From the Department of Cell Biology, The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan and the § Department of Growth Regulation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan
Received for publication, August 29, 2000, and in revised form, November 17, 2000
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ABSTRACT |
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Meltrin Various intracellular signaling and adhesion molecules
govern the cell-cell interactions during the development of
multicellular organisms. The actions of these molecules are regulated
not only by transcriptional and translational controls but also by
post-translational modifications such as phosphorylations and
proteolytic processings. Numerous membrane-anchored signaling molecules
are subjected to proteolytic processing to release their extracellular
domains. Such modifications may cause qualitative and irreversible
changes in the functions of these molecules.
ADAMs1 (a
disintegrin and metalloproteases;
also known as MDC proteins,
metalloprotease/disintegrin/cysteine-rich
proteins) are a family of membrane-anchored glycoproteins (1, 2) which play important roles in sperm-egg binding and fusion (3, 4), muscle
cell fusion (5), neurogenesis (6), and development of various
epithelial tissues (7). At present, more than 30 ADAM cDNAs have
been cloned from various species. Since more than half of these have a
catalytic site consensus sequence for metalloproteases (HEXGHXXGXXHD), they are
predicted to be catalytically active proteases. Genetic and biochemical
evidence indicate that some ADAMs participate in the processing of the
extracellular domain of membrane-anchored proteins. TACE (tumor
necrosis factor- Previously, our group and C. P. Blobel and his colleagues (15, 19)
cloned an ADAM with a conserved active site of a metalloprotease, meltrin NRGs (also known as acetylcholine receptor inducing activity, glial
growth factor, heregulin or neu differentiation factor) are a group of
growth factors that are members of the EGF family. NRGs mediate
an array of biological effects, including the synthesis of
acetylcholine receptors in skeletal muscle (19) and the stimulation of
Schwann cell growth (17). These biological effects of NRGs are mediated
by the ErbB family of tyrosine kinase receptors (20, 21). Gene
disruption studies indicate that NRGs are essential for early heart and
central nervous system development (22). A variety of different protein
isoforms are produced from the single NRG gene via
alternative splicing mechanisms. All isoforms contain an EGF-like
domain sufficient for biological activity. Although alternatively
spliced transcripts also generate some secreted isoforms (17), most
soluble NRGs are derived from membrane-anchored precursor proteins via
proteolytic cleavage of the extracellular region including EGF-like
domain. It has been reported that this processing occurs in
intracellular organellas (23). However, the nature of the processing
enzyme remains elusive.
In this study, we examined whether meltrin Immunohistochemistry--
Anti-meltrin Cell Culture--
P19 rat embryonic carcinoma cells were
cultured in minimum essential medium RT-PCR--
mRNA was extracted from P19 cells using
Micro-FastTrack 2.0 kit (Invitrogen). Two ng of mRNA was subjected
to one RT-PCR reaction. Reverse transcription was carried out using
SuperScript II reverse transcriptase (Life Technologies, Inc.). PCR
reactions were performed using an annealing temperature of 50 °C for
20 cycles (NRG and meltrin Expression Plasmids and Transfection--
The full-length
mouse NRG cDNAs were isolated using the primers corresponding to
the nucleotide sequences
(5'-GGCTCTAGACATGTCTGAGCGCAAAGAAGGCAG-3' and
5'-GGCTCTAGATTATACAGCAATAGGGTCTTGGTTAGC-3') from murine neonatal muscle and E12.5 mouse embryo trunk cDNAs. The mouse NRG cDNAs were fused with a synthetic DNA cassette coding for the hemagglutinin (HA)-epitope tag (MYPYDVPDYA) and subcloned into pEF-BOS, which has the
promoter region of the human EF-1 Western Blot Analysis--
Before harvesting the conditioned
medium, the cells were incubated in Opti-MEM (Life Technologies, Inc.)
containing CaCl2 (110 µg/ml) for 12 h. The
conditioned medium was initially filtered through a sterile filter unit
(pore size: 0.2 µm, Millipore) and then concentrated up to 100-fold
by centrifugation using a Centriplus 10 (Amicon) concentrator. Cells
were extracted in extraction buffer (10 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Nonidet P-40, 0.1% sodium deoxycholate) containing CompleteTM
protease inhibitor mixture (Roche Molecular Biochemicals). Extracts were clarified by centrifugation at 15,000 × g for 10 min. Protein concentration was determined using the Bradford method
(Bio-Rad). Approximately 20 µg of protein was loaded into each well.
After SDS-PAGE, proteins were electroblotted onto Immobilon
(Millipore). Anti-HA mouse monoclonal antibody (1:40 dilution; 12CA5,
Roche Molecular Biochemicals), anti-meltrin Cell Staining--
The cells transfected with
pEF-BOS-HA-NRG- Metabolic Labeling of Cells--
Cells were starved in medium
lacking methionine and cysteine (ICN) for 1 h and pulse-labeled
with [35S]methionine and -cysteine (EASYTAG express
protein labeling mixture, PerkinElmer Life Sciences) at 0.1 mCi/ml.
After a 1-h pulse, cells were either extracted in extraction buffer
containing CompleteTM protease inhibitor mixture or chased
with Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum. Where indicated, the secretory pathway inhibitors,
brefeldin A (10 µg/ml, Wako) and monensin (2 µM, Wako),
were added during the chase period. After a 5-h chase, cells were
extracted in extraction buffer. Cell extracts were clarified by
centrifugation at 15,000 × g for 20 min. The
supernatants were incubated with anti-HA monoclonal antibody (16B12,
Babco) for 30 min on ice. After the addition of protein G-Sepharose
beads, the extracts were incubated for 1 h on ice.
Immunoprecipitates were washed six times with extraction buffer and
subjected to SDS-PAGE.
Coincidental Expression of Meltrin
The mouse embryonic carcinoma P19 cell is a multipotential stem cell,
which differentiates into a variety of cell types including neuron and
glia. Since this cell line is used as an in vitro model for
differentiation of the nervous system, we examined the expression of
meltrin Proteolytic Processing of NRGs by Meltrin
Since DRG neurons express meltrin
To investigate whether the meltrin
Expression of E347Q meltrin
Small proportion of unprocessed membrane-anchored NRGs expose their
extracellular domains on the cell surface (23). To investigate the
effect of meltrin
We further investigated whether meltrin
Recently, it has been reported that meltrin Brefeldin A-sensitive and Monensin-insensitive Cleavage of NRG- NRGs mediate a variety of biological functions including glial
cell development, synaptogenesis, and cardiac development through the
activation of the ErbB family of tyrosine kinase receptors (33). Most
NRG isoforms encode membrane-anchored proteins that generate soluble
ligands for the ErbB family by proteolytic cleavages. It is not yet
clear, however, whether the functions of NRGs depend on actions of
processed and released soluble NRGs or whether the transmembrane form
is biologically active. Genetic disruption of only the intracellular
domain of membrane-anchored NRG isoforms results in a similar phenotype
of embryonic maldevelopment to that observed with disruption of the
entire gene (34). Furthermore, deletion of the cytoplasmic tail of
membrane-anchored NRGs completely abrogated the release of mature NRGs
(34). These results strongly suggest that the proteolytic processings
of membrane-anchored NRGs are critical regulatory mechanisms of NRG functions.
In the present study, we provided evidence that meltrin As reported previously, NRG- Meltrin Expression of H346A,H350A or Phorbol ester induces the processing of several membrane-anchored
proteins through the activation of protein kinase C (PKC). As reported
previously in other cell types, we found that phorbol ester induces the
processing and release of mature soluble NRG- In summary, we showed that meltrin /ADAM19 is a member of ADAMs
(a disintegrin and
metalloproteases), which are a family of membrane-anchored glycoproteins that play important roles in fertilization, myoblast fusion, neurogenesis, and proteolytic processing of several
membrane-anchored proteins. The expression pattern of
meltrin
during mouse development coincided well with
that of neuregulin-1 (NRG), a member of the epidermal growth factor family. Then we examined whether meltrin
participates in the proteolytic processing of membrane-anchored NRGs. When NRG-
1 was expressed in mouse L929 cells, its
extracellular domain was constitutively processed and released into the
culture medium. This basal processing activity was remarkably
potentiated by overexpression of wild-type meltrin
, which lead to
the significant decrease in the cell surface exposure of extracellular
domains of NRG-
1. Furthermore, expression of protease-deficient
mutants of meltrin
exerted dominant negative effects on the basal
processing of NRG-
1. These results indicate that meltrin
participates in the processing of NRG-
1. Since meltrin
affected
the processing of NRG-
4 but not that of NRG-
2, meltrin
was
considered to have a preference for
-type NRGs as substrate.
Furthermore, the effects of the secretory pathway inhibitors suggested
that meltrin
participates in the intracellular processing of NRGs
rather than the cleavage on the cell surface.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
converting enzyme)/ADAM17 was initially
identified as the protease responsible for the processing of pro-tumor
necrosis factor-
(8, 9). Furthermore, studies on the disruption of
the mouse TACE gene demonstrated that TACE is involved in
the processing of extracellular domains of several membrane-anchored
proteins including tumor necrosis factor p75 receptor, the adhesion
molecule L-selectin, amyloid precursor protein, and transforming growth
factor-
(7, 10). Kuzbanian/ADAM10 is involved in the neurogenesis of
Drosophila (6), and processes and releases a soluble form of
Delta, a Notch ligand (11). Recently, it has been reported that meltrin
/ADAM9 is involved in the processing of heparin-binding EGF-like growth factor (12). Mouse Kuzbanian and meltrin
also cleave amyloid
precursor protein (13, 14). These findings strongly suggest potential
roles of ADAM metalloproteases in the proteolytic processing of various
membrane-anchored proteins.
/ADAM19. Meltrin
consists of multiple domains including prodomain, metalloprotease domain, disintegrin domain, cysteine-rich domain, epidermal growth factor (EGF)-like domain, transmembrane domain, and cytoplasmic tail. During mouse embryogenesis,
meltrin
mRNA is markedly expressed in craniofacial
and dorsal root ganglia (DRG) and ventral horns of the spinal cord,
where peripheral neuronal cell lineages differentiate (16). Heart,
lung, skeletal muscle, and intestine also express meltrin
mRNA transiently (16). These expression patterns of meltrin
coincide well with that of neuregulin-1 (NRG)
(17, 18).
participates in the
processing of membrane-anchored NRGs. First, both meltrin
and NRG
proteins were expressed in DRG neurons at the same stages of mouse
embryogenesis. Next, overexpression of wild-type meltrin
significantly increased the release of soluble NRGs in culture medium
and decreased the cell surface expression of the extracellular domains
of NRG-
1. Furthermore, the processing of NRGs was abrogated by
expression of protease-deficient mutants of meltrin
. Finally, the
enhanced processing of NRGs by meltrin
was blocked by the treatment
with brefeldin A but not by monensin, which suggested the action of
meltrin
in the Golgi apparatus. Taken together, we concluded that
meltrin
(or similar ADAM proteases) participates in the cleavage of
membrane-anchored NRGs.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
antiserum used in
this study was raised in rabbits against a keyhole limpet
hemocyanin-coupled peptide (PEYRSQRVGAIISSKI) corresponding to the
extreme C-terminal sequence of meltrin
. E12.5 mouse embryos were
dissected, rinsed with phosphate-buffered saline (PBS), incubated in
PBS containing 20% sucrose at 4 °C overnight, and embedded in OCT
compound (Tissue-Tek, Miles Inc.) on a dry ice block. Cryosections (6 µm in thickness) on 3-aminopropyltriethoxysilane-coated glass slides
were prepared and fixed for 5 min in acetone at
20 °C. Antibodies
were applied overnight at 4 °C in a humidified chamber in PBS
containing 10% heat-inactivated normal goat serum at the following
dilutions: anti-meltrin
antiserum, 1:300; anti-NRG Ab-3
(NeoMarker), 1:300, and anti-neurofilament 160 (NF160, Sigma), 1:500.
The slides were washed three times for 10 min each in PBS containing
0.05% Tween 20 (PBST), then incubated in secondary antibodies in PBS
containing 10% heat-inactivated normal goat serum for 1 h at room
temperature. The slides were then washed three times in PBST and
mounted with PERMAFLOUR (Immunotech). The anti-meltrin
and anti-NRG
immunoreactivities were amplified using biotinylated anti-rabbit IgG
(1:500 or 1:1000 dilution, Vector Laboratories) and streptavidine-Cy3
(1:500 or 1:1000 dilution, Jackson ImmunoResearch). Fluorescein
isothiocyanate-conjugated anti-mouse IgG (1:500 dilution, Jackson
ImmunoResearch) was used for the anti-NF160 immunoreactivity. Imaging
was carried out using a Leica DM IRBE inverted confocal microscope
using ×10 and ×40 objectives (Leica) and TCS-NT software (Leica).
medium supplemented with 10%
fetal bovine serum. Mouse L929 fibroblasts were cultured in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum. All
cultures were maintained at 37 °C in the presence of 5%
CO2. To induce differentiation, P19 cells were cultured on
bacterial grade dishes to form aggregates for 4 days in the presence of
1 µM retinoic acid (Sigma) and then replated on tissue
culture grade dishes in growing medium.
) or 55 °C for 15 cycles
(glyceraldehyde-3-phosphate dehydrogenase). Signals are roughly
proportional to the amount of cDNA under these conditions. The
following primer pairs were used:
5'-ACATCAACATCCACGACTGGGACCAGCCATCT-3' and
5'-GCAGTAGGCCACCACACACATGATGCC-3' (NRG),
5'-GCGGAATTCGGAGGCCGGAGGTGTGGCAAC-3' and
5'-GGCGTCGACGGTGCCATCCATCTGATAATA-3' (meltrin
),
5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA-3' (glyceraldehyde-3-phosphate dehydrogenase).
chromosomal gene (24), to obtain
pEF-BOS-HA-NRGs. A couple of protease-deficient (E347Q and H346A,H350A)
meltrin
cDNAs and a metalloprotease domain-deleted (
MP)
meltrin
cDNA were constructed by mutagenesis based on a PCR
technique using mutated primers. In E347Q mutant meltrin
, glutamine
is substituted for the conserved glutamic acid at position 347. In
H346A,H350A mutant meltrin
, alanines are substituted for the
conserved histidines at positions 346 and 350. In
MP meltrin
,
amino acid residues 208-430 are deleted. The nucleotide sequences of
the mutants were confirmed by direct sequencing. The cDNAs of
wild-type and mutant meltrin
were subcloned into pEF-BOS. Wild-type
meltrin
was also subcloned into pEF-BOS (12). pBIE plasmid was
generated by deletion of the human cytomegalovirus promoter region of
pIRES2-EGFP (CLONTECH) and replaced by the promoter
region of pEF-BOS. Wild-type and
MP meltrin
cDNAs were
subcloned into pBIE to generate pBIE-meltrin
and pBIE-
MP meltrin
, respectively. These plasmids were transfected by the LipofectAMINE
PLUS method according to the manufacturer's instructions (Life
Technologies Inc.).
rabbit antiserum (1:500 dilution), and anti-C terminus of NRG rabbit polyclonal antibody (1:100
dilution; sc-348, Santa Cruz) were used as primary antibodies. After
incubation with primary antibody, the blots were incubated with
biotinylated anti-mouse or anti-rabbit IgG (1:2500 dilution, Jackson
ImmunoResearch) and then with horseradish peroxidase-conjugated streptavidin (1:5000 dilution, Amersham Pharmacia Biotech). The blots
were developed using the ECL plus system (Amersham Pharmacia Biotech).
Prestained protein molecular weight marker was from Bio-Rad.
1 together with pBIE, pBIE-meltrin
, or pBIE-
MP
meltrin
were incubated with anti-HA mouse monoclonal antibody
(1:200 dilution; 16B12, Babco) at 4 °C for 30 min, and then washed
four times with ice-cold PBS. After fixation with 4% paraformaldehyde
in PBS for 15 min, the cells were incubated with Cy3-conjugated goat
antibody to mouse IgG (1:400 dilution, Jackson ImmunoResearch) at
25 °C for 30 min. Imaging was carried out using a Leica DM IRBE
inverted confocal microscope using a ×63 oil objective (Leica) and
TCS-NT software (Leica).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and NRGs--
We previously
reported the expression pattern of meltrin
mRNA
during mouse embryogenesis (16). Meltrin
mRNA is
markedly expressed in the regions where peripheral neuronal cell
lineages differentiate including craniofacial and DRG and
ventral horns of the spinal cord. In addition, heart, lung, skeletal
muscle, and intestine express meltrin
mRNA
transiently. This expression pattern of meltrin
coincides well with that of an EGF family growth factor,
NRGs (17, 18). In this study, we further investigated the
precise expression sites of NRGs and meltrin
proteins in the
developing mouse nervous system. Adjacent transverse sections through
mouse E12.5 embryo were coimmunostained with antibodies against
neuronal marker, neurofilament 160 (NF160), and C-terminal domain of
meltrin
(Fig. 1, A-C), or
EGF-like domain of NRGs (Fig. 1, D-F). As reported
previously, high levels of NRG protein were expressed in DRG that give
rise to sensory neurons (Ref. 17, Fig. 1E) and the ventral
horns of the spinal cord that produce motor neurons (data not shown).
Similarly, strong and specific immunoreactivity for meltrin
was
observed in the DRG (Fig. 1B) and the ventral horns of the
spinal cord (data not shown). This result indicates that meltrin
protein is expressed in the regions where peripheral neuronal cell
lineages differentiate during embryogenesis. In addition,
immunostainings of both meltrin
and NRGs were detected in most
NF160-positive cells in the DRG region (Fig. 1, C and F). These results clearly demonstrate that the majority of
NF160-positive neuronal cell lineages in DRG express both meltrin
and NRGs simultaneously.
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Fig. 1.
Expression of meltrin and NRGs proteins in dorsal root ganglia during mouse
embryogenesis. Adjacent transverse sections through
forelimb of E12.5 mouse embryos were costained with antibodies against
neurofilament 160 (NF160) and meltrin
(A-C) or NRGs
(D-F). Asterisks and arrows indicate
the neural tube and the DRG, respectively. A and
D, anti-NF160 staining (green). B,
anti-meltrin
staining (red). E, anti-NRGs
staining (red). C, overlay of NF160 and meltrin
immunostaining. F, overlay of NF160 and NRGs
immunostaining. Bar, 50 µm.
and NRGs in this cell line during
differentiation. Differentiation was induced by aggregating P19 cells
in the presence of 1 µM retinoic acid for 4 days and then
the cells were dissociated and plated in the absence of retinoic acid.
Cells were harvested at the indicated times, and the cell extracts were
subjected to Western blotting using antibodies against the markers of
neurons (microtubule-associated protein-2), glial cells (glial
fibrillary acidic protein), and smooth muscle cells (smooth muscle
actin). The expression of microtubule-associated protein-2 was
increased at day 6 and then decreased gradually (data not shown). On
the other hand, the expression of glial fibrillary acidic protein was
increased during the differentiation period (data not shown). To
examine the expression level of NRGs and meltrin
mRNAs, total mRNAs were prepared from cells at the
indicated times, and subjected to RT-PCR (Fig.
2). NRGs and
meltrin
mRNAs were both expressed at very low levels
at day 0. The transcripts of these genes began to appear at day 6, then
reached a maximum level at day 8. This similarity in the
transcriptional profiles indicates a plausible interaction between
meltrin
and NRGs. Many NRGs are derived from membrane-anchored
precursor proteins via proteolytic cleavage. We therefore investigated
whether the metalloprotease activity of meltrin
is involved in the
processing of NRGs.
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Fig. 2.
Expression of meltrin and NRGs mRNAs in differentiated P19 cells.
Undifferentiated P19 cells (day 0) were cultured on bacterial grade
dishes with retinoic acid for 4 days and subsequently cultured on
culture grade dishes without retinoic acid for 6 days. Differentiated
P19 cells were harvested at the indicated times, and the total RNA
fractions of the cells were subjected to RT-PCR analysis using primer
sets for meltrin
(top panel) and NRGs (middle
panel). Glyceraldehyde-3-phosphate dehydrogenase
(G3PDH) levels served as a control for template levels
(bottom panel).
--
Alternative
splicing of a single gene gives rise to multiple isoforms of NRG. Many
of these encode transmembrane, glycosylated precursors of soluble NRGs.
In this study, we used three transmembrane isoforms of NRG (
2,
1,
and
4) and the domain structure of the NRGs used here is shown
schematically in Fig. 3A. The
extracellular portion of these NRGs contains an immunoglobulin motif
(Ig), a glycosylated spacer domain (Glyco.), and an EGF-like domain
(EGF) (25). The two major classes of NRGs diverge in the C terminus of
the EGF-like domain giving rise to the
- and
-isoforms.
Additional variation is seen in the juxtamembrane region following the
EGF domain by the insertion of one of three different sequences
(numbered 1, 2, or 4). To detect the ectodomains of NRGs released into
the culture medium, the N terminus of NRGs was tagged with HA
epitope.
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Fig. 3.
Meltrin participates in the processing of
NRG-
1. A, diagram of the
domain organization of NRG isoforms used in this study.
Boxes represent the major structural motifs of NRGs: an
immunoglobulin-like domain (Ig), a glycosylated spacer
domain (Glyco.), an EGF-like domain (EGF), and a
transmembrane domain (TM). Variation among these isoforms
occurs in the C terminus of the EGF-like domain and in the
juxtamembrane domain. Two variant C terminus of the EGF-like domain
characterize the
- and
-isoforms. Further differences arise in
the juxtamembrane domain to generate isoforms
2,
1, and
4. HA
epitope was attached to the N terminus of all NRGs. B and
C, Western blotting of CM and cell extracts from L929 cells
transfected with plasmids expressing HA-NRG-
1 and meltrin
. Cells
were transfected with the indicated plasmids and cultured for 48 h
and then CM and cell extracts were harvested as described under
"Experimental Procedures." CM was subjected to Western blotting
using anti-HA antibody to detect soluble NRG-
1 (B). Cell
extracts were subjected to Western blotting using anti-NRG C terminus
antibody (C, upper panel) and anti-meltrin
antibody
(C, lower panel). In lane 6 of the upper
panel, 5-fold amount of the extract of NRG-
1 and
MP meltrin
expressing cells was used. The migration of prestained molecular
mass standards is shown on the right. D, cells transfected
with plasmid expressing HA-NRG-
1 together with pBIE (vector),
pBIE-meltrin
(WT meltrin
), or pBIE-
MP meltrin
(
MP
meltrin
) were stained with anti-HA antibody in the nonpermeabilized
condition. Cell surface NRG-
1 was detected by anti-HA staining (red
signals in upper panels). Transfected cells were identified
by the existence of green fluorescent protein (GFP, green
signals in lower panels). Note that most of GFP-positive
cells expose the N-terminal HA-epitope on the cell surface in pBIE or
pBIE-
MP meltrin
-transfected cells but not in pBIE-meltrin
-transfected cells.
and NRG simultaneously (Fig. 1),
we first examined the processing of a neuronal type of NRG, NRG-
1.
In this study, we used mouse L929 fibroblast which expresses a low
level of endogenous meltrin
(data not shown). L929 cells
were transfected with an expression plasmid encoding NRG-
1 and then
the conditioned medium (CM) was subjected to Western blotting using
anti-HA antibody. Released soluble NRG-
1 (~46 kDa) was detected in
the CM of NRG-
1 expressing cells (Fig. 3B, lane 2). This
released polypeptide could induce the tyrosine phosphorylation of ErbB2
and -3 when added to differentiated muscle cells, C2C12 (data not
shown), which shows that this 46-kDa polypeptide is a functionally
mature NRG-
1. The broad appearance of processed NRG-
1 band might
represent the variety of multiple N-linked and O-linked glycosylation in its spacer region (26). We further investigated whether coexpression of meltrin
affects the release of
mature NRG-
1. Overexpression of wild-type meltrin
considerably increased the release of mature NRG-
1 (Fig. 3B, lane 3).
Western blotting of cell extracts using the anti-C terminus of NRGs
antibody showed that overexpression of wild-type meltrin
increased
the ratio of processed cytoplasmic tail of NRG-
1 (74 kDa, open
triangle) and decreased the ratio of full-length NRG-
1 (120 kDa, filled triangle) (Fig. 3C, upper panel, lane
3). These results strongly suggest that meltrin
could
potentiate the basal processing activity of NRG-
1.
protease activity is necessary
for the processing of NRG-
1, several mutants of meltrin
were
constructed. In E347Q and H346A,H350A meltrin
, glutamine, and
alanine residues were substituted for the glutamic acid and histidine
residues, respectively, which are essential for the metalloprotease
activity. In
MP meltrin
, metalloprotease domain is completely
deleted. Western blotting using anti-meltrin
antibody revealed two
immunoreactive species with apparent molecular masses of 125 and 100 kDa in the cell expressing E347Q meltrin
as shown in the cell
expressing wild-type meltrin
(Fig. 3C, lower panel, lanes
3 and 4). The 100-kDa form is considered to be
generated by removal of the prodomain from the 125-kDa form, probably
by a furin-like pro-protein convertase, which cleaves ADAMs at the sequence motif RXKR in a late Golgi compartment (27, 28). Western
blotting of the cells expressing H346A,H350A meltrin
revealed
mainly the 125-kDa unprocessed form (Fig. 3C, lower
panel, lane 5).
made no change in the basal processing
of NRG-
1 (Fig. 3, B, lane 4, and C, upper panel,
lane 4). This observation clearly demonstrates that protease
activity of meltrin
is essential for the increase of NRG-
1
processing. On the other hand, expression of H346A,H350A meltrin
remarkably suppressed the release of mature NRG (Fig. 3B, lane
5). At the same time, expression of H346A,H350A meltrin
increased the ratio of the unprocessed form of NRG-
1 and decreased
the ratio of its processed cytoplasmic tail in the cells (Fig.
3C, upper panel, lane 5). Expression of
MP meltrin
decreased the production of NRG-
1 by unknown reasons (data not
shown). However, Western blotting of an increased amount of the extract
revealed that expression of
MP meltrin
also increased the ratio
of the unprocessed form of NRG-
1 and decreased the ratio of the
processed form of NRG-
1 (Fig. 3C, upper panel, lane 6).
Thus, expression of these mutants of meltrin
exert dominant
negative effects on the basal processing of NRG-
1. Taken together,
these results indicate that meltrin
participates in the processing
of NRG-
1 through its metalloprotease activity.
on appearance of the extracellular domains on the
cell surface, the cells expressing HA-NRG-
1 together with or without
meltrin
were stained with anti-HA antibody under the
nonpermeabilized condition. In this experiment, another type of
expression plasmids were constructed in which wild-type or
MP
meltrin
was expressed together with green fluorescent protein (GFP)
by inserting internal ribosomal entry site sequence between cDNAs
encoding these proteins. The result shown in Fig. 3D showed that efficient exposure of the N-terminal HA-tag of HA-NRG-
1 significantly decreased in the cells expressing wild-type meltrin
.
Such an effect could not be seen in
MP meltrin
expressing cells.
Thus, enhanced processing of memrane-anchored NRG-
1 by meltrin
resulted in the decreased exposure of extracellular domains on the cell surface.
participates in the
processing of other isoforms of NRGs. The expression plasmids encoding
wild-type or H346A,H350A meltrin
were co-transfected into L929
cells with an expression plasmid encoding NRG-
2,
1, or
4. Then
the amount of released mature NRGs was determined by Western blotting
of CM (Fig. 4A). The amount of
mature NRG-
1 and -
4 was increased by overexpression of wild-type
meltrin
and decreased by expression of H346A,H350A meltrin
. On
the other hand, the amount of mature NRG-
2 was not affected by
overexpression of either wild-type or H346A,H350A meltrin
. We then
examined the effect of meltrin
on the stability of full-length NRGs
by a pulse-chase experiment. Full-length NRG-
1 is proteolytically cleaved in the presence of wild-type meltrin
(Fig. 4B, lower panel, lane 5). This cleavage is dependent on the protease
activity of meltrin
(Fig. 4B, lower panel, lane 6). On
the other hand, full-length NRG-
2 is not cleaved by meltrin
(Fig. 4B, upper panel, lane 5). Taken together, these
results demonstrate that meltrin
participates in the processing of
-type, but not
-type, NRGs.
View larger version (23K):
[in a new window]
Fig. 4.
Substrate specificity of meltrin
. A, plasmids encoding several
isoforms of NRG were transfected to L929 cells together with plasmid
encoding wild-type or H346A,H350A (mut) meltrin
. CM was subjected
to Western blotting using anti-HA antibody as described in Fig.
3B. B, L929 cells were transfected with plasmids encoding
NRG-
2 (upper panel) or NRG-
1 (lower panel)
and meltrin
. Cells were labeled for 1 h with
[35S]methionine/cysteine (PerkinElmer Life Sciences) and
immediately frozen (0) or chased with cold media for 5 h (5). Cells were extracted, and NRGs were
immunoprecipitated from extracts with anti-HA antibody. All samples
were subjected to SDS-PAGE. Arrowheads indicate the
migration of full-length NRGs. C, cells were transfected
with plasmids encoding NRG-
1 and meltrin
or meltrin
. CM was
subjected to Western blotting using anti-HA antibody.
is involved in the
processing of a membrane-anchored growth factor, heparin-binding EGF
(12). We examined whether meltrin
also participates in the
processing of NRG-
1. Coexpression of meltrin
resulted in the
release of 30- and 35-kDa HA-containing region of NRG-
1 into CM
(Fig. 4C, arrowheads). These polypeptides are much smaller than the mature soluble NRGs reported previously (20, 29). This result
indicates that meltrin
cleaves NRG-
1 in a manner different from
meltrin
.
1
by Meltrin
--
To examine whether meltrin
is localized in the
cell surface, we carried out a cell surface biotinylation analysis
using cells transfected with meltrin
-expressing plasmid. However, we could not detect any surface-exposed meltrin
(data not shown). In the same experiment, the fusion meltrin
, which has an exogenous signal sequence of human granulocyte colony-stimulating factor, was
efficiently biotinylated (data not shown), thereby excluding the
possibility that the result was due to experimental failure. These
findings indicate that meltrin
is mainly localized inside of the
cell. Since it has been reported that some portions of NRGs undergo
intracellular proteolysis (23), we investigated the subcellular
compartment in which meltrin
processes NRG-
1 using two
inhibitors of the secretory pathway, brefeldin A and monensin. In the
presence of brefeldin A, the NRG-
1 processing induced by meltrin
was completely blocked (Fig. 5,
lane 11). On the other hand, monensin did not block the
processing of NRG-
1 induced by meltrin
(Fig. 5, lane
8). Thus the processing of NRG-
1 induced by meltrin
is a
brefeldin A-sensitive and monensin-insensitive event. Brefeldin A
blocks traffic from the endoplasmic reticulum to the Golgi by
interfering with anterograde transport from the endoplasmic reticulum
to Golgi (30, 31). On the other hand, monensin is expected to interfere
with the transfer across Golgi compartments and compromise secretion
from the trans-Golgi (30, 32). Taken together, our results suggest that
meltrin
participates in the intracellular cleavage of NRG-
1
within the Golgi apparatus.
View larger version (15K):
[in a new window]
Fig. 5.
Brefeldin A-sensitive and
monensin-insensitive cleavage of NRG- 1 by
meltrin
. Plasmids encoding NRG-
1 were
transfected to L929 cells together with plasmid encoding wild-type or
H346A,H350A (mut) meltrin
. Cells were labeled for 1 h and then
chased with cold media for 5 h in the absence or presence of 10 µg/ml brefeldin A or 2 µM monensin. Cells were
extracted, and NRGs were immunoprecipitated from extracts with anti-HA
antibody. All samples were subjected to SDS-PAGE and autoradiography.
Arrowheads indicate the migration of full-length NRGs.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
participates in the processing of
-type NRGs. Initially, both
meltrin
and NRG proteins were found to be expressed in dorsal root
ganglia at the same stages during embryogenesis (Fig. 1). During
neurogenic differentiation of P19 cells, the expression of
meltrin
and NRGs mRNA was activated in a
similar fashion (Fig. 2). Next, overexpression of wild-type meltrin
potentiated the release of mature soluble NRG-
1 (Fig. 3B, lane
3) with a concomitant decrease in the cell surface expression of
extracellular domains of NRG-
1 (Fig. 3D). The protease
activity of meltrin
is indispensable for the potentiation of
NRG-
1 processing (Fig. 3, B and C, lane 4).
Furthermore, expression of H346A,H350A or
MP meltrin
remarkably
suppressed the release of soluble NRG-
1 (Fig. 3B, lanes 5 and 6) with a concomitant increase in the ratio of
full-length NRG-
1 and a decrease in the ratio of processed forms of
NRG-
1 in the cells (Fig. 3C, lanes 5 and 6).
We further confirmed the enhanced processing of NRG-
1 with meltrin
protease by the pulse-chase experiment shown in Fig. 4B.
These results clearly demonstrate that meltrin
has functional
processing activity of NRG-
1 and that the protease activity of
meltrin
is necessary for constitutive processing of NRG-
1. This
is the first report on the function of meltrin
and, at the same
time, the first report that indicates the involvement of ADAM
metalloproteases in the proteolytic processing of membrane-anchored NRGs. It is considered that meltrin
plays a pivotal role in the
development of several organs through the processing of NRGs.
2 is the predominant isoform in
mesenchymal cells, whereas NRG-
1 is the major neuronal isoform (35).
The main cleavage sites in these NRG molecules are in exon-
and
exon-
, respectively (26). While L929 cells possess endogenous
proteolytic processing activities for both
- and
-type NRGs, both
overexpression of wild-type and H346A,H350A meltrin
only affected
the cleavage of
-type NRGs. It is plausible that
-type NRG is
cleaved by a protease(s) other than meltrin
in L929 cells.
Alternatively, L929 cells may lack some regulatory factors that
cooperate with overexpressed meltrin
to cleave
-type NRG efficiently.
expressed in L929 cells was mainly localized in the Golgi
apparatus (data not shown) although intracellular localization of
meltrin
remains to be determined precisely. Examinations of the
effects of brefeldin A and monensin on the processing revealed that
meltrin
participates in the intracellular processing of NRGs,
probably in the Golgi apparatus or in monensin-insensitive secretory
pathways. Recently, several reports demonstrated that some ADAMs are
processed and activated in the trans-Golgi network (27, 28), and
localized mainly in the Golgi apparatus (13, 27). Furthermore,
Skovronsky et al. (36) have found activity of TACE and/or
Kuzbanian in the trans-Golgi network. These observations and our
results indicate that multiple ADAMs function in the trans-Golgi network as intracellular processing enzymes.
MP meltrin
markedly suppressed the
basal processing activity of NRG-
1 (Fig. 3). Genetic and biochemical
characterization of other ADAM proteases also indicated such dominant
negative effects of protease-deficient mutants (11-14, 37). In
preliminary experiments, we found that small proportion of meltrin
and NRGs expressed in L929 cells could be coimmunoprecipitated (data
not shown). H346A,H350A and
MP meltrin
might show
dominant-negative effects through the interaction with NRGs, thereby
blocking the interaction of endogenous proteases with NRGs. On the
other hand, expression of E347Q meltrin
did not affect the basal
processing activity (Fig. 3). As shown in Fig. 3C, the
prodomain of E347Q meltrin
is removed precisely while those of
H346A,H350A and
MP meltrin
are not removed. These meltrin
mutants might have different conformation from wild-type or E347Q
meltrin
, and their conformational abnormality might affect
endogenous meltrin
or similar proteases to act on NRG-
1. The
identification of the domain of meltrin
required for the dominant
negative effect on the processing will provide further insight into the
mechanism by which meltrin
recognizes and processes NRG-
1.
1 in L929 cells (Ref.
23, and data not shown). This induced processing was not suppressed by
expression of H346A,H350A mutant of meltrin
(data not shown). Our
observation indicates that meltrin
accounts for the constitutive
processing but not for the PKC-regulated processing of NRG-
1. Thus,
distinct pathways for the processing of NRG-
1 are suggested: one
pathway is dependent on meltrin
protease while, in the other
PKC-regulated pathway(s), processing is carried out by other proteases.
Several reports have demonstrated that TACE, Kuzbanian, and meltrin
take part in PKC-regulated processing (7, 10, 12, 13). As shown in Fig.
4C, meltrin
is not able to process NRG-
1 as a mature
form. Further studies are warranted to determine whether or not other
ADAMs such as TACE and Kuzbanian participate in the PKC-regulated
processing of NRG-
1.
and NRGs are simultaneously
expressed in the nervous system during development and meltrin
participates in the proteolytic processing of
-type NRG isoforms which are involved in neurogenesis and synaptogenesis. During differentiation of P19 cells the activation of the meltrin
and NRG genes preceded that of glial fibrillary
acidic protein (Fig. 2, data not shown), suggesting regulatory roles of
meltrin
in glial cell differentiation through the release of mature
NRGs. Further analysis including genetic disruption of meltrin
will be required to demonstrate the role of meltrin
in the development of the nervous system.
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FOOTNOTES |
---|
* This work was supported in part by a grant-in-aid for Scientific Research on Priority Areas (B) of The Ministry of Education, Science, Sports and Culture, research grants from the Japanese Health Science Foundation, the National Center of Neurology and Psychiatry of the Ministry of Health and Welfare of Japan, and CREST (Core Research for Evolutional Science and Technology) of Japan Science and Technology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Research Fellow of the Japan Society for the Promotion of Science.
¶ To whom correspondence should be addressed: Dept. of Growth Regulation, Institute for Frontier Medical Sciences, Kyoto University, Kawahara-cho 53, Shogo-in, Kyoto 606-8507, Japan. Tel.: 81-75-751-3826; Fax: 81-75-751-4646; E-mail: asehara@frontier.kyoto-u.ac.jp.
Published, JBC Papers in Press, December 14, 2000, DOI 10.1074/jbc.M007913200
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ABBREVIATIONS |
---|
The abbreviations used are:
ADAM, a disintegrin
and metalloprotease;
NRG, neuregulin-1;
EGF, epidermal growth factor;
DRG, dorsal root ganglia;
TACE, tumor necrosis factor- converting
enzyme;
PBS, phosphate-buffered saline;
RT-PCR, reverse
transcriptase-polymerase chain reaction;
HA, hemagglutinin;
PAGE, polyacrylamide gel electrophoresis;
CM, conditioned medium;
GFP, green
fluorescent protein;
PKC, protein kinase C.
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