title: Expression and processing of fluorescent fusion proteins of amyloid precursor protein (APP).
abstract: Processing of β-amyloid precursor protein (APP) by β- and γ-secretases in neurons produces amyloid-β (Aβ), whose excess accumulation leads to Alzheimer's disease (AD). Knowledge on subcellular trafficking pathways of APP and its fragments is important for the understanding of AD pathogenesis. We designed fusion proteins comprising a C-terminal fragment of APP (app) and fluorescent proteins GFP (G) and DsRed (D) to permit the tracking of the fusion proteins and fragments in cells. CAD cells expressing these proteins emitted colocalized green and red fluorescence and produce ectodomains, sGapp and sRapp, and Aβ, whose level was reduced by inhibitors of β- and γ-secretases. The presence of GappR in endosomes was observed via colocalization with Rab5. These observations indicated that the fusion proteins were membrane inserted, transported in vesicles and proteolytically processed by the same mechanism for APP. By attenuating fusion protein synthesis with cycloheximide, individual fluorescent colors from the C-terminus of the fusion proteins appeared in the cytosol which was strongly suppressed by β-secretase inhibitor, suggesting that the ectodomains exit the cell rapidly (t1/2 about 20min) while the C-terminal fragments were retained longer in cells. In live cells, we observed the fluorescence of the ectodomains located between parental fusion proteins and plasma membrane, suggesting that these ectodomain positions are part of their secretion pathway. Our results indicate that the native ectodomain does not play a decisive role for the key features of APP trafficking and processing and the new fusion proteins may lead to novel insights in intracellular activities of APP.
authors.affiliation: Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA. Kathleen-coughlan@ouhsc.edu
authors.forename: Kathleen
authors.initials: K
authors.lastname: Coughlan
authors.forename: Xiangping
authors.initials: X
authors.lastname: Huang
authors.forename: Xiangyuan
authors.initials: X
authors.lastname: He
authors.forename: Charlotte H Y
authors.initials: CH
authors.lastname: Chung
authors.forename: Guangpu
authors.initials: G
authors.lastname: Li
authors.forename: Jordan
authors.initials: J
authors.lastname: Tang
mesh_terms: D016564:Amyloid beta-Protein Precursor / Q000378:metabolism*; D000686:Amyloidosis / Q000378:metabolism / Q000473:pathology*; D000818:Animals; D015153:Blotting, Western; D002470:Cell Survival; D002478:Cells, Cultured; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism*; D051379:Mice; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013347:Subcellular Fractions; D000097573:Red Fluorescent Protein
chemical_list: D016564:Amyloid beta-Protein Precursor; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein