From the
Prompted by the recent discovery that neurotrophins, which are
known to be biologically active as non-covalently linked homodimers,
can also be induced to form biologically active heterodimers in
vitro, we have investigated the biosynthesis of neurotrophin
heterodimers by transfected mammalian cells. When COS cells were
cotransfected with expression plasmids for nerve growth factor (NGF),
brain-derived neurotrophic factor (BDNF), or neurotrophin-3 (NT-3), the
appropriate heterodimers were detected in the conditioned medium by
immunoprecipitation and, in the case of NGF
NGF,
The neurotrophins are initially synthesized as precursors
containing an amino-terminal propeptide of approximately 100 amino acid
residues that, in the case of NGF at least, is necessary for the
production of the properly folded protein
(7) . They are
subsequently proteolytically processed, possibly by furin
(8) or
other member(s) of the prohormone convertases, to release mature
neurotrophin consisting of roughly 120 amino acid residues/monomer.
The crystal structure of mouse NGF
(9) revealed an elongated,
predominately
Although these studies
established that different neurotrophin monomers were structurally
compatible, they did not address which, if any, heterodimers could be
formed during the normal process of folding within a cell. This issue
is especially relevant given the recent demonstrations that two
neurotrophins may be simultaneously produced by the same
cell
(17, 18) , suggesting that heterodimers may form
in vivo, and the finding that certain heterodimers are
biologically active
(19, 20) . Furthermore, it was
recently reported that BDNF
COS cells were metabolically labeled with 100
µCi of [
This
approach was also used to determine if NGF and BDNF formed heterodimers
when coexpressed in COS cells. BDNF was detected in conditioned medium
in both its mature, 14-kDa form and its 32-kDa uncleaved precursor form
(Fig. 2B, lane 5). This incomplete processing
of the BDNF precursor by COS cells was consistently observed and was in
contrast to the efficient processing of the NGF and NT-3 precursors.
However, this processing was not required for heterodimer formation
because both NGF
Because we did not have
an antibody that efficiently immunoprecipitated BDNF or NT-3, we used a
form of BDNF containing a 10-amino acid Myc epitope at its carboxyl
terminus (BDNF-Myc). The Myc epitope tag did not appear to interfere
with the mature BDNF moiety, because BDNF-Myc was able to activate TrkB
phosphorylation at levels comparable with that of purified BDNF (data
not shown). Like wild type BDNF, BDNF-Myc was incompletely processed,
and an analysis of the conditioned medium from COS cells transfected
with the BDNF-Myc expression plasmid (BDNF-Myc/pM8) showed both pro-
and mature BDNF-Myc (see Fig. 4, lane 4). When
conditioned medium from COS cells cotransfected with BDNF-Myc/pM8 and
NT3/pBJ5 was immunoprecipitated with an anti-Myc monoclonal
antibody
(28) , a complex containing BDNF-Myc and NT-3 was
detected by immunoblot (Fig. 3, lane 10), indicating
that BDNF-Myc and NT-3 were stably associated. These results are
consistent with the recent finding
(19) that the coexpression of
BDNF and NT-3 in A293 cells led to the formation of BDNF
Conditioned medium from COS cells coexpressing NGF and NT-3
contained significant quantities of NGF
The formation of the NGF
The observation that cleavage of the BDNF and NT-3
precursors was not necessary for heterodimer formation prompted us to
examine if this applied to NGF as well. We used site-directed
mutagenesis to alter the RX(K/R)R consensus recognition
sequence for furin
(34) , the putative processing enzyme within
the constitutive secretory pathway. Changing the arginine that is four
amino acid residues upstream of the dibasic cleavage site to glutamine
(the R(-4)Q mutant) prevented the formation of mature NGF and
resulted in the secretion of pro-NGF (Fig. 5, lane 3).
The identity of this product was confirmed by immunoprecipitation with
antibodies directed against a region near the amino terminus of the NGF
propeptide
(29) . When BDNF was coexpressed with the R(-4)Q
mutant or wild type NGF, a similar amount of pro-BDNF was complexed in
either case (Fig. 5, lanes 5 and 6), providing
evidence that heterodimer formation does not require proteolytic
processing of either precursor.
Initially, NGF
Although our data indicate that heterodimer formation can
occur intracellularly, the mechanism by which it occurs is not
addressed. One possibility is that heterodimer assembly occurs during
the folding process at the same stage in which homodimers are assembled
(presumably in the endoplasmic reticulum, which is the site of folding
and oligomer assembly for most secretory proteins). Alternatively, it
could occur at a later stage (for example, within secretory vesicles)
by monomer exchange between already folded homodimers. Although it
cannot be ruled out, we consider the latter possibility less likely
because it would require monomer exchange to be extremely rapid given
the relatively short time required for NGF to exit the secretory
pathway in COS cells. The former possibility would presumably require
not only that different neurotrophin monomers be structurally
compatible but also that they fold inside the cell in a similar manner.
Several lines of evidence suggest that this is the case. For instance,
it is known that a domain within the NGF propeptide is necessary for
the proper folding of mature NGF
(23) . This region is highly
conserved between the different neurotrophin propeptides, suggesting
that it serves a similar function. In addition, it is known that the
propeptide of NT-3 can substitute for the wild type propeptide in the
biosynthesis of BDNF and vice versa (19).
The incomplete processing
of the BDNF and NT-3 precursors by COS cells enabled us to detect
heterodimers containing pro-BDNF and pro-NT-3. Furthermore, by mutating
an amino acid near the NGF cleavage site, we were able to produce
heterodimers containing the NGF precursor as well. These data are
consistent with the model that heterodimer formation occurs
independently of precursor cleavage, which is believed to occur in the
trans-Golgi network. Furthermore, the poor processing of BDNF as
compared with NGF and NT-3 raises the question of whether there are
differences in how the neurotrophins are processed in vivo.
If heterodimers are formed inside the cell in the same manner as
homodimers, the relative amounts of each produced would depend on the
level of expression of each neurotrophin (monomer) and the bias that a
monomer has toward assembling as a homodimer versus a
heterodimer. Hypothetically, if there were no bias toward either, in a
cell that expressed equal amounts of two different monomers, the amount
of heterodimer formed would be twice that of either
homodimer.
Double labeling in situ hybridization studies have revealed that the mRNA for two
neurotrophins can be detected within single neurons in the hippocampus,
forebrain, and cortex
(17, 18) . Furthermore, based on
the overlap in the patterns of mRNA expression for the different
neurotrophins (for example, see Refs. 22, 36, and 37), this may occur
in other tissues and cell types as well. Our data suggest that this
condition may be sufficient for the formation of heterodimers in
vivo. A two-site ELISA approach may be useful for detecting such
heterodimers; however, such an ELISA would require greater sensitivity
than the NGF
Conceivably, if heterodimers are produced in vivo they
could serve a variety of functions. They may have activities like both
or either of the parental homodimers or novel activities, possibly by
promoting the heterodimerization of different Trk
receptors
(20) . As previously suggested, they may serve as a
mechanism to regulate the net output of biologically active
neurotrophin
(19) . Finally, a heterodimer could be sorted to a
different cellular location (for example, the dendrites versus the axon of a neuron) than a homodimer or secreted in a different
manner, as in the case of platelet-derived growth factor AB
heterodimer
(38, 39) .
In summary, our data provide
evidence that neurotrophin heterodimers are produced by different types
of transfected mammalian cells that express two neurotrophins at the
same time. Heterodimer formation occurs predominantly intracellularly
and occurs between neurotrophin precursors as well. These results
suggest that when a single cell produces multiple neurotrophins
simultaneously in vivo, both homodimeric and heterodimeric
neurotrophins are likely to be secreted.
We thank Y.-A. Barde, I. Bartke, J. M. Bishop, T.
Ebendal, A. Lowe, D. Morrissey, R. A. Murphy, C. Radziejewski, and G.
D. Yancopoulos for generously providing valuable reagents and J.
Nesbitt for advice on transfecting C6 cells. We are also grateful to P.
Barker, J. Snipes, and M. Canossa for critically reviewing this work
and to B. Barres, U. Suter, and members of our laboratory for valuable
discussions and suggestions.
NT-3, using a two-site
enzyme-linked immunosorbent assay. Heterodimer formation occurred
predominantly intracellularly and did not require precursor cleavage,
because heterodimers containing pro-NGF and pro-BDNF were detected in
the conditioned medium. When rat C6 glioma cells or mouse AtT-20
neuroendocrine cells were cotransfected with expression plasmids for
NGF and NT-3, NGF
NT-3 heterodimer was detected at levels
comparable with those of homodimeric NGF and NT-3, indicating that
heterodimer formation can occur at significant levels in a variety of
cell types. These data provide evidence that NGF, BDNF, and NT-3 are
capable of forming heterodimers when coexpressed in mammalian cells and
suggest that such heterodimers are likely to be formed in vivo when a single cell expresses multiple neurotrophins.
(
)
BDNF, and NT-3 are members of the
neurotrophins, a family of homologous proteins that play a critical
role in the development, maintenance, and regeneration of the nervous
system. These factors share similar secondary structures
(1, 2) and exist in solution as non-covalently linked
homodimers
(3) . The biological effects of the neurotrophins are
mediated by the Trk family of tyrosine kinase receptors, possibly in
conjunction with the low affinity neurotrophin receptor
(p75
) (for reviews, see Refs. 4 and 5). Trk activation
appears to require ligand-induced dimerization
(6) , and the
dimeric structure of the neurotrophins may be essential for this
process.
-strand structure containing a ``core''
formed by the dimer interface, which is stabilized by extensive
hydrophobic interactions, and a striking clustering of three disulfide
bridges referred to as the ``cystine knot'' motif. Residues
involved in the NGF core are extremely well conserved between different
neurotrophins, in contrast to the more variable solvent-exposed
``loop'' regions. The cystine knot motif is the defining
feature of a superfamily of growth factors
(10) that includes
the platelet-derived growth factor and transforming growth factor
families, members of which are biologically active as covalently linked
homo- and heterodimers in vivo. These factors share a similar
tertiary fold consisting primarily of
-strands
(11, 12, 13) and are also
initially synthesized as precursors containing a propeptide that is
known, in several instances, to be required for the folding of the
mature protein (14). Given the high degree of conservation of the
neurotrophin core and the similarities between members of the cystine
knot family, it seemed plausible that neurotrophin heterodimers could
be formed. This was confirmed by two recent studies in which purified
homodimers were denatured and refolded
together
(15, 16) . NGF, BDNF, NT-3, and neurotrophin-4/5
were capable of forming heterodimers by this method, although the
stability of these heterodimers differed.
NT-3 was formed by coinfection of
cells with viruses expressing BDNF and NT-3
(19) . The low levels
of neurotrophins present in vivo make their direct biochemical
analysis extremely difficult. Therefore, as a first step, we have
investigated aspects of neurotrophin heterodimer biosynthesis by
transfected mammalian cells. We found that NGF, BDNF, and NT-3 all
formed heterodimers with each other when coexpressed in mammalian cells
by transient transfection. We also demonstrated that heterodimer
formation occurs intracellularly and independently of precursor
cleavage. Finally, we used a specific two-site ELISA to detect
NGF
NT-3 heterodimers produced by transfected glial and
neuroendocrine cell lines.
Expression Plasmids
The cDNA for mouse NGF, rat
BDNF, and rat NT-3 were all subcloned into pBJ-5, an SR-based
expression plasmid
(21) . The construction of the NGF expression
plasmid, NGF/pBJ5, was described earlier
(7) . An EcoRI
fragment from the plasmid pSK-/BDNF containing the rat BDNF
coding sequence was inserted into the EcoRI site of pBJ-5 to
produce BDNF/pBJ5. NT-3/pBJ5 was created by digesting the plasmid
pKS-rNT-3(+) with KpnI, blunt ending, and digesting with
SacII to release a fragment containing the full rat NT-3
coding region
(22) . This fragment was ligated into PBJ-5 vector,
which had been digested with EcoRI, blunt ended, and digested
with SacII. The plasmids pSK-/BDNF and pKS-rNT-3, as
well as BDNF-Myc/pM8 were provided by G. D. Yancopoulos (Regeneron
Pharmaceuticals, Tarrytown, NY). BDNF-Myc/pM8 was constructed by a
polymerase chain reaction-based approach previously described
(23) and utilized the pCDM8 expression vector
(24) . The
R(-4)Q mutant was constructed by polymerase chain reaction-based
mutagenesis as described previously
(25) using NGF/pBJ5 as a
template. The entire coding region was sequenced by the
dideoxynucleotide method
(26) to verify that errors were not
introduced by the polymerase chain reaction process.
Antibodies
To minimize the cross-reactivity to
other neurotrophins, the BDNF and NT-3 rabbit antisera were raised
against peptides corresponding to internal sequences in which the two
neurotrophins share no amino acid identity. The peptide CEKVPVSKGQL was
used for the antibodies against BDNF, and the peptide CGEIKTGNSPV was
used for those against NT-3. The peptides were coupled to activated
keyhole limpet hemocyanin (Pierce) via a cysteine added to the amino
terminus of each peptide, and rabbits were immunized with 1 µg of
the conjugate in Freund's complete adjuvant followed by three
boosts using 0.5 µg of conjugate in Freund's incomplete
adjuvant. Both antibodies detected 5 ng of the appropriate neurotrophin
by immunoblotting and did not cross-react with 50 ng of other
neurotrophins tested. Neither antibody was effective for
immunoprecipitation nor for use in two-site ELISAs. The rat monoclonal
anti-NGF antibody MC-1
(27) , the mouse 9E10 anti-Myc
monoclonal antibody
(28) , and the rabbit polyclonal antibodies
directed against the NGF propeptide
(29) have been previously
described. The mouse anti-NT-3 monoclonal antibody used for ELISA was
provided by Y. A. Barde (Max Planck Institute, Martinsreid, Germany)
and I. Bartke (Boehringer Mannheim, Penzberg, Germany). Rabbit
polyclonal antiserum raised against purified mouse NGF was provided by
R. A. Murphy (Montreal Neurological Institute, Montreal, Canada). The
chicken polyclonal antiserum against purified NT-3 was provided by D.
Morrissey (Regeneron Pharmaceuticals).
Transfections and Immunoprecipitations
COS-7 cells
were maintained and transiently transfected by the
DEAE-dextran/chloroquine method as described previously
(7) . 15
µg of plasmid DNA was used to transfect each 10-cm plate; when two
pBJ-5-based plasmids were cotransfected, 7.5 µg of each was used,
and expression of each neurotrophin was reduced by roughly 50%. 12.5
µg of BDNF-Myc/pM8 and 2.5 µg of NT-3/pBJ5 were used when these
plasmids were cotransfected because the expression of BDNF-Myc/pM8 was
severely reduced when it was cotransfected in a 1:1 ratio with any
pBJ-5 based plasmid. When conditioned medium from COS cells expressing
BDNF-Myc was immunoblotted with anti-BDNF antibodies, a double band was
observed at 14 kDa in some experiments, which may be due to partial
cleavage of the Myc tag; this was not further investigated. For COS
cells transfected with a single expression plasmid, the yield of
neurotrophin in conditioned medium after 72 h was generally
100-180 ng/ml for NGF and 50-120 ng/ml for BDNF and NT-3.
Rat C6 glioma cells and mouse AtT-20 cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10%
fetal calf serum (Life Technologies, Inc.) with penicillin and
streptomycin added in a 5% CO atmosphere. 2
10
cells/10-cm plate were transiently transfected using 40
ml of LipofectAMINE (Life Technologies, Inc.) reagent and 5 mg of
plasmid DNA (C6 cells) or 100 ml of LipofectAMINE and 20 mg of DNA
(AtT-20 cells), following directions of the manufacturer. In some
experiments, cells were removed using trypsin 24 h after transfection,
and half the cells from a plate transfected with a neurotrophin
expression plasmid were mixed with the same number of cells transfected
with a second neurotrophin plasmid. (As a control, all other plates in
these experiments were also trypsinized.) The cells were allowed to
reattach for 24 h in full medium, at which time the medium was changed.
The medium was then metabolically labeled (see below) or assayed 72 h
post-transfection.
S]cysteine (Amersham Corp.)
60-65 h post-transfection for 1.5 or 3 h as described
previously
(7) . Under these conditions BDNF was detected in the
conditioned medium predominantly in its uncleaved precursor form;
significant amounts of unprocessed NGF and NT-3 were also consistently
detectable (for example, see Fig. 4and Fig. 5). This
reduction in cleavage efficiency as compared with unlabeled cells (for
example, see Fig. 2) may be due to a disruption of the cellular
machinery occurring when the cells were incubated in cysteine-free
medium for 3 h prior to the addition of
[
S]cysteine. Alternatively, some cleavage may
have occurred extracellularly within the conditioned medium; however,
we did not observe a change in the proportion of mature neurotrophin
when we incubated metabolically labeled medium for 6 h at 37 °C.
Figure 4:
Intracellular versus extracellular formation of heterodimers. A, COS cells
were metabolically labeled 72 h post-transfection with
[S]cysteine for 1.5 h, and the conditioned media
were immunoprecipitated with anti-NGF monoclonal antibody followed by
SDS-PAGE and autoradiography. NT-3 coimmunoprecipitated with NGF when
they were coexpressed (lane 4). To distinguish between
intracellular and extracellular heterodimer formation, the following
controls were performed. 24 h post-transfection, cells expressing NGF
or NT-3 alone were removed from their plates by trypsinization, mixed,
and allowed to reattach for 48 h (mix cells control). They were then
metabolically labeled and analyzed as above (lane 5). In mix
media control, metabolically labeled media from cells expressing NGF or
NT-3 alone were mixed before analysis (lane 6); in lane
7, this mixed media control was incubated at 37 °C for 6 h
before analysis. The migration of NT-3 monomer is shown in lane
8. The presence of comparable levels of NT-3 (monomer) in
lanes 4-7 was confirmed by immunoprecipitation with an
anti-NT-3 antibody (data not shown). B, NGF and BDNF were
subjected to an analysis similar to that in
A.
Figure 5:
Formation of heterodimer containing
uncleaved NGF and BDNF precursors. Conditioned media from metabolically
labeled COS cells transfected with the indicated expression plasmids
were immunoprecipitated with NGF antiserum and analyzed by SDS-PAGE.
Proteolytic processing of the NGF precursor was prevented by mutating
an arginine four residues upstream of the dibasic cleavage site (the
R(-4)Q mutant; lanes 3 and
6).
Figure 2:
Detection of NGFNT-3 and
NGF
BDNF heterodimers in conditioned medium from transfected COS
cells. A, COS cells were transiently transfected with vector
(mock) or expression plasmids for NGF, NT-3, or both (in a 1:1
ratio), and conditioned media from the transfectants were analyzed
directly (lanes 3-6) or after immunoprecipitation with
anti-NGF monoclonal antibody (lanes 7-10) by SDS-PAGE
and immunoblotting. Duplicate immunoblots were probed with NGF
antiserum (upper panel) or NT-3 antiserum (lower
panel). Lanes 1 and 2 contain 50 ng of purified
NGF and NT-3, respectively. B, conditioned media from COS
cells transfected with expression plasmids for NGF, BDNF, or both, were
subjected to an analysis similar to that in
A.
For experiments in which COS cell conditioned medium was analyzed by
immunoblotting, the medium was removed 24 h post-transfection and
replaced with Dulbecco's modified Eagle's medium containing
1% fetal calf serum. 72 h post-transfection the medium was removed,
centrifuged to remove debris, and concentrated using a Centricon-10
concentrator (Amicon) with a 10-kDa cutoff. The final serum
concentration was adjusted to 12%, which prevented nonspecific
interactions between the neurotrophins and agarose beads.
Immunoprecipitations were performed by adding 1 µg of anti-NGF or
anti-Myc monoclonal antibody to 500 µl of conditioned medium
(without added detergents) and incubating at 4 °C for 4 h.
Conditioned medium was then incubated for 1 h with 50 µl of
anti-rat or anti-mouse antibodies conjugated to agarose beads (Sigma).
After 4 washes in PBS, 1% Nonidet P-40, 1% deoxycholate, and 180
mM NaCl, the immunoprecipitates were eluted from the agarose
beads with SDS-sample dye
(30) . Dithiothreitol was added to a
final concentration of 100 mM, and the samples were boiled for
5 min before analysis by SDS-PAGE.
SDS-PAGE and Immunoblotting
For samples analyzed
by immunoblotting, SDS-PAGE
(31) was performed using 15%
polyacrylamide gels, and proteins were transferred to nitrocellulose
membrane (Schleicher & Schuell, Inc.) in transfer buffer (25
mM Tris, 190 mM glycine, 20% methanol)
(30) .
Membranes were blocked for 3 h in blocking buffer (PBS buffer, pH 7.4,
with 5% powdered nonfat milk), followed by a 2-h incubation with the
primary antibody diluted in blocking buffer with 0.2% Tween 20 (Sigma).
The primary antibodies were used at the following concentrations: BDNF
and NT-3 antiserum, 1:400 dilution; NGF antiserum, 1:750 dilution; and
anti-Myc monoclonal antibody, 1 µg/ml. Membranes were washed three
times in wash buffer (PBS and 0.2% Tween 20), incubated with either
protein A-peroxidase (when rabbit antiserum was used as primary
antibody) or peroxidase-conjugated goat anti-mouse antibodies (Sigma)
for 1 h, and then washed and developed using the ECL chemiluminescent
detection system (Amersham Corp.). All incubations were performed at
room temperature. Metabolically labeled samples were prepared as
described above and were subjected to 12.5% SDS-PAGE; gels were
subsequently fixed, enhanced using 2,5-diphenyloxazole, dried, and
exposed to Kodak XAR5 film. Rainbow protein molecular mass markers
(Amersham Corp.) covering the 14.3-200-kDa range were used as
standards.
ELISA
The two-site NGFNT-3 ELISA was
performed as follows. 200 ng of mouse anti-NT-3 monoclonal antibody was
fixed to each well of a 96-well Immulon 4 plate (Dynatech
Laboratories), followed by blocking for 2 h at room temperature using
3% fetal calf serum in PBS. The samples to be assayed (generally done
in triplicate) or standards consisting of purified mouse NGF
NT-3
(gift from C. Radziejewski, Regeneron Pharmaceuticals), mouse NGF
(Harlan Bioproducts), or NT-3 (provided by Regeneron Pharmaceuticals)
at concentrations of 0-50 ng/ml in antibody solution (3% fetal
calf serum and 0.05% Tween 20 in PBS) were added, followed by a 6-h
incubation. This and all subsequent incubations were done at 4 °C
with shaking. The plate was then washed four times using PBS, 0.05%
Tween 20. The rabbit anti-NGF antiserum, diluted 1:2000 in antibody
solution, or rat anti-NGF monoclonal antibody MC
-1, at a
concentration of 1 µg/ml, was added, and the plates were incubated
for 4 h. The plate was then washed as before, and either
peroxidase-conjugated anti-rabbit antiserum (Sigma) or anti-rat
antiserum (preabsorbed against mouse IgG), diluted at 1:2000, was
incubated for 4 h. The plate was washed and developed using
2,2`-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma) as a
substrate. The reaction was stopped after 15 min by adding 10 µl of
10% SDS, and an ELISA plate reader (Dynatech model MR 700) was used to
measure the optical density at 405 nm. Data analysis was carried out
using the Microplate Manager program (Bio-Rad). The two-site ELISAs
used to detect NT-3 and NGF were performed in a similar manner with the
following modifications: chicken anti-NT-3 antiserum at a 1:1000
dilution was used as the top layer in the NT-3 ELISA, followed by
peroxidase-conjugated anti-chicken antibodies (Sigma) at a 1:2000
dilution. The two-site NGF ELISA utilized MC
-1 as the bottom
layer, and rabbit anti-NGF antiserum diluted at 1:2000 as the top
layer. The NGF
NT-3 ELISA showed no cross-reactivity to purified
NGF or NT-3 at 50 ng/ml; the NGF and NT-3 ELISAs showed no
cross-reactivity to purified NT-3 or NGF, respectively, but did show
20-30% cross-reactivity to purified NGF
NT-3. The detection
limits were approximately 20-50 pg/ml for the NGF ELISA and
400-1000 pg/ml for the NT-3 and NGF
NT-3 ELISAs.
RESULTS
We initially used the following approach to detect
neurotrophin heterodimers. The NGF-specific monoclonal antibody
MC-1
(27) was used to immunoprecipitate NGF and
NGF-containing heterodimers. This immunoprecipitate was then analyzed
by immunoblotting after SDS-PAGE to determine if BDNF or NT-3 had
coimmunoprecipitated and was, therefore, stably associated with NGF. We
tested the specificity of this method using purified neurotrophin homo-
and heterodimers (Fig. 1). In Fig. 1, lanes 1-4 show samples containing 50 ng of purified NGF, BDNF, NT-3, or
NGF
NT-3 heterodimer. In lanes 5-8 are the analyses
of 100 ng of the same proteins immunoprecipitated with the anti-NGF
antibody. The samples were subjected to electrophoresis and
immunoblotted with anti-NGF, anti-BDNF, or anti-NT-3 polyclonal
antibodies as indicated. Each antiserum recognized only the appropriate
neurotrophin by immunoblot, with NGF in lane 1, BDNF in
lane 2, and NT-3 in lane 3. As expected,
purified NGF
NT-3 heterodimer was recognized by both anti-NGF and
anti-NT-3 antisera at levels that reflected the amount of each monomer
present (Fig. 1, lane 4). The NGF monoclonal antibody
immunoprecipitated both homodimeric NGF (Fig. 1, lane 5)
and purified NGF
NT-3 heterodimer (Fig. 1, lane 8)
with comparable efficiency (50-60%) but not BDNF or NT-3
(Fig. 1, lanes 6 and 7). The NGF monoclonal
antibody also immunoprecipitated purified NGF
BDNF (data not
shown).
Figure 1:
Immunoprecipitation
of NGF homodimer and NGF-containing heterodimer by NGF monoclonal
antibody MC-1. Samples containing 50 ng of purified NGF, BDNF,
NT-3, or NGF
NT-3 heterodimer (lanes 1-4) or
samples in which 100 ng of the purified neurotrophins was
immunoprecipitated with the anti-NGF monoclonal antibody MC
-1
(lanes 5-8) were prepared under reducing and denaturing
conditions and analyzed by 15% SDS-PAGE followed by immunoblotting with
NGF antiserum (top panel), BDNF antiserum (middle
panel), or NT-3 antiserum (bottom panel). Bands
corresponding to NGF and NT-3 monomers were detectable in lane
8, indicating that the antibody immunoprecipitated the
NGF
NT-3 heterodimer. Migration of protein molecular mass
standards is indicated on the right (in
kDa).
Heterodimer Formation by COS Cells
We used these methods
to determine if neurotrophin heterodimers could be detected in the
conditioned medium from cells expressing more than one neurotrophin.
Transiently transfected COS cells were used because they have been
previously shown to constitutively secrete high levels of mature,
biologically active NGF
(32) . Using plasmids encoding the prepro
forms of mouse NGF (NGF/pBJ5) or rat NT-3 (NT-3/pBJ5), we expressed the
neurotrophins, either alone or in combination, and analyzed the
conditioned medium as described above. Conditioned medium
(non-immunoprecipitated) from cells transfected with either NGF/pBJ5 or
NT3/pBJ5 (Fig. 2A, lanes 4 and 5)
contained the appropriate neurotrophin in its mature, 13-kDa (NGF) or
14-kDa (NT-3) form. When the two plasmids were cotransfected in a 1:1
ratio, both neurotrophins were present in the conditioned medium
although at reduced levels (Fig. 2A, lane 6).
When these conditioned media were immunoprecipitated with the NGF
monoclonal antibody, NT-3 was detected in the immunoprecipitate when
coexpressed with NGF (Fig. 2A, lane 10) but not
when expressed alone (Fig. 2A, lane 9),
indicating that NGF and NT-3 were stably associated as expected for an
NGFNT-3 heterodimer. The two-site NGF
NT-3 ELISA (see below)
confirmed that NGF
NT-3 heterodimer was present when NGF and NT-3
were coexpressed but not when either was expressed alone.
BDNF and NGF
pro-BDNF heterodimers were
detected after immunoprecipitation with NGF antibody
(Fig. 2B, lane 10).
NT-3
heterodimer. The analysis in Fig. 3, lane 10, also
showed the presence of pro-BDNF-Myc, suggesting the formation of
significant amounts of the pro-BDNF-Myc
NT-3 heterodimer.
Figure 3:
Detection of heterodimer containing
Myc-tagged BDNF. Conditioned media from COS cells transfected with
plasmids encoding Myc-tagged BDNF, NT-3, or both, were
immunoprecipitated with anti-Myc monoclonal antibody. The conditioned
media (lanes 3-6) and immunoprecipitate (lanes
7-10) were analyzed by SDS-PAGE followed by immunoblotting
with anti-Myc (upper panel) or anti-NT-3 (lower
panel) antibodies. The 29-kDa bands in lanes 7-10 correspond to the IgG light chain.
Intracellular Formation of Heterodimers
These
findings provide evidence that the NGF, BDNF, and NT-3 are capable of
forming heterodimers when coexpressed. However, because the medium from
these COS cells had been conditioned for 48 h, we felt it was necessary
to determine if the heterodimers had formed intracellularly or after
their secretion into the conditioned medium as a result of monomer
exchange between homodimers. To investigate the site of formation of
the NGFNT-3 heterodimer, we analyzed the conditioned medium of
transfected COS cells after metabolically labeling the cells with
[
S]cysteine for 1.5 h. Because NGF is known to
traverse the COS cell secretory pathway with a half-time of 30-45
min (33),
(
)
the time during which monomer
exchange could occur in the conditioned medium was therefore reduced to
less than 1 h. Two controls were performed to determine if monomer
exchange had in fact occurred. In the first, cells expressing either
NGF or NT-3 alone were removed from the plate by trypsinization 24 h
after transfection and mixed together (``mix cells''
control). The cells were allowed to reattach and were metabolically
labeled 72 h post-transfection. In the second control, metabolically
labeled conditioned medium from cells that expressed NGF or NT-3 alone
were mixed and incubated at 37 °C for different lengths of time
(``mix media'' control). If the formation of NGF
NT-3
heterodimers occurred exclusively intracellularly, they would be
detectable only when NGF and NT-3 were cotransfected. If NGF
NT-3
heterodimer formed exclusively by monomer exchange within the
conditioned medium, it should also be detected in both the mix cells
and mix media controls. If the presence of COS cells increased monomer
exchange within the conditioned medium, NGF
NT-3 heterodimer
formation would occur when cells coexpressed NGF and NT-3, as well as
in the mix cells control, but not in the mix media control.
NT-3 heterodimer, as
indicated by the presence of bands corresponding to both NGF and NT-3
monomers after immunoprecipitation with the NGF monoclonal antibody
(Fig. 4A, lane 4). (Note that both homodimeric
NGF and NGF
NT-3 heterodimer would contribute to the NGF band.
Therefore, if equal amounts of NGF and NGF
NT-3 were
immunoprecipitated, the intensity of the NGF band would be three times
that of the NT-3 band.) The appearance of both pro-NGF and pro-NT-3 in
this same analysis indicated that heterodimers containing the
unprocessed NT-3 precursor were formed; however, it was not possible to
determine if these heterodimers contained mature or unprocessed NGF,
because both were immunoprecipitated by the NGF monoclonal antibody.
Heterodimer formation was not observed in the mix cells or mix media
controls, demonstrating that heterodimer formation had occurred
intracellularly. However, when this medium was incubated at 37 °C
for 6 h, a small amount of NT-3 was detected, suggesting that some
monomer exchange had taken place. Note that in the samples being
directly compared in this analysis, the levels of NGF monomer present
were comparable (Fig. 4A, lanes 4-7).
Immunoprecipitation of the same samples with NT-3 antiserum indicated
that the levels of NT-3 monomer were also comparable (data not shown).
BDNF heterodimer was analyzed in a
similar manner. (The formation of BDNF
NT-3 was not examined by
metabolic labeling because BDNF and NT-3 could not be distinguished on
the basis of their electrophoretic mobilities.) Metabolically labeled
COS cells secreted BDNF predominately as uncleaved precursor, which
formed heterodimers when coexpressed with NGF (Fig. 4B,
lane 4). The mixing of cells expressing NGF and BDNF led to a
barely detectable amount of heterodimer formation
(Fig. 4B, lane 5). This amount increased when
media containing NGF and BDNF were mixed and incubated for 6 h
(Fig. 4B, lanes 6 and 7). These
findings suggest that NGF
BDNF (or at least NGF
pro-BDNF) was
formed both intracellularly and at a detectable rate within the
conditioned medium. Previously, it was reported
(15) that the
NGF
BDNF heterodimer, but not NGF
NT-3 or BDNF
NT-3
heterodimers, was formed by monomer exchange when the purified parental
neurotrophins were incubated together for 8 h at 37 °C in a pH 8
buffer. To confirm that heterodimers were being formed extracellularly
under the conditions employed in our experiments, we mixed together
equal amounts of purified NGF and NT-3 or NGF and BDNF in COS cell
medium and incubated the mixture at 37 °C for varying lengths of
time. NGF
BDNF heterodimers, detected by immunoprecipitation and
immunoblotting, formed more quickly than NGF
NT-3, although after
48 h the levels were estimated to be less than 5% of those of homodimer
(data not shown).
NGF
Because the immunoprecipitation method was
relatively insensitive, it did not permit the analysis of conditioned
medium from cell types that secreted lower levels of neurotrophins when
transfected. For this reason, we designed a two-site ELISA that
specifically detected NGFNT-3 Production by Transfected Glial and
Neuroendocrine Cells
NT-3 heterodimers but not NGF or NT-3
homodimers. An anti-NT-3 monoclonal antibody was used as the bottom
layer or ``capture'' antibody, and, following incubation with
the sample being assayed, either the NGF monoclonal
(Fig. 6A) or the NGF antiserum (Fig. 6B)
was used as the top layer antibody to detect captured (NT-3
immunoreactive) molecules that also contained the NGF moiety (see
``Materials and Methods'' for experimental details). This
method reproducibly detected purified NGF
NT-3 at concentrations
as low as 0.5-1 ng/ml (using NGF antiserum as top layer) or
2.5-5 ng/ml (using NGF monoclonal antibody as top layer) but did
not detect purified NGF or purified NT-3 at levels as high as 50 ng/ml.
Because it provided greater sensitivity, the NGF antiserum was used as
the top layer antibody in subsequent experiments.
Figure 6:
Detection of NGFNT-3 by ELISA.
Standard curves of purified NGF, NT-3, or NGF
NT-3 at
concentrations of 0.5-50 ng/ml assayed by two-site ELISA
utilizing monoclonal NT-3 antibody as the bottom (capture) layer and
monoclonal NGF antibody (A) or polyclonal NGF antiserum
(B) as the top (detection) layer. Samples were assayed in
quadruplicate. Error bars indicate ±1 S.E. In cases in
which no error bars are shown, S.E. was less than 0.002 absorbance
unit.
The NGFNT-3
ELISA was used to determine if heterodimers could be formed by cell
types that were derived from the nervous system. Rat C6 glioma cells
were chosen because they can be transfected with relatively high
efficiency and are known to be capable of producing
neurotrophins
(35) . NGF
NT-3 heterodimer was detected in
the conditioned medium of rat C6 glioma cells when they were
transiently transfected with plasmids for NGF and NT-3 but not when
they expressed either one individually (Fig. 7A). Little
or no NGF
NT-3 was detected in the mix cells or mix media
controls, suggesting that heterodimer formation occurred
intracellularly. NGF
NT-3 heterodimer was also detected in the
conditioned medium of AtT-20 cells, a mouse neuroendocrine line, after
cotransfection with NGF/pBJ5 and NT3/pBJ5 but not after transfection
with either plasmid individually (data not shown).
Figure 7:
Production of NGFNT-3 by transfected
C6 glioma cells. Conditioned media from C6 glioma cells transiently
transfected with expression plasmids for NGF, NT-3, or both, were
assayed using NGF
NT-3 ELISA (A), NGF ELISA (B),
or NT-3 ELISA (B). Mix cells and mix media controls were
performed as described in the legend to Fig. 4. NGF
NT-3 is
detected in significant quantities only when plasmids for NGF and NT-3
are cotransfected. The level of NGF
NT-3 detected in mix cells
control is approximately equal to the detection limit in this
experiment. Values represent mean of samples assayed in triplicate
±1 S.E.
To determine if
the amount of NGFNT-3 heterodimer produced by transfected C6
cells was significant compared with the amount of homodimers, we
assayed the conditioned medium with two-site ELISAs designed to detect
NGF and NT-3 homodimers. After cotransfection with NGF/pBJ5 and
NT3/pBJ5, 2.2 ± 0.1 ng/ml NGF and 7.1 ± 0.4 ng/ml NT-3
were detected in the medium, as compared with 4.6 ± 0.1 ng/ml
NGF
NT-3. Furthermore, the measured levels of homodimer are likely
to be slight overestimates of the true levels, because the NGF and NT-3
ELISAs both cross-reacted to some extent with the NGF
NT-3
heterodimer (see ``Materials and Methods'' for details).
Therefore, it can be concluded that the amount of heterodimer is at
least comparable with the levels of NGF and NT-3 homodimer present.
DISCUSSION
In this study, we have characterized the biosynthesis of
neurotrophin heterodimers by transfected mammalian cells. Each of the
neurotrophins tested was capable of forming heterodimers, which were
produced intracellularly and also occurred between the neurotrophin
precursors. Furthermore, we find that glial and neuroendocrine cell
lines transfected with plasmids for NGF and NT-3 produce NGFNT-3
in amounts that are comparable with those of the parental homodimers.
NT-3, NGF
BDNF, and BDNF-Myc
NT-3 were
detected by immunoprecipitation from medium conditioned by COS cells
expressing two neurotrophins. This method provides a way to detect
neurotrophin heterodimers (including those involving neurotrophin
precursors) without potentially disruptive chromatographic purification
steps. However, its sensitivity is restricted by the detection limit of
immunoblotting (roughly 5 ng with our antibodies) or, if metabolic
labeling is used, it requires that the different neurotrophin monomers
be electrophoretically distinguishable. In the case of NGF
NT-3,
the presence of heterodimer was confirmed using a two-site ELISA. This
method was more sensitive and permitted a quantitative comparison of
the levels of heterodimer and homodimer (see below).
Heterodimer Formation Occurs Predominately
Intracellularly
In order to determine if simultaneous
coexpression of two neurotrophins by a single cell is necessary or
sufficient for heterodimer formation, the site of heterodimer formation
was investigated. We find that NGFNT-3 and NGF
BDNF
heterodimers are formed intracellularly when the appropriate
neurotrophins are coexpressed in COS cells. A similar conclusion was
reached regarding BDNF
NT-3 formation in A293 cells
(19) .
We also detect small amounts of heterodimer formation after prolonged
coincubations of media containing NGF and NT-3 or NGF and BDNF at 37
°C. Taken together, our results and those of other investigators
suggest that coexpression of two neurotrophins within a single cell is
sufficient for the formation of heterodimers containing NGF, BDNF, or
NT-3.
(
)
However, if one neurotrophin were
expressed in excess, very little of the lesser expressed neurotrophin
would be able to homodimerize. Thus, the formation of heterodimers
could provide a mechanism by which the expression of one neurotrophin
influences the amount of a second neurotrophin produced.
NGF
We used two-site ELISAs to
compare the levels of NGFNT-3 Heterodimer Is Produced by Transfected
Glial and Neuroendocrine Cells
NT-3, NGF, and NT-3 present within the
conditioned medium of C6 glioma cells transfected with expression
plasmids for NGF and NT-3. The heterodimer was detected at slightly
lower levels than NT-3 and at higher levels than NGF. The amount of
NGF
NT-3 present was probably an underestimate of the levels
secreted, because NGF
NT-3 is less stable than other heterodimers
(15) and is likely to have undergone some dissociation during
the 24 h in which the medium was conditioned. The levels of
NGF
NT-3 heterodimer present suggest that if there is a bias
against its formation as compared with homodimer formation within the
cell, this bias is not large. NGF
NT-3 was also detected in the
conditioned medium of transfected AtT-20 cells, a neuroendocrine cell
line. The expression level in the glial and neuroendocrine cell types
tested here was 10-40-fold lower than the level in COS cells.
This suggests that the observed heterodimer formation is not an
artifact resulting from the specific cell types chosen or expression
levels attained by transfection but rather reflects an inherent
capability of different neurotrophin precursors to dimerize during the
normal folding process.
NT-3 ELISA described here, even if heterodimers were
present in quantities comparable with those of homodimeric
neurotrophins in an area of high expression such as the hippocampus.
NT-3
heterodimer, and NT-3 homodimer formed would be A
,
2AB, and B
, respectively. Therefore, if
A = B, they would be formed in a 1:2:1 ratio.
However, if NGF and NT-3 (monomers) were expressed in a 9:1 ratio, the
dimerized neurotrophins would consist of 81% NGF, 18% NGF
NT-3,
and only 1% NT-3.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.