Beta nerve growth factor binding to PC12 cells. Association kinetics and cooperative interactions

The association kinetics of 125I beta nerve growth factor (NGF) binding to the PC12 clonal cell line have been examined in detail at 0.5 and 37 degrees C. These data were examined by utilizing a reversible second-order integrated rate equation, and the results were not consistent with a simple bimolecular process. Two association rates were required to explain the results adequately. At 37 degrees C, the faster component was estimated to have a second-order association rate constant of 1.4 X 10(7) M-1 s-1, while the rate constant for the slower component (3.8 X 10(6) M-1 s-1) was about 4-fold lower. As shown by others, the temperature dependence of the dissociation kinetics indicated that while the rapidly dissociating component was only slightly slowed by lowering the chase temperature to 0.5 degrees C, the second component was slowed by about 270-fold, from 8 X 10(-4) s-1 to 3 X 10(-6) s-1. The binding data that describe the slowly dissociating component were obtained by utilizing this differential temperature dependence and revealed a concave downward Scatchard plot. The binding parameters determined from computer analysis using a nonlinear fitting program (LIGAND) suggest that this component consists of (a) an interacting class of about 4000 sites/cell that have a first stoichiometric steady-state dissociation constant of 65 pM and a second stoichiometric interaction constant of 16 pM, indicative of positively cooperative interactions, and (b) a class of sites consistent with a ratio of sites/Kd of about 11.1 sites/(cell X pM). The steady-state binding results at 37 degrees C indicated only one class of binding sites (155,000 +/- 18,000 sites/cell) that had an apparent Kd of 0.52 +/- 0.03 nM. One class of sites was also observed at 0.5 degrees C, and the receptor concentration was found to be reduced (99,000 +/- 7600 sites/cell) while the Kd was increased (1.7 +/- 0.14 nM). A significant level of positively cooperative interactions was observed frequently at 37 degrees C that was not due to a failure to reach steady-state conditions, internalization, or degradation. Since cooperativity of binding was never observed at 0.5 degrees C, a membrane event may be involved. Determination of the contribution of the different classes of NGF receptors found on PC12 cells to the biological actions of NGF requires a clear understanding of their kinetic properties and their relationship to each other. The studies presented here indicate that their interactions are more complex than previously described.     

Nerve growth factor receptors. Characterization of two distinct classes of binding sites on chick embryo sensory ganglia cells

Steady state and kinetic studies on the binding of 125I-beta nerve growth factor (NGF) to single cells from sensory ganglia of 8-day-old chick embryos show two distinct, saturable binding sites with dissociation constants of Kd(I) = 2.3 X 10(-11) M and Kd(II) = 1.7 X 10(-9) M. The difference in the affinities is due to different rate constants of dissociation (k-1(I) = 10(-3) s-1, k-1(II) = 2 X 10(-1 s-1). The association to both sites is apparently diffusion controlled (k+1(I) = 4.8 X 10(7) M-1s-1, k+2(II) = 10(7) to 10(8) M-1s-1). The binding of betaNGF to both sites is specific, since none of a number of hormones or proteins tested compete for the binding of 125I-betaNGF to either of those two sites. The heterogeneity of the binding of 125I-betaNGF is not due to heterogeneity of the 125I-betaNGF preparation nor to a negatively cooperative binding. In experiments where the dissociation of 125I-betaNGF is induced by the addition of saturating amounts of unlabeled betaNGF, the ratio of the 125I-betaNGF released with either of the two dissociation rate constants is solely dependent on the occupancy of the two sites before dissociation is started and is independent of the total occupancy of the sites during dissociation. The rate of dissociation of 125I-betaNGF from the higher affinity binding site I is accelerated by unlabeled betaNGF under conditions where the occupancy is both increased and decreased. Although the dissociation characteristics of 125I-beta NGF change with increasing times of exposure of the cells to the ligand, and 125I-beta NGF is degraded after it binds to the cells, these secondary processes do not interfere with the analysis of the binding data. At the lowest concentration of 125I-beta NGF used for the analysis less than 10% of the 125I-beta NGF is degraded. Both kinetic and steady state binding data reveal the two NGF binding sites at 2 degrees C as well as at 37 degrees C.     

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.     

Cellular receptors for type beta transforming growth factor. Ligand binding and affinity labeling in human and rodent cell lines

Type beta transforming growth factor (beta TGF) purified from human platelets to homogeneity as judged by NH2-terminal amino acid sequence analysis has been labeled with 125I to characterize its interaction with cellular receptors. Binding of 125I-beta TGF to target cells is temperature- and time-dependent, specific, saturable, and reversible. About 1.6-1.9 X 10(4) binding sites/cell with high affinity for beta TGF (Kd = 5.6-7.8 X 10(-11) M and 9.1-14 X 10(-11) M, respectively) are found in NRK-49F and BALB/c 3T3 cells. beta TGF receptors do not appear to undergo acute down-regulation by the ligand. Specific binding of 125I-beta TGF has been observed in several human, rat, and mouse fibroblast lines and in some, but not all, tumor-derived cell lines examined. 125I-beta TGF has been cross-linked to intact cells and isolated membrane preparations using disuccinimidyl suberate. Cells and isolated membranes from human, rat, and mouse origin affinity labeled with 125I-beta TGF exhibit a major labeled species of approximately 280 kilodaltons that has the properties of high affinity and specificity expected from a physiologically relevant beta TGF receptor. Minor labeled species of 70-90 kilodaltons are also labeled by 125I-beta TGF, but they correspond to molecular species with low apparent affinity (Kd approximately 10(-8) M) for 125I-beta TGF.     

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.     

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.     

Two receptor classes for epidermal growth factor on pheochromocytoma cells, distinguishable by temperature, lectins, and tumor promoters

Rat pheochromocytoma cells (clone PC12) display cell surface receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF) and therefore provide a useful model system with which to study the role of these receptors in the regulation of proliferation and differentiation. In this paper PC12 cells are demonstrated to possess two classes of EGF receptors, a high-affinity class with 7,600 sites per cell and an apparent dissociation constant (Kd) of 0.05 nM, and a low-affinity class with 62,000 sites per cell and a Kd of 14.1 nM. These findings are contrary to literature data (Huff et al., 1981; Vale and Shooter, 1983) but can be explained in part by differences in experimental conditions. Binding studies at 37 degrees C compared with room temperature demonstrated similar affinities of both classes, but during prolonged incubation at 37 degrees C, the binding capacities of both classes decreased. Furthermore the high-affinity class was sensitive to lectins, such as concanavalin A (Con A), and to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Both compounds caused a decrease of the affinity of the high-affinity class without affecting the low-affinity class. At high concentrations of Con A or TPA, a decrease of the apparent number of binding sites of the low-affinity class was also observed. The similarities between the characteristics of EGF binding and NGF binding in PC12 cells are striking and will be discussed.     

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.     

2'-Deoxy-GTP in the microtubule cytoskeleton of neuronal cells cultured with nerve growth factor.

Tubulin, widely recognized as a GTP/GDP-binding protein, has been isolated in its polymerized state from rat PC12 cells and embryonic chick dorsal root ganglion neurons by Triton X-100 detergent extraction of the cytoskeletal fraction. Perchloric acid extraction and deproteinization of this fraction permitted subsequent analysis of nucleotide identity and content by high performance liquid chromatography. PC12 cells grown in the absence of nerve growth factor (NGF) contained ADP, ATP, GDP, and GTP at levels consistent with the actin and tubulin content of the cytoskeletal fraction. Microtubules from PC12 cells cultured in the presence of NGF contain an additional nucleotide that we have identified as dGTP. Analysis of whole cell nucleotide extracts from PC12 cells grown in the absence or presence of NGF revealed no evidence for the presence of dGTP at 4 and 14 days, respectively. We have determined that embryonic chick dorsal root ganglion neurons also contain this deoxyribonucleotide, and we found virtually no ADP or ATP in the extracted dorsal root ganglion cytoskeletal fraction. On the basis of metabolic labeling studies with [14C] guanine, we have inferred that the presence of dGTP in NGF-treated PC12 cells probably arises either from binding to the nonexchangeable nucleotide site of tubulin undergoing dynamic assembly/disassembly or from binding to the exchangeable site of tubulin subsequently incorporated into highly stabilized microtubules.     

Cross talk among tyrosine kinase receptors in PC12 cells: desensitization of mitogenic epidermal growth factor receptors by the neurotrophic factors, nerve growth factor and basic fibroblast growth factor.

We have studied the effects of nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) on epidermal growth factor (EGF) binding to PC12 cells. We show that NGF and bFGF rapidly induce a reduction in 125I-EGF binding to PC12 cells in a dose-dependent manner. This decrease amounts to 50% for NGF and 35% for bFGF. Both factors appear to act through a protein kinase C(PKC)-independent pathway, because their effect persists in PKC-downregulated PC12 cells. Scatchard analysis indicates that NGF and bFGF decrease the number of high affinity EGF binding sites. In addition to their effect on EGF binding, NGF and bFGF activate in intact PC12 cells one or several serine/threonine kinases leading to EGF receptor threonine phosphorylation. Using an in vitro phosphorylation system, we show that NGF- or bFGF-activated extracellular regulated kinase 1 (ERK1) is able to phosphorylate a kinase-deficient EGF receptor. Phosphoamino acid analysis indicates that this phosphorylation occurs mainly on threonine residues. Furthermore, two comparable phosphopeptides are observed in the EGF receptor, phosphorylated either in vivo after NGF treatment or in a cell-free system by NGF-activated ERK1. Finally, a good correlation was found between the time courses of ERK1 activation and 125I-EGF binding inhibition after NGF or bFGF treatment. In conclusion, in PC12 cells the NGF- and bFGF-stimulated ERK1 appears to be involved in the induction of the threonine phosphorylation of the EGF receptor and the decrease in the number of high affinity EGF binding sites.     

A monoclonal antibody modulates the interaction of nerve growth factor with PC12 cells

A nerve growth factor (NGF) receptor interactive monoclonal antibody (192-IgG) which enhances beta-NGF binding to PC12 cells has been produced. The hybridoma clone was obtained by fusing Sp2/0- Ag14 myeloma cells with splenocytes from Balb/C mice which had been immunized with n-octyl glucoside solubilized proteins from isolated PC12 cell plasma membranes. The antibody is an IgG, which does not bind beta-NGF. It binds to the same number of sites on PC12 cells at low temperature as does beta-NGF. The 192-IgG increases the apparent affinity of beta-NGF binding to fast receptors on PC12 cells at low temperature by a factor of 2.5- to 4-fold and enhances the photoactivatable cross-linking of beta-NGF to the same receptor while decreasing the cross-linking of beta-NGF to the slow NGF receptor. At 37 degrees C 192-IgG partially inhibits the regeneration of neurites from primed PC12 cells. The 192-IgG also reduces the rate of appearance of binding to slow NGF receptors and increases the proportion of beta-NGF bound to fast receptors at 37 degrees C. These results implicate the slow receptor as the mediator of the biological response. This antibody provides a tool for examining steps in the mechanism of action of beta-NGF after binding to the receptor.     

Characterization of nerve growth factor binding to cultured neural crest cells: evidence of an early developmental form of the NGF receptor

Cultured neural crest cells undergoing differentiation have been shown to contain a subpopulation of cells with specific receptors for nerve growth factor (NGF). These cells are the potential targets of NGF during differentiation and development. This study was done to pharmacologically characterize the binding of NGF to long-term (1- to 3-week) cultures of quail neural crest cells. The data indicate that 125I-NGF binding was specific and saturable, with less than 20% nonspecific binding. Scatchard analysis revealed the presence of one type (class) of receptors with a binding constant (Kd) similar to that of the low-affinity binding site described for embryonic dorsal root and sympathetic ganglia (approximately 3.2 nM). This was corroborated by displacement experiments (Kd of 1.3 nM), in which 125I-NGF binding was measured in the presence of increasing concentrations of nonradioactive NGF. In addition, affinity labeling revealed that the 125I-NGF-receptor complex had a molecular weight of about 93K, characteristic of the low-affinity NGF receptor of PC12 cells. The NGF receptor of cultured neural crest cells was trypsin-sensitive, as is typical of the low-affinity NGF binding sites. These findings indicate that differentiating neural crest cells lack high-affinity 125I-NGF binding sites. In contrast, embryonic dorsal root and sympathetic ganglia cells, known NGF targets, have both high- and low-affinity receptors. Measurements of the differential release of surface-bound 125I-NGF indicated that a relatively small amount (about 14%) of NGF is internalized over a 1-hr period. Cultured neural crest cells which bear NGF receptors were also shown by light microscopic radioautographic techniques to incorporate [3H]thymidine. I suggest, therefore, that cultured neural crest cells which have not terminally differentiated, as judged by morphological criteria and continued proliferation, may express an early developmental form of the NGF receptor.     

Properties of the nerve growth factor receptor of a clonal line of rat pheochromocytoma (PC12) cells.

The interaction of nerve growth factor (NGF) with its receptor on cells of the PC 12 cell line was studied. All experiments were done at 0.5 °C to minimize degradation and processes requiring membrane mobility. Under these conditions, a single class of high affinity binding sites with a dissociation constant of 2.9 × 10^?9 M was observed. The number of receptors per cell was 58000. The binding was linear with the number of cells in the assay and was not displaced by proteins other than native nerve growth factor. Trypsin treatment of the cells destroyed the specific binding. The removal of divalent cations had no effect on the binding. Culturing the cells for 2 weeks in NGF prior to assay did not change the receptor number or receptor affinity and there was a similar lack of effect of the density of the culture from which the cells were taken for assay. The present findings are compared with previous studies on the dorsal root ganglia and sympathetic ganglia neurons, and the implication for the use of PC 12 as a model for the study of NGF action are discussed.     

Subunit interactions inhibit the binding of beta nerve growth factor to receptors on embryonic chick sensory neurons

The binding of the beta nerve growth factor subunit in the 7 S nerve growth factor complex to the two nerve growth factor receptors on chick embryo dorsal root ganglion cells was investigated. Under conditions where the 7 S nerve growth factor complex is maximally stable (in the presence of excess alpha and gamma subunits and of 20 to 30 microM zinc ion), no binding to either receptor was detectable. The time course of the decrease in the binding of beta nerve growth factor to the receptors upon addition of alpha and gamma subunits and zinc ion paralleled the formation of the 7 S complex. Addition of alpha and gamma subunits and zinc ion to the bisdes-arginine118-nerve growth factor, which does not re-form the 7 S complex, failed to inhibit the binding of the derivative to either receptor. While the alpha subunit alone had no effect on beta nerve growth factor binding, the gamma subunit decreased its binding in proportion to the amount of complex formed between these two subunits, suggesting that the beta . gamma complex, like the 7 S complex, does not bind to nerve growth factor receptors.     

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.     

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.     

Nerve growth factor receptors on PC12 cells: Evidence for two receptor classes with differing cytoskeletal association

PC12, an NGF responsive cell line, exhibits two classes of NGF receptors which we designate “Fast” and “Slow.” Fast receptors, accounting for 75% of specific NGF binding, are distinguished by their rapid rates for association and dissociation of ¹²⁵I-NGF. At 37°C, binding of ¹²⁵I-NGF to Fast receptors is 5-fold more rapid than to Slow receptors and dissociation of ¹²⁵I-NGF from Fast receptors is 40-fold more rapid than from Slow receptors. No evidence was obtained for a ligand-induced conversion of receptors from Fast to Slow characteristics. Scatchard analysis of binding experiments indicates that PC12 cells possess 60,000 specific receptors for NGF of which 15,000 are of the Slow class. Despite having very different kinetic constants, Slow and Fast receptors have similar equilibrium binding constants (about 2 × 10^?10 M) due to cancelling effects of differing association and dissociation rates. Brief digestion of PC12 cells with trypsin before addition of NGF inactivates essentially all Fast receptors without significantly affecting Slow receptors. Therefore Fast and Slow classes of receptors must exist prior to addition of NGF, and the observed receptor heterogeneity is not due to ligand-induced changes. ¹²⁵I-NGF bound to Slow receptors is preferentially associated with preparations of Triton X-100 insoluble cytoskeletons, while ¹²⁵I-NGF bound to Fast receptors is solubilized by this procedure. Cytoskeletally associated NGF is almost exclusively associated with the extranuclear cytoskeletal matrix rather than with the nucleus itself. Preparation of nuclei by various methods suggests that the presence of contaminating cytoskeletal elements should be considered in evaluating the existence of translocation and binding of NGF to the nucleus. Inhibition of endocytotic internalization of NGF either by lowering of temperature to O°C or by preincubation of cells with sodium azide in medium lacking glucose does not reduce the slowly released component of bound NGF, nor alter its cytoskeletal association. The possible functional roles of Slow and cytoskeletal receptors are discussed.     

Retinoic acid regulates both expression of the nerve growth factor receptor and sensitivity to nerve growth factor.

In PC12 cells, retinoic acid (RA) stimulates the expression of p75NGFR, a component of the nerve growth factor (NGF) receptor, as indicated by a rapid increase in p75NGFR mRNA, an increase in the binding of 125I-labeled NGF to p75NGFR, and an increase in the binding of NGF to low affinity sites. RA-treated cells are more sensitive to NGF, but not to either fibroblast growth factor or phorbol 12-myristate 13-acetate, showing that RA has a specific effect on the responsiveness of PC12 cells to NGF. Exposure to RA leads neither to an increase in the expression of mRNA for trk, another component of the NGF receptor, nor to an increase in binding to high affinity receptors, suggesting that an increase in the expression of p75NGFR is sufficient to make cells more sensitive to NGF. This work suggests that, in addition to having direct effects on gene expression, RA can indirectly modulate differentiation of neurons by modifying their expression of cell surface receptors to peptide growth factors.