ABSTRACT
APP (amyloid precursor protein) and LRP1 (low-density lipoprotein receptor-related protein 1) have been implicated in the pathogenesis of AD (Alzheimer's disease). They are functionally linked by Fe65, a PTB (phosphotyrosine-binding)-domain-containing adaptor protein that binds to intracellular NPxY-motifs of APP and LRP1, thereby influencing expression levels, cellular trafficking and processing. Additionally, Fe65 has been reported to mediate nuclear signalling in combination with intracellular domains of APP and LRP1. We have previously identified another adaptor protein, GULP1 (engulfment adaptor PTB-domain-containing 1). In the present study we characterize and compare nuclear trafficking and transactivation of GULP1 and Fe65 together with APP and LRP1 and report differential nuclear trafficking of adaptors when APP or LRP1 are co-expressed. The observed effects were additionally supported by a reporter-plasmid-based transactivation assay. The results from the present study indicate that Fe65 might have signalling properties together with APP and LRP1, whereas GULP1 only mediates LRP1 transactivation.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cell Nucleus/metabolism , Cytoplasm/metabolism , Low Density Lipoprotein Receptor-Related Protein-1/metabolism , Adaptor Proteins, Signal Transducing/genetics , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Amyloid beta-Protein Precursor/chemistry , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Binding Sites , Humans , Low Density Lipoprotein Receptor-Related Protein-1/genetics , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Signal Transduction , Transcriptional Activation , TransfectionABSTRACT
Previous studies identified engulfment adapter phosphotyrosine binding (PTB) domain containing 1 (GULP1) as an NPXY-motif interactor of low-density lipoprotein receptor-related protein 1 (LRP1) and suggested a potential relevance in Alzheimer's disease (AD). Since AD associated proteins amyloid-ß A4 precursor protein (APP) and LRP1 were shown to interact with the PTB domain of Fe65 and several other adapters via their intracellular NPXY-motifs, we examined a possible interaction of GULP1 PTB domain with the YENPTY-motif of APP. Here we demonstrate that GULP1 is present in human hippocampal and neocortical neurons. Confocal live cell imaging revealed that coexpressed and endogenous GULP1 colocalizes with APP in the Golgi and endoplasmic reticulum. Analysis of the interacting domains by co-immunoprecipitation of point and deletion mutants revealed that the interaction depends on the PTB domain of GULP1 and the YENPTY-motif of APP. Coexpression of GULP1 affected APP cell surface localization and suppressed generation of Aß40/42 and sAPPα. Taken together, these data identify GULP1 as a novel neuronal APP interacting protein that alters trafficking and processing of APP.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Amyloid beta-Protein Precursor/metabolism , Gene Expression Regulation/physiology , Neurons/metabolism , Adaptor Proteins, Signal Transducing/genetics , Amino Acid Motifs/genetics , Amyloid Precursor Protein Secretases/genetics , Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/genetics , Animals , Aspartic Acid Endopeptidases/genetics , Aspartic Acid Endopeptidases/metabolism , Biotinylation , Cells, Cultured , Embryo, Mammalian , Endoplasmic Reticulum/metabolism , Gene Expression Regulation/genetics , Golgi Apparatus/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hippocampus/cytology , Humans , Immunoprecipitation , Low Density Lipoprotein Receptor-Related Protein-1/metabolism , Mice , Microscopy, Confocal , Neocortex/cytology , Neurons/ultrastructure , Peptide Fragments/metabolism , Protein Transport/genetics , Protein Transport/physiology , TransfectionABSTRACT
Cleavage of APP by BACE1 is the first proteolytic step in the production of amyloid-beta (Abeta), which accumulates in senile plaques in Alzheimer's disease. Through its interaction with APP, the low-density receptor-related protein 1 (LRP1) enhances APP internalization. Recently, BACE1 has been shown to interact with and cleave the light chain (lc) of LRP1. Since LRP1 is known to compete with APP for cleavage by gamma-secretase, we tested the hypothesis that LRP1 also acts as a competitive substrate for beta-secretase. We found that the increase in secreted APP (sAPP) mediated by over-expression of BACE1 in APP-transfected cells could be decreased by simultaneous LRP1 over-expression. Analysis by multi-spot ELISA revealed that this is due to a decrease in sAPPbeta, but not sAPPalpha. Interaction between APP and BACE1, as measured by immunoprecipitation and fluorescence lifetime assays, was impaired by LRP1 over-expression. We also demonstrate that APP over-expression leads to decreased LRP1 association with and cleavage by BACE1. In conclusion, our data suggest that--in addition to its role in APP trafficking--LRP1 affects APP processing by competing for cleavage by BACE1.