Identification of critical residues within the conserved and specificity patches of nerve growth factor leading to survival or differentiation

Sidharth Mahapatra, Hrishikesh Mehta, Sang B. Woo, Kenneth E. Neet

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

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+/p75NTR+) cells by >90%, while concurrently retaining near wild-type survival activity in MG139 (TrkA+ only) and PCNA fibroblast (p75NTR-only) cells. Additionally, survival in both naive and terminally differentiated PC12 cells was shown to be intermediate between NGF and negative controls. Dose-response curves with 7-84-103 showed that the differentiation curve was shifted by about 100-fold, whereas the EC50 for survival was only increased by 3.3-fold. Surface plasmon resonance analysis revealed a 200-fold decrease in binding of 7-84-103 to TrkA. The retention of cell survival was attributed to maintenance of signaling through the Akt survival pathway with reduced MAPK signaling for differentiation. The effect of key mutations along the NGF receptor interface are transmitted inside the cell to enable the generation of survival-selective recombinant NGF muteins that may represent novel pharmacologic lead agents for the amelioration of Alzheimer disease.

Original languageEnglish (US)
Pages (from-to)33600-33613
Number of pages14
JournalJournal of Biological Chemistry
Volume284
Issue number48
DOIs
StatePublished - Nov 27 2009

Fingerprint

Nerve Growth Factor
Alzheimer Disease
Nerve Growth Factor Receptor
Mutagenesis
Surface Plasmon Resonance
PC12 Cells
Proliferating Cell Nuclear Antigen
Surface plasmon resonance
Neurites
Fibroblasts
Point Mutation
Neurons
Cell Survival
Cells
Maintenance
Ligands
Mutation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Identification of critical residues within the conserved and specificity patches of nerve growth factor leading to survival or differentiation. / Mahapatra, Sidharth; Mehta, Hrishikesh; Woo, Sang B.; Neet, Kenneth E.

In: Journal of Biological Chemistry, Vol. 284, No. 48, 27.11.2009, p. 33600-33613.

Research output: Contribution to journalArticle

@article{bc868b93b3164e6796f40021fd2ba8e3,
title = "Identification of critical residues within the conserved and specificity patches of nerve growth factor leading to survival or differentiation",
abstract = "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+/p75NTR+) cells by >90{\%}, while concurrently retaining near wild-type survival activity in MG139 (TrkA+ only) and PCNA fibroblast (p75NTR-only) cells. Additionally, survival in both naive and terminally differentiated PC12 cells was shown to be intermediate between NGF and negative controls. Dose-response curves with 7-84-103 showed that the differentiation curve was shifted by about 100-fold, whereas the EC50 for survival was only increased by 3.3-fold. Surface plasmon resonance analysis revealed a 200-fold decrease in binding of 7-84-103 to TrkA. The retention of cell survival was attributed to maintenance of signaling through the Akt survival pathway with reduced MAPK signaling for differentiation. The effect of key mutations along the NGF receptor interface are transmitted inside the cell to enable the generation of survival-selective recombinant NGF muteins that may represent novel pharmacologic lead agents for the amelioration of Alzheimer disease.",
author = "Sidharth Mahapatra and Hrishikesh Mehta and Woo, {Sang B.} and Neet, {Kenneth E.}",
year = "2009",
month = "11",
day = "27",
doi = "10.1074/jbc.M109.058420",
language = "English (US)",
volume = "284",
pages = "33600--33613",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "48",

}

TY - JOUR

T1 - Identification of critical residues within the conserved and specificity patches of nerve growth factor leading to survival or differentiation

AU - Mahapatra, Sidharth

AU - Mehta, Hrishikesh

AU - Woo, Sang B.

AU - Neet, Kenneth E.

PY - 2009/11/27

Y1 - 2009/11/27

N2 - 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+/p75NTR+) cells by >90%, while concurrently retaining near wild-type survival activity in MG139 (TrkA+ only) and PCNA fibroblast (p75NTR-only) cells. Additionally, survival in both naive and terminally differentiated PC12 cells was shown to be intermediate between NGF and negative controls. Dose-response curves with 7-84-103 showed that the differentiation curve was shifted by about 100-fold, whereas the EC50 for survival was only increased by 3.3-fold. Surface plasmon resonance analysis revealed a 200-fold decrease in binding of 7-84-103 to TrkA. The retention of cell survival was attributed to maintenance of signaling through the Akt survival pathway with reduced MAPK signaling for differentiation. The effect of key mutations along the NGF receptor interface are transmitted inside the cell to enable the generation of survival-selective recombinant NGF muteins that may represent novel pharmacologic lead agents for the amelioration of Alzheimer disease.

AB - 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+/p75NTR+) cells by >90%, while concurrently retaining near wild-type survival activity in MG139 (TrkA+ only) and PCNA fibroblast (p75NTR-only) cells. Additionally, survival in both naive and terminally differentiated PC12 cells was shown to be intermediate between NGF and negative controls. Dose-response curves with 7-84-103 showed that the differentiation curve was shifted by about 100-fold, whereas the EC50 for survival was only increased by 3.3-fold. Surface plasmon resonance analysis revealed a 200-fold decrease in binding of 7-84-103 to TrkA. The retention of cell survival was attributed to maintenance of signaling through the Akt survival pathway with reduced MAPK signaling for differentiation. The effect of key mutations along the NGF receptor interface are transmitted inside the cell to enable the generation of survival-selective recombinant NGF muteins that may represent novel pharmacologic lead agents for the amelioration of Alzheimer disease.

UR - http://www.scopus.com/inward/record.url?scp=70450250025&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=70450250025&partnerID=8YFLogxK

U2 - 10.1074/jbc.M109.058420

DO - 10.1074/jbc.M109.058420

M3 - Article

C2 - 19762468

AN - SCOPUS:70450250025

VL - 284

SP - 33600

EP - 33613

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 48

ER -