Wheat Germ Agglutinin Inhibits the Effects of Nerve Growth Factor on the Phosphorylation of Proteins in PC12h Cells

Treatment of PC12h cells in tissue culture with nerve growth factor (NGF) led to an increased incorporation of [32P]orthophosphoric acid into specific proteins. The increased phosphorylation of 60,000-dalton and 20,000-dalton proteins in the 0.2% Triton X-100 detergent-soluble fraction, of 35,000-dalton protein in the 0.2% Triton X-100 detergent-insoluble fraction, and of slow migrating protein (SMP) in the nonhistone nuclear fraction was observed upon NGF treatment. On the other hand, wheat germ agglutinin (WGA) treatment of PC12h cells induced a slightly decreased phosphorylation of these NGF-responsive proteins. Incubation of cell-free extracts from PC12h cells with [gamma-32P]ATP led to the phosphorylation of a 100,000-dalton protein. In extracts from cells treated with NGF, the labeling of the 100,000-dalton protein was substantially and selectively reduced. In contrast, treatment of PC12h cells with WGA led to an increased phosphorylation of the 100,000-dalton protein in cell-free extracts. Thus, NGF and WGA showed opposite effects on the phosphorylation of specific proteins in both intact cells and cell-free extracts. In addition, it was also observed in both systems that pre- and posttreatment of PC12h cells with WGA abolished the effects of NGF on the phosphorylation and produced a phosphorylation pattern similar to that from PC12h cells treated only with WGA. In parent PC12 cells, it has been reported that the treatment of cells with WGA inhibits NGF binding to its receptors and converts the rapidly dissociating receptors to slowly dissociating receptors. Thus, WGA in conjunction with NGF, results in the practical disappearance of rapidly dissociating receptors on cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fibroblast growth factor-induced decrease in the phosphorylation of Nsp100 mediated through a calcium-dependent mechanism and blocked by lectins

Separate treatment of PC12h cells with basic fibroblast growth factor (bFGF) and with epidermal growth factor (EGF) induced a selective decrease in the incorporation of radioactive phosphate into a 100,000-dalton soluble protein during phosphorylation with (gamma-32P)ATP of soluble extracts from the cells, as was seen previously with nerve growth factor (NGF). This 100,000-dalton soluble protein was designated in earlier studies as nerve growth factor-sensitive protein 100 (Nsp100). The inhibitory effects of bFGF and EGF on Nsp100 phosphorylation were prevented by pretreatment of PC12h cells with the calcium chelator, EGTA. Treatment of PC12h cells with the plant lectin wheat germ agglutinin (WGA), which binds to N-acetylglucosamine and sialic acid residues on glycoconjugates, blocked the inhibitory effects of bFGF, EGF, and NGF on Nsp100 phosphorylation. The blockage by WGA was reversed by the addition of the lectin-specific sugar N-acetylglucosamine to the PC12h cultures. Although pretreatment of PC12h cells with succinylated WGA, which has the ability to bind to N-acetylglucosamine but not to sialic acid residues, failed to block the inhibitory effect of NGF on Nsp100 phosphorylation as described previously, it did prevent the inhibitory effect of bFGF on this phosphorylation. These data suggest that in PC12h cells bFGF and EGF induce a decrease in the phosphorylation of Nsp100 mediated through a Ca2(+)-dependent mechanism, as in the case of NGF. Furthermore, the blockage of the bFGF-induced inhibition of Nsp100 phosphorylation by WGA and its succinylated form indicates that N-acetylglucosamine residues of bFGF receptor molecules might be involved in the mechanism by which bFGF inhibits the phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Probing the structure-function relationship of nerve growth factor

We compared the receptor binding, antigenicity, biological activation, and cell-mediated proteolytic degradation properties of mouse nerve growth factor (mNGF) and human NGF (hNGF). The affinity of hNGF toward human NGF-receptor is greater than that of mNGF, but the affinity of mNGF toward rat NGF-receptor is greater than that of hNGF. Thus, the specificity of the interaction between NGF and its receptor resides both on the NGF and on its receptor. Using a group of anti-NGF monoclonal antibodies that competitively inhibit the binding of NGF to receptor, sites differing between mNGF and hNGF were detected. Together, these results indicate that the sites on hNGF and mNGF, responsible for binding to NGF-receptor, are similar but not identical. In comparing the relative abilities of mNGF and hNGF to stimulate a biological response in PC12 cells, we observed that mNGF was better at stimulating neurite outgrowth than was hNGF, consistent with the differences observed for receptor binding affinity. However, the ED₅₀ for biological activation is approximately 100-fold lower than theK _d for receptor occupancy, and, thus, the dose-response curve is not consistent with a simple activation proportional to receptor occupancy. The data are consistent with a model requiring a low-level threshold occupancy of NGF-receptor (K _d=10^–9 M) in order to stimulate full biological activity. Finally, we observed the degradation of NGF by PC12 cells. We found that the NGF molecule is significantly degraded via a receptor-mediated uptake mechanism. Together, the data provide insight into regions of the NGF molecule involved in contacts with the receptor leading to formation of the NGF: NGF-receptor complex. Additionally, they establish the link between occupancy of receptor and biological activation and the requirement for receptor-mediated uptake in order to degrade NGF proteolytically in cultured PC12 cells.     

Wheat germ agglutinin, concanavalin A, and lens culinalis agglutinin block the inhibitory effect of nerve growth factor on cell-free phosphorylation of Nsp100 in PC12h cells

It has been shown that in PC12 and its subclone PC12h treatment of the cells with nerve growth factor (NGF) induces a selective decrease in the incorporation of radioactive phosphate into a 100,000-dalton protein, designated in an earlier study as Nsp100, in the subsequent phosphorylation of soluble extracts from cells with (gamma-32P)ATP. In the present study, we show that plant lectins, wheat germ agglutinin (WGA), concanavalin A (Con A), and lens culinaris agglutinin (LCA), inhibit the action of NGF on Nsp100 phosphorylation in PC12h cells. Treatment of the cells with WGA, which binds to N-acetylglucosamine and sialic acid residues on glycoproteins, strongly blocked the inhibitory action of NGF on the protein phosphorylation. Con A and LCA, both of which recognize the same specific sugars (mannose, glucose), displayed only a moderate blocking effect. Unlike the native lectin, succinylated WGA, which has the ability to bind to N-acetylglucosamine but not to sialic acid residues, and other lectins examined in this study did not inhibit the action of NGF on Nsp100. WGA-mediated inhibition of NGF action was reversed by the addition of N-acetylglucosamine and by the addition of a much lower concentration of a sialoglycoprotein, mucin, into the culture. Since the binding of succinylated WGA to N-acetylglucosamine residues of cell-surface glycoconjugates is not sufficient to prevent the action of NGF, WGA might act on sialic acid residues of the NGF receptor molecule to effect the inhibition of biological actions of NGF.     

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.     

Formation of signal transduction complexes during immobile phase of NGFR movements

Nerve growth factor (NGF) is a secreted neurotrophin involved in the differentiation, growth, and maintenance of neurons. Here, we have used single-molecule imaging to characterize the behavior of Cy3-tagged NGF after binding to receptor complexes on the surfaces of PC12 cells. We show that NGF-receptor complexes have two distinct diffusive states, characterized as mobile and immobile phase. The transition between the two diffusive states occurred reversibly with duration times determined by a single rate limiting process. The abrupt transition to the immobile phase often occurred simultaneously with the clustering of NGF-receptor complexes. Immobilization depended on the phosphorylation of the TrkA NGF-receptor. Using dual-color imaging, it was demonstrated that the membrane recruitment of the intercellular signaling protein occurs with NGF-receptor complexes in the immobile phase indicating signal transduction occurs during this phase. Thus, NGF signaling is performed through a repetitive random process to induce transient formation of signaling complexes.     

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.     

Nerve growth factor stimulates the phosphorylation of a 250 kDa cytoskeletal protein in cell-free extracts of PC12 cells

Nerve growth factor (NGF) rapidly stimulates the phosphorylation of a 250 kDa cytoskeletally-associated protein (pp250) by a protein kinase which is also associated with structural elements of the cell. We have solubilized these proteins and demonstrated that NGF-stimulated phosphorylation can be observed in cell free extracts of cytoskeletons from NGF-treated PC12 cells. The pp250 substrate and the 250-kinase were solubilized from PC12 cytoskeletons by treatment with 2 M urea. Phosphorylation of pp250 was maximally stimulated following treatment of the cells for 5 min with NGF. This effect was transient, diminishing with longer exposure of the cells to hormone. The 250-kinase preferred Mn²⁺ over Mg²⁺ and was inhibited by both Na⁺ and K⁺. The phosphorylation of pp250 was not affected by Ca²⁺. Upon fractionation of the urea-soluble cytoskeletal proteins by gel filtration, the 250-kinase eluted in two peaks; one peak of enzyme activity coeluting with the pp250 substrate, and a second peak of enzyme activity eluting with an apparent Mr of approximately 60 kDa. Treatment of the PC12 cells with the phorbol ester TPA also stimulated the phosphorylation of pp250, although this effect was not as great as that produced by NGF. This cell free system should be a valuable tool in the investigation of the mechanisms of NGF action.Special issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.     

Cell Density Modulates Receptor-Mediated Internalization of Nerve Growth Factor in Pheochromocytoma Cells

Abstract

Binding and fate of the nerve growth factor (NGF) in pheochromocytom a cells (clone PC12) have been measured with the use of iodine-labeled ligand and with ¹²⁵I-NGF antibodies. With such double approach it is possible to distinguish between surface bound and total NGF bound to PC12 cells. It is found that NGF-receptor complexes undergo down-regulation. This process is noticeable at low but not at high cell densities, and only in PC12 cells never exposed to NGF. Previous incubation with growth factor leads to the disappearance of down-regulation of NGF-receptor complexes. Assuming that this process is an indirect measure of NGF-receptor internalization, it is concluded that it is modulated by the cell density or by previous exposure to the factor. These findings are postulated to be relevant to the mechanism of action of NGF and to its multiple effects on target cells.     

Inhibition of the cellular actions of nerve growth factor by staurosporine and K252A results from the attenuation of the activity of the trk tyrosine kinase.

The protein kinase inhibitors staurosporine and K252A inhibit some of the cellular actions of nerve growth factor (NGF). To explore the molecular mechanisms involved, we test the ability of these agents to block one of the earliest cellular responses to NGF, protein tyrosine phosphorylation. Concentrations of 10-100 nM staurosporine and K252A inhibit NGF-dependent tyrosine phosphorylation in PC12 cells and inhibit trk oncogene-dependent tyrosine phosphorylation in trk-transformed NIH3T3 (trk-3T3 cells). In contrast, these compounds are without effect on epidermal growth factor (EGF)-stimulated tyrosine phosphorylation in PC12 cells. NGF-stimulated tyrosine phosphorylation of the pp140c-trk NGF receptor and tyrosine phosphorylation of pp70trk are also inhibited by similar concentrations of staurosporine and K252A, whereas tyrosine phosphorylation of the EGF receptor, insulin receptor, and v-src is not affected. Both staurosporine and K252A inhibit the autophosphorylation of pp70trk on tyrosine residues in an in vitro immune complex kinase reaction. Incubation of trk-3T3 cells with 10 nM staurosporine causes rounded transformed cells to revert to a normal flattened phenotype, whereas src-transformed cells are unaffected by this agent. These data suggest that staurosporine and K252A specifically inhibit the trk tyrosine kinase activity through a direct mechanism, probably accounting for the attenuation by these agents of the cellular actions of NGF.     

Epidermal growth factor receptors on PC12 cells: Alteration of binding properties by lectins

The PC12 cell line displays cell surface receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF). It has been previously shown that the lectin wheat germ agglutinin (WGA) alters the properties of NGF receptors on these cells. We now report that preincubations with either WGA or concanavalin A (Con A) decrease the binding of ¹²⁵I-EGF to PC12 cells by greater than 50%. The inhibition of binding occurred at 37°C and 4°C and could be blocked or reversed by the addition of sugars which bind specifically to WGA or Con A. Scatchard analysis revealed that these lectins decreased binding primarily by lowering the affinity of the receptor and to a lesser extent by decreasing receptor number. Succinylalion of Con A (sCon A) produced a derivative that was less effective than the native lectin in decreasing EGF binding; however, addition of an antibody against Con A restored the ability of sCon A to decrease binding. Similar to results obtained with ¹²⁵I-NGF binding, WGA but not Con A was found to increase, by scveralfold; the proportion of ¹²⁵I-EGF binding that is resistant to solubilization by Triton X-100 detergent. A potential association of the EGF receptor with cytoskeletal elements is discussed which could account for such results.     

Effects of 12-0-tetradecanoylphorbol-13-acetate (TPA) on rat pheochromocytoma (PC12) cells: Interactions with epidermal growth factor and nerve growth factor

The phorbol ester tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) specifically inhibited the binding of radioiodinated epidermal growth factor (¹²⁵I-EGF) to rat pheochromocytoma (PC12) cells in a noncompetitive fashion with an apparent K_i of 11–26 nM. Both TPA and EGF elicited similar biological responses in PC12 cells including enhanced incorporation of ³H-choline and ³²P-orthophosphate into macromolecules, induction of ornithine decarboxylase, and stimulation of the phosphorylation of a 30,000 MW nonhistone, chromosome-associated protein. These effects were also elicited by nerve growth factor (NGF) which, in contrast to the former agents, is a differentiating stimulus for the PC12 cells. The effects of TPA were additive or more than additive to the effects of NGF and EGF. When PC12 cells were induced to differentiate by treatment with NGF for 72 hours, the binding of ¹²⁵I-EGF and responses to EGF were reduced by approximately 70%. The response of PC12 cells to the tumor promoter TPA was unaffected by treatment with NGF. Thus, the qualitatively similar effects of TPA and EGF seemed to be mediated through separate receptor systems with only the EGF receptor system reduced by NGF treatment.     

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.     

Molecular investigations on the high-affinity nerve growth factor receptor.

Nerve growth factor (NGF) receptors have been investigated by means of affinity labeling with 125I-NGF and chemical cross-linking. Two distinct NGF-receptor complexes are detected on PC12 cells; these correspond to 100 kd and 158 kd for the low-affinity (LNGFR) and the high-affinity (HNGFR) receptors, respectively. Interestingly, three different antibodies directed against distinct epitopes on the LNGFR immunoprecipitate the low-but not the high-affinity NGF-receptor complex. Although the identities of the signaling molecules in the HNGFR are unknown, antibodies to the src, ras, raf-1, and yes products fail to immunoprecipitate either receptor complex, suggesting that these molecules are not a part of, or tightly coupled to, either receptor type. Phosphotyrosine residues are found exclusively on the HNGFR complex, suggesting that tyrosine phosphorylation may be one of the initiating events in the NGF-induced signal transduction cascade.     

Rapid fibroblast growth factor-induced increases in protein phosphorylation and ornithine decarboxylase activity: regulation by heparin and comparison to nerve growth factor-induced increases.

Fibroblast growth factors (FGFs), like nerve growth factor (NGF), induce morphological differentiation of PC12 cells. This activity of FGF is regulated by glycosaminoglycans. To further understand the mechanisms of FGF and glycosaminoglycan actions in PC12 cells, we studied the regulation of protein phosphorylation and ornithine decarboxylase (ODC) activity by FGF in the presence and absence of heparin. As with NGF, aFGF and bFGF increased the incorporation of radioactive phosphate into the protein tyrosine hydroxylase (TH). The increase in TH phosphorylation was localized to the tryptic peptide, T3. Both T3 and T1 phosphorylations occur in response to NGF, but there was no evidence that aFGF or bFGF stimulated the phosphorylation of the T1 peptide. This result suggests differential regulation of second messenger systems by NGF and FGF in PC12 cells. Heparin, at a concentration that potentiated aFGF-induced neurite outgrowth 100-fold (100 micrograms/ml), did not alter the ability of aFGF to increase S6 phosphorylation or ODC activity. One milligram per milliliter of heparin, a concentration that inhibited bFGF-induced neurite outgrowth, also inhibited bFGF-induced increases in S6 phosphorylation and ODC activity. These observations suggest (i) that acidic and basic FGF activate a protein kinase, possibly protein kinase C, resulting in the phosphorylation of peptide T3 of TH; (ii) that the FGFs and NGF share some but not all second messenger systems; (iii) that heparin potentiates aFGF actions and inhibits bFGF actions in PC12 cells via distinct mechanisms; (iv) that heparin does not potentiate the neurite outgrowth promoting activity of aFGF by enhancing binding to its PC12 cell surface receptor; and (v) that heparin may coordinately regulate several activities of bFGF (induction of protein phosphorylation, ODC and neurite outgrowth) via a common mechanism, most likely by inhibiting the productive binding of bFGF to its PC12 cell surface receptor.     

Epidermal growth factor, but not nerve growth factor, stimulates tyrosine-specific protein-kinase activity in pheochromocytoma (PC12) plasma membranes

Rat pheochromocytoma (PC12) cells contain specific plasma membrane receptors for both epidermal growth factor (EGF) and nerve growth factor (NGF). Whereas EGF addition to PC12 cells causes a persistent enhancement of proliferation. NGF addition induces a transient stimulation of growth, followed by growth arrest and neuronal differentiation. Despite these differences in biological response, EGF and NGF share a number of early receptor-mediated responses, which are likely te be related to their effect on cell proliferation. In this paper we show that EGF, but not NGF, is able to stimulate the phosphorylation of membrane proteins. In addition, EGF was able to stimulate phosphorylation of a synthetic peptide (RR-SRC) by PC12 membranes in a concentration-dependent manner. Kinetic analysis of the phosphorylation reaction indicated that EGF increased the Vmax from 13 to 70 pmoles/min/mg protein, while no change was observed in Km. Furthermore, EGF was able to stimulate tyrosine phosphorylation of angiotensin I and II, to the same extent as RR-SRC. In contrast no effects of NGF on peptide phosphorylation by PC12 membranes were observed. Cross-linking experiments demonstrated the presence of receptors for both NGF and EGF in PC12 membranes. These different effects of NGF and EGF on activation of membrane-associated protein-kinase activity demonstrate that NGF might be able to stimulate growth transiently without stimulating protein kinase activity.     

TrkA cross-linking mimics neuronal responses to nerve growth factor.

TrkA, a tyrosine kinase receptor, is an essential component of the nerve growth factor (NGF) response pathway. The binding of NGF to the receptor induces receptor autophosphorylation and activation of intracellular signaling pathways, resulting in diverse biological effects. We prepared polyclonal antibodies against the entire extracellular domain of rat trkA produced using a baculovirus expression system. These antibodies specifically recognize rat trkA on antigen blots and in immunoprecipitations. Both IgG and Fab fragments block binding of NGF to trkA expressed by the PC12 cell line. In NGF binding studies using anti-trkA and anti-low-affinity NGF receptor (LNGFR) immunoglobulin (Ig) G, essentially all binding of NGF can be inhibited. The results imply that > or = 97% of the NGF binding sites on PC12 cells are accounted for by trkA and the LNGFR. The binding data also argue that all low-affinity NGF binding sites on PC12 cells reflect interactions with the LNGFR, while all high-affinity sites are trkA dependent. A fraction of the high-affinity (or slow) binding sites seem to require both trkA and the LNGFR. Although the monovalent anti-trkA Fab fragments inhibited the biological effects of NGF, such as induction of tyrosine phosphorylation, and survival and neurite outgrowth of sympathetic neurons, the IgG preparation was not effective as an inhibitor. Instead, the IgG fraction by itself was almost as effective as NGF at stimulating receptor activation, cell survival, and neurite outgrowth. Thus, it appears oligomerization of trkA by antibody-induced cross-linking is sufficient to produce the known cellular effects of NGF.     

A comparison of binding properties and structure of NGF receptor on PC12 pheochromocytoma and A875 melanoma cells

Rat PC12 pheochromocytoma and human A875 melanoma cells express nerve growth factor (NGF) receptors on their surfaces. Covalent crosslinking of bound 125I-NGF to PC12 or A875 intact cells or plasma membrane-enriched fractions resulted in labelling of a peptide doublet at Mr = 110,000 and a single labelled peptide at Mr = 200,000 for each of the cell and membrane preparations. However, a difference between equilibrium binding properties of NGF-receptor on PC12 and A875 cells was observed. PC12 cells exhibited biphasic binding properties with two apparent binding sites: KD = 5.2 nM sites and KD = 0.3 nM sites. The high-affinity PC12 binding sites were trypsin resistant, and 125I-NGF dissociated slowly from them. A875 cells exhibited sites with homogeneous properties (KD = 1.0 nM), all binding sites were trypsin sensitive, and 125I-NGF dissociated rapidly in the presence of unlabelled NGF. Membrane-enriched fractions from either cell type contained binding sites with a uniform low affinity (KD = 3 nM) that were trypsin sensitive, and 125I-NGF rapidly dissociated from them. Sixty to 80 percent of binding sites in membranes could be converted to the high-affinity, trypsin-resistant state by addition of wheat germ agglutinin (WGA). The loss of high-affinity, trypsin-resistant sites from PC12 cells during preparation of plasma membrane fractions does not appear to be the result of selective isolation of low-affinity sites or proteolytic degradation since there is a loss of 125I-NGF binding immediately after cell lysis which is not blocked by protease inhibitors. Also, high-affinity, trypsin-resistant binding sites are not found associated with other cell fractions. The differences between receptor properties on PC12 cells and on A875 cells apparently are the result of differences in the respective intracellular environments. Thus, significant structural homology exists between receptors on A875 and PC12 cells. Cell components other than the binding unit of the NGF receptor may be responsible for the different properties of receptor.     

K-252a inhibits nerve growth factor-induced trk proto-oncogene tyrosine phosphorylation and kinase activity.

The rat pheochromocytoma PC12 cell line differentiates into a sympathetic neuronal phenotype upon treatment with either nerve growth factor (NGF) or basic fibroblast growth factor. The alkaloid-like compound K-252a has been demonstrated to be a specific inhibitor of NGF-induced biological responses in PC12 cells (Koizumi, S., Contreras, M. L., Matsuda, Y., Hama, T., Lazarovici, P., and Guroff, G. (1988) J. Neurosci. Res. 8, 715-721). NGF interacts with the protein product of the proto-oncogene trk and rapidly stimulates the tyrosine phosphorylation of both p140prototrk and a number of cellular substrates. Here we show that these phosphorylation events are directly inhibited in PC12 cells by K252a in a dose-dependent manner, indicating that the site of action of this inhibitor is at the NGF receptor level. K-252a inhibits p140prototrk activity in vitro, demonstrating that K-252a has a direct effect on the p140prototrk tyrosine kinase. Though many of the biochemical responses to NGF in PC12 cells are mimicked by basic fibroblast growth factor and epidermal growth factor, K-252a has no effect on the action of these growth factors in PC12 cells, demonstrating that the initial biological events initiated by NGF are distinctive during neuronal differentiation.     

Induction of neurite outgrowth by v-src mimics critical aspects of nerve growth factor-induced differentiation.

PC12 cells treated with nerve growth factor (NGF) or infected with Rous sarcoma virus differentiate into sympathetic, neuronlike cells. To compare the differentiation programs induced by NGF and v-src, we have established a PC12 cell line expressing a temperature-sensitive v-src protein. The v-src-expressing PC12 cell line was shown to elaborate neuritic processes in a temperature-inducible manner, indicating that the differentiation process was dependent on the activity of the v-src protein. Further characterization of this cell line, in comparison with NGF-treated PC12 cells, indicated that the events associated with neurite outgrowth induced by these two agents shared features but could be distinguished by others. Both NGF- and v-src-induced neurite outgrowths were reversible. In addition, NGF and v-src could prime PC12 cells for NGF-induced neurite outgrowth, and representative early and late NGF-responsive genes were also induced by v-src. However, unlike NGF-induced neurite growth, v-src-induced neurite outgrowth was not blocked at high cell density. A comparison of phosphotyrosine containing-protein profiles showed that v-src and NGF each increase tyrosine phosphorylation of multiple cellular proteins. There was overlap in substrates; however, both NGF-specific and v-src-specific tyrosine phosphorylations were observed. One protein which was found to be phosphorylated in both the NGF- and v-src-induced PC12 cells was phospholipase C-gamma 1. Taken together, these results suggest that v-src's ability to function as an inducing agent may be a consequence of its ability to mimic critical aspects of the NGF differentiation program and raise the possibility that Src-like tyrosine kinases are involved in mediating some of the events triggered by NGF.