RanBP9 promotes the physical association of APP with LRP and BACE1 and facilitates APP association with buoyant detergent-resistant raft fractions. A, RanBP9 promotes the physical association of APP with LRP. In the upper panel, HEK293T cells were transfected with combinations of LRP-L4, APP, and FLAG-RanBP9 constructs, and equal protein amounts were immunoprecipitated (IP) for LRP-L4 (11H4) and immunoblotted for APP (CT15). In the lower three panels, equal protein amounts of cell lysates were directly immunoblotted for RanBP9 (M2), LRP-L4 (1704), and APP (CT15). B, RanBP9 promotes the physical association of APP with BACE1 in HEK293T cells. In the upper panel, HEK293T cells were co-transfected with combinations of GFP-BACE1, APP, and FLAG-RanBP9-FL or FLAG-RanBP9-Δ1, and equal protein amounts were immunoprecipitated for APP (IG7) and immunoblotted for BACE1 (B279). In the lower four panels, equal protein amounts of cell lysates were directly immunoblotted for APP (CT15), BACE1 (B279), and RanBP9 (M2). C, RanBP9 promotes the association of APP with lipid rafts. CHOAPP751 and CHOAPP751-RanBP9 stable cells were lysed in CHAPS buffer and subjected to discontinuous sucrose density gradient ultracentrifugation fractionation as previously reported (6). Equal volumes from each fraction were subjected to immunoblotting for full-length (FL) APP, CTFs, flotillin, and endogenous RanBP9. L indicates total lysate before fractionation.

RanBP9 physically associates with LRP and APP in mammalian cells and in brain. A, RanBP9 binds both LRP and APP, and LRP promotes RanBP9·APP interaction. HEK293T cells were triple-transfected with combinations of FLAG-RanBP9, APP, and LRP-L4 or LRP-L4 lacking the C37 domain (ΔC37). Equal protein amounts were immunoprecipitated (IP) with anti-FLAG (M2, Sigma) antibody and immunoblotted for APP (CT15) and LRP (1704) (upper two panels). In the lower three panels equal amounts of lysates were immunoblotted for APP (CT15), LRP (1704), and FLAG-RanBP9 (M2). B, endogenous RanBP9 physically associates with endogenous APP and LRP in vivo. Wild type mouse brain homogenates were prepared from frontal cortical regions in 2% CHAPS buffer and subjected to immunoprecipitation with three APP and three LRP antibodies as indicated or normal rabbit serum and anti-FLAG (M2) antibody as negative controls. Endogenous RanBP9 was detected with anti-RanBP9 antibody. In the bottom panels immunoprecipitated proteins were immunoblotted for either APP (CT15) or LRP (11H4).

SPRY-LisH domains of RanBP9 are sufficient to strongly interact with LRP, APP, and BACE1 and increase Aβ generation. A, schematic of domain organization of RanBP9 and FLAG-tagged deletion constructs: Δ1 (residues 1–392), Δ2, (residues 408–729), Δ3 (residues 1–107), and Δ4 (residues 1–254). B, SPRY-LisH domains bind strongly to LRP and APP. HEK293T cells were transiently co-transfected with APP or LRP-L4 together with vector control (VC), FLAG-tagged RanBP9, Δ1, Δ2, or Δ3 deletion mutants. Equal protein amounts were subjected to immunoprecipitation (IP) for FLAG-RanBP9 variants (M2) and immunoblotted for APP (CT15) or LRP (1704). Equal protein amounts from lysates were also directly immunoblotted for APP (CT15), LRP (1704), and FLAG (M2). Note that transient transfection of RanBP9-FL also yields an N-terminal 60-kDa fragment (N60), which comigrates with the RanBP9-Δ1 mutant (residues 1–392). C, BACE1 coimmunoprecipitates with RanBP9 and RanBP9-Δ1. In the upper panel, HEK293T cells were co-transfected with GFP-BACE1 and FLAG-RanBP9-FL or FLAG-RanBP9-Δ1, and equal protein amounts were immunoprecipitated for FLAG (M2) and immunoblotted for BACE1 (B279). In the lower two panels, equal protein amounts of cell lysates were directly immunoblotted for RanBP9 (M2) and BACE1 (B279). D, SPRY-LisH domains are sufficient to robustly increase Aβ secretion. Conditioned medium from CHO-APP-751 cells stably expressing full-length or various deletion mutants of RanBP9 were immunoprecipitated for Aβ and quantitated by densitometry using Image J. The graph shows the means ± S.E. normalized to vector control. n = 4 each group; *, p < 0.01 by one-way analysis of variance followed by post hoc Kruskal-Wallis test.

RanBP9 robustly increases Aβ secretion by stimulating β-secretase processing of APP. A, CHOAPP751 and CHO-APP751-RanBP9 stably transfected cells were plated at near confluency, and conditioned media were collected overnight. In the first and second panels, equal protein amounts of cell lysates were subjected to immunoblotting for FLAG-RanBP9 (M2) and APP (CT15). In the third panel, conditioned media were subjected to immunoprecipitation for Aβ (Ab9), and detection with mixed 6E10/82E1 antibodies. In the fourth to sixth panels, equal amounts of conditioned media were immunoblotted for sAPPα (6E10), sAPP total (63G), and sAPPβ (18957, IBL). In the bottom two panels, equal protein amounts of cell lysates were subjected to immunoblotting for APP CTF-β (6E10) and total APP CTF (CT15). B, densitometric quantitation of Aβ blots reveals an average ∼3.5-fold increases in the Aβ levels in CHO-APP751-RanBP9-expressing cells compared with the parental CHO-APP751 cells. Mean ± S.E., n = 6 each; ***, p < 0.001 by Student's t test. C, densitometric quantitation reveals a significant decrease in sAPP-α relative to sAPP total in CHO-APP751-RanBP9 cells compared with parental CHO-APP751 cells (mean ± S.E., n = 6 each; **, p < 0.01 by Student's t test). D, densitometric quantitation reveals a significant increase in APP-CTF-β relative to total APP CTF in CHO-APP751-RanBP9 cells compared with parental CHO-APP751 cells (mean ± S.E., n = 4 each; **, p < 0.01 by Student's t test). E, RanBP9 increases β-secretase cleavage independent of the KPI domain. CHO-APP695 and CHO-APP695-RanBP9 stably transfected cells were plated at near confluency, and conditioned media were collected overnight. In the first panel, equal protein amounts of cell lysates were immunoblotted for APP (CT15). In the second and third panels, equal amounts of conditioned media were immunoblotted for sAPPβ (18957, IBL) and sAPP total (63G). In the last panel, equal amounts of conditioned medium were subjected to immunoprecipitation for Aβ (Ab9) and detection with mixed 6E10/82E1 antibodies.

Endogenous RanBP9 is pivotal for Aβ secretion in CHO cells and primary neurons. A, CHO-APP751 stable cells were transiently transfected either with control sense siRNA or RanBP9-specific siRNA twice over a 48-h interval. After 24 h of the second transfection, lysates were immunoblotted for RanBP9 (anti-RanBP9), APP (CT15), and tubulin (anti-tubulin). The conditioned medium was immunoprecipitated with Ab9 antibody and immunoblotted for Aβ (6E10/82E1 mix). B, Aβ levels quantified from control siRNA and RanBP9 siRNA-transfected CHO-APP751 cells by image J software showed highly significant reduction by RanBP9 siRNA (Student's t test, mean ± S.E., n = 4 each; ***, p < 0.001). C, primary hippocampal neurons were grown for 7–10 days until a robust network of processes formed and then were transiently transfected with control sense or RanBP9 siRNA duplexes. After two rounds of transfection, equal protein amounts of cell lysates were immunoblotted for total APP (CT15), RanBP9 (anti-RanBP9), and tubulin. Equal amounts of overnight-conditioned media were directly immunoblotted for SWE-sAPPβ (6A1; upper panel). A representative experiment is shown. D, conditioned media from control and RanBP9 siRNA-transfected primary hippocampal neurons were subjected to ELISA for Aβ40. The graph shows the means and S.E. from four independent experiments normalized to control siRNA, illustrating a ∼36% reduction in Aβ40 secretion by RanBP9 siRNA (t test; **, p < 0.01).

RanBP9 overexpression reduces cell surface APP and promotes APP internalization. A, CHOAPP751 and CHO-APP751-RanBP9 stable cells were surface-biotinylated on ice, and lysates were immunoprecipitated with anti-biotin antibody followed by detection of APP by CT15 antibody. A representative experiment is shown. B, densitometric quantification using image J software reveals a ∼52% reduction of surface levels of APP relative to total APP in the presence of RanBP9 (t = 3.94, p = 0.0028 (**), df = 10). Error bars represent S.E. C, to determine the kinetics of APP internalization, CHO-APP751 cells surface-biotinylated with cleavable biotin on ice and returned to 37 °C for the indicated times to allow internalization of labeled proteins. The remaining cell surface biotin was removed by 2-mercaptoethanesulfonic acid, and equal amounts of cell lysates were immunoprecipitated (IP) with anti-biotin antibody (internalized proteins) followed by immuno-detection for APP (CT15, upper panel). Note that the non-internalized controls (0 min) show no internalization, indicating that removal of surface biotin is complete and internalization peaks at 5–10 min. In the lower panel, equal amounts of cell lysates were directly immunoblotted for APP (CT15). D, a representative biotin surface labeling and internalization experiment from control CHO-APP751 and CHO-APP751-RanBP9 cells according procedures in C. S0 indicates total surface labeling before removal of surface biotin, whereas 0 and 10 min indicate time points of internalization. In the bottom panel and L in the top panel, equal lysate volumes were subjected to direct immunoblotting for APP (CT15). E, quantitative measurement of internalized APP after 10 min normalized to surface APP at time zero determined in the same experiments (t = 5.006, p = 0.0007 (**), df = 10). Error bars represent S.E. Note the significantly increased rate of APP internalization relative to surface APP in CHO-APP751-RanBP9 cells compared with parental CHO-APP751 cells.