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INTRODUCTION |
Agrin, a heparin sulfate proteoglycan (1, 2), is the major
signaling molecule that triggers the formation and development of the
neuromuscular junction (3-5). Motor neurons in the spinal cord
synthesize agrin and secrete it from axonal terminals into the basal
lamina of synaptic cleft (6). Agrin acts on muscle cell surface and is
responsible for the induction of several major aspects of postsynaptic
specialization at the developing neuromuscular junction. The most
studied action of agrin is the clustering of nicotinic acetylcholine
receptors (AChR)1 on the
postsynaptic muscle cell membrane (7-10). In addition, agrin is also
required for the organization of other synaptic proteins, recruitment
of cytoskeletal components, and the formation of junctional folds at
the neuromuscular synapse (11-14). The action of neural agrin is
mediated by a high affinity agrin receptor on muscle cell membrane. The
identity of the receptor remains unknown, but MuSK, a
muscle-specific receptor tyrosine
kinase, is believed to be an important component of the
receptor complex (15-17). Agrin induces rapid tyrosine phosphorylation
of MuSK in cultured myotubes. The downstream signaling cascade that
leads to AChR clustering is poorly understood. It involves tyrosine phosphorylation of the
-subunit of AChR and the recruitment of rapsyn, a 43-kDa protein, to the postsynaptic membranes of the neuromuscular junction (7, 8).
In mammals, a single agrin gene with three alternative splicing sites
(x, y, and z sites) encodes multiple isoforms of agrin in a variety of
tissues (18, 19). The AChR-clustering activity of agrin protein is
critically influenced by amino acid sequences contained within the z
site. This site is located in the third of three consecutive
laminin-like globular (G3) domains near the C-terminal end of agrin. An
exon, which encodes a short insert of eight amino acid residues
(ELTNEIP; z8), is found only in neural agrin and is required for the
AChR-clustering activity (see Fig. 1A) (20, 21). Splicing
variants of agrin expressed by non-neural tissues including muscle all
lack the z8 insert. They displayed little biological activity in
AChR-clustering assays in vitro (22-25). How alternative
splicing at the z site results in a striking difference in the
functional activity of agrin isoforms is unclear.
Although agrin is a large (>200 kDa) protein with multiple domains,
the AChR-clustering activity resides entirely in the C-terminal portion
of the protein (~75 kDa) (22, 26). This region contains four copies
of epidermal growth factor-like repeats and three copies of
globular domains (G1, G2, and G3) initially described in the C terminus
of laminin
2 chains (see Fig. 1A). Deletion analysis has
revealed that the C-terminal 21-kDa fragment of agrin, which consists
of only the G3 domain and z8 insert (AgG3z8), is necessary and
sufficient for binding to agrin receptor on muscle cells and for
inducing AChR clustering (22). The other laminin-like domains (G1 and
G2), as well as the epidermal growth factor-like repeats, are involved
in agrin binding to
-dystroglycan and heparin (18). They are
completely dispensable without significantly altering the
receptor-clustering activity of neural agrin (27).
An important factor that influences the biological activity of neural
agrin is calcium. Several studies have shown that calcium concentration
is critical for the induction of AChR clustering by motoneuron and
agrin in cultured myotubes and for the maintenance of cluster stability
(28-30). Removal of extracellular calcium completely blocked
agrin-induced activation of MuSK. Clamping of intracellular calcium
with BAPTA had little effect on MuSK activation but inhibited
tyrosine phosphorylation of AChR
-subunit (30, 31). These findings
provide circumstantial evidence that extracellular calcium may promote
agrin binding to the membrane receptor, whereas intracellular calcium
appears to regulate the downstream signaling events subsequent to the
formation of agrin-receptor complex. Nonetheless, the mechanism and
precise site of action by calcium remain unknown.
The G3 domain sequence of agrin is homologous to the globular domain of
2 laminin (see Fig. 1B). Crystallographic studies reveal
that this domain of laminin binds calcium through the side chains of
two aspartic acids and two main chain carbonyls (32, 33). Because the
aspartates are conserved in agrin G3 domain, and the z8 insert provides
two additional negatively charged residues (Glu), it has been
speculated that calcium might bind neural agrin, which contains the z8
sequence (32, 33). The present study has been undertaken to address the
following: 1) whether calcium directly binds to the G3 domain of agrin,
2) whether the z8 insert affects the protein interaction with calcium,
and 3) whether the interaction plays a role in regulating the
AChR-clustering activity of alternatively spliced isoforms of agrin protein.
We report that the G3 domain of rat neural agrin (AgG3z8) expressed in
Pichia pastoris promoted AChR clustering on the surface of
C2C12 myotubes in a calcium-dependent manner. Direct
binding of calcium to AgG3z8 was demonstrated by trypsin digestion and thermal denaturation experiments. Calcium induced a significant change
in the conformation of AgG3z8, and the effect was correlated with an
enhanced binding affinity of the protein to muscle receptor. Mutation
of the calcium-binding site in G3 domain diminished the conformational
change of neural agrin, reduced its binding affinity to muscle
membrane, and inhibited AChR-clustering activity. Conversely, the G3
domain of muscle agrin (AgG3z0) displayed little structural change in
the presence of calcium, bound poorly to muscle surface, and was
inactive in AChR-clustering assays. We conclude that calcium specifically alters the conformation of G3 domain of neural agrin. The
distinct effects of calcium on agrins with or without the z8 sequence
determine the biological activities of alternatively spliced isoforms
of the protein.
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EXPERIMENTAL PROCEDURES |
cDNAs, Expression Vectors, and Strains--
cDNA clones
encoding the C-terminal portion of the rat neural (CAg4,8) and muscle
agrin (CAg0,0) were kindly provided by Dr. Michael Ferns (McGill
University, Montreal, Quebec, Canada). The yeast expression vector
pPICZ
A and the KM71 mutS strain of P. pastoris
were purchased from Invitrogen.
Chemicals and Reagents--
Restriction and modification enzymes
for DNA cloning were purchased from either New England Biolabs
(Beverly, MA) or Invitrogen. Synthetic oligonucleotide primers
were synthesized by Integrated DNA Technology (Coralville, IA).
Nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity resin was the
product of Qiagen, Inc. (Valencia, CA). Rhodamine-
-BuTx was
purchased from Molecular Probes (Eugene, OR). 125I-Na was
obtained from Amersham Biosciences. Cell culture media and
supplements were the products of Invitrogen. General chemicals including protein sequencing grade trypsin (EC 3.4.21.4) were purchased
from Sigma.
Construction of Expression Plasmids--
Sequences encoding the
G3 domain of neural (amino acids 1756-1948) or muscle agrin (amino
acids 1756-1940) were each amplified by PCR using CAg4,8 or CAg0,0 as
the template (see Fig. 1). The forward and reverse primers used in the
PCR are 5'-AGG AAT TCC CAG TGG GGG ACC TAG AAA CAC TG-3' and 5'-CCC TCT
AGA TTA GTG GTG ATG ATG ATG GTG GGG AGT GGG GCA GGG TCT-3',
respectively. The PCR products were double-digested with
EcoRI and XbaI and ligated into identically
digested pPICZ
A. Ligation mixtures were used to transform
electrocompetent Top 10 cells. Plasmid DNAs were prepared from colonies
grown on low salt LB plates with Zeocin (25 µg/ml). Mutation of the
putative calcium-binding residue (Asp1820) to
alanine was carried out using sequence-specific oligonucleotide primers
by following the QuikChangeTM PCR mutagenesis protocol
(Stratagene, La Jolla, CA). All of the constructs were verified by
restriction digestion and dideoxynucleotide DNA sequencing (kit from
Epicentre Technologies, Madison, WI).
Protein Expression--
The pPICZ
A-AgG3z8 or -AgG3z0
construct was linearized by PmeI digestion and transformed
into P. pastoris KM71 strain by electroporation. Positive
yeast clones were selected on YPD plates with Zeocin (0.1 mg/ml). A
single colony was used to inoculate a 50-ml overnight culture in
buffered glycerol complex medium (1% yeast extract, 2% peptone, 3.4 g/liter yeast nitrogen base, 0.1 M potassium phosphate, pH
6.0, 0.4 mg/liter biotin, and 1% glycerol). The initial culture was
then expanded to 1 liter in buffered methanol complex medium (BMMY; 1%
yeast extract, 2% peptone, 3.4 g/liter yeast nitrogen base, 0.1 M potassium phosphate, pH 6.0, 0.4 mg/liter biotin). Methanol was added daily to the BMMY medium at a final concentration of
0.75% to induce and maintain protein expression. On the fourth day of
induction, the culture supernatant was collected, and proteins were
salted out in 70% ammonium sulfate by centrifugation at 6000 × g for 30 min at 4 °C. The precipitates were dissolved in
40 ml binding buffer (250 mM NaCl, 10 mM
imidazole, 50 mM sodium phosphate, pH 7.4), loaded onto a
10-ml Ni-NTA column, washed consecutively with 40 ml of binding buffer
and 40 ml of washing buffer (250 mM NaCl, 25 mM
imidazole, 50 mM sodium phosphate, pH 7.4), and finally
eluted in 40 ml of phosphate-buffered saline containing 250 mM imidazole. After the imidazole was removed by dialysis
against two liters of 5 mM phosphate buffer, pH 7.4, the
eluent was concentrated by gentle dehydration using Aquacide II
(Calbiochem-Novabiochem). The agrin protein was then further purified
using the Rotofor isoelectric focusing system (Bio-Rad). The recovered
fractions were passed through a HiPrep 26/10 desalting column to remove
the ampholytes (Amersham Biosciences).
Computer Modeling of Agrin Structure--
The structural models
were built by using the homology modeling function of Swiss-PdbViewer
V3.7 b2 (34). The crystal structure of
L2LG5 (Protein Data Bank code
1DYKA) was selected as the template, and the sequence alignment of AgG3
and
L2LG5 was adjusted according to their secondary structures (32).
The modeling request was submitted to Swiss-Model server, and the
modeling results were presented by PdbViewer.
Cell Culture and AChR-clustering Assay--
C2C12 myoblast
cultures were maintained in the growth medium (DMEM containing 20%
fetal calf serum, 2 mM glutamine, 0.5% chick embryo
extract (Invitrogen), and penicillin/streptomycin). Fusion was induced
by switching myoblasts at ~70% confluence to the fusion medium (DMEM
containing 5% horse serum, 2 mM glutamine). AChR clusters
were induced by adding AgG3 proteins to fully differentiated myotube
cultures. 5 h later, the cells were fixed in 2% paraformaldehyde (5 min), stained with rhodamine-conjugated
-bungarotoxin,
rinsed in phosphate-buffered saline, and viewed under a ×400 lens of an Olympus IX-70 fluorescent microscope. The results were photographed with a SPOT-2e camera, and the degree of AChR clustering was analyzed with NIH ImageJ software.
To remove extracellular calcium, we replaced the standard DMEM
media that contains ~2 mM calcium with DMEM that
lacked both calcium and magnesium. To increase extracellular calcium,
we added to the standard DMEM an additional 3, 8, and 98 mM
CaCl2, giving a final concentration of 5, 10, and 100 mM, respectively.
Circular Dichroism Spectroscopy--
Circular dichroism spectra
were collected at 25 °C on an Aviv 202 spectrometer. Protein was
diluted in either 6 mM Tris, pH 7.4, 15 mM NaCl
to ~0.2 mg/ml to reduce low UV absorbance in wavelength scanning
experiments or in 1× Tris-buffered saline for titration and
temperature denaturation experiments. At least six scans per sample
were recorded at 1-nm intervals from 280 to 195 nm with 1-s integration
time. Protein concentration was determined using a BCA protein assay
kit (Pierce). Mean residue ellipticity values were calculated based on
the known protein sequences. The spectra were analyzed by the CDSSTR
program using an IBasis 4 reference set (35-37). The results presented
in this study were averaged values from four separate experiments.
Calcium titration curves of 0 to 100 mM CaCl2
with 5 mM concentration steps were measured at wavelength
215 nm with 2 min of equilibration time and 1 min of average time.
Thermal denaturation curves were also measured at 215 nm, with a
1 °C temperature step, a 0.2-min equilibration time, and a 1-s
averaging time.
Limited Trypsin Digestion--
AgG3z8 or AgG3z0 protein was
first dialyzed against Tris-buffered saline with either 5 mM CaCl2 or 5 mM EDTA and 5 mM EGTA. Trypsin digestion was carried out at
enzyme/substrate ratios (w/w ratio) of 1:100, 1:50, 1:25, and 1:12.5
for 30 min at 37 °C. Digestion was stopped by boiling the proteins
in SDS sample buffer for 10 min, and the digestion pattern was analyzed
on a 15% SDS-polyacrylamide gel.
Radioligand Binding Assays--
Recombinant agrins were
iodinated using the chloramin T method described by Hunter and
Greenwood (38). The labeling was carried out by adding 1 mCi of
125I-Na (Amersham Biosciences) to 0.5 mg of protein in
potassium-phosphate buffer, pH 7.0. The reaction was started with 20 µl of a chloramin T solution (2 mg/ml). After 45 s, 20 µl of 2 mg/ml Na2S2O5 was added to
terminate the labeling reaction. Free 125I was separated
from labeled proteins by gel filtration on Sepharose-G25 columns.
Approximately 85% of the proteins were recovered after gel filtration.
The biological activity of neural agrin was retained after iodination
as assayed by their ability to induce AChR clustering in cultured C2C12 myotubes.
Iodinated agrin proteins were added at indicated concentrations to
cultured myotubes on six-well plates in the fusion medium. After
incubation for 60 min at room temperature, cells were rinsed four times
with 50 mM phosphate buffer, pH 7.4, and lysed with 0.2M NaOH. Bound radioactivity was counted using a
-counter.
 |
RESULTS |
Characterization of Agrin G3 Domain--
Using truncated fragments
of agrin expressed by mammalian cell lines, several previous studies
have shown that the C-terminal 21-kDa sequence of agrin, which contains
only the G3 domain with the z8 insert, is the minimal domain sufficient
for inducing AChR clustering in cultured myotubes (22, 39, 40). To
obtain protein materials sufficient for structural and functional
analysis, we expressed the G3 domains of neural (AgG3z8) and muscle
agrin (AgG3z0), respectively, in P. pastoris (Fig.
1). PCR-amplified rat cDNA encoding
these domains were subcloned into the yeast expression vector,
pPICZ
A. The signal sequence from yeast
-mating factor and a
hexahistidine tag were added in-frame to the 5' and 3' ends of agrin
cDNA, respectively. AgG3z8 and AgG3z0 were each secreted as a
soluble protein into the culture medium (>30 mg/liter). They were
purified to >95% by using the Ni-NTA column chromatography. On a
reducing SDS-polyacrylamide gel, the proteins migrated as a single band
of 26 and 24 kDa, respectively (Fig.
2A). The sizes were not
changed upon digestion with endoglycosidase H or PNGase F (in
10% SDS at 95 °C for 2 h), suggesting they were not modified by N-linked glycosylation (data not shown). Preparative
isoelectric focusing electrophoresis on the Rotofor apparatus separated
the samples into 20 fractions, pH 3-10. Both proteins were
concentrated in fraction 9 and 10, pH 6.0-6.5 (see Fig. 2,
B and C), in agreement with a calculated pI of
6.31 for AgG3z0 and 6.04 for AgG3z8. The yield after the purification
was ~20 mg/liter culture for each of the proteins.

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Fig. 1.
Domain structure of agrin and constructs used
in this study. A, the full-length rat agrin protein
contains three possible inserts at the X, Y, and
Z alternative splicing site. C-Ag is the
C-terminal, 75-kDa fragment of agrin protein, which displays full
AChR-clustering activity. G1, G2, and
G3 indicate the three laminin-like globular domains. The
four E boxes represent the epidermal growth factor-like
repeats. The G1 and G2 domains bind -dystroglycan and heparin. The
G3 domain with the z8 insert binds to an unidentified receptor complex
on muscle cell surface that is distinct from dystroglycan. AgG3 z8 and
AgG3z0 are the G3 domains of neural and muscle agrin, respectively,
expressed using P. pastoris for this study. B,
alignment of the primary sequences of agrin G3 domain
(AgG3z8) and the LG5 domain of 2 laminin
( L2LG5) (32). Amino acid residues were denoted using the
single letter code. The secondary structures of L2LG5 (32) are drawn
underneath the protein sequence. -Strands are indicated
by open arrows, and the -helices are indicated by a
dark bar. The z8 insert sequence (ELTNEIPA),
which is found only in neural agrin, is underlined. The two
conserve aspartic residues (Asp1820 and
Asp1889) in the hypothetical calcium-binding site are
indicated by arrowheads.
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Fig. 2.
Purification of agrin expressed in P. pastoris. A, AgG3z8 (arrow) and
AgG3z0 (arrowheads) eluted from Ni-NTA columns were
separated by 12% SDS-PAGE. B, the G3 domains of neural and
muscle agrin were further purified by isoelectric focusing
electrophoresis on Rotofor apparatus using ampholytes, pH 3-10.
Top, an aliquot of 20 µl of each fraction was examined by
SDS-PAGE; bottom, normalized protein distribution on the pH
gradients. C, superimposition of the CD spectra of AgG3z0
and AgG3z8 measured in the Tris buffer free of calcium. The data were
averaged from four separate experiments, each with six scans, and the
S.E. is shown at every 10 nm. See text for calculated composition of
protein secondary structures.
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The following two experiments were carried out to determine whether the
yeast-expressed agrin G3 domains were properly folded. First, we
measured their spectrum with a CD spectrophotometer. The composition of
protein secondary structures was calculated using the CDSSTR program
(35, 36). The spectra of neural and muscle agrins were slightly
different, but both had deep minima at 215 nm (Fig. 2C).
AgG3z8 had 30%
-sheet, 25%
-turn, and 2%
-helix. These
values are consistent with a model predicted based on the crystal
structure of the homologous domain of
2 laminin (see Fig.
1B and Fig. 3).
Similarly, AgG3z0 had a structural composition of 27%
-sheet
and 26%
-turn with no
-helix. The results of CD studies thus
suggest that the recombinant G3 domains of neural and muscle agrins
both assumed a folded conformation rich in
-structures.

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Fig. 3.
Computer modeling of agrin G3 domain
structure. The ribbon structural models were built using
Swiss-PdbViewer based on crystal structure of L2LG5 (Protein Data
Bank code 1DYK). Red, calcium; green, side chain
of aspartic acid; blue, side chain of asparagine;
yellow, disulfide bond. The z8 insert is situated in close
proximity to the putative calcium-binding site.
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Next, we assessed the function of the protein domains using an
AChR-clustering assay with C2C12 myotubes grown in standard culture
medium. In the absence of agrin, staining of myotubes with
rhodamine-conjugated
-bungarotoxin revealed few, if any, spontaneous
receptor clusters on the cell surface. Treating the cells for 3 h
with AgG3z8 at concentration as low as 0.01 µg/ml (~0.4
nM) induced the formation of numerous AChR clusters. The number of clusters approached maximal when 1 µg/ml (~100
nM) AgG3z8 was added to the culture (Fig.
4). The efficacy of yeast AgG3z8 protein
was thus comparable with that of a chick agrin G3 fragment expressed in
COS and HeLa cells (EC50 ~13 nM) (40, 41). In contrast, muscle agrin (AgG3z0) at similar concentrations failed to
induce AChR cluster formation. Only when the myotubes were treated with
extremely high concentrations (>50 µg/ml, ~2 µM) of
AgG3z0, a few receptor aggregates became visible (data not shown).
Thus, the G3 domain of neural agrin expressed in Pichia acted potently to induce AChR aggregation, whereas the muscle isoform
displayed little, if any, biological activity.

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Fig. 4.
The AChR-clustering activity of recombinant
agrins. A, purified AgG3z8 or AgG3z0 protein was added
at concentrations indicated into the culture medium of C2C12 myotubes.
4 h later, cells were fixed briefly in 0.5% paraformaldehyde,
rinsed with phosphate-buffered saline, and stained with
rhodamine-conjugated -BuTx. Scale bar, 50 µm.
B, digital photographs of the stained myotubes were
converted to grayscale mode and analyzed by using the ImageJ software.
Background signals were eliminated by adjusting the threshold. The mean
gray value of each picture was then measured to represent the degree of
AChR clustering on the cell surface.
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Extracellular Calcium Alters the Binding and Biological Activity of
G3 Domain--
Previous studies have demonstrated that calcium
enhances the binding of G1 and G2 domains of agrin to
-dystroglycan
and heparin on muscle membrane. The role of these interactions has not
been defined, but they appear to be not required for agrin-induced AChR
clustering (42-44). To determine whether calcium influences the
function of agrin G3 domain, we added 10 nM AgG3z8 to
cultured myotubes grown in medium with or without calcium. Staining
with rhodamine-conjugated
-BuTx revealed that few AChR clusters were present on the surface of myotubes in a calcium-free medium (see Fig.
5A and Fig. 4B). In
contrast, AgG3z8 induced the formation of numerous receptor clusters on
the plasma membrane of myotubes cultured in standard DMEM (~2
mM calcium). The number of clusters increased when
extracellular calcium was raised to 5 mM. The response to
agrin approached maximal at ~10 mM calcium, and it was
saturated at 100 mM calcium (Fig. 5B). In
separate experiments, myotubes treated in the same manner were labeled
with 125I-
-BuTx instead of rhodamine-
-BuTx. The
result showed that the total number of surface AChRs was not
significantly different among cells incubated with or without calcium
(data not shown). We thus exclude that the increased AChR clustering
was a result of enhanced transcription of AChR subunit genes by
calcium.

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Fig. 5.
AChR clustering induced by the G3 domain of
neural agrin requires extracellular calcium. A,
myotubes were incubated with 10 nM AgG3z8 in media
with or without calcium for 3 h. The cultures were then labeled
with rhodamine- -BuTx to visualize the AChR. Scale bar, 50 µm. B, the receptor clustering induced by AgG3z8 was
markedly increased by raising the concentration of extracellular
calcium. AgG3z0 (10 nM) was inactive in the AChR-clustering
assay regardless of calcium concentrations. The data represent the
average of results from three separate experiments. C,
radioligand binding assay was carried out to determine the affinity of
agrin to C2C12 myotubes. Myotubes grown on six-well culture plates were
incubated 125I-AgG3z8 or 125I-AgG3z0 at
concentrations indicated in medium with or without calcium for 1 h
at room temperature. Cells were then rinsed with 50 mM
phosphate buffer, pH 7.4 and lysed with 0.2 M NaOH. Bound
radioactivity was counted using a -counter. Calcium enhanced binding
of AgG3z8 to the muscle cells, but it had little effect on
AgG3z0.
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The requirement for calcium in agrin-induced synaptic specialization
may reflect calcium-dependent binding of agrin to its receptor or to some other calcium-dependent signaling
processes inside the muscle cells. By taking the advantage that the G3
domain binds specifically to muscle agrin receptor but not to
-dystroglycan and heparin, we carried out radioligand binding assay
using iodinated G3 proteins (125I-AgG3) in cultured C2C12
myotubes. In the absence of extracellular calcium,
125I-AgG3z8 bound poorly to muscle cell surface
(KD ~375 nM). In standard DMEM medium
(~2 mM calcium), however, the binding affinity of agrin
was increased by nearly two orders of magnitude (KD
~9.3 nM). At higher calcium concentrations (5 and 100 mM), a further but less dramatic increase in AgG3z8
binding was detected (see Fig. 5C and Table
I). In contrast, iodinated G3 domain of
muscle agrin (125I-AgG3z0) bound muscle cells with
significantly lower affinity than the neural isoform regardless of
extracellular calcium concentrations. These results therefore suggest
that extracellular calcium promotes direct binding of the G3 domain to
agrin receptor.
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Table I
Effects of extracellular calcium on dissociation constants of agrin
binding to muscle cells
Radioligand binding assay was carried out to determine the affinity of
agrin to C2C12 myotubes grown on six-well culture plates in the
presence of different calcium concentrations. Each dissociation
constant was calculated based on results of three independent
measurements.
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Calcium Induces a Change in the Conformation of Neural
Agrin--
The enhanced binding affinity of AgG3z8 to muscle cells
implicated that the metal ion may alter the structure of the G3 domain of agrin or its putative receptor on the postsynaptic membrane. Alternatively, calcium may play a role in promoting the assembly of an
agrin receptor complex, which involves MASC
(MuSK-associated synaptic
component), a putative muscle membrane protein, and MuSK (15, 30). To test these hypotheses, we measured CD spectra of the
recombinant proteins at various calcium concentrations. In the absence
of calcium, the G3 domains of neural and muscle agrin both displayed a
similar composition of secondary structures (Fig. 2C).
Interestingly, calcium induced a prominent change in the CD
spectrum of AgG3z8 (Fig. 6, A
and C). A change of the ellipticity at wavelength 215 nm was
apparent at 5 mM calcium (p < 0.01, n = 4). Raising the concentration of calcium to 100 mM resulted in a further reduction in the ellipticity at
215 nm. Plotting the change of [
]215 of AgG3z8 against
calcium concentration indicated that the KD for the
interaction was ~10 mM (Fig. 6C). Net
secondary structure analysis using CDSSTR revealed that at 100 mM calcium, AgG3z8 protein had a significant 11% increase in
-sheet content with a concomitant reduction in unordered
structure. In contrast, the CD spectrum of AgG3z0 exhibited little
change in the presence of calcium (Fig. 6, B and
C). It was unlikely that AgG3z0 was already saturated by
background calcium, as addition of 5 mM EDTA/EGTA did not
alter the protein spectrum (data not shown). Based on these
results, we conclude that calcium binding specifically increases the
content of
-structures in the G3 domain of neural but not muscle
agrin.

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Fig. 6.
Analysis of agrin proteins by CD
spectrophotometry. A, superimposition of CD spectra of
AgG3z8 (A) and AgG3z0 (B) measured in 0, 5, and
100 mM calcium. The data represent the mean of results from
four separate experiments, each with six scans, and the S.E. is shown
at every 10 nm. C, calcium titration curves. The change of
[ ]215 reflects calcium dependence of CD spectra of
neural agrin. The concentration of calcium that causes half-maximum
change of [ ]215 is about 10 mM.
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Muscle Agrin Binds Calcium but Does Not Undergo Conformational
Changes--
In the CD spectroscopic studies, we did not detect any
major change in the conformation of AgG3z0 protein in response to
calcium. The result suggested that this domain of muscle agrin might
not interact with calcium. Alternatively, it may bind calcium but does
not undergo a significant change in the net conformation. To test these
assumptions, we first examined whether radioactive calcium
(45CaCl2) would directly bind AgG3z0.
Unfortunately, the rather low binding affinity of the interaction (in
mM range, see below) made it difficult to obtain consistent
and accurate results. To circumvent this problem, we performed limited
trypsin digestion experiments on agrins incubated with or without
calcium. The patterns of enzymatic digestion were relatively simple on
the SDS gel because of the small size of the G3 domain. In the absence
of calcium (with 5 mM each of EDTA/EGTA), incubation of
AgG3z8 with 0.1, 0.2, or 0.4 µg of trypsin resulted in two digestion
intermediates, one at 20 kDa and the other at 18 kDa (Fig.
7A, lanes 3,
arrowheads). AgG3z0 treated in the same manner displayed one
digestion product of 20 kDa (Fig. 7A, lower
panel, lanes 3, arrowhead). Almost all (>90%) of the full-length AgG3z8 and AgG3z0 were degraded when trypsin concentration was raised to 0.8 µg (Fig. 7, A
(lanes 4-6, arrows) and B). In the
presence of calcium (5 mM), however, both the
neural and muscle proteins were partially protected from
trypsinolysis. A larger portion of the full-length AgG3z8 and AgG3z0
remained intact at various trypsin concentrations tested (Fig.
7A, lanes 8-11, arrows). Calcium also
enhanced the intensity of protein bands corresponding to the 20- and
18-kDa digestion products (Fig. 7A, lanes 8-11,
arrowheads). Moreover, additional intermediates of ~14 kDa
also appeared on the gel in the presence of calcium (Fig.
7A, lanes 8-11, asterisks). The
protective effect was more striking when the ion concentration was
increased to 100 mM (Fig. 7B). The action of
calcium on AgG3 proteins was specific, as other divalent ions including
Mg2+ did not exhibit any pronounced protection (data not
shown). These data thus suggested that the G3 domain of muscle agrin
might also interact with calcium.

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Fig. 7.
Limited trypsin digestion of AgG3
proteins. A, 10 µg of AgG4z8 or AgG3z0 were digested
with trypsin in the presence of 5 mM EDTA/EGTA or 5 mM CaCl2 at 37 °C for 30 min. Digestion
products were separated on a 15% SDS gel. B,
semi-quantitation of protein bands corresponding to the intact AgG3
proteins in each of the lanes shown in A. The
pixel intensity of each band on the gel was measured using NIH ImageJ
software and normalized against that of protein in the absence of
trypsin.
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Further evidence for interaction of both forms of agrin with calcium
was observed in thermal denaturation studies. Upon unfolding, the
ellipticity at wavelength 215 nm decreased significantly. In the
absence of calcium, the proteins were totally denatured at about
60 °C (Fig. 8). Based on the change of
ellipticity in the range of 25 to 72 °C, we estimated that the
Tm for AgG3z8 and AgG3z0 were both ~55 °C.
In the presence of 100 mM calcium, both proteins became
more stable, and the two thermal denaturation curves were shifted to
higher temperature. As a result, the Tm increased to 62 °C for neural agrin and 63 °C for muscle agrin. These data, together with those from the trypsin digestion experiments, suggest that the AgG3 protein might interact with and be stabilized by
calcium. However, calcium binding induces a conformational change only
in the G3 domain of neural agrin.

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Fig. 8.
Thermal denaturation of AgG3z0 and AgG3z8
proteins. The ellipticities at 215 nm of CD spectra of AgG3z0
(A) and AgG3z8 (B) were measured in 0 mM (open squares) or 100 mM calcium
(solid squares). Data were averaged from four measurements
and smoothed. Note that the curves for both neural and
muscle agrins were shifted to the right in the presence of
calcium.
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Mutation of Calcium-binding Site Impairs the Function of Neural
Agrin--
Our experiments described above established that calcium
binds to the G3 domain and induces a striking change in the
conformation of neural agrin. Sequence alignment and computer modeling
based on crystal structure of a homologous domain in laminin indicated that two aspartate residues (Asp1820, Asp1889)
in the G3 fragment of agrin may be part of the calcium-binding sites
(see Fig. 1B and Fig. 3). We thus replaced
residue Asp1889 with alanine by site-directed mutagenesis
and examined whether the mutation impairs the function of AgG3z8.
Limited trypsin digestion experiments revealed that calcium failed to
protect the D1889A mutant from degradation, suggesting the protein was
deficient in calcium binding (Fig.
9A). It was unlikely that the
mutation disrupted the overall folding of the G3 domain, as the CD
spectrum of mutant was indistinguishable from that of wild-type protein in a calcium-free solution. Unlike the wild-type AgG3z8, however, the
mutant protein displayed no conformational change upon addition of
calcium to the sample (Fig. 9B). Moreover, calcium failed to increase the binding affinity of the mutant G3 domain to muscle cell
membrane (see Fig. 9C and Table I). Consistent with results of the CD and binding studies, mutation of the calcium-binding residue
significantly reduced the AChR-clustering activity of neural agrin
measured in the presence of 2, 50, or 100 mM calcium (Fig.
9, D and E). We thus conclude that the
calcium-induced conformational change in the G3 domain is closely
related to receptor binding and AChR-clustering activity of neural
agrin.

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Fig. 9.
Mutation disrupting the calcium-binding site
impairs the function of AgG3z8. A, calcium was unable
to protect the AgG3z8 (D1889A) mutant from trypsin
digestion. B, the mutant protein displayed identical CD
spectra in the presence or absence of 100 mM calcium.
C, mutation of the calcium-binding residue decreased the
binding affinity of AgG3z8 to muscle cells in the presence of 2 mM extracellular calcium. D, compared with the
wild-type AgG3z8, the D1889A mutant (1 µg/ml) induced fewer AChR
clusters on myotube surface in the presence of 2 mM
calcium. Scale bar, 50 µm. E, the
AChR-clustering activity of D1889A mutant was consistently lower than
that of the wild-type AgG3z8 in the presence of higher concentrations
of calcium. **, p < 0.01 compared with wild-type
AgG3z8, n = 4.
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DISCUSSION |
Using the Pichia expression system, we have achieved
high yields of the G3 domains of neural and muscle agrin. The fragments were generated as secreted proteins, and they remained soluble even
after being concentrated to 90 mg/ml (3.5 mM). The yeast AgG3z8 induced aggregation of AChRs in C2C12 myotubes at low
nM concentrations with an activity comparable with that of
the same protein expressed in mammalian cells (40, 41). CD
spectroscopic studies indicate that the recombinant proteins have net
secondary structures rich in
-sheet as expected because of the
homology to
L2LG5 (Fig. 1B). The results of biochemical,
functional, and structural studies thus converge to the conclusion that
the recombinant G3 domains of agrin we expressed in P. pastoris are properly folded proteins, which are amenable to
future structural determination at high resolution.
It is well documented that calcium is required for induction of
postsynaptic specializations in cultured myotubes by motoneuron or
full-length agrin (28-30). The fact that calcium chelator BAPTA inhibited tyrosine phosphorylation of AChR
-subunit has led to the
idea that the ion might act as an intracellular messenger for agrin
signaling (31). Borges et al. (30) reported recently that
removal of extracellular calcium prevented activation of MuSK by agrin,
whereas clamping intracellular calcium had little effect on this
process. The data provide circumstantial evidence that extracellular
calcium may promote agrin binding to the membrane receptor or the
assembly of agrin receptor complex. Calcium has been shown to enhance
agrin binding to
-dystroglycan and heparin on muscle cell surface.
These interactions, however, are mediated by the G1 and G2 domain of
agrin, which are not required for the AChR-clustering activity. In the
present study, we uncover a novel role of calcium in mediating the
action of agrin. The metal ion induces a striking change in the CD
spectrum of neural agrin. Such change is closely related to enhanced
binding affinity and AChR-clustering activity of the protein. Thus, we
postulate that binding of the ion alters the conformation of neural
agrin and converts the protein from a low affinity, inactive precursor
to an active, high affinity ligand. By promoting interaction of agrin with its muscle receptor, extracellular calcium plays a critical role
in the postsynaptic specialization induced by agrin.
The results of our study also provide insight into the mechanism
responsible for the striking difference in biological activities of
alternatively spliced isoforms of agrin. The AChR-clustering activity
of neural and muscle agrin differs by at least 5,000-fold because of the presence of an extra eight amino acids within the G3
domain of neural agrin (see the Introduction and Fig. 1). A possible
explanation for this difference is that the z8 insert might constitute
all or part of the binding site for agrin receptor. However, we found
that a synthetic peptide of z8 sequence by itself was neither active in
receptor-clustering assay using C2C12 myotubes nor did it inhibit the
activity of neural agrin. In addition, substituted-cysteine scanning
mutagenesis experiments revealed that modification by
methanethiosulfonate reagents on cysteine residues introduced in z8
sequence did not significantly change the activity of AgG3z8
protein.2 Thus, the z8 insert
itself is unlikely to be a major element involved in direct binding to
agrin receptor on the postsynaptic membrane. Instead, results of our
present studies suggest that the interaction between the z8 sequence
and calcium determines the biological activity of alternatively splice
isoforms of agrin. The G3 domain of agrin is homologous to the globular
(LG5) domain of
2 laminin (Fig. 1B). This domain of
laminin binds calcium through the side chains of two aspartic acids and
two main chain carbonyls (32, 45). The two aspartates are conserved in
agrin. Superimposing the sequence of agrin G3 domain on the crystal
structure of laminin suggests the z8 insert is situated in close
proximity to the presumed calcium-binding site. In addition, the z8
insert contains two negatively charged residues (Glu) that may promote z8 interaction with calcium (see Fig. 1B and Fig. 3).
Consistent with these predictions, our CD studies show that calcium
specifically alters the secondary structures of the G3 domain of neural
agrin. The metal ion significantly reduces unordered structure and
increases the content of
-sheets in AgG3z8. In contrast, muscle
agrin displayed little change in the net secondary structure at the
same concentration of calcium. The KD of the
response deduced from changes of ellipticity in CD spectra of neural
agrin is ~10 mM (Fig. 6C). Because raising
calcium concentration from 2 to 5 or 10 mM increases the
number of agrin-induced AChR clusters in C2C12 myotubes (Fig. 5), our
result indicates that binding of calcium to AgG3z8 is probably not
saturated at physiological calcium concentration (2.2 mM).
Because neural and muscle agrins differ only by the z8 insert, this
short sequence must be involved, either directly or indirectly, in
mediating the conformational changes of AgG3z8 by calcium. Modeling of
agrin G3 domain based on the crystal structure of laminin suggests the
z8 insert is situated in close proximity to the presumed
calcium-binding site (Fig. 3). In preliminary experiments, moreover, we
found that substitution of the 4th residue (asparagine) in z8 insert
with alanine diminished both the calcium-induced conformational change
of AgG3z8 and its AChR-clustering activity.2 Because the
calcium-induced conformation change is quite significant (a net
increase of
-sheet by 11%), there must be structural changes that
occur outside the z8 insert, which only constitutes 4% of total
protein. Taking these together, our data are compatible with a model in
which the z8 insert is essential for agrin to undergo a conformational
change upon calcium binding. The structural change consequently brings
together the key residues required for agrin binding to its receptor.
Depicting the precise effect of calcium on the G3 domain will require
NMR or x-ray crystallographic analyses of the three-dimensional structure of agrin protein. In a preliminary NMR study, Alexandrescu et al. (46) have reported the backbone assignments for a
bacteria-expressed chick AgG3z0, but the detailed structure of neural
and muscle agrins remains to be determined. By establishing a system
that could generate large quantities of properly folded AgG3z8, our present experiments set a solid foundation for such experiments in the
near future. In addition, our work shall also facilitate discovery of
the putative agrin receptor(s) on postsynaptic membranes of the muscle
cells. Agrin is known to signal through a complex involving MuSK and a
co-receptor MASC (15, 47). The identity of MASC remains mysterious
partly because of the difficulties in obtaining sufficient quantities
of agrin protein for affinity chromatography. The approach we
introduced here makes such experiments feasible. As agrin is the only
molecule known to regulate many aspects of synaptic structure and
function, elucidating the mode of its action should contribute
significantly to our understanding of the molecular mechanisms
underlying synaptogenesis in the nervous system.