* National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892; and Regeneron
Pharmaceuticals, Inc., Tarrytown, New York 10591
The induction of acetylcholine receptor
(AChR) clustering by neurally released agrin is a critical, early step in the formation of the neuromuscular
junction. Laminin, a component of the muscle fiber basal
lamina, also induces AChR clustering. We find that induction of AChR clustering in C2 myotubes is specific
for laminin-1; neither laminin-2 (merosin) nor laminin-11 (a synapse-specific isoform) are active. Moreover,
laminin-1 induces AChR clustering by a pathway that is
independent of that used by neural agrin. The effects of
laminin-1 and agrin are strictly additive and occur with
different time courses. Most importantly, laminin-
1-induced clustering does not require MuSK, a receptor tyrosine kinase that is part of the receptor complex
for agrin. Laminin-1 does not cause tyrosine phosphorylation of MuSK in C2 myotubes and induces AChR
clustering in myotubes from MuSK/
mice that do
not respond to agrin. In contrast to agrin, laminin-1 also
does not induce tyrosine phosphorylation of the AChR,
demonstrating that AChR tyrosine phosphorylation is
not required for clustering in myotubes. Laminin-1 thus
acts by a mechanism that is independent of that used by
agrin and may provide a supplemental pathway for
AChR clustering during synaptogenesis.
THE highly specialized postsynaptic structures at the
adult neuromuscular junction arise and are maintained in part through signals conveyed by specific
components of the basal lamina (Burden et al., 1979 The clustering of preexisting AChRs beneath the nerve
terminal is thought to occur through the action of agrin, a
component of the adult synaptic basal lamina that is synthesized and released by motor neurons (McMahan,
1990 Agrin mediates AChR clustering by binding and activating a signaling receptor on the muscle surface that is specific for the neural agrin isoform (Bowen et al., 1996 Besides agrin, a number of other factors stimulate
AChR clustering in cultured muscle cells. These include
laminin (Vogel et al., 1983 The possible role of laminin in synapse formation is of
particular interest as it is present at the earliest synapses
and is colocalized with AChR clusters formed both in vivo
and in vitro (Bayne et al., 1984 Because of its potential physiological importance, we
have investigated the mechanism by which laminin-1 induces AChR clustering in C2 myotubes. We find that laminin-1, but not laminin-2 or -11, induce AChR clustering
and that the effects of laminin-1 are additive to those of
agrin. Laminin-1-induced AChR clustering does not require MuSK and is not accompanied by tyrosine phosphorylation of the AChR. The laminin-1 signaling pathway is
thus different from that used by agrin and may serve as an
additional or supplemental mechanism for AChR clustering during the formation and maintenance of the neuromuscular junction.
Antibodies and Reagents
A peptide corresponding to the last 20 amino acid residues of rat MuSK
(CSIHRILQRMCERAEGTVGV; Valenzuela et al., 1995 Production of the MuSK Mice heterozygous for a mutant MuSK allele (DeChiara et al., 1996 Muscle Cell Culture
C2C12 mouse muscle cells were cultured as described by Gordon and Hall
(1989) AChR Clustering Assay
C2 myotubes were grown on Permanox-treated 8-well chamberslides
(Nunc, Inc., Naperville, IL), and MuSK MuSK Extraction and Isolation
Mature C2 myotubes were incubated with neural agrin (C-Ag4,8), laminin-1,
or with both for 45 min at 37°C. After the treatments, the cells were rinsed
with Ca2+/Mg2+-free PBS and scraped up into an extraction buffer (1%
Nonidet P-40 or 1% digitonin; 10 mM Tris, pH 7.5; 150 mM NaCl; 5 mM
each EDTA and EGTA; 1 mM sodium orthovanadate; 1 mM each PMSF,
benzamidine, N-ethylmaleimide, sodium tetrathionate; and 20 µg/ml each
leupeptin and aprotinin). After removing insoluble material by centrifugation at 18,000 g for 10 min, the C2 extracts were incubated with a polyclonal anti-MuSK serum followed by protein A coupled to Sepharose. The Sepharose beads were washed in extraction buffer and proteins bound to the beads were removed by boiling in SDS sample buffer. The
immunoprecipitated proteins were then separated by SDS-PAGE, transferred to nitrocellulose sheets, and the blots incubated with a mixture of
phosphotyrosine antibodies, mAbs 4G10 and PY20, followed by incubation with an HRP-conjugated anti-mouse secondary antibody. Immunolabeled bands were visualized by enhanced chemiluminescence (ECL; Amersham Corp., Arlington Heights, IL). The blots were stripped with 0.2 M
glycine, pH 2.5, and 0.1% Tween20 and reprobed with the MuSK polyclonal
antiserum to confirm that similar amounts of total MuSK were isolated.
AChR Extraction and Isolation
C2 myotubes were treated and extracted as described above. AChRs were
isolated by incubating the C2 extracts with Laminin-1 Induces AChR Cluster
Formation on C2 Myotubes
Previous experiments have shown that laminin-1 can induce AChR clustering on primary rat myotubes or on G8-1
clonal rat muscle cells (Vogel et al., 1983
When C2 myotubes were treated with both neural agrin
and laminin-1, a greater number of AChR clusters was observed than with either treatment alone (Fig. 1 D). Some
AChR clusters that formed in response to treatment with
both neural agrin and laminin-1 also appeared to be longer
and more organized along the myotube edge (Fig. 1 D)
compared to clusters induced by laminin-1 or neural agrin
alone. To test whether laminin-1 and neural agrin act
through the same pathway or through independent pathways to produce AChR clusters, we determined whether
or not their effects were additive. C2 myotubes were
treated with various concentrations of neural agrin (0.15 pM to 15 nM) in the presence or absence of 40 nM of laminin-1. At all concentrations of agrin tested, including concentrations that produced a maximal effect, the addition of
laminin-1 induced a strictly additive response (Fig. 2 B).
These observations suggest that the two ligands do not
compete for the same receptor and that the pathways by
which they act do not share limiting steps.
To determine whether the laminin effect on AChR clustering is specfic for particular isoforms, we tested whether
other laminins expressed in muscle could cause AChRs to
aggregate. Accordingly, C2 myotubes were treated with either
laminin-1, -2 (merosin), or -11 (a synapse-specific isoform
containing the
Laminin-1 and Agrin Cluster AChRs with
Different Time Courses
Agrin acts quickly to aggregate surface AChRs; after the
addition of agrin to C2 myotubes, AChR clusters are first
apparent after 3 h, and their number reaches a peak within
6 to 8 h (Ferns et al., 1996
Laminin-1 Induces AChR Clustering
in MuSK Neural agrin requires MuSK, a receptor tyrosine kinase, to
induce the formation of AChR clusters. Mice with a targeted disruption of the gene encoding MuSK do not form
neuromuscular synapses (DeChiara et al., 1996
Laminin-1 Does Not Induce MuSK
Tyrosine Phosphorylation
Since MuSK activation is thought to be an early event in
the agrin signaling pathway, we next tested whether laminin-1 activates MuSK. C2 myotubes were treated for 45 min with either 4, 12, 40, or 120 nM of laminin-1, 1.5 pM to
15 nM of soluble neural agrin (Ag4,8), or with 15 nM of soluble muscle agrin (Ag0,0). Proteins were then extracted in
a detergent-containing buffer and divided into two groups.
Extracts from one group were immunoprecipitated with a
MuSK polyclonal serum, and the remaining extracts were treated to isolate AChRs as described below. After separating proteins by SDS-PAGE and transferring them to nitrocellulose, the blots were probed with a mixture of the
phosphotyrosine antibodies, mAb 4G10 and mAb PY20.
We found that under these conditions, treatment of C2
myotubes with neural agrin, even at concentrations of 15 pM, induced MuSK tyrosine phosphorylation. Thus, the
concentrations of neural agrin that induce AChR clustering and those that induce MuSK tyrosine phosphorylation
are similar. Laminin-1, however, did not induce MuSK
phosphorylation even at high concentrations (Fig. 8) or after 5, 8, 12, or 18 h of treatment (Sugiyama, J.E., and Z.W.
Hall, unpublished observations), indicating that it is probably not a functional ligand for MuSK. The blots were
later reprobed for total MuSK to confirm that equivalent
amounts of MuSK had been immunoprecipitated. The lower
bands in the MuSK immunoblot are most likely nonspecific bands recognized by the antiserum.
Laminin-1 Does Not Induce Phosphorylation
of the AChR Because tyrosine phosphorylation of the
Laminin-1 Induces AChR Aggregation on C2 Myotubes
Vogel et al. (1983) Laminin-1 and Agrin Use Different Pathways
to Cluster the AChR
Although laminin-1 and agrin both induce AChR clusters,
the two ligands use separate signaling pathways. This conclusion rests on four observations: (a) the number of
AChR clusters induced by laminin-1 and by agrin are additive, even at saturating concentrations of agrin (Fig. 2 b);
(b) although agrin-induced AChR clustering requires
MuSK (DeChiara et al., 1996 If laminin-1 and agrin shared a signaling pathway, then a
saturating response to one would not be further increased
by treatment with the other. This is clearly not the case, as
40 nM of laminin-1 increased the agrin response by a constant amount at all concentrations of agrin tested, indicating that the effects are strictly additive (Fig. 2 b). Although
this result does not exclude the possibility that the two
pathways may share nonlimiting, downstream steps, the
limiting steps of each pathway, and those preceding them, must be separate.
Neural agrin activates MuSK, a muscle-specific receptor
tyrosine kinase that is thought to be part of the agrin receptor complex (DeChiara et al., 1996 The time courses of laminin-1- and of agrin-induced
AChR clustering are quite different. Whereas agrin-induced
AChR clustering occured within a few hours (Fig. 5; Ferns
et al., 1996 Agrin treatment of C2 myotubes not only causes AChR
clustering, but also tyrosine phosphorylation of the AChR
The Molecular Mechanism of Laminin -1-Induced
AChR Clustering
The molecular mechanism of AChR clustering induced by
laminin, as in the case of agrin, is unknown. The clusters
presumably represent the redistribution of AChRs on the
myotube surface, rather than the insertion of newly synthesized AChR clusters, since surface AChRs pre-labeled
with rhodamine- Tyrosine phosphorylation appears to play a role in the
signaling pathway for AChR clustering. Immunofluorescence experiments show that phosphotyrosine staining
colocalizes with AChR clusters early in development (Qu
and Huganir, 1994 An interesting recent observation shows that low concentrations of laminin-1 (30 pM to 6 nM) cause the clustering of What Is the Physiological Role of Laminin-1-induced
AChR Clustering?
Although in vitro laminin-1 induces AChR clusters by a
pathway that is clearly distinct from that used by agrin,
how does this pathway function in vivo? In mice that lack
agrin, few AChR clusters are associated with sites of
nerve-muscle contact, indicating that agrin-induced clustering is the major mechanism for concentrating AChRs
beneath nerve terminals (Gautam et al., 1996 The idea that laminin-1 acts to increase the size of agrin-induced AChR clusters is consistent with its slower time
course, and with observations of the properties of AChR
clusters formed by the combination of laminin-1 and agrin.
At high concentrations of agrin (1.5 or 15 nM), the AChR
clusters that formed were smaller and less intensely
stained than those seen after treatment with low concentrations of agrin (15 or 150 pM). In contrast, all laminin-1-induced clusters, regardless of the concentration used,
were large, more condensed, and intensely stained. The
clusters that formed when myotubes were treated with
laminin-1 and agrin together (using either low or high concentrations of agrin) appeared as highly condensed clusters along the edges of the myotubes, similar to those observed upon treatment with laminin-1 alone. Laminin-1 may thus act to further concentrate or organize the agrin-induced AChR clusters.
If the laminin-1 pathway is found to be used, what is the
agent that activates it in vivo? Of the three isoforms that
we have examined, only laminin-1 is active, but other isoforms may be active as well. It will be of particular interest
to examine the activity of a recently identified synaptic
form of laminin containing the The fact that laminin and other agents, in addition to
neural agrin, can cause AChR clustering suggests that auxiliary pathways of clustering may operate during maturation of the postsynaptic membrane. One attractive hypothesis is that AChR clustering is initiated by neural agrin,
present in limiting amounts, and that clustering may be
amplified during development of the synapse by other, less
active but more abundant agents present in muscle, such
as laminin or muscle agrin (Godfrey et al., 1988; Hall
and Sanes, 1993
). The most prominent feature of the postsynaptic muscle membrane is the high density (>10,000/µm2)
of acetylcholine receptors (AChRs)1 on the myotube surface (Fertuck and Salpeter, 1976
). During synaptogenesis, AChRs, which are diffusely distributed on myotubes before innervation, accumulate at the nascent synapse soon
after the growth cone first contacts the myotube membrane. Shortly thereafter a defined basal lamina appears
between the nerve terminal and the muscle cell. This local
postsynaptic accumulation of AChRs is caused by two mechanisms: an increase in local receptor synthesis and
the redistribution of preexisting surface AChRs (Hall and
Sanes, 1993
).
). Agrin is a large protein with several sites of alternative splicing that affect its biological activity (Rupp et al.,
1991
; Ferns et al., 1992
, 1993
; Ruegg et al., 1992
; Tsim et
al., 1992
). The isoforms most active in clustering AChRs
are found in neurons, whilst muscle cells synthesize less active isoforms (Ferns et al., 1993
; Hoch et al., 1993
). Treatment of myotube cultures with agrin causes coaggregation
of the AChR and other components of the adult synapse,
such as acetylcholinesterase, laminin, and heparan sulfate
proteoglycan (Wallace, 1989
; Nitkin and Rothschild, 1990
).
Recently, experiments using knock-out mice have unequivocably established the importance of agrin in synaptogenesis. Muscles from agrin-deficient mice fail to make
differentiated neuromuscular junctions; although muscles
in these animals have AChR clusters, they are smaller, and
few are associated with nerve fibers (Gautam et al., 1996
).
). Although the muscle receptor that binds agrin and mediates
its action on AChR clustering has not been completely
identified (Nastuk et al., 1991
), one of its components appears to be MuSK (muscle specific kinase), a receptor tyrosine kinase specifically expressed by skeletal muscle.
MuSK is upregulated at the onset of muscle differentiation and is localized to the adult neuromuscular junction (Valenzuela et al., 1995
). Agrin treatment of cultured myotubes
induces tyrosine phosphorylation of MuSK, and in intact
myotubes, agrin can be crosslinked to MuSK, presumably
via an accessory protein (Glass et al., 1996
). Experiments
using mice homozygous for a MuSK gene disruption show
that MuSK is necessary for formation of the neuromuscular junction. MuSK-null mice do not form AChR clusters,
and myotubes made from these mutant mice fail to respond to neural agrin (DeChiara et al., 1996
; Glass et al.,
1996
). Thus, MuSK is required for agrin-induced AChR
clustering and appears to be part of the receptor complex
that binds agrin and mediates its action on muscle cells. The precise molecular mechanism by which agrin-induced
activation of MuSK leads to AChR clustering is not known
but may involve phosphorylation of the AChR itself (Wallace et al., 1991
; Qu and Huganir, 1994
; Ferns et al., 1996
).
In C2 myotubes, agrin induces a transient phosphorylation
of the
subunit of the AChR that precedes clustering;
moreover, inhibitors that block the AChR phosphorylation also block AChR clustering (Wallace, 1994
; Ferns et
al., 1996
).
), VVA-B4, a lectin that selectively binds a synapse-specific carbohydrate (Martin and
Sanes, 1995
), and two proteins, basic fibroblast growth factor and heparin-binding growth-associated molecule, that
are active when bound to polystyrene beads (Peng et al.,
1991
, 1995
). Neither the mechanism by which these factors act, nor their physiological role, is known.
; Chiu and Sanes, 1984
;
Daniels et al., 1984
; Gordon et al., 1993
). In the adult,
laminin is a major component of the muscle fiber basal
lamina, with distinct synaptic and extrasynaptic isoforms.
Laminin is a heterotrimer composed of three chains,
,
,
and
, and multiple forms of each chain are encoded by
separate genes (e.g.,
1-5,
1-3, and
1-2). The type of
laminin isoform produced is dependent upon its chain
composition (Timpl et al., 1982
; Burgeson et al., 1994
).
Several different laminin isoforms with distinct distribution patterns are found in skeletal muscle (Sanes et al.,
1990
; Lentz et al., 1997
; Miner et al., 1997
). The isoforms
that have been identified in muscle are laminin-1 (
1
1
1),
laminin-2 (merosin;
2
1
1), which is the predominant isoform found in adult muscle (Leivo and Engvall, 1988
),
and laminin-11 (a synapse-specific isoform;
5
2
1), a recently characterized heterotrimer that contains two chains,
5 and
2 (s-laminin), that are only expressed at the adult
endplate (Hunter et al., 1989
; Martin et al., 1995
; Patton,
B.L., and J.R. Sanes, personal communication). The
1
chain of laminin-1 is expressed early in skeletal muscle development (Sewry et al., 1995
), and its expression increases
in dystrophic and inflammatory myopathies, indicating that it
may play a role in muscle regeneration (Mundegar et al., 1995
).
MATERIALS AND METHODS
) was synthesized and used to produce polyclonal antibodies in rabbits by Research
Genetics (Huntsville, AL). Anti-phosphotyrosine antibodies were purchased from Upstate Biotechnology Inc. (mAb 4G10; Lake Placid, NY)
and from Transduction Laboratories (mAb PY20; Lexington, KY). The
AChR
subunit antibody, mAb 124, was a gift from John Lindstrom
(University of Pennsylvania, Philadelphia, PA). Rhodamine-conjugated
-bungarotoxin was purchased from Molecular Probes, Inc. (Eugene, OR).
Laminin-1 (EHS tumor-derived),
-bungarotoxin, and CNBr-activated Sepharose beads were purchased from Sigma Chemical Co. (St. Louis, MO).
Toxin-conjugated Sepharose beads were prepared as described previously
by Gu and Hall (1988)
. Laminin-2 (isolated from human placenta) was
purchased from GIBCO BRL (Gaithersburg, MD). Laminin-11 (isolated
from a Schwann cell sarcoma) was a gift from Arlene Chiu (Beckman Research Institute of the City of Hope, Duarte, CA). Soluble recombinant
neural (C-Ag4,8) and muscle (C-Ag0,0) agrin was prepared from transiently
transfected COS cells as previously described by Ferns et al. (1993)
, and
concentrations were determined by comparison to purified agrin (Sugiyama et al., 1994
) on Western blots.
/
Cell Line
) were
crossed with transgenic mice that were hemizygous for a temperature-sensitive large T antigen transgene under the control of a
interferon-inducible MHC promoter (Jat et al., 1991
). The resulting double heterozygotes
were crossed to obtain mice that were homozygous for the mutant MuSK
allele and hemizygous for the large T transgene. Hindlimb muscles from
both limbs were dissected at birth, placed into 1 ml of Dulbecco's modified minimum Eagle's medium (DMEM) supplemented with L-glutamine
and penicillin/streptomycin, and placed on ice. Collagense B (Sigma
Chemical Co.) was added to a final concentration of 0.25% and incubated
at 37°C for 30 min. The tissue was washed several times in DMEM and
then incubated in 0.25% trypsin for 30 min at 37°C with several gentle agitations. The tissue was disrupted by pipeting 7-10 times with a narrow tip
pipette; the cell suspension was filtered through sterile nylon mesh to remove debris. The filtrate was diluted in DMEM containing 10% fetal bovine serum, L-glutamine, penicillin/streptomycin, and 10 ng/ml
interferon (Biosource International, Camarillo, CA), and then plated onto a
10-cm tissue culture dish to allow the fibroblasts to attach. After 40 min at
30°C, the myoblast-enriched supernatant was harvested and replated in
96-well plates. The cells were grown in the presence of 10 ng/ml
interferon for ~10 d. Individual clones were expanded and tested for their ability to form myotubes when incubated at the nonpermissive temperature of
37°C in DMEM containing 2% horse serum, L-glutamine, and penicillin/ streptomycin without
interferon.
. Myoblasts were grown in DMEM containing 20% fetal calf serum,
0.5% chick embryo extract (GIBCO BRL), 2 mM glutamine, and penicillin/streptomycin. When the cells reached confluence, the growth medium
was replaced with DMEM containing 5% horse serum and 2 mM
glutamine to induce myotube differentiation. MuSK
/
myotube cultures were grown, as described above, on glass chamberslides coated with
fibronectin.
/
myotubes were grown on
8-well glass chamberslides coated with fibronectin. Controlled experiments showed that the use of fibronectin did not affect laminin-1-induced
AChR clustering. Mature myotubes were treated with soluble neural
agrin (C-Ag4,8) or with laminin-1 for 18 h (unless otherwise mentioned),
and the cells were subsequently stained with rhodamine-conjugated
-bungarotoxin (Molecular Probes, Inc.) for 1 h at 37°C to visualize
AChR clusters. The cells were then rinsed, fixed in cold methanol for 5 min, and mounted in glycerol containing p-phenylenediamine. AChR
clusters were counted in random fields (chosen under phase optics) under
400× (C2 cultures) or 200× (MuSK
/
cultures). Within each experiment (n = 1), 10 fields/treatment were counted and averaged. To measure
the intensity of rhodamine-labeled AChR clusters, random fields (10 fields/treatment per experiment) under 600× were scanned into a MetaMorph image analysis program, and an average of the measured intensity
was taken after subtracting the background intensity. To measure the length of rhodamine-labeled AChR clusters, within an experiment, all
clusters within 10 random fields (randomly chosen under phase optics)
were measured under 400× and averaged.
-bungarotoxin coupled to
Sepharose beads, followed by washing the beads in extraction buffer and
boiling in SDS sample buffer. Western blotting was performed as described above. The blots were stripped and reprobed with an antibody
against the
subunit, mAb 124, to confirm that similar amounts of total
AChR
subunit were isolated on Separose beads.
RESULTS
). We compared
laminin-1 and agrin-induced AChR clustering on C2 myotubes using laminin-1 isolated from the basement membrane of the mouse EHS tumor, and a soluble COOH-terminal truncated agrin fragment (Ferns et al., 1993
). When
cultures of C2 mouse myotubes were treated with either
40 nM (33 µg/ml) of laminin-1 or 150 pM soluble neural
agrin (C-Ag4,8), the number of AChR clusters observed after labeling with rhodamine-conjugated
-bungarotoxin
was dramatically increased compared to the small number
of spontaneous AChR clusters seen in untreated C2 myotubes (Fig. 1, A-C). These results were further analyzed by
counting the number of AChR clusters induced by various
concentrations of laminin-1. The effects of laminin-1 were
detectable at concentrations as low as 12 nM and reached
maximal levels at concentrations of ~120 nM (Fig. 2 A).
When saturating concentrations were used, laminin-1 was
nearly as effective in inducing the formation of AChR
clusters as agrin (compare Figs. 2, A and B). Clusters produced by laminin-1, however, appeared more intensely
stained than those produced by agrin, suggesting that the
density of the AChRs in the laminin-1-induced clusters
may be higher. When the fluorescence intensity of rhodamine-labeled AChR clusters was quantitated using an image
analysis program, we found that the AChR clusters induced by 40 nM laminin-1 were over two times more
brightly stained than those induced by 15 pM agrin (Fig. 3
A). In addition, the length of the laminin-1-induced
AChR clusters appeared to be greater than those induced
by higher concentrations (1.5 nM) of agrin (Fig. 3 B).
Fig. 1.
Laminin-1 induces AChR clustering and increases agrin-induced AChR clustering in an additive manner. C2 myotubes were
untreated (A), treated with 150 pM soluble neural agrin (B), 40 nM laminin-1 (C), or treated with both 150 pM neural agrin and 40 nM
laminin-1 (D). Cultures were then stained with rhodamine-conjugated -bungarotoxin. Neural agrin and laminin-1 induce the formation
of AChR clusters, while treatment with both further increases the number of AChR clusters. Bar, 25 µm.
[View Larger Version of this Image (83K GIF file)]
Fig. 2.
(A) Laminin-1 induces AChR aggregation in a dose-dependent manner. C2 myotubes were treated with concentrations of laminin-1 ranging from 1.2 to 400 nM. Laminin-1 begins
to induce AChR clustering at 12 nM and peaks at 120 nM. (B)
Laminin-1 and neural agrin act additively to induce AChR clustering. C2 myotubes were treated with varying concentrations of
neural agrin ranging from 0.15 pM to 15 nM, without (closed
squares) or with (open circles) 40 nM of laminin-1. Treatment
with both laminin-1 and agrin results in an increased number of
AChR clusters over that induced by neural agrin alone. For each
treatment, the number of AChR clusters was drawn from three
experiments by counting and averaging clusters within 10 fields/
experiment and is shown as means ± SEM (n = 3).
[View Larger Versions of these Images (11 + 15K GIF file)]
Fig. 3.
(A) Laminin-1-induced AChR clusters are more
intensely stained than agrin-induced AChRs. C2 myotubes
were treated with 15 pM neural agrin, 40 nM laminin-1, or
with the two together, and the fluorescence intensity was
measured as described in Materials and Methods. The laminin-1-induced clusters are more intensely stained than the
agrin-induced AChRs, while treatment with both agrin and
laminin-1 produced brightly stained clusters more similar to
those induced by laminin-1. Data are averaged from two different experiments (10 fields/experiment) and shown as
means ± SD (n = 2). (B) AChR clusters induced by high
concentrations of agrin are shorter than those induced by
low concentrations of agrin or by laminin-1. C2 myotubes
were treated with 15 pM or 1.5 nM neural agrin, 12 or 120 nM laminin-1, or with both 1.5 nM neural agrin and 120 nM
laminin-1. Clusters induced by high (1.5 nM) concentrations
of agrin are smaller than those induced by low (15 pM) concentrations. In contrast, both concentrations of laminin- 1-induced large AChR clusters to form. Treatment with high concentrations of both agrin and laminin-1 produced large clusters, similar in size to the laminin-induced clusters. Data are averaged from three different experiments (10 fields/experiment) and shown as means ± SEM (n = 3). Asterisks denote significant difference (*P < 0.05 or **P < 0.01) between high concentrations of agrin and other treatments according to analysis of variance (ANOVA) and a Student-Newman-Keuls test.
[View Larger Versions of these Images (13 + 17K GIF file)]
2 chain). We found that only the laminin-1
isoform could induce AChR clustering (Fig. 4). Neither
laminin-2 nor -11 was active, even at concentrations of 120 nM (the concentration of laminin-1 that produced a maximal clustering response). Both laminin-2 and -11 used in
these experiments were shown to be active in other biological assays (Kleinman, H., and A. Chiu, personal communication). The failure of laminin-11 to induce clustering is
surprising since it is localized at the adult neuromuscular
junction (Patton, B.L., and J.R. Sanes, personal communication). Induction of AChR clustering on myotubes is thus not a general property of laminins, but appears to be specific for laminin-1 (
1
1
1). As
1 is the only chain of
laminin-1 that is not shared with the other two isoforms, it
appears to confer specificity for clustering.
Fig. 4.
AChR clustering activity is specific for the laminin-1 isoform. C2 myotubes were untreated (A) or treated with 60 nM of laminin-2 (B), laminin-11 (C), or laminin-1 (D). Cultures were stained with rhodamine-conjugated -bungarotoxin and examined under
400×. Unlike laminin-1, laminin-2 and -11 were both unable to induce AChR aggregation over background levels. Bar, 25 µm.
[View Larger Version of this Image (86K GIF file)]
). To determine if laminin-1 acts
with the same time course, we treated C2 myotubes with
either neural agrin (150 pM) or laminin-1 (120 nM) for 4, 12, or 18 h. We then stained the myotubes with rhodamine-
-bungarotoxin to visualize the receptor clusters and counted
the number of AChR clusters in random fields (Fig. 5).
After 4 h of treatment, agrin-treated cultures exhibited a
large increase in the number of AChR clusters, whereas
laminin-1-treated myotubes displayed no increase over
the background level. After 12 h, however, C2 cultures
treated with laminin-1 exhibited roughly a twofold increase
in AChR cluster number, and after 18 h of treatment, the
number of laminin-1-induced clusters approached the number of clusters induced by 150 pM of neural agrin. Thus,
the induction of AChR clusters by laminin-1 occurs significantly more slowly than by neural agrin.
Fig. 5.
Agrin and laminin-1
induce AChR aggregation
over different time courses.
C2 myotubes were treated
with 150 pM soluble neural agrin (solid bars) or with 120 nM laminin-1 (striped bars)
for 4, 12, or 18 h. Whereas
agrin quickly induces receptor aggregation within 4 h
and has a peak effect by 12 h,
laminin-1 acts with a slower
time course, and half-maximal clustering does not occur until ~12 h. Data averaged
from three (Laminin-1) or
four (Agrin) experiments are
shown as means ± SEM (n = 3 or 4). Asterisks denote significant
difference between untreated and agrin-treated (**P < 0.001) or
laminin-treated (*P < 0.01) cultures according to analysis of
variance (ANOVA) and a Student-Newman-Keuls test.
[View Larger Version of this Image (24K GIF file)]
/
Myotubes
), and myotubes from such mice fail to cluster AChRs in response to
neural agrin (Glass et al., 1996
). To determine whether
MuSK is also required for laminin-1 clustering activity, we treated immortalized myotube cultures made from
MuSK
/
mouse muscle with either laminin-1 or neural
agrin and then stained them with rhodamine-
-bungarotoxin. When treated with laminin-1, the MuSK
/
myotubes
produced AChR clusters of varying sizes, ranging from
short, dense clusters on the myotube surface to long clusters along the edge of the myotube, similar to those seen on C2 myotubes (Fig. 6). Quantitation of these results by
counting clusters in random fields showed that only myotubes treated with laminin-1 were able to produce AChR
clusters (Fig. 7). The number of AChR clusters induced by
laminin-1 on the MuSK
/
myotubes, however, was not
as great as those observed on C2 myotubes (~20-fold fewer clusters compared to C2 myotubes), even when concentrations as high as 120 nM of laminin-1 were used. In
general, the MuSK
/
myotubes differed in appearance
and were thinner than the C2 myotubes. Thus, the reduced
number of AChR aggregates on the mutant myotubes could be the result of inherent differences between C2 myotubes and the immortalized MuSK
/
myotubes, or it
could be a specific consequence of the absence of MuSK.
As expected, we did not observe AChR clustering on
MuSK
/
myotubes treated with neural agrin, even at
concentrations up to 15 nM. In the absence of laminin-1, occasional bright spots and faint edge staining were observed on the MuSK
/
myotubes but did not resemble
AChR clusters. This nonspecific staining accounted for the
nominal clustering values recorded for untreated and
agrin-treated MuSK
/
myotubes (Fig. 7).
Fig. 6.
Laminin-1 induces AChR clustering on MuSK/
myotubes. MuSK
/
myotubes were untreated (A) or treated with
150 pM neural agrin (B) or 120 nM laminin-1 (C). Cultures were
stained with rhodamine-conjugated
-bungarotoxin and visualized under 400×. Only laminin-1 treatment induces AChR aggregation on MuSK
/
myotubes. Bar, 25 µm.
[View Larger Version of this Image (21K GIF file)]
Fig. 7.
Quantitation of
laminin-1-induced AChR
clustering on MuSK/
myotubes. AChR clusters observed on MuSK
/
myotubes (untreated or treated
with 120 nM laminin-1 or 150 pM neural agrin) were counted under 200×, and an
average from four experiments (10 fields/experiment) is shown as means ± SEM.
(n = 4). Note that there are
somewhat fewer MuSK
/
myotubes per field compared
to C2 myotubes, and the
magnification used for MuSK
/
cluster quantitation is different from that used for C2 cluster quantitation in Fig. 2 A (400×); thus, the number of clusters per field on MuSK
/
and C2 myotubes cannot be directly compared.
[View Larger Version of this Image (10K GIF file)]
Fig. 8.
Laminin-1 does not induce MuSK phosphorylation. C2
myotubes were incubated with either laminin-1 (4 to 120 nM) or
with neural agrin (1.5 pM to 15 nM) for 45 min at 37°C. MuSK
was isolated from cell extracts by immunoprecipitation, and isolated proteins on nitrocellulose blots were probed with anti-phosphotyrosine antibodies. Laminin-1 does not induce MuSK tyrosine phosphorylation, even at concentrations in excess of what
is required to induce AChR clustering. In contrast, strongly labeled phosphotyrosine bands corresponding to MuSK were observed in lanes containing extracts treated with 0.15 to 15 nM
neural agrin. No phosphotyrosine signal was observed in lanes
containing untreated or muscle agrin-treated extracts. The blots
were stripped and reprobed with a polyclonal antiserum against
MuSK to show that the amount of MuSK in each lane is similar.
The arrowhead indicates the MuSK-reactive band that corresponds to the single band in the phosphotyrosine blot above; the
other nonspecific bands are due to antibody crossreactions.
[View Larger Version of this Image (45K GIF file)]
Subunit
subunit of the
AChR is an early response to agrin that precedes, and may
be required for, agrin-induced AChR clustering, we were
interested to determine whether laminin-1 also caused tyrosine phosphorylation of the AChR. C2 myotubes were
incubated with either 4, 12, 40, or 120 nM of laminin-1, 1.5 pM to 15 nM of soluble neural agrin (Ag4,8), or 15 nM of
soluble muscle agrin (Ag0,0) as described above, and detergent extracts were prepared. The extracts were then incubated with
-bungarotoxin conjugated to Sepharose beads
to isolate AChRs. Bound AChR was removed from the
toxin beads with SDS and the subunits separated by SDS-PAGE and transferred to nitrocellulose blots. The blots
were assayed for tyrosine phosphorylation of the AChR
subunit as described above and then reprobed with mAb
124, an antibody against the
subunit. Immunoblots with
mAb 124 showed that equal amounts of AChR had been
isolated on the
-bungarotoxin beads. Whereas treatment
with neural agrin induced
subunit phosphorylation, neither laminin-1 treatment, at any concentration tested, nor muscle agrin treatment caused tyrosine phosphorylation
of the
subunit (Fig. 9). Thus, tyrosine phosphorylation of
the
subunit is not required for AChR clustering.
Fig. 9.
Laminin-1 does not induce tyrosine phosphorylation of
the AChR subunit. C2 myotubes were treated with neural agrin
(1.5 pM to 15 nM) or with laminin-1 (4 to 120 nM) for 45 min at
37°C. AChRs were isolated on
-bungarotoxin Sepharose beads,
and nitrocellulose blots containing AChRs were probed with
anti-phosphotyrosine antibodies. Laminin-1 did not induce
subunit phosphorylation at any concentration. The same concentrations of neural agrin that induced both AChR clustering and
MuSK phosphorylation also induced
subunit phosphorylation. Untreated and muscle agrin-treated cultures did not exhibit
subunit phosphorylation. The nitrocellulose blot was stripped and reprobed with an antibody against the
subunit (mAb 124) to ensure that similar amounts of
subunit were present in each sample.
[View Larger Version of this Image (40K GIF file)]
DISCUSSION
first demonstrated that laminin-1 purified from EHS tumors could induce AChR aggregation in
a rat clonal muscle cell line and in primary rat myotubes.
We have confirmed these results with cultured C2 myotubes, showing that soluble laminin-1 induces AChR clusters in a dose-dependent manner. At its optimal concentration (120 nM), laminin-1 induced an approximately fivefold increase in cluster number over background. The
increase in AChR cluster number induced by laminin-1
was similar to that induced by soluble neural agrin, and the
size of the clusters produced by low concentrations of both
ligands was similar. The laminin-1-induced AChR clusters
appeared by eye to be more intensely stained, and quantitative analysis of the fluorescence intensity confirmed this
impression. The increased intensity of AChR staining most
likely represents an increase in receptor density, although increased folding of the membrane could be responsible.
; Glass et al., 1996
), laminin-1-induced clustering does not (Figs. 6-8); (c) agrin- and
laminin-1-induced clustering occur with different time
courses (Fig. 5); and (d) agrin induces tyrosine phosphorylation of the AChR, whereas laminin-1 does not (Fig. 9).
We examine each of these points in turn.
; Glass et al., 1996
).
This activation appears to be the first step in the pathway
of agrin-induced AChR clustering. Thus, myotubes from
mice that are homozygous for a disruption of the MuSK
gene do not respond to agrin by clustering AChRs (Glass et al., 1996
). In contrast, laminin-1 did induce AChR clustering in MuSK
/
myotubes (Figs. 6 and 7), while treatment of C2 myotubes with laminin-1 did not induce
tyrosine phosphorylation of MuSK (Fig. 8). Thus, laminin-1-induced clustering of the AChR must occur by a pathway that is independent of MuSK activation.
), AChR clusters were not detected until at least
12 hours after laminin-1 treatment (Fig. 5). In addition to
laminin-1 and agrin, other agents have been reported to
induce AChR clustering, but those that have been tested
all do so with a time course similar to that of agrin. Thus,
treatment with either VVA-B4 lectin (Martin and Sanes,
1995
) or polystyrene beads (Baker et al., 1992
), caused clustering that was detectable within 4 h and reached a
peak well before 8 h. The slower course of laminin-induced
AChR clustering suggests that it occurs by a mechanism
that is distinct from that used by other agents that cluster
surface AChRs.
subunit (Wallace et al., 1991
; Qu and Huganir, 1994
;
Wallace, 1994
; Ferns et al., 1996
). Several lines of evidence
indicate that phosphorylation of the AChR
subunit may
be required for agrin-induced AChR aggregation. Agrin-induced phosphorylation occurs before clustering (Ferns et al., 1996
), and under a variety of conditions, agrin-induced AChR clustering and tyrosine phosphorylation are associated. Similar concentrations of agrin induce both clustering and tyrosine phosphorylation of the AChR, and tyrosine kinase inhibitors inhibit both agrin-induced AChR
clustering and
subunit phosphorylation (Wallace, 1994
; Ferns et al., 1996
). Furthermore, treatment of C2 myotubes with phosphatase inhibitors decreases the extractability of AChRs within the membrane (Meier et al., 1995
).
Thus, while circumstantial evidence links the two events,
direct experimental proof is lacking. In the case of laminin-1-induced AChR clustering, however, no evidence
was found for tyrosine phosphorylation of the AChR (Fig. 9). Although it may be required for agrin-induced cluster
formation or for cluster stability within the synaptic membrane, AChR clustering in myotubes does not require tyrosine phosphorylation of the
subunit, as aggregation
can occur in its absence.
-bungarotoxin are clustered by laminin
(Vogel et al., 1983
). Preliminary observations showing that
the appearance of the laminin-1-induced clusters is not inhibited by cycloheximide (Sugiyama, J.E., and Z.W. Hall, unpublished observations) are consistent with this interpretation. Neither the receptor nor the signaling pathway
activated by laminin-1, which results in AChR clustering,
is known. Laminin-1-binding proteins in muscle that could
potentially mediate its effect include integrins (Venstrom
and Reichardt, 1993
) and
-dystroglycan (Ervasti and Campbell, 1993
; Gee et al., 1993
). The integrins known to be
present in myotubes include those containing
7, which is
concentrated at synapses,
1, 3-7, 9,
V, and
1 (Martin et
al., 1996
). In addition, full length muscle agrin, which has
been shown to induce AChR clusters (Ferns et al., 1992
),
also binds laminin (Sugiyama, J.E., and Z.W. Hall, unpublished results) and may thus play a role in laminin-induced
AChR clustering. Further investigation will be required to
determine which, if any, of these proteins mediate the
AChR clustering effect of laminin-1 on myotubes.
), and the dispersal of agrin-induced
AChR clusters by tyrosine kinase inhibitors occurs at a
time when AChR phosphorylation has declined, suggesting that phosphorylation of other proteins may be required for cluster maintenance (Ferns et al., 1996
). Experiments with cells heterologously expressing the AChR also
suggest that tyrosine phosphorylation of proteins other
than the AChR could be required for clustering (Gillespie
et al., 1996
). Initial experiments to test the effects of tyrosine kinase inhibitors on laminin-1-induced AChR clustering have as yet yielded ambiguous results (Sugiyama,
J.E., and Z.W. Hall, unpublished observations). Thus although AChR
subunit tyrosine phosphorylation is clearly
not required for laminin-1-induced AChR clustering, the
possibility remains that tyrosine phosphorylation of other
proteins may play a role.
-dystroglycan (Cohen et al., 1997
). This finding is
of potential interest for the role of laminin in synaptogenesis, as
-dystroglycan is localized at synapses in vivo and
occurs in association with agrin-induced AChR clusters in
cultured myotubes (Campanelli et al., 1994
; Gee et al., 1994
;
Sugiyama et al., 1994
; Cohen et al., 1995
). As laminin-1 is
known to bind
-dystroglycan (Ervasti and Campbell, 1993
;
Gee et al., 1993
), self-association between laminin-1 molecules (Yurchenco and Cheng, 1994
) is postulated to be the
mechanism responsible for the clustering of
-dystroglycan. This mechanism is unlikely to cause laminin-induced
AChR clustering, however, as laminin-1 is not known to
bind the AChR directly. As the laminin-1-induced
-dystroglycan clusters were not associated with AChR clusters, their significance for synaptogenesis is unclear.
). A few
nerve-associated AChR clusters remain, however, leading
Gautam et al. (1996)
to conclude that additional pathways
must mediate nerve-induced clustering. The laminin-1
pathway could provide one explanation for the AChR clusters that appear in agrin-deficient animals. One problem with this hypothesis is that AChR clusters are not seen
in the muscles of MuSK-deficient animals, implying that
the agent responsible for AChR clustering in agrin-deficient animals requires MuSK, which laminin-1 does not.
An attractive possibility is that during synaptogenesis, the
laminin-1 pathway is not normally used to initiate AChR
clusters but builds on agrin-initiated AChR clusters in a way
that uses MuSK as a structural element. Laminin-1-induced clustering would thus be facilitated by MuSK, but would
not activate it or require it. This idea is consistent with recent experiments suggesting that MuSK is part of the primary synaptic scaffold to which AChRs are added (Apel
et al., 1997
). Under this interpretation, in MuSK
/
animals, the laminin-1 pathway would not be strongly enough
activated during synaptogenesis to initiate AChR clustering by itself, but under in vitro conditions, AChR clustering could be elicited even in the absence of MuSK by high
concentrations of laminin-1. Such an interpretation might
explain the lower level of AChR clusters observed after
laminin-1 addition to MuSK
/
myotubes, as compared
to C2 myotubes.
4 chain. Our results showing that laminin-1 (
1
1
1) is active, whereas laminin-2
(
2
1
1) and laminin-11 (
5
2
1) are not, suggest that
the clustering activity of each laminin isoform arises from the identity of the
chain. Only incomplete information is
currently available about the distribution of laminin isoforms during muscle development (Sanes et al., 1990
). Recent immunocytochemical experiments indicate that the
1 chain is present very early in muscle development, but
that it may not be localized at developing synapses (Patton, B.L., and J.R. Sanes, personal communication). The
5 chain, in contrast, is concentrated at adult endplates,
but at least within the context of
2 and
1, is inactive in
AChR clustering. Available data thus suggests that although laminin-1 activates the pathway that we have described, it may not be the functional ligand during synaptogenesis.
; Fallon and Gelfman, 1989
). It will be of interest to elucidate the
molecular mechanism of laminin-induced AChR clustering and to determine the in vivo factor which modulates
this alternative pathway.
Received for publication 13 May 1997 and in revised form 11 July 1997.
Address all correspondence to Dr. Zach Hall, Office of the Director, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892. Tel.: (301) 496-9746; Fax: (301) 496-0296.We thank Dr. Arlene Chiu for the generous gift of purified laminin-11 and
for her critical analysis of the manuscript and Dr. Hynda Kleinman for
sharing both the laminin-2 and her knowledge of the laminin field. We appreciate Dr. Josh Sanes' insightful comments and also thank Dr. Christian
Fuhrer for preparing the -bungarotoxin-Sepharose beads and for many
helpful discussions. Finally, we are grateful to Drs. Matt Daniels, Brian
Lowe, and Robin Taylor for their help with MetaMorph image analysis
and to Dr. Vikram Ramanathan for his statistical expertise.
This work was supported by the National Institutes of Health.
1. | Apel, E.D., D.J. Glass, L.M. Moscoso, G.D. Yancopoulos, and J.R. Sanes. 1997. MuSK forms a synaptic scaffold in the absence of rapsyn, but requires rapsyn to signal and recruit additional synaptic components. Neuron. 18: 623-635 |
2. | Baker, L.P., Q. Chen, and H.B. Peng. 1992. Induction of acetylcholine receptor clustering by native polystyrene beads. J. Cell Sci. 102: 543-555 [Abstract]. |
3. | Bayne, E.K., M.J. Anderson, and D.M. Fambrough. 1984. Extracellular matrix organization in developing muscle: correlation with acetylcholine receptor aggregates. J. Cell Biol. 99: 1486-1501 [Abstract]. |
4. |
Bowen, D.C.,
J. Sugiyama,
M. Ferns, and
Z.W. Hall.
1996.
Neural agrin activates a high-affinity receptor in C2 muscle cells that is unresponsive to muscle agrin.
J. Neurosci.
16:
3791-3797
|
5. | Burden, S.J., P.B. Sargent, and U.J. McMahan. 1979. Acetylcholine receptors in regenerating muscle accumulate at original synaptic sites in the absence of the nerve. J. Cell Biol. 82: 412-425 [Abstract]. |
6. | Burgeson, R.E., M. Chiquet, R. Duetzmann, P. Ekblom, J. Engel, H. Kleinman, G.R. Martin, G. Meneguzzi, M. Paulsson, J. Sanes, et al . 1994. A new nomenclature for the laminins. Matrix Biol. 14: 209-211 |
7. | Campanelli, J.T., S.L. Roberds, K.P. Campbell, and R.H. Scheller. 1994. A role for dystrophin-associated glycoproteins and utrophin in agrin induced acetylcholine receptor clustering. Cell. 77: 663-674 |
8. | Chiu, A.Y., and J.R. Sanes. 1984. Development of basal lamina in synaptic and extrasynaptic portions of embryonic rat muscle. Dev. Biol. 103: 456-467 |
9. |
Cohen, M.W.,
C. Jacobson,
E.W. Godfrey,
K.P. Campbell, and
S. Carbonetto.
1995.
Distribution of ![]() |
10. |
Cohen, M.W.,
C. Jacobson,
P.D. Yurchenco,
G.E. Morris, and
S. Carbonetto.
1997.
Laminin-induced clustering of dystroglycan on embryonic muscle cells:
comparison with agrin-induced clustering.
J. Cell Biol.
136:
1047-1058
|
11. | Daniels, M.P., M. Vigny, P. Sonderegger, H.C. Bauer, and Z. Vogel. 1984. Association of laminin and other basement membrane components with regions of high acetylcholine receptor density on cultured myotubes. Int. J. Dev. Neurosci. 2: 87-99 . |
12. | DeChiara, T.M., D.C. Bowen, D.M. Valenzuela, M.V. Simmons, W.T. Poueymirou, S. Thomas, E. Kinetz, D.L. Compton, E. Rojas, J.S. Park, et al . 1996. The receptor tyrosine kinase MuSK is required for neuromuscular junction formation in vivo. Cell. 85: 501-512 |
13. | Ervasti, J.M., and K.P. Campbell. 1993. A role for the dystrophin-glycoprotein complex as a transmembrane linker between laminin and actin. J. Cell Biol. 122: 809-823 [Abstract]. |
14. | Fallon, J.R., and C.E. Gelfman. 1989. Agrin-related molecules are concentrated at acetylcholine receptor clusters in normal and aneural developing muscle. J. Cell Biol. 108: 1527-1535 [Abstract]. |
15. | Ferns, M., W. Hoch, J.T. Campanelli, F. Rupp, Z.W. Hall, and R.H. Scheller. 1992. RNA splicing regulates agrin-mediated acetylcholine receptor clustering activity on cultured myotubes. Neuron. 8: 1079-1086 |
16. | Ferns, M.J., J.T. Campanelli, W. Hoch, R.H. Scheller, and Z. Hall. 1993. The ability of agrin to cluster AChRs depends on alternative splicing and on cell surface proteoglycans. Neuron. 11: 491-502 |
17. | Ferns, M., M. Deiner, and Z. Hall. 1996. Agrin-induced acetylcholine receptor clustering in mammalian muscle requires tyrosine phosphorylation. J. Cell Biol. 132: 937-944 [Abstract]. |
18. |
Fertuck, H.C., and
M.M. Salpeter.
1976.
Quantitation of junctional and extrajunctional acetylcholine receptors by electron microscope autoradiography
after 125I-![]() |
19. | Gautam, M., P.G. Noakes, L. Moscoso, F. Rupp, R.H. Scheller, J.P. Merlie, and J.R. Sanes. 1996. Defective neuromuscular synaptogenesis in agrin-deficient mutant mice. Cell. 85: 525-535 |
20. |
Gee, S.H.,
R.W. Blacher,
P.J. Douville,
P.D. Yurchenco, and
S. Carbonetto.
1993.
LBP120 from brain is closely related to the dystrophin-associated glycoprotein, dystroglycan, and binds with high affinity to the major heparin
binding domain of laminin.
J. Biol. Chem.
268:
14972-14980
|
21. |
Gee, S.H.,
F. Montanaro,
M.H. Lindenbaum, and
S. Carbonetto.
1994.
Dystroglycan-![]() |
22. | Gillespie, S.K.H., S. Balasubramanian, E.T. Fung, and R.L. Huganir. 1996. Rapsyn clusters and activates the synapse-specific receptor tyrosine kinase MuSK. Neuron. 16: 953-962 |
23. | Glass, D.J., D.C. Bowen, T.N. Stitt, C. Radziejewski, J. Bruno, T.E. Ryan, D.R. Gies, S. Shah, K. Mattsson, S.J. Burden, et al . 1996. Agrin acts via a MuSK receptor complex. Cell. 85: 513-523 |
24. | Godfrey, E.W., R.E. Siebenlist, P.A. Wallskog, L.M. Walters, D.L. Bolender, and D.E. Yorde. 1988. Basal lamina components are concentrated in premuscle masses and at early acetylcholine receptor clusters in chick embryo hindlimb muscles. Dev. Biol. 130: 471-486 |
25. | Gordon, H., and Z.W. Hall. 1989. Glycosaminoglycan variants in the C2 muscle cell line. Dev. Biol. 135: 1-11 |
26. | Gordon, H., M. Lupa, D. Bowen, and Z. Hall. 1993. A muscle cell variant defective in glycosaminoglycan biosynthesis forms nerve-induced but not spontaneous clusters of the acetylcholine receptor and the 43 kDa protein. J. Neurosci. 13: 586-595 [Abstract]. |
27. |
Gu, Y., and
Z.W. Hall.
1988.
Characterization of acetylcholine receptor subunits in developing and in denervated mammalian muscle.
J. Biol. Chem.
263:
12878-12885
|
28. | Hall, Z.W., and J.R. Sanes. 1993. Synaptic structure and development: the neuromuscular junction. Neuron. 10(Suppl.):99-121. |
29. | Hoch, W., M. Ferns, J.T. Campanelli, Z.W. Hall, and R.H. Scheller. 1993. Developmental regulation of highly active alternatively spliced forms of agrin. Neuron. 11: 479-490 |
30. | Hunter, D.D., V. Shah, J.P. Merlie, and J.R. Sanes. 1989. A laminin-like adhesive protein concentrated in the synaptic cleft of the neuromuscular junction. Nature (Lond.). 338: 229-234 |
31. | Jat, P.S., M.D. Noble, P. Ataliotis, Y. Tanaka, N. Yannoutsos, L. Larsen, and D. Kioussis. 1991. Direct derivation of conditionally immortal cell lines from an H-2Kb-tsA58 transgenic mouse. Proc. Natl. Acad. Sci. USA. 88: 5096-5100 [Abstract]. |
32. | Leivo, I., and E. Engvall. 1988. Merosin, a protein specific for basement membranes of Schwann cells, striated muscle, and trophoblast, is expressed late in nerve and muscle development. Proc. Natl. Acad. Sci. USA. 85: 1544-1548 [Abstract]. |
33. | Lentz, S.I., J.H. Miner, J.R. Sanes, and W.D. Snider. 1997. Distribution of the ten known laminin chains in the pathways and targets of developing sensory axons. J. Comp. Neurol. 378: 547-561 |
34. | Martin, P.T., and J.R. Sanes. 1995. Role for a synapse-specific carbohydrate in agrin-induced clustering of acetylcholine receptors. Neuron. 14: 743-754 |
35. |
Martin, P.T.,
A.J. Ettinger, and
J.R. Sanes.
1995.
Synaptic localization domain
in the synaptic cleft protein laminin ![]() |
36. |
Martin, P.T.,
S.J. Kaufman,
R.H. Kramer, and
J.R. Sanes.
1996.
Synaptic integrins in developing, adult, and mutant muscle: selective association of ![]() ![]() ![]() |
37. | McMahan, U.J. 1990. The agrin hypothesis. In Cold Spring Harbor Symposia on Quantitative Biology. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 407-418. |
38. | Mundegar, R.R., J. van Oertzen, and S. Zierz. 1995. Increased laminin A expression in regenerating myofibers in neuromuscular disorders. Muscle Nerve. 18: 992-999 |
39. | Meier, T., G.M. Perez, and B.G. Wallace. 1995. Immobilization of nicotinic acetylcholine receptors in mouse C2 myotubes by agrin-induced protein tyrosine phosphorylation. J. Cell Biol. 131: 441-451 [Abstract]. |
40. |
Miner, J.H.,
B.L. Patton,
S.I. Lentz,
D.J. Gilbert,
W.D. Snider,
N.A. Jankins,
N.G. Copeland, and
J.R. Sanes.
1997.
The laminin ![]() ![]() ![]() |
41. | Nastuk, M.A., E. Lieth, J. Ma, C.A. Cardasis, E.B. Moynihan, B.A. McKechnie, and J.R. Fallon. 1991. The putative agrin receptor binds ligand in a calcium-dependent manner and aggregates during agrin-induced acetylcholine receptor clustering. Neuron. 7: 807-818 |
42. | Nitkin, R.M., and T.C. Rothschild. 1990. Agrin-induced reorganization of extracellular matrix components on cultured myotubes: relationship to AChR aggregation. J. Cell Biol. 111: 1161-1170 [Abstract]. |
43. | Peng, H.B., L.P. Baker, and Q. Chen. 1991. Induction of synaptic development in cultured muscle cells by basic fibroblast growth factor. Neuron. 6: 237-246 |
44. | Peng, H.B., A. Afshan, Ali, Z. Dai, D.F. Daggett, E. Raulo, and H. Rauvala. 1995. The role of heparin-binding growth-associated molecule (HB-GAM) in the postsynaptic induction in cultured muscle cells. J. Neurosci. 15: 3027-3038 [Abstract]. |
45. | Qu, Z., and R.L. Huganir. 1994. Comparison of innervation and agrin-induced tyrosine phosphorylation of the nicotinic acetylcholine receptor. J. Neurosci. 14: 6834-6841 [Abstract]. |
46. | Ruegg, M.A., K.W.K. Tsim, S.E. Horton, S. Kroger, G. Escher, E.M. Gensch, and U.J. McMahan. 1992. The agrin gene codes for a family of basal lamina proteins that differ in function and distribution. Neuron. 8: 691-699 |
47. | Rupp, F., D.G. Payan, C. Magill-Solc, D.M. Cowan, and R.H. Scheller. 1991. Structure and expression of a rat agrin. Neuron. 6: 811-823 |
48. | Sanes, J.R., E. Engvall, R. Butkowski, and D.D. Hunter. 1990. Molecular heterogeneity of basal laminae: isoforms of laminin and collagen IV at the neuromuscular junction and elsewhere. 111:1685-1699. |
49. | Sewry, C.A., M. Chevallay, and F.M.S. Tomé. 1995. Expression of laminin subunits in human fetal skeletal muscle. Histochem. J. 27: 497-504 |
50. | Sugiyama, J., D.C. Bowen, and Z.W. Hall. 1994. Dystroglycan binds nerve and muscle agrin. Neuron. 13: 103-115 |
51. | Timpl, R., H. Rhode, L. Risteli, U. Ott, P. Gehron, Robey, and G.R. Martin. 1982. Laminin. Methods Enzymol. 82: 831-839 |
52. | Tsim, K.W.K., M.A. Ruegg, G. Escher, S. Kroger, and U.J. McMahan. 1992. cDNA that encodes active agrin. Neuron. 8: 677-689 |
53. | Valenzuela, D.M., T.N. Stitt, P.S. DiStefano, E. Rojas, K. Mattsson, D.L. Compton, L. Nunez, S. Park, J.L. Stark, D. Gies, et al . 1995. Receptor tyrosine kinase specific for the skeletal muscle lineage: expression in embryonic muscle, at the neuromuscular junction, and after injury. Neuron. 15: 573-584 |
54. |
Venstrom, K.A., and
L.F. Reichardt.
1993.
Extracellular matrix 2: role of extracellular matrix molecules and their receptors in the nervous system.
FASEB
J.
7:
996-1003
|
55. | Vogel, Z., C.N. Christian, M. Vigny, H.C. Bauer, P. Sonderegger, and M.P. Daniels. 1983. Laminin induces acetylcholine receptor aggregation on cultured myotubes and enhances the receptor aggregation activity of a neuronal factor. J. Neurosci. 3: 1058-1068 [Abstract]. |
56. | Wallace, B.G.. 1989. Agrin-induced specializations contain cytoplasmic, membrane, and extracellular matrix-associated components of the postsynaptic apparatus. J. Neurosci. 9: 1294-1302 [Abstract]. |
57. | Wallace, B.G.. 1994. Staurosporine inhibits agrin-induced acetylcholine receptor phosphorylation and aggregation. J. Cell Biol. 125: 661-668 [Abstract]. |
58. | Wallace, B.G., Z. Qu, and R.L. Huganir. 1991. Agrin induces phosphorylation of the nicotinic acetylcholine receptor. Neuron. 6: 869-878 |
59. | Yurchenco, P.D., and Y.-S. Cheng. 1994. Laminin self-assembly: a three-arm interaction hypothesis for the formation of a network in basement membranes. Contrib. Nephrol. 107: 47-56 |