(Received for publication, August 16, 1995; and in revised form, December 18, 1995)
From the
The synaptic basal membrane protein agrin initiates the
aggregation of acetylcholine receptors at the postsynaptic membrane of
the developing neuromuscular junction. Recently, -dystroglycan was
found to be a major agrin-binding protein on the muscle cell surface
and was therefore considered a candidate agrin receptor. Employing
different truncation fragments of agrin, we determined regions of the
protein involved in binding to
-dystroglycan and to heparin, an
inhibitor of
-dystroglycan binding. Deletion of a 15-kDa fragment
from the C terminus of agrin had no effect on its binding to
-dystroglycan from rabbit muscle membranes, even though this
deletion completely abolishes its acetylcholine receptor aggregating
activity. Conversely, deletion of a central region does not affect
agrin's clustering activity, but reduced its affinity for
-dystroglycan. Combination of these two deletions resulted in a
fragment of
35 kDa that weakly bound to
-dystroglycan, but
displayed no clustering activity. All of these fragments bound to
heparin with high affinity. Thus,
-dystroglycan does not show the
binding specificity expected for an agrin receptor. Our data suggest
the existence of an additional component on the muscle cell surface
that generates the observed ligand specificity.
During formation of the neuromuscular junction, motor neurons
provide a number of signals that promote the differentiation of the
postsynaptic membrane(1) . An early signal is the secretion of
specific isoforms of agrin, a large protein of the extracellular
matrix, and their incorporation into the basal lamina(2) .
Agrin initiates the concentration of nicotinic acetylcholine receptors
(AChRs) ()and other components of the muscle cell surface at
the site of contact with axons of motor
neurons(3, 4) . Recombinant expression of the
C-terminal half of agrin behind an artificial signal peptide produces a
soluble form of agrin, which is sufficient to induce AChR clustering in
myotube culture(5, 6, 7) . This portion of
agrin contains several interesting structural motifs (see Fig. 1A)(6, 8) . Three regions display
a considerable sequence homology to the globular domain (G-domain) of
laminin implicated in binding to proteoglycans. Intermingled with these
laminin G-domains are four EGF-like repeats.
Figure 1: Agrin deletion constructs. A, domain structure of agrin constructs used in this study. All constructs contained an artificial signal sequence. The relative AChR clustering activities of fragments N1 (100 ± 25%) and N4 (109 ± 23%) are similar(13) . The numbers of clusters observed in the presence of the other fragments are not significantly different from the number of spontaneous clusters in control cultures without agrin. B, Western blot analysis of conditioned media of COS-7 cells transfected with various agrin constructs. 2 pmol of agrin fragments N1, C2, N4, and N6 and 0.4 pmol of construct N4/C2 were separated on a 3-12% SDS-polyacrylamide gradient gel. After transfer to nitrocellulose, agrin was visualized with the polyclonal antiserum AS 11 (10) against agrin and an alkaline phosphatase-coupled secondary antibody. Positions of molecular mass markers (in kilodaltons) are shown on the left.
Alternative splicing at
three positions (X, Y, and Z) in this region generates several agrin
isoforms(7, 9) . Splicing at site Z is particularly
important: agrin variants containing no insert at this position are
widely expressed in basement membranes of many tissues and display only
weak AChR clustering activity. In contrast, inserts of 8, 11, or 19
amino acids are found only in agrin of neuronal origin (9, 10, 11) . The insert of 11 amino acids
increases agrin's clustering activity 50-fold, and inserts
of 8 or 19 amino acids
10,000-fold (12, 13) .
Studies of structure-function relationships indicate that a C-terminal 50-kDa fragment of rat agrin is sufficient for induction of AChR clustering. Additional deletion from either side of this recombinant fragment abolishes agrin's aggregating activity(13) . In chick, a similar 45-kDa fragment is necessary for full clustering activity, and at very high concentrations, a C-terminal fragment of only 21 kDa induces AChR aggregation(14) .
The mechanism by which agrin induces the
formation of AChR clusters is unknown. Recently, several groups have
reported that -dystroglycan is a major binding protein for agrin
on the muscle cell surface (15, 16, 17, 18) . Several
characteristics of this binding support the hypothesis that
-dystroglycan serves as a functional agrin receptor. (i) Binding
of agrin to
-dystroglycan is Ca
-dependent, as is
agrin's AChR aggregating activity. (ii) Binding and function of
agrin are inhibited by heparin. (iii) In mutant muscle cell lines that
are defective in processing of proteoglycans, a much higher
concentration of agrin is needed to induce AChR clustering compared
with normal muscle fibers(7, 12) . Correspondingly,
-dystroglycan in these cell lines, which has a smaller apparent
molecular weight, shows a reduced binding to agrin (16, 17, 18) .
Early in development,
-dystroglycan is widely distributed over the muscle cell surface
and becomes concentrated at developing AChR clusters upon addition of
agrin(19) . It is part of a complex including several
transmembrane and peripheral membrane proteins that are associated with
the submembrane cytoskeleton proteins dystrophin and
utrophin(20, 21) . Several members of this dystrophin
glycoprotein complex (DGC) copurify with AChRs of the electric organ of Torpedo
californica(22, 23, 24, 25) .
In the extrasynaptic region,
-dystroglycan binds to
laminin(26, 27) . It has been suggested that the DGC
forms a link between the extracellular matrix and dystrophin and
thereby stabilizes the muscle fiber(28) . In vertebrates,
dystrophin expression is restricted to the extrasynaptic regions and
the troughs of the synaptic folds. In the crests of these folds, where
the AChRs are concentrated, dystrophin is replaced by its homologue
utrophin(24, 29) . Thus, agrin is most likely linked
to utrophin via the DGC in the mature synapse, and this link may
stabilize synaptic structures.
The important question of whether
-dystroglycan mediates the AChR aggregating activity of agrin
alone or in conjunction with other surface components and therefore is
an agrin receptor is still unanswered. In an attempt to solve this
problem, several groups inhibited agrin binding to
-dystroglycan
in culture by addition of a mAb against
-dystroglycan. However,
the effects on agrin-induced AChR clustering are controversial: in some
studies, no effects on AChR aggregation could be
detected(18, 30) . One group observed a significant
reduction in the number of AChR aggregates(17) , while another
group found no change in the number of clusters, but rather a more
dispersed appearance of AChR aggregates(16) .
Here, we
addressed this question using a different approach. We assessed the
binding of previously and newly generated truncation fragments of agrin
to -dystroglycan and compared the binding pattern with the
functional activity of these fragments. Our results demonstrate that
-dystroglycan does not display the ligand specificity expected for
an agrin receptor.
COS-7 cells were transiently
transfected with these agrin constructs using the DEAE-dextran method
as described previously(5) . Dulbecco's modified
Eagle's medium was substituted for the serum-containing medium 1
day after transfection. The concentration of construct N1 in
conditioned medium collected 3 days after transfection was 50
nM(12) . Based on this estimate, concentrations of the
other agrin constructs were determined using a dot-blot
procedure(32) . For agrin-containing conditioned media, this
assay was linear between 5 and 100 ng of total protein.
The beads were washed twice with buffer A and twice with buffer A containing 150 mM NaCl, followed by two washes with 10 mM sodium phosphate buffer, pH 6.7. GammaBind Plus-Sepharose and antibodies were equilibrated in 200 mM triethanolamine, pH 8.2, and cross-linked with 10 mM dimethyl suberimidate (Pierce) in the same buffer for 45 min at room temperature. The reaction was quenched with 200 mM ethanolamine, pH 8.2. After subsequent washes with 50 mM Tris, pH 7.4 (buffer B), containing 500 mM NaCl, with buffer B containing 150 mM NaCl, and with buffer B, the beads were mixed with agrin-containing conditioned media for 2 h. In control samples, agrin was replaced by buffer B. These and all subsequent steps were performed at 4 °C.
The beads were washed with buffer B
containing 150 mM NaCl and with buffer B and incubated with
1.4 ml of diluted alkaline extract of rabbit muscle membranes overnight
on a shaking platform. Unbound fractions were collected, and the beads
were washed twice with 50 mM Tris, pH 8, 2 mM calcium
chloride (buffer C) and twice with buffer C containing 150 mM NaCl. Bound -dystroglycan was eluted by replacing
Ca
-containing buffer C with 500 µl of 50 mM Tris, pH 8, 150 mM NaCl, 20 mM EGTA for 1 h with
shaking. This step was repeated once. Finally, the agrin fragments were
eluted by boiling the beads in SDS-PAGE loading buffer.
We have previously generated a panel of truncated agrin
fragments and characterized their ability to induce aggregation of
AChRs on the surface of muscle fibers (Fig. 1A)(13) . Fragments N1 and N4 showed high
clustering activity, while the other fragments were inactive at equal
concentrations. In this study, we used several of these fragments,
which were highly expressed and efficiently secreted into the medium of
transfected cells (Fig. 1B), to identify regions of the
agrin protein required for binding to -dystroglycan and to
heparin.
Since only relatively small amounts of different agrin
fragments can conveniently be generated by transient transfection, we
developed a two-step affinity chromatography protocol to evaluate
-dystroglycan's ability to bind to different agrin
fragments. We immobilized an appropriate monoclonal antibody against
agrin on protein G beads by cross-linking. In the first step, we loaded
these beads with different agrin fragments. Because the epitopes of all
rat agrin mAbs are known(13) , it was possible to predict which
agrin fragments would bind to the beads. In a second step, these agrin
mAb beads were used as affinity matrices to detect possible binding of
-dystroglycan. To this end, an alkaline extract of rabbit skeletal
muscle was incubated with different agrin matrices as well as a control
matrix containing only mAb, but no agrin.
-Dystroglycan bound
quantitatively to N1, comprising the C-terminal half of agrin, as
judged by the almost complete removal of
-dystroglycan from the
unbound fraction and its detection in the eluate (Fig. 2).
Binding depended on agrin since the control matrix did not retain any
-dystroglycan. Binding was also Ca
-dependent
because bound immunoreactivity could be eluted by substituting 20
mM EGTA for Ca
in the elution buffer (Fig. 2B). Thus, this interaction displayed
characteristics previously described for the binding of
-dystroglycan to
agrin(15, 16, 17, 18) .
Figure 2:
Agrin construct C2 binds
-dystroglycan with similar efficiency compared with fragment N1.
mAb 30 against agrin was incorporated into affinity matrices as
described under ``Experimental Procedures.'' No
agrin(-) or
100 pmol of agrin constructs N1 and C2 were
bound to the beads and incubated with an alkaline extract of rabbit
skeletal muscle membranes. A, 50% of the unbound material and
an equal volume of the alkaline extract (AE) were separated by
SDS-PAGE and stained with a mixture of two mAbs against
-dystroglycan (
-D). B, bound
-dystroglycan was eluted by replacing Ca
with
EGTA. A Western blot of 75% of each of the eluates was stained with a
mixture of two mAbs against
-dystroglycan. C, to control
whether similar amounts of constructs N1 and C2 bound to the affinity
matrices, beads were boiled in gel loading buffer, and equal aliquots
of the supernatants were loaded onto a 3-12% gradient gel.
Nitrocellulose transfers were reacted with AS 11 against
agrin.
In the
first set of experiments, we looked at the importance of the C-terminal
sequence for -dystroglycan binding. Agrin construct C2, which
lacks the last of the three laminin G homology regions, bound to
-dystroglycan to the same extent as construct N1 (Fig. 2, A and B). Comparable amounts of both fragments could
be eluted from the beads (Fig. 2C). Therefore, we
conclude that deletion of a C-terminal fragment of agrin, which
completely abolishes agrin's AChR clustering
activity(13) , did not affect its binding to
-dystroglycan.
Next, we investigated the effects of truncations
from the N-terminal region of N1. For these experiments, we used a mAb
whose epitope is located close to the C terminus. Agrin fragment N1,
bound to mAb 86, was able to remove -dystroglycan almost
quantitatively from the muscle extract (Fig. 3A).
However, construct N4, which has a similar potency to induce AChR
aggregation compared with N1(13) , did not bind
-dystroglycan above the level of the antibody control under these
conditions (Fig. 3B). The same result was obtained with
fragment N6, for which no AChR aggregating activity has been detected.
Analysis of agrin that remained on the beads after elution of
-dystroglycan demonstrated that comparable amounts of the three
constructs had been bound to the matrices (Fig. 3C).
Figure 3:
Binding of N-terminal truncation fragments
of agrin to -dystroglycan. No agrin(-) or
25 pmol of
agrin fragments N1, N4, and N6 were bound to beads coated with mAb 86
and incubated with an alkaline extract of rabbit skeletal muscle
membranes. A, 50% of the unbound fractions and an equal volume
of the alkaline extract (AE) were separated by SDS-PAGE and
stained with a mixture of two mAbs against
-dystroglycan (
-D). B, bound
-dystroglycan was eluted by
replacing Ca
with EGTA. A Western blot of 75% of the
eluates was stained with a mixture of two mAbs against
-dystroglycan. C, the beads were boiled in gel loading
buffer, and aliquots of the supernatants were loaded onto a 3-12%
gradient gel. Nitrocellulose transfers were reacted with AS 11 against
agrin.
After prolonged development of similar Western blots, we observed
weak binding of -dystroglycan to agrin construct N4, but not to
construct N6 (data not shown). This is demonstrated in Fig. 4,
in which we used a more sensitive method for the detection of weak
immunoreactivities on Western blots. To estimate the differences in
-dystroglycan binding between constructs N1 and N4, we adsorbed
100 pmol of agrin fragment N4 as well as only 50, 25, and 10 pmol
of N1 to mAb 30 affinity beads (Fig. 4C).
Interestingly, the smallest immobilized amount of fragment N1, which
would correspond to a concentration of
7 nM in solution,
removed less than half of the
-dystroglycan present in the extract (Fig. 4, A and B). We estimate that the amount
of
-dystroglycan in the binding reactions was similar to the
smallest amount of N1. Even the small amount of N1 bound more
-dystroglycan than a 10-fold larger amount of fragment N4. We
therefore conclude that unlike C-terminal deletions, truncations from
the N terminus of N1 reduce agrin's capability of binding to
-dystroglycan.
Figure 4:
Fragments N4 and N4/C2 display weak
binding to -dystroglycan.
100 pmol of agrin constructs N4 and
N4/C2 as well as 50, 25, and 10 of fragment N1 (from left to right, as
symbolized by the triangle) were bound to affinity matrices
containing mAb 30. In a control experiment, no agrin(-) was bound
to the beads. All samples were incubated with an alkaline extract of
rabbit skeletal muscle membranes. A, 18% of the unbound
fractions and an equal volume of the alkaline extract (AE)
were separated by SDS-PAGE, transferred to nitrocellulose, and reacted
with a mixture of two mAbs against
-dystroglycan (
-D). Bound antibodies were visualized with
peroxidase-coupled secondary antibody using the enhanced
chemiluminescence system. B, bound
-dystroglycan was
eluted by replacing Ca
with EGTA. A Western blot of
85% of the eluates was treated with a mixture of two mAbs against
-dystroglycan, and immunoreactive bands were visualized by
enhanced chemiluminescence. C, the beads were boiled in gel
loading buffer, and equal aliquots of the supernatants were loaded onto
a 4-15% PhastGel. To allow a better comparison, 20% of the N4
aliquot was also loaded onto the gel. Nitrocellulose transfers were
reacted with AS 11 against agrin and visualized with alkaline
phosphatase-coupled secondary antibody.
These results suggest that the second laminin
G-domain, which is known to be important for agrin's clustering
activity(13, 14) , as well as flanking sequences might
be sufficient to mediate weak -dystroglycan binding. To directly
test this hypothesis, we generated an additional construct, N4/C2 (Fig. 1), which contained only the third and fourth EGF-like
repeats and the second laminin G-domain. When expressed in COS-7 cells,
N4/C2 was readily secreted into the medium and appeared as a band of 35
kDa on a Western blot (Fig. 1B). Fragment N4/C2 was
indeed sufficient for low affinity binding of
-dystroglycan as
similar amounts of
-dystroglycan were bound to this fragment and
to N4 (Fig. 4B). Similar to the longer C-terminal
deletion construct C2, N4/C2 was unable to induce clustering of AChR on
C2 myotubes above control levels (Fig. 1A).
Binding
of -dystroglycan to laminin (27, 35) and to
agrin(16, 17, 18) is inhibited by heparin.
Several lines of evidence implicate proteoglycans as important for
agrin-induced AChR aggregation(7, 12, 36) .
Therefore, we attempted to map the binding site on the agrin molecule
for heparin and to compare it with the
-dystroglycan site. We
assessed the retention of different agrin fragments on a heparin column (Fig. 5). All fragments, except N6, bound almost quantitatively
and required high ionic strength (>0.4 M NaCl) for elution.
Constructs N4 and N4/C2 were eluted at slightly higher concentrations
(data not shown). Thus, the second laminin G-domain present in all
binding constructs was sufficient to mediate binding to heparin.
Figure 5: Binding of agrin constructs to heparin-Sepharose. Conditioned medium containing agrin constructs N1, C2, N4, N4/C2, or N6 was diluted 3-fold to reduce the salt concentration to 50 mM and loaded onto a 5-ml heparin-Sepharose fast protein liquid chromatography column. Conditioned media of fragments N1, C2, N4, and N4/C2 before the column chromatography step were compared on a Western blot with a 5-fold higher amount of the flow-through fraction. For construct N6, equal amounts of the sample and flow-through fraction were transferred to nitrocellulose. Agrin immunoreactivity was visualized using the polyclonal antiserum AS 11.
We used truncation fragments of the C-terminal half of agrin
to determine the regions of the agrin protein necessary for binding to
the candidate agrin receptor, -dystroglycan, and to heparin, an
inhibitor of agrin-induced AChR clustering. Since the clustering
activity of these fragments has previously been
determined(13) , a comparison of the binding and activity
profiles has implications for the possible role of
-dystroglycan
as a functional agrin receptor.
Our analysis shows that at least two
regions of the agrin protein are required for -dystroglycan
binding (Fig. 6): fragment N4/C2 contained the second laminin G
homology region as well as the two C-terminal EGF-like repeats. These
regions were sufficient for low affinity binding. However, only the
presence of the first laminin G-domain and the first two EGF-like
repeats, as in construct C2, led to an agrin with high affinity
-dystroglycan binding similar to the largest construct, N1. The
difference between low and high affinity binding is substantial since
fragment N4 bound considerably less
-dystroglycan than a 10-fold
smaller amount of construct N1.
Figure 6:
Comparison of minimal agrin fragments
required for strong -dystroglycan binding, interaction with
heparin, and AChR clustering activity. The N termini of the various
constructs are marked by arrows; the C terminus of fragments
C2 and N4/C2 is represented by an arrowhead. The symbols representing different domains of agrin are as depicted in Fig. 1. The stippled line indicates regions of agrin
sufficient for low (but not high) affinity binding to
-dystroglycan.
It is likely that the second laminin
G-domain is crucial for -dystroglycan and heparin binding as
homologous domains mediate binding of laminin to
-dystroglycan and
to heparin(35) . The related third laminin G-domain did not
influence binding at all, and the first laminin G-domain possibly had
an effect on
-dystroglycan binding, but not on heparin binding. A
further refinement of our analysis would require even smaller fragments
of agrin. However, additional agrin truncation constructs are secreted
into the medium in very low amounts insufficient for affinity
chromatography(13) .
Comparing regions of the agrin molecule
necessary for -dystroglycan binding and for AChR clustering
activity, we found two important differences (Fig. 6): the C2
and N4/C2 constructs, lacking the C-terminal region of agrin, displayed
binding, but no AChR aggregating activity. Conversely, N4, a construct
lacking the first laminin G-domain and the first two EGF-like repeats,
displayed normal AChR clustering activity, but significantly reduced
binding to
-dystroglycan. Furthermore, in our experiments, we
employed two antibodies to immobilize agrin that were known to block
the AChR clustering activity of soluble agrin in culture(13) ;
surprisingly, mAb 30 (Fig. 3) and mAb 435 (data not shown) did
not prevent simultaneous binding of agrin to
-dystroglycan.
Two
observations made in previous studies had indicated discrepancies
between the ability of agrin fragments to bind to -dystroglycan
and their biological activity. First, proteolytic 70- and 95-kDa
fragments of purified agrin both bound to membranes of Torpedo electric organ(37) . In the 70-kDa fragment, the C
terminus had been removed by proteolysis at an unknown position.
Similar to our fragments C2 and N4/C2, this fragment was not able to
induce AChR clustering (38) . However, although the major
agrin-binding protein in these membranes is
-dystroglycan(15) , other proteins might have contributed
to binding of the 70-kDa fragment.
Second, a neuron-specific insert
of 8 amino acids (10, 11, 39) in a region
close to the fourth EGF-like repeat (Fig. 1A) increases
agrin's AChR clustering activity
10,000-fold(12, 13) . However, Sugiyama et al.(18) found that two agrin splice variants, with or without
this insert, equally bound to -dystroglycan. In their experiments
as well as in our study,
-dystroglycan dissociated from
-dystroglycan and the other members of the DGC was used as a
binding partner for agrin. The alkaline extraction procedure used in
our experiments might have affected the characteristics of the binding
of
-dystroglycan to different agrin fragments. However, this
appears unlikely since a previous study demonstrated that this
procedure does not affect the binding of homologous laminin G-domains
of laminin to
-dystroglycan (27) . In fact, binding of
agrin can be detected even after SDS denaturation and renaturation of
-dystroglycan on nitrocellulose (15, 17, 18) .
Interestingly, both the
insert sequence and the C terminus of agrin determine a particular
conformation of agrin that is defined by the presence of the epitope
for mAb 86(13) . Both regions do not affect agrin binding to
-dystroglycan. Thus, this conformation, which is crucial for the
clustering activity of agrin, is not required for binding to
-dystroglycan.
Taken together, our results support the idea
that -dystroglycan alone is not the functional agrin receptor. A
different or additional component that generates the observed ligand
specificity has to be postulated(18, 40) .
Three
principal scenarios can be considered for the agrin--dystroglycan
interaction. First,
-dystroglycan may in some manner collaborate
with other signaling proteins and facilitate their binding to agrin.
Receptor systems that utilize auxiliary proteoglycans are known for
several growth factors. For example, transforming growth factor-
first binds to an accessory proteoglycan, betaglycan, and later
combines with a different receptor to form a ternary complex, which is
finally converted into the signaling receptor(41) . However,
the concept of
-dystroglycan as an analogous auxiliary
proteoglycan does not readily explain why construct C2 does not act as
an inhibitor of agrin-induced AChR aggregation (13) and why the
weaker binding of construct N4 does not correlate with reduced
clustering activity. Therefore, our data favor two models in which the
active agrin fragments N1 and N4, but not inactive fragments such as
C2, bind to a yet unknown receptor other than
-dystroglycan(18) .
In a second scenario, binding of
agrin to this so far unidentified receptor alone is sufficient to
induce AChR clustering. -Dystroglycan does not participate in this
process. Instead, it merely is a structural protein involved in
stabilization of the synaptic sarcolemma. This model is entirely
consistent with our data.
A more intriguing third scenario, however,
would be a two-step model in which -dystroglycan is important for
the second phase of receptor aggregation. According to this model,
binding of agrin to the hypothetical receptor only initiates the
formation of small clusters of AChRs that are observed during the early
phase of receptor aggregation both in vivo and in
vitro(42, 43) .
-Dystroglycan can be
detected very early in these microclusters (30) . Binding of
agrin to
-dystroglycan could be required for a following
maturation step. This binding could trigger a reorganization of the
membrane cytoskeleton, for example, an association of utrophin with the
DGC. Utrophin colocalizes with mature AChR clusters, but is not
detectable in microclusters(44) . The recruitment of utrophin
or another rearrangement of the submembrane cytoskeleton could initiate
the condensation of microclusters into larger aggregates. According to
this model, this consolidation of AChR clusters is mediated by all
-dystroglycan-binding agrin fragments, including C2. In contrast,
inactive fragments such as C2 are not expected to bind to the
hypothetical receptor. Therefore, fragment C2 should inhibit neither
clustering initiation nor cluster condensation. Indeed, this fragment
does not block aggregation of AChRs(13) . The question remains
how construct N4 can induce clusters with similar efficiency compared
with N1 despite its reduced binding to
-dystroglycan. A possible
explanation is that agrin synthesized by muscle fibers can compensate
for the diminished binding of N4 to
-dystroglycan(18) .
The function of muscle-derived agrin, which does not contain an insert
at splicing site Z and therefore has a low capability of initiating
AChR clustering (7) , is unknown. Its strong binding to
-dystroglycan may suggest a role for muscle-derived agrin during
this maturation step.
To test these models, it will be necessary to
identify and isolate additional high affinity agrin-binding proteins.
Our construct N4 could be a valuable tool in this search as its binding
to the signaling receptor should be less overshadowed by its binding to
-dystroglycan.