Binding of Neurotrophin-3 to p75LNGFR, TrkA, and TrkB Mediated by a Single Functional Epitope Distinct from That Recognized by TrkC

Nerve growth factor (NGF) and neurotrophin-3 (NT-3) mediate activities such as survival, differentiation, and proliferation in various subsets of neurons. In this report, we define precisely the residues in human NGF responsible for NGF biological activity and binding specificity to the neurotrophin receptor TrkA. In earlier studies we defined five amino acid residues of NGF which confer NGF-like activity to NT-3 when replacing corresponding residues in the 120-amino acid long NT-3 molecule. Using this gain-of-function strategy we report the further dissection of this functional epitope. We also define another motif separated topographically in the NGF dimer and determined to be independently responsible for NGF specificity. The first of the two motifs determined to elicit NGF specificity is defined by the residues Val-48, Pro-49, and Gln-96, which are situated in the two top ␤-loops of NGF. The second motif is represented by residues Pro-5 and Phe-7 situated in the proximal part of the NH 2 terminus. Both motifs contain structurally important residues revealing a novel principle, where specificity for neurotrophin ligand-receptor interactions could be determined by variable residues modifying the conformation of the neurotrophin backbone. These findings will enhance further the possibility of mimicking NGF with low molecular weight compounds.

Section: Discussionsupporting

confidence: 86%

Two Restricted Sites on the Surface of the Nerve Growth Factor Molecule Independently Determine Specific TrkA Receptor Binding and Activation

Kullander

Kaplan

Ebendal

1997

“…The IC 50 of NT-3 competition of 125 I-NGF binding requires a 250-fold excess of NT-3 to block binding to 4.1 TrkA/B receptors, whereas a 25-fold excess of NT-3 is required to block binding to wild type TrkA receptors (19). These results further confirm the role of TRKA-D5 subdomain in NT-3 binding, and the 10-fold difference in NT-3 binding to TrkA WT and 4.1 TrkA/B chimera indicates that TrkB-D1-D4 subdomains do not compensate for the equivalent subdomains of TrkA.…”

Section: Discussionmentioning

confidence: 99%

“…The fact that NT-3 has a lower affinity interaction with its heterologous receptors TrkA and TrkB (13,19), and that the biological outcomes of NT-3-TrkA interactions are limited (e.g. partial survival, or limited differentiation) (6) suggests three nonexclusive possibilities as follows: (i) NT-3 as heterologous ligand may lack some docking sites on TrkA for high affinity binding and full function; (ii) the interaction may take place through epitopes that are distinct from those used by NGF; and/or (iii) receptor conformational or allosteric regulation by each ligand may be different.…”

mentioning

confidence: 99%

TrkA Receptor “Hot Spots” for Binding of NT-3 as a Heterologous Ligand

Ivanisevic

Zheng

Woo

et al. 2007

Neurotrophins signal via Trk tyrosine kinase receptors. Nerve growth factor (NGF) is the cognate ligand for TrkA, the brainderived neurotrophic factor for TrkB, and NT-3 for TrkC. NT-3 also binds TrkA as a lower affinity heterologous ligand. Because neurotrophin-3 (NT-3) interactions with TrkA are biologically relevant, we aimed to define the TrkA "hot spot" functional docking sites of NT-3. The Trk extracellular domain consists of two cysteine-rich subdomains (D1 and D3), flanking a leucinerich subdomain (D2), and two immunoglobulin-like subdomains IgC1(D4) and IgC2(D5). Previously, the D5 subdomain was defined as the primary ligand-binding site of neurotrophins for their cognate receptors (e.g. NGF binds and activates through TRKA-D5 hot spots). Here binding studies with truncated and chimeric extracellular subdomains show that TRKA-D5 also includes an NT-3 docking and activation hot spot (site 1), and competition studies show that the NGF and NT-3 hot spots on TRKA-D5 are distinct but partially overlapping. In addition, ligand binding studies provide evidence for an NT-3-binding/allosteric site on TRKA-D4 (site 2). NT-3 docking on sites 1 and/or 2 partially blocks NGF binding. Functional survival studies showed that sites 1 and 2 regulate TrkA activation. NT-3 docking on both sites 1 and 2 affords full agonism, which can be additive with NGF activation of Trk. However, NT-3 docking solely on site 1 is partially agonistic but noncompetitively antagonizes NGF binding and activation of Trk. This study demonstrates that Trk signaling is more complex than previously thought because it involves several receptor subdomains and hot spots.

“…Steady-state competitive bindings were performed at 4°C as described previously (34). Saturation bindings were performed in triplicate wells with 10 6 cells/ml and serial dilutions of radioiodinated factors in Dulbecco's phosphate-buffered saline supplemented with 1 mg/ml bovine serum albumin and 1 mg/ml glucose.…”

Section: Methodsmentioning

confidence: 99%

“…Bioassays-Biological activities in TrkA-expressing MG87-NIH3T3 fibroblasts were assayed as described previously (34). Briefly, in a 96-well plate 20,000 cells/well were incubated in serial dilutions of native or mutant NGF in serum-free medium (Dulbecco's modified Eagle's medium).…”

Section: Methodsmentioning

confidence: 99%

Differential Modulation of Neuron Survival during Development by Nerve Growth Factor Binding to the p75 Neurotrophin Receptor

Rydén¹,

Hempstead²,

Ibáñez³

1997

Self Cite

100

Nerve growth factor (NGF) supports the survival and differentiation of distinct populations of peripheral and central neurons. NGF binds to two classes of cell-surface receptors, the protein tyrosine kinase TrkA and the smaller p75 receptor lacking intrinsic catalytic activity. It has been suggested that both receptors are required for NGF high affinity binding, although TrkA appears to be sufficient for transducing most of the biological effects of NGF. Some evidence suggests that p75 could play a modulatory role on TrkA activation by an as yet unknown mechanism. In this study, we have investigated functional roles of p75 using a purified triple mutant NGF (triNGF) deficient in p75 binding but retaining significant TrkA binding and activation. The mutant was found to be as potent as wild type NGF at promoting survival of serum-deprived TrkA-expressing fibroblasts. On developing chick sensory neurons, survival responses to mutant and native NGF were indistinguishable when assayed at nanomolar concentrations. However, triNGF was 3-to 4-fold less potent than wild type NGF at lower concentrations (i.e. 10 ؊11 M). Interestingly, in PC12 cells coexpressing TrkA and p75, no high affinity binding sites for triNGF could be detected. The reduced responsiveness to triNGF in sensory neurons was increasingly evident at later developmental stages; late embryonic neurons did not respond at all to concentrations of triNGF that were saturating at earlier developmental stages. Likewise, although no difference could be seen between wild type and mutant NGF on the survival responses of embryonic rat superior cervical ganglion sympathetic neurons, the mutant was much less potent than native NGF on postnatal sympathetic neurons. In sensory neurons, the decrease in responsiveness to triNGF correlated with a developmental reduction in the expression of both p75 and TrkA. Thus, NGF binding to p75 enhances responsiveness to ligand, particularly when this is present at limiting concentrations. During development, p75 modulates responsiveness to NGF so that binding to p75 becomes increasingly important in neurons undergoing a down-regulation of NGF receptors. These results support a ligand-dependent modulatory role for p75 in NGF-mediated neuron survival consistent with p75 functioning as a TrkA regulator and/or signaling receptor.