From the Howard Hughes Medical Institute,
¶ Department of Internal Medicine and Biological Chemistry, and
§ Department of Neurology, University of Michigan Medical
Center, Ann Arbor, Michigan 48109 and
Veterans Affairs Medical
Center Geriatric Research, Education, and Clinical Center,
Ann Arbor, Michigan 48105
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ABSTRACT |
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Constitutive amyloid precursor protein (APP)
metabolism results in the generation of soluble APP (APPs) and A
peptides, including A
40 and A
42-the major component of amyloid
plaques in Alzheimer's disease brain. The phosphotyrosine binding
(PTB) domain of X11 binds to a peptide containing a YENPTY motif found
in the carboxyl terminus of APP. We have cloned the full-length
X11 gene now referred to as X11
.
Coexpression of X11
with APP results in comparatively greater levels
of cellular APP and less APPs, A
40, and A
42 recovered in
conditioned medium of transiently transfected HEK 293 cells. These
effects are impaired by a single missense mutation of either APP (Y682G
within the YENPTY motif) or X11
(F608V within the PTB domain), which
diminishes their interaction, thus demonstrating specificity. The
inhibitory effect of X11
on A
40 and A
42 secretion is amplified
by coexpression with the Swedish mutation of APP (K595N/M596L), which
promotes its amyloidogenic processing. Pulse-chase analysis
demonstrates that X11
prolongs the half-life of APP from ~2 h to
~4 h. The effects of X11
on cellular APP and APPs recovery were
confirmed in a 293 cell line stably transfected with APP. The specific
binding of the PTB domain of X11
to the YENPTY motif-containing
peptide of APP appears to slow cellular APP processing and thus reduces
recovery of its soluble fragments APPs, A
40, and A
42 in
conditioned medium of transfected HEK 293 cells. X11
may be involved
in APP trafficking and metabolism in neurons and thus may be implicated
in amyloidogenesis in normal aging and Alzheimer's disease brain.
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INTRODUCTION |
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The finding of miliary amyloid plaques in brain parenchyma is
classically recognized as a hallmark of Alzheimer's disease (AD)1 pathology, although the
role of amyloid deposition in AD is controversial. Recent data of the
effects of gene mutations linked to familial AD suggests that the
deposition of amyloid plaque in brain may play a causal role in the
cascades leading to dementia and the pathologic abnormalities seen in
AD brain: the amyloid hypothesis of AD (1-3). The major components of
amyloid plaque are A peptides, including A
40 and A
42, derived
by constitutive proteolytic cleavage of amyloid precursor protein (APP)
encoded on human chromosome 21. APP is a type I cell surface protein
with an extracellular region, a transmembrane region, and short
intracellular carboxyl-terminal cytoplasmic region. The A
sequence
encompasses half of the transmembrane domain and a short part of the
extracellular domain of APP. A
40 and A
42 are released by
- and
-secretase activities that cleave APP at the amino and carboxyl
termini of A
, respectively. By this pathway, A
and soluble APP
(APPs
) are released into the extracellular space. Alternate cleavage
of APP within the A
sequence by an
-secretase activity releases
APPs
and precludes full-length A
formation. In nonneuronal cell
lines such as HEK 293 and Chinese hamster ovary cells, secreted APP
fragments are generated primarily via the
-secretase pathway,
although some A
is generated and secreted by
-/
-secretases,
primarily in the endosomal/lysosomal pathway. In these cells,
endocytosis of cell surface APP requires the Tyr-Glu-Asn-Pro-Thr-Tyr
(YENPTY) motif found in its intracellular carboxyl terminus and is thus
necessary for A
generation (4, 5).
The cytoplasmic region of APP containing the YENPTY motif interacts
with the PTB/PI (phosphotyrosine binding-protein interaction) domain of
X11 (6), Fe65 (7, 8), and their homologous genes
X11-like and Fe65-like (9, 10). X11 and
Fe65 are highly expressed in neurons and contain a PTB domain
originally described in Shc (11, 12). The Shc PTB domain interacts with
XNPXpY motifs (where
is hydrophobic,
X is any amino acid, N is Asn, P is Pro, and pY
is phosphotyrosine) found in tyrosine kinase receptors and other
tyrosine-phosphorylated proteins. The PTB domain of Shc is likely
involved in tyrosine kinase signal transduction cascades. However, the
PTB domain is a more general protein-protein interaction domain found
in several otherwise unrelated proteins such as X11, Fe65, Numb, and
Disabled. Although the PTB domains of these proteins are homologous to
Shc, they differ by binding to nonphosphorylated partners (13, 14). The
function of the newly described PTB domains is now being examined. For
example, the PTB domain of Numb is crucial for the differentiation of
sensory organ precursors in Drosophila (15, 16). Although
the PTB domain of X11
binds specifically to the YENPTY-containing
region of APP, the functional significance of this interaction is
unknown. Deletion of the last 18 amino acids of APP encompassing the
YENPTY motif or mutation of the amino-terminal tyrosine of the YENPTY motif of APP to glycine (Y682G) impairs binding to APP. Likewise, mutation of X11 at position 608 (F608V), previously referred to as the
F479V mutation in the nonfull-length protein, impairs X11
-APP interaction (6). The importance of these specific amino acid residues was confirmed by analysis of the crystal structure of the
X11
PTB domain complexed to a peptide encompassing the YENPTY motif
of APP (17).
Recently, we and others have identified a second X11 gene in
humans. We refer to the newly isolated gene as X11 (Fig.
1). The goal of this study was to
functionally characterize the interaction between X11
or X11
with
APP. Coexpression of X11
with APP in human embryonic kidney (HEK)
293 cells results in comparatively greater levels of cellular APP
(APPc), and less APPs, A
40, and A
42 recovered in conditioned
medium of transiently transfected cells. These effects are 1)
correlated with a prolonged half-life of APPc, 2) impaired by a single
missense mutation of either APP (Y682G) or X11
(F608V), and 3)
amplified by coexpression with the Swedish mutation of APP (APPswe;
K595N/M596L) found in a pedigree of early onset familial AD. Thus,
structural and functional data implicate a normal role for X11
in
APP trafficking and metabolism via a specific protein-protein
interaction.
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EXPERIMENTAL PROCEDURES |
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Cell Culture-- Human embryonic kidney 293 were grown in Dulbecco's modified Eagle's medium containing 100 units of penicillin/ml and 100 µg of streptomycin sulfate/ml supplemented with 10% fetal calf serum.
Cell Transfection and Protein Extraction-- Cells were split one day before transfection (1 × 106 cells/6-cm plate) and transfected with 10 µg of DNA by the calcium phosphate procedure. After 48 h, cells were washed twice with phosphate-buffered saline and lysed in lysis buffer (50 mM HEPES, pH 7.5, 10% glycerol, 150 mM NaCl, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA) supplemented with protease inhibitors (aprotinin, leupeptin, and phenylmethylsulfonyl fluoride). All constructs were cloned in pRK5 vector as described previously (6). The APP695 isoform was used exclusively in this study.
For [35S]methionine labeling, cells were incubated with methionine-deficient Dulbecco's modified Eagle's medium containing 100 µCi/ml for 15 min followed by a chase in complete Dulbecco's modified Eagle's medium. After washing the cells with phosphate-buffered saline, proteins were extracted with lysis buffer. Conditioned media of transfected cells were collected before lysis, and proteins were immunoprecipitated overnight with Karen or 6E10 antibodies at 4 °C. Bound proteins were recovered on protein A-agarose beads. After extensive washing with lysis buffer, proteins were separated by SDS-PAGE and detected by immunoblot or by PhosphorImager and autoradiography. Radiolabeled proteins detected by PhosphorImager were quantitated with ImageQuant software (Molecular Dynamics).Antibodies and ELISA--
The anti-myc antibody 9E10 (Oncogene
Science) at 1 µg/ml was used for immunoblotting. The 22C11 monoclonal
antibody (Boeringer Mannheim) was directed against an epitope of the
extracellular region of APP. The polyclonal antisera 369 was directed
to the cytoplasmic carboxyl terminus of APP. Karen is a goat polyclonal antisera directed to the secreted amino-terminal domain of APP (18).
The monoclonal antibody 6E10 (Senetek) was raised to A1-17. The
A
sandwich ELISA was performed as described previously (19) using
BAN50 as the capture antibody and either horseradish peroxidase-coupled BA-27 or BC-05 as the detection antibody for A
40 or A
42,
respectively. BAN-50 is a monoclonal antibody specific for
A
1-10.
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RESULTS |
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APP Mutations Affected Recovery of APPs in Conditioned
Medium--
The YENPTY motif in the intracellular carboxyl terminus of
APP is involved in its cellular processing. For example, deletion of
this motif results in greater recovery of APPs in conditioned medium
(4, 5). Either deletion of the YENPTY sequence or mutation of the
amino-terminal tyrosine of the motif (APP Y682G) abrogates binding to
the X11
PTB domain (6). Thus, we hypothesized that the APP Y682G
mutation would recapitulate the effects of YENPTY deletion on APPs
recovery. HEK 293 cells were transiently transfected with APP, APP
Y682G, or APPswe constructs. The APPswe double mutation resulted in
comparatively greater A
and less APPs
recovery in conditioned
medium. Comparable levels of APP expression were verified by immunoblot
of cell lysates with the anti-APP antibody 369 (Fig.
2A, upper panel).
APPs
in conditioned media was immunoprecipitated and detected by
immunoblot with 6E10 (Fig. 2A, lower panel). The
APP Y682G mutation resulted in greater release of APPs
in
conditioned medium, similar to deletion of the YENPTY motif (Fig.
2A, lower panel). As expected, APPswe resulted in
a decrease in APPs
in conditioned medium. Transfected HEK 293 cells
were also labeled with [35S]methionine, and APPs
was
recovered by immunoprecipitation with 6E10, separated by SDS-PAGE, and
revealed by autoradiography (Fig. 2B). Similar to the
results of Fig. 2A, APPs
release was markedly increased
by the Y682G mutation, although comparable expression of APP was found
in cell lysates (data not shown). Thus, the X11
PTB domain binding
site in APP is functionally important for APPs
release. Interference
with this interaction either by deletion of the carboxyl terminus
or mutation of the YENPTY domain of APP resulted in greater APPs
release into conditioned medium.
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X11 Expression Impaired
-Secretase Processing of APP--
In
HEK 293 cells APP is metabolized primarily by an
-secretase activity
at the cell surface, resulting in APPs
secretion. Because a fraction
of X11
protein is localized at the cell membrane, we hypothesized
that X11
would influence APPs
release. HEK 293 cells were
transiently cotransfected with APP and myc-tagged X11
constructs or
control vector (Fig. 3A). APP
and X11
in cell lysates were detected by immunoblot. APPs
in
conditioned medium was immunoprecipitated with Karen and detected by
immunoblot with 6E10. Coexpression of APP with X11
resulted in
decreased recovery of APPs
in an X11
dose-dependent
manner. X11
also resulted in a large increase of APP in cell
lysates. Although APPs
in medium was barely detectable when
transfected with 5 µg of the X11
construct, no further increase of
APP in the cell lysate was detected compared with 1 µg of X11
. This might suggest that APP is being processed by a
-secretase pathway. Conditioned media were immunoprecipitated with Karen, and
bound proteins were detected by immunoblot with 22C11, an anti-APP
antibody directed against all APPs species. The same decrease in APPs
was documented with this antibody, ruling out this possibility (Fig.
3A). We speculate that the generation of APP may be
decreased by high expression of X11
or that the APP level reaches a
plateau in the cell. Cells were also cotransfected with APP mutations
(Fig. 3B). Coexpression of 5 µg of X11
construct with
either APP or APPswe resulted in far less APPs
in conditioned medium. This result was expected because APPswe does not influence X11
binding (data not shown). Coexpression of APP Y682G with X11
led only to a small decrease of APPs
compared with APP Y682G transfection only (Fig. 3B). Accordingly, APP Y682G retained
only 5-10% of binding activity with X11
in vivo and
in vitro (6). Collectively, this data suggested that the
interaction of the PTB domain of X11
with the intracellular region
of APP containing the YENPTY motif impaired release of APPs
.
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X11 Coexpression with APP Decreased A
40 and A
42 Recovery
in Conditioned Medium--
A
peptides, particularly A
40 and
A
42, are also produced by constitutive APP metabolism. In contrast
to
-secretase cleavage of APP, which precludes generation of
full-length A
peptides, A
40 and A
42 are generated by
- and
-secretase activities. In HEK 293 cells, A
peptides are generated
almost exclusively by an endosomal pathway, which required a functional
YENPTY motif (4). We assessed the effect of X11
coexpression with
APP on A
40 and A
42 recovery in conditioned medium (Fig.
4), as measured by a sensitive and
specific ELISA (19). Compared with cells transfected with X11
,
transfection with APP resulted in measurable A
concentrations in
medium. As expected, transfection with APPswe resulted in much greater
levels of A
in conditioned medium (Fig. 4), in parallel with
diminished APPs levels (Fig. 2). Conversely, the APP Y682G mutation
resulted in a slight decrease in A
40 and A
42 release (Fig.
4A), in parallel with a slight increase in APPs
(Fig. 2).
Coexpression of X11
with APP reduced the levels of A
40 and A
42
in medium. This inhibitory effect of X11
was amplified by
coexpression with the APPswe mutation. As predicted by the binding
data, APP Y682G metabolism was resistant to X11
effects.
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X11 Coexpression Stabilized Cellular APP--
Our data suggests
that X11
coexpression blocks the production of soluble APP
metabolites and results in apparent greater levels of cellular APP,
thus appearing to stabilize APP in the cell (Fig. 3). To test this
hypothesis, we performed a pulse-chase analysis of HEK 293 cells
transiently transfected with APP alone or with X11
. Cells were
labeled for 15 min with [35 S]methionine before chase for
up to 8 h. APP in cell lysates was immunoprecipitated by Karen
antibody, separated by SDS-PAGE, and detected by PhosphorImager
analysis and autoradiography. Radiolabeled APP was quantitated by
ImageQuant software. Similar to previous reports, APP had a half-life
of ~2 h in HEK 293 cells (Fig. 5). Coexpression of X11
prolonged APP half-life to ~4 h. To further evaluate the stabilization of cellular APP by X11
, we transiently expressed X11
in HEK 293 cells stably expressing APP and measured APP levels in cell lysates. In these experiments, X11
expression resulted in an apparent increase in the amount of APP in cell lysates.
As predicted, the X11
F608V mutation had less effect (Fig.
6A, lower panel).
Comparable amounts of X11
and X11
F608V were expressed in the
cells (Fig. 6A, upper panel).
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DISCUSSION |
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Nonneuronal cell lines are instructive model systems of APP
trafficking and metabolism. HEK 293 cells produce A, primarily A
40, via
-/
-secretase activities in an endosomal pathway,
although the primary metabolic products of APP in this cell line result from
-secretase activity (4). The intracellular carboxyl-terminal domain of APP, in particular the YENPTY consensus sequence required for
endocytosis by clathrin-coated pits, plays an important role in APP
processing and A
generation by the endosomal pathway (4, 5). X11
,
a protein highly expressed in neurons, specifically interacts with a
peptide encompassing the YENPTY motif of APP (6). We now demonstrate
that the interaction of X11
or X11
with APP has significant
effects on its metabolism in HEK 293 cells and has implied effects on
cellular trafficking of APP.
Coexpression of X11 with APP decreased the recovery of its soluble
fragments APPs
, A
40, and A
42 in conditioned medium of HEK 293 cells. These effects are specific, as demonstrated by the use of a
single point mutation within either the cytoplasmic YENPTY motif of APP
or the PTB domain of X11
, which impairs their interaction and thus,
X11
effects. The decreased recovery of soluble APP fragments in
conditioned medium was observed in parallel with an apparent increase
in APP in cell lysates. This suggested prolongation of the half-life of
APPc, which was confirmed by pulse-chase analysis. Expression of a
second member of the X11 gene family, i.e.
X11
, in HEK 293 cells had similar effects on APP processing. X11
bound as efficiently as X11
to APP in
vivo and in vitro (data not shown). Thus, the specific
interaction of the X11 PTB domain with the YENPTY motif-containing
region of APP appeared to retard its processing and prolong its
half-life, resulting in decreased recovery of soluble proteolytic
fragments. When coexpressed with APP, X11
slowed both the
-secretase pathway and the endosomal/lysosomal pathway leading to
A
generation. The mechanisms and intracellular site(s) of X11
effects on APP metabolism are unknown, but one may hypothesize that
X11
slows endosomal trafficking of APP. Alternatively, X11
may
prevent the secretion of APPs, leading to an accumulation of APP in the cell. Interestingly, recent evidence suggests increased neuronal endocytosis and thus increased A
secretion in neurons of sporadic AD
brain compared with age-matched control brain (20).
The observed effects of X11 on inhibition of A
secretion with APP
coexpression are qualitatively similar but amplified by coexpression
with APPswe. In effect, coexpression of X11
with APPswe decreased
A
40 and A
42 secretion to that seen with APP expression only.
Similar to APP, when coexpressed with APPswe, X11
retarded both the
-secretase pathway and A
generation. The Swedish mutation of APP
promoted its metabolism by
-/
-protease activities, resulting in a
5-10-fold increase in A
40 and A
42 secretion, with a concomitant
decrease in the
-secretase pathway (21, 22). There are other
important differences between APP and APPswe metabolism. For example,
in contrast to APP, transfection of an APPswe construct lacking a
cytoplasmic tail, which precludes reinternalization, did not reduce the
secretion of A
peptides. Thus, an additional
-/
-protease
pathway in Golgi-derived vesicles or the Golgi itself is present in
APPswe metabolism to A
in nonneuronal cells (23-26). X11
may
affect metabolism of APPswe by this cellular pathway as well as the
endosomal pathway of A
generation.
X11 and X11
are neuronal proteins that contain two PDZ
(PSD-95/Dlg/ZO-1) domains in addition to the PTB domain. The PDZ domains found in other neuronal membrane proteins such as the PSD-95
family and nitric oxide synthase are implicated in their membrane
clustering and localization. Clustering and localization of proteins
may serve to stabilize proteins and prolong their half-life (27). This
is similar to the effects of X11 on APP that we observed in this study.
Although the binding partners of the PDZ domain of X11
are unknown,
a heterotrimeric complex of PDZ partner/X11
/APP may be implicated in
X11
effects and APP localization. The Fe65 gene family is
also expressed primarily in neurons, and the encoded protein contains a
PTB domain that binds to APP (8) and thus may influence its metabolism.
In addition to two PTB domains, Fe65 contains in its sequence a WW protein interaction domain that binds to proline-rich sequences (28).
Thus, Fe65 and X11
may have differential effects on APP trafficking
and metabolism based on the formation of alternate and potentially
competing heterotrimeric complexes of APP with either a PDZ binding
partner of X11
or a WW binding partner of Fe65.
Neuronal processing of APP is in some ways distinct from its metabolism
in nonneuronal cells and results in greater A generation compared
with nonneuronal cells (29-32). In addition to having the more
ubiquitous
-secretase pathway at or near the cell surface and
endosomal/lysosomal processing of APP to A
, neuronal cells have
additional
/
processing of APP within the endoplasmic
reticulum/early Golgi. This neuronal exocytic pathway favors the
generation of a higher ratio of A
42 to A
40 compared with the
-/
- proteases of the endocytic pathway (31, 18, 33).
Interestingly, presenilins are localized primarily to the endoplasmic
reticulum and Golgi (34-36), suggesting that presenilin-1 or
presenilin-2 mutations linked to familial AD exert their effect on APP
metabolism, specifically increased A
42 secretion, by promotion of
-/
-cleavage within this exocytic pathway. The effects of X11 on
the metabolism of APP717 mutations or on APP metabolism coexpressed
with presenilin mutations, all of which result in a higher ratio of
A
42 to A
40 secretion (19, 37), are unknown.
It will be of interest to probe the effects of X11 coexpression in transgenic mice harboring mutations of human APP linked to familial AD. With aging, these transgenic animals develop a partial AD-like phenotype, in particular behavioral changes and amyloid deposition in brain (38-40). Examination of X11 and Fe65 expression and their binding partners in normal aging and AD brain may shed light on two unanswered questions in AD research, namely, the selective anatomic localization of amyloid plaques in brain and the increased risk of AD with aging. Finally, if the amyloid hypothesis of AD proves tenable, knowledge of the effects of X11 and Fe65 on APP metabolism may serve as the basis for novel therapeutic strategies to delay the onset or slow the progression of amyloid formation and thus the clinical dementia of AD.
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ACKNOWLEDGEMENTS |
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We thank Dr. N. Suzuki, Takeda Chemical Co., Japan, for antibodies BAN-50, BA-27, and BC-05 and Drs. B. Greenberg and S. Gandy for the antisera Karen and 369, respectively.
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FOOTNOTES |
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* The work was supported by a pilot of National Institutes of Health Grant P50 AG08671.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF047347 and AF047348.
To whom correspondence and reprint requests should be
addressed: VAMC GRECC, 2215 Fuller Rd., Ann Arbor, MI 48105. Tel.:
734-761-7686; Fax: 734-761-7489; E-mail: raymondt{at}umich.edu.
** An investigator of the Howard Hughes Medical Institute.
1
The abbreviations used are: AD Alzheimer's
disease; A, amyloid-
protein; APPc, cellular APP; APPswe, Swedish
mutation of APP; APP,
-amyloid precursor protein; APPs
, soluble
APP cleaved by
-secretase, APPs
, soluble APP cleaved by
-secretase; PDZ, PSD-95/Dlg/ZO-1; PI, protein interaction; PTB,
phosphotyrosine binding; HEK, human embryonic kidney cells; PAGE,
polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent
assay.
2 J.-P. Borg and B. Margolis, unpublished data.
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REFERENCES |
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