Human Nerve Growth Factor Receptor Research Articles

Eccrine angiomatous hamartoma (nevus):

Eccrine angiomatous hamartoma (nevus) is a rare form of congenital tumorous malformation with proliferation of eccrine secretory coils and ducts, surrounding capillary angiomatous channels and occasionally other minor elements. To date, there have been only about 24 cases reported in the literature. We report an additional case with more detailed description of the microscopic findings, including immunohistochemical observations. The patient was a 28-year-old female who presented with painless, flesh- to reddish brown-colored, violaceous or bluish subcutaneous nodules on the extremities and trunk. The tumors did not show sweating following exertion. The histologic features were comparable to the previously reported cases. The hamartomatous eccrine sweat glands and ducts and a few apocrine glands demonstrated qualitatively diminished antigens commonly found in the eccrine sweat apparatuses, such as carcinoembryonic antigen (CEA) and S-100 protein. The findings of CD34, CD44, human nerve growth factor receptor and Ulex europaeus antigens have not been previously reported. The histologic features suggested a "hamartomatous" growth rather than a true neoplastic process.

Low-affinity p75 nerve growth factor receptor expression in the embryonic monkey telencephalon: Timing and localization in diverse cellular elements

Monoclonal antibodies against the low-affinity (p75) subunit of the human nerve growth factor receptor have been used to determine the temporal appearance of this receptor and to identify the associated cellular elements in the developing occipital cortex of rhesus monkeys. Adult and fetal brains from embryos at embryonic days 45–121 were used. This embryonic time span includes periods of active neurogenesis, cell migration and initial formation of axonal connections in the cerebral cortex. The first immunolabeling in the developing cerebral wall was seen between embryonic days 56 and 64. The labeling was present in the transient subplate neurons, a small number of axonal processes and pericytes associated with blood vessels. By birth, labeled neurons of the subplate zone disappeared, but immunolabeled axonal processes could now be seen in large numbers in the cortex. These findings are consistent with the role of nerve growth factor in the coordination of cortical differentiation, but not with the initiation of neuronal proliferation, since the emergence of nerve growth factor receptor-labeled elements in the cortex occurs two to three weeks after the onset of neurogenesis in this species. Further, the diverse cellular elements labeled in the fetal cerebrum with the antibodies to the low-affinity nerve growth factor receptor suggests that a receptor or receptors associated with growth factor signaling for more than one growth factor family are recognized by these antibodies. Differential timing in the expression of families of growth factor receptors may be one mechanism by which developing neurons in the cerebral cortex could respond to the different signals which guide such processes as synaptogenesis and morphogenesis.

HSV-1 vector-mediated gene transfer of the human nerve growth factor receptor p75hNGFR defines high-affinity NGF binding

A series of recombinant herpes simplex virus (HSV-1) vectors have been constructed that encode either the full-length cDNA of the human p75 NGF receptor (p75hNGFR) or truncated forms of the receptor. Infection of cultured fibroblast cells with viral stocks results in abundant expression of all three cDNAs, as detected by affinity cross-linking, immunoblot analysis, and equilibrium binding. Furthermore, viral infection of primary neuronal cultures gives easily detectable p75 expression by immunofluorescence and affinity cross-linking. When p75 was introduced by viral infection into fibroblast cells expressing the trk proto-oncogene, a new binding site was created, consistent with high-affinity NGF binding. This site is not created by the coexpression of truncated forms of p75 that lack either the extracellular ligand binding domain or the cytoplasmic domain of the receptor, suggesting that both of these regions of the receptor are required for the formation of the high-affinity NGF binding site. Hence, these HSV-1 vectors give rise to appropriate NGF receptor binding after viral infection. The application of these HSV-1 constructs to primary neuronal culture and in vivo models of p75NGFR function is discussed.

Chromosome assignments of the human TNF p55 and p75 receptor genes.

At least two different receptor molecules have been described that are capable of binding tumor necrosis factor alpha, a cytokine that plays an important role in inflammation and antitumor activity. Comparative analyses at the nucleotide sequence level suggest that these receptors are members of a newly defined protein family that also includes human and rat nerve growth factor receptors. In this study, we determine the chromosome assignments of the human TNF alpha receptor genes, one of which may have evolved as part of a conserved Hox locus-containing chromosome segment.

Effects of NGF and glucocorticoid on NGF receptor immunolabeling of cultured rhesus adrenal chromaffin cells

Nerve growth factor (NGF) promotes the outgrowth of neurites from cultured adrenal chromaffin cells from adult rhesus monkeys [8], but little is known about the distribution, at the cellular level, of the NGF receptors (NGFR) responsible for this response. We examined changes in immunostaining for NGFR in chromaffin cells cultured for 4 weeks in the presence or absence of NGF, with or without dexamethasone (DEX), which inhibits neuritic outgrowth from these cells. Purified cultures of adrenal chromaffin cells from adult rhesus monkeys were grown for up to 9 weeks in NGF, DEX, NGF plus DEX, or control medium. Cells were immunolabeled with three different monoclonal antibodies directed against different epitopes of the human NGFR. Although the distribution of immunolabeling was not uniform from cell to cell, the overall intensity of NGFR immunolabeling varied dramatically between different growth conditions. Of greatest interest, DEX-treated cells stained the most intensely at all time points, while the intensity of immunolabeling was much fainter in NGF-treated cells and decreased with time in culture. In contrast to the intensity of labeling, the proportion of chromaffin cells with immunoreactivity increased with time in all treatment groups. Thus, GCs do not appear to antagonize the effects of NGF merely by decreasing the total number of immunoreactive NGFR on the surface of these cells.

Basic fibroblast growth factor enhances nerve growth factor receptor gene promoter activity in human neuroblastoma cell line CHP100.

The human neuroblastoma cell line CHP100 provides a useful model system in which to study the molecular mechanisms of transcriptional regulation of the low-affinity nerve growth factor receptor (NGFR) gene during neuronal development. Basic fibroblast growth factor (bFGF) induced morphological changes in CHP100 cells, including flattening of cell bodies and neurite outgrowth. bFGF also increased p75NGFR immunoreactivity, as assessed by immunocytochemistry, and increased p75NGFR mRNA levels, as assessed by Northern (RNA) blot analysis. A chimeric gene consisting of 6.7 kb of the 5'-flanking region of the human NGFR gene linked to the chloramphenicol acetyltransferase gene was constructed. In stable transformants of CHP100 cells, 10 ng of bFGF per ml induced an eightfold increase in chloramphenicol acetyltransferase activity. These results indicate that upstream elements of the NGFR gene mediate transcriptional regulation by bFGF.

Basic fibroblast growth factor enhances nerve growth factor receptor gene promoter activity in human neuroblastoma cell line CHP100.

The human neuroblastoma cell line CHP100 provides a useful model system in which to study the molecular mechanisms of transcriptional regulation of the low-affinity nerve growth factor receptor (NGFR) gene during neuronal development. Basic fibroblast growth factor (bFGF) induced morphological changes in CHP100 cells, including flattening of cell bodies and neurite outgrowth. bFGF also increased p75NGFR immunoreactivity, as assessed by immunocytochemistry, and increased p75NGFR mRNA levels, as assessed by Northern (RNA) blot analysis. A chimeric gene consisting of 6.7 kb of the 5'-flanking region of the human NGFR gene linked to the chloramphenicol acetyltransferase gene was constructed. In stable transformants of CHP100 cells, 10 ng of bFGF per ml induced an eightfold increase in chloramphenicol acetyltransferase activity. These results indicate that upstream elements of the NGFR gene mediate transcriptional regulation by bFGF.

Structural domains of the extracellular domain of human nerve growth factor receptor detected by partial proteolysis

Using partial proteolytic cleavage, the nerve growth factor (NGF) binding site and the epitopes for two anti-NGF receptor (NGFR) monoclonal antibodies were localized on the recombinant extracellular domain (RED) of the NGFR. The RED was prepared in the baculovirus-insect cell system and was purified by immunoaffinity and ion-exchange chromatography. The four cysteinerich repeat domains and some additional C-terminal sequences were resistant to proteolysis with papain or proteinase K. The M r 32,000 papain-resistant fragment (P32) and the M r 30,000 proteinase K-resistant fragment (K30) share the same N terminus as the intact RED and have C termini in the vicinity of residue 170. Even though P32 and K30 have the same N terminus and probably differ by only a small number of amino acids at the C terminus, P32, but not K30, binds 125I-NGF. As judged by Western blot analysis, two anti-NGFR antibodies (ME20.4 and NGFR5) bind to P32 but have a lesser affinity for K30. Since antibody ME20.4 inhibits NGF binding but antibody NGFR5 does not, these antibodies bind to distinct epitopes. However, these epitopes apparently are closely spaced since these antibodies compete with each other for binding to biotinylated RED. NGF, but not the control protein cytochrome c, protects RED from papain digestion. Therefore, the P32 C terminus is important for the expression of the NGF binding site and the antibody-defined epitopes, even though the NGF binding site and antibody-defined epitopes probably are not encoded by the P32 C terminus. These data suggest that complex interactions occur between different regions of the RED, and that optimum NGF binding requires the integrity of multiple RED domains, including a short sequence to the C terminus of residue 170.

Changes in nerve growth factor receptor-like immunoreactivity in the spinal cord after ventral funiculus lesion in adult cats.

Spinal motoneurons have a capability to regenerate CNS-type axons after intramedullary lesions in the adult cat. Regrowing axons have been traced through CNS-type scar tissue in the ventral funiculus of the spinal cord and into adjacent ventral root fascicles. This scar tissue, which appears to support and sustain regenerating axons, has been shown to have a persistent defect in the blood-brain barrier. It has been suggested that the blood-brain barrier may play a vital role in CNS regeneration by regulating the access of blood-borne trophic factors to the lesion area. In the present study, the binding of antibodies to the human nerve growth factor receptor in the cat spinal cord was examined with immunohistochemical methods 2 days to 8 weeks after a ventral funiculus lesion. The results show that, while no neurons in the ventral horn of the control material contained nerve growth factor receptor-like immunoreactivity as revealed by fluorescence microscopy, affected motoneurons expressed nerve growth factor receptor after ventral funiculus lesion. Nerve growth factor receptor-like immunoreactivity associated to both capillaries and interstitium was present in the scar tissue. Electron microscopic examination of sections labelled with the immunogold-silver method showed that perivascular nerve growth factor receptor-like immunoreactivity was located exclusively to non-pericytic perivascular cells. These cells were abundant in the expanded capillary perivascular spaces adjacent to the traumatic lesion. Similar cells, with or without relation to blood vessels, were observed in the scar tissue and in the pia mater. In a separate set of specimens it was observed that a ventral funiculus lesion combined with ventral root avulsion, which removes denervated PNS tissue, resulted in an expression of nerve growth factor receptor-like immunoreactivity which was similar to the one observed after ventral funiculus lesion only. The results of the present study show that affected motoneurons and cells in the scar tissue express nerve growth factor receptor after ventral funiculus lesion which implies that neurotrophic factors related to nerve growth factor may be of importance for the regenerative response.

An internal deletion in the cytoplasmic tail reverses the apical localization of human NGF receptor in transfected MDCK cells.

A cDNA encoding the full-length 75-kD human nerve growth factor receptor was transfected into MDCK cells and its product was found to be expressed predominantly (80%) on the apical membrane, as a result of vectorial targeting from an intracellular site. Apical hNGFR bound NGF with low affinity and internalized it inefficiently (6% of surface bound NGF per hour). Several mutant hNGFRs were analyzed, after transfection in MDCK cells, for polarized surface expression, ligand binding, and endocytosis. Deletionof juxta-membrane attachment sites for a cluster of O-linked sugars did not alter apical localization. A mutant receptor lacking the entire cytoplasmic tail (except for the five proximal amino acids) was also expressed on the apical membrane, suggesting that information for apical sorting was contained in the ectoplasmic or transmembrane domains. However, a 58 amino acid deletion in the hNGFR tail that moved a cytoplasmic tyrosine (Tyr 308) closer to the membrane into a more charged environment resulted in a basolateral distribution of the mutant receptor and reversed vectorial (basolateral) targeting. The basolateral mutant receptor also internalized 125I-NGF rapidly (90% of surface bound NGF per hour), exhibited a larger intracellular fraction and displayed a considerably shortened half-life (approximately 3 h). We suggest that hNGFR with the internal cytoplasmic deletion expresses a basolateral targeting signal, related to endocytic signals, that is dominant over apical targeting information in the ecto/transmembrane domains. These results apparently contradict a current model that postulates that basolateral targeting is a default mechanism.

Localization of nerve growth factor receptor immunoreactivity in the trigeminal nucleus of kittens

A monoclonal antibody raised against the human nerve growth factor receptor (NGFr) was used to map the distribution of NGFr-immunoreactivity (IR) in the trigeminal nuclear complex of 8- to 10-week-old, immature felines. Somata and fibers show NGFr-IR within the trigeminal ganglion and the mesencephalic trigeminal nucleus. NGFr-IR is also found in fibers within the trigeminal root entry zone, the spinal trigeminal tract, and in fibers and terminals within all the central trigeminal sensory nuclei. The NGFr-IR found within the trigeminal sensory nuclei typically occurs in circumscribed zones that vary in position for the different subnuclei. NGFr-IR is found in the dorsomedial and ventrolateral subdivisions of the main sensory nucleus, in the dorsomedial and occasionally in ventral positions within pars oralis, in dorsal and ventral regions within pars interpolaris, and primarily in outer lamina II with fibers that project to lamina V within pars caudalis/medullary dorsal horn. These results show some overlap with the central distribution of trigeminal primary afferent nociceptive fibers such as those found from the tooth pulp and overlap with the central distribution of such peptides as calcitonin gene-related peptide and substance P, but NGFr-IR is more restricted. Thus, it appears that NGFr-IR is associated with the endings of primary afferent fibers in the brain stem, and that these fibers may represent a certain subclass of primary afferent nociceptors. It is speculated that fibers showing NGFr-IR may have the ability to alter their response to peripheral deafferentation when compared to fibers lacking NGFr-IR.

The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis

Mouse anti-Fas monoclonal antibody has a cytolytic activity on human cells that express the antigen. Complementary DNAs encoding the cell surface antigen Fas were isolated from a cDNA library of human T cell lymphoma KT-3 cells. The nucleotide sequence of the cDNAs revealed that the molecule coding for the Fas antigen determinant is a 319 amino acid polypeptide (Mr36,000) with a single transmembrane domain. The extracellular domain is rich in cysteine residue, and shows a similarity to that of human tumor necrosis factor receptors, human nerve growth factor receptor, and human B cell antigen CD40. Murine WR19L cells or L929 cells transformed with the human Fas antigen cDNA were killed by the anti-Fas antibody in the process known as apoptosis.

Nerve growth factor receptor immunoreactivity in the new world monkey (Cebus apella) and human cerebellum.

The present study used the NGFR-5 monoclonal antibody raised against human nerve growth factor receptor (NGFR) to determine the extent of NGFR immunoreactivity within the embryonic and young adult Cebus apella cerebellum as well as the human cerebellum. Immunohistochemically processed tissue revealed NGFR expressing Purkinje cell somata, axons, and dendrites, the latter being observed within the molecular layer of both adult species. Within all regions of the cerebellum we observed both darkly and lightly immunostained Purkinje cells. The proximal axons of these cells, which were visualized for short distances within the granular cell layer, appeared to contain bulbous aggregates of reaction product. In sagittal sections, the full extent of the Purkinje cell dendritic tree was observed in the more lightly stained portions of the cerebellum. In situ hybridization experiments revealed NGFR mRNA within Purkinje cells in a pattern similar to that seen with immunohistochemistry. The distribution of NGFR immunoreactivity within the cerebellum exhibits a general topographic organization with the heaviest and most consistent staining occurring within the archi- and neocerebellum and weaker staining within the paleocerebellum. In fetal Cebus monkey cerebellum obtained at gestational day 50 and 70, NGFR immunoreactivity was observed as a band composed of developing Purkinje cell neurites. These profiles were seen in the paleo- and neocerebellum, but not the archicerebellum. The present investigation is the first demonstration of NGFR immunoreactive profiles in the adult monkey and human cerebellum. These findings suggest that nerve growth factor may influence locomotor and vestibular behaviors that are mediated by cerebellar circuity. The precise mode of action for the NGF/NGFR system within the cerebellum remains to be determined.

Axons induce differentiation of neurofibroma Schwann-like cells

Neurofibromatosis type 1 (NF-1, von Recklinghausen's disease) is characterized by the focal accumulation of Schwann-like cells (SLC) to form subcutaneous and plexiform neurofibromas and schwannomas. The aim of the present study was to determine whether NF-SLC are competent to differentiate in the presence of axons. Five dermal neurofibromas from five patients with NF-type 1 were enzymatically dissociated and the resultant cells were co-cultured with fetal rat dorsal root ganglion neurons. The cultures were studied by indirect immunofluorescence microscopy using antibodies against galactocerebroside (galC), P0 glycoprotein, human nerve growth factor receptor (NGFR) and human myelin-associated glycoprotein (MAG). SLC were strongly NGFR+ but galC- and MAG-SLC for the 2 weeks of coculture. After 3 weeks in vitro, SLC-NGFR was down-regulated but some of the spindle shaped cells had become galC+. MAG-SLC first appeared after 5 weeks in vitro but P0 glycoprotein was never detected when studied up to 6 weeks. Our data demonstrate that axons induce SLC to down-regulate surface NGFR and to express some myelin components in a qualitatively normal fashion.

Granulocyte colony-stimulating factor gene transfer suppresses tumorigenicity of a murine adenocarcinoma in vivo.

We have investigated the effect of granulocyte colony-stimulating factor (G-CSF) delivery at the site of tumor growth by transducing, via retroviral vector, the human (hu) G-CSF gene into the colon adenocarcinoma C-26 and assaying the ability of transduced cells to form tumors when injected into syngeneic mice. As a control, the same tumor cells were infected with retroviruses engineered to transduce an unrelated gene, the human nerve growth factor receptor, or carry the neomycin resistance gene only. Only cells transduced with the huG-CSF were unable to develop tumors, although huG-CSF was expressed and produced at low level as estimated by both RNA analysis and enzyme-linked immunosorbent assay, indicating that G-CSF can exert an antitumor effect at a physiological dose. Implication of G-CSF as mediator of tumor inhibition was proven by reversing the nontumorigenic phenotype of G-CSF-expressing cells with anti-huG-CSF monoclonal antibody injected at the tumor site. No tumors were formed by injecting C-26 infected cells into nu/nu mice, while neoplastic nodules appeared after injection into sublethally irradiated mice; such tumors, however, regressed when mice normalized their leukocyte counts after irradiation. Tumors were also formed after injection of a mixture of infected and uninfected C-26 cells, although critical delay in tumor formation occurred when infected cells were 10 times more represented in the mixture. Histological examination of tissues surrounding the site of injection showed infiltration of neutrophilic granulocytes, whose number correlated with that of G-CSF-expressing C-26 cells in the injected mixture. These results indicate that G-CSF may have a potent antitumoral activity when released, even at low doses, at the tumor site. The antitumoral effect is mediated by recruitment and targeting of neutrophilic granulocytes to G-CSF-releasing cells.

Nerve growth factor receptor immunoreactivity on the tunica adventitia of intramuscular blood vessels in childhood muscular dystrophies

Muscle tissues from cases of childhood neuromuscular disorders were examined immunohistochemically and immunoelectrophoretically using a monoclonal antibody against the human nerve growth factor receptor (NGFR). Strong NGFR immunoreactivity on the tunica adventitia of blood vessels and proliferating peripheral nerve endings in biopsied muscle specimens from muscular dystrophy patients was observed, but it was almost completely absent in specimens from non-diagnostic controls and cases of other neuromuscular disorders. This suggests a process in the sympathetic nervous system involving blood vessels in muscular dystrophies. Immunoblot analysis failed to show a band corresponding to 70–75 kd, the reported molecular size of the NGFR, but showed a clear band corresponding to 25 kd in muscular dystrophy patients, which is assumed to be a detached amino-terminal domain of the NGFR.

Ultrastructural Localization of Nerve Growth Factor Receptor in Acoustic Neurinoma

Light and electron microscopic immunohistochemical studies on nerve growth factor (NGF) receptors in acoustic neurinoma cases were made using a monoclonal antibody to human NGF receptor. Immunoreactivity of the NGF receptor was found exclusively on the cytoplasmic membrane of schwannoma cells. Reaction product was highly concentrated at the interdigitated distal end of the cell processes, but it was discontinuous in the perinuclear area of the cell body. Basal lamina was located outside the immunoreacted cytoplasmic membrane of the cell body, but it was not observed at the tip of the interdigitated cell processes. These findings suggest that NGF receptor expression has some relationship with the formation of interdigitated schwannoma cell processes. Other possibilities for the functional significance of the schwannoma NGF receptor are also discussed.

Monoclonal antibodies to the cell surface and a soluble form of the human nerve growth factor receptor

Monoclonal antibodies (designated IIIG5, VIID1, VIIIC8, and XIF1) have been produced that bind to the human nerve growth factor receptor (NGF-R) as well as to a soluble, truncated form of the receptor (NGF-Rt). The antibodies were generated against partially purified NGF-Rt from the conditioned medium of E9b cells, a transfected mouse fibroblast cell line (Ltk-) that expresses large numbers of the low affinity form of the human NGF-R on its cell surface (Chao MV, Bothwell MA, Ross AH, Koprowski H, Lanahan AA, Buck CR, Sehgal A [1986]: Science 232:518-521). Hybridomas were screened by radiometric immunosorbent assay (RISA) and by immunoprecipitation of solubilized cell surface receptor covalently cross-linked to [125-I]-NGF. Four positive lines were cloned by limiting dilution and were found to secrete monoclonal antibodies of the IgGl,k subclass. All monoclonal antibodies bound to both NGF-R and NGF-Rt. Two monoclonal antibodies (VIID1, XIF1) immunoblotted the NGF-R from E9b cell preparations resolved on non-reducing sodium dodecyl sulfate (SDS)-polyacrylamide gels. The antibodies immunoprecipitated NGF-R from both E9b cells and from SH-SY5Y human neuroblastoma cells. The monoclonal antibodies bound to monkey (rhesis and cynomolgus) NGF-Rt, but did not cross-react with NGF-R from chick or rat. Results of antibody competition studies demonstrated that three antibodies bound to a similar or overlapping epitope on the NGF-Rt and one monoclonal antibody (IIIG5) recognized a distinct receptor epitope. Antibodies that bound to different sites on the receptor were used to develop a sensitive 2-site RISA. The 2-site RISA can be used to rapidly quantitate NGF-R and NGF-Rt in large numbers of biological samples in the absence of added [125-I]-labeled NGF.

Nerve growth factor receptor-like immunoreactivity in nerve fibers in the spinal and medullary dorsal horn of the adult monkey and cat: correlation with calcitonin gene-related peptide-like immunoreactivity

The distribution of nerve growth factor receptor (NGFr)-like immunoreactivity and of calcitonin gene-related peptide (CGRP)-like immunoreactivity was studied in nerve fibers of the spinal and medullary dorsal horn of the monkey and the cat, using double-labelling immunohistochemistry with polyclonal CGRP antiserum and a monoclonal antibody against human NGFr. In both species there was an extensive distribution overlap between NGFr-LI and CGRP-LI. However, lack of NGRr-LI in many CGRP-immunoreactive axons suggests that not all CGRP-positive primary afferent fibers possess NGFr.

Introduction of nerve growth factor (NGF) receptors into a medulloblastoma cell line results in expression of high- and low-affinity NGF receptors but not NGF-mediated differentiation.

Expression of the cloned human nerve growth factor receptor (NGFR) cDNA in cell lines can generate both high- and low-affinity binding sites. Since the inability to respond appropriately to differentiation factors such as NGF may contribute to determining the malignant phenotype of neuroblastomas, we sought to determine whether the same is true of medulloblastomas. To generate a human central nervous system neuronal cell line that would respond to NGF, we infected the medulloblastoma cell line D283 MED with a defective retrovirus carrying the cDNA coding for the human NGFR. The resultant cells (MED-NGFR) expressed abundant low- and high-affinity NGFRs, and NGF treatment induced a rapid transient increase of c-fos mRNA in the NGFR-expressing cells but not in the parent line or in cells infected with virus lacking the cDNA insert. However, the MED-NGFR cells did not internalize the NGFR at high efficiency, nor did they differentiate in response to NGF. Three important conclusions emerge from this study: (i) internalization of NGFRs is not necessary for some early rapid transcriptional effects of NGF; (ii) an unknown factor(s) that cooperates with the cloned NGFR in allowing high-affinity NGF binding is found in a primitive central nervous system cell line; and (iii) NGFRs introduced into and expressed by D283 MED (i.e., MED-NGFR) cells are partially functional but are unable to induce differentiation in these primitive neuron-like tumor cells, implying that high-efficiency receptor-mediated endocytosis of NGF and its receptor may be a necessary step in the cascade of events leading to NGF-mediated differentiation.