Visualization of APP dimerization and APP-Notch2 heterodimerization in living cells using bimolecular fluorescence complementation.
We previously demonstrated that the amyloid precursor protein (APP) interacts with Notch receptors. Here, we confirmed the APP/Notch1 endogenous interaction in embryonic day 17 rat brain tissue, suggesting the interaction was not as a result of over-expression artifacts. To investigate potential homodimeric and heterodimeric interactions of APP and Notch2 (N2), we have visualized the subcellular localization of the APP/N2 complexes formed in living cells using bimolecular fluorescence complementation (BiFC) analysis. BiFC was accomplished by fusing the N-terminal fragment or the C-terminal fragment of yellow fluorescent protein (YFP) to APP, N2, and a C-terminally truncated form of N2. When expressed in COS-7 cells, these tagged proteins alone did not produce a fluorescent signal. The tagged APP homodimer produced a weak fluorescent signal, while neither full-length N2, nor a truncated N2 alone, produced a visible signal, suggesting that N2 receptors do not form homodimers. The strongest fluorescent signal was obtained with co-expression of the C-terminal fragment of YFP fused to APP and the N-terminal fragment of YFP fused to the truncated form of N2. This heterodimer localized to plasma membrane, endoplasmic reticulum (ER), Golgi and other compartments. The results were confirmed and quantified by flow cytometry. The BiFC method of specifically visualizing APP/Notch interactions can be applied to study APP and Notch signaling during development, aging and neurodegeneration.
Chen
Ci-Di
CD
Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Oh
Sun-Young
SY
Hinman
Jason D
JD
Abraham
Carmela R
CR
D000544:Alzheimer Disease / Q000378:metabolism / Q000503:physiopathology; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D001921:Brain / Q000378:metabolism*; D019556:COS Cells; D002451:Cell Compartmentation / Q000276:immunology; D002462:Cell Membrane / Q000276:immunology; D002522:Chlorocebus aethiops; D019281:Dimerization; D005455:Fluorescent Antibody Technique / Q000379:methods*; D005456:Fluorescent Dyes / Q000138:chemical synthesis; D007425:Intracellular Membranes / Q000276:immunology; D008164:Luminescent Proteins / Q000737:chemistry; D009474:Neurons / Q000378:metabolism*; D015388:Organelles / Q000276:immunology; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D051883:Receptor, Notch2 / Q000378:metabolism*; D015398:Signal Transduction / Q000276:immunology; D013194:Staining and Labeling / Q000379:methods*; D013347:Subcellular Fractions
D016564:Amyloid beta-Protein Precursor; D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; C497764:NOTCH2 protein, human; D051883:Receptor, Notch2; C054562:yellow fluorescent protein, Bacteria