Identification of Critical Residues within the Conserved and Specificity Patches of Nerve Growth Factor Leading to Survival or Differentiation
| Autor: | Kenneth E. Neet, Sidharth Mahapatra, Hrishikesh M. Mehta, Sang B. Woo |
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| Rok vydání: | 2009 |
| Předmět: |
Models
Molecular Neurite Cell Survival MAP Kinase Signaling System Cellular differentiation Cell Spodoptera Biology Tropomyosin receptor kinase A PC12 Cells Receptor Nerve Growth Factor Biochemistry Cell Line Nerve Growth Factor Neurites medicine Animals Amino Acid Sequence Amino Acids Receptor trkA Fibroblast Molecular Biology Protein kinase B Binding Sites Dose-Response Relationship Drug Mechanisms of Signal Transduction Cell Differentiation Cell Biology Fibroblasts Surface Plasmon Resonance Molecular biology Recombinant Proteins Protein Structure Tertiary Rats Cell biology medicine.anatomical_structure Nerve growth factor nervous system Cell culture Mutagenesis Site-Directed Mutant Proteins Protein Binding |
| Zdroj: | Journal of Biological Chemistry. 284:33600-33613 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m109.058420 |
| Popis: | Afflicted neurons in Alzheimer disease have been shown to display an imbalance in the expression of TrkA and p75(NTR) 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 by90%, while concurrently retaining near wild-type survival activity in MG139 (TrkA(+) only) and PCNA fibroblast (p75(NTR+)-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 EC(50) 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. |
| Databáze: | OpenAIRE |
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