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.     

p75(NGFR) and TrkA receptors collaborate to rapidly activate a p75(NGFR)-associated protein kinase.

The role of the low affinity nerve growth factor receptor (p75(NGFR)) in NGF-mediated signaling is not yet understood. Here we show by co-immunoprecipitation that NGF activates a protein kinase that is directly associated with p75(NGFR) in dorsal root ganglion (DRG) cells and PC12 cells in culture. Two proteins of 120 and 104 kDa constitute the majority of this activity. In PC12 cells, TrkA activation was necessary to elicit p75(NGFR)-associated kinase activity. Although NGF binding to p75(NGFR) was not necessary for kinase activation, it accelerated the activation of the kinase at low NGF concentrations. Deletion analysis showed that a 43 amino acid region in the cytoplasmic domain of p75(NGFR) was responsible for this effect. These findings show that p75(NGFR) accelerates TrkA-mediated signaling and, in addition, demonstrate that p75NGFR and TrkA collaborate to activate a previously undescribed p75(NGFR)-associated protein kinase.     

Large deletions in the cytoplasmic kinase domain of the epidermal growth factor receptor do not affect its laternal mobility

The lateral diffusion coefficients of various epidermal growth factor (EGF) receptor mutants with increasing deletions in their carboxy-terminal cytoplasmic domain were compared. A full size cDNA construct of human EGF receptor and different deletion constructs were expressed in monkey COS cells. The EGF receptor mutants expressed on the cell surface of the COS cells were labeled with rhodamine-EGF, and the lateral diffusion coefficients of the labeled receptors were determined by the fluorescence photo-bleaching recovery method. The lateral mobilities of three deletion mutants, including a mutant that has only nine amino acids in the cytoplasmic domain, are all similar (D approximately equal to 1.5 X 10(-10) cm2/s) to the lateral mobility of the "wild-type" receptor, which possess 542 cytoplasmic domain of EGF receptor, including its intrinsic protein kinase activity and phosphorylation state, are not required for the restriction of its lateral mobility.     

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.     

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.     

Effect of coexisting cells on the NGF-inducing neurite growth from PC12h-R

Possible roles of coexisting cells in inducing neurite growth from a nerve cell were studied. Nerve growth factor (NGF)-inducing neurite growth from PC12h-R (a cell line derived from cultured nerve cells) was investigated at various cell densities. At the cell density 10²10⁴ cells/ml neurites appeared even without NGF. In contrast, no neurite appeared without NGF in single cell culture. The neurite growth observed in plural cell culture without NGF was only partially inhibited by antibody to NGF receptor (Ab-NGFR). However, the effect of the used medium alone was mostly inhibited by Ab-NGFR. These results suggest that the neurite inducing potency of coexisting cells is via different sites than the NGF receptor.Abbreviations Ab-IgG-FITC anti-mouse-IgG labeled with fluorescein isothiocyanate - Ab-NF monoclonal antibody to neurofilament 160 kD - Ab-NGFR monoclonal antibody to NGF receptor - BDNF brain-derived neurotrophic factor - D-medium medium for differentiation culture - DMEM Dulbecco's modified Eagle's medium - M-medium medium for multiplication culture - NGF nerve growth factor - NGFR NGF receptor - NT-3 neurotrophin-3 - PC12 pheochromocytoma cell line - PC12h-R subclone of PC12 - Sup-D supernatant of D-medium     

NGF Gene Expression in Dividing and Non-Dividing Cells from AAV-Derived Constructs

NGF expression in COS cells when driven by pTR.NGF (CMV promoter, AAV TRs) was more effective than either pc.NGF (CMV promoter, no AAV TRs) or dlk.NGF (AAV promoters and TRs). This NGF was able to differentiate PC12 cells. Differentiated PC12 cells transfected with pTR.NGF released NGF into medium. The fraction of pTR.NGF transfected PC12 cells that extended neurite-like processes 7 days post-transfection was similar to the transfection efficiency, suggesting that transfected cells were selectively differentiated by locally released NGF. pTR.NGF-transfected primary cultures of either neurons or glia did not express exogenous NGF. These results indicate that NGF can be released by dividing and non-dividing cells, but not neonatally derived brain cells.     

Multiple defects of the nerve growth factor receptor in human neuroblastomas

Neuroblastoma is a tumor of postganglionic sympathetic origin, and nerve growth factor (NGF) is normally required for the survival and differentiation of sympathetic neuroblasts. Since the biological activity of NGF is mediated by the NGF receptor (NGFR), we hypothesized that defects in the NGF/NGFR pathway may play a role in maintenance of the undifferentiated state of neuroblastomas. To test this hypothesis, we examined the structure of the NGFR at the DNA, RNA, and protein levels in a panel of 10 neuroblastoma cell lines. In addition, we examined the function of the NGFR in these lines by analysis of NGF binding kinetics, as well as by the ability of NGF to induce c-fos expression and neurite outgrowth. Southern blot analysis showed that all 10 cell lines possess apparently normal NGFR genes. Northern blot and ligand binding/immunoprecipitation assays revealed four receptor-positive cell lines (NGP, NLF, SK-N-SH, and LA-N-6), with NGFR mRNA and protein of expected sizes (3.8 kilobases and Mr approximately 75,000, respectively). NGF binding assays and Scatchard analyses were performed on the four NGFR-positive lines. The NGP line possesses only low-affinity receptor (Kd approximately 3.5 x 10(-9)), whereas the other three lines express both low- and high-affinity forms (Kd approximately 10(-9) and Kd approximately 10(-11), respectively). However, none of the 10 lines exhibited a response to NGF treatment as assayed by c-fos mRNA induction and neurite extension.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nerve Growth Factor Receptors in Human Neuroblastoma Cells

Receptors for the nerve growth factor protein (NGFR) present in the human neuroblastoma cell line LAN-1 were characterized. LAN-1 cells display high-affinity (type I, with KD value of 5.9 X 10(-11) M) and low-affinity (type II, with KD value of 9.2 X 10(-9) M) binding to NGF. NGFR were fractionated by preparative isoelectric focusing in a granulated gel (PEGG). High-affinity binding was found in the 5.9-6.2 pH region of the PEGG, and low-affinity binding in the 4.6-4.8 and 8.8-9.3 pH ranges. After further analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) we observed both 92.5- and 200-kDa molecular species associated with NGF binding activity. The 200-kDa protein was found in fractions displaying high-affinity NGF binding and the 92.5-kDa protein in fractions displaying low-affinity NGF binding. Equilibrium binding analysis of NGF in PEGG fractions confirmed the presence of two specific saturable binding sites with KD values similar to those observed for whole dissociated cells. When NGFR II activity from the acidic region of the PEGG chromatogram was incubated with NGFR II from the basic region of the PEGG chromatogram, there was no change in NGF binding or in the number of apparent NGF receptors. However, incubation of these same fractions with a fraction having only NGFR I showed an apparent increase in high-affinity NGF binding and a decrease in low-affinity NGF binding. Immunoprecipitation of this "mixed" fraction and analysis on SDS-PAGE under reduced and nonreduced conditions showed 200-kDa and 92.5-kDa proteins under nonreduced conditions and a 92.5-kDa protein under reduced conditions. Our findings are consistent with the hypothesis that there are two distinct NGF receptors in NGF-responsive cells. The interconvertibility of low- and high-affinity receptors and the possible existence of a modulator type protein or of "silent" type receptors are also in agreement with our findings.     

Role of Nerve Growth Factor in Oxidanl Homeostasis: Glutathione Metabolism

Abstract— Free radicals are generated in the CNS by ongoing oxygen metabolism and biological events associated with injury and inflammation. Increased free radical levels may also persist in some chronic neurological diseases and in the aged. Nerve growth factor (NGF) is a member of the neurotrophin family of proteins that can regulate neuronal development, maintenance, and recovery from injury. NGF protected rat pheochromocytoma PC12 cells, an adrenal chromaffin-like NGF-responsive cell line, from the oxidant stress accompanying hydrogen peroxide treatment by stimulating GSH levels and enzymes in the GSH metabolism cycle and in the GSH/GSH peroxidase antioxidant redox system, a ubiquitous cellular antioxidant system. Specifically, NGF increased γ-glutamylcysteine synthetase (GCS) activity, the rate-limiting enzyme for GSH synthesis, by 50% after 9h and GSH levels by 100% after 24 h of treatment. NGF stimulated GSH peroxidase by 30% after 3 days and glucose 6-phosphate dehydroge-nase by 50% after 2 days. Treatment with NGF and cyclo-heximide, or actinomycin D, which inhibit protein and RNA synthesis, respectively, blocked the NGF stimulation of GCS and glucose 6-phosphate dehydrogenase. Increased GSH levels due to NGF treatment were responsible for the significant protection of PC12 cells from hydrogen peroxide-induced stress. Pretreatment of PC12 cells with NGF for 24 h rescued cells from the toxic effects of the extracellular hydrogen peroxide generated by the glucose/glucose oxidase system but did not rescue cells that were subjected to GSH deprivation due to treatment with 10 μMl -buthionine-(S,R)-sulfoximine, an inhibitor of GCS. However, treatment with 10 μMl -buthionine-(S,R)-sulfoximine alone did not affect PC12 cell viability, NGF stimulation of neurite extension, and NGF induction of GCS, GSH peroxidase, and glucose 6-phosphate dehydrogenase activity. When GSH levels were measured in PC12 cells that were treated for 24 h with other neurotrophins and growth factors, such as brain-derived neurotrophic factor, neurotro-phin-3, epidermal growth factor, insulin-like growth factor-I, and basic fibroblast growth factor, only epidermal growth factor was found to increase GSH levels by 30%. Whereas NGF increased GSH levels in the human neuro-blastoma SK-N-SH-SY5Y and the human melanoma A-875 in serum-free medium, addition of fetal calf serum to the medium abolished the NGF effects on GSH levels in the NGF-responsive cell lines, SK-N-SH-SY5Y, A-875, and the CNS C6 rat glioma subclone 2BD.     

Evidence for endocytosis-dependent proteolysis in the generation of soluble truncated nerve growth factor receptors by A875 human melanoma cells.

We have identified nerve growth factor receptor (NGFR) on the cell surface and a truncated nerve growth factor receptor (NGFRt) in the conditioned medium of NGFR-negative cells that have been transfected with either the gene or the cDNA for the full-length receptor. By using cell surface iodination or metabolic labeling of A875 human melanoma cells, coupled with immunoprecipitation, we have determined the half-life of the cell-associated receptor to be approximately 7 h. Concomitant with receptor degradation is the accumulation of NGFRt in the extracellular medium. Approximately one-fifth of the labeled receptor can be recovered as the truncated species. These data support the hypothesis that NGFRt is generated by proteolysis of previously intact receptor. Furthermore, although no specific protease inhibitor assayed could affect this processing, NGFR degradation and truncation were retarded by treatment with: 1) the weak base amines, ammonium chloride or methylamine; 2) the carboxylic ionophore, monensin; or 3) the vacuolar ATPase inhibitor, bafilomycin A1, all agents that dissipate endosomal/lysosomal proton gradients via alternate mechanisms. Incubation of cells at 4 degrees C precluded NGFR degradation and truncation. The presence of ligand did not alter the time course of receptor truncation.     

Insulin effect on translational diffusion of lipids and proteins in the plasma membrane of isolated rat hepatocytes

The effects of insulin (10(-10)-10(-8) mol/l) on lateral diffusion of three fluorescent lipid probes, 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)aminocaproyl phosphatidylcholine (NBD-PC), 5-(N-hexadecanoyl)aminofluorescein (F-C16), 5-(N-dodecanoyl)aminofluorescein (F-C12), and of fluorescein isothiocyanate-labeled proteins in the plasma membrane of intact rat hepatocytes were studied by the technique of fluorescence recovery after photobleaching. The absolute lateral diffusion coefficients of the lipid analogues NBD-PC, F-C16 and F-C12 at 21 degrees C were 2.5 X 10(-9) cm2/s, 5.4 X 10(-9) cm2/s and 19 X 10(-9) cm2/s, respectively. The diffusion coefficient mean of proteins labeled with fluorescein isothiocyanate was 6.4 X 10(-10) cm2/s. Insulin at 10(-9) and 10(-8) mol/l reduced the lateral diffusion coefficient for F-C12- and F-C16-labeled cells by 20% and for NBD-PC-labeled cells by 30% (P less than 0.025). The insulin effect was specific as tested by cell incubation with proinsulin and desoctapeptide insulin (10(-8) mol/l) and was detectable after 7 min of insulin preincubation. In contrast to lateral diffusion of lipid probes, lateral mobility of unselected membrane proteins was not altered by insulin. The observed modulation of lipid dynamics in the plasma membrane of intact hepatocytes, by which a variety of membrane functions can be influenced, may be an important step in the mechanism of insulin action.     

Sustained and transient downregulation of Src kinases in response to nerve growth factor and epidermal growth factor, respectively

Within a search for novel points of divergence in the signalling of differentiative nerve growth factor (NGF) and mitogenic epidermal growth factor (EGF) in PC12 cells, in the present study, I comparatively analysed the impact of these growth factors on the in situ activation status of Src kinases. By probing total cell extracts and anti-P-Tyr immunoprecipitates with a phosphorylation site-specific antibody targeting a conserved site of positive regulation of the overall family of Src kinases, a sustained and a transient downregulation of the immunoprecipitable subpopulation of nominally active Src kinases is detected in response to NGF and EGF, respectively. As the recovery of immunoreactive Src kinases is greater from cells being lysed while in suspension than from adherent cells, the possibility that NGF reduces the stability of Src kinases by upregulating cell adhesiveness is preliminarily explored and discussed.     

Threshold levels of ERK activation for chemotactic migration differ for NGF and EGF in rat pheochromocytoma PC12 cells

In a previous study, we show that stimulation of chemotaxis in rat pheochromocytoma PC12 cells by nerve growth factor (NGF) and epidermal growth factor (EGF) requires activation of the RAS-ERK signaling pathway. In this study, we compared the threshold levels of ERK activation required for EGF and NGF-stimulated chemotaxis in PC12 cells. The threshold ERK activity required for NGF to stimulate chemotaxis was approximately 30% lower than that for EGF. PD98059 treatment inhibited EGF stimulation of growth and chemotaxis; however, stimulation of chemotaxis required an EGF concentration approximately 10 times higher than for stimulation of PC12 cell growth. Thus, ERK-dependent cellular functions can be differentially elicited by the concentration of EGF. Also, treatment of PC12 cells with the PI3-K inhibitor LY294002 reduced ERK activation by NGF; thus, higher NGF concentrations were required to initiate chemotaxis and to achieve the same maximal chemotactic response seen in untreated PC12 cells. Therefore, the threshold NGF concentration to stimulate chemotaxis could be adjusted by the crosstalk between the ERK and PI3-K pathways, and the contributions of PI3-K and ERK to signal chemotaxis varied with the concentrations of NGF used. In comparison, LY294002 treatment had no effect on ERK activation by EGF, but the chemotactic response was reduced at all the concentrations of EGF tested indicating that NGF and EGF differed in the utilization of ERK and PI3-K to signal chemotaxis in PC12 cells. (Mol Cell Biochem 271: 29–41, 2005)     

Multiple trkA proteins in PC12 cells bind NGF with a slow association rate.

Rat pheochromocytoma (PC12) cells express two distinct nerve growth factor receptors (NGFRs), p75NGFR and trkA (p140trk). In addition to these receptors, by using 125I-mNGF affinity labeling and BS3 chemical cross-linking of PC12 cell protein, we have identified two additional trkA protein bands with apparent molecular weights of 220,000 and 300,000. These bands contain trkA, but were not immunoprecipitated by p75NGFR-specific antisera, suggesting that they do not represent trkA/p75NGFR protein complexes. The 220-kDa trkA band apparently represents trkA with alternate post-translational modification. The appearance of the 300-kDa trkA band was dependent on cross-linker concentration and could be diminished in the presence of reducing agents, suggesting that it represents a trkA dimer. All trkA bands were phosphorylated on tyrosine residues when bound to mNGF, suggesting that they participate in NGF-induced signal transduction. NGF binding kinetics to all three trkA bands were indistinguishable, with slow dissociation rates, and a slow association rate that required approximately 1 h to reach equilibrium levels at 4 degrees C. All three trkA bands bound the related neurotrophin brain-derived neurotrophic factor and neurotrophin-3 with a profile characteristic of trkA.     

Expression of nerve growth factor receptor immunoreactivity in the rat choroid plexus.

The presence and localization of nerve growth factor receptors (NGFr) in the choroid plexus of the adult rat has been investigated immunohistochemically using an anti-rat NGFr monoclonal antibody (192-IgG). A moderate to strong immunoreaction was observed in the epithelial cells of the choroid plexus, whereas the choroidal blood vessels and connective tissue remained unlabelled. Moreover, no sex-differences were encountered in the NGFr immunoreaction intensity and Bouin fixative was more effective than 10% formaldehyde evidenciating the NGFr immunostain. Occasionally, ependymal cells displaying NGFr immunoreactivity were observed. Present data demonstrate that the choroid plexus of the rat contain NGFr, probably low-affinity NGFr, and suggest an involvement of NGF in the regulation of cerebrospinal fluid secretion, but the importance of these findings, if any, must be investigated in future studies.     

Structure and developmental expression of the chicken NGF receptor

The nucleotide and deduced amino acid sequence of a cDNA clone of the chicken NGF receptor (NGFR) is reported and is compared with sequences of mammalian NGF receptors. A model is presented in which monodentate or bidentate binding of NGF dimers to repeated cysteine-rich sequence elements of the receptor yields low- or high-affinity NGF binding, respectively. In situ hybridization is used to characterize expression of NGFR in developing chick from 40 hr to 10 days of embryogenesis. NGFR mRNA expression is detected in premigratory neural crest cells, in epibranchial placode cells, and in all sensory, sympathetic and parasympathetic derivatives of these structures. In the embryonic CNS, NGFR mRNA is detected in the mantle zone but not the periventricular germinal zone throughout most of the neural tube. By Embryonic Day 8, NGFR mRNA is detected in a substantial fraction of cells in every brain region, with highest levels present in developing motor neurons. NGFR mRNA also is transiently expressed in many mesenchymal cell populations including cells in branchial arch, sclerotome, muscle anlagen, and feather follicles. The functional significance of wide-spread embryonic expression of the NGF receptor is discussed.     

The trk proto-oncogene encodes a receptor for nerve growth factor.

Two classes of receptors with distinct affinities for nerve growth factor (NGF) have been identified. The low affinity receptor (Kd approximately 10(-9) to 10(-8) M) is a cysteine-rich glycoprotein encoded by the previously characterized LNGFR gene. The structural nature of the high affinity receptor (Kd approximately 10(-11) to 10(-10) M) has yet to be established. In this study we show that the product of the human trk proto-oncogene (gp140trk) binds NGF with high affinity. Moreover, NGF could be chemically cross-linked to the endogenous gp140trk present in rat PC12 pheochromocytoma cells as well as to gp140trk ectopically expressed in mouse fibroblasts and in insect Sf9 cells. High affinity binding of NGF to gp140trk can occur in the absence of low affinity LNGFR receptors, at least in nonneural cells. Addition of NGF to PC12 cells elicits rapid phosphorylation of gp140trk on tyrosine residues and stimulates its tyrosine kinase activity. These results indicate that gp140trk is a functional NGF receptor that mediates at least some of the signal transduction processes initiated by this neurotrophic factor.