Nerve Growth Factor Binding Sites Research Articles

The Conundrum of the High-Affinity NGF Binding Site Formation Unveiled?

The homodimer NGF (nerve growth factor) exerts its neuronal activity upon binding to either or both distinct transmembrane receptors TrkA and p75NTR. Functionally relevant interactions between NGF and these receptors have been proposed, on the basis of binding and signaling experiments. Namely, a ternary TrkA/NGF/p75NTR complex is assumed to be crucial for the formation of the so-called high-affinity NGF binding sites. However, the existence, on the cell surface, of direct extracellular interactions is still a matter of controversy. Here, supported by a small-angle x-ray scattering solution study of human NGF, we propose that it is the oligomerization state of the secreted NGF that may drive the formation of the ternary heterocomplex. Our data demonstrate the occurrence in solution of a concentration-dependent distribution of dimers and dimer of dimers. A head-to-head molecular assembly configuration of the NGF dimer of dimers has been validated. Overall, these findings prompted us to suggest a new, to our knowledge, model for the transient ternary heterocomplex, i.e., a TrkA/NGF/p75NTR ligand/receptors molecular assembly with a (2:4:2) stoichiometry. This model would neatly solve the problem posed by the unconventional orientation of p75NTR with respect to TrkA, as being found in the crystal structures of the TrkA/NGF and p75NTR/NGF complexes.

Neurotrophin receptor homolog‐2 regulates nerve growth factor signaling

The neurotrophin receptor homolog (NRH2) is closely related to the p75 neurotrophin receptor (p75NTR); however, its function and role in neurotrophin signaling are unclear. NRH2 does not bind to nerve growth factor (NGF), however, is able to form a receptor complex with tropomyosin-related kinase receptor A (TrkA) and to generate high-affinity NGF binding sites. Despite this, the mechanisms underpinning the interaction between NRH2 and TrkA remain unknown. Here, we identify that the intracellular domain of NRH2 is required to form an association with TrkA. Our data suggest extensive intracellular interaction between NRH2 and TrkA, as either the juxtamembrane or death domain regions of NRH2 are sufficient for interaction with TrkA. In addition, we demonstrate that TrkA signaling is dramatically influenced by the co-expression of NRH2. Importantly, NRH2 did not influence all downstream TrkA signaling pathways, but rather exerted a specific effect, enhancing src homology 2 domain-containing transforming protein (Shc) activation. Moreover, downstream of Shc, the co-expression of NRH2 resulted in TrkA specifically modulating mitogen-activated protein kinase pathway activation, but not the phosphatidylinositol 3-kinase/Akt pathway. These results indicate that NRH2 utilizes intracellular mechanisms to not only regulate NGF binding to TrkA, but also specifically modulate TrkA receptor signaling, thus adding further layers of complexity and specificity to neurotrophin signaling.

How Do TrkA and p75 Interact?

Nerve growth factor (NGF) binds to two structurally unrelated cell surface receptors, the p75 neurotrophin receptor (p75) and the receptor tyrosine kinase TrkA. p75 increases TrkA’s responsiveness to NGF, and coexpression of the two receptors appears to lead to the formation of binding sites with higher affinity for NGF than that of either receptor alone. This suggests that the high-affinity NGF-binding site may involve a complex containing both p75 and TrkA. Wehrmann et al . determined the crystal structure of the complete TrkA extracellular domain in complex with NGF and found that this structure was not incompatible with the existence of a 1:2:1 TrkA-NGF-p75 ternary complex. To investigate this possibility in a cellular context, the authors determined interactions between proteins (p75 and a truncated form of TrkA) fused to fragments of β-galactosidase (which become functional when brought together) that were expressed in C2C12 myoblasts. NGF promoted formation of TrkA homodimers but not that of TrkA-p75 heterodimers (which also did not exist in untreated cells); p75 formed oligomers in the absence of ligand, and NGF did not affect this oligomerization. When corrected for background binding, Scatchard analysis of 125 I-NGF binding in both PC12 cells and C2C12 cells expressing TrkA and p75 constructs failed to reveal the higher-affinity NGF binding site. Thus, the authors conclude that functional interactions between p75 and TrkA likely depend on downstream signaling rather than physical complexes involving the two receptors. In discussing this research, Barker suggests that p75 could pass NGF to TrkA in a ternary complex that exists too briefly to be detected by the β-galactosidase complementation assay. T. Wehrman, X. He, B. Raab, A. Dukipatti, H. Blau, K. C. Garcia, Structural and mechanistic insights into nerve growth factor interactions with the TrkA and p75 receptors. Neuron 53 , 25-38 (2007). [PubMed] P. A. Barker, High affinity not in the vicinity? Neuron 53 , 1-4 (2007). [PubMed]

Mechanisms of neurotrophin receptor signalling

Regulation of cell survival decisions and neuronal plasticity by neurotrophins are mediated by two classes of receptors, Trks (tropomyosin receptor kinases) and p75, the first discovered member of the tumour necrosis factor receptor superfamily. The p75 receptor participates with the TrkA receptor in the formation of high-affinity nerve growth factor-binding sites to promote survival under limiting concentrations of neurotrophins. Activation of Trk receptors leads to increased phosphorylation of Shc (Src homology and collagen homology), phospholipase C-gamma and novel adaptor molecules, such as the ARMS (ankyrin-rich membrane spanning)/Kidins220 protein. Small ligands that interact with G-protein-coupled receptors can also activate Trk receptor kinase activity. Transactivation of Trk receptors and their downstream signalling pathways raise the possibility of using small molecules to elicit neuroprotective effects for the treatment of neurodegenerative diseases. Like amyloid precursor protein and Notch, p75 is a substrate for gamma-secretase cleavage. The p75 receptor undergoes an alpha-secretase-mediated release of the extracellular domain followed by a gamma-secretase-mediated intramembrane cleavage. Cleavage of p75 may represent a general mechanism for transmitting signals as an independent receptor and as a co-receptor for other signalling systems.

Neurotrophin‐3 down‐regulates trkA mRNA, NGF high‐affinity binding sites, and associated phenotype in adult DRG neurons

Neurotrophin-3 (NT-3) binds to multiple trks, not only its initially identified receptor trkC. Recent studies in our laboratory show that NT-3 negatively regulates nociceptive phenotype associated with the trkA subpopulation. Due to the extensive overlap in trkA and trkC expression it is uncertain whether there is a direct influence of NT-3 on trkA in adult sensory neurons. Thus, the aim of this study was to examine whether NT-3 might alter trkA and associated neuronal phenotype outside of the trkC subpopulation. The effect of a seven-day intrathecal infusion of NT-3 on intact, uninjured adult rat dorsal root ganglion neurons was investigated. Serial sections were processed for receptor radioautography or in situ hybridization to identify and colocalize neurons expressing high-affinity nerve growth factor (NGF) binding sites, substance P (SP), trkC, or trkA mRNAs and to examine the influence of NT-3 on these populations. NT-3 does not appear to alter trkC expression, but evokes a notable reduction in trkA, high-affinity NGF binding sites, and SP levels. It is unlikely that signalling by trkC greatly influences this response because the down-regulation of SP occurs most notably in trkA neurons that lack trkC. Moreover, we have shown here that message levels of two trkA isoforms are differentially modulated by NT-3; infusion results in greater down-regulation of the noninsert containing isoform. These findings suggest a clinically relevant role for NT-3 as an antagonist to NGF, but also raise the caution that not just trkC-positive neurons are influenced following exposure to the neurotrophin.

Identification and Structure of the Nerve Growth Factor Binding Site on TrkA

Nerve growth factor (NGF) is involved in the development and maintenance of the nervous system and has been implicated as a possible therapeutic target molecule in a number of neurodegenerative diseases, especially Alzheimer's disease. NGF binds with high affinity to the extracellular region of a tyrosine kinase receptor, TrkA, which comprises three leucine-rich motifs (LRMs), flanked by two cysteine-rich clusters, followed by two immunoglobulin-like (Ig-like) domains. We have expressed the second Ig-like domain as a recombinant protein in E. coli and demonstrate that NGF binds to this domain with similar affinity to the native receptor. This domain (TrkAIg2) has the ability to sequester NGF in vitro, preventing NGF-induced neurite outgrowth, and in vivo, inhibiting NGF-induced plasma extravasation. We also present the three-dimensional structure of the TrkAIg2 domain in a new crystal form, refined to 2.0 Å resolution.

Retroviral transfer of the ?-nerve growth factor gene into murine neuroectodermal tumor cells modulates cell proliferation rate, neurite formation, and NGF binding site expression

The response of wild-type and genetically engineered neuroectodermal tumor (NET) cells to exogenous and endogenously synthesized nerve growth factor (NGF) was investigated. Differences in cell proliferation rate, neurite formation, and expression of NGF binding sites were quantitatively determined. Ecotropic retroviral vectors were used to transfer the genes for beta-galactosidase (beta-GAL) and NGF into wild-type C-1300 and Neuro-2A murine neuroblastoma (MNB) and rat pheochromocytoma (PC-12) cells. Conditioned media obtained from NET cells infected with the NGF gene contained biologically active NGF, whereas media from beta-GAL infected cells did not. Infection with the NGF vector induced a short-term decrease in cell proliferation rate and increased neurite formation in wild-type, substrate-adherent PC-12 and Neuro-2A MNB cells (P > 0.05). Incubation of wild-type C-1300, Neuro-2A MNB, and PC-12 cells with NGF (0-200 ng/ml) for 5 days significantly reduced proliferation rates in a concentration-dependent manner and increased neurite extrusion. All NGF-NET cells had a significantly diminished response to the antiproliferative action of exogenous NGF. Ligand binding assays with 125I-NGF demonstrated a marked reduction in the number of NGF binding sites on NGF-NET cells compared to wild type. The attenuated response of NGF-NET cells to exogenous NGF correlated positively with the down-regulation of NGF binding sites. In conclusion, beta-NGF gene transfer into wild-type NET cells induces the synthesis and secretion of NGF, temporarily decreases cell proliferation rate, increases neurite extrusion, down-regulates NGF binding sites, and reduces NET cell responsiveness to NGF. A putative role for NGF may be the modulation of NET cell proliferation and differentiation.

Retroviral transfer of the β-nerve growth factor gene into murine neuroectodermal tumor cells modulates cell proliferation rate, neurite formation, and NGF binding site expression

The response of wild-type and genetically engineered neuroectodermal tumor (NET) cells to exogenous and endogenously synthesized nerve growth factor (NGF) was investigated. Differences in cell proliferation rate, neurite formation, and expression of NGF binding sites were quantitatively determined. Ecotropic retroviral vectors were used to transfer the genes for β-galactosidase (β-GAL) and NGF into wild-type C-1300 and Neuro-2A murine neuroblastoma (MNB) and rat pheochromocytoma (PC-12) cells. Conditioned media obtained from NET cells infected with the NGF gene contained biologically active NGF, whereas media from β-GAL infected cells did not. Infection with the NGF vector induced a short-term decrease in cell proliferation rate and increased neurite formation in wild-type, substrate-adherent PC-12 and Neuro-2A MNB cells (P > 0.05). Incubation of wild-type C-1300, Neuro-2A MNB, and PC-12 cells with NGF (0–200 ng/ml) for 5 days significantly reduced proliferation rates in a concentration-dependent manner and increased neurite extrusion. All NGF-NET cells had a significantly diminished response to the antiproliferative action of exogenous NGF. Ligand binding assays with 125I-NGF demonstrated a marked reduction in the number of NGF binding sites on NGF-NET cells compared to wild type. The attenuated response of NGF-NET cells to exogenous NGF correlated positively with the down-regulation of NGF binding sites. In conclusion, β-NGF gene transfer into wild-type NET cells induces the synthesis and secretion of NGF, temporarily decreases cell proliferation rate, increases neurite extrusion, down-regulates NGF binding sites, and reduces NET cell responsiveness to NGF. A putative role for NGF may be the modulation of NET cell proliferation and differentiation. J. Neurosci. Res. 59:265–275, 2000 © 2000 Wiley-Liss, Inc.

Neurotrophin binding to the p75 neurotrophin receptor is necessary but not sufficient to mediate NGF-effects on APP secretion in PC-12 cells.

In the present study the pheochromocytoma cell line (PC-12) was used as a model system to determine the role of the two neurotrophin receptors in the regulation of amyloid precursor protein (APP) secretion by nerve growth factor (NGF). To stimulate TrkA and/or p75NTR signaling in PC-12 cells, we used NGF, brain-derived neurotrophic factor (BDNF), and NGF in the presence of an excess of BDNF or the monoclonal antibody 192IgG, to block p75NTR binding to NGF. Our results demonstrate that NGF stimulates APP secretion in a dose dependent fashion with maximum effects at 10 ng/ml, known to saturate high-affinity NGF binding sites. Treatment of PC-12 cells with varying concentrations of BDNF, 1-1,000 ng/ml, did not alter APP secretion, suggesting that binding to p75NTR alone is not sufficient to affect APP secretion. When blocking NGF binding to p75NTR with BDNF or 192IgG, on the other hand, NGF effects on APP secretion were abolished. These findings suggest that in cells expressing p75NTR and TrkA receptors, binding of NGF to the p75NTR is required to mediate NGF effects on APP secretion. Our data are also consistent with a proposed function of the p75NTR in receptor recruitment and "presentation" of NGF to receptors.

Immunoglobulin-like domains define the nerve growth factor binding site of the TrkA receptor.

Nerve growth factor (NGF) is involved in the development and maintenance of the nervous system. NGF binds with high affinity to the extracellular region of the tyrosine kinase receptor TrkA. This domain comprises leucine and cysteine rich motifs, followed by two immunoglobulin like (Ig-like) domains. We describe the expression and purification of recombinant Ig-like domains. Fluorescence and circular dichroism spectroscopy show that the protein is folded into a compact globular structure and contains mainly beta-sheet secondary structure. Recombinant protein binds to NGF and can inhibit NGF bioactivity both in vitro and in vivo.

Nerve Growth Factor Binding Site on TrkA Mapped to a Single 24-Amino Acid Leucine-rich Motif

The extracellular domains of the TrkA nerve growth factor (NGF) receptor and its homologs harbor a modular mosaic of potential ligand binding motifs, namely two immunoglobulin (Ig)-like modules and an LRM3 cassette consisting of a tandem array of three leucine-rich motifs (LRMs) flanked by cysteine-rich clusters (Schneider, R., and Schweiger, M. (1991) Oncogene 6, 1807-1811). Identification of a structural motif capable of specifically recognizing the various neurotrophins was achieved by assessing their affinities to isolated recombinant modules of TrkA and TrkB. In both receptors the LRM3 cassette alone could mediate the respective neurotrophin selectivities and affinities. Further tracking down of this NGF-binding site in TrkA strikingly revealed that a single LRM of 24 amino acids could bind NGF selectively with nanomolar affinity. Since this is the first example of a single LRM with a highly specific, well defined function, it might serve as a valuable tool to elucidate the general structural requirements of substrate recognition and high affinity binding in the large superfamily of LRM-containing proteins.

Developmental changes in nerve growth factor (NGF) binding and NGF receptor proteins trkA and p75 in the facial nerve.

This study characterizes the temporal-spatial distribution of nerve growth factor (NGF) low (p75) and high-affinity (trkA) receptors in the facial nerve and geniculate ganglion (GG) of developing quail embryos (E-3 to E-14). We used 125I-labeled NGF (125I-NGF) to study binding dynamics in a temporal series of isolated primordia and an autoradiographic series of staged specimens to characterize the occurrence and distribution of NGF receptors in this cranial nerve and its ganglion. In addition, expression of trkA and p75 protein-like immunoreactivity in the facial nerve and GG was studied by Western blot, in order to distinguish between high- and low-affinity NGF receptors respectively. The quantitative study of binding show that isolated facial primordia ranging from E-3 to E-14 exhibit different levels of specific binding. High initial binding levels were observed on E-3 specimens, then an initial decrease on day 4 (E-4) followed by a steady increase from days E-4 to E-7. Maximum 125I-NGF binding was achieved on E-7, followed by a steady decline in binding on days 8 (E-8) and 9 (E-9), reaching near background levels on day 10 (E-10) of development and until the oldest stage assayed (E-14). Most of the cells bearing NGF receptors appeared to be non-neuronal crest-derived cells, but some placode-derived neurons and motor fibers of the VIIth cranial nerve transiently expressed the ability to bind 125I-NGF. The temporal pattern of p75 expression matches the pattern of quantitative binding of NGF, while the trkA expression is restricted to a few stages mainly E7 and E9, implying that most of the binding detected is via low-affinity receptors, except for a proportion of high-affinity receptors present at stages of maximum binding. This temporal pattern of NGF binding sites suggests that cells within the VIIth cranial nerve are responsive to and/or dependent upon NGF in vivo, so NGF may play a biological role during normal development of the facial nerve. In view of the developmental events that parallel the occurrence and type of NGF binding sites, we suggest that this role may be to modulate from earlier chemotaxis and cell proliferation to much later events, such as neuronal differentiation and neuron-glia interactions. The significance of these findings in regeneration during adult life remain to be investigated.

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.

NGF Increases Brain Astrocyte Number in Culture

Nerve growth factor (NGF) promotes survival and maintenance of peripheral and central neurons. In previous studies, we found that low-affinity NGF binding sites were associated with flat nonneuronal cells dissociated and cultured from the embryonic septum. These cells were also labeled with a monoclonal antibody to the NGF receptor, 192 IgG, suggesting that low-affinity NGF receptors are associated with a nonneuronal population. To define the potential effect of NGF on these nonneuronal cells, rat Embryonic Day 17 (E17) septa were dissociated and cultured in fully defined medium in the presence of NGF. Glial fibrillary acidic protein (GFAP) was used as a marker for the astrocyte population. NGF elicited a dramatic ninefold increase in the number of GFAP-positive cells after 7 days. NGF similarly increased astrocyte number in cultures grown from E18 hippocampi and E16 substantia nigra, suggesting that NGF acts on glia from multiple brain regions. To begin defining the mechanism of NGF action on astrocytes, we examined [3H]thymidine incorporation, which increased significantly, but by only 50%, upon exposure to the trophic factor. We tentatively conclude that NGF increases astrocyte number in culture by stimulating mitosis as well as enhancing survival or differentiation.

Differential expression of nerve growth factor receptors leads to altered binding affinity and neurotrophin responsiveness.

The low-affinity p75 neurotrophin receptor is believed to participate with the Trk receptor tyrosine kinase in the formation of high-affinity binding sites for nerve growth factor (NGF). To investigate the functional significance of the two NGF receptors, a truncated p75 receptor was stably expressed in PC12 rat pheochromocytoma cells, yielding cells with greatly reduced levels of wild-type p75 and normal Trk levels. Although these cells were capable of normal differentiation by NGF, very few high-affinity NGF binding sites were detected. These findings indicate that high-affinity binding may be functionally dissociated from biological responses. Furthermore, an increased responsiveness to neurotrophin 3 was observed, as manifested by increased neurite outgrowth. These results suggest that a correct ratio of p75 and p140trk is required to create high-affinity sites and that p75 expression may assist in the discrimination between related but different neurotrophin factors.

CGRP immunoreactive neurons in rat dorsal root ganglia do not all contain low-affinity NGF receptor immunoreactivity

Nerve growth factor (NGF) and its homologues bind to the low affinity NGF receptor (LNGFR). We have determined whether the LNGFR colocalizes with calcitonin gene-related peptide (CGRP) as this peptide is restricted to neurons exhibiting high affinity binding sites for NGF. LNGFR and CGRP immunoreactivities were detected in 57% and 49% of spinal ganglion neurons respectively with different soma areas and only partial colocalization. Our data suggest that the LNGFR may not be essential for a functional neurotrophin response.

Nerve growth factor binding sites on hepatic parenchymal cells.

Nerve growth factor (NGF) binding sites on rat hepatocytes (HCs) in culture for 24 to 48 h were characterized using 125I-NGF. Specific binding of 125I-NGF to HCs was saturable. Scatchard analysis indicated a single population of binding sites with a Kd of 5.5 nM and a Bmax of 540 fmol/mg protein. In isolated hepatocyte membranes, specific binding of 125I-NGF was also apparent with Kd and Bmax values of 10.8 nM and 3740 fmol/mg protein, respectively. Specific binding of 125I-NGF to HCs was displaced by excess, unlabeled NGF but not by up to 1000-fold excess of either insulin or epidermal growth factor. Internalization/sequestration of 125I-NGF into HCs was measured as radioactivity present in solubilized cells after exposure to high salt and acid. These studies indicated 83 +/- 11% of 125I-NGF was accumulated by internalization/sequestration at a concentration of 1 nM 125I-NGF. Internalization was reduced to 43 +/- 4% when incubations were carried out at 4 degrees C. These results indicate the presence of a specific, low-affinity binding site for NGF on hepatocytes in culture.

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.

The low-affinity p75 nerve growth factor (NGF) receptor mediates NGF-induced tyrosine phosphorylation.

Protein tyrosine phosphorylation is a potential mechanism for initial signaling in PC12 cells during differentiation in response to nerve growth factor (NGF). NGF-induced tyrosine phosphorylation has been found to be initiated by the trk protooncogene, which participates in the formation of high-affinity NGF binding sites. In contrast to transfection of wild-type low-affinity p75 NGF receptors, transfection of p75NGFR with mutations in the cytoplasmic domain resulted in an inability of NGF to elicit tyrosine phosphorylation of intracellular substrates, indicating that p75NGFR is involved in initiating phosphorylation events by NGF. Even though the p75NGFR receptor does not possess any inherent tyrosine kinase activity, these experiments demonstrate that the p75NGFR has a potential role in NGF-induced tyrosine phosphorylation.

Disruption of cysteine-rich repeats of the p75 nerve growth factor receptor leads to loss of ligand binding

Nerve growth factor (NGF) binds to a low affinity cell surface receptor (p75NGFR) which contains four extracellular repeats, rich in cysteine residues and negatively charged. We have made mutations in the receptor cDNA by inserting linkers in specific domains of the receptor. Nearly all the mutations caused a change in the predicted charge, and resulted in either an insertion or deletion in the primary sequence. Stably transfected fibroblasts were assayed for NGF binding by affinity cross-linking with 125I-NGF. Appropriate expression of the mutated receptors was monitored by rosetting with monoclonal antibodies and by metabolic labeling followed by immunoprecipitation. Although the mutant receptors were recognized by monoclonal antibodies, insertions and deletions in the third and fourth cysteine-rich regions of the receptor had a detrimental effect upon NGF binding. Insertions made outside the cysteine-rich region or in the cytoplasmic domain did not inhibit the ability of 125I-NGF to bind to the receptor, as assessed by affinity cross-linking. A chimeric human-rat NGF receptor transfected into fibroblasts indicates that NGF binding and monoclonal antibody recognition sites are separated but contained within the four cysteine repeats.