Journal of Molecular Biology
Volume 414, Issue 5, 16 December 2011, Pages 681-698
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Structural Model for p75NTR–TrkA Intracellular Domain Interaction: A Combined FRET and Bioinformatics Study

https://doi.org/10.1016/j.jmb.2011.09.022Get rights and content

Abstract

Nerve growth factor (NGF) is a member of the neurotrophins, which are important regulators of embryonic development and adult function in the vertebrate nervous systems. The signaling elicited by NGF regulates diverse activities, including survival, axon growth, and synaptic plasticity. NGF action is mediated by engagement with two structurally unrelated transmembrane receptors, p75NTR and TrkA, which are co-expressed in a variety of cells. The functional interactions of these receptors have been widely demonstrated and include complex formation, convergence of signaling pathways, and indirect interaction through adaptor proteins. Each domain of the receptors was shown to be important for the formation of TrkA and p75NTR complexes, but only the intramembrane and transmembrane domains seemed to be crucial for the creation of high-affinity binding sites. However, whether these occur through a physical association of the receptors is unclear. In the present work, we demonstrate by Förster resonance energy transfer that p75NTR and TrkA are physically associated through their intracellular (IC) domains and that this interaction occurs predominantly at the cell membrane and prior to NGF stimulation. Our data suggest that there is a pool of receptors dimerized before NGF stimulus, which could contribute to the high-affinity binding sites. We modeled the three-dimensional structure of the TrkA IC domain by homology modeling, and with this and the NMR-resolved structure of p75NTR, we modeled the heterodimerization of TrkA and p75NTR by docking methods and molecular dynamics. These models, together with the results obtained by Förster resonance energy transfer, provide structural insights into the receptors' physical association.

Graphical Abstract

Research Highlights

► p75NTR and TrkA are physically associated through their IC domains. ► This interaction occurs predominantly at the cell membrane, prior to NGF stimulation. ► The three-dimensional structure of the TrkA IC domain was obtained by homology modeling. ► TrkA and p75NTR heterodimerization was evaluated by molecular dynamics.

Introduction

Nerve growth factor (NGF) is a member of the mammalian neurotrophin (NT) protein family, which also encompasses BDNF, NT3, and NT4/5. NTs are known to mediate the maintenance and survival of the peripheral and central nervous systems as well as several forms of synaptic plasticity.1, 2, 3 Thus, NTs are of great therapeutic interest for the treatment of a number of neurodegenerative and psychological diseases.

NTs exert their cellular effects through the actions of two structurally different receptors, the Trk receptor tyrosine kinase and the p75 NT receptor (p75NTR). All NTs recognize p75NTR, but NGF binds preferentially to TrkA, while BDNF and NT-4 bind to TrkB, and NT-3 binds to TrkC. Upon binding of NTs, Trks dimerize, which leads to the autophosphorylation of several tyrosine residues in their cytoplasmic domain, which, in turn, triggers the Ras/MEK/ERK and PI3K/Akt intracellular (IC) signaling pathways, among others.4 NT binding to p75NTR initiates apoptosis in the absence of Trks but promotes survival when co-expressed.5, 6, 7

Trk receptors share a common structural organization: an extracellular (EC) domain that consists of a cysteine-rich cluster followed by three leucine-rich repeats, another cysteine-rich cluster and two immunoglobulin-like domains, a single transmembrane domain, and a cytoplasmic domain that contains a tyrosine kinase domain plus several tyrosine-containing motifs. Phosphorylation of cytoplasmic tyrosines in Trk receptors regulates tyrosine kinase activity and provides phosphorylation-dependent recruitment sites for adaptor molecules and enzymes that mediate the initiation of the IC signaling cascades.4 p75NTR belongs to the tumor necrosis factor receptor family and binds all NTs with similar nanomolar affinities.5, 8 p75NTR is a glycoprotein with a unique transmembrane helix. Its EC sequence contains four cysteine-rich domains characteristic of the tumor necrosis factor receptor superfamily.9, 10 The IC region consists of a flexible juxtamembrane domain followed by a globular domain known as the death domain.11 Unlike Trk receptors, p75NTR lacks catalytic activity, and thus, the signal transduction proceeds via recruitment of cytoplasmic effectors. Some of the major signaling events triggered by p75NTR include activation of NF-κB, c-jun kinase and caspases.12

Several lines of evidence suggest functional interactions between TrkA and p75NTR in the signal transduction triggered by NGF.4, 5, 13 Firstly, p75NTR enhances the response of Trks to NTs. In this sense, antibodies against p75NTR reduced NGF-mediated TrkA phosphorylation in PC12 cells and primary neurons, whereas the co-expression of p75NTR and TrkA in heterologous expression systems enhanced NGF-induced TrkA phosphorylation.14 Furthermore, p75NTR increases the specificity of the Trk receptors for particular ligands. In this case, function-perturbing antibodies to p75NTR enhance the response of TrkA to NT3, consistent with the notion that p75NTR acts to suppress TrkA responses to this ligand.15 These functional collaborations have physiological relevance, as, for example, primary dorsal root sensory neurons and sympathetic neurons derived from p75NTR null animals show a decrease in survival responses to NGF.16 The existence of p75NTR and TrkA complexes has been addressed by different biochemical assays, including cross-linking and co-immunoprecipitation,13, 17, 18 and it has been suggested that co-expression of these receptors leads to the generation of high-affinity binding sites for NGF.19 Whereas the EC, transmembrane and IC domains have been implicated in p75NTR–TrkA complex formation,13, 20 only the transmembrane and IC domains have been shown to participate in the formation of the high-affinity sites.19 However, the nature of these binding sites and whether they occur via a direct physical association of p75NTR and TrkA remain to be clarified.

Recent crystallographic studies determined the three-dimensional (3D) structure of the glycosilated p75NTR EC domain complexed with NT3 and the TrkA EC domain complexed with NGF. Both studies describe a 2:2 receptor:ligand stoichiometry,21, 22 which is supported by other biophysical assays.23 The superimposition of NT3-p75NTR and NGF-TrkA structures showed that the binding sites on the ligands were mutually exclusive and, therefore, discarded the possibility of a physical interaction of p75NTR and TrkA through their EC domains.22 Therefore, provided there is a physical interaction, it might take place through their IC domains. We addressed this question by evaluating TrkA and p75NTR IC domain interaction by Förster resonance energy transfer (FRET). We constructed TrkA-CFP and p75-YFP receptor chimeras and transfected them into PC12 cells and, comparatively, evaluated the interaction of endogenous receptors by immunolabeling. We observed a direct physical association of p75NTR and TrkA at the cell membrane and prior to NGF stimulation, for both transfected and endogenous receptors. We extended these results to COS-7 cells and hippocampal neuronal cultures.

After these findings, we constructed a 3D model for the interaction. The structure of the p75NTR death domain has been previously determined by nuclear magnetic resonance.11 However, no information was available for the TrkA IC domain. Thus, we firstly obtained the structure of the TrkA kinase domain in both the active and the inactive states by homology modeling and then modeled TrkA homodimers and TrkA–p75NTR heterodimers by docking methods and molecular dynamics simulations. The resulting structural models serve to gain insight into the association of these receptors and might provide a platform for further de novo structure-based design of novel drugs that might prevent or alleviate neurodegenerative disorders onset and/or progression.

Section snippets

TrkA and p75NTR interact physically through their IC domains

We evaluated the physical interaction of p75NTR and TrkA by FRET in differentiated PC12 cells. We observed that the FRETeff between TrkA-CFP and p75-YFP was significantly higher than that of the negative control (cells transfected only with TrkA-CFP; FRETeff: 0.22 versus 0.15, respectively, Fig. 1c) and that the higher FRET efficiency (> 0.4) was confined to the cell membrane and the endosomes (Fig. 1a). Interestingly, we observed that the FRETeff was high prior to NGF stimulation and decreased

Discussion

In the present study, we investigated the interaction of NGF receptors TrkA and p75NTR. The major finding of this work is that TrkA and p75NTR associate physically through their IC domains. Based on this result and previous references, we modeled the 3D association of TrkA and p75NTR IC domains.

Previous studies suggest the existence of preformed TrkA–p75NTR heterodimers. Co-precipitation of endogenous TrkA and p75NTR in PC12 cells indicates that the receptor complex forms in a

Cell lines and neuronal cultures

PC12 cells (gift of Dr. Osvaldo Uchitel, Laboratory of Physiology and Molecular Biology, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires) were grown in high glucose Dulbecco's modified Eagle's medium (DMEM) F12 with glutamine, 6% HS, 6% fetal calf serum, and 80 μg/mL gentamicin (complete media)28 at 37 °C in 5% CO2. Cells were grown on Lab-Tek Chambered Borosilicate Coverglass System (Nunc) or on coverslides, coated with poly-d-lysine. Differentiation was induced by the

Acknowledgements

We thank Dr. Osvaldo D. Uchitel for the kind gift of PC12 cells, Dr. Coluccio Leskow for β2-Chimerin-YFP, Dr. Mike Fainzilber for TrkA cDNA, and Dr. Francisca Bronfman for p75-YFP construct. We thank Dr. Alfredo Caceres for assistance in the establishment of hippocampal cell cultures and critical reading of the manuscript.

We emphatically thank Dr. Thomas Jovin for his advice and support. The cloning of TrkA-CFP and TrkA-YFP was conducted by Dr. M. M. Echarte, a former member of our laboratory,

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    M.F.I. and S.G. contributed equally to this work.

    Department of Neuroscience, Physiology and Pharmacology, University College London, London WC1E 6BT, UK.

    §

    Department of Cell and Developmental Biology, University College London, London WC1E 6BT, UK.

    Departamento de Fisiología, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires C1121ABG, Argentina.

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