FE65 constitutes the functional link between the low-density lipoprotein receptor-related protein and the amyloid precursor protein

Abstract

Increasing evidence has implicated the low density lipoprotein receptor-related protein (LRP) and the adaptor protein FE65 in Alzheimer's disease pathogenesis. We have shown previously that LRP mediates beta-amyloid precursor protein (APP) processing and affects amyloid beta-protein and APP secretion and APP-c-terminal fragment generation. Furthermore, LRP mediates APP processing through its intracellular domain. Here, we set out to examine whether this interaction is of direct or indirect nature. Specifically, we asked whether adaptor proteins such as FE65 influence the LRP-mediated effect on APP processing by forming a protein complex. In coimmunoprecipitation experiments, we confirmed the postulated APP-FE65 and the LRP-FE65 interaction. However, we also showed an LRP-FE65-APP trimeric complex using pull-down techniques. Because FE65 alters APP processing, we investigated whether this effect is LRP dependent. Indeed, FE65 was only able to increase APP secretion in the presence of LRP. In the absence of LRP, APP secretion was unchanged compared with the LRP knock-out phenotype. Using RNA short interference techniques against FE65, we demonstrated that a reduction in FE65 protein mimics the LRP knock-out phenotype on APP processing. These results clearly demonstrate that FE65 acts as a functional linker between APP and LRP.

Figure 1.

Interaction between FE65 and APP or LRP by coimmunoprecipitation. a, APP can be coimmunoprecipitated with FE65 from HEK 293 cells expressing FE65-Flag and APP695. Using CT15 Ab, APP is present in cell lysates of APP695-expressing cells (lanes 2, 4) and in FE65-Flag immunoprecipitates of cells coexpressing APP695 and FE65-Flag (lane 5). Note that in the absence of FE65-Flag, no APP was immunoprecipitated with the anti-Flag Ab (lane 3). b, FE65 can be coimmunoprecipitated with LRP from HEK293T cells expressing FE65-Flag and LRP-CT. Using anti-FE65Ab, FE65 is present in LRP immunoprecipitates of cells expressing LRP-CT, LRP-CTΔ 1, and LRP-CTΔ 2(lanes 4, 3, 2). Note that in the absence of LRP-CT(lane 5) or presence of LRP-CT Δ 1Δ 2(lane 1), only little FE65 can be coprecipitated with LRP. This is because of the fact that these cells do express endogenous LRP. c, Using anti-Flag Ab (top panel), FE65 can be detected in cell lysates of FE65-Flag-transfected cells (lanes 1-5). Endogenous light chain LRP (β) and transfected LRP-CT constructs were detected in cell lysates using the anti-LRP Ab 1704 (bottom panel). IP, Immunoprecipitation; M2, anti-Flag monoclonal antibody; MW, molecular weight.

Figure 2.

APP43CT-glutathione S-transferase pull-down of LRP-FE65 complex. a, Lysates from LRP-deficient mouse embryonic fibroblast (MEF) cells stably overexpressing LRP-CT were transfected with FE65 (lanes 5, 6) or FE65ΔPID2 expression vector (lanes 3, 4) or untransfected controls (lanes 1, 2). Lysates were incubated with APP43CT-glutathione S-transferase. Bound proteins were coprecipitated by the addition of glutathione-agarose. Proteins were detected with polyclonal anti-LRP Ab 1704. Note that only in the presence of functional FE65 was the LRP-CT fragment pulled down by APP43CT-glutathione S-transferase. The FE65 and FE65ΔPID2 expression in LRP-deficient MEF cells stably overexpressing LRP-CT is shown in b using an Ab against the WW domain in FE65. MW, Molecular weight.

Figure 3.

LRP-ST-glutathione S-transferase pull-down of in vitro translated FE65-APP695 complex. a, APP695 was in vitro translated in the presence of [³⁵S]-Methionine and incubated with in vitro translated FE65 (lanes 5, 6) or FE65ΔPID2 (lanes 7, 8). As control, [³⁵S]-APP695 was incubated without FE65 or FE65ΔPID2 (lanes 3, 4). After incubation with LRP-ST-glutathione S-transferase fusion protein or GST fusion protein alone (lanes 1 and 2), bound proteins were coprecipitated by the addition of glutathione-agarose. Radiolabeled APP695 was detected by exposure to x-ray film, and the radioactive decays were quantified using phosphorimaging techniques. Note that only in the presence of functional FE65, ∼30% of the radiolabeled APP695 was pulled down by LRP-ST-glutathione S-transferase. The in vitro translated FE65 and FE65ΔPID2 proteins used in these experiments are shown in b. MW, Molecular weight.

Figure 4.

LRP expression mediates FE65-induced APPs secretion. Mouse embryonic fibroblasts lacking the LRP gene (LRP-/-) and corresponding LRP-expressing control fibroblasts (LRP+/-) were stably transfected with APP751 and with or without FE65. APPs was immunoprecipitated using the monoclonal antibodies 1G7-5A3 and the samples immunoblotted with an APP pAb (863) as described in Materials and Methods. a, Endogenous LRP levels were immunoblotted with LRP C-terminal Ab 1704. Transfected FE65 levels were immunoblotted with FE65 Ab against the WW domain. Transfected APP751 levels were immunoblotted with APP C-terminal Ab CT15. b, APPs secretion is significantly increased (threefold) after FE65 expression in LRP-expressing cells (lanes 3, 4) compared with untransfected cells (lanes 1, 2), whereas LRP-/- cells show no increase in APPs secretion after FE65 overexpression (lanes 7, 8) compared with untransfected LRP-deficient cells (lanes 5, 6). c, LRP-deficient cells expressing FE65 stimulated with PMA (lanes 3, 4) secrete more APPs than unstimulated cells (lanes 1, 2). MW, Molecular weight.

Figure 5.

FE65 deletion mutant mediates APP processing. a, HS683 neuroglioma cells stably transfected with APP695 were transiently transfected with FE65 or FE65ΔPID2 (top). FE65 levels were immunoblotted with FE65 Ab against the last 15 amino acids of FE65. APPs was immunoprecipitated using the monoclonal antibodies 1G7-5A3, and the samples were immunoblotted with an APP pAb (863) as described in Materials and Methods. APPs secretion is significantly increased after transfection with FE65- or FE65ΔPID2-transfected cells (lanes 2, 3) compared with control (lane 3). b, APP can be coimmunoprecipitated with LRP from HS683 cells expressing APP695. Using 26D6 Ab, LRP is present in APP immunoprecipitates (lane 2). Corresponding lysate is shown in the left lane (lane 1). Note that both full-length (α+β) and furin-cleaved β-subunit of LRP are detected in APP immunoprecipitates using the anti-LRP Ab 1704 for Western blot analysis. LRP immune complex was significantly decreased after transfection with FE65 or FE65ΔPID2 (lanes 3, 4) compared with control (lane 2). MW, Molecular weight; IP, immunoprecipitation.

Figure 6.

FE65 RNAsi treatment increased APPs secretion. Mouse embryonic fibroblasts lacking the LRP gene (LRP-/-) and corresponding LRP-/- cells expressing the truncated LRP-CT construct were incubated with FE65 RNAsi constructs for 6 d. Transfected LRP-CT levels were immunoblotted with LRP C-terminal Ab 1704 (middle panel). Endogenous FE65 levels were immunoblotted with FE65 Ab against the last 15 aminoacids of FE65(bottom panel).APPs was immunoprecipitated using the monoclonal antibodies 1G7-5A3, and the samples were immunoblotted with an APP pAb (863) as described in Materials and Methods (top panel). APPs secretion is significantly increased after FE65 RNAsi treatment in LRP-/- cells expressing the LRP-CT (lanes 7, 8) compared with controls (lanes 5, 6). APPs secretion remains unaltered in LRP-/- cells after FE65 RNAsi treatment (lanes 3, 4) compared with controls (lanes 1, 2).

Figure 7.

Stabilization of AICD by FE65 is LRP independent. Transfected APP-C50-Myc was immunoprecipitated using an anti-MycAb from CHO cell line 13-5-1 deficient in LRP (lanes 1, 2, 5, 6) and LRP expressing CHO-K1 control cell line (lanes 3, 4, 7, 8). APP-C50-Myc was detected using the CT15 anti-APP Ab. Note that cells lacking LRP (13-5-1) show no significant difference in APP-C50-Myc expression neither in the cytoplasmic fraction nor in the nuclear fraction compared with LRP-expressing control CHO-K1 cells.