Pedodiversity: concepts and measures

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.

Section: Discussionmentioning

confidence: 99%

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

Rydén¹,

Hempstead²,

Ibáñez³

1997

100

“…Studies of structure-function relationships in the neurotrophins have identified amino acid residues important for their interaction with cell surface receptors (26). The binding site of neurotrophins to p75…”

Section: Lngfrmentioning

confidence: 99%

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

Rydén

Ibáñez

1996

Neurotrophins regulate differentiation and survival of vertebrate neurons through binding to members of the Trk family of receptor tyrosine kinases and to a common low affinity receptor, p75 LNGFR. The specificity of neurotrophin action is determined by their selective interaction with the different members of the Trk family; TrkA, TrkB, and TrkC serve as cognate receptors for nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 (NT-3), respectively. Unlike nerve growth factor and brain-derived neurotrophic factor, NT-3 can to some extent also bind and activate noncognate TrkA and B receptors, although the physiological relevance of these interactions is unclear. Previous studies established that neurotrophins use an extended surface for binding to cognate Trk receptors, while binding to p75LNGFR is mediated by a localized cluster of positively charged residues. Here we show that the binding site of NT-3 to its non-preferred receptors TrkA and TrkB is dominated by two positively charged residues, Arg-31 and His-33, previously shown to constitute a main determinant of binding to p75 LNGFR. Simultaneous mutation of these two residues into Ala completely abolished NT-3 binding and signaling through TrkA and greatly diminished binding and activation of TrkB. However, NT-3 binding and signaling through its cognate receptor TrkC was unaffected by the mutation. These results show that binding of NT-3 to p75 LNGFR , TrkA, and TrkB is mediated by a common determinant, which is distinct from that recognized by TrkC and also different and more localized than the one recognized by TrkA and TrkB in their cognate ligands. Thus, although homologous regions in all neurotrophins are used for binding to Trk receptors, a given Trk may actually contact different residues in different neurotrophins. The mutant NT-3 described here may be of greater advantage than native NT-3 when a trophic activity needs to be specifically targeted to TrkC-expressing neurons and provides a monospecific neurotrophin for future therapeutic development.Vertebrate neurons are dependent on the continuous supply of a group of proteins generally called neurotrophic factors for their development, differentiation, and survival. The neurotrophins are structurally and functionally related neurotrophic factors that function in the developing and adult nervous system (1-3). In mammals, the neurotrophin family presently includes four members, nerve growth factor (NGF) 1 (4), brainderived neurotrophic factor (BDNF) (5, 6), neurotrophin-3 (NT-3) (7-12), and neurotrophin-4 (NT-4) (13, 14), also called NT-5 (15), which share approximately 50% amino acid sequence identity. An additional neurotrophin molecule with structural and functional similarities to NGF has recently been isolated from the platty fish and named NT-6 (16), despite being only the third neurotrophin identified in this species. The neurotrophins are non-covalently linked homodimers of two highly basic 120-residue long polypeptide chains. Each protomer contains three disulfide brid...

“…Several approaches have been used to elucidate which sequences of the neurotrophins are required to specifically bind and activate their cognate receptors (18). A first approach used chemical modification of NGF where residues receptive for different agents could be blocked and the effect analyzed (for review, see Ref.…”

mentioning

confidence: 99%

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

Kullander

Kaplan

Ebendal

1997

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.