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INTRODUCTION |
Amyloid plaques are one of the major hallmarks of Alzheimer's
disease (AD)1 pathology. The
plaque core is largely composed of an approximately 4-kDa peptide
referred to as A
(1, 2). Because mutations linked to AD have been
shown to increase secretion of A
(3), secreted A
is believed to
play a causative role in AD etiology. The precursor to A
is the
Alzheimer's amyloid protein precursor (APP) (4, 5). APP is a type I
integral membrane protein, the majority of which is found in the
ER/Golgi (6). A fraction of APP is transported to the plasma membrane,
then routed through the endosomal/lysosomal system (6-12).
At least three unidentified proteases, known as the
-,
-, and
-secretases, process APP (7, 13-16). The combination of
and
cleavages generates A
.
-Secretase cleaves APP within the A
domain, releasing
APPs, the large extracellular domain. While
APPs is generated primarily at or en route to the
plasma membrane (17), A
is formed in both the secretory and
endocytic pathways (8). It has been suggested that the majority of
secreted A
is made in the endocytic pathway (8).
Several studies have shown that the cytoplasmic tail of APP is
important for the regulation of APP metabolism and localization. The
carboxyl terminus of APP contains the sequence YENPTY. NPXY is a
consensus sequence for endocytosis of low density lipoprotein receptors
(18). Deletion of portions of APP that contain the YENPTY sequence
results in increased secretion of APPs and decreased secretion of A
(8, 19-22). The effects of these deletions are thought to be the result of increased APP at the cell surface. Mutation
of the second tyrosine in the YENPTY sequence to alanine also increases
APPs secretion but has no effect on A
secretion (23).
These observations suggest that secretion of A
and APPs may be regulated independently by signals in the cytoplasmic tail of
APP.
FE65 is a brain-enriched protein of unknown function (24) that binds to
the cytoplasmic domain of APP. FE65 contains two types of
protein-protein interaction domains: a WW domain in the amino terminus
and tandem phosphotyrosine interaction domains (PIDs) in the carboxyl
terminus. WW domains recognize poly-proline sequences (25), whereas
PIDs typically recognize phosphorylated NPXY sequences (26).
FE65 was first shown to interact with APP in the yeast two-hybrid
system (27, 28). Subsequently, several studies have shown that FE65
binds directly to the YENPTY sequence in the cytoplasmic domain of APP
through its carboxyl-terminal-most PID (28-31). Although it was
previously thought that PIDs bind to tyrosine-phosphorylated sequences, the FE65-APP interaction is phosphorylation-independent (29,
31). Because NPXY sequences are known to be involved in molecular
targeting and FE65 binds to the YENPTY sequence in APP, we hypothesized
that FE65 is an important regulator of A
secretion. In this study,
we have investigated the effects of FE65 on the metabolism and
trafficking of APP.
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EXPERIMENTAL PROCEDURES |
FE65 Constructs--
Rat FE65 was subcloned into pcDNA3
(Invitrogen, Carlsbad, CA), a mammalian expression vector, by
polymerase chain reaction. The 5' polymerase chain reaction primer
encoded an amino-terminal FLAG epitope tag.
Antibodies--
Polyclonal antibodies to FE65 were raised by
immunizing rabbits with the WW domain of FE65 (25) fused to glutathione
S-transferase. The antibodies were tested for their
specificity in immunoblots and immunoprecipitations by competition with
antigen. Two polyclonal antibodies demonstrating high affinity and
specificity (170 and 173) were combined and affinity purified on a
CNBr-activated Sepharose-WW domain column (Amersham Pharmacia Biotech).
Fig. 1, a and b
show the specificity of the antibodies for immunoblotting and
immunoprecipitation, respectively. In both cases, the antibodies
recognized a band around 100 kDa that was greatly increased in
intensity upon transfection with FE65 cDNA. 5A3/1G7 (8), a mixture
of monoclonal APP antibodies used for the immunofluorescence studies,
were a generous gift of E. H. Koo. 369, a polyclonal antibody, was
used to immunoprecipitate holoAPP (32). Monoclonal A
antibodies 6E10
and 4G8 were used for immunoprecipitation and ELISA (33).

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Fig. 1.
Characterization of FE65-overexpressing MDCK
cell lines and FE65 antibodies. MDCK-695 and MDCK-695/FE65 cells
were examined by immunoblotting (a) and immunoprecipitation
(b) with 170/173, FE65 antibodies, and immunoprecipitation
with 369 (c), an APP antibody.
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Stable Cell Lines--
To determine the effects of FE65 on APP,
a cell line was needed that expressed low levels of endogenous FE65,
therefore permitting manipulation of FE65 protein levels by exogenous
expression. For this purpose, Madin-Darby canine kidney cells (MDCK),
which overexpress the 695-amino acid isoform of APP (MDCK-695) were
stably transfected with FE65 cDNA. MDCK-695 cells were a generous
gift of C. Haass (34). Cells were stably transfected with FE65 in 10-cm
diameter plates using the calcium phosphate transfection system (Life
Technologies, Inc., Grand Island, NY), essentially following the
manufacturer instructions. Each plate was transfected with 15 µg of
FE65 in pcDNA3 and 5 µg of pPUR (CLONTECH), a
selection vector containing a puromycin resistance gene. After
selection with 2. 5 µg/ml puromycin (CLONTECH),
individual clones were isolated using cloning rings. Clonal cell lines
with high expression of FE65 (MDCK-695/FE65) were identified by
immunoblotting (Fig. 1a) and immunoprecipitation (Fig.
1b) with FE65 antibodies. These cells were maintained in Dulbecco's Modified Eagle's medium (DMEM) containing 200 µg/ml G418
(Life Technologies, Inc.) and 1 µg/ml puromycin. Fig. 1c shows APP expression in representative MDCK-695 and MDCK-695/FE65 cell
lines. Levels of APP expression did not vary with FE65 expression. Control cell lines, obtained by transfection of MDCK-695 cells with
vector, were simultaneously prepared by puromycin selection. No
significant differences were seen in these control cells compared with
MDCK-695 cells (data not shown). In parallel, cell lines were prepared
that stably overexpressed FE65 but not APP (MDCK-FE65).
Transient Transfection--
MDCK and MDCK-FE65 cells were
transiently transfected with APP-751 cDNA. The transfections were
performed with LipofectAMINE (Life Technologies, Inc.) as described in
the manufacturer instructions. Briefly, a 10-cm diameter plate of cells
was transfected with 10 µg of APP in pcDNA3 for 8-12 h. Cells
were assayed approximately 48 h post-transfection.
Metabolic Labeling and Immunoprecipitation--
Metabolic
labeling and immunoprecipitation were performed essentially as
described (33). Briefly, cells were plated at a density of 5.0 × 104 cells/cm2 and then grown for approximately
16 h. After washing, they were incubated with
[35S]methionine (NEN Life Science Products) in
methionine-free DMEM for 2 h at 37 °C, followed by a 2-h chase
at 37 °C in complete DMEM.
APPs was
immunoprecipitated from the chase medium with 6E10, and A
with 6E10
plus 4G8, followed in both cases by agarose-linked goat anti-mouse IgG
(American Qualex, La Mirada, CA). Immunoprecipitates were separated by
SDS-polyacrylamide gel electrophoresis and quantified by
PhosphorImager. Total labeled cellular holoAPP was determined by lysing
cells with 1% Nonidet P-40 in PBS immediately after labeling, followed
by immunoprecipitation with 369 and Sepharose-linked protein A
(Amersham Pharmacia Biotech) (32). Values obtained for
APPs and A
were normalized to this total labeled APP
in each experiment.
ELISA--
For the sandwich ELISA, cell medium was changed to
fresh serum-free DMEM. After incubation for 4 h at 37 °C,
conditioned medium was collected and subjected to a sandwich ELISA (35)
for A
using 6E10 as the capture antibody. For detection, samples
were incubated with biotinylated 4G8 followed by alkaline
phosphatase-labeled anti-biotin. A
was quantified with a Dynatech
plate reader. The values obtained were normalized to total cellular
holoAPP determined by immunoblotting with 369 and
125I-protein A (Amersham Pharmacia Biotech) followed by
PhosphorImager quantification.
Immunofluorescence--
Cells were plated at 3.5 × 104 cells/cm2 on glass coverslips. Except where
indicated, the cells were briefly pre-permeabilized with 0.02% saponin
in 80 mM PIPES/KOH, pH 7.0, 1 mM
MgCl2, 1 mM EGTA, and 30% glycerol and then
fixed in 4% paraformaldehyde in PBS with 0.12 M sucrose
for 10 min at 4 °C. The coverslips were then incubated in PBS with
0.2% Triton X-100 for 2 min at room temperature, rinsed in PBS, and
blocked with 10% bovine serum albumin in PBS. Primary antibodies were
incubated for 1 h at room temperature or overnight at 4 °C, and
secondary antibodies were added for 1 h at room temperature in PBS
with 1% bovine serum albumin. The secondary antibodies used were goat
anti-mouse IgG and goat-anti-rabbit IgG for APP and FE65, respectively,
conjugated to rhodamine red-X and Oregon green-488 (Molecular Probes,
Eugene, OR). Coverslips were mounted with DABCO in polyvinyl alcohol. For surface immunofluorescence, cells were blocked and incubated with
primary antibody before fixation. In all cases, immunofluorescence was
eliminated by omission of primary antibody or by competition with
antigen (data not shown). Immunofluorescence was examined by
deconvolution light microscopy (Deltavision) and confocal laser scanning microscopy (Zeiss LSM510).
Subcellular Fractionation--
Iodixanol gradients were formed
by layering successively less concentrated solutions of Optiprep (Life
Technologies, Inc.) in a centrifuge tube and then placing the tube on
its side for 3-4 h essentially as described in the manufacturer
instructions. Optiprep was chosen because it is an iso-osmotic medium;
it resolves ER, Golgi, and lysosomes extremely well; and continuous
gradients can be made very easily and reproducibly without high speed
centrifugation. MDCK-695/FE65 cells were gently scraped from 15-cm
diameter plates and collected by centrifugation. They were then
homogenized in 10 mM Hepes, pH 7.4, 1 mM EDTA,
0.25 M sucrose with a metal Dounce homogenizer. Nuclei,
unbroken cells, large pieces of plasma membrane, and heavy mitochondria
were removed by centrifugation at 3000 × g. The
supernatant (PNS) was then centrifuged at 17,000 × g for 15 min. The 17,000 × g pellet was loaded on a
preformed 10-30% continuous iodixanol gradient and centrifuged at
100,000 × g for 1 h in a swinging bucket rotor.
The 17,000 × g supernatant was loaded on a preformed
0-40% continuous iodixanol gradient and centrifuged at
85,000 × g for 45 min. The gradients were unloaded by
aspiration of 1-ml fractions from the top of the gradient. After
centrifugation of the fractions at 100,000 × g to
collect the membranes and remove the iodixanol, the 100,000 × g pellets were resuspended, boiled in sample buffer, and
analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting.
Surface Biotinylation--
MDCK cells were plated at 3.0 × 106 cells per 10-cm diameter dish. After washing twice with
Hanks' balanced salt solution (HBSS; Sigma), cells were incubated with
0.5 mg/ml NHS-LC-biotin (Pierce) in HBSS at 4 °C for 30 min.
Unreacted biotin was eliminated by two washes with DMEM containing 10%
fetal calf serum and two washes with HBSS. The cells were then lysed
with 1% Nonidet P-40, and equal amounts of protein were subjected to
immunoprecipitation with the APP antibody, 369. The immunoprecipitated
material was then immunoblotted with anti-biotin monoclonal antibody
(Sigma) and 125I-protein A. Cell surface APP was quantified
by PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
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RESULTS |
FE65 Alters the Proteolytic Processing of APP--
When measured
by sandwich ELISA, A
secretion increased 4.2 ± 0.5-fold
(mean ± S.E., n = 12, p < 0.0001) in MDCK-695/FE65 cells when compared with MDCK-695 cells
(Fig. 2a). Qualitatively similar results were observed by pulse-chase labeling followed by
immunoprecipitation (Fig. 2b). In all cases, values were
normalized to holoAPP; therefore, the observed differences were not
because of variation in total APP levels.

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Fig. 2.
FE65 causes an increase in secretion of
A from MDCK-695 cells
( FE65) and MDCK-695/FE65 cells
(+FE65). a, conditioned medium (4 h)
was subjected to a sandwich ELISA for A using 6E10 as the capture
antibody and 4G8 as the detection antibody. The data represent
means ± S.E. (n = 12). b, cells were
pulsed with [35S]methionine for 2 h, followed by a
2-h chase to look at processing of newly synthesized APP. A was
immunoprecipitated from the chase medium with a 4G8/6E10 mixture and
visualized by PhosphorImager. The autoradiogram represents results
typical of those observed in three experiments with one FE65-expressing
clone and two experiments with an additional FE65-expressing clone. *,
p < 0.0001.
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Immunoprecipitation of
APPs from conditioned medium of
[35S]methionine pulse-labeled MDCK-695 and MDCK-695/FE65
cells demonstrated that overexpression of FE65 increased secretion of
APPs. During a 2-h chase, MDCK-695/FE65 cells secreted
1.7 ± 0.1-fold (n = 11, p < 0.0005) more
APPs than did MDCK-695 cells
(Fig. 3a). Stable
overexpression of FE65 enhanced release of
APPs from an independent MDCK cell line (MDCK-FE65) transiently transfected with the
751-amino acid isoform of APP by 2.2 ± 0.1-fold
(n = 6, p < 0.0001), as compared with
MDCK cells similarly transfected with APP-751 (Fig. 3b).
Therefore, FE65 regulates APP metabolism independent of APP
isoform.

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Fig. 3.
FE65 causes an increase in secretion of
APPs. MDCK-695
( FE65) and MDCK-695/FE65 (+FE65) cells
(a) or MDCK ( FE65) and MDCK-FE65
(+FE65) cells that had been transiently transfected with
APP-751 (b) were pulsed with [35S]methionine
for 2 h, followed by a 2-h chase. APPs was
immunoprecipitated from the chase medium with 6E10 and quantified by
PhosphorImager. The data represent means ± S.E. (a,
n = 11; b, n = 6). *,
p < 0.0005; **, p < 0.0001.
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Large increases in
APPs secretion are often accompanied
by decreases in A
secretion, probably because of substrate depletion (33, 36-38). In contrast, lesser increases in
APPs can
be accompanied by an increase in A
(38). In the present study, FE65
increased both
APPs and A
secretion. To determine
whether substrate depletion occurred upon overexpression of FE65, we
determined the approximate percentage of APP molecules secreted
as either
APPs or A
. Over the time course of these
studies, approximately 20% of APP labeled during the chase was
converted to
APPs and less than 10% to A
, consistent
with the idea that holoAPP substrate was not rate-limiting.
APP and FE65 Co-localize in Perinuclear Organelles--
FE65 and
APP co-immunoprecipitate from homogenates of MDCK-695/FE65 cells (data
not shown; and see Ref. 39) and other cell types (28, 31). To determine
where FE65 and APP interact in intact cells, MDCK-695/FE65 cells were
double labeled with a mixture of polyclonal FE65 antibodies, 170 and
173, and a mixture of APP monoclonal antibodies, 5A3 and 1G7 (8), and
then examined by confocal microscopy. The two proteins co-localized in
perinuclear organelles (Fig. 4) that may
be ER/Golgi compartments and/or endosomes. The strong co-localization
of APP and FE65 supports the hypothesis that APP and FE65 interact
in vivo.

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Fig. 4.
APP and FE65 co-localize in the perinuclear
region of MDCK cells. MDCK-695/FE65 cells were double-labeled with
APP and FE65 antibodies for confocal laser scanning immunofluorescence
microscopy. a, labeling with APP monoclonal antibodies;
b, labeling with FE65 polyclonal antibodies; c,
overlay of images in panels a and b.
Yellow represents overlap between the APP and FE65
immunofluorescence. The images shown are representative of more than
four experiments.
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If the binding of FE65 to APP has a true physiological role in the
brain, then FE65 and APP should co-localize in neural-derived cells
that express both proteins at endogenous levels. H4 human neuroglioma
cells were examined with the same antibodies that were used for the
MDCK cells. FE65 and APP fluorescence again overlapped in juxta-nuclear
organelles and at some edges of H4 cells
(Fig. 5). These data suggest that the
effects of FE65 on APP processing are mediated by the interaction of
APP and FE65 in either the ER/Golgi or in the recycling pathway. They
also suggest that the interaction may be an important regulator of APP
processing in neurons, which express high endogenous levels of both
proteins.

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Fig. 5.
FE65 and APP co-localize in neural-derived
cells when expressed at endogenous levels. H4 human neuroglioma
cells were double labeled with APP and FE65 antibodies for
deconvolution immunofluorescence microscopy. a, labeling
with APP monoclonal antibodies; b, labeling with FE65
polyclonal antibodies; c, overlay of images in panels
a and b. Yellow represents overlap between the APP and
FE65 immunofluorescence. Nuclei are counter-stained with DAPI
(blue). The images shown are representative of at least two
experiments.
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To further confirm that the interaction of FE65 and APP is likely to
occur in MDCK cells in vivo and to characterize the
compartments in which they interact, MDCK-695/FE65 PNS was separated
into a 17,000 × g pellet and supernatant followed by
separation on iodixanol gradients. The 17,000 × g
pellet contains Golgi, lysosomes, light mitochondria, peroxisomes, and
ER, whereas the 17,000 × g supernatant contains small
vesicles, endosomes, and soluble proteins. FE65 and APP co-localized in
the least dense organelles of the 17,000 × g pellet
(Fig. 6 a and b).
There was a strong, sharp peak of both APP and FE65 in fraction 1, with
a smaller, broader peak in fractions 3-6. Fraction 1 contains
relatively pure Golgi (40, 41) and was highly enriched in rab6, a Golgi
marker (data not shown). Fractions 3-6 are highly enriched in ER (40,
41). Most of the APP seen in these fractions appeared to be immature as
would be expected for ER-associated APP (Fig. 6a).

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Fig. 6.
FE65 and APP co-fractionate in iodixanol
gradients. MDCK-695/FE65 cells were separated into a 17,000 × g pellet and supernatant. The 17,000 × g
pellet was fractionated on a 10-30% continuous iodixanol gradient
(a and b), whereas the 17,000 × g supernatant was fractionated on a 0-40% continuous
iodixanol gradient (c and d). Membranes were
precipitated from the fractions collected from each gradient and
analyzed by immunoblotting with antibodies to APP (a and
c) or FE65 (b and d). The fractions
are, from left to right, increasingly dense. The results shown are
representative of three experiments.
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APP and FE65 also co-localized in the least dense fractions from the
17,000 × g supernatant (Fig. 6, c and
d). Although these gradients have not been characterized
well in the literature, fraction 1 was highly enriched in both rab11
and EEA1 (data not shown). Rab11 localizes to secretory vesicles,
endosomes, and TGN (42), whereas EEA1 is an early endosomal marker
(43). Therefore, fraction 1 contained endosomes. It may have also
contained other membranes, such as small vesicles derived from the TGN, although it was not rab6 immunoreactive. Together our data suggest that
there is a strong co-localization of APP and FE65 in the ER/Golgi and
possibly in endosomes.
FE65 Alters the Subcellular Localization of APP--
To visualize
APP at the cell surface, MDCK-695 and MDCK-695/FE65 cells were labeled
with the APP antibodies for immunofluorescence without prior
permeabilization. FE65 overexpression caused an increase in high
intensity surface APP labeling (Fig. 7).
To ensure that the plasma membranes were intact, the cells were also
labeled with the FE65 antibodies 170/173. No fluorescence was observed (data not shown), indicating that the APP labeling was indeed extracellular. FE65 overexpression did not alter the APP immunostaining patterns in permeabilized MDCK-695 and MDCK-695/FE65 cells (data not
shown). Thus the FE65-dependent changes in the amount of
APP localized to the plasma membrane were specific to that pool of APP.

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Fig. 7.
FE65 causes translocation of APP to the
plasma membrane. MDCK-695 (a) and MDCK-695/FE65
(b) cells were labeled with APP monoclonal antibodies
5A3/1G7 without prior permeabilization to label only APP at the cell
surface. The images were colorized using a Zeiss glow scale table,
which colors pixels in decreasing order of intensity as white, yellow,
red, and black. The images shown are representative of multiple
randomly selected fields from three experiments and were collected by
confocal laser scanning microscopy.
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To quantify the effects of FE65 on surface APP, MDCK-695 and
MDCK-695/FE65 cells were surface biotinylated. After biotinylation, total APP was immunoprecipitated. The fraction of this APP that was
biotinylated at the cell surface was identified by immunoblotting with
anti-biotin antibodies. FE65 overexpression caused a 2.4 ± 0.37-fold (n = 9) increase in APP at the cell surface
(Fig. 8). These data suggest that an
FE65-dependent translocation of APP to the plasma membrane
was responsible for the observed changes in the proteolytic processing
of APP.

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Fig. 8.
FE65 increases APP at the cell surface.
a, immunoprecipitation and immunoblotting of cell surface
biotinylated APP from MDCK-695 cells ( FE65) and
MDCK-695/FE65 cells (+FE65). b, quantification of
APP at the plasma membrane by PhosphorImager. The data represent
means ± S.E. (n = 9). *, p = 0.0013.
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DISCUSSION |
We have shown here that FE65 and APP are present in the same
subcellular compartments and that FE65 expression increases the amount
of APP at the plasma membrane. This translocation of APP to the cell
surface is associated with a dramatic increase in A
secretion
concomitant with a smaller increase in secretion of
APPs.
Our data suggest that the observed increase in APP metabolism may be
because of an increased flux of APP through the secretory pathway. Such
an increase in trafficking would result in a larger fraction of APP
reaching the plasma membrane. Increased flux of APP through the
secretory pathway to the cell surface is predicted to result in
increased
-secretase cleavage of APP and, therefore, secretion of
APPs. In addition, routing of more APP to the cell surface increases the amount of APP available for endocytosis. Because
much of the A
secreted by cultured cells is generated in the
endocytic pathway (8), the observed increase in APP at the plasma
membrane is a plausible source of the increased A
secretion.
Many studies have shown that deletion of the YENPTY sequence of APP
results in increased APPs secretion and decreased A
secretion (8, 19, 20, 22), whereas we have shown that overexpression of
FE65, which binds to this sequence, can increase secretion of both
APPs and A
. The difference in the effects of deletion of
the YENPTY sequence versus FE65 overexpression is easily
explained because the deletion is expected to inhibit efficient
endocytosis of APP. If the cell surface APP is endocytosed at a
decreased rate, less A
will be produced. If FE65 does not interfere
with endocytosis, its overexpression would not be expected to affect A
secretion in the same way as the deletion. It is interesting to
note that mutation of the second tyrosine in the YENPTY sequence, which
does not affect FE65 binding, also does not affect A
secretion (23).
FE65 may produce its effects on secretion of proteolytic fragments of
APP by targeting of some other molecule to APP through its other
protein-protein interaction domains. In addition to the PID that binds
to APP, FE65 contains a WW domain and another, more amino-terminal, PID
(27). One of several proteins that binds to the WW domain has recently
been identified as mena (44), a member of the abl tyrosine kinase
pathway that is known to localize to focal adhesions (45). It will be
interesting to determine whether mena or other FE65 binding partners
affect APP processing as well.
The results of the present study suggest that agents that inhibit the
interaction of FE65 with APP might decrease A
secretion in the
brain. Such a reduction in A
secretion might then result in
decreased accumulation of amyloid plaques. Thus, discovery of an agent
that inhibits the interaction of FE65 with APP could provide a novel
approach to slowing or preventing the progression of Alzheimer's disease.