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First published online 6 January 2009
doi: 10.1242/jcs.034058

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Subcellular localization and dimerization of APLP1 are strikingly different from APP and APLP2

Daniela Kaden¹, Philipp Voigt^2,*, Lisa-Marie Munter¹, Karolina D. Bobowski¹, Michael Schaefer^2,* and Gerd Multhaup^1,

¹ Institut für Chemie und Biochemie, Freie Universität Berlin, 14195 Berlin, Germany
² Molekulare Pharmakologie und Zellbiologie, Neurowissenschaftliches Forschungszentrum, Charité-Universitätsmedizin Berlin, 14195 Berlin, Germany

View larger version (64K): [in this window] [in a new window]   Fig. 1. Subcellular localization of APP family proteins. Human APLP1, APLP2 and APP full-length proteins were C-terminally fused to YFP or CFP. (A) Schematical view. The respective plasmids were transiently transfected in HEK293 cells either alone (B) or in combination with one of the other APP family members (C). Note that APLP1 accumulates in intracellular compartments when coexpressed with APP or APLP2 (C, first two panels compared to APLP1 alone). (D) Cell surface biotinylation of APLP1 upon APP and APLP2 coexpression. HEK293 cells were transiently co-transfected with the indicated constructs and cell surface proteins were labeled by sulfo-NHS-SS-biotin. Shown is the total (lysate) and the cell-surface-localized APLP1-YFP of two different transfections. Calnexin was used as a loading control and to verify the absence of endomembranes from biotinylation. Molecular weight standard is indicated on the right. APLP1 was stained with a polyclonal APLP1-specific antibody (42464). (E) APLP1-YFP is enriched in cell-cell contacts. Additional images are shown in supplementary material Fig. S4. (B,C,E) Cells were imaged by cLSM 1 day after transfection. Representative images of at least three independent transfections are shown. (F) Analysis of dimer/oligomer formation in trans direction. APP, APLP1 and APLP2 fused to either FLAG or YFP tags were expressed separately in HEK293 cells and then the cells were mixed 1 day before harvesting. Co-immunoprecipitations were carried out with the FLAG antibody and co-purified proteins were detected by GFP antibody. Molecular weight standard is indicated on the right. (G) Velocity of APLP1-YFP lateral plasma membrane diffusion was determined by FRAP analysis in areas of cell-cell contacts and non-contact sites. Bars, 10 µm. *, P<0.001, Wilcoxon test; C, control IP without antibody; FL, full-length; LC, lysates from control; LIP, lysates from IP; IP, immunoprecipitation with FLAG antibody; nc, non-contact sites; PM, plasma membrane; SP, signal peptide.

View larger version (25K): [in this window] [in a new window]   Fig. 2. Homodimerization of APLP1 and APLP2. FRET efficiencies (E) in transiently transfected HEK293 cells were determined by measuring the recovery of CFP fluorescence during YFP photobleaching. Cells were excited at 410 and 515 nm for CFP and YFP detection, respectively. YFP was bleached by illumination at 512 nm for 2.1 seconds in each acquisition cycle. (A) Timecourse of donor fluorescence recovery (FCFP) during selective photobleaching of the acceptor (FYFP). Shown are single cells (gray lines) and mean fluorescence traces (black lines) of a representative measurement of APLP1-CFP and APLP1-YFP. (B) Linear regression analysis of donor recovery versus fractional acceptor photobleach to extrapolate the donor fluorescence in the absence of acceptor. Depicted is the mean of the experiment shown in A. (C,D) Black bars denote FRET efficiencies for APLP1-CFP and APLP1-YFP (C) as well as APLP2-CFP and APLP2-YFP (D). Open bars represent the same measurements but with increasing amounts of the respective FLAG-tagged fusion protein. As negative control APLP1-YFP was coexpressed with glycophorin A (GypA-CFP; C, gray bar) and APLP2-YFP was coexpressed with EGF-receptor-CFP (D, gray bar). Numbers represent the relative CFP and YFP fluorescence intensities in single cells expressing the respective fusion proteins that were determined and averaged ([YFP]r and [CFP]r) to verify comparable protein expression. Depicted are the means and s.e.m. of three to four independent transfections (with four measurements each on four to six cells).

View larger version (39K): [in this window] [in a new window]   Fig. 3. Homo- and heteromultimerization of APP family proteins. (A) FRET analysis of human APP, APLP1, APLP2 hetero- (black bars) and homomultimerization (gray bars). YFP- and CFP-tagged fusion proteins were coexpressed in HEK293 cells in the indicated combinations. The relative CFP and YFP fluorescence intensities in single cells expressing the respective fusion proteins were determined and averaged ([YFP]r and [CFP]r) to verify comparable protein expression. Depicted are means and s.e.m. of three independent transfections (n=4 with four to six cells each). Similar FRET efficiencies (E) were obtained if donor and acceptor were exchanged (data not shown). (B) Co-immunoprecipitations of human APP, APLP1 and APLP2. HEK293 cells were transfected with plasmids coding for FLAG-tagged APP, APLP1 and APLP2 and the corresponding YFP plasmids, as indicated. The FLAG fusion proteins were immunoprecipitated with anti-FLAG antibodies, and co-purified constructs were detected with protein-specific antibodies (IP: -FLAG) 42464 for APLP1, 8/1 for APLP2 and W0-2 for APP. As loading control, cell lysates were analyzed with M2-FLAG monoclonal antibody (load: FLAG) and the protein-specific antibodies (load). Note, the YFP tag leads to a molecular weight shift of the detected YFP-tagged APP/APLP forms by about 25 kD.

View larger version (19K): [in this window] [in a new window]   Fig. 4. Analysis of APP processing by sandwich ELISA under conditions of APLP coexpression. Full-length APP (FL-APP lysate) from lysates of APP-transfected cells or sAPP from conditioned media were captured using an anti-Myc antibody and detected by biotinylated W0-2. Aβ40 and Aβ42 were captured using specific C-terminal antibodies and detected by biotinylated W0-2. The respective control was set as 100% (black bars) (means ±s.e.m., n=3-5; gray bars indicate APLP1/APP, white bars APLP2/APP co-transfections). Asterisks indicate significant differences to control (*P<0.05 and **P<0.005, Student's t-test).

View larger version (34K): [in this window] [in a new window]   Fig. 5. Fluorescence imaging of N-terminal deletion mutants. (A) Schematic representation of N-terminal deletion mutants. For all deletion constructs the signal peptide sequence of APP was introduced in frame with the remaining domains. (B) Confocal imaging of the N-terminal deletion mutants. Either YFP- or CFP-tagged mutants were expressed in HEK293 and cells were imaged by cLSM 1 day after transfection. Representative images from at least three independent transfections are shown. For coexpression with the corresponding wild-type constructs see supplementary material Fig. S5. (C) Analysis of homointeractions of N-terminal deletion mutants in comparison to FL forms. The YFP- and CFP-fusion proteins were expressed in HEK293 cells and analyzed by FRET. The relative CFP and YFP fluorescence intensities in single cells expressing the respective fusion proteins were determined and averaged ([YFP]r and [CFP]r) to verify comparable protein expression. Depicted are the means and s.e.m. of three to four independent transfections (with four measurements each on three to five cells). Bars, 10 µm. Striped square: loop region. *P<0.05, **P<0.001, Student's t-test. FL, full-length.

View larger version (24K): [in this window] [in a new window]   Fig. 6. Heteromultimerization analysis of N-terminal deletion mutants with full-length APP family members. Either full-length variants or the deletion mutants GFLD, E1 and E1-AcD of APP (A) and APLP1 (B) were coexpressed with the corresponding FL protein and analyzed by FRET. Black bars indicate coexpression with APP-FL, dark gray bars with APLP1-FL and light gray bars with APLP2-FL proteins. The relative CFP and YFP fluorescence intensities in single cells expressing the respective fusion proteins were determined and averaged ([YFP]r and [CFP]r) to verify comparable protein expression. Depicted are the means and s.e.m. of three to four independent transfections (with four measurements each on three to five cells). FL, full-length.

View larger version (29K): [in this window] [in a new window]   Fig. 7. Analysis of APP loop mutants. (A) Loop peptide sequence (amino acids 86 to 116 of the GFLD) with (wild type) and without (mutant sequence) the disulfide bridge. Single mutants are denoted with loop-S1 and loop-S2, and the double mutant with loop-SD. (B) Confocal imaging of APP loop mutants. YFP-tagged mutants were expressed in HEK293 cells and imaged by cLSM 1 day after transfection. Representative images of at least three independent transfections are shown. (C) Cell surface biotinylation of APP loop mutants. HEK293 cells were transiently transfected with the APP loop mutants and cell surface proteins were labeled by sulfo-NHS-SS-biotin, purified via neutravidin agarose and analyzed by western blot. Shown are western blots of the whole-cell lysates and the neutravidin eluate (cell surface). Molecular weight standards are indicated on the right. (D) FRET analysis of APP loop mutants. The mutant or WT YFP- and CFP-fusion proteins were expressed in HEK293 cells in different combinations and analyzed by FRET. Note that the presence of one loop is not sufficient to rescue the impaired dimerization (second bar versus third and fourth bar). The relative CFP and YFP fluorescence intensities in single cells expressing the respective fusion proteins were determined and averaged ([YFP]r and [CFP]r) to verify comparable protein expression. Depicted are the means and s.e.m. of three to four independent transfections (with four measurements each on three to five cells). Bars, 10 µm. WT, wild type.

View larger version (25K): [in this window] [in a new window]   Fig. 8. Schematic representation of APP-, APLP2- and APLP1-cis dimerization. The E1 domain mediates homo- and heterophilic interactions of APP and APLP2 mainly through the GFLD, especially the loop region (striped square) as indicated by the arrows. By contrast, the GFLD is dispensable for dimerization of APLP1. The E2 domain of APLP1 substitutes the function of the E1 domain in initiating the interaction. However, for APP and APLP2 dimerization of the E1 domain is required to initiate the contact, which is stabilized by the E2 domain. The model of the E2 domain has been modified according to (Wang and Ha, 2004). PM, plasma membrane.

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