Differential Activity of the Nerve Growth Factor (NGF) Antagonist PD90780 [7-(Benzolylamino)-4,9-dihydro-4-methyl- 9-oxo-pyrazolo[5,1-b]quinazoline-2-carboxylic Acid] Suggests Altered NGF-p75 Interactions in the Presence of TrkA

The neurotrophin nerve growth factor (NGF) binds to two receptor types: the tyrosine kinase receptor TrkA and the common neurotrophin receptor p75. Although many of the biological effects of NGF (such as neuronal growth and survival) are associated with TrkA activation, p75 also contributes to these activities by enhancing the action of TrkA when receptors are coexpressed. The NGF antagonist PD90780 [7-(benzolylamino)-4,9-dihydro-4methyl-9-oxo-pyrazolo[5,1-b]quinazoline-2-carboxlic acid] interacts with NGF, preventing its binding to p75. In this study, the actions of this compound are further explored, and it is found that PD90780 is not able to inhibit the binding of either brain-derived neurotrophic factor or neurotrophin-3 to p75, consistent with the direct interactions of the antagonist with NGF. In addition, we demonstrate that the ability of PD90780 to inhibit NGF-p75 interactions is lower when receptors are coexpressed, compared with when p75 is the only neurotrophin receptor expressed. These results suggest that the interaction between NGF and the p75 receptor is altered when TrkA is coexpressed. This alteration can be exploited in the development of antagonists that will selectively inhibit the pro-apoptotic actions of p75 when expressed in the absence of TrkA, although having less effect on the pro-survival effects of p75 mediated by enhanced TrkA activation. Neurotrophins function to regulate the survival, growth, and differentiation of neurons. Nerve growth factor (NGF), the first discovered and most extensively researched member of this protein family, selectively binds to a tyrosine kinase receptor, TrkA (Kaplan et al., 1991; Klein et al., 1991). NGFTrkA interaction results in the phosphorylation of the receptor and initiates an intracellular cascade of reactions that encourage prosurvival events within a neuron (for review, see Kaplan and Miller, 1997). NGF is also able to bind to a second receptor type, the common neurotrophin receptor p75. Like TrkA, this receptor is capable of promoting the trophic actions of NGF (Hempstead et al., 1991; Verdi et al., 1994), generally thought to be mediated through an enhancement of TrkA activation. Alternatively, binding and subsequent activation of the p75 receptor by NGF is also able to promote proapoptotic effects through signaling mechanisms independent of TrkA. This has been noted in mature rat oligodendrocytes expressing p75, but not TrkA, where NGF has been found to induce cell death (Casaccia-Bonnefil et al., 1996) and via p75 activation in mouse ganglion cells (Bamji et al., 1998). In addition, cell death in the developing retina has This research was supported by the Canadian Institutes for Health Research (MOP-42403 to G.M.R. and MOP-7757 to R.J.R.) and by the Neuromuscular Research Partnership Program (an alliance of the Amyotrophic Lateral Sclerosis Society of Canada, the Muscular Dystrophy Association, and the Canadian Institutes for Health Research; JNM-48405 to G.M.R.). G.M.R. is a Queen’s National Scholar. 1 Current Address: Department of Medicinal Chemistry, AstraZeneca R&D Lund, S-22187 Lund, Sweden. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.104.066225. ABBREVIATIONS: NGF, nerve growth factor; BDNF, brain-derived neurotrophic factor; HKR, HEPES-Krebs-Ringer; BS, bis-(sulfosuccinimidyl) suberate; TBS, Tris-buffered saline; PAGE, polyacrylamide gel electrophoresis; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; SNHS, sulfo-N-hydroxy-sulfosuccinimide; NT-3, neurotrophin-3; CI, confidence interval; PD90780, 7-(benzolylamino)-4,9-dihydro-4-methyl-9-oxopyrazolo[5,1-b]quinazoline-2-carboxylic acid; Ro 08-2870, 2,3,4,10-tetrahydro-7,10-dimethyl-2,4-dioxobenzo[g]pteridine-8-carboxaldehyde; ALE-0540, N-{5-nitro-1H-benz[de]isoquinoline-1,3(2H)-dione}-2-aminoethanol. 0022-3565/04/3102-505–511$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 310, No. 2 Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 66225/1156928 JPET 310:505–511, 2004 Printed in U.S.A. 505 at A PE T Jornals on O cber 9, 2016 jpet.asjournals.org D ow nladed from been attenuated by a reduction in NGF (Frade et al., 1996; Frade and Barde, 1998) and was reduced in the embryonic spinal cords of mice with deletions in either NGF or p75 genes (Frade and Barde, 1999). Although NGF is capable of binding to both TrkA and p75, its affinity for each is dependent upon the presence of the alternate receptor. The TrkA receptor possesses a higher binding affinity for NGF when p75 is coexpressed compared with when TrkA is expressed in the absence of p75 (Hempstead et al., 1991; Dechant et al., 1993; Mahadeo et al., 1994). Consistent with this, it has also been noted that preventing the binding of NGF to p75 can reduce binding of NGF to TrkA (Barker and Shooter, 1994). Recent research has supported the notion that the presence of p75 affects high-affinity NGF binding to TrkA, observing that high-affinity binding was dependent upon the ratio of both receptors (Esposito et al., 2001). This binding was not altered by mutations in the extracellular NGF binding domain of p75; however, mutations in either cytoplasmic or transmembrane domains of p75 and TrkA revealed that high-affinity binding is likely mediated through interactions within these domains. This supports previous findings that p75 and TrkA are associated as a hetero-oligomer on the cell surface (Huber and Chao, 1995; Gargano et al., 1997; Ross et al., 1998). The importance of the p75 cytoplasmic domain has also been shown in research that found that overexpression of this region results in substantial neuronal cell death (Majdan et al., 1997). In another study, experiments involving TrkA receptor chimeras revealed that the presence of p75 alters the binding site for NGF on TrkA, thereby affecting the high-affinity binding of NGF to this receptor (Zaccaro et al., 2001). Although previous studies looking at interactions between receptors have focused on the effects of NGF binding to TrkA in the presence of p75, the effects of coexpression upon NGF binding to p75 have remained unclear. Our laboratory has previously demonstrated that the affinity of NGF for p75 decreases when TrkA is also expressed (Ross et al., 1998). From these findings, it was hypothesized that this receptor-dependent relationship could be elucidated further through comparative concentration effect studies that utilize a NGF antagonist capable of selectively inhibiting the binding of NGF to the p75 receptor. One such compound was characterized in a study where its ability to inhibit the binding of NGF to p75 was demonstrated using recombinant extracellular p75 binding domains (Spiegel et al., 1995). The antagonist effects of this nonpeptidic compound, a pyrazoloquinazolinone (PD90780), were demonstrated to result from an interaction between PD90780 and NGF, rather than an interaction between PD90780 and p75. These authors confirmed their observations in the cell-free assay system using an ovary cell line that was transfected with the p75 receptor. In both experimental models, the TrkA receptor was not present; therefore, any potential influence it might have had on the ability of PD90780 to inhibit NGF binding to p75 was not evaluated. In the present study, the ability of PD90780 to inhibit NGF-p75 binding in the presence of the TrkA receptor was investigated. This was evaluated using rat pheochromocytoma (PC12) cells that express both p75 and TrkA receptors, PC12 cells that express only p75, as well as with truncated p75. These experiments, therefore, permitted a comparison of the antagonist effects of PD90780 on NGF binding with p75 when the receptor is coexpressed with TrkA, expressed in the absence of a Trk receptor, and when it is in a soluble state. Materials and Methods Radiolabeled Neurotrophin and Receptor Preparation. The iodination of NGF (mouse 2.5s; Cedarlane Labs, Toronto, ON) and rhBDNF (Alomone Labs, Jerusalem, Israel) was performed as described previously for NGF (Sutter et al., 1979) with modification (Ross et al., 1997). The ability of PD90780 to block neurotrophin binding to the p75 receptor was evaluated under various receptor conditions. This was accomplished with the use of PC12 cells (TrkA and p75), PC12 cells (p75 only), and truncated p75. The two cell types were cultured in RPMI 1640 medium with 10% fetal calf serum. Recovery of the cells was permitted with the replacement of the medium with calcium-magnesium free balanced salt solution followed by a 15-min incubation at 37°C. Cells were centrifuged, and pellets were suspended in HKR buffer (10 mM HEPES, pH 7.35 containing 125 mM NaCl, 4.8 mM KCl, 1.3 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 1 g/l glucose, and 1 g/l bovine serum albumin). In the case of truncated p75, the culture medium used to grow PC12 cells was removed and centrifuged to ensure it was free of cells. This medium contained p75 extracellular domains previously sloughed by the cells (molecular weight approximately 50 kDa; DiStefano and Johnson, 1988). Chemical Cross-Linking of I-NGF to TrkA and/or p75 in the Presence of Antagonists and Immunoprecipitation. I-NGF (0.1 nM) alone or in combination with NGF (100 nM), BDNF (10 nM), or PD90780 (100 M; Parke-Davis Pharmaceuticals, Ann Arbor, MI) was incubated with PC12 cells at a concentration of 10 cells/ml in HKR buffer in 1-ml volume for 2 h at 4°C with rocking. After binding, 20 l of the cross-linker bis-(sulfosuccinimidyl)suberate (BS) was added (final concentration of 0.4 mM) to each sample and incubated at room temperature for 30 min. The cells were washed three times with TBS, after which reducing SDS sample buffer was added to the pelleted cells to dissolve the proteins, or in the case of immunoprecipitations, prepared as described below. Cell samples undergoing immunoprecipitations for TrkA or p75 were solubilized in lysis buffer (TBS containing 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, and 1 g/ml leupeptin) and incubated for 40 min at 4°C. After centrifugation, the lysates were removed to a new tube, and either rabbit polyclonal anti-Trk cytoplasmic domain antibody or rabbit polyclonal anti-p75 antibody (9992) (antisera against glutathione S-transferase-fusion protein containing the cytoplasmic domain of p75; obtained from Dr. M. Chao) was added to the soluble proteins to isolate the respective receptors. The samples were left to incubate at 4°C overnight. Antibody complexes were removed through application and incubation with 70 l of a 50% slurry of immobilized Protein G (Pierce Chemical, Rockford, IL) for 2 h at 4°C. The solid phase was washed with lysis buffer three times, with distilled water once, and then the proteins were dissolved in SDS sample buffer. Proteins from cross-linking and immunoprecipitation experiments were separated via 6% SDS-polyacrylamide gel electrophoresis (PAGE). Chemical Cross-Linking of I-NGF to p75 and Concentration Effect Assays. I-NGF was incubated at 4°C for 2 h with or without PD90780. PC12 or PC12 cells were added at 10 cells/ml, and samples were incubated at 4°C for 2 h with rocking. Bound I-NGF and p75 proteins were cross-linked with final concentrations of 5 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 2 mM sulfo-N-hydroxy-sulfosuccinimide (SNHS) (20 l of each) and incubated with rocking at room temperature for 30 min. Samples were washed with TBS [10 mM Tris(hydroxymethyl)-aminomethane [pH 8.0] and 150 mM NaCl] three times before 506 Colquhoun et al. at A PE T Jornals on O cber 9, 2016 jpet.asjournals.org D ow nladed from the addition of reducing SDS sample buffer to dissolve the proteins. The proteins were then separated on a 6% SDS-PAGE gel. In the experiments involving truncated p75, I-NGF or I-BDNF was exposed to the same concentrations of PD90780 and incubated with medium containing truncated p75 before cross-linking with EDC/SNHS. The reaction was quenched by adding 15 l of 1 M glycine followed by 10 min of mixing. The samples were then immunoprecipitated using 192 IgG, which recognizes the extracellular domain of p75 (Calbiochem, San Diego, CA). Neurotrophin Receptor Binding. Neurotrophins NGF, BDNF, and NT-3 were iodinated, and PC12 and PC12 cells were cultivated and recovered as previously described. Tubes were set up containing single data points that held iodinated neurotrophin (0.5 nM), PD90780 (10 M), a final concentration of 10 cells/ml and NGF (at 50 nM for nonspecific binding) as required, and were then incubated at 4°C for 2 h. Aliquots (100 l) were layered on top of 200 l of 10% glycerol in HKR buffer in 0.4-ml tubes. Samples were then centrifuged at 5000 rpm for 2 min, after which the tip containing the cell pellet was cut off and radioactivity present was determined. TrkA Phosphorylation Assay. Modification of methods described permitted determination of TrkA phosphorylation (Ross et al., 1998). NGF (40 pM) was incubated with varying concentrations of PD90780 (3, 30, or 300 M) for 2 h in HKR buffer. PC12 cells used at 10 cells/ml were incubated with NGF and PD90780 solutions for 15 min at 37°C. Samples were washed once with cold PBS and once with cold TBS and then lysed with solutions containing 500 M orthovanadate and immunoprecipitated with anti-Trk antibody as previously described. An SDS-PAGE run on 6% gel followed by Western blot analysis performed with antiphosphotyrosine antibody (4G10; UBI, Lake Placid, NY) and visualized with ECL (Amersham, Baie d’Urfé, Quebec) permitted the resolution of isolated phosphoproteins. The resulting bands were quantified via densitometry anal-