* Department of Pharmacology, Department of Biophysical Chemistry, Biozentrum, University of Basel, CH-4056 Basel,
Switzerland
Agrin is a heparan sulfate proteoglycan that is required for the formation and maintenance of neuromuscular junctions. During development, agrin is secreted from motor neurons to trigger the local aggregation of acetylcholine receptors (AChRs) and other proteins in the muscle fiber, which together compose the postsynaptic apparatus. After release from the motor neuron, agrin binds to the developing muscle basal lamina and remains associated with the synaptic portion throughout adulthood. We have recently shown that full-length chick agrin binds to a basement membrane-like preparation called MatrigelTM. The first 130 amino acids from the NH2 terminus are necessary for the binding, and they are the reason why, on cultured chick myotubes, AChR clusters induced by full-length agrin are small. In the current report we show that an NH2-terminal fragment of agrin containing these 130 amino acids is sufficient to bind to MatrigelTM and that the binding to this preparation is mediated by laminin-1. The fragment also binds to laminin-2 and -4, the predominant laminin isoforms of the muscle fiber basal lamina. On cultured myotubes, it colocalizes with laminin and is enriched in AChR aggregates. In addition, we show that the effect of full-length agrin on the size of AChR clusters is reversed in the presence of the NH2-terminal agrin fragment. These data strongly suggest that binding of agrin to laminin provides the basis of its localization to synaptic basal lamina and other basement membranes.
Efficient synaptic transmission requires a high local
specialization of pre- and postsynaptic cells. At the
neuromuscular junction (NMJ),1 these specializations include aggregates of acetylcholine receptors (AChRs)
and acetylcholinesterase (AChE) in the muscle cell membrane and accumulated vesicles containing the neurotransmitter acetylcholine in the nerve terminal (for review see
Hall and Sanes, 1993 Collagen type IV and laminin are the major components
of cell basement membranes. Both molecules assemble
from three separate chains. Individual chains are encoded
by a family of homologous genes giving rise to collagen
type IV and laminin isoforms that differ in their chain
composition (Timpl, 1996 Among the best characterized molecules associated with
synaptic basal lamina is agrin, a heparan sulfate proteoglycan (HSPG) with an apparent molecular mass on SDSPAGE of 400-600 kD (Denzer et al., 1995 The local immobilization of motor neuron-derived agrin
in the developing muscle basal lamina is thought to be important to maintain postsynaptic structures throughout
adulthood (McMahan, 1990 In a first attempt to characterize the binding of agrin to
extracellular matrix (ECM), we have recently shown that
full-length chick agrin binds selectively to MatrigelTM (Kleinman et al., 1982 Cell Culture, Transfection, Protein Labeling,
Immunoprecipitation, and Immunoblot
Culturing of primary chick myotubes and transfections of COS-7 (Gluzman, 1981
Expression Constructs
cDNA constructs pcAgrin and pc Antiserum against Antiserum 648 against the Protein Purification
Recombinant cAgrin7 was obtained from stably transfected HEK 293 cells,
whereas VF agrin was derived from eyes of day 14 chick embryos (Ruegg
et al., 1989 Solid-Phase Radioligand Binding Assay
Proteins were diluted to 10 µg/ml with 50 mM sodium bicarbonate, pH 9.6 (coating buffer), and immobilized on 96-well plates (Becton-Dickinson,
Bedford, MA) by incubation overnight at 4°C. Remaining binding sites
were blocked for 1 h with TBS containing 3% BSA, 1.25 mM CaCl2, and
1 mM MgCl2 (blocking solution). 125I-cAgrin7 or 125I-cN257Fc, diluted in
blocking solution, was added and incubated for 3 h. After washing with
TBS, 1.25 mM CaCl2, 1 mM MgCl2 four times, bound radioactivity in each
well was counted with a gamma counter. Solubilization with SDS sample
buffer and subsequent analysis on SDS-PAGE followed by silver staining
confirmed that the proteins were indeed coated onto the plastic surface.
Alternatively, mAb 5B1 diluted to 10 µg/ml in coating buffer was first
immobilized by overnight incubation at 4°C. Remaining binding sites were
saturated with blocking solution for 1 h. Then 3 µg/ml agrin was added.
After 1 h of incubation, the plates were washed three times with blocking
solution and processed with 125I-laminin-1 as described above.
To calculate half-maximal binding (EC50) of cAgrin7 to laminin-1, individual data points of the dose-response were fit by the following equation:
Y = (X/EC50)/(1+X/EC50) × P1. This equation assumes a single class of
equivalent and independent binding sites, where Y represents cpm, X represents the concentration of agrin, and P1 represents cpm at saturation.
Immunocytochemistry
Double staining of agrin in COS-7 cells: COS cells, transiently transfected
with cDNAs encoding cAgrin7 and c Primary chick myotubes were incubated with 200 nM c21B8 (Gesemann
et al., 1995 Competition Experiment and AChR Aggregation
Chick muscle cells were preincubated with 500 nM of cN257Fc for 4 h at
37°C. Conditioned medium from transiently transfected COS cells containing 100 pM cAgrin7A4B8 or c The NH2-terminal End of Agrin Is Sufficient to Bind
to ECM
We have recently shown that recombinant full-length
chick agrin (cAgrin; Fig. 1 a) binds to a solubilized mixture
of ECM molecules, called MatrigelTM. In contrast to fulllength agrin, a fragment that lacks the first 130 amino acids
from the NH2 terminus (c To see whether the NH2-terminal region of agrin alone
is sufficient to bind to MatrigelTM, we engineered a cDNA
construct that encodes an agrin fragment comprising the
NH2-terminal region and the first follistatin-like domain.
To facilitate detection and purification of the fragment, it
was fused to the constant region of a mouse IgG (Fc; Bowen
et al., 1996 The binding to MatrigelTM was assayed using transiently
transfected COS cells that were grown on tissue culture
dishes coated with this basement membrane preparation.
As previously shown (Denzer et al., 1995
Laminin-1 Is a Binding Partner for Agrin
MatrigelTM consists of a mixture of several ECM molecules, such as collagen type IV, laminin-1, nidogen/entactin,
and perlecan (Kleinman et al., 1982
The strength of the binding of agrin to laminin-1 was
measured by dose-response curves using 125I-cAgrin7. On
immobilized laminin-1, half-maximal binding (EC50) was
reached at ~5 nM (Fig. 4), suggesting a rather strong interaction of agrin with laminin-1. This binding is of similar
strength as the binding of laminin-1 to nidogen (Kd ~1 nM;
Fox et al., 1991
Agrin Expressed in Developing Chick Retina Binds
to Laminin-1
The results presented so far show that recombinant chick
agrin, expressed in mammalian cells, binds to laminin-1. If
this binding were important for agrin's function in vivo,
agrin purified from tissue should have the same binding
properties. To test this, agrin was isolated by anion exchange chromatography from the vitreous fluid of embryonic day 14 chick eyes, a rich source for agrin and other
molecules secreted from retinal cells (e.g., Ruegg et al., 1989
To test directly whether VF agrin is capable of binding
to laminin-1, we performed a modified solid-phase radioligand binding assay. In this assay, purified cAgrin7 or VF
agrin was immobilized on microtiter plates that had been
first coated with the antiagrin mAb 5B1 (Reist et al., 1987 The Laminin-binding Domain Is Highly Conserved in
Mouse and Human Agrin
The NH2-terminal region required for the binding of agrin
to laminin-1 has so far only been described in chick (Denzer et al., 1995
Agrin Binds to Laminin Isoforms Expressed at the NMJ
The data presented so far show that agrin binds to mouse
laminin-1. The laminins are a family of heterotrimeric glycoproteins composed of Since we were interested in whether agrin's NH2-terminal domain would also mediate the tethering to muscle cell
basal lamina, we measured binding of cAgrin7 and cN257Fc
to laminin-2 and -4. The source for laminin-2 and -4 was a
laminin preparation isolated from adult chick heart after
EDTA extraction and sequential purification by wheat
germ agglutinin Sepharose and immunoaffinity chromatography (Brandenberger and Chiquet, 1995
Many proteins that are highly concentrated at the NMJ
in vivo are also found in AChR clusters in vitro (for review
see Bowe and Fallon, 1995
To look at the relationship between the localization of
cN257Fc and laminin, antiserum 648, directed against the Binding of Agrin to Laminin Alters the Size of the
AChR Aggregates
AChR aggregates induced by the active full-length splice
variant cAgrin7A4B8 are more than twofold smaller than
those induced by the fragment c
Agrin is a large, multidomain protein that is associated
with basement membranes in many tissues (for review see
Denzer et al., 1996 The Laminin-binding Domain of Agrin
Defines a Novel Module
At the outset of the current work, it was known that the
binding of recombinant chick agrin to MatrigelTM requires
the first 130 amino acids at the NH2 terminus of full-length
agrin (Denzer et al., 1995 Although cN257Fc includes the first follistatin-like domain, this domain is not necessary for the binding to laminin-1 (Denzer, A.J., and M.A. Ruegg, unpublished observation). The stretch preceding the follistatin-like domain is
the most highly conserved region of agrin (Fig. 6). This
high homology and the fact that this region in chick agrin
mediates binding to all laminin isoforms so far tested (i.e.,
laminin-1, -2, and -4) strongly suggest it being important
for the integration of agrin into the scaffold of ECM molecules formed by the self-assembled laminins and collagen
type IV. Since no homology to any so far defined modules
was found using BLAST or FASTA algorithms (Altschul
et al., 1990 The binding of the NtA domain of agrin to laminin-1 is
confined to a particular region in the upper part of the triple coiled-coil domain of laminin-1, as shown by the binding of cN257Fc to proteolytic fragments of laminin-1 and
visualization of this interaction by electron microscopy after rotary shadowing (Denzer, A.J., T. Schulthess, C. Fauser, B. Schumacher, J. Engel, and M.A. Ruegg, manuscript in preparation). These results show that the binding
of agrin to laminin-1 is mediated by specific domains in
both molecules and they support the result of this study
(Fig. 4) that the agrin-laminin interaction is of high affinity.
Binding of Agrin to Laminin and Its Role
in AChR Aggregation
Induction and maintenance of postsynaptic differentiation
at nerve-muscle contacts in vivo is triggered by neural
agrin in a restricted area of muscle fibers (McMahan,
1990 We have shown that the size of agrin-induced AChR aggregates is affected by the NtA domain of agrin (Denzer et
al., 1995 We also think that the binding to laminin is the molecular basis that, after degeneration of the nerve terminals
and the muscle fibers, AChR-aggregating activity and agrinlike immunoreactivity is maintained at former synaptic
sites for several weeks (Burden et al., 1979 Binding of Agrin to Laminin Isoforms and
Neuromuscular Junction Development In Vivo
Upon formation of primary muscle fibers, Similarly, the phenotype of mice that are deficient of the
For the current studies, we used agrin isoforms that include a 7-amino acid insert at the NH2-terminal splice site.
Motor neurons in the developing chick spinal cord contain
agrin transcripts encoding this agrin variant, whereas the
majority of agrin mRNA in nonneuronal cells codes for
agrin without the insert (Denzer et al., 1995 Little is known about the function of agrin isoforms
without AChR-aggregating activity. Since these inactive isoforms bind more strongly to ). Differentiation of both pre- and
postsynaptic cells is directed by molecules that are localized to the synaptic portion of the muscle cell basal lamina
(Sanes et al., 1978
; Burden et al., 1979
). The capability of
the synaptic basal lamina to control and maintain synaptic differentiation makes it distinct from the extrasynaptic
basal lamina.
). The collagen type IV molecules and the laminins are thought to form independent networks by self assembly that are linked by entactin/nidogen (Yurchenco and O'Rear, 1994
). This scaffold builds
the framework with which several other proteins like perlecan, fibulin-1, and fibulin-2 associate (for review see
Timpl and Brown, 1996
). While perlecan and nidogen are
found both at synaptic and extrasynaptic sites of the muscle basal lamina, specific isoforms of collagen type IV and
laminin are localized to the NMJ (Sanes, 1995
). Other molecules that are concentrated at the NMJ include the
neuregulins, AChE, and agrin, some of which have been
shown to regulate different aspects of synapse formation
(for review see Ruegg, 1996
).
; Tsen et al.,
1995a
). When added to cultured muscle cells, agrin induces the aggregation of AChRs and several other proteins that are also enriched at the NMJ in vivo (Nitkin et al.,
1987
). Several lines of evidence have led to the hypothesis that agrin is released from the nerve terminal of motor axons and causes the local accumulation of AChRs and
other postsynaptic proteins in muscle fibers (McMahan,
1990
). Consistent with this hypothesis, agrin-deficient mutant mice lack AChR clusters, and no functional NMJs are
formed (Gautam et al., 1996
). In chick agrin, three sites of
alternative splicing have been described. One site is located near the NH2 terminus (Denzer et al., 1995
; Tsen et al.,
1995b
), and two sites, called A and B (y and z in rodents),
are located in the COOH-terminal half of the protein. Splicing at site B strongly influences agrin's AChR-aggregating activity. Agrin isoforms with amino acid inserts at
the B-site are highly active in inducing myotubes to aggregate AChRs, while agrin isoforms lacking inserts at this
site are only marginally or not at all active (Ruegg et al.,
1992
; Ferns et al., 1993
; Gesemann et al., 1995
). Transcripts encoding the individual splice variants are differentially distributed. While AChR-aggregating isoforms are
selectively expressed by neuronal cells, isoforms lacking
inserts at site B are predominantly expressed by nonneuronal cells (for review see McMahan et al., 1992
; Bowe and
Fallon, 1995
).
). This becomes evident as
AChR-aggregating activity and agrin-like immunoreactivity remain localized to synaptic basal lamina for several
weeks after degeneration of the cells at the NMJ (Burden et al., 1979
; Reist et al., 1987
). In addition to the NMJ,
agrin-like immunoreactivity is also detected in basement
membranes of other nonneuronal tissues, such as blood
capillaries, kidney, and lung (Reist et al., 1987
; Godfrey et al.,
1988a
; Magill-Solc and McMahan, 1988
; Rupp et al., 1991
).
Hence, agrin must strongly bind to components of synaptic
basal lamina and other basement membranes.
), a solubilized basement membrane extracted from the Engelbreth-Holm-Swarm mouse sarcoma.
This binding required a 130-amino acid-long region at the
NH2-terminal end of agrin (Denzer et al., 1995
). AChR
clusters on cultured myotubes were also affected by this
region as clusters induced by full-length agrin were more
numerous but considerably smaller than those induced
by any fragment of agrin without this NH2-terminal region
(Denzer et al., 1995
). We now show that a fragment comprising this region of agrin is sufficient to bind to MatrigelTM and that the binding to this basement membrane
preparation is mediated by laminin-1. The fragment also
binds to laminin-2 and -4, the laminin isoforms expressed
by muscle fibers, and it is concentrated in AChR clusters
on cultured chick myotubes. Moreover, an excess of this
fragment is sufficient to reverse the effect of full-length
chick agrin on the size of AChR clusters. Searches on databases revealed that the NH2-terminal region is highly
conserved in mouse and human agrin. Based on these
data, the 130 amino acids from the NH2 terminus of agrin
define a laminin-binding domain.
Materials and Methods
) or HEK 293 cells (Graham et al., 1977
) were carried out as
described by Gesemann et al. (1995)
. Iodination of purified protein was
performed as described (Gesemann et al., 1996
). 35S labeling, immunoprecipitation, and immunoblots were essentially done as described (Denzer
et al., 1995
). Recombinant cAgrin7 and c
N15Agrin (see Fig. 1 a) in the
supernatant of transiently transfected COS cells were immunoprecipitated with the antiserum raised against c
N15Agrin (Denzer et al., 1995
),
whereas cN257Fc and r21Fc (see Fig. 1 a) were directly bound to protein
A-Sepharose (Pharmacia, LKB Biotechnology Inc., Piscataway, NJ). 35Slabeled proteins were analyzed by SDS-PAGE on a 3-12% gradient gel
followed by fluorography. For immunoblots, 3 µg of purified cAgrin7 or
agrin purified from vitreous fluid (VF agrin) and 5 µg of total chick heart
laminins (Brandenberger and Chiquet, 1995
) was separated by SDSPAGE on a 3-12% gradient gel, transferred to nitrocellulose membrane,
and analyzed as described in Denzer et al. (1995).
Fig. 1.
Structure and biochemical analysis of agrin constructs. (a) Structural organization of chick agrin and of fragments used in this
study. Symbols and designations of individual domains are according to Bork and Bairoch (1995. Trends Biochem. Sci. 20): FS, follistatin-like module; EG, EGF-like module; LE, laminin EGF-like module; SEA, module first found in sea urchin sperm protein, enterokinase
and agrin; LamG, laminin G-like module. Furthermore, the fragment of the constant region of mouse immunoglobulin gamma heavy
chain (Fc), the chick agrin signal sequence (SS), the hemagglutinin signal sequence (SH), the serine/threonine rich regions (S/T), potential N-linked glycosylation sites, conserved GAG side chain attachment sites, and the sites of alternative mRNA splicing are indicated.
The NH2-terminal region of agrin characterized in this study is named NtA domain. Notes: (1) Inserts at splice sites A and B are not
specified. They are mentioned in the text if relevant for the experiment; and (2) the construct cN15Agrin was previously called cFull
(Denzer et al., 1995
; Gesemann et al., 1995
) and covers the coding region of chick agrin as described by Tsim et al. (1992)
. (b) Autoradiogram of the 35S-labeled agrin fragments depicted in a after precipitation from conditioned medium of transiently transfected COS
cells. cAgrin7 and c
N15Agrin were immunoprecipitated with anti-c
N15Agrin antibodies (Denzer et al., 1995
). cN257Fc and r21Fc were
precipitated with protein A-Sepharose. Proteins were separated by 3-12% SDS-PAGE. The two protein bands of cN257Fc are probably
due to inefficient stop of protein translation at the COOH-terminal end of the construct. Molecular masses in kD of standard proteins
are indicated.
[View Larger Versions of these Images (37 + 19K GIF file)]
N15Agrin have been described previously (Denzer et al., 1995
). The sequence encoding parts of the constant
region of mouse immunoglobulin gamma heavy chain (Fc) were obtained
by PCR using the primers sFc_XhoI_BamHI (GGCAGCTCGAGGATCCTCGTGCCCAGGGATTGTGGTTG) and asFc_XbaI (GGCCCTCTAGATCATTTACCAGGAGAGTGGG). As template, the Fc part
of mouse immunoglobulin (Bowen et al., 1996
) was used. Amplification
introduced an XhoI and a BamHI site at the 5
end and a XbaI site at the
3
end. The PCR product was digested with XhoI and XbaI and ligated into
the expression vector pcDNAI (InVitrogen, San Diego, CA) to yield pFc. To
generate pcN257Fc, a second PCR was performed using EcoRI_s-289
(GCATAGAATTCGGCTGCGGGCGATGGG) and as469_BamHI
(CACGAGGATCCCCTCTGCACAGGGGTCCTTG) as primers and pcAgrin7A4B8 (Denzer et al., 1995
) as template. The product coded for the
NH2-terminal domain of chick agrin and contained an EcoRI site at the 5
end and a BamHI site at the 3
end. The BamHI site enabled the subsequent in-frame fusion of this PCR fragment to the Fc sequences by digestion with EcoRI and BamHI and ligation into pFc. pr21Fc was similarly
constructed. The cDNA part encoding ray agrin was isolated by PCR using
the primers EcoRI_s3426 (AGCTTGAATTCAGCCAGTGGAAGTGAATC) and as3967_BamHI (CACGAGGATCCCTTTCTTGGCTGGACAGTG), and r100A4B8 (Gesemann et al., 1995
) as template. After digestion
of the PCR product with EcoRI and BamHI, it was ligated into pFc. The sequence of pcN257Fc and r21Fc was verified by DNA sequencing.
and
Chains of Chick Laminins
/
chains of chick laminin-2 and -4 was generated as follows. 1 mg of chick heart laminin-2 and -4, purified as described
(Brandenberger and Chiquet, 1995
), was loaded on a preparative 3-15%
gradient SDS-PAGE, run under reducing conditions, and blotted onto nitrocellulose (BA-85; Schleicher & Schuell Inc., Keene, NH). The protein
band of 200 kD, containing the
and the
chains, was cut out, suspended
in 0.2 ml PBS, and sonicated on ice until homogenization. The sample was
mixed with 0.2 ml Freund's complete adjuvant (GIBCO BRL, Gaithersburg, MD) and injected intracutaneously into one rabbit. The rabbit was
boosted 1 mo later with the same preparation, suspended in incomplete
Freund's adjuvant. Antiserum 648 was obtained 14 d later. In immunoblots,
the antiserum recognized the 200-kD band of laminin isoforms from chick
heart, chick gizzard, and mouse laminin-1 (Brandenberger and Chiquet,
1995
; Perris et al., 1996
). As the only chain common to all these laminins is
the
1 chain, antiserum 648 must recognize at least this subunit. As the immunogen also contained the
chain, the antiserum is likely to recognize this subunit as well.
). 100 ml of serum-free conditioned medium or 50 ml of vitreous fluid was passed over a Mono Q-Sepharose (Pharmacia Diagnostics
AB, Uppsala, Sweden). The column was washed with 20 mM Tris-HCl,
pH 7.2, 500 mM NaCl, and bound proteins were eluted with a linear gradient of NaCl from 500 mM to 2 M. Individual fractions with agrin immunoreactivity, as determined by an ELISA (Gesemann et al., 1995
), were
analyzed on SDS-PAGE (3-12% gradient) and visualized by silver staining (Morrissey, 1981
). Agrin-containing fractions were pooled and dialyzed three times against PBS. Agrin concentration of such a preparation
was determined by ELISA. cN257Fc and r21Fc from transiently transfected COS-7 cells were purified with protein A-Sepharose (Pharmacia)
according to the manufacturer's advice. Purity of recovered protein was
checked by SDS-PAGE and silver staining. The protein concentration
was determined as described (Lowry et al., 1951
), using the DC Protein
assay kit (BioRad Labs, Hercules, CA) and BSA as a standard. Mouse
laminin-1, purified from mouse Engelbreth-Holm-Swarm sarcoma as described (Timpl et al., 1979
), was provided by Th. Schulthess (Biozentrum,
University of Basel, Basel, Switzerland). Mouse nidogen, isolated from
conditioned medium of stably transfected HEK 293 cells (Fox et al., 1991
), and mouse perlecan (Timpl et al., 1979
) were provided by Dr. R. Timpl
(Max Planck Institute, Munich, Germany). Human collagen type IV (Weber
et al., 1984
; Ries et al., 1995
) was obtained from Dr. K. Kühn (Max Planck
Institute) and chick
-dystroglycan, isolated from skeletal muscle (Brancaccio et al., 1995
), was a gift of Dr. A. Brancaccio (Biozentrum, University of Basel). Chick laminin isoforms (laminin-2 and -4) were purified
from chick heart as described by Brandenberger and Chiquet (1995)
. Only
laminin preparations functional in a neurite outgrowth assay (Brandenberger and Chiquet, 1995
) were used for agrin-binding studies.
N15Agrin, were grown on MatrigelTM (Becton-Dickinson) and stained for agrin as described elsewhere
(Denzer et al., 1995
). Cells transfected with cDNAs encoding cN257Fc and
r21Fc were also stained as described in Denzer et al. (1995), except that
fluorescein-conjugated goat anti-mouse IgG (1:200; Cappel, Organon Teknika Corp., West Chester, PA) was used for the extracellular staining and
that, before permeabilization and staining with rhodamine-conjugated goat
anti-mouse IgG (1:200; Cappel), residual binding sites were blocked with
unlabeled goat anti-mouse IgG (1:50; Cappel).
) for 16 h at 37°C. To localize cN257Fc, 20 nM of this fragment
was included. Cultures were rinsed with culture medium, and to visualize
individual components, they were stained with the following reagents:
AChRs with 4 × 10
8 M rhodamine-
-bungarotoxin (Molecular Probes,
Eugene, OR); cN257Fc with 5 µg/ml biotinylated goat anti-mouse IgG (Molecular Probes) followed by 3 µg/ml fluorescein-conjugated streptavidin
(Jackson ImmunoResearch Laboratories, Inc., West Grove, PA);
/
subunits of laminin with antiserum 648 (1:1,000) followed by Cy3TM-conjugated goat anti-rabbit IgG (1:200; Jackson ImmunoResearch Laboratories, Inc.); and
2 chain of laminin with mAb C4 (10 µg/ml) followed by
5 µg/ml biotinylated goat anti-mouse IgG and 3 µg/ml fluorescein-conjugated streptavidin. The first incubation was done for 1 h at 37°C. Cultures were then washed with culture medium and fixed for 30 min at room temperature with 4% paraformaldehyde, 11% sucrose in 0.1 M potassium phosphate, pH 7.2. After rinsing the cells with PBS and PBS + 20 mM glycine, the cultures were incubated for 1 h with the secondary reagents indicated before, diluted in PBS + 2% normal goat serum (PBSN). After rinsing the cells with PBS, cultures were either dehydrated with 95% ethanol
at
20°C or incubated once more with the tertiary reagent diluted in
PBSN. Coverslips were mounted with VectashieldTM (Vector Laboratories, Inc., Burlingame, CA), and cultures were examined with a microscope equipped for epifluorescence (Leica Inc., Deerfield, IL).
N15AgrinA4B8 was added for 12 h at
37°C. AChRs were visualized and analyzed as described in Gesemann et al.
(1995)
. Since cAgrin7A4B8-induced AChR clusters were very small, aggregates with the longer axis of
1 µm were included.
Results
N15Agrin; Fig. 1 a) did not
bind to this ECM preparation (Denzer et al., 1995
). In this
130-amino acid-long stretch, a site of alternative mRNA
splicing is found (Denzer et al., 1995
; Tsen et al., 1995b
). Both splice variants are capable of binding to MatrigelTM,
although with different binding strengths (Denzer et al.,
1995
). In the current report we have investigated the binding of the splice variant that includes the seven-amino
acid-long insert (Fig. 1 a). This splice variant is selectively
expressed by embryonic chick motor neurons (Denzer et
al., 1995
) and is also highly expressed in embryonic chick
brain (Tsen et al., 1995b
).
), giving rise to the fragment termed cN257Fc
(Fig. 1 a). A chimeric construct of the most COOH-terminal, 21-kD fragment of agrin from the marine ray (Smith
et al., 1992
) and the Fc part (r21Fc; Fig. 1 a) was used as
a control. All recombinant proteins were efficiently synthesized and secreted from COS cells as shown by precipitation of 35S-labeled proteins from conditioned medium of
transiently transfected cells (Fig. 1 b). The high apparent molecular mass of 400-600 kD of cAgrin7 and c
N15Agrin on
SDS-PAGE is a result of heparan sulfate glycosaminoglycan (HS-GAG) chains that are attached to the 225-kD core
protein (Denzer et al., 1995
). The chimeric constructs, cN257Fc and r21Fc, were also secreted from the transfected cells, and they displayed the expected molecular
mass of ~69-65 and 61 kD, respectively (Fig. 1 b). SDSPAGE of the chimeric proteins under nonreducing conditions demonstrated that the intermolecular disulfide bonds
of the Fc region dimerize the fragments (data not shown).
), cAgrin7 was deposited on MatrigelTM (Fig. 2, a and a
), and no extracellular agrin-like immunoreactivity was observed with COS
cells expressing c
N15Agrin (Fig. 2, b and b
). Extracellular deposits of the recombinant proteins, cN257Fc and
r21Fc, were visualized with fluorescein-conjugated goat
anti-mouse IgG antibodies added to nonfixed, nonpermeabilized cells. The cells were subsequently permeabilized
and stained with rhodamine-conjugated goat anti-mouse IgG antibodies to identify transfected cells. As shown in
Fig. 2, c and c
, cN257Fc was deposited on MatrigelTM
around the transfected COS cells, which demonstrates that
the NH2-terminal region is sufficient to bind to this ECM
mixture. The binding is not caused by the Fc part, as no
deposits were seen with r21Fc-transfected cells (Fig. 2, d
and d
).
Fig. 2.
The NH2-terminal end of agrin is sufficient to bind to MatrigelTM. COS-7 cells, which had been transiently transfected with cDNAs encoding the agrin constructs indicated on the left of each row, were grown on MatrigelTM. Deposition of recombinant protein onto MatrigelTM (right column) was monitored by staining living cells with the appropriate antibodies. To identify agrin-expressing cells,
antibodies were added after permeabilizing the cells (left). To detect cAgrin7 and cN15Agrin deposits, antiagrin antiserum followed by
a fluorescein-conjugated secondary antibody was used (a
and b
). cN257Fc and r21Fc deposits were visualized with a fluorescein-conjugated goat anti-mouse IgG (c
and d
). Permeabilized cells were incubated with antiagrin mAb 5B1 followed by a rhodamine-conjugated secondary antibody (a and b) or with rhodamine-conjugated goat anti-mouse antibody (c and d). Only cells synthesizing cAgrin7 and cN257Fc show extracellular deposits of recombinant protein (a
and c
). Bar, 40 µm.
[View Larger Version of this Image (49K GIF file)]
). To identify the components to which agrin binds, a solid-phase radioligand
binding assay was used (Gesemann et al., 1996
). Laminin-1
and perlecan were purified from the Engelbreth-HolmSwarm mouse tumor (Timpl et al., 1979
), and collagen
type IV was isolated from human placenta (Weber et al.,
1984
; Ries et al., 1995
). After coating the purified proteins
onto the plastic surface of a microtiter plate, wells were incubated with 5 nM 125I-cAgrin7 or 125I-cN257Fc and bound
radioactivity was measured. Both agrin constructs bound
to laminin-1 (Fig. 3), while no or only little binding was seen to perlecan and collagen type IV (data not shown).
Since laminins bind to nidogen with high affinity (Kd ~1
nM; Fox et al., 1991
), most laminin preparations also contain nidogen (Paulsson et al., 1987
). To test whether the
binding of agrin to laminin-1 was due to nidogen, we also
measured binding of cAgrin7 and cN257Fc to recombinant
mouse nidogen, expressed in stably transfected HEK 293 cells (Fox et al., 1991
). No binding was observed for both
agrin constructs (Fig. 3). Hence, the binding of agrin to the
laminin-nidogen complex is based on a direct interaction
with laminin-1. As expected from previous experiments
(Bowe et al., 1994
; Campanelli et al., 1994
; Gee et al., 1994
;
Sugiyama et al., 1994
; Gesemann et al., 1996
), cAgrin7 also
bound to
-dystroglycan, purified from chick skeletal muscle (Brancaccio et al., 1995
; Fig. 3 a). In contrast, the NH2terminal fragment, cN257Fc, did not bind (Fig. 3 b). This is
consistent with the notion that the binding site to this peripheral membrane protein resides in its COOH-terminal
half of agrin (Bowe et al., 1994
; Campanelli et al., 1994
;
Gee et al., 1994
; Sugiyama et al., 1994
; Gesemann et al.,
1996
). Accordingly, only the laminin-binding site is common to cN257Fc and cAgrin7. In addition, no binding of
125I-r21Fc was observed to any of the components tested
(data not shown), which excludes a contribution of the Fc
part in the binding of cN257Fc to laminin-1.
Fig. 3.
Agrin binds to
laminin-1. Solid-phase radioligand binding assay with
purified proteins. Mouse
laminin-1, nidogen, and chick -dystroglycan were immobilized in microtiter plates and
subsequently incubated with
125I-cAgrin7 or 125I-cN257Fc.
Each value represents binding after subtraction of the
background (BSA-coated
wells). The values given are
the mean ± SD from one
representative experiment
with three independent measurements in the case of 125IcN257Fc and two measurements for 125I-cAgrin7. (a) 5 nM of 125I-cAgrin7 bound to
-dystroglycan and to laminin-1, but not to nidogen. Background counts were 89 ± 15 cpm. (b) 5 nM 125IcN257Fc bound to laminin-1 but not to
-dystroglycan and nidogen. Background counts were 65 ± 3 cpm. These experiments show that the binding of agrin to laminin is mediated by the NH2-terminal end of agrin. Counts measured in a and b cannot be compared because cAgrin7 and cN257Fc are iodinated to different extents.
[View Larger Version of this Image (16K GIF file)]
). At 4 nM 125I-cAgrin7, 90.3 ± 0.5% (mean ± SEM; n = 3) of the binding was competed with 100 nM unlabeled cN257Fc, indicating that the majority of the binding of full-length agrin to laminin-1 is mediated by the
NH2-terminal end. Since agrin is an HSPG (Denzer et al.,
1995
; Tsen et al., 1995a
) and laminin-1 is known to bind to
heparin (Ott et al., 1982
; Yurchenco et al., 1993
), the residual binding of cAgrin7 to laminin-1 could be due to the
binding of the HS-GAG side chains of agrin to the heparin-binding site of laminin-1. Indeed, only the HS-GAG
side chain-carrying construct cAgrin7, but not cN257Fc,
binds to the elastase fragment E3, which contains the major heparin-binding site of laminin-1 (Ott et al., 1982
; data
not shown). In summary, our data show that laminin-1, the laminin isoform present in MatrigelTM, is a basement membrane binding partner for agrin. Furthermore, they show
that the interaction is mediated by the NH2-terminal end
of agrin, and we therefore propose that this part of agrin
forms a structural unit (see also Discussion).
Fig. 4.
Binding of agrin to laminin-1 is of high affinity. Doseresponse curve of the binding of 125I-cAgrin7 to laminin-1. Halfmaximal binding was reached at ~5 nM, suggesting a rather
strong binding of agrin to laminin-1. The binding curve shown results from one representative experiment. Background values
(BSA-coated wells) are subtracted from each data point. Values
shown are the mean ± SEM from three measurements.
[View Larger Version of this Image (11K GIF file)]
;
Halfter, 1993
; Denzer et al., 1995
). As a control, cAgrin7
from conditioned medium of stably transfected HEK 293 cells was purified by the same procedure. When agrin-containing fractions were assayed by Western blot, agrin-like
protein from vitreous fluid and cAgrin7 had a high apparent molecular mass (Fig. 5 a, left), which is consistent with
previous results (Denzer et al., 1995
). The bands with an
apparent molecular mass of ~115 kD are probably due to
proteolytic degradation similar to what has been reported
elsewhere (e.g., Nitkin et al., 1987
; Godfrey, 1991
; Rupp
et al., 1991
; Denzer et al., 1995
). The same samples were
also probed with antiserum 245, raised against native chick heart laminin (Brubacher et al., 1991
). No laminin-like immunoreactivity was observed in the samples containing
cAgrin7, derived from stably transfected HEK 293 cells,
whereas immunoreactive bands were visible in the sample
containing agrin from the vitreous fluid (Fig. 5 a, right).
The strong immunoreactive band with the apparent molecular mass of 200 kD most likely corresponds to the
/
subunits of laminin isoforms, while the faint bands with
the molecular masses of 400 and 150 kD could represent
subunits and nidogen, respectively (Brubacher et al., 1991
).
The copurification of chick laminin isoforms from the vitreous fluid with agrin by a procedure that is selective for
highly charged anions is unexpected and may reflect the
binding of laminin to agrin. Consistent with this idea,
Halfter (1993)
reported copurification of laminin with a
chick HSPG, which later was shown to be agrin (Tsen et al.,
1995a
).
Fig. 5.
Chick agrin synthesized in vivo
contains the laminin-binding domain. (a)
Western blot of recombinant cAgrin7 and
agrin purified from vitreous fluid (VF agrin).
Transferred protein were stained with antiagrin antibodies ( agrin) or antilaminin antibodies (
laminin). Agrin purified from vitreous fluid has approximately the same
apparent molecular mass as recombinant
full-length agrin. The band detected at ~115
kD most likely results from proteolytic degradation. No laminin-like immunoreactivity
is associated with recombinant agrin (right).
In contrast to this, the agrin-containing fractions from vitreous fluid are positive for
laminin-like protein. Based on their apparent molecular mass and the specificity of the
antiserum used (Brubacher et al., 1991
),
they may represent nidogen (~150 kD), the
and
chain (200 kD), and
chain (400 kD). Molecular masses of standard proteins
are given in kD. (b) cAgrin7 and agrin from
vitreous fluid were bound to immobilized antiagrin mAb 5B1 and incubated with 125I-
laminin-1. The data result from one representative experiment and are the mean ± SD of three measurements. Background counts (no agrin added; 132 ± 15 cpm) was subtracted. 100 nM cN257Fc (+ cN257Fc) inhibits binding of 125I-laminin-1 to cAgrin7 and VF agrin by more than 97%, indicating that the
NH2-terminal domain is responsible for the binding.
[View Larger Versions of these Images (41 + 21K GIF file)]
).
To these wells, 5 nM of 125I-laminin-1 was added and
bound radioactivity was measured. As shown in Fig. 5 b,
125I-laminin-1 bound to cAgrin7 and VF agrin. In both
cases, binding was competed by more than 97% by including 100 nM of unlabeled cN257Fc during the incubation
with 125I-laminin-1 (Fig. 5 b). These experiments show
that agrin synthesized and secreted by cells of the chick
retina can bind to laminin-1 and that this binding is also
mediated by the NH2-terminal domain of agrin. Although
the same amount of agrin was added to the 5B1-coated microtiter wells, the amount of 125I-laminin-1 bound to VF
agrin was only half of that bound to cAgrin7. The most
likely explanation for this difference is that some of the
binding sites in VF agrin may already be occupied by laminin isoforms from the vitreous fluid that copurified with
VF agrin (see Fig. 5 a). In summary, chick agrin synthesized in vivo has the same laminin-binding properties as
recombinant agrin, and its binding is also mediated by the
NH2-terminal domain.
). Full-length cDNA encoding rat agrin lacks
this region and instead, the first follistatin-like domain is
preceded by a sequence that has been proposed to serve as
signal sequence (Rupp et al., 1991
). To see whether the
NH2-terminal region of chick agrin is found in other species, we searched for homologous sequences using the
BLAST algorithm (Altschul et al., 1990
). Four expressed
sequence tags, isolated from different tissues in mouse and
in human (Lennon et al., 1996
), closely matched this amino acid sequence. Sequencing of the clones confirmed that
the deduced amino acid sequences of mouse and human
agrin are highly homologous to each other and to chick
agrin (Fig. 6). The homology starts at residue 26 of chick
agrin, which corresponds to the predicted cleavage site for
the signal sequence (Denzer et al., 1995
). In the stretch
from residue 26 to 149, 96% of the amino acids are identical between mouse and human, and 90% are identical between chick and the mammalian sequences (Fig. 6). This is
by far the most highly conserved region in agrin (see also
Tsim et al., 1992
), suggesting that this domain may also
confer binding to laminin in mammals.
Fig. 6.
The laminin-binding domain is present in mouse and
human agrin. Deduced amino acid sequences of the expressed sequence tags (Lennon et al., 1996) from mouse and human agrin
were aligned to the first 173 amino acids of chick agrin. In addition, the first 67 amino acids of rat agrin are shown. Amino acids
identical to chick agrin are denoted with dots. The small letters in
the chick sequence represent the proposed signal sequence (Denzer et al., 1995
) with the initiator methionine (bold) and the proposed signal sequence cleavage site (*). Underlined amino acids
show the position of the tryptic peptide derived from a HSPG of
bovine kidney (Hagen et al., 1993
). Homology between chick and
human starts after the signal sequence cleavage site of chick (Cys
26). Amino acids between Cys 26 and Glu 149 are almost 90%
identical between human, mouse, and chick. Note that the amino
acid sequences of mouse and human agrin that precede the proposed signal peptide cleavage site (*) are not homologous to
chick and consist mainly of hydrophobic amino acids. Thus, as in
chick these stretches might be part of a signal sequence (lowercase letters). The homology between chick and rat begins at Asp
157 immediately after the boundary where chick is alternatively
spliced. The human and mouse sequences lack the seven-amino
acid-long insert at this splice site (dashes). These sequence data
are available from GenBank/EMBL/DDBJ under accession number U84406 (human agrin) and U84407 (mouse agrin).
[View Larger Version of this Image (27K GIF file)]
,
, and
chains (Burgeson et al.,
1994
; Timpl, 1996
). Each laminin is characterized by its
chain composition, for example laminin-1 is a trimer with
the
1, the
1, and the
1 chains. Recent data suggest that
laminin-1 is not expressed in muscle basal lamina but instead is replaced by laminin-2 and -4 (Schuler and Sorokin,
1995
). Laminin-2 (
2,
1,
1) is present early in development throughout the extracellular matrix of muscle fibers.
In adult muscle, it is expressed in the extrasynaptic region
of the basal lamina. Laminin-4 (also called s-laminin; Hunter
et al., 1989
), in which the
1 chain is replaced by the
2
chain, is expressed later in development and localizes to
the synaptic portion of the muscle cell basal lamina (Sanes
et al., 1990
).
). As shown
by Western blot analysis using antiagrin antibodies, this
laminin preparation contained substantial amounts of agrinlike protein (Fig. 7 a). As this laminin preparation did not
contain
-dystroglycan, detected by a transfer overlay assay using iodinated chick agrin (data not shown), we conclude that the copurification of agrin with laminin isoforms from cardiac muscle is most likely a consequence of
the association of agrin with the laminins. To test directly
whether laminin-2 and -4 are binding proteins for agrin,
we performed a solid-phase radioligand binding assay using purified laminin isoforms. With the same amount of
laminin-2 and -4 coated on the microtiter wells, 5 nM of
125I-cAgrin7 gave a clear signal on laminin-2 and a fivefold
stronger signal on laminin-4 (Fig. 7 b). Like cAgrin7, the
NH2-terminal fragment, cN257Fc, also bound approximately four times more strongly to laminin-4 than to laminin-2 (Fig. 7 c). The weaker signal on laminin-2 was not
due to a contamination with laminin-4, since this isoform
represents less than 5% in the laminin-2 preparation (Brandenberger et al., 1996
). A difference in the coating
efficiency between laminin-2 and -4 was also excluded because equal amounts were immobilized when tested with
an mAb specific for the
2 chain (Brandenberger et al.,
1996
). In summary, full-length chick agrin binds to both
laminin-2 and -4, but at this particular concentration the
interaction with the extrasynaptic laminin-2 is of lower apparent affinity.
Fig. 7.
Agrin binds to
laminin isoforms expressed
in muscle. (a) Immunoblot
using antiagrin antibodies of
laminin isoforms purified from EDTA extracts of adult
chick heart by immunoaffinity column with the anti-1
subunit-specific mAb 11B7
(Brandenberger and Chiquet, 1995
). Agrin-like protein with the same apparent
molecular mass as cAgrin7 is
detected in the laminin preparation. Since no
-dystroglycan was detected in the
same laminin preparation (data not shown), the copurification of agrin suggests that
laminins are associated with
agrin in muscle tissue. (b and
c) Agrin binds directly to purified chick laminin-2 and -4. Equal amounts of the two
laminins were immobilized on microtiter plates and incubated either with 5 nM of iodinated cAgrin7 (b) or cN257Fc (c). Values given
are the result of one representative experiment and represent the mean ± SD of three measurements where the background (BSAcoated wells) has been subtracted. Background values were 163 ± 24 cpm in b and 75 ± 11 cpm in c. The binding of cAgrin7 to laminin-4 is
fivefold stronger than to laminin-2 (b) and a similar difference in the binding (fourfold) is observed with cN257Fc (c). Note that the difference in the counts measured with 125I-cAgrin7 and 125I-cN257Fc is due to different iodination efficiencies.
[View Larger Versions of these Images (20 + 27K GIF file)]
). These include proteins of the
ECM, such as HSPG (Wallace, 1989
) and laminin (Nitkin
and Rothschild, 1990
). Consequently, the laminin-binding
fragment, cN257Fc, should colocalize with AChR clusters.
To test this, AChR aggregation was induced on cultured
chick myotubes with 200 nM c21B8, the minimal COOHterminal agrin fragment required for AChR aggregation
(Gesemann et al., 1995
), and 20 nM of cN257Fc was included. After 16 h, AChR clusters were visualized with
rhodamine-
-bungarotoxin and myotube-bound cN257Fc
was stained with biotinylated goat anti-mouse IgG followed by fluorescein-conjugated streptavidin. As shown in
Fig. 8 (first row), cN257Fc localized to the agrin-induced
AChR clusters. cN257Fc also displayed a more widespread
distribution, often along the edge of the myotubes (Fig. 8,
arrowhead). In myotubes, where AChR aggregation had
been induced with c21B8, but no cN257Fc was included, no
specific staining was seen (Fig. 8, second row). These results illustrate that binding sites for the NH2-terminal fragment of agrin are concentrated in AChR clusters but are
also found on the remaining surface of the myotube.
Fig. 8.
Binding sites for agrin on cultured chick myotubes colocalize with AChRs and laminin. Cultured chick myotubes were induced to form AChR clusters with 200 nM c21B8. Simultaneously,
20 nM cN257Fc were included. AChR aggregates were stained by
rhodamine--bungarotoxin, and cN257Fc bound to the myotubes
was visualized with biotinylated goat anti-mouse IgG followed by
fluorescein-conjugated streptavidin. cN257Fc is concentrated in
AChR clusters and is distributed along the edges of the myotubes
(arrowhead). No staining is seen in the absence of this fragment
(
cN257Fc). Consistent with the idea that cN257Fc binds to laminin, the distribution of myotube-bound cN257Fc resembles the
staining pattern obtained with anti-
/
-specific antiserum 648 (
/
). The
2 chain of laminin, also called s-laminin, is concentrated
in AChR clusters. In light of the colocalization of cN257Fc and
AChR clusters, laminin-4 (
2,
2,
1) is a binding partner of
agrin in these clusters. Bar, 40 µm.
[View Larger Version of this Image (74K GIF file)]
and
chains of laminin-2 and -4, was used. Examination of
many myotubes showed that the distribution of myotubebound cN257Fc matched laminin-like immunoreactivity
(Fig. 8, third row). In addition, staining with the mAb
11B7, directed against the
1 chain of chick laminins (Brandenberger and Chiquet, 1995
), gave the same staining pattern (data not shown). Since these antibodies do not distinguish between laminin-2 and -4, we also applied mAb
C4, recognizing the
2 chain, to localize the synapse-specific laminin isoform on myotubes (Hunter et al., 1989
). In
Fig. 8 (fourth row), staining with the
2-specific mAb C4 is
shown. As reported for spontaneous AChR clusters on mouse C2 myotubes (Martin et al., 1995
), the
2 chain was
concentrated in AChR clusters on chick myotubes. Interestingly, no or only little laminin
2-like immunoreactivity
was detected outside of the clusters. This differs from the
pattern obtained with mAbs specific for the
1 or the
2
chain of laminin, where immunoreactivity was also seen
along the edge of myotubes (data not shown). In summary, these experiments demonstrate that the binding
sites for agrin coincide well with the distribution of laminin on cultured myotubes, and they suggest that laminin-4
mediates binding of agrin to AChR clusters.
N15AgrinA4B8 lacking the
first 130 NH2-terminal amino acids (Denzer et al., 1995
).
Aggregation of AChRs induced by agrin is mainly based
on the lateral migration of diffusely distributed molecules
(Godfrey et al., 1984
). We have speculated that the binding of cAgrin7A4B8 to ECM influences this process by immobilizing agrin in the ECM. If this were so, inhibition of the binding of cAgrin7A4B8 to muscle laminins should
result in AChR clusters with the same size as AChR clusters induced by c
N15AgrinA4B8. Myotubes incubated
with cAgrin7A4B8 had considerably smaller AChR aggregates than those incubated with c
N15AgrinA4B8 (Fig. 9 a).
When available laminin-binding sites for full-length agrin
were blocked by 500 nM cN257Fc, cAgrin7A4B8-induced
AChR clusters became indistinguishable from those induced by c
N15AgrinA4B8 (Fig. 9 a). In contrast to this,
500 nM of cN257Fc did not alter the shape of AChR aggregates induced by c
N15AgrinA4B8. Quantification of this
effect with a computerized image analysis system confirmed that the presence of cN257Fc increased the average
size of AChR aggregates induced by cAgrin7A4B8 approximately twofold, while the average size of c
N15AgrinA4B8induced AChR clusters was not affected (Fig. 9 b). These
experiments provide evidence that the smaller size of the
AChR clusters induced by full-length agrin is mainly
based on its binding to laminin isoforms expressed by the
myotubes.
Fig. 9.
The NH2-terminal, laminin-binding domain of agrin
causes AChR clusters to be small. (a) Fluorescence micrographs of
cultured chick myotubes labeled with rhodamine--bungarotoxin.
Myotubes were grown for 12 h in the presence of conditioned
medium of transiently transfected COS cells containing either
100 pM cAgrin7A4B8 or c
N15AgrinA4B8. AChR clusters induced
by cAgrin7A4B8 are considerably smaller than those induced by
c
N15AgrinA4B8. In the presence of cN257Fc, the size of
cAgrin7A4B8-induced AChR clusters increases. (b) Quantification
of the effect on the size of the AChR aggregates. The presence of
500 nM cN257Fc (+ cN257Fc) increases the size of the AChR
clusters induced by cAgrin7A4B8 twofold and makes them indistinguishable from AChR clusters induced by c
N15AgrinA4B8. In
contrast, addition of 500 nM cN257Fc does not alter the size of
c
N15AgrinA4B8-induced AChR aggregates. The result (mean ± SEM) of one representative experiment is shown where the size of AChR clusters in 20 myotube segments was determined. Bar,
50 µm.
[View Larger Versions of these Images (55 + 36K GIF file)]
Discussion
). In the current study, we investigated
the molecular basis of agrin's binding to basement membrane. We found that agrin binds with high affinity to
laminin isoforms, including those that are concentrated at
the NMJ, and we have mapped the main laminin-binding
site to the NH2-terminal end of agrin. Agrin has been shown
to be a key regulator of synaptic differentiation at the NMJ
(McMahan, 1990
; Gautam et al., 1996
), and thus we will mainly discuss the significance of the binding of agrin to
laminin in this process.
). We now find that laminin-1,
one component of MatrigelTM, serves as a binding partner
for chick agrin. The binding of recombinant cAgrin7 (Fig.
1 a) to laminin-1 is of high affinity (EC50 of ~5 nM; Fig. 4)
and is mainly carried by the NH2-terminal end of agrin as
an excess of the NH2-terminal fragment, cN257Fc, competes the binding of full-length agrin by more than 90%.
; Pearson and Lipman, 1988
), we propose to call
this novel module NtA domain (for NH2-terminal domain
in agrin; see also Fig. 1 a).
). The simplest explanation for this local action of
neural agrin is that it becomes trapped in the extracellular
matrix near its release site, the tip of the motor neuron's
axon. Although we cannot exclude that other molecules
are also involved in this process, we propose that the local
immobilization of neural agrin is mainly mediated by its
binding to laminin isoforms expressed by muscle fibers. Consistent with this view, the fragment cN257Fc binds to
purified laminin-2 and -4 (Fig. 7), the two main laminin
isoforms expressed in developing muscle fibers (Chiu and
Sanes, 1984
; Sanes et al., 1990
). In addition, agrin-like protein copurifies with laminin isoforms (Fig. 7 a), and
cN257Fc colocalizes with laminin and AChR clusters on
cultured myotubes (Fig. 8). Similarly, laminin-like immunoreactivity colocalizes with agrin-like protein in chick embryo hindlimb muscle in vivo and in vitro, and both
proteins are enriched in AChR clusters (Godfrey et al.,
1988b
; Nitkin and Rothschild, 1990
). The binding of neural
agrin to laminin would also explain earlier observations
that motor neuron-derived agrin associates with the earliest AChR clusters in frog nerve-muscle cocultures (Cohen
and Godfrey, 1992
) and that it is deposited onto culture
substrates containing laminin (Cohen et al., 1994
).
; Fig. 9). On cultured chick myotubes, the size of
c
N15AgrinA4B8-induced AChR clusters is indistinguishable from the size of the clusters induced by c21B8, the
minimal fragment required for AChR aggregation that
does not bind to
-dystroglycan (see Figs. 8 and 9; Gesemann et al., 1996
). Hence, unlike the binding of agrin to
-dystroglycan, the NtA domain has a clear effect on the
size of the AChR clusters. One explanation for this phenomenon may be that agrin becomes immobilized on the
muscle cell surface by its binding to laminin and thereby
prevents small AChR clusters from fusing (see also Discussion in Denzer et al., 1995
).
; Reist et al.,
1987
). At least in vitro, the tight association of agrin with
muscle cell basal lamina requires the NtA domain; cultured chick myotubes that are incubated for only 30 min
with AChR-aggregating full-length agrin (cAgrin7A4B8), subsequently washed and further incubated for 15 h in
agrin-free culture medium, show agrin-induced AChR aggregates. In contrast to this, no AChR clusters are induced
with the same paradigm using c
N15AgrinA4B8 or any
other active, COOH-terminal agrin fragment (Denzer, A.J.,
and M.A. Ruegg, unpublished data). Our observation that
c
N15AgrinA4B8 is not capable of inducing AChR clusters
by short-term incubation is similar to results of Wallace
(1988), who showed that maintenance of AChR clusters in
vitro needs the continuous presence of agrin. Since his
agrin preparation mainly contained proteolytic fragments
of the COOH-terminal half of agrin (Nitkin et al., 1987
),
these fragments also lack the NtA domain.
1-containing
laminin isoforms are expressed throughout the muscle cell
basal lamina (Chiu and Sanes, 1984
; Schuler and Sorokin,
1995
). At later stages of synaptogenesis, the
2 chain of
laminin (s-laminin) accumulates in synaptic basal lamina,
and the
1 chain is displaced from this region and continues to be expressed extrasynaptically (Chiu and Sanes,
1984
; Hunter et al., 1989
; Sanes et al., 1990
). In contrast to
the
chains, the
2 and
1 chains are found throughout
the muscle fiber basal lamina (Sanes et al., 1990
). These
results strongly suggest that laminin-2 (
2,
1,
1) and
laminin-4 (
2,
2,
1) are the laminin isoforms in the extrasynaptic and synaptic basal laminae, respectively. We
find that, at one particular concentration, laminin-4 binds
more strongly to agrin than laminin-2 (Fig. 7). Hence,
agrin may preferentially bind to laminin-4, and this may be
the basis of the tight association of neural agrin with synaptic basal lamina throughout adulthood.
2 chain of laminin may, at least partially, be based on alterations of agrin. In these mice, NMJs are formed on
schedule, but maturation of pre- and postsynaptic specializations is impaired, and terminal Schwann cells penetrate
partially the synaptic cleft (Noakes et al., 1995
). Although
the loss of the
2 chain is compensated by continued expression of the
1 chain at the NMJ (Martin et al., 1996
),
this may not be sufficient to tether agrin to the NMJ. Since
agrin has been shown to influence the formation of presynaptic specializations (Campagna et al., 1995
; Gautam
et al., 1996
), alterations in the binding of agrin to the NMJ
may also explain the abnormalities in presynaptic specialization in the
2-deficient mice.
; Tsen et al.,
1995b
). Interestingly, the cDNA clones encoding mouse
and human agrin, which are derived from nonneuronal tissue, all encode the splice variant lacking the insert (Fig. 6).
Preliminary results show that both splice variants bind to laminin-1 (Denzer, A.J., and M.A. Ruegg, unpublished
observation). Since there appears to be a difference in the
binding of the two splice variants to MatrigelTM (Denzer et
al., 1995
), it will be interesting to see whether splicing at
this site influences binding of agrin to different laminin
isoforms.
-dystroglycan than AChRaggregating isoforms and since they also bind to laminin,
the inactive isoforms might have a structural role to link
the basement membrane with the underlying cytoskeleton
via the dystrophin-glycoprotein complex (for reviews see
Campbell, 1995
; Worton, 1995
). We have now generated the tools that will enable studies on the physiological significance of the binding of agrin to laminin isoforms and to
the different binding proteins expressed on the muscle cell
membrane, like
-dystroglycan.
Received for publication 15 October 1996 and in revised form 28 January 1997.
Address all correspondence to Markus A. Ruegg, Department of Pharmacology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland. Tel.: 41 61 267 2246 or 2213. Fax: 41 61 267 2208. E-mail: rueegg{at}ubaclu.unibas.chWe thank Beat Schumacher for excellent technical assistance and Angelo Marangi for determining the sequence of the mouse cDNAs. We thank Drs. J.L. Bixby (Miami University School of Medicine, Miami, FL) for providing the construct encoding the Fc part of mouse IgG and P. Sonderegger (University of Zurich, Switzerland) for the vitreous fluid. We are indebted to Dr. A. Brancaccio, Dr. K. Kühn, T. Schulthess, and Dr. R. Timpl for providing us with purified proteins. In addition, we are grateful to Dr. T. Meier for his comments on the manuscript.
This work was supported by grants to M.A. Ruegg and M. Chiquet from the Swiss National Science Foundation, and grants to M.A. Ruegg from the Swiss Foundation for Research on Muscle Diseases and the Rentenanstalt/Swiss Life.
AChE, acetylcholinesterase; AChR, acetylcholine receptor; ECM, extracellular marix; HS-GAG, heparan sulfate glycosaminoglycan; HSPG, heparan sulfate proteoglycan; NMJ, neuromuscular junction; NtA, NH2-terminal domain in agrin; VF, vitreous fluid.