* Department of Physiology, University of Basel, Vesalgasse 1, CH-4051 Basel, Switzerland; Department of Research, Stiftung
Tumorbank Basel, University Women's Clinic, CH-4031 Basel, Switzerland; and § Department of Pharmacology, Biozentrum,
University of Basel, CH-4056 Basel, Switzerland
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Abstract |
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The neural isoforms of agrin can stimulate
transcription of the acetylcholine receptor (AChR) subunit gene in electrically active muscle fibers, as does
the motor neuron upon the formation of a neuromuscular junction. It is not clear, however, whether this induction involves neuregulins (NRGs), which stimulate
AChR subunit gene transcription in vitro by activating
ErbB receptors. In this study, we show that agrin-
induced induction of AChR
subunit gene transcription is inhibited in cultured myotubes overexpressing an inactive mutant of the ErbB2 receptor, demonstrating involvement of the NRG/ErbB pathway in agrin-
induced AChR expression. Furthermore, salt extracts
from the surface of cultured myotubes induce tyrosine phosphorylation of ErbB2 receptors, indicating that
muscle cells express biological NRG-like activity on
their surface. We further demonstrate by RT-PCR
analysis that muscle NRGs have Ig-like domains required for their immobilization at heparan sulfate proteoglycans (HSPGs) of the extracellular matrix. In extrasynaptic regions of innervated muscle fibers in vivo,
ectopically expressed neural agrin induces the colocalized accumulation of AChRs, muscle-derived NRGs,
and HSPGs. By using overlay and radioligand-binding
assays we show that the Ig domain of NRGs bind to the
HSPGs agrin and perlecan. These findings show that
neural agrin can induce AChR subunit gene transcription by aggregating muscle HSPGs on the muscle fiber
surface that then serve as a local sink for focal binding
of muscle-derived NRGs to regulate AChR gene expression at the neuromuscular junction.
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Introduction |
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THE high density accumulation of acetylcholine receptor (AChR)1 channels at the neuromuscular
junction (NMJ), required for impulse transmission
across the synapse, is the result of transcriptional activation of AChR subunit genes in the subsynaptic muscle nuclei (Brenner et al., 1990; Sanes et al., 1991
) and of the insertion of their gene products, the AChR channels, in the
synaptic muscle membrane (for review see Sanes, 1997
).
The AChRs are stabilized in the subsynaptic membrane
by anchoring to the cytoskeleton via an elaborate subsynaptic apparatus of highly specialized molecular composition (Fallon and Hall, 1994
; Apel and Merlie, 1995
; Carbonetto and Lindenbaum, 1995
). Both the transcription of
AChR genes and the differentiation of the subsynaptic apparatus are under the control of molecules originating
from the motor neuron and associated with the synaptic
portion of the muscle fiber's basal lamina (BL) (McMahan, 1990
; Brenner et al., 1992
; Jo and Burden, 1992
).
The neural signal proposed to activate AChR gene transcription in muscle is acetylcholine receptor-inducing activity (ARIA; Martinou et al., 1991; Corfas et al., 1993
;
Chu et al., 1995
; Fischbach and Rosen, 1997
), a member of
the neuregulin (NRG) family of growth and differentiation factors (Falls et al., 1993
) arising in several isoforms
from a single gene, nrg-1, by alternative mRNA splicing.
ARIA/NRG precursors are expressed in motor neurons (Falls et al., 1993
) as transmembrane glycoproteins and
are cleaved near their transmembrane domains for release
(for reviews see Lemke, 1996
; Fischbach and Rosen,
1997
). The mature form of ARIA/NRG is characterized by
an Ig-like domain that binds heparin (Falls et al., 1993
) and
by a conserved EGF-like domain sufficient to activate receptors of the ErbB family of receptor tyrosine kinases. Neuregulin binding to ErbB receptor heterodimers induces
their tyrosine phosphorylation and activates AChR subunit
gene transcription in cultured myotubes (Si et al., 1996
;
Tansey et al., 1996
; Altiok et al., 1997
).
The neural signal controlling nerve-dependent aggregation of AChR channels in the subsynaptic muscle membrane is agrin (McMahan, 1990), a multidomain heparan
sulfate proteoglycan (HSPG) with binding affinities for
-dystroglycan, laminin, and heparin (for review see Denzer et al., 1996
). Unlike motor neurons, muscle fibers do
not synthesize agrin isoforms active in AChR aggregation (Ferns et al., 1992
; Ruegg et al., 1992
; Hoch et al., 1993
;
Gesemann et al., 1995
). Agrin is associated with the synaptic BL of the NMJ (Reist et al., 1987
) presumably by its
binding to laminin (Denzer et al., 1997
, 1998
). Neuregulins
are also bound to synaptic BL (Goodearl et al., 1995
; Jo
et al., 1995
; Sandrock et al., 1995
), but the mechanism of
this immobilization is not known.
Recent experiments demonstrate, however, that active
agrin not only causes the redistribution of cell surface
AChRs in cultured myotubes and in vivo but that agrin
also induces expression of the AChR subunit gene in the
absence of nerve-derived NRGs; the expression is resistant to muscle activity as it is at normal synapses (Jones et
al., 1996
, 1997
). In primary myotube cultures, AChR gene
transcription induced by agrin depends on its binding to
the culture substrate, but conspicuously, does not depend
on its AChR aggregating activity (Jones et al., 1996
).
These findings led us to propose that agrin rather than
NRGs may be the key neural factor regulating subsynaptic
AChR gene transcription in the muscle fiber. If NRGs are
involved in this process they may be derived from the muscle fibers, under the local control of matrix-bound agrin
(Jones et al., 1996
, 1997
). Consistent with this hypothesis, motor neuron-specific agrin isoforms alone, upon expression in extrasynaptic regions of innervated muscle fibers,
induce ectopic accumulation of the NRG receptors, ErbB2
and ErbB3 (Rimer, M., I. Cohen, T. Lømo, S.J. Burden,
and U.J. McMahan, 1996. Soc. Neurosci. Abstr. 1689; Meier
et al., 1997
). Furthermore, muscle cells express transcripts
encoding ARIA/NRG isoforms (Moscoso et al., 1995
; Ng
et al., 1997
). However, it is not known whether muscle
cell-derived NRGs are biologically active.
In this paper, we have tested the hypothesis that muscle
cells are a source of functional ARIA/NRG-like biological
activity that could be locally concentrated at the muscle
surface by agrin to activate AChR subunit gene transcription. We found all elements required for such a process:
(a) in cultured myotubes, transcription of AChR subunit
gene induced by substrate-bound agrin is inhibited when
myotubes overexpress an inactive human mutant of ErbB2,
HER2KM, as is transcription induced by NRG; (b) NRG
transcripts are expressed in innervated muscle and NRG-like biological activity can be extracted from cultured muscle cells; (c) NRG-like immunoreactivity colocalizes with
aggregates of newly synthesized AChRs induced in nerve-free segments of innervated muscle fibers by ectopic neural agrin; (d) as proposed by Loeb and Fischbach (1995),
the Ig-like domains of muscle derived NRGs mediate
direct interaction with glycosaminoglycan (GAG) side
chains of HSPGs. Indeed, the HSPGs agrin and perlecan, but not other glycoproteins of the postsynaptic apparatus
bind NRGs in overlay and radioligand-binding assays; (e)
in addition to agrin, other HSPGs are accumulated in the
muscle BL by agrin, thus providing additional binding sites
for the localization of NRGs. Taken together, these findings support the hypothesis that agrin regulates synapse-specific AChR gene expression by localizing muscle derived NRGs and activating ErbB receptors.
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Materials and Methods |
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The term neuregulin (NRG) is used in this paper whenever we wish to address transcripts or proteins encoded by the nrg-1 gene (Fischbach and
Rosen, 1997) irrespective of species or isoform. Human, rat, and chick
NRG isoforms are referred to as heregulin(s) (HRGs), Neu differentiation
factor (NDF), or ARIA, respectively (Lemke, 1996
; Fischbach and Rosen,
1997
). Products of the nrg-2 gene were not considered as they appear not
to be expressed in muscle (Carraway et al., 1997
; Chang et al., 1997
).
AChR Subunit Transcription in C2C12 Cells
Overexpressing HER2 or HER2KM
Recombinant full-length neural cAgrin7A4B8 was immobilized on 35-mm
culture dishes by precoating dishes with 65 µl of 20 µg/ml laminin from
EHS tumor (Sigma Chemical Co., St. Louis, MO) followed by spreading
60 µl of 5 µg/ml Agrin7A4B8 diluted in PBS, and then incubation overnight
at 4°C. After rinsing, 120,000 C2C12 cells were added to each well and co-transfected with 3 µg of the AChR -promoter luciferase reporter construct pLCF216
(Jones et al., 1996
), 250 ng of pCH110 (Pharmacia Biotechnology Inc., Piscataway, NJ) encoding
-galactosidase, and 250 ng of
either pHER2/neu or pHER2KM (Wallasch et al., 1995
) using the calcium
phosphate method (Chen and Okayama, 1987
) in serum-free medium
(Jones et al., 1996
). To exclude unspecific effects on gene regulation after
HER2 and HER2KM transfection, we analyzed muscle creatine kinase
(MCK) transcription using a 6.5-kbp MCK promoter fragment from
pUC118MCK6.5 subcloned into pGL2 (Promega, Heidelberg, Germany).
Cells were analyzed 72 h later for luciferase activity using a Luminometer (Turner Designs, Sunnyvale, CA) and relative luciferase units were normalized to
-galactosidase expression. To test for transfection efficiency
and protein expression, COS cells were transfected accordingly, proteins
extracted in SDS sample buffer and analyzed on Western blots for the expression and tyrosine phosphorylation of HER2 using the antibodies described below.
Reverse Transcriptase-PCR
Extrasynaptic segments of innervated adult rat diaphragm were dissected
and polyadenylated mRNA was isolated according to the method of Hengerer (1993). First strand cDNA was synthesized using Superscript reverse
transcriptase (Life Technologies, Inc., Gaithersburg, MD) according to
the manufacturer's instructions with 50 ng of mRNA primed with 2 pmoles of the 5'-GCAGTAGGCCACCACACACATGATGCC-3' oligonucleotide (TM-antisense) (Microsynth, Balgach, Switzerland), which
corresponds to the transmembrane (TM) region of rat NDF (Wen et al.,
1992
). The first-strand cDNA was then extracted with phenol/chloroform,
precipitated, and then resuspended in 20 µl of water. To amplify an NDF
transcript spanning the TM and Ig domains, 10 µl was amplified in a volume of 50 µl with an air thermal cycler (Idaho Technology, Idaho Falls,
ID) using a cycle protocol of 94°C for 15 s, 58°C for 15 s, and 72°C for 35 s
for 35 cycles and the TM-antisense primer together with an Ig-sense primer 5'-CTCTGGAGAGTATATGTGCAAAGTGATCAGC-3' that
recognizes the 3' part of the NDF Ig-domain (Wen et al., 1992
). To examine whether the resulting Ig/TM product included
or
splice isoforms
and to examine the spacer insert between the Ig and EGF domains (for review see Lemke, 1996
), the reaction mix was diluted 1:100 and 1 µl was
amplified in a 10 µl volume using 35 cycles of 94°C for 1 s, 58°C for 1 s,
72°C for 10 s. Primer combinations were as follows: to examine the spacer
insert we used the Ig-sense primer together with the EGF-antisense (5'-CCGTGAAGCACTCGCCCCCATTCACACAG-3') primer. To detect
- and
-specific isoforms, either a
-specific primer (5'-CCAAAACTACGTAATGGCCAGCTT-3') or an
-specific primer (5'-TGTACCCATGAAAGTCCAAACCCA-3') was used in combination with the
TM-antisense primer. PCR products were resolved on a 2% agarose gel
and transferred to nylon membrane (Boehringer Mannheim GmbH, Mannheim, Germany), which was then hybridized overnight at 42°C with
a PCR-generated NDF probe labeled with Digoxygenin-11-dUTP (Boehringer Mannheim GmbH) and spanning the Ig-TM domains, ensuring recognition of all NDF isoforms. Amplified NDF DNA was visualized using
alkaline phosphatase-conjugated anti-DIG antibody and the chemiluminescence substrate CDP-StarTM (Boehringer Mannheim GmbH) according
to the manufacturer's instructions.
Agrin and Heregulin
Chick full-length agrin, cAgrin7A4B8, and cAgrin7A0B0 (Denzer et al., 1995),
as well as the COOH-terminal agrin fragment c95A0B0 (Gesemann et al.,
1995
), were purified from conditioned medium of stably transfected HEK
293 cells as described elsewhere (Gesemann et al., 1995
; Denzer et al., 1997
).
HRG1(1-246) cDNA encoding a full-length extracellular domain of the
human orthologue of chicken ARIA (Falls et al., 1993
; Jeschke et al.,
1995
) was subcloned into the bacterial expression vector pFLAG-1 (International Biotechnologies Inc., Cambridge, UK), and then used for transformation of Escherichia coli XL-1 blue. Expression of recombinant protein was induced with 1 mM isopropylthio-
-D-galactoside (IPTG),
homogenate was enriched for inclusion bodies, and then extracted with
6 M urea followed by extensive dialysis against PBS and purification with
anti-FLAG M2 affinity gel (International Biotechnologies Inc., New Haven, CT). Recombinant HRG
1(177-246) DNA containing a His tag and
FLAG epitope (Jeschke et al., 1995
) was expressed in bacteria and enriched from periplasmic extract on a His-Trap affinity column (Pharmacia
Biotechnology Inc.) according to standard protocols. Cloning of the novel
HRG-isoform HRG
and the isolated Ig domain, HRG
BbsI, will be described in detail in a separate paper (see Results under Binding of Neuregulin to Agrin Isoforms). Briefly, HRG
derived from a cDNA library obtained from MDA-MB-231 cells was cloned into the bacterial expression
vector pQE30 (Qiagen, Hilden, Germany) resulting in pQE30/HRG
.
The coding sequence of HRG
was subcloned into pEGFP to provide
pEGFP/HRG
, from which pEGFP/HRG
BbsI was constructed by linearization with BbsI, fill-in with T4 DNA polymerase, and subsequent digestion with SmaI and religation. Finally, plasmid pQE30/HRG
BbsI was
obtained by replacing the SpeI/HincII fragment of pQE30/HRG
with the
corresponding restriction fragment from plasmid pEGFP/HRG
BbsI.
Bacteria (E. coli XL1 blue) transformed with pQE30/HRG
or pQE30/
HRG
BbsI were induced with 0.4 mM IPTG for 5 h, inclusion bodies
were solubilized in 6 M urea and purified over a cation exchange column
(SP Sepharose Fast Flow; Pharmacia Biotech Sevrage, Uppsala, Sweden)
using HPLC equipment. The resulting HRG
(fraction A) was dialyzed
against 50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20, and used for
most overlay assays and for radiolabeling. For some experiments, this enriched HRG
/fraction A preparation was further purified by Ni+-NTA
(Qiagen) affinity chromatography (HRG
, fraction C). HRG
BbsI was
purified on His-Trap affinity column in the presence of 6 M urea and
stored at 4°C. As a control for nonspecific binding resulting from the presence of a His tag we subcloned the BamHI-HindIII insert of pQE16
(Qiagen) encoding dihydrofolate reductase (DHFR) cDNA ligated to a
6× His tag at its 3' end into the BamHI-HindIII cut plasmid pQE30. This
provided the control protein DHFR flanked with COOH- and NH2-terminal His tags (His-DHFR-His). The NH2-terminal His tag (Arg-Gly-Ser-
[His]4) was recognized by the RGS-His antibody (Qiagen). His-DHFR-His was extracted from bacterial inclusion bodies with 6 M urea and used
without further purification.
Extraction and purification of recombinant proteins was monitored by
SDS-PAGE and Coomassie brilliant blue staining. Protein concentrations
of HRG and His-DHFR-His were determined by comparing absorptions
of Coomassie-stained protein bands with calibration curves obtained with
BSA as standard. His- and FLAG-tagged heregulins were analyzed further on immunoblots under standard conditions using anti-His antibodies
(Qiagen) and anti-FLAG M1 antibody (International Biotechnologies
Inc.) for the detection of HRG
1(177-246) or anti-FLAG M2 antibody for
the detection of HRG
1(1-246) combined with peroxidase-conjugated anti-
mouse antibodies (Jackson ImmunoResearch Laboratories, Inc., West
Grove, PA) and chemiluminescence detection using Super SignalTM substrate (Pierce Chemical Co., Rockford, IL). Relative concentrations of
HRG
1(1-246), HRG
1(177-246), and HRG
BbsI were adjusted to obtain
comparable signals on immunoblots. Fig. 1 shows the heregulin isoforms
used in this study and their detection by anti-His or anti-FLAG antibodies.
|
Extraction of NRG-like Biological Activity from Myotubes and ErbB2 Immunoprecipitation
Extracts from extracellular matrix (ECM) of chick brain and spinal cord
cells have been shown previously to contain NRG (Loeb and Fischbach,
1995). Primary rat myotubes cultured according to Brenner et al. (1992)
for 6 d and C2C12 myotubes cultured for 5-6 d in differentiation medium
were used to test muscle cells for their content of NRG-like biological activity. After washing in ice cold PBS, three to four 30-mm plates were extracted sequentially with 1 ml salt extraction solution (PBS supplemented
with 1 mM MgCl2, 2.5 mM CaCl2, and NaCl to 1.0 or 1.5 M) by gentle agitation on ice. After centrifugation at 4°C, salt extracts were dialyzed against 0.5× PBS for several hours and then dialyzed against serum-free DME overnight at 4°C. To monitor NRG-like biological activity, fresh
cultures of 6-d-old primary rat myotubes or C2C12 cells differentiated in
30-mm plates were stimulated with 1 ml of this dialyzed, conditioned salt
extract for 8 min at 37°C. For comparison, cultures were either incubated
with dialysis medium (DME) as unstimulated control or with dialysis medium supplemented with saturating amounts of recombinant HRG
1(177-246)
(stimulated control). After washing in ice-cold PBS, membrane proteins
were extracted with 1 ml detergent extraction buffer (50 mM Tris-HCl,
pH 7.5, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 50 mM NaF, 10 mM
sodium-molybdate, 1 mM sodium-orthovanadate, 1% (vol/vol) Triton X-100,
5 µg/ml leupeptin, 5 µg/ml aprotinin, and 1 mM PMSF) and extracts
cleared from cell debris by centrifugation at 4°C. ErbB2 was specifically immunoprecipitated with 0.1 µg/ml anti-ErbB2 antibody (C18; Santa Cruz
Biotechnology Inc., Santa Cruz, CA) and 10 µl protein A-Sepharose beads for 2-3 h at 4°C. Control experiments showed that this antibody does not cross-react with either ErbB3 or ErbB4 in such detergent- extracts (not shown). After washing three times with 1 ml detergent extraction buffer and once with 1 ml PBS, excess liquid was removed, and
then beads were boiled in 20 µl SDS sample buffer. Samples were separated on 6% SDS-PAGE and transferred to nitrocellulose. In our gel system ErbB2 protein migrated as an ~200-kD band; this was confirmed by
immunoblotting with anti-ErbB2 antibodies. For detection of tyrosine
phosphorylation, blots were preincubated with 3% BSA in TTBS (25 mM
Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% [vol/vol] Tween-20), incubated
with peroxidase-conjugated anti-phosphotyrosine antibody 4G10 (1:3,000;
Upstate Biotechnology Inc., Lake Placid, NY) followed by chemiluminescence detection. Densitometric analyses of Western blots were carried out
using ImageQuant software and data from different experiments were
combined by normalizing the results of each experiment to saturating
HRG-stimulated ErbB2 phosphorylation.
Immunoblots and Transfer Blot Overlay Assays
Binding of heregulin was assayed for the chick agrin constructs, cAgrin7A0B0
or cAgrin7A4B8. Proteins were separated by SDS-PAGE on 3-12% gradient
gels under reducing conditions and transferred to nitrocellulose (Towbin
et al., 1979). Routinely we loaded 100 ng/lane of agrin for immunoblots
and up to 250 ng/lane of agrin for transfer blot overlay assays. To test
whether agrin proteins were efficiently transferred, blots were incubated
with anti-agrin antibodies (No. 3240) raised against the c95 fragment of
agrin (Gesemann et al., 1995
), followed by peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) and chemiluminescence detection (Pierce Chemical Co.). Transfer blot overlay assays were carried out essentially as described (Yamada et al., 1996
). Briefly,
unspecific binding sites were blocked with 10 mM triethanolamine, pH
7.6, 140 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 0.01% Tween 20 (TLBB) supplemented with 5% (wt/vol) nonfat milk powder (MTLBB) for 45 min
before they were overlaid with heregulin protein diluted in MTLBB for
1.5 h. In some experiments, MTLBB was supplemented with 10 mM
-mercaptoethanol during incubation with HRG. For detection of His-tagged heregulin proteins, blots were washed three times for 10 min, incubated with anti-His antibody (Qiagen) diluted (1:3,000) in MTLBB for 60 min. After three 10-min washes in TLBB buffer, bound proteins were detected with peroxidase-conjugated goat anti-mouse antibody (1:3,000 in
MTLBB; Jackson ImmunoResearch Laboratories) followed by chemiluminescence. For detection of bound FLAG-tagged heregulins, anti-FLAG M1
(detection of HRG
1[177-246]) and anti-FLAG M2 (detection of HRG
1[1-246])
primary antibodies were used. For the removal of heparan sulfate glycosaminoglycan side chains, cAgrin7A4B8 and cAgrin7A0B0 at concentrations
of 30 µg/ml were diluted threefold with 15 mM Tris-HCl, pH 7.5, 75 mM
sodium acetate, pH 7.5, 1 mM CaCl2, and reaction volumes of 30-35 µl
were incubated with 0.5 U of heparitinase (Heparinase III, No. H8891;
Sigma Chemical Co., St. Louis, MO) for 8 h at 25°C. Reaction was stopped
by the addition of SDS sample buffer and deglycosylated proteins were
used for overlay assays as described. Untreated controls were incubated in
the same buffer but without enzyme added. To test for the influence of
Ca2+ on the binding of HRG to agrin, some overlay assays were performed in Ca2+/Mg2+-free TLBB and MTLBB solutions supplemented
with 10 mM EDTA.
Solid Phase Radioligand-binding Assay
HRG (fraction A; 130 µl of a 1.4 mg/ml protein solution) was iodinated
using the chloramine T method (Hunter and Greenwood, 1962
) according
to Gesemann et al. (1996)
. Samples of 125I-HRG
were separated on SDS-PAGE and analyzed by a PhosphorImager (Molecular Dynamics, Inc.,
Sunnyvale, CA). Microtiter plates (Becton Dickinson Co., Mountain
View, CA) were coated with 1 µg/ml Agrin7A0B0 and Agrin7A4B8 or 10 µg/ml
of c95A0B0 (Gesemann et al., 1995
), laminin-1 prepared from mouse Engelbreth-Holm-Swarm sarcoma (Timpl et al., 1979
), tenascin (Chiquet et al.,
1991
), fibronectin (purified from conditioned fibroblast medium; Chiquet
et al., 1991
), or perlecan (for review see Timpl, 1993
; Timpl and Brown,
1995
) diluted in 50 mM sodium bicarbonate, pH 9.6, overnight at 4°C.
-Dystroglycan prepared from chicken lung (Gesemann et al., 1997
) and
MatrigelTM (Kleinman et al., 1982
), were each diluted 1:10 for coating. After coating, wells were blocked with TBS containing 3% BSA, 1.25 mM CaCl2, 1 mM MgCl2 (blocking solution) and incubated for 3 h with 125I-HRG
diluted 1:200 in blocking solution (final concentration of ~40 nM).
After washing with TBS, 1.25 mM CaCl2, and 1 mM MgCl2 four times,
bound radioactivity in each well was counted with a gamma counter.
Heparan sulfate glycosaminoglycan side chains were removed before
the incubation with 125I-HRG by treating cAgrin7A0B0-coated wells with
0.5 U heparitinase in heparitinase buffer (see above).
Ectopic Expression of Agrin and Immunocytochemistry
Full-length cAgrin7A4B8 or the agrin fragment cN257C95A4B8, a fusion construct of the NH2-terminal laminin-binding domain of agrin (cN257) with
the c95A4B8 fragment (Gesemann et al., 1995; Denzer et al., 1997
; construct
provided by D. Hauser and M.A. Ruegg, Biozentrum, Basel, Switzerland), was expressed at extrasynaptic regions of innervated rat soleus
muscle by direct injection of expression plasmid as described previously
(Jones et al., 1997
). 4-8 wk after injection, muscle was excised, stained
with rhodamine-
-bungarotoxin (Molecular Probes, Inc., Eugene, OR),
and then processed as whole-mount preparations for anti-HSPG immunostaining or cut into 12-µm frozen sections and stained for the presence of
NRG aggregates at sites of ectopic AChR synthesis and accumulation.
For staining of HSPG accumulation at sites of ectopic agrin-induced
AChRs, dissected muscle was incubated in mAb C17 (Eldridge et al.,
1986
) diluted 1:100 in 5% horse serum, 1% BSA in PBS for 2 h, washed,
and then incubated with BODIPY goat anti-mouse (1:250; Jackson ImmunoResearch Laboratories) for 2 h. Whole-mount preparations were
viewed on a laser scanning microscope (Leica, Heerbrugg, Switzerland).
For detection of NRG we used the rabbit antiserum sc-348 (Santa Cruz Biotechnology Inc.) specific for membrane-associated NRG precursors with no cross-reactivity to mature forms of NRGs (Moscoso et al., 1995
).
We also generated a rabbit antiserum anti-HRG No. 76990 raised to bacterially expressed HRG
1(1-246). For this, rabbits were initially injected
with 50 µg of HRG
1(1-246) antigen expressed in bacteria, extracted from
inclusion bodies and dissolved in 2.4 ml Freund's complete adjuvant.
Boost injections were carried out with antigen mixed with Freund's incomplete adjuvant, and the IgG fraction from the rabbit serum was obtained by affinity chromatography using the Econo-Pac Serum IgG purification column (Bio-Rad Laboratories, Hercules, CA) according to the
manufacturer's instructions. For immunocytochemistry, cryosections were
blocked for 5 min with 5% horse serum, 1% BSA in PBS with 0.1% (vol/
vol) Triton X-100 and primary antibodies diluted 1:100 (anti-HRG[622-641]; sc-348) or 1:250 (anti-HRG
1[1-246]; No. 76990) in incubation buffer (5%
horse serum, 1% BSA, in PBS with 0.01% [vol/vol] Triton X-100) for 2 h.
After washing, sections were exposed to unconjugated goat anti-rabbit
IgG (1:250 in dilution buffer; Cappel, Cochranville, PA) for 2 h, washed,
and then incubated with FITC-conjugated rabbit anti-goat IgG (1:250 in
dilution buffer; Jackson ImmunoResearch Laboratories) for 2 h.
The concern has been raised (Cohen et al., 1997; Rimer et al., 1997
)
that an inflammatory response by the use of chicken agrin in rat muscle may
be involved in the induction of ectopic AChR clusters by ectopic agrin.
However, unlike the injections of chicken agrin plasmid into the muscle
bulk, which apparently causes inflammation (Cohen et al., 1997
; Rimer et
al., 1997
), the intracellular injection of much smaller amounts of agrin
plasmids into single muscle fibers as carried out in our present and previous experiments (Jones et al., 1997
) does not produce any sign of immunological response. Specifically, histological examination of soleus cross-sections in the region of agrin plasmid injection showed no signs of edema, muscle fiber dilatation, or influx of polymorphonuclear granulocytes or of
erythrocytes, as can be observed in acute antibody-mediated rejection;
nor was there influx of mononuclear cells such as lymphoblasts, lymphocytes, or macrophages, as can occur during a cellular rejection. Perhaps,
the immunological response upon plasmid injection to the muscle bulk was
elicited by uptake of the cDNA by other cells than muscle and their subsequent presentation of chicken agrin epitopes (Donnelly et al., 1997
).
![]() |
Results |
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Previous experiments demonstrated that agrin alone is
sufficient to initiate transcription of the AChR subunit
gene (Jones et al., 1996
, 1997
) and accumulation of adult
AChR channels (Jones et al., 1997
; Rimer et al., 1997
) in
muscle. This prompted the hypothesis that NRGs supplied
by the nerve terminal are not required to control AChR
gene expression. Instead, nerve-derived agrin might initiate AChR subunit gene transcription by recruitment of
muscle-derived NRGs and their localization to synaptic
BL (Jones et al., 1996
). The experiments described below
were designed to test this hypothesis.
Blockade of the NRG/ErbB Pathway Blocks Agrin-induced AChR Gene Transcription
If agrin indirectly increases AChR gene expression by recruiting muscle-derived NRG, thus activating the ErbB receptor signaling pathway, inhibition of ErbB receptor activation would be expected to abolish agrin-induced gene
transcription. We tested this prediction by overexpressing
an inactive HER2 receptor construct (HER2KM) in
C2C12 myotubes and monitoring the effect on agrin-induced
AChR subunit gene induction. HER2KM is a kinase inactive single point mutant of HER2, the human orthologue of rat ErbB2, and HER2KM is thought to form inactive heterodimers with HER3/ErbB3, thereby blocking
activation of the ErbB signaling pathway in the myotubes (Wallasch et al., 1995
).
An AChR subunit promoter luciferase reporter gene
construct (Jones et al., 1996
) was transiently transfected
into C2C12 myoblasts cultured on a laminin substrate impregnated with recombinant full length chick Agrin7A4B8.
After differentiation into myotubes, luciferase activity was
increased approximately fourfold above that in control
cells grown on laminin alone (see Fig. 2). A similar increase in luciferase activity was seen, when parallel cultures were treated with saturating concentrations of a fragment of recombinant heregulin
1 (HRG
1[177-246]), which
is in the range of induction previously reported by others
(Tansey et al., 1996
). In contrast, cotransfection of the
AChR
-promoter luciferase reporter construct with a
plasmid encoding inactive HER2KM significantly reduced the induction of luciferase activity, both that by substrate-bound agrin and that by HRG
1, to levels not different
from that in control cultures. HER2 and HER2KM acted
specifically on the activation of the AChR
subunit promoter, as no effects were observed on the levels of muscle
creatine kinase (MCK) expression.
|
The reduction in agrin- and HRG1-induced luciferase
activities by HER2KM was not a nonspecific effect arising
from overexpression of HER2KM. First, unlike HER2KM,
cotransfection of an expression plasmid encoding active,
wild-type HER2 did increase luciferase activity even in the
absence of agrin. This increase produced by HER2 alone
may be due to the formation of autophosphorylated HER2 homodimers, as transfection of HER2 alone into
COS cells produced
when compared with transfection
with HER2KM
a substantial level of phosphorylated
HER2, despite the absence of ligand (data not shown;
Wallasch et al., 1995
). Second, expression of the two plasmids was similar, as tested in COS cells by Western blotting HER2 and HER2KM protein levels (not shown).
Expression of Neuregulins Containing Ig and EGF Domains in Muscle
It has been proposed by Loeb and Fischbach (1995) that
immobilization of NRGs to the ECM might be via their Ig-like domains binding to HSPGs. For maximal induction of
AChR gene transcription by ErbB receptor activation, isoforms of NRGs are required (Fischbach and Rosen,
1997
). We therefore examined whether NRG
isoforms are expressed in innervated adult muscle. To determine
whether active NRGs containing the Ig and EGF domains
are expressed in adult skeletal muscle, we isolated mRNA
from the extrasynaptic region of innervated rat diaphragm. Using reverse transcriptase PCR (Fig. 3 A), NRG
transcripts spanning the Ig domain through to the transmembrane domain were amplified. The resulting products (Fig. 3 B, left panel) were then subjected to a second round
of PCR using primers which specifically amplified either
- or
-NRG isoforms or amplified the spacer insert between the immunoglobulin and EGF domain. In this way
we could amplify
-specific, but not
-specific isoforms that
are cotranscribed with the Ig domain (Fig. 3 B, right
panel). Further, the calculated molecular weight of the
isoform suggested it to be
1 (148 bp) or
2 (124 bp), but
not
4 (205 bp; Wen et al., 1994
). This is in agreement with experiments carried out in adult chicken muscle in which
the expression of
1-NRG is predominant but very little
-NRG is transcribed (Ng et al., 1997
). Three PCR products were amplified using the Ig-EGF primers, which
flanked the alternatively spliced spacer domain. Based
upon the expected molecular weight, the predominant spacer domain isoform in nonsynaptic region of rat diaphragm is the 34-amino acid (277 bp) variant, although
lower amounts of both the 17-amino acid (226 bp) and
0-amino acid (175 bp) isoforms were also amplified (Fig. 3
B, right panel).
|
Taken together, these results suggest that neuregulin transcripts in extrasynaptic regions of innervated adult muscle encode a protein containing functional domains essential for binding to HSPGs and activating the ErbB receptor tyrosine kinases.
Neuregulin-like Biological Activity Can Be Extracted from Cultured Myotubes
We next tested whether NRG-like biological activity is indeed secreted by muscle cells. For this, primary rat myotube cultures were extracted with high salt in physiological
buffer. This treatment did not induce obvious cell lysis as
determined by visual inspection at the end of the salt
buffer incubation, indicating that this mainly removed cell
surface- and ECM-immobilized components. Dialyzed extracts were subsequently applied onto fresh myotube cultures for ErbB receptor stimulation. Treatment of cultures
with the salt extracts stimulated tyrosine phosphorylation
of ErbB2 fourfold, whereas treatment with saturating, recombinant HRG1(177-246) produced an
20-fold increase
(Fig. 4, A and B). Since NRGs activate ErbB2/ErbB3 heterodimers at the NMJ (Altiok et al., 1995
; for review see
Fischbach and Rosen, 1997
), this suggests that the primary
cultures contained NRG-like activity. To exclude the possibility that the extracted NRG originated from fibroblasts
contaminating our primary cultures, we also examined salt
extracts of myotubes formed from the C2C12 cell line. As
with the primary cultures, phosphorylation of ErbB2 was
observed when such extracts were applied to fresh C2C12
myotubes (Fig. 4 C). From these experiments we conclude
that cultured myotubes express NRG-like biological activity as monitored by ErbB2 phosphorylation. However, it is not clear how these NRGs would become localized to the
synapse during NMJ formation.
|
Neural Agrin Induces Accumulation of NRG at Ectopic Postsynaptic Specializations
At normal neuromuscular synapses NRGs are accumulated (Jo et al., 1995), and ectopic expression of agrin in innervated muscle fibers induced accumulations of ErbB receptors (Meier et al., 1997
). This prompted us to test
whether agrin might induce the local accumulation of muscle-derived NRG. For this purpose, we injected innervated
rat soleus muscle in vivo with expression plasmids encoding
neural cAgrin7A4B8 (Jones et al., 1997
). Double labeling of
ectopic, agrin-induced AChR aggregates with rhodamine-
-bungarotoxin and with antibodies specific for NRGs revealed the accumulation of muscle-derived neuregulins at
sites of ectopic AChR aggregation (Fig. 5). Antisera raised
against either the extracellular or intracellular domains of
NRG resulted in similar staining patterns. This indicates
that membrane bound NRG precursors are accumulated at sites of AChR synthesis and aggregation in the nerve-free region of muscle fibers expressing agrin.
|
Binding of Neuregulin to Agrin Isoforms
Since NRGs bind to heparin (Falls et al., 1993) and agrin is
a HSPG (Denzer et al., 1995
; Tsen et al., 1995
), we examined whether recombinant NRG isoforms directly bound
to recombinant chick agrin. When tested in transfer blot
overlay assays, both HRG
1(1-246) and HRG
, a novel
NRG isoform (Schoumacher, F., H. Mueller, and U. Eppenberger, manuscript in preparation), bound to immobilized full-length neural cAgrin7A4B8, an ~400-600-kD glycoprotein (Fig. 6, A and B). These results indicate that
HRG isoforms bind directly to immobilized full-length
neural agrin and that this interaction does not require the
presence of the complete EGF-like domain of HRG.
|
Next, we examined whether HRG binds to Agrin7A0B0,
a muscle-specific full-length isoform that lacks AChR aggregating activity (Ferns et al., 1992
, 1993
; Gesemann et al.,
1995
), but induces AChR subunit gene expression in cultured myotubes (Jones et al., 1996
). In solid phase radioligand-binding assays (Fig. 6 C), as well as in transfer blot
overlay assays (Fig. 7 A) we detected binding of HRG
to
Agrin7A0B0, but only when Ca2+ was present (data not
shown). We then asked whether NRGs bind to other, non-agrin glycoprotein components of muscle synaptic membrane, such as
-dystroglycan, or of the BL, such as perlecan, tenascin, fibronectin, and laminin-1. In overlay assays,
HRG
did not bind to immobilized
-dystroglycan purified from chicken lung, nor to laminin-1, and nidogen separated under reducing conditions on SDS-PAGE (data
not shown). This was confirmed by solid phase radioligand-binding assays in which the proteins were coated in non-denatured and non-reduced configuration to microtiter wells (Fig. 6 C). Similar results were obtained for fibronectin, tenascin, and MatrigelTM. 125I-HRG
did, however, exhibit strong affinity for the muscle ECM component
perlecan (Timpl, 1993
). A possible explanation for the lack of binding to MatrigelTM, which contains perlecan,
might be the reduced accessibility of perlecan incorporated into the laminin and collagen matrix. Thus, HRG
exhibits only a weak nonspecific binding to BSA and to
non-HSPG proteins, suggesting that this low level of binding most likely reflects nonspecific interactions. Specific
binding was only seen for the HSPGs agrin and perlecan. Therefore, we asked whether their binding to NRGs might
be mediated by their negatively charged GAG side chains,
as proposed by Loeb and Fischbach (1995). This would be
consistent with the previous finding that neuregulins bind
heparin, a polymer of sulfated glycosaminoglycans (Falls
et al., 1993
).
|
The Ig-like Domain of Neuregulins Mediates Binding to the GAG Chains of Agrin
As expected for NRG binding to agrin and perlecan via
GAG chains, overlay, and radioligand-binding assays
showed that binding of HRG to immobilized full-length
agrin isoforms cAgrin7A4B8 and cAgrin7A0B0 was completely inhibited by heparin at concentrations as low as 2 µg/ml, the lowest concentration tested (data not shown). Similar inhibition was also seen for perlecan at heparin
concentrations 20 µg/ml (data not shown). In addition, enzymatic digestion of Agrin7A0B0 with heparitinase, an enzyme that cleaves GAG side chains from proteoglycans,
resulted in a shift to lower apparent molecular weight, as
reported earlier (Denzer et al., 1995
), and in substantially
reduced binding of HRG
to cAgrin7A0B0 depleted of
GAG chains (Fig. 7 A). This result was confirmed by solid
phase radioligand-binding assays where treatment of
cAgrin7A0B0-coated wells with heparitinase reduced binding of 125I-HRG
to the wells ~20-fold (n = 4; data not
shown), comparable to binding to immobilized BSA. Finally, if binding of NRGs to agrin were mediated by GAG
chains, then NRGs should not bind to shorter COOH-terminal fragments of agrin-lacking GAG chains. Therefore, we tested binding of 125I-HRG
to immobilized agrin-c95A0B0, a COOH-terminal fragment lacking conserved
GAG side chain attachment sites (Denzer et al., 1995
;
Tsen et al., 1995
). HRG
clearly bound to both full-length agrin isoforms, although with different apparent affinity
most likely representing different levels of glycosylation
because of clonal selection of agrin-producing HEK 293 cells. Binding of 125I-HRG
to c95A0B0, however, was comparable with nonspecific binding to BSA (Fig. 7 B).
Taken together, these experiments demonstrate that
binding of HRG to agrin is restricted to a region located
NH2-terminal to the first laminin G-like domain (Gesemann et al., 1995), and is mediated by GAG side chains.
They also demonstrate that the region of agrin required
for HRG binding does not overlap with the region of agrin
that is sufficient to cause AChR aggregation.
Indirect evidence led to the conclusion that interaction
of neuregulins with components of the synaptic BL might
be mediated by the Ig-like domain of NRGs (Loeb and
Fischbach, 1995). To test this directly, we expressed truncated HRG protein containing either the Ig-like domain,
HRGBbsI, or the EGF-like domain, HRG
1(177-246), and
tested whether these isolated domains bound immobilized
full-length agrin isoforms. Overlay assays (Fig. 7 C) revealed binding of HRG
BbsI, i.e., the Ig-like domain, but
not the HRG
1(177-246) EGF-like domain to agrin. This experiment demonstrates that the Ig-like domain of neuregulins is sufficient to account for the binding to the synaptic
BL proteoglycan agrin.
Agrin Induces the Accumulation of HSPGs at the Muscle Surface
The experiments described so far are consistent with a
mechanism whereby agrin induces AChR gene transcription by binding muscle-derived NRGs by their Ig domains
to its GAG side chains. The following observations suggest, however, that neural agrin may not be the sole binding partner for muscle NRGs: (a) ectopic NRG accumulations expressed in response to neural agrin only partially overlaps with ectopic agrin deposits (see also Meier et al.,
1997); and (b) neural agrin induces the accumulation of
muscle-derived HSPGs on the muscle surface. This was
seen in experiments in which chick agrin pcN257C95A4B8, a
fusion construct consisting of the ~25-kD laminin-binding
NtA domain (Denzer et al., 1997
) ligated to c95A4B8 (Gesemann et al., 1995
) was injected into myofibers. This agrin
construct lacks the region that carries GAG chains but is
sufficient to bind to laminin and to induce the synthesis and accumulation of extrasynaptic AChRs when injected
as expression plasmids into extrasynaptic regions of innervated rat muscle fibers (Fig. 8). Double labeling of
pcN257C95A4B8-injected muscle with Rh-
-BGT and antibodies specific for rat HSPGs (Eldridge et al., 1986
) revealed close colocalization of ectopic AChR aggregates and
HSPGs. This suggests that nerve-derived agrin isoforms
expressed ectopically in innervated muscle induce the accumulation of HSPGs that can serve as local sink for the
immobilization of muscle-derived NRGs. The nature of the
HSPGs that are accumulated by agrin is presently unknown.
|
![]() |
Discussion |
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---|
We have demonstrated previously that expression of neural agrin locally induces the transcription of AChR subunit
genes (Jones et al., 1996, 1997
), a function classically attributed to NRGs released from motor neurons; the same
sites also contain ErbB receptors (Rimer, M., I. Cohen, T. Lømo, S.J. Burden, and U.J. McMahan. 1996. Soc. Neurosci. Abstr. 1689; Meier et al., 1997
). Based on these findings we proposed that synapse-specific AChR subunit
gene expression by agrin is induced indirectly via muscle-derived NRGs binding to agrin-induced components of
the synaptic BL (Jones et al., 1996
, 1997
). The present
findings further support this hypothesis: the induction of
AChR gene expression by agrin can be blocked by inhibiting the ErbB receptor pathway by overexpression of an inactive mutant of HER2/ErbB2. NRG-like immunoreactivity is accumulated at the sites of agrin-induced AChR gene
activation and AChR accumulation. We further show that
recombinant NRG isoforms of the type expressed in muscle bind directly to the HSPGs agrin and perlecan but not
to several other components found at the muscle basal
lamina. Binding is to the GAG chains of these HSPGs and
is mediated by the Ig-like domain of NRGs. Synaptic accumulation of NRGs may thus be due, at least in part, to
binding to agrin itself. Other HSPGs are accumulated in
the synaptic BL under the control of agrin, providing further binding sites for NRGs. Thus, agrin induces on the
muscle fiber surface all components for regulating AChR
gene expression according to the above model.
Expression of NRGs in Muscle
In the absence of NRGs from other potential sources,
NRGs mediating agrin-induced AChR gene transcription
in nerve-free myotube cultures must be supplied by the
muscle cells themselves. Indeed, cultured muscle cells synthesize NRG-like biological activity, which can be extracted from the cell surface and induces phosphorylation of the ErbB2 receptor tyrosine kinase. An indirect action
of agrin via the accumulation of NRG derived from muscle could also explain why agrin's action on AChR expression depends on its binding to substrate (Jones et al.,
1996): substrate-bound agrin, but not soluble agrin, could
focally enrich and present muscle-derived NRGs on the
muscle surface, thus activating AChR gene expression.
Previous work has identified NRG transcripts in muscle,
but these studies were done using non-innervated clonal
cell lines (Moscoso et al., 1995) that, unlike adult muscle,
express substantial amounts of AChR
subunit transcripts
and adult-type AChR channels independently of innervation (Pinset et al., 1991
; Shepherd and Brehm, 1994
).
Moreover, it has not been addressed whether active neuregulin
isoforms are colinearly transcribed with the Ig
domain (Ng et al., 1997
), a prerequisite for binding of muscle NRGs to HSPGs in the BL. Our study now demonstrates that not only cultured myotubes but also adult muscle fibers express NRG mRNA encoding isoforms with an
Ig domain. Such transcripts were resolved both in synaptic
(data not shown) and extrasynaptic regions of adult innervated muscle, as predicted earlier (Jones et al., 1997
). In
agreement with results obtained from Sol8 myotubes
(Moscoso et al., 1995
) or chicken muscle (Ng et al., 1997
)
muscle-derived NRGs probably represent NRG
1 and
NRG
2 isoforms arising from alternative mRNA splicing
at a conserved site located between the EGF domain and
the transmembrane domain (Lemke, 1996
; Fischbach and
Rosen, 1997
). NRG
isoforms have been shown to be
most potent in activation of ErbB receptor heterodimers
and in the induction of AChR
subunit gene transcription
(Fischbach and Rosen, 1997
). These findings suggest that
the protein products of the muscle-derived NRG transcripts are biologically active and that they contain domains required for binding to synaptic HSPGs.
Requirements for NRG Immobilization in the Synaptic Basal Lamina
The expression of NRG-like activity by cultured muscle cells suggests that (a) the transcripts resolved by reverse transcriptase-PCR in muscle are translated into biologically active NRGs, and (b) that muscle-derived NRGs can be associated with the ECM through ionic interactions, as has been proposed for ARIA/NRG released from cerebellar and spinal cord cells (Loeb and Fischbach, 1995). However, their conclusion that ARIA binds to unidentified HSPGs via the Ig domain was derived indirectly, i.e., based on the heparin inhibition of ErbB phosphorylation by recombinant NRGs. In the present work, we demonstrate the importance of the GAG chains directly by showing that binding between NRGs and agrin is inhibited by enzymatic removal of GAGs, and accordingly, that NRGs do not bind to an ~95-kD COOH-terminal agrin fragment lacking conserved GAG attachment sites. We have identified two potential binding partners at the NMJ, agrin and perlecan, and have specifically excluded other glycoproteins of the NMJ for NRG binding.
Of the potential NRG-binding HSPG partners identified in this paper, i.e., cAgrin7A4B8, cAgrin7A0B0, and perlecan, neither cAgrin7A0B0 nor perlecan were enriched at extrasynaptic AChR clusters induced by ectopic neural agrin
in vivo (Meier et al., 1997; Moll, J., M.A. Ruegg, and H.R.
Brenner, unpublished observation), and thus, are not involved in NRG accumulation at such sites. However, this
does not exclude a role for cAgrin7A0B0 in NRG accumulation during synaptogenesis, as in nerve-muscle cocultures,
muscle-derived agrin is focally expressed at sites of neurally induced AChR clusters (Lieth and Fallon, 1993
). Perlecan, on the other hand, is expressed at high levels in nonsynaptic BL throughout the muscle, and thus would not
appear to contribute to synaptic localization of NRGs.
Whereas neural agrin alone can induce AChR gene expression via NRG accumulation, its GAG chains do not
provide the only binding sites for NRGs at the synapse. In
addition, agrin induces one other HSPG that in turn may
contribute to localize NRGs to the synapse. Specifically,
NRGs are also accumulated at AChR aggregates induced
by a COOH-terminal fragment of full-length agrin, agrin cN257C95A4B8, a construct that lacks GAG chains. Consistent with this, deposits of full-length agrin extend further
along the muscle fiber surface (Meier et al., 1997) than
NRGs, which are tightly colocalized with AChR aggregates and agrin-induced HPSGs (see Figs. 5 and 8). Furthermore, CBA-1 agrinA4B19, another COOH-terminal agrin fragment lacking GAG chains, induces AChR
subunit gene transcription both in vitro and in vivo (Jones et al.,
1996
, 1997
). Accumulation of newly synthesized HSPGs
by agrin was previously shown in cultured myotubes (Wallace, 1989
). Finally, focal accumulation of NRGs by agrin
might be further enhanced if nerve-released agrin not only
caused the accumulation of NRG-binding HSPGs but in
addition, also induced NRG gene transcription from myonuclei underlying the postsynaptic membrane.
In contrast to neural agrin, muscle cAgrin7A0B0 does not
induce ectopic synaptic membrane (Meier et al., 1997),
yet in cultured myotubes its potency to induce AChR
subunit gene transcription is similar to that of neural
agrin7A4B8. This apparent discrepancy might reflect the
considerably higher levels of expression of MuSK, NRGs,
and ErbB receptors in cultured myotubes over that seen in
the non-synaptic segments of mature muscle (Moscoso et al.,
1995
; Jones, G., and H.R. Brenner, unpublished data). As a consequence, the accumulation of constitutively expressed NRGs to muscle agrin's GAG chains and the activation of constitutively expressed ErbB receptors would
be sufficient to stimulate AChR gene expression in cultured myotubes. In contrast, in adult muscle the ectopic induction and accumulation of NRG and ErbB receptors may depend on the activation of MuSK, which is phosphorylated by neural but not by muscle agrin (Glass et al.,
1996
).
In conclusion, our data demonstrate at sites of ectopic agrin deposits, the presence of the major molecular components required for agrin to stimulate AChR gene expression indirectly via the accumulation of muscle-derived NRGs. According to this model, agrin induces local deposits of muscle HSPGs in the muscle BL. Muscle-derived NRGs then bind with their Ig domains to accumulated HSPGs, creating a local source of this differentiation factor. NRGs would then stimulate muscle ErbB receptors that are accumulated in synaptic muscle membrane under the direction of neural agrin.
Alternative Pathways for Agrin-induced AChR Expression?
Whereas this model can explain how agrin induces AChR
gene expression involving NRGs in the absence of a nerve
terminal, two questions arise. First, could agrin's action to
stimulate the expression of AChR genes also be mediated
directly via a cognate receptor in the muscle membrane,
such as MuSK (Valenzuela et al., 1995), a muscle-specific
receptor tyrosine kinase phosphorylated by agrin and mediating AChR aggregation (Glass et al., 1996
)? Indeed, deletion of the MuSK gene inhibits NMJ formation including synapse-specific AChR gene transcription (De Chiara et al.,
1996), and deletion of the rapsyn gene, which abolishes the
synaptic aggregation of ErbB receptors, does not affect
synapse-specific expression of AChR genes (Gautam et
al., 1995
). On the other hand, direct involvement of MuSK
in agrin-induced expression of AChRs is not supported,
since only neural isoforms of agrin that cluster AChRs
also cause phosphorylation and activation of MuSK (Glass et al., 1996
). In contrast, the ability of substrate-bound
agrin isoforms to activate AChR gene expression is independent of their AChR clustering activity (Jones et al.,
1996
). Furthermore, AChR gene expression can be induced
in cultured myotubes both by COOH- and by NH2-terminal fragments of agrin (Jones et al., 1996
; and unpublished
results), making binding to a specific, cognate receptor appear unlikely. The second question is whether or not (during the formation of the normal NMJ) NRGs released
from motor nerve terminals are important for the expression of AChR subunit genes at the synapse, as originally
proposed (Martinou et al., 1991
; Falls et al., 1993
). The reduction of AChR levels observed in subsynaptic membrane of mice heterozygous for a deletion of Ig(+)-NRG isoforms (Sandrock et al., 1997
) is consistent with both a
pre- and a postsynaptic origin of BL-bound NRG. Thus,
deleting expression of NRGs selectively either in the muscle fibers or in the motor neurons will be required to determine the relative roles of nerve- and muscle-derived
NRGs in activating AChR gene transcription during synapse formation.
![]() |
Footnotes |
---|
Received for publication 17 October 1997 and in revised form 6 February 1998.
Address all correspondence to Dr. H.-R. Brenner, Department of Physiology, University of Basel, Vesalgasse 1, CH-4051 Basel, Switzerland. Tel.: (+41) 61 267 35 42. Fax: (+41) 61 267 35 59. E-mail: brenner{at}ubaclu.unibas.chWe thank Drs. M. Jeschke and N. Hynes for cDNA encoding recombinant
HRG1; Dr. R. Timpl for perlecan; Dr. A. Brancaccio for purified
-dystroglycan; T. Schulthess for laminin-1; Dr. M. Chiquet for tenascin and fibronectin; Drs. C. Wallasch and A. Ullrich for the pHER2 and
pHER2KM constructs; Dr. R. Zuellig for the plasmid pUC118MCK6.5,
which contains the muscle creatine kinase promoter; and Dr. M.A. Ruegg
for agrin expression constructs and for comments on the manuscript. We
also thank Dr. L. Landmann for preparing the laser scanning confocal images of anti-HSPG-stained preparations, and Dr. H.-J. Schuurman, Novartis Basel, for help with histology. The mAb C17 developed by Dr. J. Sanes was obtained from the Developmental Studies Hybridoma Bank
maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA, under contract NO1-HD-7-3263 from the NICHD.
We also thank M. Lichtsteiner for excellent technical assistance.
This work was supported by grants from the Swiss National Science Foundation, the Ott-Fonds of the Swiss Academy of Medical Sciences, the Sandoz Stiftung and the Freie Akademische Gesellschaft, Basel (to H.R. Brenner). F. Schoumacher was supported by grants from the Swiss National Science Foundation to Dr. U. Eppenberger and Dr. H. Mueller, and in part by the Stiftung Tumorbank. A.J. Denzer was supported by the Sandoz Stiftung.
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Abbreviations used in this paper |
---|
AChR, acetylcholine receptor; ARIA, acetylcholine receptor-inducing activity; BL, basal lamina; DHFR, dihydrofolate reductase; ECM, extracellular matrix; GAG, glycosaminoglycan; HRG, heregulin; HSPG, heparan sulfate proteoglycan; MCK, muscle creatine kinase; NDF, Neu differentiation factor; NMJ, neuromuscular junction; NRG, neuregulin; TM, transmembrane.
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