PMC

Maturation of BRI2 and APP is necessary for their interaction. (A) Schematic representation of Flag-BRI2-myc, which has a Flag tag at the N-terminus and a myc tag at the C-terminus. (B) HeLa cells transfected with APP and Flag-BRI2-myc were immunoprecipitated with anti-HA, anti-Flag, rabbit control (RP) or anti-APPct antibodies. Mouse monoclonal anti-HA and rabbit polyclonal (RP) were used as negative controls for mouse monoclonal αFlag and αAPPct in immunoprecipitation experiments. (C). HeLa cells were transfected as in (B). The total lysates were immunoprecipitated with anti-HA, anti-myc, RP and αAPPct. APP and Flag-BRI2-myc were detected with 22C11 and anti-Flag immunoblots, respectively.

BRI2 inhibits APP processing on the plasma membrane and in endocytic vesicles. (A) HeLa cells transfected with APP or APP plus Flag-BRI2 were biotinylated, incubated at 37 °C for the indicated times, lysed and precipitated with streptavidin beads. The precipitants were probed with either αAPPct, which recognize both APP CTFs C83 and C99. (B) Quantification of C99 and C83 levels of the experiment shown in A. The peak point of C83 was defined as 100 s. (C) Cells were transfected, incubated at 37 °C for indicated times. The cell lysates and culture supernatants were precipitated with streptavidin beads as in A. The lysate precipitants were probed for APP, APP CTFs (C99 and C83), C99 (with 6E10 that only recognizes C99), and BRI2. The media precipitants were probed for sAPPα and sAPPβ. (D) Quantification of sAPPα and sAPPβ. The maximum point of each sAPPα and sAPPβ were set to 100.

A model depicting the anti-amyloidogenic functions of BRI2. N-glycosylated () imAPP localizes in the endoplasmic reticulum (ER) and cis-Golgi. N- and O-glycosylated () mAPP resides in compartments following trans-Golgi and on the plasma membrane (). Immature BRI2 is processed by a convertase in the secretory pathway at the carboxyl-terminus between R243 and E244 (; ) to generate the soluble Bri2–23 peptide and a membrane-bound mBRI2. APP and BRI2 interact only after both molecules have matured. While mBRI2 inhibits processing of APP by all secretases (reducing therefore every APP metabolite including Aβ), Bri2–23 can antagonize formation of Aβ oligomers. The absence of BRI2, like in Bri2 KO mice, results in increased levels of APP metabolites (upper left panel). Molecules and molecules regions are not drawn to scale.

BRI2 does not alter APP trafficking and only mBRI2 is transported to cell membranes. (A) Schematic drawing of wild type BRI2. Immature BRI2 (imBRI2) is cleaved by a furin-like convertase to mature BRI2 (mBRI2). The cleavage releases a C-terminal 23 amino acid peptide (Bri2–23) from imBRI2. Cytoplasmic, luminal, and BRICHOS (B) domains are indicated. (B) HeLa cells were transfected with Flag-BRI2 or APP as indicated. Cell surface proteins were biotinylated and precipitated with streptavidin beads. Immunoblot analysis of total lysate (T), unbound (U), and bound (B) proteins with 22C11, anti-Flag and αAPPct antibodies. α-Tubulin was not found in the bound fraction demonstrating that intracellular proteins were not biotinylated. (C) Iodixanol gradient fractionation shows that imAPP is concentrated in ER fractions (where the ER-resident protein calnexin fractionates) while mAPP is found in the Golgi complex fraction (marked by the Golgi complex marker GM130) and early endosomal fractions (where EEA1 is enriched). BRI2 expression does not alter APP localization (second panel from the top).

Efficient export of BRI2 out of ER is required to inhibit APP processing. (A) Schematic representation of Flag-BRI2FR (K242 and R243 are replaced by alanines). (B) Pulse-chase experiments of HeLa cells transfected with either Flag-BRI2 or Flag-BRI2FR. (C) HeLa cells were transfected with Flag-BRI2FR, surface biotinylated, lysed and the lysates were precipitated with streptavidin, as in . (D) BRI2 localizes to the cell surface and displays no overlap with the ER marker, ER-GFP. In contrast, BRI2D23 accumulates intracellularly and exhibits colocalization with ER-GFP. HEK cells were transfected with Flag-BRI2 or Flag-BRI2Δ23 together with ER-GFP, and stained with anti-Flag. Scale bar = 10 μm. (E) BRI2Δ23 is poorly presented on the cell surface. HeLa cells were transfected with Flag-BRI2 or Flag-BRI2Δ23. After surface biotinylation and lysis, cell lysates were precipitated with streptavidin beads. T, U and B fractions were probed for α-tubulin and BRI2 or BRI2Δ23 by immunoblot with DM1A and anti-Flag, respectively. α-Tubulin was not biotinylated. Eight times more sample was loaded in the B fractions as compared to T and U fractions. (F) HeLa cells were transfected with Flag-BRI2 or Flag-BRI2FR together with APP. The cell lysates were immunoprecipitated with the indicated antibodies. Total lysates (T) and precipitants were probed for APP, BRI2, and APP CTFs with 22C11, anti-Flag, and αAPPct, respectively. (G) HeLa cells were transfected with pcDNA3, Flag-BRI2, or Flag-BRI2Δ23, together with APP. The total cell lysates (T) and anti-Flag immunoprecipitates (anti-Flag IP) were probed for APP (22C11), BRI2 (anti-Flag), and APP CTF (αAPPct) by immunoblots.

mBRI2 and mAPP interact on the plasma membrane and in endocytic vesicles. (A) HeLa cells transfected with APP or APP plus Flag-BRI2 were surface biotinylated. The total lysates (T) were immunoprecipitated with anti-Flag, and eluted with the Flag peptide. The eluate (E) was further precipitated with streptavidin beads, and unbound (U) and bound (B) fractions were collected, and probed for APP, APP CTFs, and BRI2. Material loaded in the B fraction is eight times more than the E and the U fraction. (B) The experiments were performed as in A except that after labeling with biotin, cells were incubated at 37 °C for the indicated times and the biotin remaining on the surface was removed with glutathione after the incubation and before the cells were lysed. The efficacy of biotin removal is confirmed by the absence of mAPP in the B fraction at time 0. (C) Mouse dermal fibroblasts were prepared from the tail skin of the mice that have the indicated genotypes. The total lysates (T) were prepared and immunoprecipitated with an anti-BRI2 antibody. APP, APP CTFs, BRI2, and calnexin in the total lysates and precipitants were detected in western blots with 22C11, αAPPct, anti-BRI2, and anti-calnexin. Calnexin is used as a loading control.