding kinetics, this is only apparent when MuSK is
expressed in muscle cells, suggesting that the protein
must form a complex with one or more accessory pro-
teins (Glass et al., 1996). The MuSK protein is also nec-
essary for the early prepatterning of AChRs that is
observed in the absence of motor axons (Figure 8.15).
The lasting importance of MuSK activity is clearly seen
in a group of patients suffering from the autoimmune
disease, myasthenia gravis. In most of these patients,
auto-antibodies against the AChR are made, leading to a
decrease in synaptic transmission and muscle weakness.
However, some patients make autoantibodies against
the MuSK protein, and this also leads to severe prob-
lems with neuromuscular transmission (Hoch et al., 2001).
The transduction process from MuSK activation to
receptor clustering is not worked out, but activation of
a Src-like kinase and GTP-binding proteins are proba-
bly required (Wallace, 1994; Ferns et al., 1996; Weston et
al., 2000; Mittaud et al., 2001). One crucial peripheral
membrane protein, called Rapsyn, is clearly required
to mediate AChR clustering (Figure 8.20A) (Sealock et
al., 1984). Messenger RNA for Rapsyn is present in
muscle cells prior to AChR cluster formation, and the
protein co-localizes with newly formed clusters in vivo
(Noakes et al., 1993). Rapsyn contains one domain that
mediates self-association, a second that associates with
the main intracellular loop of AChRs, and a third that
interacts with a-dystroglycan (Ramaroa et al., 2001).
When AChR subunits were introduced into cells that
do not ordinarily express this molecule, no clusters
formed. However, the co-expression of Rapsyn is suf-
ficient to promote AChR clustering (Frohner et al.,
1990; Phillips et al., 1991). The phenotype of Rapsyn-
deficient mice is fully consistent with a primary role
in cluster formation (Gautam et al., 1995). AChR
mRNA and protein are restricted to the central region
of muscle fibers but do not aggregate at the site of
neural contact. Rapsyn probably acts as more than just
an intermediate signal for MuSK. The presence of
Rapsyn is able to induce MuSK clusters in a fibroblast
expression system, and it is also able to activate tyro-
sine kinase activity (Gillespie et al., 1996). Several
other constituents accumulate at the synaptic cleft,
including acetylcholinesterase and s-laminin. There-
fore, many signaling pathways regulate postsynaptic
The dystrophin-associated glycoprotein, a-dystro-
glycan, is a second postsynaptic Agrin-binding pro-
tein (Figure 8.20A) (Gee et al., 1994; Campanelli
et al., 1994; Bowe et al., 1994; Sugiyama et al., 1994).
However, Agrin mutants that have poor affinity for a-
dystroglycan are nonetheless able to induce AChR clus-
ters (Meier et al., 1996; Hopf and Hoch, 1996). Therefore,
a-dystroglycan is probably not directly involved in
this part of synaptogenesis. The absence of dystrophin
in Duchenne's muscular dystrophy leads to reduced
expression of associated proteins in the sarcolemma,
resulting in damage during contraction, poor calcium
homeostasis, and eventual necrosis (Davies et al.,
1995). Agrin also binds to laminin, a major component
of the basal lamina (Figure 8.20A). Laminin can also
induce AChR clustering in cultured muscle cells in a
MuSK-independent manner (Sugiyama et al., 1997).
It seems likely that intercellular signaling influences
AChR function. Calcitonin gene-related peptide
(CGRP) is released from motor terminals and is able to
rapidly increase the mean open time of AChR channels
in Xenopus nerve-muscle cultures (Lu et al., 1993). This
effect appears to be mediated through an elevation of
cAMP in the muscle cell and is blocked by inhibitors
of cAMP-dependent protein kinase (PKA). A second
protein kinase, PKC, is also able to modulate the kinet-
ics of low-conductance AChRs (Fu and Lin, 1993).
Therefore, phosphorylation of “immature” AChRs
may prolong their open state, thereby increasing the
size of transmitter-evoked postsynaptic potentials at
the time of innervation.
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