Neuronal Fe65 is a central adapter for the intracellular protein network of Alzheimer’s disease related amyloid precursor protein (APP). It contains a unique tandem array of phosphotyrosine-binding (PTB) domains that recognize NPXY internalization motifs present in the intracellular domains of APP (AICD) and the low-density lipoprotein receptor-related protein LRP1 (LICD). The ternary APP/Fe65/LRP1 complex is an important mediator of APP processing and affects β-amyloid peptide production. Here we dissect by biochemical and biophysical methods the direct interactions within the ternary complex and reveal a phosphorylation-dependent insulin receptor substrate (IRS-) like interaction of the distal NPVY4507 motif of LICD with Fe65-PTB1. Structured summary of protein interactionsAPP-AICD and FE65 bind by nuclear magnetic resonance (View interaction)Src phosphorylates LRP1-LICD by protein kinase assay (View interaction)LRP1-LICD physically interacts with FE65 and APP-AICD by pull down (View interaction)FE65-PTB1 and LRP1-LICD bind by nuclear magnetic resonance (View interaction)LRP1-LICD binds to FE65-PTB1 by pull down (View interaction)APP-AICD binds to FE65-PTB2 by pull down (View interaction)