Structure-function studies of nerve growth factor: functional importance of highly conserved amino acid residues.

Ibáñez, Carlos F.; Hallböök, Finn; Ebendal, Ted; Persson, Håkan

doi:10.1002/j.1460-2075.1990.tb08265.x

Cited by 67 publications

(9 citation statements)

References 47 publications

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“…NGF is secreted as a propeptide of 305 residues (ϳ35 kDa), pro-NGF, and subsequently cleaved at the N terminus by serine proteases to generate a smaller 120-amino acid mature polypeptide (ϳ13 kDa) (23,24). The folded monomer is stabilized by three disulfide bridges that form the core cystine-knot motif, characteristic of all neurotrophins (25,26).…”

Section: Afflicted Neurons In Alzheimer Disease Have Been Shown To DImentioning

confidence: 99%

Identification of Critical Residues within the Conserved and Specificity Patches of Nerve Growth Factor Leading to Survival or Differentiation

Mahapatra¹,

Mehta²,

Woo

et al. 2009

Journal of Biological Chemistry

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Afflicted neurons in Alzheimer disease have been shown to display an imbalance in the expression of TrkA and p75NTR at the cell surface, and administration of nerve growth factor (NGF) has been considered and attempted for treatment. However, wild-type NGF causes extensive elaboration of neurites while providing survival support. This study was aimed at developing recombinant NGF muteins that did not support neuritogenesis while maintaining the survival response. Critical residues were identified at the ligand-receptor interface by point mutagenesis that played a greater importance in neuritogenesis versus survival. By combining point mutations, two survival-selective recombinant NGF muteins, i.e./7-84-103 and KKE/7-84-103, were generated. Both muteins reduced neuritogenesis in PC12 (TrkA ؉ /p75 NTR؉ ) cells by >90%, while concurrently retaining near wild-type survival activity in MG139 (TrkA Neurotrophins are a family of closely related proteins that have diverse functions ranging from neuronal survival and differentiation to regulation of axonal and dendritic outgrowth, synapse formation, cell migration, and cellular proliferation (1-5). The family of neurotrophins was established with the discovery of its founding member, nerve growth factor (NGF) 3 (3, 6, 7). Brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and neurotrophin 4/5 (NT-4/5) possess a high sequence homology (ϳ50%) and adopt similar tertiary structures (7-12). Neurotrophins bind selectively to a family of 140-kDa Tropomyosin-receptor-kinases (Trk), i.e. NGF to TrkA, brain-derived neurotrophic factor and NT-4 to TrkB, and NT-3 to TrkC, via interactions with a nanomolar affinity (K d ϳ10 Ϫ9 M) (13-16). All neurotrophins can also bind a 75-kDa common neurotrophin receptor, p75 NTR , via similar affinity interactions (17, 18).NGF is a target-derived polypeptide synthesized by the hippocampus and retrogradely transported within axons of cholinergic interneurons (striatum), magnocellular cholinergic neurons (basal forebrain), and Purkinje cells (cerebellum) (19 -22). NGF is secreted as a propeptide of 305 residues (ϳ35 kDa), pro-NGF, and subsequently cleaved at the N terminus by serine proteases to generate a smaller 120-amino acid mature polypeptide (ϳ13 kDa) (23,24). The folded monomer is stabilized by three disulfide bridges that form the core cystine-knot motif, characteristic of all neurotrophins (25,26). Each monomer is composed of two pairs of antiparallel ␤-strands connected by four ␤-hairpin loops (25). NGF possesses highly conserved hydrophobic residues along the elongated central axis of the molecule, which facilitates its tight association into a noncovalent homodimer (K d Ͻ10 Ϫ13 M) (25,27). The solvent-exposed ␤-hairpin loops (I-V), along with the N and C termini, are highly variable across neurotrophins and play a functional role in establishing specificity in receptor activation (12, 17, 26, 28 -31).The crystal structure of a symmetric complex of dimeric NGF bound to a pair of TrkA-d 5 domains identified two critical pat...

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Section: Afflicted Neurons In Alzheimer Disease Have Been Shown To DImentioning

confidence: 99%

Identification of Critical Residues within the Conserved and Specificity Patches of Nerve Growth Factor Leading to Survival or Differentiation

Mahapatra¹,

Mehta²,

Woo

et al. 2009

Journal of Biological Chemistry

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“…However, from the published data not all of them appear to be important for binding. The exchange of Trp21 to phenylalanine or leucine, for example, did not affect the biological activity of NGF [53,54]; similarly, mutation of Phe86 to alanine yielded nearly fully active protein [55]. In contrast, the importance of Ile31 for high-affinity binding is well established, since mutation of this residue to alanine reduces TrkA binding to NGF by 4-fold [56].…”

Section: The 'Conserved Patch'mentioning

confidence: 99%

“…7), with the C terminus projecting toward a groove formed by loops 2 and 4 of NGF. Furthermore, these two loops display a high degree of sequence diversity among the neurotrophins, and swapping experiments in which loops or individual residues were exchanged between NGF and BDNF [53] or between NGF and NT-3 [58] demonstrated that they carry important specificity determinants for ligand-receptor recognition. Therefore, a third patch, not observed in the crystal structure of the NGF-TrkA-d5 complex and consisting of the linker segment of the receptor interacting with loops L2 and L4 of NGF, is likely to complete the interface between NGF, and intact TrkA.…”

Section: A Third Patch Important For Specificity?mentioning

confidence: 99%

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Nerve growth factor: structure and function

Wiesmann

Vos²

2001

CMLS, Cell. Mol. Life Sci.

169

135

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Neurotrophins are critical for the development and maintenance of the peripheral and central nervous system. These highly homologous, homodimeric growth factors control cell survival, differentiation, growth cessation, and apoptosis of sensory neurons. The biological functions of the neurotrophins are mediated through two classes of cell surface receptors, the Trk receptors and the p75 neurotrophin receptor (p75NTR). Nerve growth factor (NGF), the best characterized member of the neurotrophin family, sends its survival signals through activation of TrkA and can induce cell death by binding to p75NTR. Recent domain deletion and mutagenesis studies have identified the membrane-proximal domain of the Trks as necessary and sufficient for ligand binding. Crystal structures of this domain of TrkA, TrkB, and TrkC, and an alanine scanning analysis of this domain of TrkA and TrkC have allowed identification of the ligand-binding site. The recent crystal structure of the complex between NGF and the ligand-binding domain of TrkA defines the orientation of NGF in the signaling complex, and eludicates the structural basis for binding and specificity in the family. Further structural work on NGF-TrkA-p7SNTR complexes will be necessary to address the many remaining questions in this complex signaling system.

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“…The hydrophilicity, accessibility, and conservation of these residues suggest a common role for this loop region in all neurotrophins, possibly in modulating binding to cell-surface receptors. A previous study showed that mutation of Trp-76 to phenylalanine had no major effect on receptor binding and biological activity (18). A more recent study found reduced affinity for TrkA and decreased biological activity after replacement of His-75 with alanine (19).…”

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confidence: 98%

A Second Determinant of Binding to the p75 Neurotrophin Receptor Revealed by Alanine-scanning Mutagenesis of a Conserved Loop in Nerve Growth Factor

Rydén

Ibáñez

1997

Journal of Biological Chemistry

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In the neurotrophin family, variable regions contain solvent-accessible residues important for receptor binding specificity, whereas many of the conserved residues are buried in hydrophobic cores or in the dimer interface. A stretch of six amino acids (from Asp-72 to Asn-77) in nerve growth factor (NGF) represents an exception to this general rule. These residues are highly conserved and yet form an exposed hydrophilic loop region away from other known determinants of receptor binding. We have investigated the functional importance of this region in NGF using alanine-scanning mutagenesis. Individual mutation of Asp-72, Lys-74, or His-75 to alanine (mutants D72A, K74A, and H75A, respectively) reduced the binding affinity for the p75 neurotrophin receptor by 4 -10-fold. Only the D72A mutant showed an additional impairment in binding to the TrkA receptor, which was accompanied by reduced biological activity in PC12 cells, indicating a structural and/or conformational effect of this mutation. Replacement of Ser-73 or Asn-77 with alanine (mutants S73A and N77A, respectively) had no measurable effects on receptor binding. The triple mutant K74A/H75A/N77A exhibited properties that were consistent with the combined effects of the individual mutations, namely impaired binding to p75 without deficits in its interaction with TrkA. In contrast, in the triple mutant D72A/S73A/K74A, the simultaneous replacement of Asp-72 and Lys-74 with alanine had a compensatory effect such that binding to both p75 and TrkA was comparable to that of wild-type NGF, despite the deficits seen in the individual replacements. This molecule, however, was produced at low levels, and its biological activity in sympathetic ganglion explants was reduced, which, together with results from TrkA phosphorylation assays, indicated a reduced stability during prolonged culture conditions. Taken together, these data reveal a second region of interaction with the p75 receptor in NGF with the positively charged residues Lys-74 and His-75 as candidate points of contact. In addition, Asp-72 appears to be a structurally important side chain for stabilizing the conformation of the loop through interactions with neighboring residues.

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Structure-function studies of nerve growth factor: functional importance of highly conserved amino acid residues.

Cited by 67 publications

References 47 publications

Identification of Critical Residues within the Conserved and Specificity Patches of Nerve Growth Factor Leading to Survival or Differentiation

Identification of Critical Residues within the Conserved and Specificity Patches of Nerve Growth Factor Leading to Survival or Differentiation

Nerve growth factor: structure and function

A Second Determinant of Binding to the p75 Neurotrophin Receptor Revealed by Alanine-scanning Mutagenesis of a Conserved Loop in Nerve Growth Factor

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