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The Journal of Neuroscience, March 15, 1999, 19(6):1998-2007
Metabolic Stabilization of Muscle Nicotinic Acetylcholine
Receptor by Rapsyn
Zuo-Zhong
Wang1,
Askale
Mathias1,
Medha
Gautam2, and
Zach W.
Hall1
1 Laboratory of Cell Biology, National Institute of
Mental Health, National Institutes of Health, Bethesda, Maryland 20892, and 2 Department of Pharmacological and Physiological
Science, St. Louis University Medical School, St. Louis, Missouri
63104
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ABSTRACT |
Although the metabolic half-life of muscle endplate acetylcholine
receptor (AChR) changes during development and after denervation in the
adult, little is known about the molecular mechanisms that influence
receptor stability. We have investigated the effect on AChR turnover of
its interaction with rapsyn, a 43 kDa peripheral membrane protein that
is closely associated with the AChR in muscle cells and is required for
its clustering at endplates. Both in transfected COS cells and in
cultured myotubes from rapsyn-negative and rapsyn-positive mice, we
have found that the presence of rapsyn slows the turnover of AChRs by
as much as twofold. The effect was similar for both embryonic
( 2   ) and adult (2 ) AChRs and for AChRs whose subunit lacked a putative tyrosine
phosphorylation site. Neither colchicine nor cytochalasin D altered
AChR turnover or prevented the rapsyn effect. Mutant rapsyn proteins
whose N-terminal myristoylation signal was eliminated, or whose C
terminus or zinc-finger domains were deleted, failed to change the rate
of receptor turnover. Each of these mutations affects the association
of the AChR with rapsyn, suggesting that AChR stability is altered by
interaction between the two proteins. Our results suggest that, in
addition to its role in AChR clustering, rapsyn also functions to
metabolically stabilize the AChR.
Key words:
nicotinic receptors; rapsyn; 43 kDa protein; receptor
turnover; acetylcholine; neuromuscular junction; endplate; myotubes
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INTRODUCTION |
In developing and adult muscle,
acetylcholine receptors (AChRs) at the neuromuscular junction undergo
dramatic changes in their metabolic stability that correspond to the
innervation state of muscle fibers. Thus, in uninnervated myotubes, at
newly formed synapses, and in denervated muscle fibers, AChRs turn over
at a relative rapid rate (t1/2  1 d),
whereas at mature adult synapses the AChRs turn over much more slowly
(t1/2 10 d) (Berg and Hall, 1974 ; Chang
and Huang, 1975; Fambrough, 1979; Steinbach et al., 1979; Salpeter,
1987). Recent investigation of AChRs at denervated endplates has
revealed other populations of AChRs with intermediate turnover times
(Andreose et al., 1993; O'Malley et al., 1993). Although the
physiological modulation of AChR stability has been long-known,
relatively little is understood about the molecular interactions that
influence AChR stability. Interactions of receptor subunits with
cytoskeletal components have been postulated to be important
(Fambrough, 1979; Steinbach et al., 1979; Xu and Salpeter, 1995, 1997),
but little evidence is available about the significance of interactions
with specific proteins.
One protein that is known to interact with the AChR is rapsyn, a
peripheral membrane protein found at synapses in Torpedo electric
organs and vertebrate muscle cells in 1:1 stoichiometry with the AChR
(for review, see Froehner, 1991). Rapsyn, which associates with the
inner face of the membrane through an amino-terminal myristoyl group
(Phillips et al., 1991b), has recently been shown to play a key role in
AChR clustering. Thus, rapsyn can induce AChR clustering in
heterologous cells (Froehner et al., 1990; Phillips et al., 1991a), and
rapsyn-deficient mice fail to form AChR clusters at synapses in
embryonic myotubes (Gautam et al., 1995). At developing neuromuscular
junctions, rapsyn appears concurrently with AChR clusters at the very
beginning of synaptogenesis (Burden, 1985; Noakes et al., 1993).
Chemical cross-linking studies and structural analysis using electron
microscopy and x-ray diffraction suggest that rapsyn may
interact directly with the cytoplasmic domains of the AChR (Sealock et
al., 1984; Mitra et al., 1989), either through a specific interaction
with the subunit (Burden et al., 1983) or through interactions with
each of the subunits (Maimone and Merlie, 1993; Yu and Hall, 1994).
Rapsyn is also thought to interact with other postsynaptic proteins
such as those in the utrophin-associated complex of proteins at the
neuromuscular junction (Phillips et al., 1993; Apel and Merlie, 1995;
Apel et al., 1995). Because of its multiple interactions, rapsyn is
postulated to play an adaptor role, mediating the interaction of the
AChR with cytoskeletal elements at the synapse (Walker et al., 1984; Froehner, 1991; Apel and Merlie, 1995; Glass and Yancopoulos, 1997).
Because of its close association with the AChR, we have investigated
whether rapsyn influences the metabolic stability of the receptor. We
find, both in heterologous cells and in muscle cells, that association
with rapsyn slows the degradation of the AChR and thus increases its
metabolic stability.
A preliminary account of these results has been published previously
(Wang and Hall, 1996).
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MATERIALS AND METHODS |
cDNAs and expression vectors. Full-length cDNA clones
coding for the (Isenberg et al., 1986), (Buonanno et al.,
1986), (Yu et al., 1986), and subunits (Lapolla et al., 1984)
of mouse muscle nicotinic AChR were gifts from Drs. J. P. Merlie (Washington University, St. Louis, MO) and N. Davidson (California Institute of Technology, Pasadena, CA). The full-length cDNA for the
mouse subunit was obtained from Dr. P. D. Gardner [University of Texas Health Science Center, San Antonio, TX; see Gardner (1990)]. A cDNA construct encoding the extracellular ligand binding domain of
the 7 neuronal AChR and the transmembrane domains and cytoplasmic loop of the 5-HT3 serotonergic receptor
(7-5HT3 V201 chimera) was a gift from Dr. J. W. Patrick [Baylor College of Medicine, Houston, TX; see Dineley and
Patrick (1998); Eisele et al., 1993]. A full-length cDNA clone
encoding mouse rapsyn was kindly provided by Dr. S. Froehner
[University of North Carolina, Chapel Hill, NC; see Froehner (1989)].
Each of the cDNAs was subcloned into an SV40-based expression vector,
pSM (Brodsky et al., 1990). Anti-rapsyn antibodies mAb 1579A
and mAb1234A were generous gifts from Dr. S. Froehner (Scotland et al.,
1993).
Transfection of cells. COS cells grown in DMEM-H21
supplemented with 10% fetal bovine serum were transfected using an
adenovirus-aided, DEAE-dextran transfection procedure (Forsayeth and
Garcia, 1994, Wang et al., 1996a,b). Cells at ~70% confluence in a
60 mm dish were incubated for 2 hr at 37°C with a transfection
mixture containing plasmid DNAs in DMEM-H21 (2.5 ml), an E1 defective
adenovirus lysate (0.5 ml), and DEAE-dextran (120 µg). The amounts of
plasmid DNA used in the transfection mixture for , , or ,
and subunits were (in µg): 1.32, 0.66, 1.0, and 0.26, respectively. They were empirically determined to produce maximal cell
surface AChR expression. For coexpression of rapsyn in the cells, 2 µg of plasmid DNA encoding rapsyn was added per 60 mm dish, except
where indicated otherwise. After 2 hr of incubation, the transfection
mixture was removed, and the cells were rinsed with PBS twice before
being returned to the incubator in 4 ml of growth medium. After 24 hr
at 37°C, the cells were trypsinized and plated into three wells of
24-well plates. Surface AChR binding was measured after an additional 24 hr in culture.
Culture of rapsyn-deficient mouse myotubes. The
rapsyn / muscle cell line was derived using
rapsyn/ mutants bearing an immortalizing
transgene consisting of a temperature-sensitive SV40 large T antigen
(tsA58) under the control of a -interferon (-IFN)-inducible mouse
H-2kb Class I MHC promoter (Jat et
al., 1991). Mice bearing the immortalizing transgene were obtained from
matings between rapsyn+/ mice and rapsyn
heterozygotes that were also positive for the SV40 T antigen transgene
(M. Gautam, unpublished data). Cells were placed on
gelatin-coated plastic dishes and maintained at 33°C in DMEM
containing 20% fetal bovine serum, 3% chick embryo extract, and 4 U/ml mouse -IFN. After 2-3 d, cells were switched to 37°C in DMEM
with 2% horse serum and no -IFN to promote myotube formation.
Surface 125I--bungarotoxin binding and receptor turnover
assays were performed 24-48 h after the myotubes were switched to the
fusion medium.
Surface AChR assay and receptor turnover. Surface AChR
expression was determined by incubating cells for 90 min at 37°C in fresh growth medium containing 4 nM
125I--bungarotoxin (-BuTx, 220 Ci/mmol; Amersham,
Arlington Heights, IL). Nonspecific binding was measured by including
excess unlabeled -BuTx (1 µM) in the assay medium or
by assaying sham-transfected cells. At the end of the incubation
period, unbound 125I--BuTx was removed by washing the
cells three times with PBS. Cells were solubilized in 0.1N NaOH, and
radioactivity was counted with a Wallac gamma counter.
The degradation rate of surface AChR was measured by labeling the
receptor with 125I--BuTx for 90 min. Unbound
125I--BuTx was removed by washing the cells three times
with PBS. The cells in growth medium were then returned to the 37°C
CO2 incubator. At various intervals thereafter, duplicate
aliquots of culture medium were collected and replaced with fresh
medium. At the end of the experiments, cells were solubilized in 0.1N NaOH. Radioactivity in both the aliquots and cells was measured with a
Wallac gamma counter. The total radioactivity on the cell surface at
the beginning of the experiment was calculated by addition of that
found in the medium and solubilized cells. The data were used to
calculate the turnover rates, and degradation curves were fitted by
linear regression and normalized to 100% at time 0 after surface labeling.
Mutagenesis of rapsyn. A full-length mouse cDNA encoding
wild-type rapsyn was cloned into the mammalian expression vector pSM under the control of an SV40 early promoter. This clone
was used to create rapsyn mutants by site-directed mutagenesis as described (Phillips et al., 1993). To make a myristoylation-defective rapsyn (RapsynA2), we mutated the
Gly2 residue in the wild-type rapsyn to
Ala2 and left the remainder of the polypeptide
sequence unchanged. Rapsyn 16-254, a leucine
zipper deletion mutant, was created by deleting the cDNA sequences
encoding amino acids 16-254 with an in-frame fusion of
Gln15 to Ile255. The C-terminal
truncation mutant rapsyn255-412 was made by
replacing amino acid 255 with a stop codon to cause early termination
of the polypeptide at amino acid 254. To disrupt the two tandem zinc
finger domains, we constructed the mutant rapsynH Q by changing amino acids 384 and 387 from
histidine to glutamine. To eliminate the putative serine
phosphorylation site in the C terminus of rapsyn, we made mutant
rapsynSA by replacing serine 405 and 406 with
alanine (Scotland et al., 1993).
Gel electrophoresis and immunoblotting. Transfected COS
cells were extracted with a Triton X-100-containing buffer as described (Wang et al., 1996a). Cell lysates were denatured by boiling for 5 min
in SDS gel-loading buffer containing -mecaptoethanol,
electrophoresed on a 12.5% SDS-polyacrylamide gel (Laemmli, 1970), and
then transferred to nitrocellulose membranes. After preincubation in
PBS/0.3% Tween 20 containing 3% bovine serum albumin to block
nonspecific binding, the membranes were incubated with primary
antibodies against rapsyn in the same buffer and washed with PBS/Tween
20. Antibodies bound to the membrane were detected with horseradish
peroxidase-conjugated secondary antibodies and enhanced
chemiluminescent reagents (ECL, Amersham).
Immunofluorescent staining. Transfected COS cells grown on
chamber slides were incubated with rhodamine-conjugated -BuTx for 90 min, fixed for 10 min in 2% paraformaldehyde, and washed in cold PBS.
After a short incubation in PBS containing 10% bovine serum and 0.05%
saponin at 4°C for 20 min to permeabilize the membrane, cells were
incubated at 4°C for 1 h with a monoclonal rat antibody to the
subunit of the mouse AChR. After a brief rinse in cold PBS, cells
were incubated with an FITC-conjugated goat anti-rat secondary
antibody. The slides were examined under a fluorescence microscope
equipped with the appropriate filters.
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RESULTS |
Rapsyn slows turnover of AChRs in transfected cells
We first examined the effect of rapsyn on the metabolic half-life
of the mouse muscle nicotinic AChR expressed on the surface of
transfected COS cells. COS cells were transfected with cDNAs for all
four subunits of either the embryonic (2) or
adult (2) forms of the AChR, either with or
without the inclusion of rapsyn cDNA. Forty-eight hours after
transfection, the surface AChRs were labeled with
125I--bungarotoxin, and AChR degradation was followed by
the subsequent appearance of radioactivity in the medium, as described
by Gu et al. (1990). In both cases, the inclusion of rapsyn cDNA
significantly increased the half-life of AChRs on the cell surface. For
adult AChRs, the half-life of the receptor in the absence of rapsyn was
11 h; in the presence of 1 µg of rapsyn cDNA per 60 mm
dish, the half-life was increased to 17 h. The half-life of the
embryonic form of the AChR was similarly increased by coexpression of
rapsyn (Fig. 1A). We
observed no significant difference between the turnover times of the
adult and embryonic forms of the receptor, either with or without
rapsyn (see Discussion).
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Figure 1.
Metabolic stabilization of surface AChR by
coexpression of rapsyn in COS cells. A, Cotransfection
of mouse rapsyn cDNA (1 µg/60 mm dish) significantly reduced the
degradation rate of surface AChRs labeled by
125I--bungarotoxin. Transfected COS cells in culture
were labeled with 4 nM 125I--BuTx for 90 min, washed, and incubated in growth medium. The radioactivity released
into the media was measured at the times indicated, and the data were
used to calculate the turnover rates. Degradation curves were fitted by
linear regression and normalized to 100% at time 0 after surface
labeling. Metabolic stabilization of surface AChR by rapsyn was seen in
cells transfected with subunit cDNAs encoding either embryonic-type
(2) or adult-type (2)
AChRs. B, The effect on AChR
(2) turnover was more prominent with increased
amounts of rapsyn cDNA cotransfected per 60 mm dish. Each data
point represents the mean ± SEM of four separate
experiments.
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The increase in half-life of the AChR depended monotonically on the
amount of rapsyn cDNA that was added, up to a value of 28 h, seen
with 4 µg of rapsyn cDNA per 60 mm dish, the maximum amount that
could be used without reducing surface expression of the AChR (Fig.
1B). This continued increase suggests that with larger amounts of rapsyn cDNA, either a larger fraction of the AChR was
present in a form that turned over more slowly or that all molecules
turned over at progressively slower rates with increased expression of
rapsyn. Our data do not distinguish between these two possibilities.
The effect of rapsyn is specific
Other proteins were coexpressed with the adult form of the AChR to
determine whether they would have the same effect as rapsyn. The
proteins chosen were CD8, an integral membrane protein normally found
on the surface of lymphocytes (Littman, 1987), and two peripheral membrane proteins that are present at the neuromuscular junction, syntrophin and -dystroglycan. Syntrophin is a 58 kDa protein that,
like rapsyn, is associated with the cytoplasmic side of the membrane
(Adams et al., 1993; Froehner et al., 1997); -dystroglycan is a
heavily glycosylated protein that is attached to the extracellular matrix on the outside of the membrane (Ibraghimov-Beskrovnaya et al.,
1992). Both -dystroglycan and syntrophin are part of the
dystrophin-associated complex of proteins that link the cytoskeleton and the extracellular matrix; both have also been implicated in the
molecular mechanisms underlying the formation and maintenance of AChR
clusters (Fallon and Hall, 1994; Apel and Merlie, 1995; Froehner et
al., 1997). When coexpressed with the subunits of the adult form of the
AChR, none of the three proteins altered the AChR turnover time (Fig.
2).
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Figure 2.
The effect of rapsyn on metabolic stability of the
AChR is specific because other membrane proteins fail to change the
rate of receptor degradation. When coexpressed in COS cells, lymphocyte
surface molecule CD8 was incapable of stabilizing the AChR
(A). Coexpression of postsynaptic muscle proteins,
1-syntrophin (B), and dystroglycan
(C) neither affected the turnover rate of surface
AChR (2) nor changed the stabilizing effect of
rapsyn on the receptor. For each 60 mm culture dish, 4 µg of each
cDNA encoding CD8, -syntrophin, and dystroglycan were used for
transfection, respectively. The amount of rapsyn cDNA cotransfected was
4 µg in B and 2 µg in C. Each data point
represents the mean of three determinations.
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To determine whether the metabolic stabilization induced by rapsyn is
specific for muscle-type AChRs, we have sought to examine the effect of
rapsyn on turnover of neuronal 7 AChR. When 7 subunit is
expressed in COS cells, the surface receptor level is below the
detection limit of the 125I--BuTx binding assay,
although toxin binding sites and pentameric receptor can be found in
whole cell lysates (Cooper and Millar, 1997; Dineley and Patrick,
1998). To circumvent this problem, we have used a previously
characterized chimeric construct 7-5HT3 (V201). This
construct encodes the extracellular ligand binding domain of the 7
nicotinic receptor fused at valine 201 to the transmembrane domains and
the cytoplasmic loop of the 5HT3 receptor (Eisele et al.,
1993). When expressed in COS cells, the chimeric receptor is
transported to the cell surface, where it can be detected by
125I--BuTx binding. In addition, the chimeric protein
migrates as a pentamer in a sucrose gradient (Dineley and Patrick,
1998). Using the degradation of bound, radioactive toxin as an assay, we tested whether coexpressed rapsyn would alter the turnover time of
this protein. We found that it did not. Both when expressed with or
without rapsyn, the 7-5HT3 (V201) receptor was degraded with a half-life of ~8 h (Fig.
3A). These results suggest
that the stabilization effect of rapsyn does not extend to other
membrane-associated receptor proteins. Immunocytochemical experiments
showed that coexpression with rapsyn also did not alter the surface
distribution of the 7-5HT3 protein, suggesting that the
two are not associated (data not shown).
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Figure 3.
The effect of rapsyn is specific for the
muscle-type AChRs and is independent of tyrosine phosphorylation of
AChR subunits. A, Degradation of an 7-5HT3
chimeric receptor on COS cell surface was unaffected by coexpression of
rapsyn (2 µg cDNA/60 mm dish). The 7-5HT3 chimera was
made by fusing the NH2-terminal extracellular domain of the
neuronal nicotinic 7 subunit at valine 201 to the complementary
C-terminal domain of the serotonergic 5-HT3 receptor
subunit (see Results; also see Eisele et al., 1993; Dineley and
Patrick, 1998). B, Rapsyn was effective in slowing the
degradation of surface AChRs whose subunit lacked the putative
tyrosine phosphorylation site in the cytoplasmic loop
[(p0)]. The seven amino acids
GTDEYFI containing the site of tyrosine
phosphorylation on the cytoplasmic loop between the third and
fourth transmembrane domains were mutated to those of the subunit
PSRDKQE (containing no tyosine residue) (Yu and Hall, 1994).
C, Herbimycin A, a potent inhibitor of tyrosine kinases, did
not alter the decreased turnover rate of surface AChRs induced by
rapsyn. The drug (1 µM) was added to the culture medium
6 h before 125I--BuTx labeling and was continuously
present in the labeling and growth media thereafter. Control cells were
treated similarly with the solvent DMSO without the drug. Each data
point represents the averaged results of three determinations.
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Tyrosine phosphorylation of AChR subunit is not required for
the effect of rapsyn
Rapsyn can be cross-linked in situ to the subunit
of the AChR (Burden et al., 1983), suggesting that the two polypeptides are in close association, presumably through interactions of rapsyn with the long cytoplasmic domain connecting transmembrane regions M3
and M4 of the subunit. The loop contains a putative site of
tyrosine phosphorylation (Huganir and Miles, 1989; Wagner et al.,
1991). When this site was eliminated by replacing the amino acids
GTDEYFI with the corresponding amino acids from the subunit, which has no site of tyrosine phosphorylation [PSRDKQE; Yu and Hall (1994)], rapsyn-induced stabilization of surface AChR metabolism was unaffected (Fig. 3B). Moreover, treatment of
transfected COS cells with a potent tyrosine kinase inhibitor,
herbimycin A (1 µM), did not alter the effect of rapsyn
on surface AChR turnover (Fig. 3C). Because herbimycin A has
been shown to effectively block the clustering of muscle AChR and
prevent the phosphorylation of the subunit (Ferns et al., 1996), we
conclude that neither AChR clustering nor tyrosine phosphorylation of
the receptor is essential for metabolic stabilization of AChRs by
rapsyn in COS cells.
Metabolic stabilization is not inhibited by agents that disrupt
the cytoskeleton
The cytoskeleton of muscle cells may play an important role in
maintaining rapsyn-induced AChR clusters by serving as an anchor for
the rapsyn-receptor complex (for review, see Froehner, 1991; Apel and
Merlie, 1995). It remains unclear whether rapsyn binds to the
cytoskeleton directly or through links provided by other postsynaptic
proteins such as utrophin or the dystrophin-glycoprotein complex
(Phillips et al., 1993; Apel et al., 1995). Previous studies, however,
have shown that receptor clusters are stabilized by actin-containing filaments and that the movement of receptors in the plane of the membrane requires microtubules (Bloch, 1983; Connolly, 1984;
Walker et al., 1984). To examine whether rapsyn-induced metabolic
stabilization of the AChR in COS cells involves interaction with the
cytoskeleton, we tested the effects of microfilament- and
microtubule-disrupting drugs on receptor turnover. Neither cytochalasin
D (2 µg/ml), which disrupts actin filaments, nor colchicine (10 µM), which depolymerizes microtubules, affected the
turnover rate of the AChR. In addition, neither of these drugs
interfered with the metabolic stabilization effect of rapsyn on surface
receptors (Fig. 4).
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Figure 4.
Cytochalasin D (2 µg/ml), which disrupts actin
filaments, and colchicine (10 µM), which depolymerizes
microtubules, failed to change the degradation rate of surface AChR
(A) and the stabilizing effect of rapsyn on the
receptor (B). The drugs were applied to the culture
medium immediately after the cells were labeled with
125I--BuTx for determination of surface AChR degradation
rate. Two micrograms of rapsyn cDNA were used for cotransfection
experiments in B. Each data point represents the averaged
results of three measurements.
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The rapsyn domains involved in AChR clustering are also required
for metabolic stabilization of the receptor
Three domains of rapsyn have been identified as being essential
for its ability to stimulate AChR clustering (Phillips et al., 1991b;
Scotland et al., 1993). At its N terminus, rapsyn is normally modified
by the addition of an N-linked myristate moiety to
glycine2 (Fig.
5A). When the signal for
myristoylation is removed by mutation of the glycine to alanine, the
mutated protein associates poorly with the membrane and is inactive in
clustering the AChR. The carboxyl region of rapsyn contains a
zinc-finger domain that has also been shown to be necessary for
clustering, and the central portion of the protein contains a leucine
zipper motif, which is necessary for AChR interaction (Fig.
5A,B). We examined mutations in each of these regions for
their effect on the metabolic stability of the AChR.
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Figure 5.
Schematic diagram of wild-type and mutant forms of
rapsyn protein. A, G represents glycine residue, the site
for myristoylation. The zig-zag line before G
indicates the myristate moiety. A indicates the substitution
of alanine to prevent myristoylation. The potential leucine zipper
(striated bar), zinc fingers (black bar), and
serine phosphorylation site (S) were also
illustrated. B, The two histidine residues at positions 384 and 387 were mutated to glutamine (Q) to disrupt the
tandem zinc-finger domains of rapsyn. Serines 405 and 406 were
substituted by alanines to mutate the putative phosphorylation site in
the C terminus of rapsyn.
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When oligonucleotide-directed mutagenesis was used to change the
N-terminal Gly2 of rapsyn to
Ala2, AChR clustering was drastically reduced as
reported (Fig. 6C) (Phillips
et al., 1991b), and the mutated rapsyn no longer affected the turnover
rate of the AChR (Fig. 6, bottom left). Mutant
rapsyn255-412, which lacked 158 residues on the
C terminus of the protein, impaired the clustering activity of rapsyn
(Fig. 6E). Likewise, mutation of the two histidine
residues at positions 384 and 387, which disrupts the tandem
zinc-finger domains in the C terminus of rapsyn, efficiently prevented
metabolic stabilization of the AChR and disrupted clustering (Fig.
6F). Thus the myristoylation signal, the C-terminal
domain, and the zinc-finger motif are all required for the effect of
rapsyn on AChR turnover. In contrast, mutant
rapsyn16-254, which lacked the leucine zipper
domain, still retained some receptor clustering function as shown by
immunofluorescent staining of the transfected COS cells (Fig.
6D) and had a partial effect on AChR turnover (Fig.
6, bottom left). Thus, the leucine zipper motif is not an
absolute requirement for metabolic stabilization by rapsyn.
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Figure 6.
Mutagenesis of mouse rapsyn protein delineates
domains involved in metabolic stabilization of surface AChRs.
A-G, Cells were transfected with cDNAs encoding , ,
, and subunits of the AChR alone (A), or in
combination with each of the cDNAs for wild-type rapsyn
(B), mutant rapsynA2
(D), rapsyn16-254
(D), rapsyn255-412
(E), rapsynHQ
(F), or rapsynSA
(G). Two days after transfection, cells were fixed
and stained with a monoclonal rat antibody to the subunit of the
AChR (mAb210), followed by FITC-conjugated secondary antibody.
Bottom, Effects of rapsyn mutants on the degradation rate of
surface AChRs in COS cells. The cells were transfected with cDNAs
encoding the adult-type AChR (2 subunits) alone
or in combination with each of the mutant rapsyn cDNAs as indicated (4 µg/60 mm dish). Each data point represents the averaged results of
four determinations.
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The C-terminal region of rapsyn contains a putative serine
phosphorylation site by PKA or PKC (Froehner, 1991; Scotland et al.,
1993). To examine its role in receptor clustering and stabilization, we
mutated the site by changing serines 405 and 406 to alanines (Fig.
5B). When coexpressed with the AChR in COS cells, this
mutant protein induced clustering and also slowed the degradation of surface AChR (Fig. 6G and bottom right). We
conclude that the serine phosphorylation sites are not essential for
metabolic stabilization of the AChR by rapsyn.
Surface AChRs in myotubes of rapsyn-deficient mice are
metabolically unstable
Recent genetic studies have shown that the AChR fails to become
clustered at the postsynaptic membrane of neuromuscular junctions in
mice with a targeted disruption of the rapsyn gene (Gautam et al.,
1995). To examine whether the deficiency of rapsyn also alters the rate
of receptor turnover, we compared AChR degradation rates in cultured
muscle cells of homozygous ( /) and heterozygous (+/) rapsyn
knockout mice. The myoblasts obtained from these mice carry a
temperature-sensitive, SV40 large T antigen under control of the class
I MHC promoter (Jat et al., 1991). In the presence of -interferon,
the myoblasts can be maintained in an undifferentiated state at the
permissive temperature (33°C). When switched to fusion medium and
grown at 37°C, they differentiate into multinucleated myotubes (M. Gautam, unpublished data). In rapsyn+/
myotubes, spontaneous AChR clusters were present but very few in
number. When soluble, recombinant agrin was added to the culture medium, the number of receptor clusters increased dramatically. In
contrast, rapsyn/ myotubes had neither
spontaneous nor agrin-induced receptor clusters (data not shown). Thus,
when the metabolic stability of AChRs was examined, the surface AChRs
labeled with 125I--bungarotoxin in
rapsyn+/ cells were found to have a half-life of
~37 h, comparable to those of noninnervated, embryonic muscle and
cultured C2 myotubes. In contrast, the AChR half-life in
rapsyn/ muscle cells was reduced to ~19 h
(Fig. 7). Thus, in muscle cells, as well
as in heterologous cells, the presence of rapsyn alters the metabolic
stability of AChRs.
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Figure 7.
The surface AChRs on cultured myotubes of
rapsyn-deficient (rapsyn/) mice degrade
significantly faster than those of control
(rapsyn+/) mice. The myotubes in culture were
labeled with 4 nM 125I--BuTx for 90 min,
washed, and incubated in growth medium. The radioactivity released into
the media was measured at the times indicated, and the data were used
to calculate the turnover rates. Degradation curves were fitted by
linear regression and normalized to 100% at time 0 after surface
labeling. Each data point represents the mean ± SEM of four
separate determinations.
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|
| |
DISCUSSION |
The primary finding of this paper is that rapsyn modifies the
metabolic turnover rate of cell surface AChR. This effect is seen both
in heterologous COS cells, when rapsyn and the AChR are coexpressed,
and in cultured muscle cells, when wild-type myotubes are compared with
cells derived from genetically altered mice that lack rapsyn. The
effect appears to be specific, because coexpression of other synaptic
muscle proteins in COS cells, including -dystroglycan and
syntrophin, as well as the unrelated lymphocyte membrane protein CD8,
did not change the rate of degradation of the AChR.
In COS cells, as in Xenopus oocytes and quail fibroblasts,
rapsyn also changes the distribution of AChRs at the cell surface (Froehner et al., 1990; Phillips et al., 1991a; Yu and Hall, 1994). When expressed alone, rapsyn occurs at or near the cell surface in
aggregates; in contrast, AChRs are diffusely distributed when expressed
alone, but occur in clusters that are coextensive with the rapsyn
aggregates when the two proteins are expressed together. Experiments
with mutated forms of rapsyn indicate that alterations that affect the
ability of rapsyn to cluster AChRs, such as removal of the
myristoylation site or deletion of the zinc-finger or leucine zipper
domains (Phillips et al., 1991b; Scotland et al., 1993), also affect
the ability of rapsyn to alter AChR turnover (Figs. 5, 6). One
interpretation of these findings is that the alteration in AChR
metabolic stability caused by rapsyn is the result of clustering, as
suggested by a recent research report (Phillips et al., 1997). The two
effects appear to be separate, however, because rapsyn has the same
effect in muscle cells in which most AChRs are not clustered (Fig. 7).
In addition, it has been long known that AChRs in clusters formed at
sites of nerve contact in cultured myotubes have the same turnover rate
as diffusely distributed surface AChRs (Salpeter et al., 1982;
O'Malley et al., 1993). Furthermore, in contrast to recent results of
Phillips et al. (1997), we observed that the addition of neural agrin
to C2 myotubes in culture only slightly enhanced the metabolic
stability of surface AChRs (t1/2 = 13.4 ± 0.2 hr in control vs 14.3 ± 0.5 hr in agrin-treated group;
p > 0.05), although neural agrin caused the formation
of numerous AChR clusters on cell membrane (Z. Wang and Z. Hall,
unpublished observations). Thus, the effect of rapsyn on turnover time
is not secondary to cluster formation, but is a distinct effect,
independent of clustering.
The results with the mutated forms of rapsyn suggest that similar
domains in the protein mediate the effects of rapsyn on AChR turnover
and AChR clustering. In both cases, direct interaction between rapsyn
and the AChR is likely, suggesting that these domains are necessary for
association of the two proteins. Recently, Ramarao and Cohen
(1998) have identified residues 298-331 of rapsyn protein as a
potential coiled-coil domain and established that mutants disrupting
this region prevent AChR clustering. Although we have not tested
whether the sequence 298-331 is required for metabolic stabilization
by rapsyn, our result with rapsyn255-412, which
lacked this putative domain, is consistent with the data of this recent
report. Drugs that disrupt actin filaments or microtubules did not
affect turnover, suggesting that the changes in AChR degradation are
not attributable to an overall change in cytoskeletal organization.
Although direct evidence is lacking, several observations suggest that
AChRs that are unclustered, as well as those in clusters, are
associated with rapsyn (Noakes et al., 1993; Peng and Froehner, 1985).
The 1:1 correspondence of AChRs and rapsyn (LaRochelle and Froehner,
1986, 1987) and the coprecipitation of rapsyn and the AChR in C2
myotubes in culture (C. Fuhrer and Z. Hall, unpublished observations) are both consistent with this idea. The ability of rapsyn
to affect the turnover rate of AChRs in uninnervated myotubes in which
clusters are rare (Fig. 7) provides further support for the idea that
rapsyn is associated with both clustered and unclustered AChRs.
The association of one protein with another is known to affect
metabolic stability in a number of cases. For instance, association of
the and subunits of AChR in the endoplasmic reticulum during receptor assembly stabilizes the subunit by decreasing its
degradation rate (Blount et al., 1990). Such change may be brought
about by a conformational change of the protein after its association, which renders it resistant to protease degradation. A conformational change of the AChR after association with rapsyn is unlikely, however,
because the interactions do not change the channel properties of the
receptor and its ligand-binding activity (Brennan et al., 1992). A
second alternative is that rapsyn obscures a sequence on the AChR
that serves as a degradation signal for the receptor. Such
signals are present in other membrane receptor proteins, including
members of the EGF receptor, where they are thought to play a critical
role in mediating receptor endocytosis, thus affecting the metabolic
stability of membrane proteins (Kornilova et al., 1996; Kurten et al.,
1996). Alternatively, the change in turnover time may occur because the
two proteins, AChR and rapsyn, when associated, are metabolized
together. This possibility is unlikely, however, because the half-life
of rapsyn was found to be short (~3 h), and the presence of AChR does
not affect the degradation rate of rapsyn in muscle cells (Frail et
al., 1989).
Although rapsyn is thought to bind directly to the AChR, little is
known about the receptor domains involved in mediating the interaction.
Rapsyn in situ can be chemically cross-linked to the subunit of the AChR (Burden et al., 1983), and studies on AChR
clustering suggest that each of the subunits of the AChR can interact
with rapsyn, presumably via the long cytoplasmic loop between
transmembrane regions III and IV (Maimone and Merlie, 1993; Yu and
Hall, 1994). Specific sequences that mediate the interaction have not
been detected. Our finding that when the N-terminal domain of the
nicotinic 7 subunit was expressed on the surface as an
7-5HT3 chimeric receptor, rapsyn coexpression had no
effect on either its surface distribution or its degradation rate (Fig.
3A) is consistent with the idea that rapsyn interacts with
an intracellular domain of the AChR. Mutation of a presumptive phosphorylation sequence on the subunit did not alter the effect of
rapsyn on either AChR distribution (Yu and Hall, 1994) or AChR turnover
(Fig. 3B). These results, plus the observation that
herbimycin A was without effect, suggest that tyrosine phosphorylation
is not required for the interaction of rapsyn with the AChR. Recently it has been possible to coimmunoprecipitate rapsyn and the AChR from
extracts of C2 myotubes, which will allow further investigation of the
interaction (C. Fuhrer and Z. Hall, unpublished observations).
Rapsyn was equally effective in slowing degradation of both adult
(2) and embryonic
(2) forms of the AChR, indicating that
its interaction is not dependent on the presence of either or subunit. We also found in the present study that surface AChRs
containing either the or subunits have indistinguishable half-lives (~11 h) when expressed in COS cells. This result coincides with those from two other laboratories, using Xenopus
oocytes or CHO cells (Kopta and Steinbach, 1994; Liu et al., 1994),
respectively, but differs from an earlier report from our own
laboratory in which we looked at AChRs expressed in COS cells (Gu et
al., 1990) and a recent report on AChRs in muscle cells (Sala et al.,
1997). Our own interpretation of these conflicting results is that
there is a small intrinsic difference between the turnover times of the
two AChR types that is variably detected because of differences in
cell-type and experimental conditions.
During development, the embryonic form of the AChR at endplates, which
undergoes rapid turnover (half-time ~1 d) equivalent to that seen in
unclustered AChRs, is replaced by the adult form of the AChR, with a
concomitant change in turnover to an approximately 10-fold lower rate
(half-time ~10 d). After denervation of the endplate in the adult,
the -AChR changes its half-time of turnover to ~3 d and is then
replaced by -AChRs with a turnover time of 1 d (Burden, 1977;
for review, see Fambrough, 1979; Salpeter, 1987). These experiments
indicate that AChR turnover time at the endplate depends on subunit
composition and on other factors controlled by innervation. Although
rapsyn is presumably an integral part of the complex of proteins that
regulate the metabolic stability of AChRs at the endplate, variations
in rapsyn concentration are unlikely to be an active regulatory
element. Rapsyn is present at AChR clusters at the very beginning of
synaptogenesis (Burden, 1985; Noakes et al., 1993), and message levels
for both proteins are coordinately regulated during development
(Froehner, 1989; LaRochelle et al., 1990). In the absence of rapsyn,
AChR clusters cannot form, and AChRs do not become stabilized. Our
results showing the effects of association with rapsyn on AChR
stability may have broader relevance to understanding the effects of
other receptor or ion channel-associated cytoskeletal elements such as
PSD 95, GRIP, gephrin, and ankyrin on the metabolism of membrane
receptors and ion channels (Sheng and Kim, 1996; Colledge and Froehner, 1998; Gee et al., 1998).
| |
FOOTNOTES |
Received Aug. 18, 1998; revised Dec. 22, 1998; accepted Jan. 6, 1999.
This work was supported by grants from the Muscular Dystrophy
Association, the National Institute of Neurological Disorders and
Stroke, and the intramural research program of the National Institute
of Mental Health. We thank Drs. N. Davidson, S. Froehner, P. Gardner,
J. Lindstrom, J. Merlie, and K. Campbell for antibodies and cDNAs. We
also thank Dr. Joshua Sanes for helpful discussions, and members of the
Hall laboratory for comments and advice.
Correspondence should be addressed to Dr. Zuo-Zhong Wang at his present
address: Department of Neurobiology, University of Pittsburgh School of
Medicine, 3500 Terrace Street, E1440 BST, Pittsburgh, PA 15261.
Dr. Hall's present address: Department of Physiology, University of
California School of Medicine, 513 Parnassus Avenue, San Francisco, CA
94143-0410.
| |
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Top
Abstract
Introduction
