Characterization of the binding properties and retrograde axonal transport of a monoclonal antibody directed against the rat nerve growth factor receptor

We have demonstrated in vitro and in vivo the specific binding of a monoclonal antibody to the rat nerve growth factor (NGF) receptor. Previous work had shown that this antibody, designated 192-IgG, does not compete with NGF for binding to the NGF receptor of PC12 cells, but instead interacts with the receptor to increase NGF binding to PC12 cells (Chandler, C. E., L. M. Parsons, M. Hosang, and E. M. Shooter, 1984, J. Biol. Chem., 259:6882-6889). In the present study, a solid-phase separation assay verified the specific formation of a ternary complex of 192-IgG, the NGF receptor, and NGF: 125I-labeled 192-IgG precipitated from solution only when incubated with both solubilized NGF receptor and NGF covalently linked to a solid phase (Sepharose 4B). Filtration assays using plasma membrane preparations of various tissues showed strict correlation of 125I-192-IgG and 125I-labeled NGF binding; only membranes obtained from superior cervical ganglion bound significant amounts of the monoclonal antibody and NGF. Injection of 125I-192-IgG into the rat anterior eye chamber led to accumulation of intact antibody molecules in the ipsilateral superior cervical ganglion, indicating retrograde axonal transport of 125I-192-IgG from the neuronal termini, located at the iris, to the cell bodies situated in the ganglion. The time course and saturation characteristics of 125I-192-IgG retrograde transport were very similar to those previously reported for 125I-NGF transport, indicating that 192-IgG can be internalized and transported by the same mechanisms as is NGF. Consistent with results of the in vitro binding assays, 192-IgG and NGF failed to compete for retrograde transport and were actually co-transported. Retrograde axonal transport of 192-IgG appears to be species specific, since 125I-192-IgG was transported in the rat, but not in mice, gerbils, hamsters, or guinea pigs. These results establish monoclonal antibody 192-IgG as a specific probe for the rat NGF receptor in vitro and in vivo.     

A single Mr approximately 103,000 125I-beta-nerve growth factor-affinity-labeled species represents both the low and high affinity forms of the nerve growth factor receptor

Both high and low affinity receptors for nerve growth factor (NGF) have been described, but only the former appear to mediate NGF actions and uptake. To specifically characterize the molecular identity of the high affinity site and to compare it with the low affinity site, the water-soluble carbodiimide EDC was used to cross-link 125I-NGF to NGF receptors on: rat PC12 cells, PC12nnr5 cells (PC12 mutants that have only low affinity NGF binding), SH-SY5Y human neuroblastoma cells (which have only high affinity binding sites), and cultured rat sympathetic ganglion cells. A variety of criteria were used to distinguish the two classes of affinity-labeled receptors: competition with unlabeled NGF, dissociation rate, and selective solubilization by 0.1% Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that cross-linking generated only a single Mr approximately 103,000 125I-NGF affinity-labeled species which represents both the low and high affinity forms of the receptor. The 125I-NGF X receptor complexes formed with both affinity classes of the receptor were quantitatively immunoprecipitated by the monoclonal anti-NGF-receptor antibody 192-IgG and both showed identical shifts in mobility when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. These findings indicate that both high and low affinity NGF receptors possess apparently identical NGF-binding moieties. The differences between the kinetic and functional properties of the two receptor types may therefore result from their interactions with other membrane components or with cytoplasmic proteins.     

Selective destruction of nerve growth factor receptor-bearing cells in vitro using a hybrid toxin composed of ricin A chain and a monoclonal antibody against the nerve growth factor receptor

A hybrid toxin composed of ricin A chain and a monoclonal antibody directed against the rat nerve growth factor (NGF) receptor (192-IgG) was prepared using the heterobifunctional cross-linking agent N-succinimidyl-3-(2-pyridyldithio)-propionate and purified by affinity chromatography. Characterization studies showed that the hybrid, 192-s-s-A, displaced bound 125I-labeled 192-IgG from rat superior cervical ganglion (SCG) membranes with an IC50 3-5 times lower than that of unconjugated 192-IgG. When incubated with cultured rat SCG neurons, 192-s-s-A inhibited protein synthesis in a concentration-dependent fashion. The effect of 192-s-s-A on these neurons was reversed by coincubation with an excess of 192-IgG. The IC50 of 192-s-s-A on protein synthesis in rat SCG neurons was 4 nM. Intact ricin and ricin A chain inhibited protein synthesis in these neurons with IC50 values of 5 pM and 500 nM, respectively. The 192-s-s-A hybrid had no effect on mouse SCG neurons or a human melanoma cell line known to have NGF receptors. This is consistent with the finding that 192-IgG recognizes only the rat NGF receptor. Also, 192-s-s-A did not inhibit protein synthesis in primary cultures of rat skeletal muscle or Vero cells, which do not have cell surface receptors for NGF. 192-s-s-A was able to inhibit protein synthesis in PC12 cells but the potency was 10-100 times less in these cells compared to rat SCG neurons. Ricin and A chain were also 10-100 times less potent in PC12 cells than neurons. Rat SCG neurons exposed to 192-s-s-A lost their refractile appearance under phase-contrast optics, showed granular degeneration of neurites, and died. Thus the decreased protein synthesis caused by the hybrid toxin correlated with the morphological destruction of the neurons. 192-s-s-A represents a potentially powerful tool by which to selectively destroy NGF receptor-bearing cells in vitro. The hybrid toxin may prove useful as an in vivo toxin.     

The Nerve Growth Factor Receptor on PC 12 Cells: Interconversion Between Two Forms with Different Binding Properties

PC12 cells possess two classes of nerve growth factor (NGF) receptors on their surfaces which can be distinguished by kinetic criteria. The majority class binds and releases 125I-NGF at a relatively rapid rate and has been called fast. The second class of receptors has been called slow because of relatively slower rates of binding and release of 125I-NGF, and also may be distinguished from fast receptors by their cytoskeletal association and resistance to trypsin. PC12 cell plasma membranes were prepared and shown to have only the fast class of receptors. These membranes were fused to receptorless 3T3 cells with polyethylene glycol. The resultant fused cells were shown to possess NGF receptors, essentially all of which behave like slow receptors. Immunofluorescence microscopy was used to monitor the introduction of PC12 cell membrane and NGF receptors into 3T3 cells. Results obtained with C10-2, a monoclonal antibody specific for a major PC12 cell-surface antigen. show that up to 90% of 3T3 cells receive PC12 membrane and that the PC12 membrane becomes integrally incorporated into the 3T3 cell plasma membrane. It is suggested that an association of receptors with cytoskeleton may be involved in the conversion of fast to slow receptor behavior, and that the differing proportion of fast and slow NGF receptors in PC12 and 3T3 cells reflects the differing cytoskeletal organization of these cells.     

A monoclonal antibody which inhibits epidermal growth factor binding has opposite effects on the biological action of epidermal growth factor in different cells

An epidermal growth factor (EGF) receptor-interactive monoclonal antibody (151-IgG) that inhibits EGF binding to PC12 rat pheochromocytoma cells and to various other cell types has been produced. The hybridoma clone was obtained by fusing Sp2/O-Ag14 myeloma cells with splenocytes from Balb/C mice which had been immunized with n-octyl glucoside-solubilized protein from isolated PC12 cell plasma membranes. The antibody is an IgG which binds to protein A. 151-IgG did not bind EGF. At 0.5 degrees C 151-IgG was directly competitive for EGF binding to PC12 cells. It also inhibited EGF binding to bovine corneal endothelial cells, rabbit corneal fibroblasts, human foreskin fibroblasts, and normal rat kidney cells, and it slightly enchanced EGF binding to SW 3T3 cells. PC12 cells have the same number of binding sites for 151-IgG as for EGF (approximately 27,000 sites/cell). 151-IgG inhibited the photoactivatable cross-linking of EGF to a protein of Mr 170,000 in PC12 cells. 151-IgG inhibited the EGF-stimulated incorporation of [3H]thymidine into quiescent bovine corneal endothelial cells, rabbit corneal endothelial cells, epithelial normal rat kidney cells, and SW 3T3 cells while it enhanced the EGF-stimulated [3H]thymidine incorporation into quiescent human foreskin fibroblasts. 151-IgG by itself possessed intrinsic EGF-like activity for human fibroblasts but not for the other cells tested. This suggests that there is a difference in EGF receptors and/or processing in these normal cell types.     

Inhibition of beta nerve growth factor binding to PC12 cells by alpha nerve growth factor and gamma nerve growth factor

Pheochromocytoma (PC12) cells have been found to differ from dorsal root ganglionic cells with respect to the modulation of the beta nerve growth factor (beta NGF) binding properties elicited by alpha NGF and gamma NGF. In contrast to our previous results with intact dorsal root ganglionic cells in which only high-affinity binding was blocked, alpha NGF and gamma NGF were found to block competitively all steady-state binding of iodinated beta NGF to PC12 cells at both 37 and 0.5 degrees C. The EC50 that was found for the alpha NGF displacement was 9-10 microM, and the gamma NGF effect had an EC50 of 200 nM, in the predicted range based upon the apparent Kd for dissociation of the alpha beta or the beta gamma complex in solution. The concurrence of the binding EC50 and the Kd for each complex indicates that the formation of alpha beta or beta gamma complexes in solution competes with the process of PC12 receptor binding with 125I-beta NGF. Experiments were carried out examining the dissociation kinetics following the addition of excess unlabeled beta NGF or alpha NGF at both 37 and 0.5 degrees C. Three dissociation components were observed with alpha NGF, in contrast to the two normally found with beta NGF. Lowering the chase temperature to 0.5 degrees C changed the relative contributions made by each component without dramatically changing any of the rate constants. The "slow" receptor was further examined by the dependence on 125I-beta NGF concentration of the slowest component with a chase of either excess alpha NGF or excess gamma NGF at 0.5 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular characteristics of nerve growth factor receptors on PC12 cells

Cross-linking of 125I-nerve growth factor (NGF) to PC12 cells with the photoreactive heterobifunctional agent N-hydroxysuccinimidyl-4-azidobenzoate results in the labeling of two major bands with Mr 158,000 and 100,000 and a minor band with Mr 225,000 as determined by polyacrylamide gel electrophoresis under denaturing and reducing conditions. Binding of 125I-NGF to and cross-linking into all these species is abolished in the presence of excess unlabeled NGF but not in the presence of unlabeled epidermal growth factor, insulin, or bovine pancreatic trypsin inhibitor. When PC12 cells with bound 125I-NGF are incubated in excess unlabeled NGF at 0 degree C prior to cross-linking, only the Mr 158,000 species remains. In addition, binding of 125I-NGF to the Mr 158,000 complex is trypsin-resistant, whereas binding to the Mr 100,000 complex is not. These experiments identify the Mr 158,000 species as the slow NGF-receptor complex (chase stable at 0 degree C) and the smaller Mr 100,000 species as the fast NGF-receptor complex (trypsin sensitive). Furthermore, 125I-NGF bound to the former but not to the latter species is displaced by very-low concentrations of NGF, showing that at least a significant fraction of the high-molecular-weight slow receptor is also a high-affinity receptor. This identification is supported by the finding that chick sensory neurons which possess both high- and low-affinity receptors exhibit two major labeled bands with Mr 145,000 and 105,000 as a result of cross-linking with 125I-NGF, whereas a cell population enriched in non-neuronal cells, which possess only low-affinity receptors, exhibits only the Mr 105,000 component. A shift in molecular weight of both species after pretreatment with neuraminidase indicates that both complexes contain sialoglycoproteins and rules out the possibility that differences in sialic acid content are responsible for the difference in molecular weight of the two complexes. The relative amount of the labeling of these two complexes is not affected by the presence of protease inhibitors nor by a variation of 5000-fold in cross-linker concentration. These results place some limits on possible models for the NGF receptors and their interconversion.     

The internalization of nerve growth factor by high-affinity receptors on pheochromocytoma PC12 cells.

The internalization and subsequent fate of the two populations of nerve growth factor (NGF) receptors on pheochromocytoma PC12 cells were explored either by identifying the relative amounts and sizes of the receptors, after incubation of cells with [125I]NGF, by cross-linking with a photoreactive heterobifunctional reagent or by following the topological distribution of the cross-linked receptors with time. The ratio of the slow, high-affinity to the fast, low-affinity NGF receptor decreased over a 5-h incubation with [125I]NGF in a process which did not involve proteolytic conversion of the slow to the fast receptor. During this period the cross-linked slow receptor moved from a trypsin-labile to a trypsin-stable site suggestive of internalization. In contrast, the cross-linked fast NGF receptor remained trypsin sensitive for at least 2 h of incubation, indicative of a constant cell surface localization. The internalized [125I]NGF in the cross-linked slow NGF receptor was not degraded, indicating that cross-linking, by preventing the acid pH-induced dissociation of the NGF-receptor complex in the endosomes, blocks normal sorting of [125I]NGF to the lysosomes. The cross-linked receptor was not recycled to the cell surface. If this reflects the properties of the unmodified receptor then another process, possibly receptor conversion, is required to replenish slow NGF receptors in the cell surface.     

Phosphorylation of nerve growth factor receptor proteins in sympathetic neurons and PC12 cells. In vitro phosphorylation by the cAMP-independent protein kinase FA/GSK-3

We have examined phosphorylation of nerve growth factor (NGF) receptor in cultured sympathetic neurons and PC12 cells. Dissociated rat superior cervical ganglion neurons or PC12 cells were incubated with 32Pi to label cellular phosphoproteins. Membrane proteins were solubilized, and NGF receptor proteins were immunoprecipitated with the monoclonal antibody 192-IgG. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography showed that NGF receptor components of Mr = 80,000 and Mr = 210,000 were phosphorylated. Phosphorylation of neither species was affected by treating the cells with NGF or phorbol 12-myristate 13-acetate. When the 80,000-Da protein was subjected to complete trypsin proteolysis and then analyzed by reverse phase liquid chromatography, two 32P-labeled peptides were resolved. The more hydrophobic peptide accounted for most of the 32P and contained only phosphoserine; the other peptide contained phosphoserine and phosphothreonine. No phosphotyrosine was detected in the receptor proteins. When receptor molecules from nonlabeled PC12 cells were immunoprecipitated and then incubated in vitro with [gamma-32P]ATP and the cAMP-independent protein kinase FA/GSK-3, phosphorylation occurred predominantly on serine and to a lesser extent on threonine. However, the immunoprecipitated receptor proteins neither autophosphorylated nor were they detectably phosphorylated by cAMP-dependent protein kinase, casein kinase II, or protein kinase C (the Ca2+/phospholipid-dependent enzyme). We conclude that binding units of the NGF receptor are phosphorylated constitutively in at least two sites in intact cells and that they can be phosphorylated by FA/GSK-3 in vitro.     

A comparison of nerve growth factor binding protocols with native and mutant PC12 cells

A comparison has been made of various methods for measuring binding of nerve growth factor (NGF) to PC12 cells in suspension, on plates, and by a combination of the two. Results indicated that the extensive washing in the plate binding assay removed some cell surface ligand, underestimated the fast receptor binding, and overestimated the proportion of internalized ligand. In addition, the binding and internalization by a nonresponding PC12 mutant cell line has been studied. The nonresponding mutants had fewer total NGF receptors (10–50%) than normal cells in any binding assay. However, when measured in the suspension assay, the mutant cells showed both fast and slow binding receptors, in proportion approximately equivalent to those found on native PC12 cells. The PC12 nonresponders in suspension were also found to internalize and degrade low levels of NGF, in proportion to their reduced receptor number. Different results concerning PC12 wild type and mutant cells that have been reported in the literature may be due to the particular binding assay protocol that was used.     

Internalization of nerve growth factor by PC12 cells. A description of cellular pools

Binding and internalization of nerve growth factor (NGF) by responsive cells is a complex process. We have incubated rat pheochromocytoma cells (PC12) with 125I-NGF at 37 degrees C and measured the association of ligand after removal of subsets of bound ligand by different methods. Chase with unlabeled NGF at either 4 or 37 degrees C, acid stripping, nonionic detergent stability, and combinations of these protocols were utilized. These variations of the binding assay were able to distinguish ligand bound to fast versus slow cell surface receptors, NGF bound to slow receptors at the cell surface versus cell interior, and soluble ligand versus cytoskeletally attached NGF. Quantitative and temporal relations among five cellular pools were defined. Experiments with the inhibitors chloroquine, cytochalasin B, and colchicine defined pools of NGF in terms of the route through the cell from the plasma membrane to the lysosome. Chloroquine caused accumulation of NGF only in the pool that was not associated with the cytoskeleton, implicating the involvement of this pool in supplying ligand to the lysosome. Results with cytochalasin B and colchicine suggest that both microfilaments and microtubules are involved in pathways leading to NGF degradation. A semiquantitative model for the movement of NGF through the cell is presented based on these observations.     

Modification of nerve growth factor receptor properties by wheat germ agglutinin

PC12 is a nerve growth factor (NGF) responsive cell line which exhibits two classes of NGF receptors distinguishable by different kinetic rate constants, sensitivity to trypsin and resistance to Triton detergent solubilization. Whereas incubation of PC12 cells with wheat germ agglutinin (WGA) prior to addition of 125I-NGF inhibits binding of NGF to both classes of receptors, treatment with WGA subsequent to incubation with NGF does not inhibit NGF binding but causes the class of NGF receptors which exhibit rapid or "Fast" dissociation kinetics prior to lectin treatment to be converted to the form which exhibits "Slow" dissociation kinetics. This WGA-mediated receptor conversion is lectin specific, blocked by N-acetyl-D-glucosamine, occurs at similar rates at 4 and 37 degrees C, and is not impaired by a metabolic poison. NGF receptors converted by WGA, like pre-existing Slow receptors, are resistant to trypsinization and remain associated to Triton X-100 extracted "cytoskeletons." Very similar results were obtained for NGF receptors on a human melanoma cell line A875. These results suggest that Fast and Slow receptors are two interconvertible forms of a single protein, rather than distinct proteins. The significance of the generality of these properties for NGF receptors from diverse species and cell types is discussed.     

Binding, sequestration, and processing of epidermal growth factor and nerve growth factor by PC12 cells

The rat PC12 pheochromocytoma cell line exhibits biological responses to both nerve growth factor (NGF) and epidermal growth factor (EGF). The existence of receptors and biological responses on a common cell for these two well-characterized polypeptide growth factors makes this an attractive system for comparison of ligand binding and processing. Both NGF and EGF are bound to PC12 cells in a competable form at 4 degrees C. At 37 degrees C both ligands are "sequestered," but at different rates and to different extents. While sequestration happens rapidly and nearly quantitatively for bound EGF, the dissociation reaction appears to compete favorably with NGF sequestration. Both EGF and NGF are degraded by PC12 cells. Sequestered EGF, however, is degraded to a greater extent than sequestered NGF.     

Relationship among types of nerve growth factor receptors on PC12 cells

We analyzed the kinetics and thermodynamics of 125I-nerve growth factor (125I-NGF) binding to NGF-receptor on PC12 cells. We used conditions of pseudo-first order kinetics and techniques to quantitate internalized complexes, "slow" or high affinity binding complexes, and cell surface "fast" or low affinity complexes. Two possible models were examined: binding to two independent receptors at the cell surface (i.e. high and low affinity forms of NGF-receptor) and a model for consecutive formation of fast, low affinity binding followed by slow, high affinity binding or internalization. Our data are consistent with the consecutive model only. The rates of association and dissociation of NGF with slow, high affinity sites and internalized, acid wash-resistant sites are indistinguishable from each other. We also analyzed, in detail, the two assays primarily used to distinguish slow binding complexes from internalized complexes. Scatchard analysis of total binding and dissociation of pre-equilibrated 125I-NGF in the presence of unlabeled NGF at high concentration (cold wash). Neither of these assays shows any evidence that the slow, high affinity binding step is different from internalization of the 125I-NGF-receptor complex. Based on this analysis, there are only two detectable forms of NGF-receptor on PC12 cells: complexes on the surface of the cells with a binding affinity of 0.5 nM at 37 degrees C and complexes internalized by the cells. Furthermore, the data are consistent with a model in which NGF-receptor is internalized constitutively and independently of occupancy by NGF. We also examined the fate of internalized 125I-NGF. In the first 60 min after contact with PC12 cells, no degradation of 125I-NGF was observed. Moreover, a significant amount of 125I-NGF recirculates to the cell surface and is released as intact, Mr = 13,000 NGF. The cells were also stimulated by NGF in a primary neurite outgrowth assay with an ED50 of 2-16 pM under conditions of low initial cell numbers in a large extracellular volume of NGF-containing medium. Thus, low level occupancy of the cell surface receptors, Kd = 0.5 nM, for several days is sufficient to stimulate neurite outgrowth. This indicates the presence of spare NGF-receptors on the surface PC12 cells.     

Alteration of binding properties and cytoskeletal attachment of nerve growth factor receptors in PC12 cells by wheat germ agglutinin

Incubation of PC12 cells preloaded with 125I-nerve growth factor (NGF) reveals rapidly and slowly dissociating binding components indicative of a heterogeneous population of receptors. If the cells are previously exposed to wheat germ agglutinin (WGA) for 30 min, NGF now binds to an apparently homogeneous receptor population which exhibit slow dissociation kinetics. Total binding is also reduced by 50%. If WGA is added subsequent to 125I-NGF, total binding is not diminished, but rapidly dissociating receptors occupied with NGF are all converted to the slowly dissociating form. This conversion of receptors occurs rapidly, reaching completion within 2 min at 37 degrees or 4 degrees C, and is unaffected by metabolic energy poisons, suggesting that WGA- induced slowly dissociating receptors are not the product of internalization. The effects of the lectin are blocked by the sugar N- acetyl-D-glucosamine, and the lectin-induced slowly dissociating receptors are converted back to rapidly dissociating receptors by addition of this same sugar. WGA also affects the association of the NGF receptor with the Triton X-100 cytoskeleton. Greater than 90% of bound 125I-NGF becomes associated with Triton X-100 insoluble cytoskeletons in the presence of the lectin, compared with less than 20% before lectin addition. Cytoskeleton association of the NGF receptor by WGA shows similar kinetics as the conversion of rapidly to slowly dissociating receptors. This interaction may be involved in the alteration of NGF-receptor binding properties produced by this lectin.     

Characteristics of Partially Purified Nerve Growth Factor Receptor

Receptors for the nerve growth factor protein (NGF) have been isolated from three cell types [embryonic chicken sensory neurons (dorsal root sensory ganglia; DRG), rat pheochromocytoma (PC12) and human neuroblastoma (LAN-1) cells] and have been shown to be similar with respect to equilibrium dissociation constants. The present results demonstrate that there are multiple molecular weight species for NGF receptors from DRG neurons and PC12 cells. NGF receptors can be isolated from DRG as four different molecular species of 228, 187, 125, and 112 kilodaltons, and PC12 cells as three molecular species of 203, 118, and 107 kilodaltons. The NGF receptors isolated from DRG show different pH-binding profiles for high- and low-affinity binding. High-affinity binding displays a bell-shaped pH profile with maximum binding between pH 7.0 and 7.9, whereas low-affinity binding is constant between pH 5.0 and 9.1, with a twofold greater binding at pH 3.6. At 22 degrees C, the association rate constant was found to be 9.5 +/- 1.0 X 10(6) M-1 s-1. Two dissociation rate constants were observed. The fast dissociating receptor has a dissociation rate constant of 3.0 +/- 1.5 X 10(-2) s-1, whereas the slow dissociating receptor constant was 2.4 +/- 1.0 X 10(-4) s-1. The equilibrium dissociation constants calculated from the ratio of dissociation to association rate constants are 2.5 X 109-11) M for the high-affinity receptor (type I) and 3.2 X 10(-9) M for the low-affinity receptor (type II). These values are the same as those determined by equilibrium experiments on the isolated receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

p75 and TrkA Receptor Signaling Independently Regulate Amyloid Precursor Protein mRNA Expression, Isoform Composition, and Protein Secretion in PC12 Cells

Abstract: The pheochromocytoma PC12 cell line was used as a model system to characterize the role of the p75 neurotrophin receptor (p75^NTR) and tyrosine kinase (Trk) A nerve growth factor (NGF) receptors on amyloid precursor protein (APP) expression and processing. NGF increased in a dose-dependent fashion neurite outgrowth, APP mRNA expression, and APP secretion with maximal effects at concentrations known to saturate TrkA receptor binding. Displacement of NGF binding to p75^NTR by addition of an excess of brain-derived neurotrophic factor abolished NGF's effects on neurite outgrowth and APP metabolism, whereas addition of brain-derived neurotrophic factor alone did not induce neurite outgrowth or affect APP mRNA or protein processing. However, treatment of PC12 cells with C2-ceramide, an analogue of ceramide, the endogenous product produced by the activity of p75^NTR-activated sphingomyelinase, mimicked the effects of NGF on cell morphology and stimulation of both APP mRNA levels and APP secretion. Specific stimulation of TrkA receptors by receptor cross-linking, on the other hand, selectively stimulated neurite outgrowth and APP secretion but not APP mRNA levels, which were decreased. These findings demonstrate that in PC12 cells expressing p75^NTR and TrkA receptors, binding of NGF to the p75^NTR is required to mediate NGF effects on cell morphology and APP metabolism. Furthermore, our data are consistent with NGF having specific effects on p75^NTR not shared with other neurotrophins. Lastly, we have shown that specific activation of TrkA receptors—in contrast to p75^NTR-associated signaling—stimulates neurite outgrowth and increases nonamyloidogenic secretory APP processing without increases in APP mRNA levels.     

Targeting of Liposomes to Cells Bearing Nerve Growth Factor Receptors Mediated by Biotinylated Nerve Growth Factor

We have used biologically active derivatives of beta-nerve growth factor (NGF), modified by biotinylation via carboxyl groups, to target the specific binding of liposomes to cultured rat and human tumor cells bearing NGF receptors. Liposomes, to be used for targeting, were prepared by conjugating streptavidin to phospholipid amino groups on liposomes prepared by reverse-phase evaporation. Approximately 2,000 streptavidin molecules were incorporated per liposome. Addition of biotinylated NGF, but not of unmodified NGF, could mediate the subsequent binding of radiolabeled streptavidin-liposomes to rat pheochromocytoma PC12 cells in suspension at 4 degrees C. In contrast, incubation with biotinylated NGF did not mediate the binding of hemoglobin-conjugated liposomes. Under optimal incubation conditions, approximately 570 streptavidin-liposomes were specifically bound per cell. Biotinylated NGF was also used to obtain specific binding of streptavidin-liposomes containing encapsulated fluorescein isothiocyanate-labeled dextran to PC12 cells or human melanoma HS294 cells. When HS294 cells were incubated at 37 degrees C following targeted liposome binding at 4 degrees C, the cell-associated fluorescence appeared to become internalized, displaying a perinuclear pattern of fluorescence similar to that observed when lysosomes were stained with acridine orange. Trypsin treatment abolished cell-associated fluorescence when cells were held at 4 degrees C but did not alter the fluorescence pattern in cells following incubation at 37 degrees C. When liposomes containing carboxyfluorescein, a dye capable of diffusing out of acidic compartments, were targeted to HS294 cells, subsequent incubation at 37 degrees C resulted in diffuse cytoplasmic fluorescence, suggesting that internalized liposomes encounter lysosomal or prelysosomal organelles.     

Binding and degradation of nerve growth factor by PC12 pheochromocytoma cells

The specific binding of various concentrations of 125I-labeled nerve growth factor (NGF) to PC12 cells at 37 degrees C reached maxima after 90 min and then declined to 25% of maximal binding after 10 h. Decreased binding was accompanied by degradation of 125I-NGF and the appearance of acid-soluble biologically inactive 125I (mainly 125I-monoiodotyrosine) in the medium as well as a decrease in the number of surface NGF receptors. The time-dependent decrease in binding and the degradation of 125I-NGF were inhibited by low temperature and the lysosomotropic agent chloroquine while degradation was inhibited by metabolic energy inhibitors in the absence of glucose. Chloroquine also produced an increase in the accumulation of 125I-NGF which was not readily removed from the cells. These data suggest that 125I-NGF bound to PC12 cells is efficiently internalized by receptor-mediated endocytosis and degraded by the lysosomes. It appears from other data that this process does not produce the intracellular signals regulating neurite outgrowth.     

Long-term, heterologous down-regulation of the epidermal growth factor receptor in PC12 cells by nerve growth factor

Cells of the rat pheochromocytoma clone PC12 possess receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF), thus enabling the study of the interaction of these receptors in the regulation of proliferation and differentiation. Treatment of the cells with NGF induces a progressive and nearly total decrease in the specific binding of EGF beginning after 12 h and completed within 4 d. Three different measures of receptor show that the decreased binding capacity represents, in fact, a decreased amount of receptor: (a) affinity labeling of PC12 cell membranes by cross-linking of receptor-bound 125I-EGF showed a 60-90% decrease in the labeling of 170- and 150-kD receptor bands in cells treated with NGF for 1-4 d; (b) EGF-dependent phosphorylation of a src-related synthetic peptide or EGF receptor autophosphorylation with membranes from NGF-differentiated cells showed a decrease of 80 and 90% in the tyrosine kinase activity for the exogenous substrate and for receptor autophosphorylation, respectively; (c) analysis of 35S-labeled glycoproteins isolated by wheat germ agglutinin-Sepharose chromatography from detergent extracts of PC12 membranes showed a 70-90% decrease in the 170-kD band in NGF-differentiated cells. These findings permit the hypothesis that long-term heterologous down-regulation of EGF receptors by NGF in PC12 cells is mediated by an alteration in EGF receptor synthesis. It is suggested that this heterologous down-regulation is part of the mechanism by which differentiating cells become insensitive to mitogens.