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

• Published in: The Journal of biological chemistry 2009-11, Vol.284 (48), p.33600-33613
• Main Authors: Mahapatra, Sidharth, Mehta, Hrishikesh, Woo, Sang B., Neet, Kenneth E.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino Acids - chemistry; Amino Acids - genetics; Amino Acids - metabolism; Animals; Binding Sites - genetics; Cell Differentiation - drug effects; Cell Differentiation - physiology; Cell Line; Cell Survival - drug effects; Cell Survival - physiology; Dose-Response Relationship, Drug; Fibroblasts - cytology; Fibroblasts - drug effects; Fibroblasts - metabolism; MAP Kinase Signaling System; Mechanisms of Signal Transduction; Models, Molecular; Mutagenesis, Site-Directed; Mutant Proteins - chemistry; Mutant Proteins - metabolism; Mutant Proteins - pharmacology; Nerve Growth Factor - genetics; Nerve Growth Factor - metabolism; Nerve Growth Factor - pharmacology; Neurites - drug effects; Neurites - physiology; PC12 Cells; Protein Binding; Protein Structure, Tertiary; Rats; Receptor, Nerve Growth Factor - chemistry; Receptor, Nerve Growth Factor - metabolism; Receptor, trkA - chemistry; Receptor, trkA - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Recombinant Proteins - pharmacology; Spodoptera; Surface Plasmon Resonance
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M109.058420

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.