J Biol Chem, Vol. 273, Issue 52, 34933-34940, December 25, 1998

A TrkA-selective, Fast Internalizing Nerve Growth Factor-Antibody Complex Induces Trophic but Not Neuritogenic Signals^*

H. Uri Saragovi^§¶, WenHua Zheng, Sergei Maliartchouk, Gianni M. DiGugliemo, Yogesh R. Mawal, Amine Kamen^**, Sang B. Woo, A. Claudio Cuello, Thomas Debeir, and Kenneth E. Neet

From the  Department of Pharmacology and Therapeutics, ^§ Oncology/Cancer Center, and  Department of Cell Biology, McGill University and the ^** Biotechnology Research Institute, National Research Council, Montréal, Quebec H3G 1Y6, Canada, and the  Department of Biological Chemistry, Finch University of Health Sciences/The Chicago Medical School, North Chicago, Illinois 60064

	ABSTRACT

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Nerve growth factor (NGF) is a neurotrophin that induces neuritogenic and trophic signals by binding to TrkA and/or p75 receptors. We report a comparative study of the binding, internalization, and biological activity of NGF versus that of NGF in association with an anti-NGF monoclonal antibody (mAb NGF30), directed against the C termini of NGF. NGF·mAb complexes do not bind p75 effectively but bind TrkA with high affinity. After binding, NGF·mAb complexes stimulate internalization faster and to a larger degree than NGF. NGF·mAb-induced activation of TrkA, Shc, and MAPK is transient compared with NGF-induced activation; yet NGF and NGF·mAb afford identical trophic responses. In contrast, NGF induces Suc-1-associated neurotrophic activating protein phosphorylation and neuritogenic differentiation, but NGF·mAb does not. Thus, an absolute separation of trophic and neuritogenic function is seen for NGF·mAb, suggesting that biological response modifiers of neurotrophins can afford ligands with selected activities.

	INTRODUCTION

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Nerve growth factor (NGF)¹ is the prototype member of the neurotrophin family of ligands, which includes brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5 (NT-3 and NT-4/5) (1). NGF is important for the survival and differentiation of certain sensory, sympathetic, and cholinergic neurons and many other cell types (2-5). Two NGF receptor proteins have been cloned, termed p75 and TrkA. NGF-responsive cells can express either or both receptor types.

NGF and other neurotrophins bind p75 with low affinity (K_d ~10⁹ M) (6), except NT-3 which binds p75 with high affinity in some cells (7). The p75 receptor belongs to the tumor necrosis factor family, and a pro-apoptotic function is revealed in some cells that express p75 receptor in the absence of TrkA (reviewed in Ref. 8).

TrkA receptors bind NGF with intermediate to high affinity (K_d = 10¹⁰ to 10¹¹ M) (9-11). TrkA receptors possess an intrinsic tyrosine kinase catalytic activity that mediates two distinct biological outcomes typically associated with NGF: trophic survival and neuritogenic differentiation. The trophic activity of NGF is revealed by its ability to prevent apoptotic cell death (12, 13). The neuritogenic activity of NGF is revealed by its promotion of cellular differentiation and axon elongation (4).

The TrkA receptor mediates all of the trophic and neuritogenic activities of NGF whether or not p75 is expressed (13-15) or bound by the ligand (16, 17). However, co-expression of p75 affects ligand affinity and TrkA efficacy (11, 13) and also affects ligand-induced internalization (18).

NGF-mediated activation of the TrkA tyrosine kinase is the earliest step leading to signal transduction (17, 20). Tyrosine phosphorylation (Tyr(P)) of specific intracellular residues of TrkA creates docking sites for adaptor signaling molecules. Phospholipase C-, Shc, and PI3-kinase adaptors activate trophic responses via p21^ras/MAPK (17, 19-22), although MAPK activation can occur via cAMP-dependent protein kinase A (24). Phospholipase C- and SNT activation via TrkA are obligatory for neuritogenesis (20-23).

Cellular commitment to trophic or neuritogenic outcomes may be related to the kinetics of TrkA-mediated activation of MAPK. For example, NGF binding to TrkA causes a relatively slow but sustained activation leading to cellular differentiation (23-26), but epidermal growth factor (EGF) binding to EGF receptors (EGFR) causes a rapid but transient activation of MAPK (ERKs) leading to cellular proliferation (26, 27). Furthermore, signals leading to proliferation or differentiation may be affected by altering the kinetics of internalization (28). TrkA/NGF reportedly internalize in clathrin-coated vesicles or in caveoli, thought to deliver the activated receptor to the cell soma located at some distance from nerve terminals (29, 30).

Here we report interesting signaling properties of a complex between NGF and an anti-mouse NGF monoclonal antibody (mAb NGF30) that binds the C-terminal region of NGF. The NGF·mAb complex binds TrkA with high affinity but does not bind p75 efficiently, suggesting a role in receptor binding to the C-terminal region of NGF. The NGF·mAb complex induces faster internalization of ligand and TrkA than does NGF, and the NGF·mAb complex induces full trophic signals but does not induce any neuritogenic signals. The biological properties of the NGF·mAb complex as a ligand of TrkA may be due to conformational restrictions in NGF caused by mAb NGF30.

	MATERIALS AND METHODS

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Cell Lines-- PC12 cells are rat pheochromocytomas that express ~40,000 p75/cell and low levels of TrkA (p75⁺⁺⁺ TrkA⁺) (4). The 6-2.4 cells are PC12-transfected with human trkA cDNA and express high levels of receptors/cell of each p75 and TrkA (p75⁺⁺⁺ TrkA⁺⁺⁺). The nnr5 cells are a subline of PC12 that express intermediate levels of p75 and undetectable TrkA (p75⁺⁺ TrkA). E25 cells are NIH3T3 fibroblasts transfected with human trkA cDNA, and express high levels TrkA/cell (p75 TrkA⁺⁺⁺) (9). B104 cells are rat neuroblastomas that express 40,000-50,000 p75/cell and no Trk (p75⁺⁺⁺ TrkA). The 4-3.6 cells are B104 cells transfected with human trkA cDNA, and express 40,000-50,000 receptors/cell of each TrkA and p75 (p75⁺⁺⁺ TrkA⁺⁺⁺). C10 cells are a subline of 4-3.6 cells that express 40,000-50,000 TrkA/cell and no p75 (p75 TrkA⁺⁺⁺) (13). EL4 cells are mouse thymomas negative for p75 and TrkA (p75 TrkA).

Septal Neuronal Cultures, Treatments, and Choline Acetyltransferase Activity-- Cell cultures were established from the septal area of 17-day-old rat embryos using established procedures (31) and were treated with control media, NGF alone, or NGF·NGF30 1 day after plating. Choline acetyltransferase activity was evaluated after 4, 6, and 8 days in vitro (32).

Production of Extracellular Domain of TrkA (TrkA-ECD)-- Human TrkA-ECD in TNMFH +10% fetal bovine serum (Life Technologies, Inc.) was propagated using serum-free Sf9 cells. At 72 h post-infection the supernatant was harvested and purified to >99% purity as described (33).

Antibodies-- Rat anti-mouse -NGF mAb NGF30 (IgG2a) (34), mouse anti-rat p75 mAb MC192 (IgG1), and mouse anti-human TrkA mAb 5C3 (IgG1) (35) ascites were purified to >98% with protein G-Sepharose (Pharmacia, Baie d'Urfe, Quebec, Canada). Purified mAb NGF30 and rat IgG were digested with papain (100 µg/ml Life Technologies, Inc. Toronto, Ontario, Canada), to obtain monovalent Fabs (35). Fabs were repurified on KappaLock-Sepharose (U. S. Biochemical Corp.) and protein G-Sepharose and dialyzed against phosphate-buffered saline. Anti-NGF mAb 27/21 was purchased (Boehringer Mannheim).

Labeling of Antibodies-- Fluorescein (FITC) conjugation was performed with NHS-fluorescein (Pierce), and horseradish peroxidase (HRP) coupling was performed with EZ-Link^TM Activated Peroxidase Kit (Pierce); biotinylation was performed with NHS-Biotin (Pierce) as per manufacturer's instructions. All labeled agents were repurified.

NGF Species-- Recombinant human NGF, wild type mouse -NGF (Prince Laboratories, Toronto), and various recombinant mouse NGF mutants (36) were used. Concentrations were determined by high pressure liquid chromatography and by silver staining SDS-polyacrylamide gel electrophoresis (data not shown). Protomers of recombinant NGFs are 120 amino acids in length, whereas wild type mouse -NGF are 118 amino acids due to proteolytic activation (data not shown).

Enzyme-linked Immunosorbent Assays (ELISA)-- Test or control proteins were immobilized onto 96-well microtest plates (Becton Dickinson, Lincoln Park, NJ). For testing mAb NGF30 or mAb NGF30-biotin, 25-50 ng/well of the indicated NGF or control bovine serum albumin (BSA, fraction V, Boehringer Mannheim) were immobilized. For testing NGF·NGF30 complex, 6 ng/well of recombinant TrkA-ECD or control BSA were immobilized. Wells with test agents were blocked with binding buffer (BB, phosphate-buffered saline with 1% BSA) for 1 h. Then mAb NGF30, NGF30-biotin, NGF·NGF30, or rat IgG control (50 ng/well) were added in BB for 30 min. Wells were washed 5 times with BB; HRP-coupled secondary reagents were added for 30 min (goat anti-rat Ab, goat anti-mouse Ab, or avidin), and plates were washed in BB. Peroxidase activity was determined colorimetrically using 2,2-azinobis(3-ethylbenzthiazoline sulfonic acid) (Sigma). The optical density was measured at 414 nm in a Microplate reader (Bio-Rad). Assays were repeated at least three times, n = 4. Throughout, human NGF was used as negative control, and anti-TrkA mAb 5C3 was used to control the presence of TrkA-ECD on plates. Similar data were obtained with sequential binding to TrkA-ECD (NGF, washing, mAb NGF30) or with pre-associated complexes of NGF·NGF30.

Sandwich ELISA-- 25 ng of the test or control mAb (mAb 27/21 or mAb NGF30, respectively) were immobilized onto 96-well plates. Both mAbs were immobilized with similar efficacy (data not shown). After blocking the wells with BB, 25 or 750 ng of soluble NGF (~6- or ~180-fold molar excess with respect to immobilized mAb) were added to each well, excess NGF was washed off, and mAb 27/21-HRP or mAb NGF30-biotin followed by avidin-HRP were then added. Washing and peroxidase reactions and optical density readings were as described above.

FACScan Analysis-- Cells (2 × 10⁵) in 100 µl of FACScan binding buffer (phosphate-buffered saline, 0.5% BSA, and 0.1% NaN₃) were immunostained as described (35). Saturating NGF·NGF30, 5C3, MC192, or control non-binding IgGs were added to cells for 1 h at 4 °C, excess primary antibody was washed off, and cells were immunostained with fluoresceinated goat anti-rat IgG (FITC-G--rat) or goat anti-mouse IgG (FITC-G--M) secondary antibody. As cellular controls cells not expressing NGF receptors were used, and for background staining control mAb NGF30 + FITC-G--Rat was used without NGF. In direct FACScan binding assays FITC-labeled mAb NGF30 (NGF30-FITC) was used. The same binding data were obtained whether sequential binding to TrkA (NGF, washing, mAb NGF30) or pre-associated complexes of NGF·NGF30 were used. Cells were acquired on a FACScan, and bell-shaped histograms were analyzed using the LYSIS II program.

Receptor Internalization-- 4-3.6 cells were incubated in media with NGF plus rat IgG control or NGF·NGF30 complexes at 4 °C for 1 h and then allowed to internalize at 37 °C for the times indicated. After washing at 4 °C, cells were analyzed by FACScan for cell surface TrkA and p75 expression using directly labeled 5C3-FITC and MC192-FITC respectively.

Ligand Binding and Internalization-- ¹²⁵I-NGF (73.1 mCi/mg; NEN Life Science Products) binding assays and Scatchard plot analysis were done on B104 or 4-3.6 cells as described (35). Serial dilutions of ¹²⁵I-NGF in BB were pre-mixed with rat IgG (control) or with the indicated molar excess of mAb NGF30 or NGF30 Fab. A 1000-fold molar excess unlabeled NGF was used to assess nonspecific background (always <10% of binding). For ¹²⁵I-NGF internalization, 4-3.6 cells (3 × 10⁷ cells/ml) were incubated at 4 °C for 1 h with 2 nM ¹²⁵I-NGF (alone, with mAb NGF30, NGF30 Fabs, or rat IgG control) and then shifted to 37 °C. At different times 3 × 10⁶ cells were washed with citric acid buffer (10 mM sodium citrate, 150 mM NaCl, pH 4.0) to remove surface ¹²⁵I-NGF. <10% of bound ¹²⁵I-NGF is resistant to acid wash under conditions that do not allow internalization (NaN₃ and 4 °C throughout).

Proliferation and Survival Assays-- Cells (5,000-10,000 cells/well) were added to 96-well plates (Becton Dickinson, Lincoln Park, NJ) and cultured either in media containing 5% fetal bovine serum or in serum-free media + 0.1% BSA (SFM). Ligands consisted of serial dilutions of rat IgG (control), mAb NGF30 alone, wild type mouse NGF, NGF mutants, or NGF·NGF30 complexes. The proliferative/survival profile of the cells was quantitated using the tetrazolium salt reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma) and optical density (OD) readings as described (13). Assays were done 5 times, each assay n = 4-8.

Western Blots-- Blots were performed as described (13). Briefly, cells were detergent-solubilized, and protein concentrations were determined. Lysates were immunoprecipitated (Shc with anti-Shc antisera (gift of Dr. J. J. Bergeron); TrkA with anti-TrkA antisera, and SNT with P13-Suc1-agarose beads (21) (gifts of Dr. D. Kaplan)). Samples were fractionated by SDS-polyacrylamide gel electrophoresis under reducing conditions, transferred to PDVF membranes (Xymotech Biosystems, Montréal, Quebec, Canada), and immunoblotted with anti-phosphotyrosine (-Tyr(P)) antibody 4G10 (Upstate Biotechnology, Lake Placid, NY) or other primary antibodies indicated. Blots were visualized using the enhanced chemiluminescence (ECL) system (NEN Life Science Products).

MAPK Assay-- cell lysates were immunoprecipitated with anti-MAPK polyclonal antibody (Santa Cruz Biotechnology). Precipitates were resuspended in kinase buffer and subjected to in vitro phosphorylation with [³²P]ATP and myelin basic protein (MBP) as substrate (37). MAPK activity was determined by exposing gels to x-ray films followed by densitometry and by directly measuring the radioactivity of excised MBP and MAPK bands.

	RESULTS

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mAb NGF30 Binds Near Amino Acid Arg-118 of Murine NGF-- A panel of mouse NGF mutants was used as targets in ELISAs to map the epitope recognized by mAb NGF30. mAb NGF30 bound equally well to wild type mouse NGF and to mouse recombinant mutants H75A, H84Q, and 9-13 (Table I, rows 1-4). Mouse NGF double mutant H75A/H84Q was bound but at significantly lower levels (~80% of wild type NGF; Table I, row 5). mAb NGF30 binds wild type rat NGF (data not shown) but does not bind mouse mutant NGF R118A, recombinant human NGF, or recombinant human NT-3 (Table I, rows 6-8). NGF amino acids 119 and 120 are not relevant for mAb NGF30 binding because wild type NGF^1-118 is bound as well as recombinant mouse NGF^1-120.

Lack of binding by mAb NGF30 to mutant R118A suggests that this residue is directly bound by the mAb. Conformational changes in the mutant are unlikely to account for the data because the mAb works in denaturing Western blots (34). However, reduced binding to mouse H75A/H84Q double mutant suggests conformational hindrance upon the C terminus.

Stoichiometry of mAb NGF30-NGF Interactions-- We tested whether bivalent mAb NGF30 interacts with both protomers of dimeric NGF. A sandwich ELISA was used wherein immobilized mAb NGF30 was used to capture NGF, and NGF30-biotin/avidin-HRP was used to reveal binding sites not occupied by the capture mAb. If both NGF epitopes are occupied with the capture mAb, no subsequent binding of mAb NGF30-biotin would occur. This sandwich ELISA was used previously to study anti-NGF mAb 27/21 which binds one epitope of an NGF protomer and leaves the epitope of the other NGF protomer exposed and available for binding (38).

mAb NGF30 with captured NGF did not allow substantial binding of NGF30-biotin (Table II, row 1), compared with background binding in the absence of NGF (Table II, row 2), or to nonspecific binding to BSA (Table II, row 4). In contrast, immobilized NGF was efficiently bound by mAb NGF30-biotin (Table II, row 3). Similar data were obtained when 180-fold molar excess of NGF was used in an attempt to drive NGF30 binding two NGF dimer molecules (data not shown). These results indicate that mAb NGF30 excludes the binding of a second mAb NGF30 and suggest that the stoichiometry of one antibody dimer and one NGF dimer is preferred thermodynamically. In contrast, control mAb 27/21 was effective as capture in sandwich ELISAs (Table II, row 5). mAb 27/21-HRP bound to NGF·mAb 27/21 complexes as efficiently as it bound immobilized NGF (Table II, row 6). mAb NGF30 did not bind NGF·mAb 27/21 complexes (not shown), suggesting that the mAbs may block each other's binding.

NGF and NGF·NGF30 Mediate Identical Trophic Survival-- NGF can rescue TrkA-expressing cells from death in a serum deprivation model of apoptosis. Ligand-induced survival of various cell lines cultured in serum free media (SFM) was studied by the MTT method (Table III). Cells expressing TrkA and p75 (PC12, 4-3.6, and 6-2.4 cells) were optimally protected from apoptotic death in SFM by 2 nM mouse NGF (Table III, row 1), as well as by NGF·NGF30 (Table III, rows 6, 7, and 8). This was also true for cells that express TrkA in the absence of p75 (C10 and E25 cells). In contrast, cells that express p75 in the absence of TrkA (B104 and nnr5) were not protected from apoptotic death in SFM by NGF or by NGF·NGF30 at different molar ratios nor was there accelerated death induced by these ligands (data not shown).

There were no differences in the trophic protection afforded by mouse NGF in the presence or absence of mAb NGF30, even at high molar excess of mAb (2 nM NGF + 64 nM NGF30) (Table III, row 5). Monovalent Fabs of mAb NGF30 had no effect on NGF-mediated trophic protection, even when added at 40-fold molar excess (data not shown).

Various controls demonstrated specificity. First, 64 nM rat IgG did not alter the optimal protective effect of 2 nM mouse NGF (Table III, rows 1 versus 4). Second, a combination of 2 nM human NGF + 64 nM NGF30 afforded the same survival as 2 nM human NGF (Table III, rows 9 versus 10). This result is consistent with lack of mAb NGF30 binding to human NGF (see Table I). Third, 64 nM rat IgG or 64 nM NGF30 mAb alone had no effects on any cell line in SFM (Table III, rows 2 and 3).

NGF·NGF30 Does Not Induce Neuritogenic Differentiation-- PC12 or 6-2.4 cells cultured in serum-containing media differentiate in response to mouse NGF, projecting neuritic processes that can be measured morphometrically. Culture of PC12 cells with 2 nM NGF differentiated most cells, compared with no NGF added (Fig. 1A). Whereas cells did differentiate in culture with 2 nM NGF + 24 nM monovalent NGF30 Fab (Fig. 1D), cultures with 2 nM NGF + 4 nM mAb NGF30 did not differentiate (Fig. 1C).

View larger version (108K): [in this window] [in a new window]   Fig. 1.   mAb NGF30 prevents NGF-induced neuritogenesis. PC12 cells were cultured for 72 h with no NGF (A), with 2 nM mouse NGF (B-D), or 2 nM human NGF (E and F). Abs were co-cultured as follows: 24 nM rat IgG control (B), 4 nM NGF30 mAb (C), 24 nM NGF30 Fabs (D), or 24 nM NGF30 mAb (F). Pictures are representative from >4 independent assays. Similar data were obtained with 6-2.4 cells (data not shown).

Various controls demonstrated the specificity of the neuritogenic block. First, co-treatment with 2 nM NGF + 24 nM rat IgG did not affect differentiation (Fig. 1B) compared with NGF alone (data not shown). Second, neuritogenesis in response to 2 nM human NGF (Fig. 1E) was not affected by 24 nM mAb NGF30 (Fig. 1F). This result is consistent with mAb NGF30 not binding human NGF (Table I). Third, cultures in serum-containing media with 24 nM mAb NGF30, 24 nM NGF30 Fabs, 24 nM rat IgG or higher concentration of these agents, either alone or in the presence of NGF, did not reveal any toxicity (data not shown). Fourth, all of these results were repeated using 6-2.4 cells (data not shown).

It is improbable that bivalent NGF30 mAb acts simply by hindering or blocking NGF action, because it inhibits neuritogenic signals without affecting trophic signals (Table III), and monovalent NGF30 Fabs do not affect any of the activities of NGF.

NGF·NGF30 Does Not Increase Choline Acetyltransferase Activity-- To study the block to NGF-induced cellular differentiation further, rat embryonic septal cultures were tested. Septal cultures increased choline acetyltransferase activity ~2.1-fold in response to NGF. Co-culture of NGF with a 2-fold molar excess of mAb NGF30 reduced choline acetyltransferase activity to base line. No differences in cell numbers or health were seen in NGF versus NGF·NGF30-treated cultures (data not shown). These data extend earlier findings of lack of differentiation by septal neurons in response to NGF·NGF30 (34).

mAb NGF30 Blocks NGF Binding to p75 but Not to TrkA-- Binding of NGF·NGF30 to cells expressing NGF receptors was studied by FACScan analysis, assessing the presence of NGF30-FITC (Table IV). Cells expressing TrkA and p75, TrkA only, p75 only, or neither receptor were studied. The preformed complex of 2 nM NGF + 4 nM NGF30-FITC bound TrkA⁺⁺⁺ cells efficiently (Table IV, rows 1 and 2) but did not bind TrkA p75⁺⁺⁺ B104 cells (Table IV, row 3). No specific binding of NGF30-FITC to cell surfaces was detected in the absence of NGF, and there was no binding to TrkA p75 EL4 cells (Table IV, row 4). The same data were obtained in assays using NGF and mAb binding sequentially or using preformed NGF·NGF30 complexes as ligands.

View this table: [in this window] [in a new window]   Table IV mAb NGF30 prevents NGF-p75 but not NGF-TrkA interactions Cells were analyzed by direct FACScan for binding of ligands: preformed complexes (NGF + NGF30-FITC) or sequential binding (NGF  NGF30-FITC). Cells exposed to NGF30-FITC in the absence of NGF act as background. For each condition 5,000 cells were acquired. Data are mean channel fluorescence of bell-shaped histograms (LYSIS II, Becton Dickinson, CA). Representative of two independent experiments.

The data suggest that mAb NGF30 blocks or hinders the p75 binding domain of NGF. The data further indicate that preformed NGF·NGF30 complexes bind to TrkA independently of p75 expression and that NGF docked onto TrkA can still be bound by mAb NGF30. In contrast, NGF docked onto p75 cannot be bound by mAb NGF30 or the mAb causes NGF to dissociate from p75. Thus, as to NGF binding there is a reciprocal block between mAb NGF30 and p75 but not between mAb NGF30 and TrkA.

This notion was strengthened by Scatchard analysis that demonstrated ablation of ¹²⁵I-NGF binding to TrkA p75⁺⁺⁺ B104 cells in the presence of mAb NGF30 (Fig. 2). In independent assays ¹²⁵I-NGF-p75 interactions exhibited a K_d ~25 nM, and ~35,000 NGF molecules/cell were detected. Complexes of ¹²⁵I-NGF·NGF30 binding exhibited decreased binding, and ~2,000 NGF molecules/cell were detected. Interestingly, the affinity of these p75 receptors for NGF was K_d ~4 pM. Competition with a 1000-fold excess of unlabeled NGF ablated all ¹²⁵I-NGF binding to B104 cells (Fig. 2). In contrast, Scatchard plot analysis of C10 cells (TrkA⁺⁺⁺ p75) revealed that mAb NGF30 does not hinder TrkA-¹²⁵I-NGF binding (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Scatchard plot analysis of 125I-NGF binding to B104 cells expressing p75 receptor only. 125I-NGF was mixed with rat IgG control (open squares), mAb NGF30 (open triangles), or 1000-fold excess unlabeled NGF (filled circles). Binding to B104 cells (TrkA p75+++) was then analyzed by Scatchard plot.

The interactions of NGF·NGF30 with TrkA were further studied in ELISAs testing binding to immobilized extracellular domain of TrkA (TrkA-ECD) (Table V). NGF·NGF30 bound TrkA-ECD specifically (Table V, rows 1-5). Only at very high molar ratios of mAb NGF30 to NGF (64·2) there was lower binding to TrkA-ECD (Table V, row 6), suggesting that some interference occurs at this ratio. The presence of immobilized TrkA-ECD on plates was controlled with anti-TrkA mAb 5C3 (Table V, row 11). Comparable data were obtained by adding NGF and mAb NGF30 sequentially (Table V) or by adding NGF·NGF30 complexes preformed in solution (data not shown).

In control assays mAb NGF30 did not bind TrkA-ECD in the absence of mouse NGF (Table V, row 10). mAb NGF30 did not bind TrkA-ECD in the presence of human NGF (Table V, row 7), as expected from earlier results (Table I). Human NGF bound TrkA-ECD and competed for the binding of NGF·NGF30 (Table V, row 8), but efficient competition occurred at 25-fold molar excess of human NGF. The affinity of NGF for TrkA-ECD is ~2 nM (data not shown, and Ref. 33). The affinity of NGF·NGF30 for Trk-ECD was estimated to be also ~2 nM.

Signal Transduction Studies-- Early signaling events induced upon NGF binding include TrkA tyrosine phosphorylation (Tyr(P)), the Tyr(P) of adapter molecules Shc and SNT, and internalization of the ligand-receptor complex. To assess the mechanism by which the mAb splits the trophic and neuritogenic activities of NGF, TrkA-mediated signals induced by NGF versus NGF·NGF30 at 1:2 molar ratio were compared.

NGF·NGF30 Induces TrkA Tyrosine Phosphorylation with Altered Kinetics-- TrkA Tyr(P) was studied on 4-3.6 cells after a time course of ligand stimulation, by TrkA immunoprecipitation and Western blotting with anti-Tyr(P) mAb 4G10. NGF and NGF·NGF30 induced comparable TrkA Tyr(P) after 3 min (Fig. 3, lanes 1 and 2) or 15 min (Fig. 3, lanes 4 and 5). In contrast, after 45 min NGF induced sustained TrkA Tyr(P) (Fig. 3, lane 8), whereas TrkA Tyr(P) induced by NGF·NGF30 was transient and almost disappeared (Fig. 3, lane 7).

View larger version (50K): [in this window] [in a new window]   Fig. 3.   Different TrkA phosphorylation induced by NGF and NGF·NGF30. 4-3.6 cells were treated with 2 nM NGF (lanes 2, 5, and 8), with NGF·NGF30 (at 2-fold molar excess mAb) (lanes 1, 4, and 7), or were untreated (lanes 3, 6, and 9). After the indicated times, cells were lysed in detergent, and TrkA was immunoprecipitated. Samples were analyzed by Western blotting with anti-Tyr(P) mAb 4G10.

No TrkA Tyr(P) was detected at any time in untreated control cells (Fig. 3, lanes 3, 6, and 9). Blots were subsequently stripped and reprobed with anti-TrkA antisera to ensure that equal amounts of p140 TrkA protein were present (data not shown). This control also demonstrated that the p110-phosphorylated band is indeed TrkA as reported previously (39).

mAb NGF30 Prevents NGF-induced SNT Activation-- The Tyr(P) of SNT was assessed after a 5-min ligand stimulation of PC12 cells. Studies were done by affinity isolation of SNT with Suc-1-agarose beads and Western blotting with anti-Tyr(P) mAb 4G10. Treatment with NGF resulted in the expected SNT Tyr(P) (Fig. 4A, lane 2). Treatment with NGF·NGF30 did not induce SNT Tyr(P) (Fig. 4A, lane 3) compared with untreated control cells (Fig. 4A, lane 1). These results are consistent with absence of differentiation when cultured with NGF·NGF30 (Fig. 1).

View larger version (44K): [in this window] [in a new window]   Fig. 4.   Different phosphorylation of SNT and Shc induced by NGF and NGF·NGF30. A, PC12 cells were untreated (lane 1), treated with 2 nM NGF (lane 2), or NGF·NGF30 (at 2-fold molar excess mAb) (lane 3) for 5 min. Cells were lysed in detergent, and SNT was immunoprecipitated. B, same as in A, except that cells were ligand-treated for 12 min, and Shc was immunoprecipitated. Samples were analyzed by Western blotting with anti-Tyr(P) mAb 4G10.

mAb NGF30 Reduces but Does Not Abolish the Tyr(P) of Shc-- The Tyr(P) of Shc was assessed after a 12-min ligand stimulation of PC12 cells (Fig. 4B). Studies were done by immunoprecipitation with anti-Shc antibodies and Western blotting with anti-Tyr(P) mAb 4G10. NGF induced Shc Tyr(P) (Fig. 4B, lane 2), whereas treatment with NGF·NGF30 induced Shc Tyr(P) at ~2.5-fold reduced levels (Fig. 4B, lane 3). Control untreated cells had no detectable Shc Tyr(P) (Fig. 4B, lane 1). Blots were subsequently stripped and reprobed with anti-Shc antibody to ensure that equal amounts of Shc protein were present (data not shown). Results are consistent with trophic survival of cells cultured with NGF or NGF·NGF30 in SFM (Table III) and suggest that only partial Shc activation is required for full trophic support.

The three bands indicated with dashed arrows (Fig. 4B) are proteins co-precipitated with Shc which are tyrosine-phosphorylated specifically in response to ligand treatment. Based on their molecular mass the proteins may be TrkA (140 kDa), PI3-kinase (85 kDa), and either TrkA or PI3-kinase (110 kDa). We have not characterized these proteins.

Kinetics of Ligand-dependent Activation of MAPK-- The kinetics of MAPK activation were gauged after a time course of treatment of 4-3.6 cells with NGF or NGF·NGF30 (Fig. 5). Analysis was done by immunoprecipitation of MAPK and study of its activity in in vitro kinase assays using myelin basic protein (MBP) as an exogenous substrate.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   NGF and NGF·NGF30 activate MAPK with different kinetics. PC12 cells were treated with the indicated ligands for the times indicated, lysed in detergent, and MAPK was immunoprecipitated. MAPK activity was tested in in vitro kinase assays using MBP as an exogenous substrate. Quantitation was by densitometry and by counting incorporated 32P counts after fractionation in SDS-polyacrylamide gel electrophoresis. n = 3. Similar data was obtained using 4-3.6 cells (data not shown).

The kinetics and efficacy of MAPK activation by NGF or NGF·NGF30 were different. For both ligands the t_1/2 of MAPK activation was ~3 min, and optimal MAPK activation was induced by 15 min. However, NGF·NGF30 activated only ~80% of MAPK compared with NGF. After 45 min NGF signals sustained 100% MAPK activation, whereas NGF·NGF30 signals caused transient MAPK activation which decreased to ~30%.

mAb NGF30 Enhances Internalization of NGF and TrkA-- We hypothesized that changes in activation kinetics might be due to differential internalization. Thus, ligand internalization (Fig. 6A) and NGF receptor internalization (Fig. 6B) were tested in two distinct assays.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   mAb NGF30 enhances the rate and quantity of NGF and TrkA internalization. A, 125I-NGF was untreated or bound with NGF30, NGF30 Fabs, or rat IgG. Then 4-3.6 cells were exposed to 2 nM 125I-NGF equivalents and allowed to internalize at 37 °C. Data shown are specific acid-resistant 125I-NGF in cpm. Representative of two independent experiments. B, 4-3.6 cells were bound for 1 h at 4 °C with 2 nM NGF or NGF·NGF30 and were then shifted to 37 °C to allow internalization. Receptor density on the surface was quantitated by direct FACScan immunofluorescence using saturating FITC-labeled mAbs anti-TrkA (squares) or anti-p75 (triangles). For each condition 5,000 cells were acquired, and bell-shaped histograms were analyzed (LYSIS II, Becton Dickinson, CA). Internalization was calculated from the decrease in linear mean channel fluorescence with respect to untreated cells. Average ± S.E. of three independent experiments.

First, internalization of ¹²⁵I-NGF was assayed by counting ligand that become resistant to acid wash. The internalization rate of ¹²⁵I-NGF was increased by mAb NGF30 (Fig. 6A). The t_1/2 for NGF·NGF30 internalization was ~3 min, and the t_1/2 for internalization of untreated NGF was ~6 min. Monovalent mAb NGF30 Fabs or control rat IgG did not affect ligand internalization rates. In addition, an increase in the absolute number of ¹²⁵I-NGF internalized was induced by mAb NGF30. After 20 min ~12,000 cpm of ¹²⁵I-NGF were internalized versus ~30,000 cpm of ¹²⁵I-NGF·NGF30 complexes.

Second, the density of receptors on the cell surface were measured by a quantitative FACScan assay after ligand treatment of 4-3.6 cells. Ligand-induced loss of cell-surface receptors is interpreted as receptor internalization (Fig. 6B). NGF induced the internalization of TrkA receptors with a t_1/2 ~8 min. A complex of NGF·NGF30 induced faster TrkA internalization, with a t_1/2 ~4 min. Furthermore, NGF induced the internalization of only ~20% of the total cell-surface TrkA receptors, whereas NGF·NGF30 doubled receptor internalization to ~41% of the surface TrkA. The p75 receptors did not internalize substantially in response to either ligand (Fig. 6B).

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

We report on the first split of neurotrophic and neuritogenic activities of NGF. The split is caused by association of NGF to bivalent anti-NGF mAb NGF30. In contrast, there are no detectable differences in the biological function of NGF and NGF·monovalent NGF30 Fab complexes; both ligands induce trophic and neuritogenic effects.

To analyze the mechanisms leading to ligand-induced bioactivity, we compared receptor binding, internalization, and signal transduction (see Table VI for summary). There are three factors that could account for the different bioactivity of NGF·NGF30 as follows: (i) altered receptor binding, (ii) conformational effects of bivalent mAb NGF30 upon NGF, and (iii) altered ligand internalization.

Arguably, NGF·NGF30 complexes do not necessarily remain bound in solution, and low amounts of NGF free of mAb NGF30 may be found. However, a differential trophic and neuritogenic response to low concentrations of NGF has not been observed (data not shown, also see Ref. 13).

Receptor Binding Studies-- NGF, NGF·NGF30, and NGF·NGF30 Fab complexes bind to TrkA receptors selectively and with comparable affinity. In contrast, while NGF binds p75 with low affinity (K_d ~10⁹ M), neither NGF·NGF30 nor NGF· monovalent NGF30 Fabs bind p75 efficiently.

Since mAb NGF30 and NGF30 Fabs do not block NGF-TrkA interactions, the data suggest that the C-terminal region of NGF may not be required for binding and activating TrkA. This notion is consistent with other studies (16, 36, 40-44). However, our conclusion is partly at odds with reports suggesting that the C termini of NGF is critical for TrkA binding (41, 45) because trophic support is unaffected by mAb NGF30, but our conclusion is partly consistent with those reports inasmuch as neuritogenesis is ablated by mAb NGF30.

Conversely, since mAb NGF30 and NGF30 Fabs block NGF-p75 interactions, the data indicate that the C termini of NGF (or adjacent domains) are critical for p75 binding. This is consistent with previous observations (16, 46, 47). Also, important NGF-p75 interactions are mediated by NGF amino acids 30-35 within the A'-A" -turn (39-41); and these residues in one NGF protomer seem to pack closely with the C terminus of the other protomer within the NGF dimer (42, 43).

Conformational Effects of mAb NGF30 upon NGF and TrkA-- One mechanism by which bivalent mAb NGF30 may affect NGF biology differently than monovalent NGF30 Fabs may be related to the packing or the tertiary structure of NGF. Predicted distances for the NGF dimer would allow bivalent mAb NGF30 to bind both C termini simultaneously (1 bivalent mAb binding 1 NGF dimer). Indeed, our data strongly suggest that mAb NGF30 binds both NGF protomers.

NGF is a "flexible" molecule, with some unstructured domains that do not resolve crystallographically (42). Bivalent mAb NGF30 could "freeze" the C termini of NGF in a restricted conformation, but monovalent NGF30 Fabs lack the hinge region and do not freeze NGF. We hypothesize that flexible NGF, flexible NGF·NGF30 Fabs, and "frozen" NGF·NGF30 bind TrkA in different ways that result in different biological outcomes. It is postulated that there are two key regions for ligand-binding sites on TrkA, TrkA "hot spots" (48, 49); arguably frozen NGF·NGF30 and flexible NGF could interact differently with each receptor hot spot. Thus, NGF trophic and neuritogenic signals may be intrinsic to the flexible structure of the ligand (50).

Our results are analogous to studies using TrkA receptor mutants activated by wild type NGF. TrkA mutants of the juxtamembrane extracellular domain (21, 51, 52), the juxtamembrane intracellular domain (21, 52), or intracellular tyrosines (e.g. Y785F/Y490F) (17, 19) result in receptors that do not mediate neuritogenesis. However, our study is unique in that our results were obtained with wild type TrkA receptors.

Does Altered p75 Binding Account for NGF·NGF30 Biology?-- Our data and published literature argue against the notion that altered NGF-p75 interactions can cause a split in the trophic and neuritogenic functions of NGF. First, monovalent NGF30 Fabs inhibit NGF-p75 interactions without affecting neuritogenesis. Second, trophic and neuritogenic signals are mediated via TrkA exclusively (14-17). Third, NGF mutants that do not bind p75 do induce differentiation (40, 44).

Thus, p75 blocking does not per se explain changes to NGF function. However, it is possible that lack of NGF-p75 interactions result in faster internalization events because p75 hinders ligand internalization (18). Consequently, blocking NGF-p75 interactions could indirectly cause changes to NGF function (see below).

One unexpected and interesting observation is that when the low affinity NGF-p75 interactions are ablated with mAb NGF30, a small number of remaining high affinity p75-binding sites (K_d ~10¹² M) are unmasked. These putative high affinity p75-binding sites for NGF may be allosteric (53) or may be analogous to high affinity p75-binding sites for NT-3 (7). However, their significance is unclear at this time.

Signal Transduction-- NGF affords sustained activities that can lead to growth or to differentiation, whereas EGF affords transient activities that lead only to growth (23). NGF and NGF·monovalent NGF30 Fabs induce neuritogenesis by sustained activation of MAPK and SNT phosphorylation (20-22, 26), but NGF·NGF30 did not afford these signals.

NGF·NGF30 induces more transient TrkA phosphorylation, lower Shc phosphorylation, and more transient MAPK activation. Partial Shc/MAPK activation by NGF can induce full trophic signals, but it is not sufficient for neuritogenic signals. The data suggest that neuritogenic TrkA signals are more sensitive to Shc/MAPK than TrkA trophic signals or that neuritogenic signals can not be compensated by phospholipase C-, PI3-kinase, or ERKs (17-24, 26, 27). Hence, the differences in NGF and NGF·NGF30 signal transduction via TrkA may be analogous to NT-4 and brain-derived neurotrophic factor signal transduction via TrkB, where NT-4 is more dependent on Shc (54).

Ligand Internalization-- Faster and more extensive internalization of TrkA and of NGF·NGF30 were detected, and it is possible that internalization rates may account for the split of trophic and neuritogenic functions. Receptor internalization is an essential step for growth factor function (23, 28) and can either down-regulate signaling or bring the activated receptor in contact with specific substrates (27-30).

One explanation for faster internalization is that NGF·NGF30 does not interact with p75. However, our data argue against this possibility because NGF·monovalent NGF30 Fabs do not interact with p75 either, yet they afford internalization rates comparable to NGF.

Three lines of evidence suggest that rapidly internalizing ligands may be trophic, whereas slowly internalizing ligands may be neuritogenic. First, ligand-bound EGFR is more rapidly internalized than TrkA and only leads to trophic growth (27); but a slowly internalizing EGFR mutant causes differentiation in PC12 cells (55). Second, regulation of protein phosphorylation with staurosporine or K252a affords sustained EGF-induced phosphorylation of EGFR and neuritogenic differentiation in PC12 cells (23, 56). Third, defects in NGF-TrkA transport or internalization are thought to result in neurite retraction and neurodegeneration (57).

Our data are consistent with the evidence above and suggest that rapidly internalizing TrkA ligands are trophic, whereas slowly internalizing TrkA ligands are neuritogenic. Hence, it seems that NGF·NGF30 activates TrkA signal transduction more like the manner in which EGF activates signal transduction via EGF receptors.

It may be desirable to develop novel fast-internalizing TrkA ligands or to use biological modifiers of NGF such as mAb NGF30. These agents could be useful in neuropathies to maintain neurons without inducing de novo sprouting which could be non-functional or pain-causing.

	ACKNOWLEDGEMENTS

We are grateful to Drs. D. R. Kaplan and J. J. Bergeron (McGill University) and E. Bogenmann (Children's Hospital of Los Angeles) for reagents; and N. Beglova, R. Tom, D. Messenio-Jones, M. Gagnon, and N. Lavine for excellent discussions and assistance.

	FOOTNOTES

^* This work was supported by Medical Research Council of Canada grants (to H. U. S. and A. C. C.) and by National Institutes of Health Grant NS24380 (to K. E. N.). This is National Research Council publication 57673.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Scholar of the Pharmaceutical Manufacturer's Association of Canada and the Medical Research Council of Canada. To whom correspondence should be addressed· McGill University, Department of Pharmacology and Therapeutics, 3655 Drummond St., 1320, Montréal, Quebec, Canada H3G 1Y6, E-mail· Uri{at}pharma.mcgill.ca.

¹ The abbreviations used are· NGF, nerve growth factor; ELISA, enzyme-linked immunosorbent assays; wt, wild type; HRP, horseradish peroxidase; BSA, bovine serum albumin; Tyr(P), tyrosine phosphorylation; MAPK, mitogen-activated protein kinase; mAb, monoclonal antibody; Ab, antibody; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FITC, fluorescein isothiocyanate; PI3, phosphatidylinositol 3-kinase; NT, neurotrophin; SNT, Suc-1-associated neurotrophic activating protein; SFM, serum-free media; MBP, myelin basic protein.

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