Demonstration by fluorescence resonance energy transfer of two sites of interaction between the low-density lipoprotein receptor-related protein and the amyloid precursor protein: role of the intracellular adapter protein Fe65 - PubMed (original) (raw)

A Kinoshita et al. J Neurosci. 2001.

Abstract

Amyloid-beta, the major constituent of senile plaques in Alzheimer's disease, is derived from the amyloid precursor protein (APP) by proteolysis. Kunitz protease inhibitor (KPI) containing forms of APP (APP751/770) interact with a multifunctional endocytic receptor, the low-density lipoprotein receptor-related protein (LRP), which modulates its proteolytic processing affecting production of amyloid-beta. We used fluorescence resonance energy transfer (FRET) using labeled LRP and APP in H4 cell line to examine the subcellular localization and the molecular domains involved in the APP-LRP interaction. KPI-containing forms of APP (APP770) demonstrated FRET with LRP that was sensitive to the LRP inhibitor receptor-associated protein (RAP), suggesting an interaction between the extracellular domains of APP770 and LRP. APP695 also interacts with LRP to lesser degree (as measured by extracellular domain probes), and this ectodomain interaction is not altered by RAP. By using C-terminally tagged LRP and APP, we demonstrate a second site of interaction between the C termini of both APP695 and APP770 and the C terminus of LRP, and that the interactions at these regions are not sensitive to RAP. We next examined the possibility that the C-termini APP-LRP interaction was mediated by Fe65, an adaptor protein that interacts with the cytoplasmic tails of LRP and APP. FRET studies confirmed a close proximity between the amino Fe65 phosphotyrosine binding (PTB) domain and LRP cytoplasmic domain and between the carboxyl Fe65 PTB domain and the APP cytoplasmic domain. These findings demonstrate that LRP interaction with APP occurs via both extracellular and intracellular protein interaction domains.

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Figures

Fig. 1.

Fig. 1.

Reagents used in this study. This scheme shows the constructs used in this study. The membrane-spanning regions are shown in black. Both APP695 and APP770 are tagged at the C terminus with either myc or DsRed. The N-terminus truncated APPC99 is tagged with myc at the C terminus. Full-length LRP is tagged at the C terminus with either myc or EGFP (shown as GFP). The Fe65 construct is tagged either with EGFP at the N terminus or myc at C terminus. For the study of ectodomain interactions, anti-APP antibody (8E5) and anti-LRP antibody (R829), both of which are raised against the extracellular domain of APP or LRP, respectively, are used. For intracytoplasmic interaction, the combination of myc (detected with Cy3) and EGFP or the combination of DsRed and EGFP is used.

Fig. 2.

Fig. 2.

Extracellular domains of APP and LRP are closely associated with each other. H4 cells were cotransfected with APP770-myc and LRP-myc, immunostained with anti-APP (8E5) and anti-LRP (R829) antibodies, and visualized with Cy3 and FITC, respectively. Here is shown a typical example of FRET between ectodomains of APP770 and LRP.A, Cy3 (APP770) signal after 568 nm excitation.B, A discrete area of the cell was photobleached using intense 568 nm laser. C, FITC signal (LRP) using 488 nm excitation before photobleaching. D, FITC signal (LRP) using 488 nm excitation after photobleaching of the acceptor fluorophore (Cy3) with intense 568 nm laser light. An increase in donor fluorescence is observed within the discrete area that was photobleached, showing the presence of FRET. In this example, the FITC signal increased by 51%.

Fig. 3.

Fig. 3.

Extracellular domains of APP and LRP displayed FRET at the cell surface. H4 cells were cotransfected with APP770-myc and LRP-myc, immunostained with anti-APP (8E5) and anti-LRP (R829) antibodies without permeabilization, and visualized with Cy3 and FITC, respectively. APP and LRP on the cell surface were observed.A, Cy3 (APP770) signal using 568 nm excitation.B, Discrete area of the cell was photobleached using intense 568 nm laser. C, FITC signal (LRP) using 488 nm excitation before photobleaching. D, FITC signal (LRP) using 488 nm light after photobleaching the acceptor (Cy3) with intense 568 nm laser light. An increase in donor fluorescence is observed within the discrete area that was photobleached, showing the presence of FRET on the cell membrane. In this example, the FITC signal increased by 38%.

Fig. 4.

Fig. 4.

The FRET ratio increase of ectodomains is shown as a percentage of increase of donor fluorescence between APP (APP770 and APP695) and LRP. Both APP770 and APP695 demonstrated increases in donor fluorescence significantly above zero (p < 0.0001; one-group t test), but the magnitude of the FRET ratio increase of APP695 was significantly less than that of APP770 (p < 0.0001; ANOVA; Fisher's PLSD_post hoc_ test). Cotransfection with RAP significantly decreased the FRET ratio between APP770 and LRP (p = 0.00012; ANOVA; Fisher's PLSD_post hoc_ test) but not APP695 and LRP. The APP695-LRP ectodomain interaction was not affected by RAP. The FRET ratio increase on the cell membrane is almost the same as that of the routinely fixed and permeabilized cells. Cotransfection with RAP also significantly decreased the FRET ratio between APP770 and LRP at the cell surface (p < 0.0001; ANOVA; Fisher's PLSD_post hoc_ test).

Fig. 5.

Fig. 5.

Intracellular domains of APP and LRP are closely associated. H4 cells were transiently transfected with APP770-myc and LRP-EGFP expression constructs. Cells were immunostained by anti-myc antibody conjugated with Cy3. A, Cy3 (APP770) signal using 568 nm excitation. B, Discrete area of the cell was photobleached using intense 568 nm laser. C, EGFP signal (LRP) using 488 nm excitation before photobleaching.D, EGFP signal (LRP) using 488 nm excitation after photobleaching of the acceptor (Cy3) fluorophore with intense 568 nm light. An increase in donor fluorescence is observed within the discrete area that was photobleached, showing the presence of FRET. In this example, the EGFP signal increased by 40%.

Fig. 6.

Fig. 6.

The FRET ratio increase of intracellular domain is shown as a percentage of increase of donor fluorescence between APP and LRP. The FRET ratio increase in donor fluorescence (EGFP) after photobleaching of the acceptor molecule (APP-myc) was measured. The percentage of increase in donor fluorescence was shown in the graph. FRET was present between all of the APP constructs (APP770, APP695, and APPC99) tagged at the C terminal and LRP-EGFP, the ratio being significantly above zero (p < 0.0001; one-group t test). Cotransfection of RAP significantly decreased the FRET ratio increase of APP770 (p = 0.0001; ANOVA; Fisher's PLSD_post hoc_ test), but there was no significant effect of RAP on APP695 or APPC99-LRP interactions.

Fig. 7.

Fig. 7.

The interaction between Fe65-APP and Fe65-LRP was investigated by FRET. The interaction between Fe65-APP and Fe65-LRP was measured by FRET ratio increase after photobleaching of acceptor molecules. A close interaction was observed between C-terminally tagged Fe65 [shown as _Fe65(C)_] and APP770, whereas N-terminally tagged Fe65 [shown as _Fe65(N)_] and APP770 showed a much smaller interaction (p < 0.0001; ANOVA; Fisher's PLSD post hoc test). In contrast, C-terminally tagged Fe65 and LRP showed much less FRET than N-terminally tagged Fe65 and LRP (p < 0.0001; ANOVA; Fisher's PLSD post hoc test).

Fig. 8.

Fig. 8.

This scheme shows a hypothetical model of interactions of APP, LRP, and the cytoplasmic adaptor protein Fe65. Extracellularly, there may be an interaction between the ligand binding domain of LRP and KPI domain of APP, which is RAP-sensitive. Intracellularly, Fe65 may bind both APP and LRP. We show both interactions occurring in this figure. APP binding to LRP may cause endocytosis of APP and thus modulate amyloid-β (_A_β) synthesis.

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