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Previous Article | Next Article
Volume 17, Number 16, Issue of August 15, 1997 pp. 6031-6037
Copyright ©1997 Society for NeuroscienceOptimal Nerve Growth Factor Trophic Signals Mediated by Synergy of TrkA and p75 Receptor-Specific Ligands
Sergei Maliartchouk1 and H. Uri Saragovi1, 2 1 Department of Pharmacology and Therapeutics and 2 Cancer Centre, McGill University, Montréal, Québec, Canada H3G 1Y6
ABSTRACT
Nerve growth factor (NGF) receptor-mediated signaling was studied using specific monoclonal antibodies (mAbs) as ligands that discriminate between the receptors TrkA and p75. mAb-induced trophic signals were compared with the signals of the natural ligand NGF. In cells expressing TrkA but no p75 receptors (TrkA+ p75
Key words: NGF; receptor; TrkA; p75; trophic signals; agonist; ligand; mAb), binding of TrkA with mAb 5C3 leads to optimal signals. In cells expressing both TrkA and p75 (TrkA+ p75+), binding of TrkA with mAb 5C3 leads to significant but suboptimal signals, and optimal trophic signals are obtained by concomitant binding of TrkA and p75 with mAbs 5C3 and MC192. In TrkA+ p75+ cells, binding of anti-p75 mAb MC192 also enhances the trophic effect of suboptimal concentrations of NGF. In contrast, in cells expressing p75 receptors singly (TrkA
INTRODUCTION
Nerve growth factor (NGF) is a 26 kDa dimeric polypeptide that binds two receptors characterized on the basis of their binding affinity. One NGF receptor is a 140 kDa protein (p140 TrkA) with intrinsic tyrosine kinase enzymatic activity. NGF binds TrkA with intermediate affinity (Kd 10
; Kaplan et al., 1991
Coexpression of TrkA and p75 on the cell surface leads to the formation of a limited number of high-affinity NGF binding sites (Kd ~10
Although expression of TrkA alone is sufficient for cellular responses (Nebreda et al., 1991
One problem in elucidating the molecular structure of the functional NGF receptor and in determining the individual role of each receptor and a putative cross-modulation between TrkA and p75 has been the difficulty in obtaining high-affinity ligands that discriminate completely between the receptors. Mutant neurotrophins that bind Trk receptors preferentially over p75 function like wild-type neurotrophins in biological assays (Ibáñez et al., 1992
We have previously described a monoclonal antibody (mAb) 5C3 that binds a restricted epitope of TrkA with high affinity and acts as a full agonist (when compared with NGF) on cells that express TrkA but do not express p75 (LeSauteur et al., 1996
The data support the hypothesis that NGF-trophic signals are mediated by TrkA and that unbound p75 negatively modulates TrkA trophic function. More importantly, the data show that optimal agonistic ligand mimicry for a multireceptor complex can be achieved by a combination of the natural ligand and an anti-receptor antibody, or by a combination of two antibodies against different receptors. This information will be useful in the design of artificial agonists in multireceptor systems, including neurotrophin receptors.
MATERIALS AND METHODS
Cell cultures. Rat PC12 pheochromocytomas cells express p75 and TrkA; B104 rat neuroblastoma cells express ~50,000 surface p75 receptors/cell and none of the Trks (TrkA
Antibodies as NGF receptor ligands. Anti-rat p75 mAb MC192 (IgG1) (Chandler et al., 1984 20°C. mAb 5C3 is agonistic and can fully substitute for NGF in E25 cells expressing TrkA but not p75 (LeSauteur et al., 1996
Binding assays with directly labeled mAbs 5C3 and MC192 demonstrated that each antibody binds to its receptor with relative affinity and saturation profiles regardless of whether the other receptor is expressed and bound. For example, mAb 5C3 binds similarly to TrkA+ p75
Protection from cell death. Five thousand cells/well in protein-free media (PFHM-II, Life Technologies) containing 0.1% BSA (crystalline fraction V, Sigma, St. Louis, MO) were added to 96-well plates (Falcon, Mississagua, Ontario, Canada). The cultures were untreated or supplemented with serial dilutions of neurotrophins (positive control), test mAbs, or mouse IgG (negative control). The survival profile of the cells was quantitated using the MTT colorimetric assay (Mosmann, 1983
Some survival experiments were also performed in the presence of various concentrations of the tyrosine kinase inhibitor K252a (kindly provided by Dr. WenHua Zheng, McGill University). The concentrations of K252a used were reported previously (Dobrowsky et al., 1995
DNA fragmentation and apoptosis. Apoptotic death was confirmed by analysis of DNA fragmentation patterns by extraction of genomic DNA as described (Sambrook et al., 1989
Tyrosine phosphorylation assays. The tyrosine phosphorylation of TrkA was assayed after a 15 min treatment of 4-3.6 cells with the indicated agent(s). Analysis was performed by Western Blot of whole cell lysates with the enhanced chemoluminescence detection system (ECL, Amersham, Oakville, Ontario) as described (LeSauteur et al., 1996
RESULTS
Functional consequences of NGF receptor binding
Cells undergo apoptotic death when cultured in SFM (Table 1). B104 cells expressing p75 but not TrkA were not protected by p75 ligands [neurotrophins NGF and BDNF (lanes 2-9) or by various concentrations of anti-p75 mAb MC192 (lanes 10 and 11)]. Lack of significant p75 ligand-induced protection in SFM was independent of TrkA expression, and apoptotic death occurred in p75+ TrkA+ PC12 cells (Table 1, lanes 10 and 11) and in p75+ TrkA+ 4-3.6 cells (Table 1, lanes 6-11). In contrast, NGF binding to TrkA protected cells from apoptotic death in SFM (Table 1, lanes 2-5). NGF-mediated protection of PC12 and 4-3.6 cells was dose dependent and consistently suboptimal at ~1-10 pM (Table 1, lanes 4 and 5). Standard cell culture conditions containing 5% serum (Table 1, lane 12) afford both proliferation and survival. Therefore, higher readings are detected when compared with 1 nM NGF, which in SFM preferentially acts as a survival factor.
Table 1. p75 binding does not protect from apoptotic death in SFM
Treatment in SFM cultures PC12 (TrkA+ p75+) B104 (TrkA 4-3.6 (TrkA+ p75+) 1 Mouse IgG 0 ± 2.3 0.5 ± 0.7 0 ± 2.2 2 1 nM NGF 100 ± 5.1 100 ± 4.6 3 100 pM NGF 80 ± 5.5 1 ± 1.9 87.4 ± 4.5 4 10 pM NGF 40 ± 3.4 2 ± 1.7 52.3 ± 4.7 5 1 pM NGF 12 ± 1.1 10.1 ± 5.5 6 2 nM BDNF Not tested 1 ± 1.3 2.1 ± 1.7 7 200 pM BDNF Not tested 2.5 ± 1.3 0.9 ± 1.2 8 20 pM BDNF Not tested 1.6 ± 1.3 3.4 ± 2.2 9 2 pM BDNF Not tested 0 ± 0.7 0.5 ± 3.7 10 MC192 10 nM 2 ± 1.2 0.7 ± 0.8 1.2 ± 2.3 11 MC192 1 nM 0.6 ± 2.3 3.7 ± 3.1 12 5% serum 157 ± 0.9 100 ± 7.7 148 ± 7.2 PC12, B104, and 4-3.6 cells were cultured in serum-free media (SFM) supplemented with test or control ligands as indicated. Cell protection was quantitated after 48 hr by measuring OD using the MTT colorimetric assay. Data were standardized relative to optimal NGF treatment (PC12 and 4-3.6 cells). B104 cells do not respond to NGF, thus in this assay they were standardized with respect to 5% serum. A representative experiment is shown (average ± SD; n = 4) from more than three independent experiments.
Next, cells expressing p75 and human or rat TrkA receptors were used to test potential synergy of mAb MC192 as a p75 ligand and suboptimal NGF doses (5 pM) as a preferential high-affinity ligand. MAb MC192 alone affords very limited (or insignificant) protection in SFM (Table 1; Table 2, lanes 4-6); 5 pM NGF alone affords suboptimal cell protection ranging from ~30 to 50% (Table 1; Table 2, lane 3).
Table 2. Concomitant p75 and TrkA binding protects cells from apoptotic death
Treatment added to SFM cultures PC12 (TrkA+ p75+) 4-3.6 (TrkA+ p75+) 1 Mouse IgG 0 ± 1.3 0 ± 1.5 2 1 nM NGF 100 ± 6.5 100 ± 4.8 3 5 pM NGF 28 ± 8.4 48 ± 3.5 4 MC192 10 nM 2 ± 1.5 1 ± 4.1 5 MC192 1 nM 6 ± 2.7a 6 ± 3.5a 6 MC192 0.1 nM 1 ± 2.0 1 ± 4.2 7 5 pM NGF + MC192 10 nM 49 ± 3.3 85 ± 6.5 8 5 pM NGF + MC192 1.0 nM 86 ± 7.4 108 ± 5.6 9 5 pM NGF + MC192 0.1 nM 26 ± 5.1 55 ± 4.5 Assays were performed as described in Table 1 legend. mAb MC192 synergizes with suboptimal (5 pM) NGF in protecting PC12 and 4-3.6 cells from apoptotic death in SFM (lanes 7 and 8). a The small increase in survival induced by mAb MC192 is statistically significant.
NGF (5 pM) + mAb MC192 synergized to significantly increase cell protection in SFM (Table 2, lanes 7-9). This protection was dependent on the concentration of mAb MC192 and was maximal at 0.2 µg/ml (1 nM) (Table 2, lane 8). MAb MC192 concentrations ranging from 0.1 nM to 1 µM were tested, but only some concentrations are shown for clarity. At 2 µg/ml (10 nM) or higher concentrations, mAb MC192 afforded limited synergy (Table 2, lane 7), and at 0.02 µg/ml (0.1 nM) or lower concentrations it did not synergize with NGF (Table 2, lane 9). Thus, a bell-shaped dose-response resulted wherein low or high concentrations of mAb do not afford synergy with 5 pM NGF.
Similar tests were performed with 4-3.6 cells (human TrkA+ p75+) and C10 cells (a sorted subclone of 4-3.6 cells that expresses human TrkA but is p75
Table 3. Concomitant ligand binding of p75 and TrkA synergizes in trophic signals
Treatment added to SFM cultures 4-3.6 (TrkA+ p75+) C10 (TrkA+ p75 1 Mouse IgG 0 ± 3.4 0 ± 1.7 2 1 nM NGF 100 ± 5.3 100 ± 3.7 3 100 pM NGF 89 ± 6.6 35 ± 3.1 4 10 pM NGF 52 ± 3.6 7 ± 3.1 5 1 pM NGF 4 ± 4.4 0 ± 2.4 6 5 nM MC192 16 ± 5.7a 1 ± 1.2 7 0.5 nM MC192 8 ± 4.0 0 ± 1.8 8 5 nM 5C3 42 ± 3.0 79 ± 5.2 9 0.5 nM 5C3 20 ± 5.5 64 ± 5.3 10 5 nM 5C3 + 5 nM MC192 78 ± 2.7 73 ± 3.8c 11 5 nM 5C3 + 0.5 nM MC192 118 ± 3.1b 59 ± 4.9c 12 0.5 nM 5C3 + 5 nM MC192 65 ± 6.8 62 ± 1.6c 13 0.5 nM 5C3 + 0.5 nM MC192 96 ± 2.3 64 ± 1.4c Assays were performed as described in Table 1 legend. Binding of p75 and TrkA with mAbs MC192 and 5C3, respectively (lanes 9-12), synergize in protecting 4-3.6 cells from apoptotic death, whereas binding of TrkA with mAb 5C3 alone (lanes 8 and 9) affords suboptimal protection. In contrast, C10 cells are better protected by binding TrkA with mAb 5C3 alone (lanes 8 and 9). a The small increase in survival induced by mAb MC192 is statistically significant. b The survival higher than 100% is statistically significant from 1 nM NGF. c Not statistically significant from each other.
Combinations of mAbs 5C3 and MC192 afforded optimal 4-3.6 cell protection (Table 3, lanes 10-13), which is comparable with that afforded by optimal NGF (Table 3, lane 2). Synergy by combination of mAbs 5C3 and MC192 is demonstrated by significantly higher protection than treatment with either mAb alone (Table 3, lanes 6-9). Interestingly, although binding of TrkA with mAb 5C3 alone affords only ~20-40% protection to 4-3.6 cells, similar treatment of C10 cells affords 65-80% protection in SFM (Table 3, lanes 8 and 9). MAb 5C3 concentrations ranging from 0.01 to 5 µg/ml (0.05-250 nM) were tested, but only some concentrations are shown for clarity.
Consistent with C10 cells lacking surface p75, the combination of mAbs MC192 and 5C3 does not enhance the effect of mAb 5C3 alone (Table 3, lanes 10-13). As expected, C10 cells are less responsive to low doses of NGF than 4-3.6 cells (Table 3, lanes 3-5) because they lack detectable p75. Furthermore, no synergy was observed in C10 cells when mAb MC192 and 5 pM NGF were tested in combination (data not shown).
To assess whether trophic signals leading to cell survival in SFM were mediated via a tyrosine kinase activity, the K252a inhibitor was used (Table 4). As expected, K252a inhibited trophic survival induced by 1 nM NGF. K252a also inhibited trophic survival induced by optimal concentrations of mAb 5C3 or by optimal combinations of mAbs 5C3 + MC192. Inhibition by K252a was dose dependent. The highest concentration of K252a tested (500 nM) was not toxic to 4-3.6 cells (data not shown), and this dose has been used previously (Dobrowsky et al., 1995
Table 4. K252a inibits NGF receptor-mediated trophic signals
K252a (nM) % Cell survival in SFM supplemented with NGF 5C3 5C3 + 192 0 100 ± 9 50 ± 3 112 ± 4 50 60 ± 4 32 ± 3 67 ± 5 500 32 ± 4 13 ± 2 43 ± 2 Assays were performed as described in Table 1 legend. 4-3.6 cell survival in SFM was achieved by incubation with the indicated ligands. Optimal ligand concentrations were used as per Table 3 (1 nM NGF, 5 nM 5C3 mAb, and 5 nM 5C3 + 0.5 nM MC192 mAbs). Cells were challenged with various concentrations of K252a, and % survival was calculated using 1 nM NGF as 100% standard. K252a inhibits both NGF and mAb-mediated survival in a dose-dependent manner and to a similar relative degree.
Analysis of the degradation pattern of genomic DNA confirmed the apoptotic nature of cell death in SFM for 4-3.6 and PC12 cells (Fig. 1) and for B-104 cells (data not shown). The absence or presence of DNA degradation correlated conclusively with protection or lack of protection from death for all treatments and for all cell lines (Tables 1-3). 
Fig. 1. Changes in apoptotic DNA degradation. Genomic DNA was extracted from (A) 4-3.6 or (B) PC12 cells cultured as indicated for 48 hr in SFM. Equal amounts from each sample were resolved on a 1.5% agarose gel and visualized with ethidium bromide. Standard molecular markers (M) are shown. A typical apoptotic DNA ladder is seen for 4-3.6 cells, but PC12 DNA is more smeared and difficult to isolate as a ladder (Xia et al., 1995
[View Larger Version of this Image (74K GIF file)]
In 4-3.6 cells, no DNA degradation is seen after culture with 5% serum or with mAbs 5C3 + MC192, although a small amount of DNA degradation is seen for 4-3.6 cells treated with mAb 5C3 (Fig. 1A). In contrast, extensive apoptotic DNA degradation is seen when 4-3.6 cells are cultured with SFM or mAb MC192 alone (Fig. 1A).
In PC12 cells, no DNA degradation is seen after culture with 5% serum or with 5 pM NGF + 10 nM mAb MC192. PC12 cells treated with 5 pM NGF alone do have limited DNA degradation (Fig. 1B), as expected, because this concentration of NGF affords suboptimal survival. PC12 cells cultured with SFM or mAb MC192 alone show extensive DNA degradation (Fig. 1B). TrkA tyrosine phosphorylation
To further analyze the signaling mechanism of the antibody-based ligand combinations, TrkA tyrosine phosphorylation (PY) was studied. This was performed by Western blot analysis of whole cell lysates with antibodies against phosphotyrosine (-PY) or with antibodies that bind phosphotyrosinylated TrkA within the Shc recognition/docking site [phosphotyrosine 490 of TrkA (
Initial experiments were designed to resolve the concentration of mAb 5C3 that affords optimal PY of TrkA (Table 5). A 15 min treatment with mAb 5C3 at 1 µg/ml (5 nM) induced optimal TrkA PY and TrkA PY490 in C10 (TrkA+ p75
Table 5. TrkA tyrosine phosphorylation in response to MAb 5C3
Cells C10 Cells 4-3.6 Cells PY total PY490 PY total PY490 1 No ligand 11 1 4 1 2 NGF 1 pM 12 1 7 5 3 NGF 10 pM 12 1 44 33 4 NGF 100 pM 36 45 93 61 5 NGF 1 nM 100 100 100 100 6 5C3 0.05 nM 10 1 5 1 7 5C3 0.5 nM 40 40 32 21 8 5C3 5 nM 91 71 45 43 9 5C3 50 nM 35 49 39 21 Cells were untreated (lane 1) or treated with the indicated concentrations of NGF (lanes 2-5) or mAb 5C3 (lanes 6-9), for 15 min at 37°C. Ligand concentrations were selected on the basis of survival assays (e.g., Table 3). Equal amounts of protein from whole cell lysates were resolved by SDS-PAGE and analyzed by Western blotting with antiphosphotyrosine (anti-PY) or with
As expected, TrkA phosphorylation in response to low NGF concentrations (Table 5, lanes 2-4) was decreased in C10 cells compared with 4-3.6 cells, because C10 cells do not express p75 receptors. In contrast, TrkA phosphorylation in response to mAb 5C3 was always stronger in C10 cells compared with 4-3.6 cells (Table 5, lane 8).
Using the optimal NGF and mAb 5C3 concentrations above, we studied TrkA PY after treatment of cells with various combinations of the ligands (Fig. 2). A 15 min treatment of 4-3.6 cells (TrkA+ p75+) with both 5C3 and MC192 mAbs (Fig. 2A,B, lane 5) induced TrkA PY comparable with that induced by optimal NGF doses (Fig. 2A,B, lane 2). MAb 5C3 alone (Fig. 2A,B, lane 3) caused significant changes in TrkA PY; however, mAb 5C3-induced TrkA PY is lower than that induced by NGF or by combinations of mAbs 5C3 and MC192. Treatment with mAb MC192 alone did not cause significant changes in TrkA PY. 
Fig. 2. Optimal TrkA tyrosine phosphorylation by concomitant binding of p75 and TrkA. 4-3.6 cells were untreated (lane 1) or treated with 1 nM NGF (lane 2), 5 nM mAb 5C3 alone (lane 3), 0.5 nM mAb MC192 alone (lane 4), or a combination of both mAbs (lane 5) for 15 min at 37°C. Ligand concentrations were selected from survival assays (e.g., Table 3) and pilot experiments (e.g., Table 4). Equal amounts of protein from whole cell lysates were resolved by SDS-PAGE and analyzed by Western blotting. A, Anti-phosphotyrosine (anti-PY) blot. Short thick arrow indicates p140 TrkA. Notable changes in tyrosine phosphorylation of other cellular proteins can be seen induced by NGF, mAb 5C3, or 5C3 + MC192 (thick dashed arrow), by NGF or mAb 5C3 only (short thin arrow), or by all treatments (thin dashed arrow). B,
[View Larger Version of this Image (29K GIF file)]
Other cellular proteins of sizes ranging from 40 to 125 kDa are also tyrosine-phosphorylated in response to these ligands. Interestingly, the effect on these unidentified substrates is ligand specific. For example, NGF, mAb 5C3, or 5C3 + MC192 (but not MC192 alone) causes the PY of a ~120 kDa phosphoprotein (Fig. 2A, thick dashed arrow), whereas only NGF or mAb 5C3 causes the PY of a ~110 kDa phosphoprotein (Fig. 2A, short thin arrow). All treatments cause the PY of a ~40 kDa phosphoprotein (Fig. 2A, thin dashed arrow). With the exception of the ~40 kDa phosphoprotein, mAb MC192 alone did not cause significant and reproducible increases in PY of other proteins within the 15 min treatment (Fig. 2A, lane 4). More importantly, mAb MC192 did not affect TrkA PY in a significant and reproducible manner (Fig. 2A,B, lane 4; see statistical analysis in C).
Densitometry of the TrkA band of five anti-PY blots as in Figure 2A revealed a significant increase in total PY induced by a combination of mAbs 5C3 and MC192 (91% of that induced by optimal NGF) (Fig. 2C). The total PY increase induced by treatment with mAb 5C3 alone (56% of that induced by optimal NGF) is significantly higher than untreated control (p = 0.029), and it is also significantly different from total PY increases induced by mAb combinations (p = 0.022).
Densitometry of the TrkA band of five
DISCUSSION
Binding of TrkA [with various concentrations of NGF (in PC12 and 4-3.6 cells) or with anti-human TrkA mAb 5C3 (in 4-3.6 cells)] leads to significant trophic signals, as assessed by cell protection in SFM, by increased receptor PY, and by reduced apoptosis and DNA degradation. The signals leading to cell survival in SFM are mediated by a K252a inhibitable tyrosine kinase activity, likely TrkA.
Concomitant binding of TrkA (with the ligands above) and of p75 (with mAb MC192) increase trophic signals synergistically, to levels equivalent to optimal NGF concentrations. When mAbs 5C3 and MC192 are combined, there is a small but significant higher 4-3.6 cell survival over optimal NGF. This is likely attributable to the mAbs being more stable in culture at 37°C than NGF and perhaps to receptor/ligand recycling. The possibility of a small amount of cell division is unlikely, because BrdU incorporation in response to mAb 5C3 or NGF in SFM is undetectable (data not shown).
Synergy of mAb MC192 and NGF in protection from apoptosis can be explained partially by increased binding of NGF to p75 receptors (Chandler et al., 1984
Functional synergy between p75 ligands and TrkA ligands (in cells expressing both receptors), together with decreased TrkA-mediated signals in TrkA+ p75+ cells compared with TrkA+ p75
First, decreased trophic signals in response to TrkA binding by mAb 5C3 were detected in 4-3.6 cells (TrkA+ p75+) when compared with C10 cells (TrkA+ p75
Second, synergistic effects occur between TrkA ligands and mAb MC192 only when the concentration of MC192 is optimized to achieve bivalent binding of all or most receptors. At low concentrations (subsaturating), mAb MC192 does not synergize with TrkA ligands. At very high mAb MC192 concentrations, poor synergy is observed, likely because of high dose inhibition (the probability of mAb binding in a monovalent fashion). This is consistent with reports that high doses of mAb MC192 (8 µg/ml; ~40-fold higher than our optimal concentrations) can antagonize the effect of NGF on PC12 cells (Barker and Shooter, 1994
Third, protection from apoptotic death in SFM was very limited or undetectable after binding of p75 alone with NGF (in B104 cells) or with MC192 mAb (in B104, PC12, and 4-3.6 cells) and undetectable after binding with BDNF (in B104 and 4-3.6 cells). The simplest interpretation is that detectable p75 trophic signals in SFM require pre- or coactivation of TrkA. This would be consistent with reports of a protein kinase that associates with p75 receptors only after TrkA activation (Canossa et al., 1996
The mechanism by which p75 controls TrkA function probably does not involve TrkA-p75 heterodimers, because they are not likely to be induced by binding of the mAb-based ligands; however, the possibility that receptor heterodimers preexist on the cell membrane and are not ligand dependent cannot be ruled out (Wolf et al., 1995
Previously, polyclonal anti-TrkA antiserum was used to achieve ~70% of the neuronal survival afforded by optimal NGF (Clary et al., 1994
Although p75 has been reported to signal in the absence of TrkA binding (for review, see Chao, 1994
Analysis of TrkA PY, particularly the Shc docking site PY490, confirmed that higher activity is induced after concomitant binding of TrkA and p75. This likely is attributable to increased kinase kinetics, to lower tyrosine phosphatase activity, or to sustained phosphorylation of PY490 (Segal et al., 1996
On the basis of our Western blot experiments, the putative negative modulation by p75 seems to be released within a few minutes. Thus, it is unlikely that this modulation involves NF -
(Carter et al., 1996
Important changes in the PY of cellular proteins other than TrkA are also seen induced by ligands that afford optimal protection from apoptotic death. Some of these proteins are tyrosine-phosphorylated in a ligand-specific manner. The identification of these phosphoproteins may reveal differences or specificities in signal transduction induced by NGF versus antibody-based ligands and will aid in understanding whether the putative negative modulation of TrkA is direct or indirect via adapter or regulatory proteins.
Very few anti-receptor mAbs with agonistic activity exist (Taub and Greene, 1992
Partial conformational changes are expected from the fact that mAb 5C3 likely docks onto a region of TrkA and affects the receptor differently than NGF (Perez et al., 1995
Structural analysis of mAb 5C3-TrkA and NGF-TrkA complexes may reveal the nature of the differences and perhaps putative receptor conformational changes that occur on ligand binding. Furthermore, medulloblastomas engineered to express TrkA undergo apoptotic death after NGF treatment (Muragaki et al., 1997
An important and novel concept is the demonstration that functional agonism in a multireceptor system could be optimally achieved by a combination of a natural ligand and an anti-receptor antibody or by two antibodies against different constituents of the complex. This information might be useful in the design of artificial receptor agonists and antagonists, particularly for neurotrophin or other multireceptor systems.
Our work will continue using monovalent fragments of the mAbs to assess the role of dimerization. Future work will focus on how different NGF receptor-ligand complexes affect early events of neurotrophin signaling, internalization, and activation of second messengers.
FOOTNOTES
Received Nov. 8, 1996; revised May 16, 1997; accepted May 28, 1997.
We thank Drs. E. Bogenmann (University of California Los Angeles), R. Segal (Beth Israel Hospital, Boston), and P. Barker and WenHua Zheng (McGill University) for cells and reagents; P. Barker and R. Segal for discussions and reviewing this manuscript; and N. Lavine and S. C. Das for technical assistance. This work was supported by a grant from the Medical Research Council (MRC) of Canada to H.U.S. H.U.S. received a Pharmaceutical Manufacturer's Association of Canada-MRC Scholar Award, and S.M. received a Glaxo-Wellcome Studentship in Pharmacology.
Correspondence should be addressed to Dr. H. Uri Saragovi, McGill University, Department of Pharmacology and Therapeutics, 3655 Drummond Street, #1320, Montréal, Québec, Canada H3G 1Y6.
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