APP binds to the EGFR ligands HB-EGF and EGF, acting synergistically with EGF to promote ERK signaling and neuritogenesis

Abstract

The amyloid precursor protein (APP) is a transmembrane glycoprotein central to Alzheimers disease (AD) with functions in brain development and plasticity, including in neurogenesis and neurite outgrowth. Epidermal growth factor (EGF) and heparin-binding EGF-like growth factor (HB-EGF) are well described neurotrophic and neuromodulator EGFR ligands, both implicated in neurological disorders like Schizophrenia and AD. Here we show that APP interacts with these two EGFR ligands and characterize the effects of APP-EGF interaction in ERK activation and neuritogenesis. HB-EGF was identified as a novel APP interactor in a yeast two-hybrid screen of a human brain cDNA library. Yeast co-transformation and co-immunoprecipitation assays confirmed APP interaction with HB-EGF. Moreover, co-immunoprecipitation also revealed that APP binds to cellular pro-EGF. Overexpression of HB-EGF in HeLa cells, or exposure of SH-SY5Y cells to EGF, both resulted in increased APP protein levels. EGF and APP were also observed to synergistically activate the ERK signaling pathway, crucial for early neuronal differentiation. Immunofluorescence analysis of cellular neuritogenesis in conditions of APP overexpression and EGF exposure, confirmed a synergistic effect in promoting the number and the mean length of neurite-like processes per cell. Synergistic ERK activation and neuritogenic effects were completely blocked by the EGFR inhibitor PD 168393, implying EGF-induced activation of EGFR as part of the mechanism. This work shows novel APP protein interactors and provides a major insight into the APP-driven mechanisms underlying neurite outgrowth and neuronal differentiation, with potential relevance for AD and for adult neuroregeneration.