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Design and characterization of a nerve growth factor (NGF) mutein that favors survival over differentiation
Author(s) -
Mehta Hrishikesh,
Mahapatra Sidharth,
Neet Kenneth E
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.703.17
Subject(s) - nerve growth factor , phosphorylation , tropomyosin receptor kinase a , protein kinase b , neurotrophin , mapk/erk pathway , microbiology and biotechnology , chemistry , biology , medicine , receptor , biochemistry
Neurotrophins are required by neurons for differentiation and survival. NGF is involved in survival of the cholinergic neurons of the basal forebrain which are involved in the pathogenesis of Alzheimer's disease. We previously designed a mutein of NGF (F7A, H84A, R103A) which has a lower potential than wtNGF with respect to neuritogenesis in PC12 cells, but supports cell survival. Earlier experiments were performed with unpurified protein from Sf21 insect cell supernatant. We now report the survival and signaling selectivity of purified 7‐84‐103 mutein. NGF initiates signaling through TrkA with downstream signals for differentiation via MAPK phosphorylation and survival via both MAPK and Akt phosphorylation. We compared the phosphorylation of TrkA, MAPK, and Akt with that of wtNGF and another NGF mutein, ▵9/13 that does not bind TrkA. Our data show insignificant differences in the phosphorylation of TrkA and MAPK between buffer and 7‐84‐103 or ▵9/13 (2nM). However, 7‐84‐103 shows significantly higher levels of Akt phosphorylation, about 40% of wtNGF. At 10nM, the 7‐84‐103 mutein shows equivalent levels of phosphorylation of TrkA, MAPK, and Akt compared to 1nM NGF. Thus at moderate concentrations, the 7‐84‐103 NGF mutein supports survival via increased activation of the Akt pathway over the MAPK pathway but, at higher concentrations the mutein signals through both like wtNGF. (Supported by USPHS Grant NS 24380)