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First published online 24 October 2007
doi: 10.1242/dev.010702
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Molecular Neurobiology Program, The Helen L. and Martin S. Kimmel Center for Biology and Medicine at the Skirball Institute of Biomolecular Medicine, NYU Medical School, 540 First Avenue, New York, NY 10016, USA.
* Author for correspondence (e-mail: burden{at}saturn.med.nyu.edu)
Accepted 11 September 2007
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: AChR clustering, Junctional folds, Neuromuscular junction, Synaptogenesis, Chrnb1
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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-amino butyric acid and acetylcholine, are tyrosine phosphorylated in
vivo, but the role of this post-translational modification in neurotransmitter
receptor function is poorly understood
(Huganir et al., 1984
;
Qu et al., 1990;
Moon et al., 1994;
Wan et al., 1997;
Swope et al., 1999). The
nicotinic acetylcholine receptor (AChR) is a pentamer composed of four
different subunits, and two of these subunits, ß and 
, are
tyrosine phosphorylated in vivo (Qu et
al., 1990; Mittaud et al.,
2001). Tyrosine phosphorylation of the AChR is stimulated by
agrin, a 
200 kDa protein that is synthesized by motor neurons and
released from nerve terminals (Wallace et
al., 1991). Agrin stimulates MuSK, a muscle-specific receptor
tyrosine kinase that is concentrated in the postsynaptic membrane and critical
for synapse formation (DeChiara et al.,
1996; Glass et al.,
1996). Activation of MuSK leads to the rapid tyrosine
phosphorylation and subsequently to the clustering of AChRs. In mice lacking
MuSK, AChRs are expressed, but they are neither tyrosine phosphorylated nor
clustered (DeChiara et al.,
1996; Glass et al.,
1996; Smith et al.,
2001). Taken together, these findings suggest that AChR tyrosine
phosphorylation may have an important and possibly requisite role in
clustering AChRs at synapses.
Consistent with this notion, prior studies, which analyzed transfected
wild-type cultured myotubes reported that mutant AChR pentamers, which contain
a ß-subunit that cannot be tyrosine phosphorylated, cluster two-fold less
efficiently than wild-type AChRs (Meyer
and Wallace, 1998; Borges and
Ferns, 2001). In addition, these mutant AChRs are extracted more
readily than wild-type AChRs with a non-ionic detergent
(Borges and Ferns, 2001). These
data indicate that tyrosine phosphorylation of the AChR ß-subunit has a
role in clustering and stabilizing AChRs in cultured myotubes, but the role of
AChR tyrosine phosphorylation in synaptic differentiation has not been
studied.
To determine the role of AChR ß tyrosine phosphorylation in clustering and anchoring AChRs at synapses, we generated mice with targeted mutations in the three tyrosines in the large intracellular loop of the AChR ß-subunit. Mice lacking AChR ß-subunit tyrosine phosphorylation survive postnatally, but their neuromuscular synapses are simplified and contain a reduced number of AChRs, indicating that tyrosine phosphorylation of the AChR ß-subunit has an important role in organizing and stabilizing AChRs at synapses.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Isolation of AChR complexes
Whole leg muscles from P0 mice were homogenized in lysis buffer [50 mM
sodium chloride, 30 mM triethanolamine pH 7.5, 50 mM sodium flouride, 5 mM
EDTA, 5 mM EGTA, 2 mM sodium orthovanadate, 1 mM N-ethylmaleimide, 1
mM sodium tetrathionate, 1 µg/ml pepstatin plus Complete protease
inhibitors (Roche, Basel, Switzerland)] with a PT 10/35 Polytron (Kinematica
AG, Littau-Lucerne, Switzerland) at 4°C. NP-40 was added to a final
concentration of 1%, and the extract was incubated with rocking for 30 minutes
at 4°C. Insoluble proteins were removed by centrifugation, and the
supernatant was pre-cleared for 1 hour at 4°C against streptavidin-agarose
(Sigma, St Louis, MO). The supernatant was collected and incubated for 1 hour
at 4°C with biotinylated-
-bungarotoxin (-BGT; Invitrogen,
Carlsbad, CA, USA). AChR complexes were precipitated overnight with
streptavidin-agarose, followed by washing (three washes for 3 minutes each) in
lysis buffer containing 1% NP-40. Western blots were probed with antibodies to
phosphotyrosine (4G10, 1:1000; Upstate USA, Charlottesville, VA). The blots
were subsequently stripped and reprobed with antibodies to the AChR
ß-subunit (mAb124, 1:5000; a gift from J. Lindstrom, University of
Pennsylvania, Philadelphia, PA).
AChR surface expression
P0 mouse diaphragms were dissected, and AChRs were labeled with 20 nM
125I--BGT (Perkin Elmer, Waltham, MA) for 1 hour at 37°C
in oxygenated L15 medium. Background binding, in the presence of 10 µM
non-radioactive -BGT, was 10% of the binding without competitor.
Muscles were washed (five washes for 50 minutes) in oxygenated L15 medium.
Bound 125I--BGT was measured in a Wallac 1470 gamma counter
(Perkin Elmer), and the muscle was weighed.
Immunostaining sections and whole mounts of muscle
Tibialis anterior muscles from P30 mice were mildly fixed (in 0.1%
paraformaldehyde in PBS) for 1 hour at 4°C, rinsed twice at 4°C in
PBS, cryoprotected (in 30% sucrose-PBS) overnight at 4°C, and embedded in
TissueTek (Sakura, Tokyo, Japan). Frozen sections (10 µm) were stained with
the following antibodies: p-AchRß1-Tyr 390 (1:200; sc-17087; Santa Cruz
Biotechnology, Santa Cruz, CA), MuSK (1:1000; #83033), rapsyn (1:500; #232),
APC (1:200; sc-895 Santa Cruz Biotechnology), Abl (1:200; sc-131 Santa Cruz
Biotechnology), utrophin (1:10; Vector Labs, Burlingame, CA) and dystroglycan
(1:100; gift from Kevin Campbell, University of Iowa, Iowa City, IA), and
AChRs were labeled with Alexa Fluor 594--BGT (Invitrogen). The images
were acquired with a 63x (1.4 NA) objective. AChRs, axons and nerve
terminals from P0 and P30 diaphragms were stained, and synaptic AChR levels
and size were measured as described previously
(Jaworski and Burden, 2006).
Briefly, we stained diaphragm muscle with Alexa Fluor 594--BGT,
collected confocal image stacks at the same sub-saturating amplifier gain for
both genotypes using a 40x (1.3 NA) objective, and measured the Alexa
Fluor 594--BGT-stained area. We quantified actual levels from
unprocessed images but adjusted image levels for display purposes.
Quantification of the pattern (stripes and gaps) of AChR staining at
individual synapses and assignment to one of three categories was done
initially without knowledge of the genotype; after the data were acquired,
using a 63x (1.4 NA) objective, the genotype was identified.
Electron microscopy
Levator auris muscles in terminally anesthetized mice were exposed and
injected with 10 µg/ml tetrodotoxin in PBS to prevent muscle contraction,
and subsequently fixed in vivo with 4% paraformaldehyde/1% glutaraldehyde in
110 mM sodium phosphate buffer, pH 7.3. After dissection, fixation was allowed
to continue at room temperature for 90 minutes. The muscle was washed (five
times for 25 minutes) in iso-osmotic phosphate buffer (150 mM sodium chloride,
5 mM potassium chloride, 10 mM sodium phosphate, pH 7.3), and subsequently
stained for acetylcholinesterase activity to locate the muscle area containing
synapses (Karnovsky and Roots,
1964). Following three washes (for 15 minutes) with 100 mM Tris,
pH 7.2, 160 mM sucrose, the muscle was washed again (five washes for 25
minutes) in isosmotic phosphate buffer. Following treatment with osmium (4%
osmium tetroxide in 140 mM sodium phosphate, pH 7.3) for 1 hour, the muscle
was washed (twice for 1 hour) in water. The muscle was stained en bloc with
saturated uranyl acetate for 1 hour, dehydrated in ethanol and embedded in
Epon.
Electrophysiology
Diaphragm muscles from P30 mice were dissected in oxygenated high
Mg2+-low Ca2+ Tyrode's solution (125 mM sodium chloride,
5.37 mM potassium chloride, 24 mM sodium bicarbonate, 12 mM magnesium
chloride, 0.5 mM calcium chloride and 5% dextrose). Glass microelectrodes were
pulled with a Sutter P-2000 micropipette puller (Novato, CA) and filled with 3
M potassium chloride to give a resistance of between 40-60 MV. Recordings of
miniature end-plate potential (mepp) amplitude were made with a Dagan IX-1
(Minneapolis, MN) intracellular preamplifier connected to an ADInstruments
Powerlab 8/30 data acquisition system (Colorado Springs, CO). Muscle fibers
were impaled near the endplate, and the average rise-time (baseline to peak)
was statistically similar and less than 3 mseconds for both genotypes. Mepp
recordings were only made from fibers with resting membrane potentials between
-70 and -85 mV and deviated no more than 15% during the duration of the
recording. The data represent at least 30 mepps per muscle fiber.
Isolation of immortalized muscle cells
We isolated two immortalized muscle cell lines from wild-type mice that
carried the H-2Kb-tsA58 transgene and two muscle lines from the
Chrnb1 mutant (hereafter referred to as
AChR-ß3F/3F) mice that carried the
H-2Kb-tsA58 transgene (Jat et al.,
1991). Muscle cells were grown as described previously
(Smith et al., 2001).
AChR clustering assay
Myotubes were treated for 16 hours with 500 pM agrin (R&D, Minneapolis,
MN), rinsed three times (in PBS) and fixed for 10 minutes (1% paraformaldehyde
in PBS). After washing (twice for 10 minutes in PBS), the muscle was incubated
in 0.1 M glycine (in PBS) for 10 minutes rinsed twice in PBS and incubated for
30 minutes in 1% BSA in PBS. AChRs were labeled with Alexa Fluor
594--BGT (Invitrogen; 1:1000, in 1% BSA-PBS) for 1 hour and washed
(three washes for 30 minutes in PBS). Myotubes were permeabilized (in PBS
containing 0.1% Triton X-100) for 5 minutes and stained with Alexa Fluor
488-phalloidin (1:250; Invitrogen) (in 1% BSA-PBS) for 20 minutes to label
actin. Myotubes were washed (three washes for 15 minutes in PBS) and mounted
in VectaShield (Vector Labs). Images of phalloidin-stained myofibers were
collected using a Zeiss LSM 510 microscope (Oberkochen, Germany), with a
20x (0.7 NA) objective, and the number and size of AChR clusters in
these myotubes was determined using the Volocity (Improvision, Lexington, MA)
software package. The data represent the average from two cell lines for each
genotype.
AChR extraction
Differentiated myotubes were treated with 500 pM agrin in culture medium
for 3 hours at 37°C. 125I--BGT was added to fresh medium
containing 500 pM agrin. Myotubes were incubated for a further 1 hour at
37°C and washed (three washes for 15 minutes in oxygenated L15 medium).
AChRs were extracted by incubating myotubes in L15 medium containing 0.05%
Triton X-100; the extraction solution was collected and replaced every 2
minutes for a total of 6 minutes. The remaining, unextracted AChRs were
collected by incubating myotubes in L15 medium containing 1% SDS. The amount
of AChR extracted with Triton X-100 and SDS was measured in a gamma counter.
The data represent the average from two cell lines for each genotype. In some
experiments, myotubes were pretreated with 20 nM staurosporine (Sigma) or
vehicle (0.002% DMSO) for 16 hours prior to treating the myotubes with
agrin.
Statistics
Statistical analyses were performed using Minitab 15 (Minitab, State
College, PA). All values are given as mean±standard error. Parametric
tests were used only when the data were normally distributed as determined by
the Kolmogorov-Smirnov test. Non-normal data were analyzed using the
appropriate nonparametric test as indicated in the figure legend.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Because we were concerned that the two other tyrosine residues (Y357, Y442) in
the main intracellular loop of the AChR ß-subunit might be phosphorylated
if phosphorylation of Y390 were prevented, we used site-directed mutagenesis
and homologous recombination in ES cells to replace all three tyrosine
residues in the main intracellular loop with phenylalanine
(Fig. 1A,B; Materials and
methods). We confirmed gene targeting by PCR, Southern blotting and DNA
sequencing (Fig. 1B and data
not shown). Mice lacking these tyrosine phosphorylation sites
(AChR-ß3F/3F) are born alive, nurse
normally, and are otherwise indistinguishable from wild-type littermates (see
Fig. S1 in the supplementary material).
To determine whether mutation of these three tyrosine residues prevents
AChR ß-subunit tyrosine phosphorylation, we labeled AChRs in detergent
lysates from P0 limb muscles with biotinylated--bungarotoxin
(-BGT), isolated AChR complexes with streptavidin-agarose and probed
western blots with antibodies to phosphotyrosine.
Fig. 1C shows that AChRs in
AChR-ß3F/3F mutant mice, unlike AChRs in
control mice, are not tyrosine phosphorylated. In addition, we stained frozen
sections of muscle from wild-type and
AChR-ß3F/3F mutant mice with antibodies
against a phosphopeptide specific to AChR-ßY390-P and found
that these antibodies label synaptic sites in wild-type but not AChR-
ß3F/3F mutant mice
(Fig. 1D). Taken together,
these data indicate that tyrosine phosphorylation of the AChR ß-subunit
is eliminated in AChR-ß3F/3F mice.
Synapses are smaller and contain fewer AChRs in AChR-ß3F/3F mice
AChRs at embryonic synapses are arranged in small ovoid plaques of uniform
AChR density (Sanes and Lichtman,
2001). To determine whether synapse formation is impaired in
AChR-ß3F/3F mice, we stained whole mounts of
diaphragm muscle from P0 mice with Alexa594--BGT to label postsynaptic
AChRs and antibodies to Neurofilament and Synaptophysin to label presynaptic
axons and nerve terminals. Fig.
2 shows that AChRs at synapses in wild-type and
AChR-ß3F/3F mice are organized in small
ovoid plaques, apposed by nerve terminals, indicating that this simple
arrangement of AChRs at developing synapses is not dependent upon tyrosine
phosphorylation of the AChR ß-subunit
(Fig. 2A).
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30% (100±4.7%, n=4 for
wild-type; 71.1±4.5%, n=6 for
AChR-ß3F/3F mice), whereas AChR density is
unchanged in AChR-ß3F/3F mice
(Fig. 2B). These defects in
AChR clustering are not due to a decrease in AChR expression, since the total
number of surface AChRs, measured by 125I--BGT binding, is
comparable in AChR-ß3F/3F and wild-type mice
(see Fig. S1D in the supplementary material).
The structure of neuromuscular synapses becomes more complex during
postnatal development, as ovoid AChR plaques are transformed into complex,
pretzel-like shapes, characteristic of adult synapses
(Sanes and Lichtman, 2001). At
P30, synapses in AChR- ß3F/3F mice are
40% smaller than in wild-type mice (100±8.6%, n=3 for
wild-type mice; 56.8±4.1%, n=5 for
AChR-ß3F/3F mice), indicating that the
decrease in synaptic size observed at P0 is not caused by a delay in synaptic
differentiation (Fig. 2D). In
addition, by P30 the density of synaptic AChRs is also reduced by 40%
(100±6.3%, n=3 for wild-type mice; 61.2±5.1%,
n=5 for AChR- ß3F/3F mice), leading
to an approximately threefold reduction in the total number of AChRs at each
synapse (Fig. 2D).
Since miniature end-plate potential (mepp) size is proportional to the
density of synaptic AChRs, we measured the size and frequency of mepps in
muscle from wild-type and AChR-ß3F/3F mutant
mice in order to obtain a second, independent measure of AChR density.
Fig. 2E-G shows that the
frequency of mepps is comparable in mutant and control mice, whereas the
amplitude of mepps is reduced by 30% in
AChR-ß3F/3F mutant mice (0.95±0.05
mV, n=35 synapses from wild-type mice; 0.68±0.04 mV,
n=35 synapses from AChR- ß3F/3F
mice). Thus, these data demonstrate that AChR ß-subunit tyrosine
phosphorylation is critical to cluster AChRs at their normal number and
density at synaptic sites.
Morphological aberrations in AChR-ß3F/3F synapses
Concomitant with the formation of a complex, pretzel-shaped terminal arbor,
the postsynaptic membrane becomes invaginated into deep and regularly spaced
postjunctional folds (Salpeter,
1987). AChRs are concentrated at the crests and along the upper
portions of these postjunctional folds
(Fertuck and Salpeter, 1976).
When viewed en-face by light microscopy, this arrangement leads to a regular,
striped appearance of AChRs at neuromuscular synapses
(Anderson and Cohen, 1974). To
determine whether AChR ß-subunit tyrosine phosphorylation is necessary
for this aspect of postsynaptic development, we collected confocal images of
synapses, stained with Alexa Fluor 594--BGT and compared the
organization of AChRs at wild-type and
AChR-ß3F/3F mutant synapses in P30 mice
(Fig. 3A-H). At synapses in
wild-type mice, the synaptic area is organized into well-defined AChR stripes.
Moreover, most synaptic branches contain few (two or less) gaps in AChR-rich
areas (Fig. 3I,J). In contrast,
the synaptic area in AChR- ß3F/3F mice is
largely devoid of well-defined AChR stripes, and most terminal branches are
interrupted by five or more gaps in AChR staining. These aberrations in
synaptic morphology suggest that tyrosine phosphorylation of AChR
ß-subunit is necessary for the normal development and/or maintenance of
neuromuscular cytoarchitecture.
These alterations in AChR organization could be caused by defects in the
formation of postjunctional folds. To investigate this possibility we examined
the structure of neuromuscular synapses from P30 wild-type and
AChR-ß3F/3F mice by electron microscopy. The
organization of the neuromuscular junction in
AChR-ß3F/3F mice appears largely normal
(Fig. 3K-N): first, nerve
terminals contain synaptic vesicles, some of which are focused across from the
mouths of postjunctional folds; second, the synaptic basal lamina is
interposed between presynaptic and postsynaptic membranes; third,
postjunctional folds, which are lined by basal lamina, are readily apparent;
fourth, the crests and upper portions of the postjunctional folds are often
thick and darkly stained, probably because of the dense packing of AChRs
(Fertuck and Salpeter, 1976)
(Fig. 3M,N). We quantified the
number of postjunctional folds in three ways: first, we calculated a
fold-density by dividing the number of postjunctional folds, defined by
invaginations lined with basal lamina, independent of whether these
invaginations had visible mouths that open to the synaptic cleft, by the
length of the synaptic cleft; second, we calculated a fold-index by dividing
the total length of postjunctional fold membrane, independent of whether these
folds had visible mouths that open to the synaptic cleft, by the length of the
synaptic cleft; third, we calculated the density of postjunctional folds with
openings, or mouths, to the synaptic cleft
(Fig. 3O). According to the
first and second methods, the number and length of postjunctional folds are
normal in AChR-ß3F/3F mutant mice. According
to the third method, however, the number of postjunctional folds is reduced by
40% (0.38±0.02 mouths/µm, n=57 for wild-type mice;
0.24±0.01 mouths/µm, n=75 for AChR-
ß3F/3F mice; Fig.
3O). Thus, in AChR-ß3F/3F mutant
mice, postjunctional folds have fewer openings to the synaptic cleft than in
wild-type mice, although the entire length of fold membrane appears normal
(Fig. 3P,Q).
Localization of proteins to the postsynaptic membrane in AChR-ß3F/3F mice
Agrin stimulates the clustering of multiple synaptic proteins, in addition
to AChRs, in cultured myotubes, raising the possibility that the accumulation
of these proteins at synapses may depend upon AChR ß-subunit tyrosine
phosphorylation. We stained frozen sections of muscle from P30 mice with
antibodies to adenomatous polyposis coli (APC), rapsyn, MuSK, Abl1, utrophin
and dystroglycan and found that each of these proteins is concentrated at
synaptic sites in AChR-ß3F/3F mice
(Fig. 4). Thus, the
accumulation of these postsynaptic proteins at synapses is not dependent upon
AChR ß-subunit tyrosine phosphorylation.
Attenuation of AChR clustering in AChR-ß3F/3F myotubes
To determine whether tyrosine phosphorylation of the AChR ß-subunit
has a role in clustering AChRs in response to agrin, we generated muscle cell
lines from wild-type and AChR-ß3F/3F mice
and examined their response to agrin. We stimulated wild-type and
AChR-ß3F/3F myotubes with agrin for 16
hours, labeled AChRs with Alexa Fluor 594--BGT, and measured the number
and size of AChR clusters (Fig.
5A). The number of spontaneous, agrin-independent AChR clusters is
similar in mutant and wild-type myotubes
(Fig. 5B). Agrin stimulation
induces a 3.5-fold increase in the number of AChR clusters in wild-type
myotubes but smaller, 1.4-fold increase in the number of AChR clusters in
AChR-ß3F/3F myotubes
(Fig. 5B). Wild-type and
AChR-ß3F/3F myotubes contain large and small
AChR clusters, and the number of AChR clusters of all sizes is reduced in
AChR-ß3F/3F mutant myotubes
(Fig. 5B). These results
indicate that tyrosine phosphorylation of the AChR ß-subunit is not
required to cluster AChRs per se, but is necessary to fully respond to agrin
and to maximally cluster AChRs.
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; Borges and Ferns,
2001; Moransard et al.,
2003). To determine whether tyrosine phosphorylation of the AChR
ß-subunit is necessary to stabilize AChRs, we treated wild-type and
AChR-ß3F/3F myotubes with agrin for 3 hours,
labeled AChRs with 125I--BGT for 1 hour in the presence of
agrin, extracted labeled AChRs with non-ionic detergent for several minutes
and measured the rate of AChR extraction. In wild-type myotubes, agrin
stimulation causes a decrease in the rate of AChR extraction
(Fig. 5C). By contrast, in
AChR-ß3F/3F myotubes, agrin does not alter
the rate of AChR extraction (Fig.
5C), indicating that tyrosine phosphorylation of the AChR
ß-subunit is necessary to stabilize AChRs following agrin stimulation.
Moreover, staurosporine treatment destabilizes AChRs in wild-type myotubes but
fails to accelerate the rate of AChR extraction from
AChR-ß3F/3F myotubes, indicating that
staurosporine destabilizes AChRs by inhibiting tyrosine phosphorylation of the
AChR ß-subunit (Fig.
5C).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Below, we discuss the implications of these results: first, the role of AChR
ß-subunit tyrosine phosphorylation in clustering AChRs at synapses;
second, the unexpected finding that synapses are smaller in mice lacking AChR
ß tyrosine phosphorylation; third, the role of AChR ß-subunit
tyrosine phosphorylation in linking AChRs to the cytoskeleton and organizing
postjunctional folds.
AChR ß-subunit tyrosine phosphorylation has a role in clustering AChRs at synapses
Previous studies showed that AChR pentamers that contain a mutant
ß-subunit, which cannot be tyrosine phosphorylated, cluster two-fold less
efficiently than wild-type AChRs (Borges
and Ferns, 2001). Because the myotubes analyzed in these studies
co-expressed wild-type as well as mutant AChR ß-subunit, it remained
possible that the wild-type AChR pentamers formed a scaffold that facilitated
the clustering of mutant AChR pentamers and obscured a more dramatic role of
AChR ß tyrosine phosphorylation in clustering AChRs. We find that AChR
clustering is reduced to a similar extent (2.5-fold reduction) in myotubes
that express only mutant AChR pentamers. Thus, AChR ß-subunit tyrosine
phosphorylation contributes to, but is not essential for, AChR clustering.
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-subunits have redundant roles and that tyrosine
phosphorylation of the AChR is essential for clustering AChRs. Nonetheless,
the tyrosine phosphorylation sites in the ß- and -subunits are
embedded in different sequences, indicating that the phosphorylated subunits
are unlikely to recruit the same adaptor protein
(Colledge and Froehner, 1997).
Notably, the tyrosine phosphorylation site in the -subunit conforms to
a binding site for SH2 domains, whereas the tyrosine phosphorylation site in
the ß-subunit is not predicted to bind SH2 or PTB domains. For this
reason, phosphorylation of the different subunits is unlikely to have a
redundant role.
In wild-type myotubes, agrin induces the formation of AChR micro-clusters,
in a Rac-dependent manner, and AChR macro-clusters, in a Rho-dependent manner
(Weston et al., 2000;
Weston et al., 2003). We find
that the number of micro- and macro-clusters are reduced to the same extent in
AChR- ß3F/3F mutant myotubes. Our findings
are consistent with two possibilities: (1) Rac- and Rho-dependent pathways
each depend upon AChR-ß3F/3F tyrosine
phosphorylation or (2) micro- and macro-clusters are similarly unstable in the
absence of AChR- ß3F/3F tyrosine
phosphorylation.
Synapses are smaller in mice lacking AChR ß tyrosine phosphorylation
Because AChRs containing a mutant AChR ß-subunit cluster less
efficiently in cultured myotubes (Borges
and Ferns, 2001) (Fig.
5A,B), we were not surprised to find that the density of AChRs is
reduced at synapses in AChR-ß3F/3F mutant
mice. We did not anticipate, however, that synaptic size would be reduced in
AChR- ß3F/3F mutant mice. Notably, the
density of synaptic AChRs is reduced in utrophin mutant mice, yet the size of
utrophin-deficient neuromuscular synapses is normal
(Deconinck et al., 1997;
Grady et al., 1997). Thus, a
reduction in the density, or packing of synaptic AChRs, does not necessarily
lead to a reduction in synaptic size. Neuromuscular synapses are smaller but
contain a normal density of synaptic AChRs in humans carrying mutations in
DOK7 (Beeson et al.,
2006; Slater et al.,
2006). DOK7 is an adaptor protein that is recruited to tyrosine
phosphorylated MuSK and necessary for signaling downstream from MuSK,
including AChR tyrosine phosphorylation
(Okada et al., 2006). Thus, it
is possible that a failure to phosphorylate AChRs in patients harboring
DOK7 mutations is responsible for the decrease in synaptic size.
The role of AChR ß-subunit tyrosine phosphorylation in linking AChRs to the cytoskeleton and organizing postjunctional folds
Our studies in AChR-ß3F/3F muscle cell
lines suggest that the defects in AChR clustering at synapses may be caused,
at least in part, by a failure to link mutant AChRs to an underlying
cytoskeleton. Rapsyn, which is essential for clustering AChRs, binds the AChR
ß-subunit and associates more efficiently with AChRs following agrin
stimulation (Burden et al.,
1983; Gautam et al.,
1995; Moransard et al.,
2003). APC is also reported to bind the AChR ß-subunit, to
associate more efficiently with AChRs following agrin stimulation, and to be
required for clustering AChRs in cultured muscle cells
(Wang et al., 2003). Although
these data raise the possibility that alterations in the binding of rapsyn
and/or APC to AChRs might underlie the synaptic defects observed in
AChR-ß3F/3F mice, we find that APC and
rapsyn are concentrated at synaptic sites in the absence of AChR
ß-subunit tyrosine phosphorylation. Although we cannot exclude the
possibility that reduced amounts of rapsyn and/or APC are present at synapses
in AChR-ß3F/3F mutant mice, these data
demonstrate that the accumulation of rapsyn and APC at synapses does not
depend upon AChR ß-subunit tyrosine phosphorylation.
The reductions in AChR density and postjunctional fold formation in
AChR-ß3F/3F mice are strikingly similar to
the synaptic defects observed in utrophin mutant mice
(Deconinck et al., 1997;
Grady et al., 1997). Utrophin
and AChRs colocalize at the tops of postjunctional folds and form a complex in
cultured muscle fibers (Bewick et al.,
1992; Fuhrer et al.,
1999). These data suggest that tyrosine phosphorylation of the
AChR ß-subunit and utrophin might function in the same pathway. Utrophin,
however, remains concentrated at synapses in
AChR-ß3F/3F mice, and the AChR
ß-subunit is tyrosine phosphorylated in utrophin mutant mice (data not
shown). Thus, these data do not support the idea that the defects in
AChR-ß3F/3F mice are due to a failure to
recruit utrophin or that the synaptic defects in utrophin mutant mice are due
to a lack of AChR ß-subunit tyrosine phosphorylation.
How might tyrosine phosphorylation of the AChR ß-subunit regulate the
formation of postjunctional folds? Our data are consistent with the
possibility that tyrosine phosphorylation of the AChR ß-subunit provides
a docking site for a protein(s) that is involved in forming and/or stabilizing
postjunctional folds. Alternatively, others have suggested that the dense
packing of conically shaped AChRs may be sufficient to induce folding of the
postsynaptic membrane (Unwin,
2005) raising the possibility that the reduced density of synaptic
AChRs in AChR-ß3F/3F mice might lead to a
reduction in the number of postjunctional folds
(Slater et al., 1997).
Interestingly, synapses in patients carrying mutations in DOK7
have fewer postjunctional folds (Beeson et
al., 2006; Slater et al.,
2006). Since tyrosine phosphorylation of the AChR ß-subunit
depends upon DOK7, these findings raise the possibility that the reduced
number of postjunctional folds in patients harboring mutations in
DOK7 may be due, at least in part, to a failure to fully tyrosine
phosphorylate the AChR ß-subunit
(Okada et al., 2006).
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/23/4167/DC1
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ACKNOWLEDGMENTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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2 test. (J) Two or fewer gaps are evident at category 1
synapses; three or four gaps are evident at category 2 synapses; five or more
gaps are evident at category 3 synapses. AChR-
ß3F/3F mice have fewer category 1 synapses and more
category 3 synapses than wild-type mice. Wild type: 40 category 1 synapses, 16
category 2 synapses, 3 category 3 synapses;
AChR-ß3F/3F: 11 category 1 synapses, 26
category 2 synapses, 18 category 3 synapses; P<0.005,
