From the Department of Biology, Emory University, Atlanta, Georgia
30322 and the Department of Cell Biology, Neurobiology,
and Anatomy, University of Cincinnati College of Medicine,
Cincinnati, Ohio 45267
Received for publication, June 28, 2000, and in revised form, October 18, 2000
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ABSTRACT |
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Two neuregulin-1 isoforms highly expressed in the
nervous system are the type III neuregulin III- Neuregulin-1 gene products are cell-cell signaling proteins
that are ligands for receptor tyrosine kinases of the ErbB/HER subfamily (for reviews, see Refs. 1-4). Signaling events mediated by
neuregulins (NRGs)1 have been
shown to be essential for normal development of the nervous system and
heart (5-9). Roles for NRG-1 proteins in the development of other
organs and in the adult have also been suggested.
Gene transcripts encoding at least 14 different NRG isoforms have been
identified (2). Based on the structure of their N-terminal region,
NRG-1 isoforms can be divided into three types (Refs. 1 and 10; see
"Neuregulin Isoforms and Nomenclature" and Fig. 1A). The
NRG isoforms originally called neu differentiation factor (11 and 12) and heregulin (13) have a type I N terminus. The N terminus of
chick ARIA (14) is most similar to the mammalian type I N terminus. The
isoforms originally called glial growth factor (15, 16) that include a
"kringle domain" have a type II N terminus. The isoforms originally
called SMDF, nARIA, or CRD-NRG (17-21) have a type III N terminus. The
type I and type II isoforms contain an Ig-like domain in the N-terminal
region (Ig-NRGs), whereas the type III isoform contain a cysteine-rich domain (CRD-NRGs). Studies of the temporal and spatial expression patterns of NRG isoforms suggest that type I/II and type III NRGs serve
distinct functions (10, 18). This inference has been confirmed by
"knockouts" that have specifically deleted the Ig-NRGs (6) or the
CRD-NRGs (Ref. 8; see also Ref. 10). With respect to the type III NRGs,
analysis of the knockout animals indicates that type III NRGs play a
critical role in the interactions of peripheral nerve axons with muscle
and with Schwann cells. Defects in animals lacking type III NRGs
include retraction of nerve terminals from newly formed synapses,
absence of Schwann cells from peripheral nerves, and loss of motor and
sensory neurons (8).
The cellular processing of type I NRGs has been the subject of several
investigations (22-25). Most type I NRG isoforms include a hydrophobic
stretch of amino acids C-terminal of the epidermal growth factor
(EGF)-like) domain that serves as a transmembrane (TM) domain. These
type I isoforms are synthesized as transmembrane "proproteins" from
which a paracrine signal can be produced by proteolytic cleavage in the
stalk region (Fig. 1B). The release of the ectodomain into
the extracellular space may require transport of the uncleaved
proprotein to the cell surface, followed by stalk cleavage and
"shedding" of the ectodomain from the surface. Alternatively, after
arriving at the surface, the proprotein may be internalized into
endocytotic pathway compartments in which proteolytic processing occurs
with subsequent secretion of the ectodomain fragment (24). Release of
the type I NRG ectodomain into culture medium is accelerated by
activation of protein kinase C (12, 22, 24) and blocked by shortening
the cytoplasmic tail to fewer than ~90 amino acids (7, 26) or by
deletion of specific segments within this 90-amino acid stretch
(26).
NRG III- The III- Neuregulin Isoforms and Nomenclature--
The isoform
designation "III-
Based on the results presented in this study, we propose that the
III-
We refer to neuregulin proteins with both an ectodomain epitope and
cytoplasmic tail epitope as "proproteins," in recognition of their
potential to be biosynthetic precursors to cleaved bioactive products.
However, this does not exclude the possibility that these
"proproteins" are themselves bioactive signaling proteins.
Throughout this paper, "neuregulin" and the abbreviation "NRG"
refer only to the proteins encoded by the nrg-1 gene. Three related genes (nrg-2, nrg-3, and
nrg-4) have now been identified, but the proteins encoded by
these genes are not discussed.
Creation of Neuregulin Constructs--
All neuregulin proteins
analyzed in this study included an HA epitope tag in the ectodomain
(Fig. 1D). The sole exception is untagged I- Cell Culture and Transfection--
COS-7 cells were cultured in
Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal
bovine serum. PC 12 cells (N21 strain; Ref. 33) were a gift from
Richard Burry (Ohio State University). PC12 cells were cultured in DMEM
containing 10% horse serum and 5% FetalClone (Hyclone) with 50 ng/ml
NGF (Life Technologies, Inc.). Schwann cells were dissociated and
purified from neonatal rat sciatic nerves (56). Purified Schwann
cells were cultured on poly-L-lysine-coated slides in DMEM
supplemented with 10% fetal calf serum, 2 µM forskolin
(Calbiochem), and 5 ng/ml glial growth factor (a gift from Mark
Marchionni (Cambridge Neuroscience)). PC12 and COS-7 cells were
maintained in 5% CO2 at 37 °C. Schwann cells were
maintained in 7.5% CO2 at 37 °C. For
immunocytochemistry, cells were plated and transfected in eight-well
Permanox chamber slides (Nunc, Naperville, IL). For biochemical
experiments, cells were plated in 100-mm culture dishes.
Plasmids were prepared for transfection using Qiagen MaxiPrep kits.
Transfection of COS-7 cells using DEAE-dextran was performed as
described previously (34) using 0.2 µg of DNA/well and 10 µg of
DNA/100-mm dish. Transfection of PC 12 cells was performed using
LipofectAMINE 2000 (Life Technologies) according to the manufacturer's
instructions. PC12 cells were transfected 1 day after plating (~90%
confluent); 1 µg of DNA was used per well. Schwann cell cultures were
transfected when 50-80% confluent. Transfection of Schwann cells was
performed with FuGene reagent (Roche Molecular Biochemicals) according
to the manufacturer's directions. For each well, 0.75 µl of FuGene
was mixed with 25 µl of serum-free DMEM and then incubated with 0.5 µg of DNA for 15 min at room temperature prior to adding to the media
of the cultured cells. COS-7 and PC 12 cells were processed 72 h
after transfection. Schwann cells were analyzed 48 h after transfection.
Membrane Association Analysis--
COS-7 cells transfected with
the III-
Triton X-114 partitioning experiments were performed using a
modification of Bordier's original protocol (35). The membrane pellet
(see above) was resuspended in 960 µl of hypotonic lysis buffer and
mixed with 240 µl of 8% (v/v) Triton X-114 in 10 mM Tris, pH 7.5, 150 mM NaCl (final volume 1.2 ml; final
Triton X-114 concentration of 1.6%). This mixture was centrifuged at
125,000 × g for 1 h at 4 °C using a Beckman
TLA 100.3 rotor to produce a soluble and an insoluble (pellet)
fraction. To induce phase separation, 200 µl of the soluble fraction
was warmed to 28 °C for 3 min. The warmed mixture was then layered
onto a 6% sucrose cushion (300 µl) in a microcentrifuge tube and
centrifuged at 200 × g for 5 min. This resulted in a
three-phase solution: aqueous, top; sucrose cushion, middle; detergent,
bottom. The aqueous phase was mixed with an equal volume of 2× sample
buffer. The detergent phase was adjusted to 200 µl with lysis buffer
and then mixed with an equal volume of 2× sample buffer.
Lysate Preparation and Western Blot Analysis--
To prepare
lysates for Western blot analysis, cells in 100-mm dishes were rinsed
twice with HBS (20 mM Hepes, pH 7.4, 150 mM
NaCl, 2 mM CaCl2) and lysed by scraping in 400 µl of lysis buffer (1% SDS, 20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl). The lysate was passed
through a 27-gauge needle four times to shear DNA and cleared of
insoluble material by centrifugation at 15,000 × g for
20 min at 4 °C. The samples analyzed in Fig. 2 were total cell
lysates, not cleared lysates. These samples were prepared by harvesting
the cells directly in 400 µl of 2× SDS sample buffer/dish.
Western blot analysis was performed as described elsewhere (34).
Samples were heated for 5 min at 95 °C prior to being loaded on a
gel for analysis. The antibody dilutions used were as follows:
Experiments illustrated in the figures were repeated three times (Figs.
2-4, 6, and 7) or twice (Figs. 5 and 8). Each repetition gave results
similar to those illustrated. For quantitation of cell surface or
released NRG, samples to be compared were analyzed on a single blot
along with multiple dilutions of a standard sample. Films were scanned,
and band "volume" (summed pixel density) was measured using the NIH
Image software. A standard curve was created by plotting the pixel
density for each dilution of the standard sample as a function of the
relative amount loaded. The relative amount of NRG in each test sample
was calculated based on this standard curve.
Cell Surface Biotinylation--
For each 100-mm dish of
transfected COS-7 cells, the cells were washed twice with 5 ml of
ice-cold HBS and then incubated in 3 ml of HBS containing 0.5 mg/ml
sulfo-NHS-LC-biotin (Pierce) for 45 min at 4 °C. Following removal
of the sulfo-NHS-LC-biotin solution, residual sulfo-NHS-LC-biotin was
quenched by incubating with 10 mM glycine in HBS for 5 min.
Cleared lysate (prepared as described above) was mixed with an equal
volume of TENT buffer (20 mM Tris HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100) and
incubated with 50 µl of streptavidin-conjugated agarose beads (Pierce) for 1 h at 4 °C with end-over-end mixing. The agarose beads were washed three times with lysis buffer and eluted in 2× SDS
sample buffer. A gel sample was also prepared from the cleared lysate
to assess the total amount of NRG proteins (cell surface plus internal)
expressed in each dish.
Analysis of Conditioned Medium and Phorbol 12-Myristate
13-Acetate (PMA) Stimulation--
48 h after transfection, COS-7 cells
were washed once with PBS, and the standard growth medium was replaced
with Opti-MEM I (Life Technologies). 12 h later, this medium was
replaced with 5 ml of fresh Opti-MEM I per 100-mm dish with or without
100 nM PMA, and the cells were returned to the incubator.
Following a 30-min (Fig. 6) or 2-h (Fig. 8) incubation at 37 °C, the
conditioned medium was collected and filtered through a 0.22-µm
filter (Millipore Corp.) to remove floating cells. The medium was then
concentrated 40 fold by ultrafiltration using Centricon-10 units
(Millipore). To assess the effect of PMA on cell surface NRG, the same
treatment protocol was followed. At the end of the 30-min incubation
with PMA, the cells were rinsed twice with ice-cold HBS, and
biotinylation with sulfo-NHS-LC-biotin was performed as described above.
Immunocytochemistry--
To assess cell surface NRG, cells were
washed twice with ice-cold serum-free DMEM and then incubated in
primary antibody solutions for 30 min on ice, washed again with
ice-cold serum-free DMEM, and incubated in secondary antibody solution
for 30 min on ice. Cells were then washed and fixed with 2%
paraformaldehyde containing 0.1% Triton X-100 in PBS for 20 min on
ice. Fixed cells were blocked in 10% normal donkey serum diluted in
PBS (blocking buffer) for 30 min at room temperature. For labeling of
intracellular epitopes, these fixed and permeabilized cells were
incubated in primary antibody solution for 1 h at room temperature
and then washed in PBS (3 times for 5 min each). Cells were then
incubated in the appropriate secondary antibody solution for 1 h
at room temperature. Cells were washed with PBS and mounted with
Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA). As a
negative control to show that cell surface label was not due to
antibody internalization, cells were incubated in the
Images were collected using a Bio-Rad 1024 laser-scanning confocal
system (Bio-Rad) coupled to a Zeiss Axioskop microscope. Images shown
in the figures were obtained using a 20× Plan-Neofluar (NA 0.5) or
40× Plan-Neofluar (NA 0.75) lens. A Z-series of images was collected
for each preparation at a step of 0.5 µm. The images shown were
compiled from a stack of five individual Z-series images using the
Bio-Rad Lasersharp software. Images were further processed using
Adobe Photoshop. For each cell type, all images of the same fluorophore
were collected and processed identically.
To compare the processing of NRG III-1a and the type I
neuregulin I-
1a. The sequence of these two isoforms differs only in
the region that is N-terminal of the bioactive epidermal growth
factor-like domain. While the biosynthetic processing of the
I-
1a isoform has been well characterized, the processing of
III-
1a has not been reported. In this study, we compared III-
1a
and I-
1a processing. Both III-
1a and I-
1a were synthesized as
transmembrane proproteins that were proteolytically cleaved to produce
an N-terminal fragment containing the bioactive epidermal growth
factor-like domain. For I-
1a, this product was released into the
medium. However, for III-
1a, this product was a transmembrane
protein. In cultures of cells expressing III-
1a, the amount of
neuregulin at the cell surface was much greater, and the amount in the
medium was much less than in cultures expressing I-
1a. Phorbol ester
treatment and truncation of the cytoplasmic tail had markedly different effects on III-
1a and I-
1a processing. These results demonstrate an important role for the N-terminal region in determining neuregulin biosynthetic processing and show that a major product of III-
1a processing is a tethered ligand that may act as a cell surface signaling molecule.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3 was the first reported NRG isoform with a type III
N-terminal region (17). This isoform, originally referred to as SMDF,
has a
3 type of EGF-like domain, and thus lacks the transmembrane
domain C-terminal of the EGF-like domain (Fig. 1A). Subsequent to the isolation of the III-
3 isoform, a III-
1a
isoform (Fig. 1A) was discovered (Refs. 18-20; see also
Ref. 27). Based on RNA studies (10, 12, 18-20, 28-30), it is likely
that the III-
1a isoform is one of the most abundant NRG isoforms in
the late embryonic and postnatal nervous system and that the III-
3 isoform is relatively rare.
1a and I-
1a isoforms differ in their N-terminal regions
but are identical in their EGF-like, "stalk," transmembrane, and
cytoplasmic regions (Fig. 1A). Based on the similarity in structure of the III-
1a and I-
1a isoforms, schematic diagrams (4,
8) have represented the topology of III-
1a as similar to I-
1a
(Fig. 1, B and C). This model of III-
1a
topology suggests that III-
1a, like I-
1a, will be cleaved in the
stalk region and that the consequence of this cleavage will be release
of the entire N-terminal fragment into the extracellular fluid. To test this model and to determine whether the cellular mechanisms that regulate I-
1a topology and biosynthetic processing also govern III-
1a, we have compared III-
1a processing to I-
1a processing using biochemical and immunocytochemical techniques.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1a" refers to a NRG with a "type III"
N-terminal region, a "
" type EGF-like domain, a "1" type
sequence at the carboxyl terminus of the EGF-like domain, and an
"a" type cytoplasmic tail (see Fig. 1A for
illustration). NRG isoforms can differ in the sequence of their
N-terminal region (type I, II, and III), EGF-like domain (
or
),
the C-terminal end of the EGF-like domain (1, 2, 3, or 4), and
cytoplasmic tail (a, b, c, or none). In NRG isoforms with
1,
2,
or
4 EGF-like domains, there is a stalk region, transmembrane
domain, and cytoplasmic tail C-terminal of the EGF-like domain. (The
stalk is the sequence between the EGF-like domain and the transmembrane
domain.) These features are not found in NRG isoforms with a
3
EGF-like domain. Most NRGs in the nervous system have a
-type
EGF-like domain and a-type tail (12, 28). The amino acid sequence of
rat NRG I-
1a can be found in Ref. 12, and the amino acid sequence of the rat type III N terminus can be found in Ref. 19.
1a proprotein has a cytoplasmic tail at both its N terminus and
C terminus. However, to be consistent with the published literature,
the term "cytoplasmic tail" used without qualification refers to
the cytoplasmic region C-terminal to the EGF-like domain. For example,
III-
1a has the "a-tail" type of cytoplasmic tail.
1a (labeled
"native") used in the experiment illustrated in Fig. 2. Standard
recombinant DNA techniques (31, 32) were used to create expression
vectors for NRG proteins with an HA epitope tag in the ectodomain, just
N-terminal of the EGF-like domain. The sequences of I-
1a and
III-
1a differ at their N-terminal ends but are identical from a
serine residue located 14 amino acids N-terminal of the first cysteine
residue of the EGF-like domain through their C termini (Fig.
1A). The PCR-based strategy used to insert the HA tag
changes the sequence between this serine and cysteine from
STSTSTTGTSHLIKC to STSTSTTGTSIDYPYDVPDYASLHLIKC (underlined residues represent HA epitope tag). All HA-tagged constructs used contained this identical sequence within the
ectodomain. A similar polymerase chain reaction-based strategy was used
to introduce a FLAG epitope tag at the N terminus of an HA-tagged III-
1a construct for examination of the membrane orientation of the
III-
1a N-terminal fragment. In this FLAG-III-
1a construct, the
N-terminal sequence was changed from MEIYSP ... to
MDYKDDDKEFGGMEIYSP ... (underlined sequence
represents FLAG tag). The fidelity of all sequences amplified by
polymerase chain reaction was verified by DNA sequencing. The vector
backbone for all constructs was pcDNA 3.1 (Invitrogen). The tail
deletion constructs and NRG I-
1c construct included a C-terminal Myc
epitope tag. The Myc tag was not utilized in this study. Details
regarding the primers used and the specific procedures employed to
create these constructs are available upon request.
1a construct were washed twice with PBS (0.1 M
phosphate buffer, pH 7.4, 150 mM NaCl). One ml of hypotonic
lysis buffer (20 mM Hepes, 2 mM
MgCl2, pH 7.4) was added per 100-mm dish of transfected
cells. The cells were collected in this buffer using a rubber policeman
and passed through a 21-gauge needle 30 times. Nuclei and unbroken
cells were removed by centrifugation at 200 × g for 10 min at 4 °C. The supernatant from this low speed spin
(Input in Fig. 5A) was centrifuged at
125,000 × g for 1 h at 4 °C using a Beckman
TLA 100.3 rotor. To analyze the proportion of cellular III-
1a
proprotein and its products bound to membranes, the supernatant
(S2) and membrane pellet (P2) from this high
speed spin were prepared in SDS sample buffer. For membrane extraction
experiments, the membrane pellet was extracted on ice for 30 min in
hypotonic lysis buffer with 1 M KCl or with 50 mM Na2CO3, pH 12. Following
extraction, the samples were centrifuged at 125,000 × g for 1 h at 4 °C to separate the soluble
(S; supernatant) and insoluble (I; pellet) fractions. An equal percentage of the starting material for supernatant and pellet fractions was analyzed by Western blotting.
-HA
monoclonal antibody 16B12 (raw ascites fluid; Berkeley Antibody Company, Richmond, CA), 1:3000;
-NRG a-tail rabbit polyclonal antibody SC348 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), 0.1 µg/ml; horseradish peroxidase-conjugated goat anti-rabbit (Pierce),
1:50,000; horseradish peroxidase-conjugated donkey anti-mouse (Jackson
ImmunoResearch Laboratories), 1:50,000. Blots were developed using
Renaissance chemiluminescence substrate (PerkinElmer Life Sciences). Prestained standards used on Western blots (BenchMark protein ladder; Life Technologies) were calibrated against unstained molecular weight markers (Bio-Rad). A standard curve (distance migrated
versus log Mr) was created
based on the migration of the marker proteins, and molecular weights
were assigned to each band of interest according to the position of the
center of the band.
-a-tail and
secondary antibodies prior to permeabilization, as described above.
When
-a-tail was applied prior to permeabilization, no labeling of
cells was detected (not shown). Primary and secondary antibodies for
cell surface label were diluted in serum-free DMEM. Primary and
secondary antibodies for intracellular staining were diluted in
blocking buffer. Antibody dilutions were as follows:
-HA monoclonal
16B12, 1:500;
-FLAG monoclonal M2, 17 µg/ml;
-NRG a-tail rabbit
polyclonal antibody SC348, 0.5 µg/ml; fluorescein
isothiocyanate-conjugated donkey anti-mouse, 1:100;
lissamine-rhodamine sulfonyl chloride-conjugated donkey
anti-rabbit, 1:1000. Secondary antibodies were purchased from Jackson
ImmunoResearch Laboratories, Inc.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1a with that of I-
1a,
we constructed cDNAs encoding NRG isoforms with an HA epitope tag
inserted immediately N-terminal of the EGF-like domain (Fig. 1D). The use of a shared tag
allowed us to directly infer the relative amount of III-
1a and
I-
1a ectodomain from signal strength in immunoassays. The position
chosen for the HA tag corresponded to the position used in a previous
study of transforming growth factor-alpha (TGF
) processing (36). By
using antibodies to the HA tag and to an epitope located in the
cytoplasmic a-tail, we were able to detect both the N-terminal fragment
(NTF) and the C-terminal fragment (CTF) of NRG proproteins
proteolytically cleaved in the stalk region. We have also tested a
similarly constructed HA-tagged III-
3. Our results with the
HA-tagged I-
1a and III-
3 proteins are consistent with previous
studies of native I-
1a and III-
3 isoforms and studies of these
isoforms with epitope tags located at the extreme N or C terminus (12,
22, 24, 25, 37). Therefore, the processing of the HA-tagged NRG
proteins does not seem to differ substantially from processing of the
native forms of these proteins.
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Fig. 1.
Structure of the
I- 1a, III-
1a, and
III-
3 NRG isoforms and constructs used in this
study. A, structural regions of the full-length
I-
1a, III-
1a, and III-
3 proteins. See "Neuregulin Isoforms
and Nomenclature" for discussion of NRG isoform names. III-
1a and
I-
1a differ only in their N-terminal region; their sequence is
identical from the EGF-like domain through the carboxyl terminus. The
sequence of III-
1a and III-
3 is identical from the N terminus to
just beyond the end of the EGF-like domain. The III-
3 isoform
terminates 20 amino acids following the last cysteine of the EGF-like
domain and thus lacks the stalk, transmembrane, and cytoplasmic regions
shared by III-
1a and I-
1a. Based on hydrophobicity analysis,
III-
3 and I-
1a each have one potential transmembrane domain
(black boxes). III-
1a contains both of these
potential transmembrane domains. The amino acids that define the
1 and 3 EGF-like domain subtypes are shown
below the diagrams of III-
1a and III-
3, respectively.
Numbers above each diagram indicate the position
of the first or last amino acid of various structural regions within
the protein. B, membrane orientation and processing proposed
for the I-
1a isoform. The EGF-like domain in the NRG ectodomain is
alone sufficient for high potency activation of the cognate receptors,
ErbB2, -3, and -4. I-
1a is synthesized as an
Nout/Cin transmembrane protein. Proteolytic
cleavage in the "stalk" region (arrow) produces an NTF
containing the bioactive EGF-like domain and a CTF, also referred to as
the "a-tail remnant." The NTF is efficiently released into the
medium. C, model for III-
1a membrane orientation and
processing derived from I-
1a model. This model predicts that the CRD
is within the ectodomain and that stalk cleavage creates a soluble
ectodomain fragment containing both the EGF-like domain and CRD domain.
D, constructs used in this study. An HA epitope tag (*) was
introduced immediately N-terminal of the EGF-like domain. The
FLAG-III-
1a construct included, in addition, an N-terminal FLAG tag.
For one series of experiments, we created constructs encoding NRGs with
cytoplasmic tail truncations. Instead of the 374-amino acid a-tail,
these forms had tail lengths of 79 amino acids (III-
1-T79), 45 amino
acids (III-
1-T45), or 13 amino acids (III-
1-T13 and I-
1-T13).
In this series of experiments, we also tested I-
1c, an NRG form with
a 157-amino acid tail. The location of the epitope for the a-tail
antibody (
-a-tail) used is indicated by a thick
underbar. The number of the first and last amino acid in
some regions of the proteins is indicated above the
diagrams.
The III-1a Proprotein Is Proteolytically Processed to a 76-kDa
N-terminal and a 60-kDa C-terminal Fragment--
To determine the size
of full-length III-
1a and of fragments that might be derived from
this, we transfected COS-7 cells with a III-
1a expression vector and
analyzed cell lysates by Western blot. Bands of 140 and 110 kDa were
labeled by both the
-HA and
-a-tail antibodies (Fig.
2A, lanes
2 and 2'; Fig. 2B, lane
2). Since the 140- and 110-kDa proteins have both an
ectodomain (HA) and a-tail epitope, these are III-
1a proprotein
forms. The predicted size of the peptide encoded by the III-
1a
cDNA is 78 kDa (including 1.5 kDa of mass contributed by the HA
tag), which is considerably smaller than the observed size of the
III-
1a proproteins. The larger than predicted mass of these
proprotein forms might be due to posttranslational modifications such
as glycosylation.
|
We also observed a band of 76 kDa that labeled only with -HA and a
broad 60-kDa band that labeled only with the cytoplasmic domain
antibody. These results suggest that the 76-kDa protein is an NTF and
the 60-kDa protein is a CTF derived from the III-
1a proprotein by
proteolytic cleavage. Consistent with this interpretation, the sum of
these fragment sizes is 136 kDa, approximately equal to the size of the
140-kDa protein labeled by both antibodies. The III-
1a CTF is
similar in size to the I-
1a CTF (Fig. 2B). Since the
cleavage site of I-
1a has been shown to be in the ectodomain stalk
region (23, 25), the similar size of the III-
1a and I-
1a CTFs
indicates that III-
1a is also processed by cleavage in the stalk
region. The 76-kDa NTF band was much more intense than the 140- and
110-kDa proprotein bands, indicating that the NTF, not the proprotein,
is the predominant ectodomain containing NRG form in cells expressing
III-
1a. It is noteworthy that proteins similar in size to the
III-
1a NTF (76 kDa) and larger proprotein form (140 kDa) have been
detected in Western blot analyses of brain and spinal cord samples
developed with pan-NRG antibodies (20, 38, 39).
Consistent with previously published results (24), Western analysis of
lysates from cells expressing I-1a revealed prominent bands of 110 and 95 kDa representing the I-
1a proprotein and a broad band of
45-kDa protein representing the I-
1a NTF (Fig. 2, A and
B, lanes 4 and 5). We also
routinely observed a series of fainter bands extending from ~43 to 30 kDa on blots developed with
-HA but not on blots developed with
-a-tail. These bands may represent proteolytic degradation products
of the I-
1a proprotein or NTF.
Western analysis of lysates from cells expressing III-3 (SMDF) was
also consistent with published results (37). In these lysates, a band
of 83 kDa, representing full-length III-
3, was labeled by the
-HA
antibody (Fig. 2A, lane 3). The
sequence of the III-
1a NTF, which is created by cleavage of the
III-
1a proprotein in the stalk region (see above), should be
identical to the sequence of III-
3 except in the region C-terminal
of the EGF-like domain (Fig. 1A). The slightly larger size
of the full-length III-
3 relative to the III-
1a NTF (83 versus 76 kDa) might be due to differences in glycosylation.
Consistent with this possibility, the ectodomain sequence unique to the
3 type of EGF-like domain is rich in serine.
In summary, Western blot analysis indicates that the III-1a
proprotein is proteolytically processed in the stalk region and that
the predominant cellular form that includes the ectodomain differs
between III-
1a- and I-
1a-expressing cells. In
III-
1a-expressing cells, the major form that includes the ectodomain
is the NTF resulting from stalk cleavage, but in I-
1a-expressing
cells, it is the proprotein. The bands seen in lysates of III-
1a-,
I-
1a-, and III-
3-expressing cells are listed in Table
I. Table I also summarizes information
from experiments described below that define NRG protein fragments at
the cell surface and released into medium.
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The NTF Produced by Cleavage of the III-1a Proprotein
Accumulates at the Cell Surface--
To determine whether the
N-terminal region of NRG isoforms affects the amount of NRG ectodomain
exposed at the cell surface, we first compared the cellular
distribution of III-
1a and I-
1a using immunocytochemical methods.
Since neurons (10, 17-19, 30) and Schwann cells (20, 21) are
known to express NRGs, we examined NRG distribution in
NGF-differentiated PC12 cells (cells that have neuronal
characteristics) and primary cultures of Schwann cells as well
as in COS-7 cells. Transfected PC12 cells (Fig. 3, A and B),
Schwann cells (Fig. 3, C and D), and COS-7 cells (Fig. 3, E and F) gave similar results.
Nonpermeabilized cells (
) expressing III-
1a were strongly labeled
with
-HA, whereas no specific surface labeling of I-
1a-expressing
cells was detected. Since NRG activity has been demonstrated in axonal
membranes (40, 41), it is noteworthy that the surface of neurites as
well as the cell body was strongly labeled in PC12 cells expressing
III-
1a (Fig. 3A). Permeabilized cells (+) expressing
III-
1a and I-
1a were labeled by
-a-tail (Fig. 3,
A-F) or
-HA (not shown) with similar intensity,
suggesting that the total amount of NRG is roughly similar in cells
expressing III-
1a and I-
1a. The larger cell surface accumulation
of III-
1a than I-
1a for all three cell types examined suggests
that cell biological mechanisms common to many cell types govern these
differences.
|
Cell surface biotinylation (Fig. 4) was
used to determine more quantitatively the relative amounts of the NRG
proproteins and their products exposed at the cell surface. In
III-1a-expressing cells, most of the NRG that was biotinylated was
the 76-kDa NTF (Fig. 4A). In long exposures of blots
developed with the
-a-tail antibody, faint bands representing cell
surface 140-kDa proprotein and 60-kDa CTF were also detected (not
shown), but the 110-kDa proprotein band was not observed.
|
Although for I-1a-expressing cells, cell surface NRG was not
observed by immunocytochemistry (Fig. 3), it was detected in the
biotinylation experiments (Fig. 4A). This is consistent with the results of others, who have detected low levels of type I NRGs at
the cell surface by immunohistochemistry or cell surface biotinylation
(22, 24). The most abundant NRG forms at the surface of the
I-
1a-expressing cells were the 45-kDa NTF and the 110-kDa proprotein
(Fig. 4A). In the experiment shown in Fig. 4, the
predominate form was the 45-kDa form, but in other experiments, the
amount of 110-kDa proprotein at the surface exceeded the amount of the
45-kDa form (see, for example, Fig. 7). The I-
1a NTF might accumulate at the cell surface due to interaction between the Ig-like
domain of the type I NRG ectodomain and cell surface heparin sulfate
proteoglycans (42). We did not detect the 95-kDa I-
1a proprotein at
the cell surface.
The relative amounts of NRG ectodomain exposed at the cell surface of
III-1a- and I-
1a-expressing cells were estimated by quantitating
the biotinylated protein bands (Fig. 4C). Assuming the two
proteins were equally biotinylated, there was ~25 times more
III-
1a ectodomain at the cell surface than I-
1a ectodomain. Furthermore, not only was there much more NRG ectodomain at the surface
of III-
1a-expressing cells, but also a larger proportion of the
cellular NRG was at the surface, since III-
1a lysates contained less
NRG than the I-
1a lysates (Fig. 4B). Results of experiments in which cell surface proteins were digested with trypsin
are consistent with this
interpretation.2 In these
experiments, a large percentage of the cellular III-
1a was degraded,
but only a tiny percentage of the total cellular I-
1a 110- and
45-kDa forms were degraded
In summary, both immunocytochemical and biochemical experiments
indicate that cells expressing III-1a have much more NRG ectodomain
exposed at their cell surface than cells expressing I-
1a, and this
difference appears to be largely due to the accumulation of the NTF of
III-
1a in the plasma membrane.
The N-terminal Product Produced by Cleavage of the III-1a
Proprotein Is a Transmembrane Protein--
The accumulation of the
III-
1a NTF at the cell surface (Figs. 3 and 4) and the presence of a
potential transmembrane domain within the CRD (Fig. 1A)
suggested the possibility that the III-
1a NTF is a transmembrane
protein. To test this hypothesis, we first assessed the proportion of
cellular III-
1a associated with membranes. After high speed
centrifugation, none of the NTF in a cleared cell lysate was found in
the supernatant, the fraction that contains the soluble, cytoplasmic
proteins (Fig. 5A). Next we
determined whether the NTF behaves as an integral or a peripheral
membrane protein. We incubated a membrane fraction prepared from cells expressing III-
1a in 1 M potassium chloride or sodium
bicarbonate, pH 12, and then separated the membrane-associated proteins
from soluble proteins by centrifugation. With both of these treatments, the III-
1a NTF remained associated with the membranes (Fig.
5B), the behavior expected for an integral membrane protein.
The NTF also largely partitioned into the detergent phase in Triton
X-114 phase partitioning experiments (Fig. 5B), further
evidence that the NTF is an integral membrane protein. Finally, we
examined whether the NTF has intracellular as well as extracellular
epitopes. When a III-
1a protein with an N-terminal FLAG epitope tag
was expressed in COS-7 cells, the FLAG tag could only be detected in
permeabilized cells (Fig. 5C), indicating that this epitope is cytoplasmic, whereas an HA epitope tag located just N-terminal of
the EGF-like domain is exposed on the cell surface (Fig. 3). Together,
these data indicate that the III-
1a NTF is an
Nin/Cout transmembrane protein.
|
The III-1a Ectodomain Is Inefficiently Shed into the Medium
Compared with the I-
1a Ectodomain--
Previous studies have
demonstrated that the ectodomain of I-
1a is efficiently released
from cells (24) and that this release is accelerated by treatment with
phorbol esters (22, 24, 25), compounds that activate protein kinase C. To compare the release of the III-
1a and I-
1a ectodomain, medium
conditioned by I-
1a- and III-
1a-expressing cells was assayed for
NRG protein by Western blot. In medium conditioned by
I-
1a-expressing cells, an HA-immunoreactive peptide of 45-kDa was
observed (Fig. 6A,
lane 1). In medium conditioned by
III-
1a-expressing cells, a faint 63-kDa band was detected (Fig.
6A, lane 3). A soluble NRG ectodomain
fragment of this size could be produced by proteolytic cleavage of the
76-kDa III-
1a NTF between the HA tag and the TM domain within the
CRD (see Fig. 1). Strikingly, the concentration of NRG in the medium
conditioned by III-
1a-expressing cells for 30 min was only ~10%
of the NRG concentration in medium conditioned by I-
1a-expressing
cells (Fig. 6C). Thus, while there is much more III-
1a
ectodomain at the cell surface (Figs. 3 and 4), there is much less
III-
1a ectodomain in conditioned medium.
|
The effects of PMA treatment on NRG release (Fig. 6) and cell surface
NRG (Fig. 7) were very different for
cells expressing III-1a and I-
1a. Treatment of I-
1a-expressing
cells with PMA resulted in a dramatic increase in the amount of NRG in
conditioned medium (Fig. 6A, lane 1 versus lane 2; Fig. 6C) and
a dramatic decrease in the amount of NRG at the cell surface (Fig. 7,
lane 6 versus lane
5), but similar treatment of III-
1a-expressing cells with
PMA had little effect on either the release of NRG into the medium
(Fig. 6A, lane 3 versus
lane 4; Fig. 6C) or the amount of cell
surface NRG (Fig. 7, lane 3 versus
lane 2). These results demonstrate that the
mechanisms regulating NRG release and cell surface accumulation are
different for III-
1a and I-
1a.
|
Truncation of the Cytoplasmic Tail Has Little Effect on the Cell
Surface Accumulation and Proteolytic Processing of III-1
NRGs--
For type I TM-NRGs, truncation of the cytoplasmic tail to
less than ~90 amino acids has been shown to block release of the ectodomain into the medium (7, 26). Since III-
1a and I-
1a have
identical sequence in the EGF-like, stalk, TM, and cytoplasmic tail
regions (Fig. 1A), we investigated whether shortening of the
cytoplasmic tail would similarly affect III-
1a and I-
1a cell
surface accumulation and release.
The amount of NRG at the cell surface and released into the medium was
similar for COS-7 cells expressing III-1a (374-amino acid tail) and
III-
1 proteins with a truncated tail (Fig.
8A, lanes
3-6, and C, lanes 2-5).
However, the amount of cell surface and released NRG was markedly less
in cultures of cells expressing type I NRG with a truncated tail
(I-
1-T13) than cultures of cells expressing longer, naturally
occurring tail forms (I-
1a and I-
1c) (Fig. 8A,
lanes 8-10, and C, lanes
6-8). Thus, while the cytoplasmic tail is essential for the
cell surface localization and release of NRGs with a type I N terminus,
it does not serve a similar function for NRGs with a type III N
terminus.
|
![]() |
DISCUSSION |
---|
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---|
Based on the identity of the III-1a and I-
1a sequence in the
regions known to be important for processing of I-
1a, it has been
assumed that the topology and processing of III-
1a would be similar
to I-
1a (see Fig. 1, B and C). Our results
show that this is not the case. Cultures expressing III-
1a had ~25
times more NRG at the cell surface and ~10 times less NRG in the
medium than parallel cultures expressing I-
1a. Truncation of the
cytoplasmic tail had little effect on the accumulation of NRG at the
surface of III-
1a-expressing cells, and treatment with phorbol
esters had little effect on the release of soluble NRG from these cells.
Taken together, our data support the alternative model for the membrane
topology and processing of III-1a illustrated in Fig.
9. Proteolytic cleavage in the stalk
region (Fig. 9, site 1) creates an NTF that is an
Nin/Cout transmembrane protein. Since in
immunohistochemical experiments epitopes located both at the N and C
terminus of the III-
1a proprotein are only detected following cell
permeabilization (Figs. 3 and 5), we propose that the proprotein passes through the membrane twice.
|
In cells expressing III-1a, the amount of 76-kDa N-terminal fragment
is large relative to the amount of proprotein (Fig. 2), indicating that
most of the NRG in these cells has been cleaved at site 1 but not site
2. Cleavage of III-
1a at both site 1 and site 2 is required for
release of the 63-kDa ectodomain fragment into culture medium. Since
the only cell-associated III-
1a cleavage product observed was the
76-kDa NTF, the model depicts cleavage at site 1 as preceding cleavage
at site 2. A candidate site for cleavage 2 is the peptide bond between
Gly117 and Leu118, since when a NRG
III-
3-immunoglobulin chimera was expressed in 293 cells, the
released ~63-kDa protein had the corresponding leucine as its N
terminus (43).
It is noteworthy that, in contrast to the model shown in Fig.
1C, the model suggested by our data (Fig. 9) predicts that
most of the sequence of the CRD is within the membrane or cytoplasm and
is not a component of the released ectodomain. We expect the model of
Fig. 9 to hold also for the processing of III-2a, the other major
type III NRG known to be expressed in the nervous system, since this
isoform is identical to III-
1a except that it lacks the eight
1-specific amino acids at the C-terminal end of the EGF-like domain.
The amino acid sequence of the III-1a NTF is identical to the
sequence of the full-length III-
3 except very near the carboxyl terminus (Fig. 1). A previous study of III-
3 (37) and our own data2 demonstrate that this isoform has many
characteristics similar to those that we have observed for the
III-
1a NTF. When expressed in fibroblastic cells, full-length
III-
3 is an Nin/Cout transmembrane protein
that accumulates at the cell surface. Cleavage of III-
3 at a site
similarly located to site 2 (Fig. 9) results in the release of a
III-
3 ectodomain fragment, but the levels of this fragment in medium
are low compared with the levels of released I-
1a ectodomain
fragment in medium from parallel cultures. The similarity in behavior
of III-
3 and the III-
1a NTF suggests that if the biological
activities of III-
3 differ from the activities of III-
1a, these
differences will be attributable to activities of the intact III-
1a
proprotein or C-terminal fragment.
Limitations imposed by the available NRG antibodies have prevented
detailed study of NRG processing in cells natively expressing NRG
proteins. Thus, previous studies of NRG processing have employed COS-7,
CHO, or 293 cells transfected with NRG expression constructs (22, 24,
25, 37). Similarly, in this study we compared the processing of NRG
III-1a with I-
1a in COS-7 cells transfected with NRG expression
vectors. While such expression studies have the potential disadvantage
that some aspects of NRG processing might differ in natively expressing
cells, the use of transfected fibroblastic cells for biochemical
studies has proven to be a very fruitful approach in examining the
biosynthetic processing of TGF
and other transmembrane ligands (44).
Further, cell surface NRG was greater for III-
1a- than
I-
1a-expressing cells, regardless of whether these NRGs were
expressed in COS-7 cells, PC12 cells, or Schwann cells (Fig. 3). That
this central result of our study holds true for each cell type tested
suggests that the processing of III-
1a is similar in COS-7 cells,
neuronal cells, and Schwann cells.
Several characteristics of transmembrane type I NRG topology and
processing fit a model previously established for TGF and other EGF
receptor ligands that are synthesized as transmembrane proproteins (44,
45). For both TGF
and NRG I-
1a, stalk cleavage results in release
(or "shedding") of a soluble ectodomain product, and this release
is activated by protein kinase C (Figs. 6 and 7; Refs. 22, 24, 25, 46,
47). Also for both, transport of the protein to the cell surface and
subsequent ectodomain release are regulated by the cytoplasmic
tail (Fig. 8; Refs. 7, 26, 36, 48, and 49). Since the sequence of the
NRG III-
1a and I-
1a EGF-like domain, stalk, transmembrane domain,
and cytoplasmic tail are identical (Fig. 1), it might have been
expected that III-
1a would fit the same pattern, but it does not.
Activation of protein kinase C by treatment with phorbol esters caused
little change in the amount of III-
1a at the cell surface or in the medium. This indicates that cleavage at site 2 (Fig. 9), unlike stalk
cleavage (site 1), is insensitive to protein kinase C. Therefore, release of the III-
1a ectodomain is not governed by the same proteolytic machinery as release of I-
1a, TGF
and a number of other transmembrane proteins. The failure of tail truncation to substantially reduce cell surface III-
1 indicates that the
mechanisms responsible for trafficking III-
1a and I-
1a to the
cell surface also differ.
One known biochemical difference between type I and type III NRGs that is likely to have significant cell biological consequences is that type I NRGs bind strongly to heparin sulfate proteoglycans, whereas type III NRGs do not (42, 50). Consistent with this, evidence indicates that the released ectodomain of type I TM-NRGs is deposited in the basal lamina of neuromuscular synapses and the extracellular matrix of brain (42, 50, 51). A second reported difference between these classes of NRGs is that soluble, recombinant type III NRG is more potent than type I NRG in inducing nicotinic AChR subunit gene expression in cultured sympathetic neurons (18). The results presented here add new evidence that the cell biological properties of type III NRGs differ substantially from the properties of type I NRGs.
The differences between cells expressing III-1a and I-
1a with
respect to cell surface and released NRG suggest the hypothesis that
III-
1a is preferentially employed in vivo for cell-cell communication requiring a juxtacrine (direct contact) mode of interaction, whereas I-
1a is preferentially employed for paracrine signaling. In fact, the small amount of III-
1a ectodomain in culture
medium (Fig. 6) raises the question of whether release of the type III
ectodomain has any physiological role. To date, two cell-cell
interactions mediated by membrane-associated NRG activity have been
identified. One is the induction by differentiating neuroblasts of
glial commitment in neural crest stem cells (52). The second is the
induction of Schwann cell proliferation by the axonal membranes of
sensory neurons (40, 53-55). The N-terminal fragment of III-
1a (or
the closely related III-
2a) is a strong candidate for being the NRG
form mediating these interactions, since both neural crest-derived
neuroblasts and sensory neurons express type III NRGs (19, 52) and
since Schwann cells along peripheral nerves are markedly depleted in
the type III NRG knockout (8). Future studies must elucidate whether
type III NRGs are active only in direct contact cell-cell interactions
in vivo and determine the novel mechanisms regulating type
III NRG biogenesis.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Duanzhi Wen (Amgen, Inc.) for providing NRG cDNA clones. We thank Nancy Ratner (University of Cincinnati) and members of the Falls laboratory for many helpful comments on the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM 56337 and by a grant from the Emory University Research Committee.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.
§ To whom correspondence should be addressed: Dept. of Biology, Emory University, Atlanta, GA 30322. Tel.: 404-727-0520; Fax: 404-727-2880; E-mail: dfalls@emory.edu.
Published, JBC Papers in Press, October 20, 2000, DOI 10.1074/jbc.M005700200
2 J. Wang and D. Falls, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
NRG, neuregulin;
CRD, cysteine-rich domain;
CTF, C-terminal fragment;
DMEM, Dulbecco's modified Eagle's medium;
EGF, epidermal growth factor;
NTF, N-terminal fragment;
PMA, phorbol 12-myristate 13-acetate;
TGF, transforming growth factor-
;
TM, transmembrane;
HA, hemagglutinin;
PBS, phosphate-buffered saline;
HBS, Hepes-buffered saline;
NHS, N-hydroxysuccinimide.
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