Internalization Of Nerve Growth Factor Research Articles

Cell signaling associated with internalization of 67 kDa laminin receptor (67LR) by soluble laminin and its implication for protection against neurodegenerative diseases.

Cell signaling associated with internalization of 67 kDa laminin receptor (67LR) by soluble laminin and its implication for protection against neurodegenerative diseases.

Nerve Growth Factor-induced p75-mediated Death of Cultured Hippocampal Neurons Is Age-dependent and Transduced through Ceramide Generated by Neutral Sphingomyelinase

Binding of nerve growth factor (NGF) to the p75 neurotrophin receptor (p75) in cultured hippocampal neurons has been reported to cause seemingly contrasting effects, namely ceramide-dependent axonal outgrowth of freshly plated neurons, versus Jun kinase (Jnk)-dependent cell death in older neurons. We now show that the apoptotic effects of NGF in hippocampal neurons are observed only from the 2nd day of culture onward. This switch in the effect of NGF is correlated with an increase in p75 expression levels and increasing levels of ceramide generation as the cultures mature. NGF application to neuronal cultures from p75(exonIII-/-) mice had no effect on ceramide levels and did not affect neuronal viability. The neutral sphingomyelinase inhibitor, scyphostatin, inhibited NGF-induced ceramide generation and neuronal death, whereas hippocampal neurons cultured from acid sphingomyelinase(-/-) mice were as susceptible to NGF-induced death as wild type neurons. The acid ceramidase inhibitor, (1S,2R)-d-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol, enhanced cell death, supporting a role for ceramide itself and not a downstream lipid metabolite. Finally, scyphostatin inhibited NGF-induced Jnk phosphorylation in hippocampal neurons. These data indicate an initiating role of ceramide generated by neutral sphingomyelinase in the diverse neuronal responses induced by binding of neurotrophins to p75.

Modulation of nerve growth factor internalization by direct interaction between p75 and TrkA receptors

Modulation of nerve growth factor internalization by direct interaction between p75 and TrkA receptors

Retrograde axonal transport of signal transduction proteins in rat sciatic nerve

Neurons require a mechanism to transmit stable signals over the large distance from the nerve growth cone or terminal to the cell body, in order that information from the target tissue can be relayed to the cell body where it is required. Nerve growth factor (NGF), a target-derived neurotrophic factor, is thought to signal over this distance by receptor mediated internalization of NGF, followed by retrograde axonal transport of the NGF-receptor complex. In this paper we show, by immunohistochemistry of rat sciatic nerve, accumulation of phosphotyrosine immunoreactivity only on the distal side of a nerve crush, suggesting axonal transport of tyrosine kinases and/or tyrosine phosphorylated proteins primarily in a retrograde direction. Furthermore, we also show retrograde axonal transport of phosphoinositide 3-kinase, ERK, MEK and MEK kinase, of which all but MEK kinase are known to be activated downstream of tyrosine receptor kinase activation. The retrograde transport of these proteins suggests that they may be involved in transmission of signals along the axon, relaying neurotrophic factor receptor activation at the nerve terminal to the nerve cell body.

P75 nerve growth factor receptor modulates p140trkA kinase activity, but not ligand internalization, in PC12 cells.

The biological activity of nerve growth factor (NGF) has been shown to be mediated by the p140trkA receptor tyrosine kinase, while the role of the p75 NGF receptor (p75NGFR) is still unresolved. Here we have investigated the relative contribution of p140trkA and p75NGFR to early consequences of NGF binding: ligand internalization, p140trkA autophosphorylation, and tyrosine phosphorylation of Shc, phospholipase C gamma-1 (PLC gamma-1), and extracellular signal-regulated kinases (ERKs). It was found that NGF internalization was neither prevented by blocking p140trkA activity using the protein kinase inhibitors methylthioadenosine, staurosporine, and K-252a, nor by inhibiting NGF binding to p75NGFR with antibodies. However, when NGF binding to p140trkA was reduced by the use of a synthetic peptide corresponding to amino acids 36-53 of human p140trkA, internalization of NGF was decreased. Thus, at least in PC12 cells, internalization appears to require binding of NGF to p140trkA, but occurs irrespective of p140trkA kinase activity and ligand occupancy of p75NGFR. The NGF triple mutant Lys-32/Lys-34/Glu-35 to Ala, which has been demonstrated to bind to p140trkA, but not to p75NGFR, induced tyrosine phosphorylation more rapidly than wild-type NGF. Likewise, NGF-induced tyrosine phosphorylation was accelerated when NGF binding to p75NGFR was prevented with REX-IgG. These findings indicate that NGF bindign by p75NGFR may modulate NGF-induced p140trkA kinase activity.

A low-affinity nerve growth factor receptor antibody is internalized and retrogradely transported selectively into cholinergic neurons of the rat basal forebrain

The mechanism by which nerve growth factor transduces its signal in responsive cells is yet to be clearly defined. However, it has been suggested that the internalization of nerve growth factor, the first step in the retrograde flow of nerve growth factor, is a property of the high-affinity receptors, p140 trka. Here we show that when a monoclonal antibody (MC 192), which immunoprecipitates p75 NGFR (the low-affinity 75,000 mol. wt nerve growth factor receptor protein) and not p140 trka, was administered into the dorsal hippocampal formation of the rats, it was internalized and retrogradely transported to the cell bodies residing in the medial septum-diagonal band complex. The topographic organization and the localization of these neurons containing retrogradely transported p75 NGFR antibody were strikingly similar to those nerve cells immunostained for choline acetyltransferase in the immediately-adjacent section, indicating that the neurons which contained p75 NGFR antibody were cholinergic neurons. A double-label immunocytochemistry confirmed this conclusion. On the other hand, none of the parvalbumin-positive GABAergic neurons contained retrogradely transported p75 NGFR antibody. Moreover, in contrast to specific transport of p75 NGFR antibody into cholinergic neurons, when wheat germ agglutinin-colloidal gold was injected into the hippocampus at the same levels, it was taken up and retrogradely transported into both choline acetyltransferase-positive cholinergic and parvalbumin-immunoreactive GABAergic neurons in the medial septum-diagonal band complex.

Nerve growth factor (NGF) receptor on rat glial cell lines. Evidence for NGF internalization via p75NGFR.

Two types of nerve growth factor (NGF) receptors have been described: high affinity (class I) and low affinity (class II). Biological responses to NGF are thought to be mediated by class I receptors, whereas the role of class II receptors is less clear. While some neuronal cells express both receptor types, only class II receptors have been detected on glial cells. Two glial cell lines, peripheral Schwannoma D6P2T and central 33B glioma cells, were employed to investigate the properties of class II receptors in the absence of class I receptors. These cell lines were found to express NGF receptors identified as class II by a low nanomolar dissociation constant, rapid dissociation kinetics at 4 degrees C, and trypsin sensitivity. The receptor was found to bind brain-derived neurotrophic factor with similar affinity as NGF. The responsible binding molecule appeared in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a heterogeneously glycosylated protein of 60-80 kDa with a tendency to aggregate. All receptor bands affinity-labeled with radioiodinated NGF were immunoprecipitated with anti-p75NGFR antibody, but not with anti-p140prototrk antiserum. In these cells, which express p75NGFR as only NGF receptor, a time- and temperature-dependent appearance of a nondisplaceable, trypsin-resistant, acid wash-stable ligand fraction, followed by an increase of trichloroacetic acid-soluble radiolabel in the medium was observed. This sequestration resembled receptor-mediated internalization with subsequent degradation of NGF. Whether this ligand processing indicates a functional role of p75NGFR in glial cells remains to be shown.

Ethanol enhances growth factor-induced neurite formation in PC12 cells

Ethanol can injure the nervous system by disturbing the growth of neural processes. PC12 cells, which form neurites in response to nerve growth factor (NGF), fibroblast growth factor (FGF), and cAMP analogues, were used to study mechanisms by which ethanol alters process outgrowth. Ethanol potentiated NGF-induced nuurite outgrowth in cells cultured on different substrata and in serum-containing or defined medium. Ethanol did not increase NGF receptor binding or internalization of NGF. Neurite outgrowth induced by basic FGF was also increased by ethanol but outgrowth induced by forskolin was not. Ethanol potentiated NGF-induced expression of Thy-1, but not of neural cell adhesion molecule (N-CAM), indicating that some, but not all actions of NGF are enhanced by ethanol. In some brain regions, chronic exposure to ethanol increases the growth of dendrites. This has been explained as a compensatory response of surviving neurons to the loss of neighboring cells, and not as a direct effect of ethanol. The present findings suggest that, in some cells, ethanol directly promotes growth factor-mediated neurite formation. This could harm the nervous system by disturbing the balanced development and organization of synapses.

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.

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.

Internalization of nerve growth factor by pheochromocytoma PC12 cells: absence of transfer to the nucleus

The intracellular distribution of 125I-labeled nerve growth factor (NGF) in rat pheochromocytoma PC12 cells was studied by quantitative electron microscopic (EM) autoradiography and by subcellular fractionation. PC12 cells were grown as monolayer cultures in medium supplemented with serum in the presence of 125I-NGF. EM autoradiography showed that 125I-NGF was localized at the plasma membrane and cytoplasmic compartments but did not accumulate in the nuclear chromatin or in the nuclear membrane compartment of cells analyzed after 1 hr and 1, 2, and 8 d of incubation with 125I-NGF. 125I-NGF also was not detected in nuclear subcellular fractions prepared from cells grown in serum-supplemented medium either in suspension for 1 d or in monolayer cultures for 1 to 8 d. In contrast, and in confirmation of the results of Yankner and Shooter (Yankner, B. A., and E. M. Shooter (1979) Pro. Natl. Acad. Sci. U. S. A. 76: 1269-1273), about 60% of the cell-bound 125I-NGF was found in the nuclear pellet after cell fractionation if the cells had been kept previously in suspension for 1 d in phosphate-buffered saline supplemented with 0.2% glucose, 0.1% bovine serum albumin, and 125I-NGF. The ultrastructure of PC12 cells grown under such conditions, however, revealed signs of varying degrees of damage. Autoradiography of the nuclear pellet from these cells showed the grains to be located mainly over damaged nuclei or over cell debris between nuclei. It is concluded that NGF, after binding to specific receptors at the plasma membrane, is transferred to membrane-confined cytoplasmic compartments but does not have to be transferred further to the nuclear membrane or to the nuclear chromatin as a prerequisite for its physiological action.

Sequestration of 125I-labeled beta nerve growth factor by embryonic sensory neurons.

Nerve growth factor (NGF) binds to two specific receptors on sensory nerve cells. These two receptors are characterized by different equilibrium dissociation constants. The higher affinity (type I) receptors have an equilibrium dissociation constant of 3.3 X 10(-11) M. The lower affinity (type II) receptors have an equilibrium dissociation constant of 1.7 X 10(-9) M. These two receptors are not a result of negative cooperativity, but apparently are different receptors. At 22 degrees C the rate of association is 1 X 10(7) M-1 S-1 and the rates of dissociation are 6.5 X 10(-4) S-1 (type I) and 3.2 X 10(-2) S-1 (type II). After binding, a time-dependent process occurs that makes that NGF inaccessible to the external milieu (sequestered). The sequestration process is energy-dependent, but apparently temperature-independent. The data suggest that only the type I receptors are involved in the sequestration process. This process is similar to that observed on sympathetic neurons and may be the first step in the internalization of NGF by responsive cells.

Cell density modulates receptor-mediated internalization of nerve growth factor in pheochromocytoma cells.

Binding and fate of the nerve growth factor (NGF) in pheochromocytoma 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.