©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Biosynthesis of Neurotrophin Heterodimers by Transfected Mammalian Cells (*)

John V. Heymach, Jr.(§) and Eric M. Shooter (¶)

From the (1) Department of Neurobiology, Stanford University School of Medicine, Stanford, California 94305-5401

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 NGFNT-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, NGFNT-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.


INTRODUCTION

NGF,() 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.

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 -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.

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 BDNFNT-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 NGFNT-3 heterodimers produced by transfected glial and neuroendocrine cell lines.


MATERIALS AND METHODS

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.

COS cells were metabolically labeled with 100 µCi of [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 NGFBDNF 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 NGFNT-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 NGFNT-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 NGFNT-3. The detection limits were approximately 20-50 pg/ml for the NGF ELISA and 400-1000 pg/ml for the NT-3 and NGFNT-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 NGFNT-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 NGFNT-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 NGFNT-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 NGFBDNF (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 NGFNT-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 NGFNT-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 NGFNT-3 ELISA (see below) confirmed that NGFNT-3 heterodimer was present when NGF and NT-3 were coexpressed but not when either was expressed alone.

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 NGFBDNF and NGFpro-BDNF heterodimers were detected after immunoprecipitation with NGF antibody (Fig. 2B, lane 10).

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 BDNFNT-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-MycNT-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 NGFNT-3 heterodimers occurred exclusively intracellularly, they would be detectable only when NGF and NT-3 were cotransfected. If NGFNT-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, NGFNT-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.

Conditioned medium from COS cells coexpressing NGF and NT-3 contained significant quantities of NGFNT-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 NGFNT-3 heterodimer would contribute to the NGF band. Therefore, if equal amounts of NGF and NGFNT-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).

The formation of the NGFBDNF heterodimer was analyzed in a similar manner. (The formation of BDNFNT-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 NGFBDNF (or at least NGFpro-BDNF) was formed both intracellularly and at a detectable rate within the conditioned medium. Previously, it was reported (15) that the NGFBDNF heterodimer, but not NGFNT-3 or BDNFNT-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. NGFBDNF heterodimers, detected by immunoprecipitation and immunoblotting, formed more quickly than NGFNT-3, although after 48 h the levels were estimated to be less than 5% of those of homodimer (data not shown).

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.

NGFNT-3 Production by Transfected Glial and Neuroendocrine Cells

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 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 NGFNT-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 NGFNT-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) . NGFNT-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 NGFNT-3 was detected in the mix cells or mix media controls, suggesting that heterodimer formation occurred intracellularly. NGFNT-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 NGFNT-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. NGFNT-3 is detected in significant quantities only when plasmids for NGF and NT-3 are cotransfected. The level of NGFNT-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 NGFNT-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 NGFNT-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.

Initially, NGFNT-3, NGFBDNF, and BDNF-MycNT-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 NGFNT-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 NGFBDNF heterodimers are formed intracellularly when the appropriate neurotrophins are coexpressed in COS cells. A similar conclusion was reached regarding BDNFNT-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.

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.() 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.

NGFNT-3 Heterodimer Is Produced by Transfected Glial and Neuroendocrine Cells

We used two-site ELISAs to compare the levels of NGFNT-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 NGFNT-3 present was probably an underestimate of the levels secreted, because NGFNT-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 NGFNT-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. NGFNT-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.

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 NGFNT-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.

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.


FOOTNOTES

*
This work was supported by Grant NS 04270 from the National Institutes of Health, by Grant PRG-94-138 from the Alzheimer's Association, and by Contracts 92-15942 and 93-18647 from the State of California, Department of Health Services. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by National Institutes of Health Neonatal and Developmental Biology Training Grant HD-07249.

To whom correspondence should be addressed. Tel.: 415-723-6638; Fax: 415-725-0388.

The abbreviations used are: NGF, nerve growth factor; BDNF, brain-derived neurotrophic factor; NT-3, neurotrophin-3; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; BDNF-Myc, form of BDNF containing a 10-amino acid Myc epitope at its carboxyl terminus.

J. V. Heymach, Jr., and E. M. Shooter, unpublished data.

Theoretically, if the association between monomers is unbiased and if A and B are the relative amounts of NGF and NT-3 monomers present prior to dimerization, the relative amounts of NGF homodimer, NGFNT-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% NGFNT-3, and only 1% NT-3.


ACKNOWLEDGEMENTS

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.


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