From the Departments of Pharmacology and
Biophysical Chemistry, Biozentrum, University of Basel,
Klingelbergstrasse 70, CH-4056 Basel, Switzerland
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Agrin is a basement membrane-associated proteoglycan that induces the formation of postsynaptic specializations at the neuromuscular junction. This activity is modulated by alternative splicing and is thought to be mediated by receptors expressed in muscle fibers. An isoform of agrin that does not induce postsynaptic specializations binds with high affinity to dystroglycan, a component of the dystrophin-glycoprotein complex. Transcripts encoding this agrin isoform are expressed in a variety of non-muscle tissues. Here, we analyzed the tissue distribution of agrin and dystroglycan on the protein level and determined their binding affinities. We found that agrin is most abundant in lung, kidney, and brain. Only a little agrin was detected in skeletal muscle, and no agrin was found in liver. Dystroglycan was highly expressed in all tissues examined except in liver. In a solid-phase radioligand binding assay, agrin bound to dystroglycan from lung, kidney, and skeletal muscle with a dissociation constant between 1.8 and 2.2 nM, while the affinity to brain-derived dystroglycan was 4.6 nM. In adult kidney and lung, agrin co-purified and co-immunoprecipitated with dystroglycan, and both molecules were co-localized in embryonic tissue. These data show that the agrin isoform expressed in non-muscle tissue is a high-affinity binding partner of dystroglycan and they suggest that this interaction, like that between laminin and dystroglycan, may be important for the mechanical integrity of the tissue.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Agrin is a heparan sulfate proteoglycan that is required for the induction and regeneration of pre- and postsynaptic specializations at neuromuscular junctions (NMJ1; Refs. 1 and 2). When applied to cultured chick myotubes, agrin leads to the formation of protein aggregates containing acetylcholine receptors (AChRs), acetylcholinesterase, and several other molecules that are also found at the neuromuscular junction in vivo (3). The aggregating activity of agrin is strongly influenced by alternative mRNA splicing at two sites, called A and B in chick (y and z in rodents). While agrin isoforms that contain inserts at these sites are highly active in an AChR aggregation assay, an agrin isoform lacking the inserts, designated agrinA0B0, is inactive (4-6). Agrin mRNA encoding isoforms with AChR-aggregating activity are expressed in motor neurons, while cells in non-nervous tissue synthesize agrin transcripts lacking inserts at sites A and B (agrinA0B0; Refs. 4 and 7-12). Consequently, agrin-like immunoreactivity is detected in numerous non-neuronal tissues (13, 14).
In skeletal muscle and the electric organ of Torpedo
californica, the major binding protein of agrin is
-dystroglycan (15-18).
-Dystroglycan originates from a precursor
protein that becomes cleaved post-translationally giving rise to
-dystroglycan, a heavily glycosylated peripheral membrane protein
that binds to laminin and agrin, and the transmembrane protein
-dystroglycan (19). Both molecules associate with several other
proteins to form the dystrophin-glycoprotein complex (DGC; reviewed in
Ref. 20). Mutations in genes that encode different members of the DGC
cause several muscular dystrophies (reviews: 21, 22), suggesting that
the DGC links the subsarcolemmal cytoskeleton and the muscle fiber's
basement membrane to confer mechanical stability. Unlike some of
the other components of the DGC, dystroglycan is expressed in a variety
of tissues (19), and the recent observation that dystroglycan-deficient
mice, which die early in development, fail to form a functional
Reichert's membrane (23) suggests that dystroglycan is involved in
organizing many basement membranes.
Laminin-1 and laminin-2 bind strongly to -dystroglycan (24),
suggesting that this interaction provides the main linkage of the DGC
to basement membranes. Mutations in the gene coding for the
2 subunit of laminin result in severe muscular
dystrophies (25-27). Moreover, antibodies directed against laminin and
-dystroglycan perturb development of kidney (28, 29).
As a first step toward identifying possible functions of the
non-AChR-aggregating agrin isoform agrinA0B0, we have
investigated its binding to -dystroglycans isolated from different
tissues. We find that agrinA0B0 binds to
-dystroglycan
isolated from adult chick lung, kidney, and skeletal muscle with a
dissociation equilibrium constant of ~ 2 nM. Binding
to
-dystroglycan from adult chick brain was approximately 2-fold
lower. In addition, agrin co-localized with
-dystroglycan in
developing chicken kidney and lung. In conclusion, these data provide
evidence that agrin and
-dystroglycan bind strongly to each other
outside of the NMJ, suggesting that agrin may have a similar function
as the laminins in linking the basement membrane to the DGC.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Protein Purification, Labeling, and Immunoprecipitations--
A
95-kDa COOH-terminal agrin fragment, c95A0B0, expressed in
COS-7 or 293 cells was purified by immunoaffinity column as described (6) using the monoclonal antibody 5B1 (13). Agrin fragments were
iodinated as described elsewhere (30). Purification of -dystroglycan
was carried out as described elsewhere (31) with the following
modifications: about 15 g of freshly frozen tissue was homogenized
in 40 ml of 50 mM Tris-HCl, pH 7.5 containing 0.2 M NaCl and 1 mM phenylmethylsulfonyl fluoride
(buffer A) and incubated overnight with 10 ml of DEAE-Sephacel
(Pharmacia Biotech Inc.). The resin was washed three times with 30 ml
of buffer A, and bound proteins were eluted from the Sephacel with 20 ml of 50 mM Tris-HCl, pH 7.5, and 0.5 M NaCl
(buffer B). The eluate was incubated with 2 ml of wheat germ
agglutinin-Sepharose. After three washes with 20 ml of buffer B,
elution from wheat germ agglutinin-Sepharose was achieved by adding 0.3 M N-acetylglucosamine. Eluted proteins were then
dialyzed against 25 mM Tris-HCl, 137 mM NaCl,
2.7 mM KCl, pH 7.4 (TBS) and concentrated using CentriconTM
filters (Amicon). For immunoprecipitations, 5 g of freshly frozen
tissue was homogenized in 20 ml of buffer A containing 1% Triton
X-100. After centrifugation at 39,000 × g for 15 min,
the supernatant was passed through a filter paper. 750 µl of this
filtrate was subjected to immunoprecipitations using 4 µl of
anti-agrin antiserum (32). After incubation overnight at 4 °C,
immunocomplexes were precipitated with protein A-Sepharose as described
elsewhere (6). The presence of
-dystroglycan in the
immunoprecipitates was analyzed by SDS-PAGE followed by Western blot
analysis.
SDS-PAGE and Immunoblotting--
Adult chicken tissues (about
1 g) were homogenized on ice with a 15-ml Dounce homogenizer in 10 ml of PBS containing a mixture of protease inhibitors (32). Homogenized
tissues were subjected to SDS-PAGE on a 3-12% gradient gel. Proteins
(~80 µg/lane) were transferred to nitrocellulose membranes as
described elsewhere (33). In agrin blots the transfer was carried out
in the absence of methanol. After blotting, membranes were washed once
with water and then blocked with PBS containing 3% milk powder and
0.05% Tween 20 (PBSM/T) for 2 h. Agrin
immunoreactivity was detected by incubating blots with the anti-agrin
antiserum raised against recombinant chick agrin (32) at a dilution of
1:2,000 in PBSM/T. -Dystroglycan was detected by the
monoclonal antibody 8D5 (NovoCastra; Ref. 34), diluted 1:20 in
PBSM/T. Incubation time for both reactions was 90 min at
room temperature. Nitrocellulose membranes were washed three times with
PBSM/T and then subjected to a 45-min incubation with
appropriate horseradish peroxidase-conjugated secondary antibodies at a
dilution recommended by the manufacturer (Jackson ImmunoResearch
Laboratories). After three washes with PBSM/T and one wash
with PBS, immunoreactive protein bands were visualized by the ECL
detection method (Amersham Corp.).
Transfer Overlay and Solid-phase Radioligand Binding
Assays--
Transfer overlay assays were done as described (30).
Solid-phase radioligand binding assays were based on a protocol
described in (30) with minor modifications. Briefly, -dystroglycan
was immobilized on microtiter plates by overnight incubation at 4 °C in 50 mM sodium bicarbonate, pH 9.6. Remaining binding
sites were saturated by a 1-h incubation with TBS containing 1.25 mM CaCl2, 1 mM MgCl2
(TBS+Ca/+Mg) and 3% BSA. After blocking, wells were
incubated with 125I-c95A0B0 at room temperature
for 3 h in TBS+Ca/+Mg containing 3% BSA. The wells
were washed four times with TBS+Ca/+Mg, and bound
radioactivity was counted with a
counter. Each data point
represents the average of triplicate wells ± S.D. Unspecific binding (binding in the presence of 2 µM unlabeled
c95A0B0) was subtracted from each data point. Curves were
fitted by nonlinear regression analysis, assuming a single class of
equivalent binding sites, using the equation counts/min = (P × c/(Kd + c)), where counts/min represents radioactivity, Kd the
binding dissociation constant, c the concentration of
125I-c95A0B0, and P counts/min at
saturation.
Immunofluorescence--
Staining of frozen sections (12 µm)
from embryonic chicken lung or kidney were done as follows. Tissues
were cryoprotected by embedding them in PBS containing 20% sucrose
before they were frozen in Tissue-Tek (Miles). Unfixed cryostat
sections were blocked with PBS containing 3% BSA and 5% normal goat
serum for 1 h and incubated with diluted (1:2,000) anti-agrin
antibodies (32) for 1 h. After four washes with PBS, agrin
immunoreactivity was visualized using Cy3-conjugated secondary antibody
(Jackson ImmunoResearch Laboratories) at a dilution of 1:1,000.
-Dystroglycan immunoreactivity in lung was detected by subsequent
incubation of blocked slices with the monoclonal antibody 8D5
(NovoCastra; Ref. 34) followed by incubation with biotinylated
secondary antibodies (Jackson ImmunoResearch Laboratories) for 1 h. Conditioned medium from the hybridoma 8D5 was diluted 2-fold,
whereas the biotinylated secondary antibodies were used at a dilution
of 1:1,000. After four washes with PBS, sections were incubated with
fluorescein isothiocyanate-conjugated streptavidin for 45 min and again
washed four times with PBS.
-Dystroglycan in kidney slices was
stained by a slightly different protocol. Sections were blocked with
PBS containing 5% BSA, followed by a 1-h incubation with the
monoclonal antibody 8D5. After four washes with PBS, goat anti-mouse
IgG (Jackson ImmunoResearch Laboratories) at a dilution of 1:1,000 were
added for 45 min.
-Dystroglycan immunoreactivity was then visualized
using Cy3-conjugated rabbit anti-goat IgG (1:1,000; Jackson
ImmunoResearch Laboratories). After antibody incubation, slices were
again washed, mounted on glass coverslips with Citifluor (City
University), and analyzed under a Leica microscope equipped with
epifluorescence optics.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
As a first step we analyzed the tissue distribution of agrin and
one of its binding proteins, -dystroglycan, in the adult chick. To
this end, homogenates of brain, skeletal muscle, kidney, lung, and
liver were separated by SDS-PAGE, blotted to nitrocellulose, and
assayed for agrin and
-dystroglycan immunoreactivity by Western blot
analysis. As shown in Fig. 1, agrin-like
immunoreactivity with an apparent molecular mass between 400 and 600 kDa, was clearly detected in lung, kidney, and brain whereas no signal
was seen in skeletal muscle and liver homogenates. Upon longer exposure agrin-like immunoreactivity with the high molecular mass could also be
detected in skeletal muscle while we were not able to detect it in
liver homogenates (data not shown). The tissue distribution of agrin in
adult chicken brain, liver, and skeletal muscle is similar to that
observed in the embryo, except that lower levels were detected in adult
skeletal muscle (32). One explanation for this may be that in adult
muscle, agrin expression is restricted to the NMJ (8, 13). Agrin-like
immunoreactivity was also associated with discrete bands around 100 kDa. Proteins of similar size were purified from the electric organ of
T. californica (35) and were also observed in embryonic
chick and rat tissue (32, 36). These bands may represent proteolytic
fragments derived from full-length agrin.
|
Since no antibodies are available that reliably recognize
-dystroglycan from chicken, we probed the same homogenates for the
presence of
-dystroglycan, which is derived from the same precursor
molecule and forms a tight complex with
-dystroglycan (19). As shown
in Fig. 1,
-dystroglycan was most abundant in brain, followed by
lung, kidney,and skeletal muscle, but
-dystroglycan was not detected
in liver homogenates. Hence, the amount of
-dystroglycan in these
tissues correlates with that of agrin except in skeletal muscle where
-dystroglycan was very abundant, but only a little agrin-like
immunoreactivity was found.
Because lung, kidney,and muscle mainly express agrin transcripts
encoding the splice variant lacking inserts at sites A and B
(agrinA0B0; Refs. 7, 8, 11, and 12), we next determined whether agrinA0B0 binds to -dystroglycan isolated from
these tissues. To this end, binding of agrinA0B0 to
-dystroglycan preparations isolated from a variety of tissues was
analyzed by transfer overlay assays. As ligands, we used either
iodinated full-length agrin (cAgrin7A0B0) or the 95-kDa
COOH-terminal fragment, c95A0B0 (see Ref. 32 for
nomenclature). As shown in Fig. 2,
-dystroglycan from all the tissues, except liver, gave a strong
binding signal. No difference in
-dystroglycan binding was observed
using recombinant full-length chick agrin (cAgrin7A0B0) or
its COOH-terminal half (c95A0B0; Fig. 2), indicating that
the NH2-terminal part of agrin does not substantially
contribute to the binding. While
-dystroglycan from lung, kidney,
and skeletal muscle had an apparent molecular mass of 180 kDa,
-dystroglycan from brain was substantially smaller. Differences in
the Mr of
-dystroglycan have been observed by others using laminin-1 as a ligand (31, 37, 38). Since the predicted
molecular mass of the
-dystroglycan core protein is much smaller
(~74 kDa; Ref. 19), the difference is most likely due to its
extensive post-translational glycosylation (15, 38-40). The signals on
transfer overlay assays were detected with as little as 2.5 nM iodinated ligand, suggesting a high-affinity binding of
agrin to
-dystroglycan from all the tissues examined.
|
Since transfer overlay assays do not allow accurate measurement of
binding affinity, we used radioligand binding assays to determine
equilibrium constants for the binding of recombinant 125I-c95A0B0 to immobilized -dystroglycan.
As shown in Fig. 3, binding of
c95A0B0 to
-dystroglycan from skeletal muscle, lung,
kidney, and brain was detected at low nanomolar concentrations. We
measured Kd values between 2 and 5 nM
(Table I), demonstrating that
agrinA0B0 binds to
-dystroglycan with high affinity.
Binding of 125I-c95A0B0 to
-dystroglycan
from all tissues was Ca2+-dependent and was
competed to more than 90% with 2 µM unlabeled c95A0B0 (data not shown). The approximately 2-fold higher
Kd for brain-derived
-dystroglycan is most likely
due to its different glycosylation. In this context it is interesting
to note that the binding of laminin-1 and laminin-2 to
-dystroglycan
involves the carbohydrate moiety of
-dystroglycan (39-41).
|
|
Our data strongly suggest that agrin is a high-affinity binding partner
for -dystroglycan in kidney and lung. To see whether
-dystroglycan is associated with agrin in situ, we next
tested the
-dystroglycan preparations from lung and kidney for the
presence of agrin-like protein by Western blot analysis. As shown in
Fig. 4A, agrin-like
immunoreactivity was indeed detected in these preparations, but it had
a reduced apparent molecular mass compared with agrin detected in
tissue homogenates (see Fig. 1). As the binding site for dystroglycan
is localized in the COOH-terminal part of agrin (see Fig. 2), the lower
Mr of agrin found in
-dystroglycan
preparations may be due to proteolytic cleavage within the
amino-terminal portion of the molecule. Cleavage within this region
would release a fragment that lacks the NtA domain of agrin, which is
responsible for the binding of agrin to laminins (42). As the binding
of agrin to laminin is of high affinity (42), proteolytic degradation
may be requisite to extract agrin from tissue under the mild conditions used to purify
-dystroglycan. To assure that the presence of agrin-like immunoreactivity in the
-dystroglycan preparation reflected an association of both molecules in situ, we
tested whether immunoprecipitation with anti-agrin antibodies from
total tissue homogenates would also enrich for
-dystroglycan. As
shown in Fig. 4B,
-dystroglycan was indeed present in
these immunoprecipitations. These experiments thus show that agrin and
-dystroglycan are associated with each other in situ in
adult kidney and lung.
|
To see whether agrin and dystroglycan could interact with each other
during development, we next stained consecutive sections with
anti-agrin or anti--dystroglycan antibodies. As shown in Fig.
5, agrin-like immunoreactivity was highly
concentrated in basement membranes of lung bronchioles and kidney
tubules. At the light microscopic level,
-dystroglycan showed the
same tissue distribution (Fig. 5, right column). No staining
was detected with an agrin preimmune serum or in the absence of the
-dystroglycan antibody (data not shown). In summary, these data
indicate that agrin and
-dystroglycan may also interact with each
other during kidney and lung development.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous work on the binding of agrin and dystroglycan dealt
mainly with the functional consequences of this interaction in the
formation of the NMJ. It has been shown that the agrin isoform lacking
inserts at sites A and B (agrinA0B0), which is inactive in
AChR aggregation, binds to skeletal muscle -dystroglycan with higher
affinity than the AChR-aggregating isoform agrinA4B8 (18, 30, 43). Moreover, the region of agrin sufficient for AChR aggregation
is distinct from the binding site for
-dystroglycan, making it
unlikely that the binding of agrin to
-dystroglycan is involved in
the signaling mechanisms initiated by agrin (30, 43).
In a solid-phase radioligand binding assay agrinA0B0 binds
to skeletal muscle -dystroglycan with a dissociation constant of
less than 2 nM (Table I). Using the same binding assay, a Kd value of 8 nM was determined for
laminin-1 and a mixture of laminin-2 and laminin-4 (24). Hence, agrin
seems to bind to muscle
-dystroglycan with higher affinity than the
laminins. We also noticed this difference in the binding affinities in
inhibition experiments. While an excess of unlabeled
agrinA0B0 was always very efficient in inhibiting the
binding of iodinated laminin-1 to
-dystroglycan, the same
concentration of unlabeled laminin-1 inhibited the binding of iodinated
agrinA0B0 only
partially.2
In mature muscle fibers, agrin is concentrated at the NMJ and only
little is detected in extrasynaptic regions (Fig. 1; Refs. 8 and 13).
Agrin is therefore unlikely to be the major binding partner of
-dystroglycan in muscle fibers outside of the NMJ; instead,
laminin-2 may have this role. The situation may, however, be different
in adult kidney and lung, where agrin is expressed at high levels (Fig.
1). Lung and kidney, like muscle fibers, express transcripts encoding
agrinA0B0 (11, 12). Our study now shows that
agrinA0B0 binds to
-dystroglycan from both tissues with
high affinity (Kd values ~2 nM) and
that agrin is complexed with dystroglycan in vivo (Figs. 4
and 5). Hence, the major binding protein for
-dystroglycan in adult
kidney and lung may be agrin and not the laminins.
In kidney, the binding of laminin-1 to -dystroglycan has been
suggested to be important during epithelial morphogenesis. This is
mainly based on perturbation studies using polyclonal antibodies
directed against proteolytic fragments E8 or E3 of laminin-1 (28) or
the function-blocking monoclonal antibody IIH6 directed against
-dystroglycan (29). In those studies, the antibodies perturbed the
differentiation of mesenchyme into epithelium in organ cultures of
embryonic kidneys. Here we show co-localization of
-dystroglycan and
agrin in developing chick kidney tubules and lung bronchioles (Fig. 5).
Although we used anti-
-dystroglycan antibodies instead of
anti-
-dystroglycan antibodies, it is likely that the tissue
distribution of the two proteins does not differ. Both proteins
originate from the same precursor molecule (19), they tightly associate
with each other, and in other species, no differences in the tissue
distribution of
- and
-dystroglycan have been observed. We
therefore conclude that agrin is highly expressed in developing chick
kidney and lung and that it co-distributes with
-dystroglycan. This
co-localization hence suggests that binding of agrin to
-dystroglycan may also be possible during embryogenesis. Although we
have not looked at the tissue distribution of agrin and
-dystroglycan at early stages of kidney and lung development, we
hypothesize that part of the perturbation of kidney development with
the monoclonal antibody IIH6 (29) might be due to inhibition of the
agrin-dystroglycan interaction. Hence, it would be interesting to study
the effect of anti-agrin antibodies on the development of kidney.
We have also determined the binding affinity of agrinA0B0
to -dystroglycan isolated from adult brain. We find that this
binding is of more than 2-fold lower affinity than that to
-dystroglycan from skeletal muscle, kidney, and lung (Table I). The
weaker binding of agrin is paralleled by a decrease in the apparent
Mr of brain
-dystroglycan, suggesting an
involvement of carbohydrates in the binding (37, 44). Consistent with
this, glycosylation of
-dystroglycan is critically involved in the
binding of laminin-1 (39-41). Again, the functional significance of
the binding of agrin to brain-derived
-dystroglycan is not yet
clear. Agrin-like immunoreactivity in the brain is highly concentrated
in the basal lamina of certain microvessels that serve as selective
filtration barriers. The endothelial cells lining the blood capillaries
express the splice variant agrinA0B0, which binds most
strongly to
-dystroglycan (12). Blood capillaries in the nervous
system also express
-dystroglycan, utrophin, and
2
chain-containing laminins (45-47). The presence of agrin and
dystroglycan in blood capillaries and the finding that agrin expression
is high when a functional blood-brain barrier is established (48)
suggest a role of the dystroglycan-agrin binding in blood-brain barrier
formation. Alternatively, the binding of agrin to
-dystroglycan may
play a role in the maintenance of synaptic structures as DGC-like
immunoreactivity is detected at synapses in the central nervous system
(47, 49). Similarly, agrin-like immunoreactivity has been found at
synapses in the chick retina (50).
No gross abnormalities in the tissues examined here have been reported
in the agrin-deficient mice (2). In these knockout mice, only the exons
encoding site B were removed. As a consequence, even in homozygous
knockout animals, agrin isoforms lacking the B-site were still
expressed, although at a much lower level. As the binding affinity of
agrin to -dystroglycan is high, the low level of agrin may have been
sufficient for a functional interaction with
-dystroglycan. Animals
that are completely deficient in agrin may be required to reveal the
functional consequences of the loss of the binding of agrin to
-dystroglycan.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Micarna SA, in particular to Mr. Carquillat, for providing us with fresh chick tissue. We thank Dr. J. Engel for many helpful discussions during the course of this work. We are grateful to Drs. A. J. Denzer and R. A. Kammerer for their suggestions and to D. M. Hauser and Dr. W. B. Adams for reading the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant 31-33697.92 from the Swiss National Science Foundation and by the Swiss Foundation for Research on Muscle Diseases and the Rentenanstalt/Swiss Life.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: The Salk Institute MNL-O, 10010 North Torrey Pines Rd., La Jolla, CA 92037.
¶ These authors contributed equally to this work.
** To whom correspondence should be addressed: Dept. of Pharmacology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland. Tel.: 41-61-267-2246 or 41-61-267-2213; Fax: 41-61-267-2208; E-mail: rueegg{at}ubaclu.unibas.ch.
1 The abbreviations used are: NMJ, neuromuscular junctions; AChR(s), acetylcholine receptors; DGC, dystrophin-glycoprotein complex; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; BSA, bovine serum albumin.
2 M. Gesemann and M. A. Ruegg, unpublished observation.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|