The amyloid precursor protein (APP) is a transmembrane glycoprotein central to Alzheimer's 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 coimmunoprecipitation assays confirmed APP interaction with HB-EGF. Moreover, coimmunoprecipitation 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.

The amyloid precursor protein (APP) is a transmembrane glycoprotein central to Alzheimer's 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 coimmunoprecipitation assays confirmed APP interaction with HB-EGF. Moreover, coimmunoprecipitation 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.Keywords: APP interactors and signaling, HB-EGF and EGF ligands, EGFR activation, ERK signaling, neuronal differentiation and regeneration heparin-binding domain, and a diphtheria toxin binding domain; these last two are distinct features of HB-EGF, not present in EGF [17,18]. Importantly, both EGF and HB-EGF soluble and membrane-bound forms (their precursor proteins) have been described as biologically active, with membrane-bound forms competing with mature proteins for receptor binding [19].Both EGF and HB-EGF bind directly to the epidermal growth factor receptor (EGFR; also known as ERBB1 or HER1), and HB-EGF can additionally bind ERBB4 (or HER4) [20].Binding of the mature growth factor to EGFR induces receptor dimerization and autophosphorylation of the receptor tyrosine residues, which in turn recruits phosphotyrosine binding proteins and initiates different signaling events. The major pathways activated by EGFR include Ras stimulation of the RAF-MEK-ERK1/2 cascade, PI3K stimulation of Akt and PLC-gamma, and JAK stimulation of the STAT pathway [21]. EGF and HB-EGF are well described mitogens for multiple cell lines, ranging from epithelial cells and fibroblasts to neural stem cells [22,23]. Given their neurotrophic and neuromodulatory roles, these molecules also promote cell differentiation, survival and pl...