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Effects of Alanine‐Scanning Mutagenesis of the Variable Regions of Nerve Growth Factor on Survival, Differentiation, and Intracellular Signaling

Neurotrophins enable neurons to survive, grow, interact, and differentiate through specific high affinity interactions with a family of tyrosine kinase receptors and the p75NTR common receptor. Over a decade of mutagenesis data have clearly implicated the N‐terminus, C‐terminus, and variable regions I‐V in playing crucial roles in the interaction between nerve growth factor (NGF) and its receptor, TrkA. Crystallographic data augment these findings by identifying two distinct patches, a conserved patch and a specificity patch, establishing contacts with the TrkA‐d5 region. However, no study has concurrently looked at all 7 regions to determine whether certain residues play a more discriminating role than others in receptor activation for discrete signal transduction pathways. The current study aimed at using alanine‐scanning mutagenesis to synthesize point mutants, spanning the N‐terminus, variable regions I–V, and C‐terminus of NGF, for the assessment of distinct effects on survival, differentiation, and intracellular signaling in three separate cell lines, i.e. PC12 cells (TrkA+/p75+), MG139 cells (TrkA+/p75−), and PCNA cells (TrkA−/p75+). Our data show a consistent decrease in neuritogenesis in PC12 cells, with the greatest reduction seen in F7A, H84A, and R103A, i.e. up to an 18% decrease. In contrast, survival in MG139 cells and death in PCNA cells were clearly comparable to wild‐type NGF across mutants. This strengthens our assessment that minor alterations to the NGF backbone can selectively lead to discernible changes in differentiation, while leaving cellular survival unperturbed, leading to a possibility of key mutations along these regions producing a signaling mechanism‐specific neurotrophin.