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P2‐142: Analysis of the dimerization and metabolism of different APP isoforms
Author(s) -
Ben Khalifa Naouel,
Constantinescu Stefan N.,
Tasiaux Bernadette,
Van Hees Joanne,
Courtoy Pierre J.,
Vandersmissen Patrick,
Renauld JeanChristophe,
Smith Steven O.,
Octave JeanNoël,
KienlenCampard Pascal
Publication year - 2009
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1016/j.jalz.2009.04.453
Subject(s) - bimolecular fluorescence complementation , förster resonance energy transfer , green fluorescent protein , gene isoform , chemistry , transmembrane domain , fluorescence , ectodomain , complementation , microbiology and biotechnology , biophysics , biochemistry , hek 293 cells , protein–protein interaction , transmembrane protein , biology , gene , receptor , mutant , physics , quantum mechanics
a transport adaptor of the Golgi-localized gamma-ear-containing ARFbinding (GGA) family, has been shown to interact with BACE via its VHS-domain. We analyzed the differential roles of GGA1, GGA2 and GGA3 upon co-localization and interaction with BACE and effects upon APP-processing. In addition we tested the hypothesis that serine-phosphorylation of GGA1 and GGA3 affects BACE-interaction. Methods: We applied confocal imaging and fluorescence lifetime imaging microscopy (FLIM) for colocalization and interaction studies, an electrochemiluminescence-based assay to measure the influence of all three GGA upon APP processing, westernblotting to measure intracellular levels of APP cleavage fragments and insitu-hybridization to visualize correlated expression of GGAs and BACE in rat brain. Results: All three GGAs colocalize with BACE1 at perinuclear compartments. FLIM revealed a donor-lifetime decrease indicating interaction between all three GGAs and BACE. Control experiments with GGA and BACE mutants show that the VHS domain of the GGA proteins and the DXXLL-motive in the BACE protein are necessary for this interaction. Mutants of GGA1 and GGA3 which represent non-phosphorylated forms of these proteins showed decreased lifetime whereas autoinhibited pseudo-phosphorylated mutants reversed this. Elisa and Westernblotting revealed an increase of intracellular sAPP upon overexpression of any GGA. However a decrease in sAPP secretion was observed. This effect was neither reversible with D-VHS mutants nor with GGA1/3 phosphorylation-mutants.Insitu-hybridization revealed spatial and time correlated expression of GGAs and BACE in postnatal and adult rats. Conclusions: These results indicate that all GGAs have related functions on BACE interaction, controlled by concentration, location and phosphorylation. Beside the interaction with BACE, we suggest additional BACE-independent influence of GGAs on APP transport and processing, and therefore an essential role in APP cleavage and subsequent Abeta generation.