From the Laboratory of Neurobiophysics, School of Pharmaceutical
Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan
 |
INTRODUCTION |
A principal component of amyloid plaques in Alzheimer's disease
(AD)1 is a 38-43-amino acid
-amyloid peptide (A
) (1-3), which is derived from the
-amyloid precursor protein (APP) (4-11). Production and
accumulation of A
is a major event believed to be involved in the
etiology of AD (reviewed in Refs. 12 and 13). APP is an integral
membrane protein with a short intracellular carboxyl terminus. Normal
proteolytic processing of APP generates two fragments, a large
extracellular amino-terminal domain (sAPP
) and a truncated carboxyl-terminal fragment (CTF
), both of which are products of
cleavage within the A
domain. Another form of proteolytic processing
occurring at
-site(s) gives rise to limited production of
carboxyl-terminal fragments (CTF
) and A
in unidentified
intracellular compartments of the protein secretory pathway (14).
Previous studies suggest that the
-secretase responsible for
cleavage of APP at the
-site is active in the trans-Golgi
network or other end-stage compartments of the protein secretory
pathway (15, 16). Studies utilizing the APP of familial Alzheimer's
disease (FAD), such as APP with the "Swedish" double
mutation (K595N/M596L, numbering for the APP695 isoform), suggest that
APP cleavage at the
-site by
-secretase occurs in the
medial-Golgi network and in unidentified compartments
proximal to the plasma membrane (17, 18), although a recent report
suggests that the endoplasmic reticulum is the site of generation of
A
42 (but not of A
40) in neurons (19). Furthermore, it is
generally held that the increased production of CTF
always gives
rise to more A
, although the molecular mechanisms of CTF
production by
-secretase from wild-type APP and of subsequent A
production following the cleavage of CTF
by
-secretase are still
unknown in sporadic AD patients, who constitute the majority of AD
cases.
The APP cytoplasmic domain is thought to be responsible for the
intracellular transport of APP. Two internalization signals, NPTY
(APP770 amino acids 759-762 and APP695 amino acids 684-687) and YTSI
(APP770 amino acids 728-731 and APP695 amino acids 653-656), have
been identified (20, 21). The NPXY motif in the cytoplasmic domain of membrane proteins is believed to mediate interactions between
internalized proteins and the clathrin cage of the clathrin-coated vesicle (22). The YXXI motif conforms to a potential
4-residue tyrosine-based internalization signal consensus sequence
(21). Furthermore, the sorting signal(s) in the cytoplasmic domain of APP are thought to play an important role in the distribution of APP
within the protein secretory pathway (15), although the sorting signal
has not been well analyzed. Therefore, using site-directed mutagenesis,
we performed further analysis of the sorting signal(s) in the
cytoplasmic domain of APP, which may be responsible for the regulation
of APP metabolism such as the production of CTF
and/or A
.
Identification and characterization of the signal(s) in the APP
cytoplasmic domain are important for our understanding of the molecular
mechanism that directs APP into a protein secretory pathway where
CTF
and/or subsequent A
production occurs without an FAD
mutation.
In this study, we introduced an alanine-scanning mutation into the
cytoplasmic domain of APP and established many cell lines that
expressed stably transfected APP carrying a single amino acid mutation
in its cytoplasmic domain. Among the cell lines, cells that expressed
APP carrying a mutation at amino acid 747 of APP770 (position 672 of
APP695) generated a greater amount of CTF
, but this did not result
in an increase of A
secretion, indicating that the intracellular
increase in CTF
is not necessarily related to an increase in A
production. This effect was not due to a simple intracellular
accumulation of CTF
resulting from a lack of
-secretase. These
results suggest that the basic amino acid at position 747 of APP770
(position 672 of APP695) plays an important role in the direction of
APP into the normal secretory pathway, in which APP is cleaved
preferentially at the
-site. Thus, APP carrying this mutation may be
metabolized in a different protein secretory pathway distinct from the
normal one. Our findings provide a new insight into what regulates the
processing of CTF
by
-secretase and the nature of the compartment
in which
-secretase cleavage occurs in the case of sporadic AD.
 |
EXPERIMENTAL PROCEDURES |
Introduction of Substitution Mutation and Plasmid
Construction--
cDNA encoding human APP770 (10) was cloned from
a
ZAP HeLa cDNA library by immunoscreening with anti-APP
antibody, G-369 (23). A PCR of a human brain cDNA library
(CLONTECH) was performed with primers APP695
cDNA-(681-700) (forward, 5'-GTTCCGAGGGGTAGAGTTTG-3') and APP695
cDNA-(1922-1940) (reverse, 5'-GCATCCATCTTCACTTCAG-3'). A
1.2-kilobase PCR product was digested with
XcmI/BglII, and the resulting fragment (APP695
cDNA-(990-1916)) was recombined into the larger fragment
from XcmI/BglII digestion of APP770 to delete Kunitz-type protease inhibitor (exon 7) and APP770-specific (OX-2, exon
8) domains of APP770 and to construct a complete APP695 cDNA (see
Fig. 1a) (5). The cDNAs were subcloned into pcDNA3
(Invitrogen) at HindIII/XbaI sites. A sequence of
the APP770 extracellular domain between amino acids 379 and 666 was
deleted by exclusion of the XhoI/BglII fragment
(p
APP770wt) (see Fig. 1a) (24). Site-directed mutations
were introduced using PCR (25) with the following primers: primer 1, 5'-GCCGCGGTCACCCCAGAGGAGGCCCACACCTGTCC-3' (the
underlined nucleotides were changed to produce an EcoO65I site (T to G) and the Arg-to-Ala mutation (CG to GC)) and primer 2, 5'-ATTTAGGTGACACTATAGAATAG-3' (SP6 promoter primer), in the presence of
plasmid p
APP770wt and Pwo DNA polymerase (Boehringer Mannheim). Primer 3, 5'-TCTGGGGTGACCGCGGCGTCAACCTCCACC-3'
(the underlined nucleotide was changed to produce an
EcoO65I site (A to C)), and primer 4, 5'-TAATACGACTCACTATAGGG-3' (T7 promoter primer), were used in PCR in
the presence of plasmid p
APP770wt and Pwo DNA polymerase.
Both PCR products were digested with EcoO65I, ligated, and
then inserted into pcDNA3 at the HindIII/XbaI
sites. The resulting plasmid, p
APP770R747A, encodes an amino
acid sequence identical to that of
APP770wt except for the
substitution of Ala for Arg747. The introduction of the
EcoO65I site did not change the amino acid sequence of the
APP protein. A similar procedure was used to introduce other single
amino acid substitutions into the APP cytoplasmic domain. The sites of
restriction enzymes and the positions of primers used in this study are
shown in Fig. 1a.
Detection of Intracellular Carboxyl-terminal Fragments of
APP--
293 cells were transfected with plasmid DNA, and several
independent clones stably expressing APP were isolated for each
construct. Identical results for APP metabolism were obtained from
several independent clones, although the level of APP expression varied among the clones. The cells were lysed, and the CTFs of APP were immunoprecipitated (26) using a polyclonal anti-APP cytoplasmic domain
antibody, UT-421, or a monoclonal anti-A
antibody, 2D1 (24). The
immunoprecipitates were subjected to SDS-PAGE (15% (w/v)
polyacrylamide) using a modified version (27) of the procedure of
Laemmli (28). Samples were transferred to a nitrocellulose membrane
(BA83, Schleicher & Schull), and the membrane was probed with antibody
UT-421 and 125I-protein A (IM144, Amersham Pharmacia
Biotech), and then analyzed by autoradiography using a Fuji BAS 2000 imaging analyzer or by ECL (Amersham Pharmacia Biotech).
Pulse-Chase Study--
Pulse-chase labeling of cells was carried
out with [35S]methionine (1 mCi/ml; NEG-072, NEN Life
Science Products). 293 cells expressing APP695wt and APP695 R672A were
labeled metabolically for 30 min, followed by a chase period as
indicated. The chase was initiated by replacing the labeling medium
with medium containing excess unlabeled methionine. CTF and APP were
immunoprecipitated from the cell lysate using antibody UT-421 and
separated by SDS-PAGE (15% (w/v) polyacrylamide) (27). sAPP was
isolated from the medium by immunoprecipitation with monoclonal
anti-APP extracellular domain antibody 22C11 (Boehringer Mannheim) and
separated by SDS-PAGE (7.5% (w/v) polyacrylamide) (26). Radioactivity
in CTF
, CTFs
, sAPP, and APP was analyzed using the Fuji BAS 2000 imaging analyzer.
ELISA Analysis--
The conditioned medium from cells (2 × 10 6 cells) was collected 18-20 h after a medium change.
A
40 and A
42 were quantified using sandwich ELISA with three types
of A
-specific monoclonal antibodies as described previously (24).
Briefly, wells were coated with the A
40 (4D1) or A
42 (4D8)
end-specific monoclonal antibodies (0.3 µg of IgG in
phosphate-buffered saline (140 mM NaCl and 10 mM sodium phosphate, pH 7.2)), washed with
phosphate-buffered saline containing 0.05% (v/v) Tween 20 (washing
buffer), blocked with bovine serum albumin (3% (w/v) in
phosphate-buffered saline), and washed with washing buffer, and then a
sample (100 µl) diluted suitably with washing buffer containing 1%
(w/v) bovine serum albumin (dilution buffer) was incubated together
with a standard amount of synthetic A
-(1-40) and A
-(1-42)
peptides (synthesized at the W. M. Keck Foundation Biotechnology
Resource Laboratory, Yale University). After washing, wells were
treated with biotinized 2D1 (12. 5 ng in dilution buffer), washed, and
incubated with 100 µl of a streptavidin-horseradish peroxidase
complex (1:2000 dilution; RPN1051, Amersham Pharmacia Biotech). The
plates (96 wells) were further washed, and 100 µl of ABTS solution
(KPL 5062-01, Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD)
was added to the wells and incubated at room temperature. Reactions
were stopped by adding 100 µl of 1% (w/v) SDS, and the absorbance at 405 nm was measured.
 |
RESULTS |
Production of Intracellular Carboxyl-terminal Fragments (CTF
and
CTF
) in Cells That Express APP Carrying a Single Amino Acid Mutation
in Its Cytoplasmic Domain--
It is well known that APP is cleaved
preferentially at the
-site to produce CTF
(Fig.
1a) (29). Intracellular
products of APP cleaved at the
-site are extremely rare in healthy
cells if APP does not carry an FAD mutation such as the
Swedish double amino acid substitution (30). However, the
production of intracellular CTF
(Fig. 1a) and the
secretion of A
increase in the sporadic type of AD even if the
resident APP gene carries an entire sequence (31, 32). In the case of
sporadic AD, one of the most reasonable explanations for the increased
production of CTF
and the secretion of A
is abnormality in the
protein secretory pathway. To explore the signals that direct APP into
the protein secretory pathway, we introduced a series of single amino
acid substitutions into the 735-755-amino acid region of the
cytoplasmic domain of
APP770, which lacks the 287-amino acid region
between amino acids 379 and 666 of APP770, but undergoes identical
intracellular metabolism as endogenous APP in transfected cells (24).
The mutants were then examined for their level of production of APP
carboxyl-terminal fragments, CTF
and CTF
(Fig. 1). The two known
functional internalization signal sequences, NPTY and YTSI
(underlined in Fig. 1b), were excluded from the
analysis because they have already been well characterized (21).

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Fig. 1.
Structure of APP and its carboxyl-terminal
fragments (a) and amino acid sequence of the APP
cytoplasmic domain (b). a: part i,
structure of APP770. The shaded box represents
the -amyloid domain, and arrows indicate sites cleaved by
-, -, and -secretases. The positions of primers 1-4, used to
produce the EcoO65I site and to induce the site-directed
mutations, are indicated. Part ii, structure of
CTF . Part iii, structure of CTF .
Arrows 1-4 indicate potential heterogeneous
cleavage sites of -secretase. TM, transmembrane domain.
b: amino acid sequence of the cytoplasmic domain of
APP770wt. Underlining indicates the signal sequences for APP
internalization (20, 21). R indicates the
position of Arg747 of APP770 and the position of
Arg672 of APP695. The amino acid sequence of the
cytoplasmic domain is identical among the three major isoforms, APP770,
APP751, and APP695.
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293 cells (human transfected primary embryonal kidney) were transfected
with wild-type (p
APP770wt) and mutant (p
APP770mt) plasmids (see
"Experimental Procedures" for plasmid construction), and then
several independent clones of cells expressing stably transfected
APP770 were isolated. CTF was recovered from 293 cells expressing
wild-type (
APP770wt) or mutant (
APP770mt) APP by
immunoprecipitation with polyclonal anti-APP cytoplasmic domain antibody UT-421 (epitope(s) exists within APP770-(759-770)) and was
detected by immunoblot analysis using the same antibody (Fig. 2). CTF
, which is a product of
cleavage by
-secretase, was detected in 293 cells expressing
APP770wt, (Fig. 1a (part
ii) and arrows in Fig. 2 (a and
b)). The CTF
from endogenous APP was below the detectable
levels (data not shown). CTF
with a higher molecular mass than
expected was detected (Fig. 1a (part
iii) and arrowheads in Fig. 2 (a and
b)) in 293 cells expressing the
APP770 R747A (substitution of Ala for Arg747) mutant protein. A weak
CTF
band (20-30% of the amount of CTF
in terms of
radioactivity) was also detectable in cells expressing
APP770wt
after longer exposure of the autoradiogram (Fig. 2, b and
c) or after metabolic labeling of protein with
[35S]methionine (see Fig. 5a). A moderate
amount of CTF
(~50% of the amount of CTF
in terms of
radioactivity) was also detectable in cells expressing
APP770 L749A
(substitution of Ala for Leu749) and
APP770 K751A
(substitution of Ala for Lys751) mutants
(arrowheads in Fig. 2 (b and c)).
Although the differences in the production level of CTF
among the
substitution mutants reflected the expression level of
APP770mt in
the cell, none of the alanine-scanning mutants, except for
APP770
R747A, containing a substitution within the 735-755-amino acid region
of APP770, led to greater CTF
production (~130% of the amount of
CTF
) when compared with
APP770wt (see the ratio CTF
/CTF
in
Fig. 2c and data not shown for the site between
Val735 and Thr743). This result suggests that
the argininyl residue at amino acid 747 of APP770, or possibly the
domain consisting of 5 amino acids from Arg747 to
Lys751 of APP770, plays an important role in APP cleavage
at the
-site.

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Fig. 2.
Production of carboxyl-terminal fragments
from APP770wt and APP770mt.
a, the CTFs of APP770wt and APP770mt carrying the
alanine-scanning mutation from amino acids 744 to 755 are indicated.
The arrow indicates CTF , and arrowheads
indicate CTFs . b, shown is a longer exposure of the
autoradiogram in a. The sizes (in kDa) of the molecular mass
standards are indicated. c, CTFs detected with
125I-protein A were quantified with an imaging analyzer.
The ratio of CTF to CTF was determined and compared with the
production level of CTFs . Results are the average of duplicate
studies, and error bars are indicated.
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The CTF
derived from
APP770 R747A was identified by monoclonal
anti-A
antibody 2D1, which recognizes a human-specific amino acid
sequence, FRH (APP770 amino acids 676-678 and APP695 amino acids
600-602), between the
- and
-secretase sites (Fig.
3). In Fig. 3, we show that CTF was
recovered by immunoprecipitation with antibodies UT-421 and 2D1. When
the immunoprecipitates were subjected to electrophoresis, both CTF
(arrow) and CTF
(arrowheads) were detected
with antibody UT-421. CTF was clearly competed out by the addition of
antigen peptide, APP770-(751-770). Antibody 2D1 recognized four CTF
species (arrowheads 1-4), but not CTF
(arrow), and these four CTF
species were competed out by
the addition of the A
-(1-15) (APP770-(672-687)) peptide. These
results confirm that CTF
derived from
APP770 R747A is a product
formed by the action of
-secretase and that it contains the
-amyloid domain.

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Fig. 3.
Characterization of carboxyl-terminal
fragments from APP770 R747A. CTFs were
immunoprecipitated (I.P.) from cells expressing APP770
R747A with the indicated antibodies (UT-421 and 2D1) in the presence
(+) or absence ( ) of competitor peptides (10 µM). CTF
was probed with antibody UT-421 and detected by ECL. The
arrow indicates CTF . Arrowheads
1-4 are heterogeneous CTF species (see Fig.
1a).
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To further analyze the function of the amino acid at position 747 of
APP770, we substituted another basic amino acid, lysine (
APP770
R747K), and an acidic amino acid, glutamic acid (
APP770 R747E) for
Arg747 (Fig. 4a).
Identical substitution mutations were also introduced into the entire
length of APP695, a neuron-specific isoform (Fig. 4b), as
described under "Experimental Procedures." The 293 cells stably
expressing these APP proteins with a mutation at amino acid 747 of
APP770 (position 672 of APP695) were also isolated and analyzed for
intracellular CTF
production. The production of the heterogeneous
CTF
species was also clearly observed when Arg747 of
APP770 (Arg672 of APP695) was replaced by Glu (
APP770
R747E and APP695 R672E), but not when replaced by Lys (
APP770 R747K
and APP695 R672K) (arrowheads in Fig. 4). Because only small
quantities of CTF
3 and CTF
4 were usually
detected (Figs. 2 and 3), all CTF
species cannot be observed in Fig.
4. The substitution of the acidic amino acid at position 747 of
APP770 (position 672 of APP695) with a basic amino acid seemed to
have more drastic effects on the production of the CTF
species
compared with that seen after substitution with alanine. The slight
upward mobility shift of the substitution mutants of CTF
(arrows) and CTF
(arrowheads) may be due an
alteration of the charge of CTF (Figs. 2 and 4) because this shift was
not observed in
APP770 R747K or APP695 R672K, where the basic amino acid was replaced by other basic amino acids (Fig. 4). There was no
difference between the
APP770 and APP695 isoforms with respect to
these mutations. This result indicates that the basic amino acid at
position 747 of
APP770 (position 672 of APP695) is essential for the
cleavage of APP at the
-site and that the substitution of a
non-basic amino acid with a basic amino acid alters APP metabolism in
the protein secretory pathway.

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Fig. 4.
Production of carboxyl-terminal fragments
from APP770 and APP695 carrying a mutation at amino acid
747 of APP770 (amino acid 672 of APP695). Arginine
at amino acid 747 of APP770 (amino acid 672 of APP695) was changed
to alanine ( APP770 R747A and APP695 R672A), lysine ( APP770 R747K
and APP695 R672K), and glutamic acid ( APP770 R747E and APP695
R672E). a, CTFs from APP770wt (wt) and
APP770mt; b, CTFs from APP695wt (wt) and
APP695mt. CTFs were detected with 125I-protein A. Arrows indicate CTF , and arrowheads indicate
CTF species. The sizes (in kDa) of the molecular mass standards are
indicated.
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Intracellular Generation and Degradation of CTFs--
To examine
for eventual differences between the intracellular generation and
degradation of CTF
and CTF
deriving from APP695 R672A and those
derived from APP695wt, a pulse-chase study was performed (Fig.
5). Cells expressing APP695 R672A and
APP695wt were labeled with [35S]methionine and chased for
the indicated times (Fig. 5a). The radioactivities of CTF
and CTF
1-4 were quantified, and the relative ratios
with respect to the maximum level were calculated (Fig. 5b
for CTF
and Fig. 5c for CTF
1-4). CTF
(squares) and CTF
(circles) derived from
APP695 R672A (closed symbols) and APP695wt (open
symbols) were produced and degraded at almost the same rate (Fig.
5, b and c). These results clearly indicate that
both CTF
and CTF
derived from APP695 R672A are generated and
degraded in the same way as those derived from APP695wt, except for
APP695 R672A, which was more efficiently cleaved by
-secretase. Thus, the increase in the level of CTF
resulting from the APP695 R672A mutation, which was detected by immunoblot analysis (Figs. 2-4),
is not due to a simple intracellular accumulation of CTF
. The CTFs
from APP695 R672A, as well as those from APP695wt, are subject to
further degradation.

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Fig. 5.
Intracellular rates of generation and
degradation of the carboxyl-terminal fragments from APP695wt and APP695
R672A. APP was pulse-labeled with [35S]methionine
and chased for the indicated times. a, shown are
autoradiograms of CTF and CTF 1-4 from APP695wt
(left) and APP695 R672A (right). b and
c, the incorporation of radioactivity into CTF was
determined as described under "Experimental Procedures," and the
relative ratios of the levels of CTF (b) and CTF 1-4
(c) to maximum levels, which were assigned a reference value
of 1.0, were calculated. Results are the average of duplicate assays.
Error bars are indicated.
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Secretion of sAPP from Cells Expressing APP695 R672A--
Because
the large extracellular amino-terminal domain truncated at
- or
-sites (sAPP
/
) is believed to be always secreted into the
medium, we examined the secretion of sAPP
/
derived from APP695
R672A and compared it with that derived from APP695wt. The culture
medium of the pulse-chase studies (Fig. 5) was collected; sAPP
/
was recovered by immunoprecipitation with anti-APP extracellular domain
antibody 22C11; and the immunoprecipitates were subjected to SDS-PAGE
(7.5% (w/v) polyacrylamide), followed by autoradiography (Fig.
6a). It was not possible to
distinguish sAPP
from sAPP
on SDS-PAGE as described previously
(33). The arrow in Fig. 6a indicates a band
containing both sAPP
and sAPP
. The sAPP
/
from APP695 R672A
(closed circles) appeared slightly earlier than that from APP695wt (open circles) in the medium
(Fig. 6b). The secretion of sAPP
/
reached the maximum
level after 3 h (APP695 R672A) or after 5 h (APP695wt). The
slight difference may be due to the fact that sAPP from APP695 R672A
probably contains more sAPP
than sAPP from APP695wt. This result
suggests the possibility that sAPP
may be secreted earlier than
sAPP
, although we did not detect any remarkable differences between
the metabolic rates of intracellular APP695 (Fig. 6c) and
CTFs (Fig. 5). The secretion level of sAPP
/
from APP695 R672A
seemed to be low when compared with the level of APP expression (Fig.
6a). Therefore, the relative ratio of the level of
sAPP
/
to that of intracellular APP was determined at different
time points during the pulse-chase experiment (Fig. 6d). The
secretion level of sAPP
/
from APP695 R672A was lower (~50% at
3 h and ~30% at 5 h of APP695wt) compared with that from
APP695wt when the values were standardized with the level of APP
expression (Fig. 6d). This may be a reason why the secretion
of sAPP from APP695 R672A reached a plateau at 3 h. This result
suggests that sAPP
/
from APP695 R672A is not always secreted into
the medium and that a certain amount of sAPP
/
from APP695 R672A
is subject to intracellular degradation prior to secretion.

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Fig. 6.
Secretion rate of sAPP, intracellular
metabolic rate of APP, and secretion level of sAPP. sAPP /
was recovered from the medium, and APP was recovered from the cell
lysate of the pulse-chase experiment (see Fig. 5) by
immunoprecipitation. a, shown are autoradiograms of
sAPP / from APP695wt (left) and APP695 R672A
(right). b and c, the incorporation of
radioactivity into sAPP / and APP was determined as described
under "Experimental Procedures," and the relative ratios of the
levels of sAPP / (b) and APP (c) are
indicated relative to the maximum levels, which were assigned a
reference value of 1.0. d, the level of sAPP /
secretion was normalized to the level of APP expression and assigned an
sAPP/APP ratio. Results are the average of duplicate assays, and
error bars are indicated.
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Secretion of A
from Cells Expressing APP695mt--
If CTF
is
always cleaved by
-secretase, cells expressing APP695 R672A should
generate a greater amount of A
, as the pulse-chase study of Fig. 5
indicates that CTF
from APP695 R672A did not accumulate
intracellularly, instead but was subject to further metabolism. To
examine this possibility, the amount of A
in the medium was analyzed
with sandwich ELISA using three types of monoclonal anti-A
antibodies: 2D1, which recognizes the human-specific amino acid
sequence FRH (amino acids 600-602) of APP695 in the A
sequence; 4D1
(epitope is the terminal of A
40), which recognizes APP derivatives truncated at A
40; and 4D8 (epitope is the terminal of A
42), which
recognizes APP derivatives truncated at A
42 (24). Combinations of
antibodies 2D1 and 4D1 or 4D8 in sandwich ELISA allows the specific
measurement of the amount of A
40 or A
42 secreted into the medium.
We established additional 293 cell lines that stably expressed APP695
carrying an FAD mutation and APP695 carrying both the FAD mutation and
an R672A substitution. We did this because it is well known that APP
carrying an FAD mutation is subject to active
-site cleavage and
subsequent secretion of A
species following the cleavage of CTF
by
-secretase (30, 34, 35). When 293 cells (~2 × 106 cells) expressing APP695wt were cultured in 3 ml of
medium for 24 h and 100 µl of the medium was quantified for
A
, usually 40-50 fmol of A
40 and 20-30 fmol of A
42 were
detected. Because the amount of A
secretion is affected by the level
of APP expression in the established cell lines, we have indicated the
secretion level of A
as a ratio of the level of APP expression that
was determined from the intracellular APP content (Fig.
7). The APP695 R672A mutation suppressed
the production of A
40 more efficiently than that of A
42. The data
suggest that the APP695 R672A mutation does not contribute to the
generation the A
40 and A
42, at least in 293 cells, despite the
fact that a greater amount of CTF
was generated intracellularly.
This result also indicates that the increase in CTF
does not always
result in increased secretion of A
and that CTF
derived from the
APP695 R672A mutation is subject to degradation prior to cleavage by
-secretase.

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Fig. 7.
Secretion of A from cells expressing
APP695wt and APP695mt. A 40 (a) and A 42
(b) in the medium were quantified by sandwich ELISA with
A 40 (4D1) and A 42 (4D8) end-specific antibodies. To estimate the
level of APP695 expression, APP from cells stably transfected with
plasmids was immunoprecipitated, detected by immunoblotting with
antibody UT-421 following SDS-PAGE (7.5% (w/v) polyacrylamide gel),
and quantified using a Fuji BAS 2000 imaging analyzer (data not shown).
The level of APP695 expression (a relative ratio) was normalized to the
amount of APP from a clone expressing APPwt. The amount of
A (fmol/100 µl of medium) was divided by the relative APP695 ratio
and is expressed as the A /APP695 ratio. wt, cells
expressing APP695wt; R672A, cells expressing the APP695
R672A mutation; V642F, cells expressing APP695 V642F
(London mutation); R672A/V642F, cells expressing
a double mutation of APP695 R672A and APP695 V642F; sw,
cells expressing the Swedish mutation (a double mutation of
APP695 K595N and APP695 M596L); R672A/sw, cells expressing a
double mutation of APP695 R672A and the Swedish mutation.
Results are the average of several independent studies with independent
clones (n = 4-7), and error bars
indicate S.D. *, p < 0.01; ***, p < 0.001.
|
|
The Swedish mutation (APP695sw), which is thought
to enhance the production of A
by increasing its sensitivity to
-secretase (30, 34, 36, 37), caused ~a 23-fold increase in A
40
and a 6-fold increase in A
42 production. Another FAD mutation, the "London (Hardy)" mutation (APP695 V642F) (38, 39), also
caused a remarkable increase in A
40 and A
42 secretion. The
results of this study are consistent with these previous reports
describing the effect of the FAD mutation on A
production (35).
Interestingly, the level of A
40 secretion from cells carrying a
double mutation of R672A and FAD, Swedish
R672A/sw and London R672A/V642F, was less than
that from cells carrying the original FAD mutation alone (Fig.
7a). This result indicates that the R672A mutation induces a
decrease in the A
40 secretion from cells carrying the FAD mutation, as in the case for wild-type APP. An identical double mutation of R672A
and FAD caused no remarkable decrease in A
42 secretion (Fig.
7b), which suggests that the secretion of A
42 may be
different from that of A
40. Furthermore, these observations support
the notion that APP carrying the R672A mutation is cleaved in a
secretory pathway that differs from the normal secretory pathway, where APP carrying the FAD mutation is cleaved.
 |
DISCUSSION |
Previous studies in cells expressing APP with a truncated
cytoplasmic domain have not been able to clarify the function of this
cytoplasmic domain in detail because the cytoplasmic domain is thought
to contain multiple signal domains regulating APP metabolism (15, 40).
Therefore, we tried to explore the intracellular sorting signals in the
APP cytoplasmic domain with an alanine-scanning mutation, a single
amino acid substitution of alanine for the original amino acid. We
focused on the production of intracellular CTFs in cells expressing APP
carrying the alanine-scanning mutation because an alteration of the
protein secretion system should first of all induce CTF
generation
and because the APP cytoplasmic domain may function to direct APP into
the normal secretory pathway. In this study, we found that the basic
amino acid at position 747 of APP770 (position 672 of APP695) in the
cytoplasmic domain plays an important role in the cleavage of APP at
the
-site. This basic amino acid seems to be essential for the
normal metabolism of APP in the normal secretory pathway, in which APP
is cleaved preferentially by
-secretase rather than by
-secretase
when APP does not carry the FAD mutation (Fig.
8). The increased production of CTF
derived from APP695 R672A did not result in increased secretion of A
and sAPP. The most likely interpretation of these observations is that
CTFs
do not migrate to the compartment of the normal secretory
pathway and are subject to degradation prior to cleavage by
-secretase (Fig. 8). The alternative possibility, that the argininyl
residue at amino acid 672 of APP695 is critical for
-secretase
recognition, does not seem plausible because the production of A
42,
at least, was not affected by the R672A mutation of APP695 and because
a double mutation of R672A and FAD increased A
secretion above the
level seen with the R672A mutation alone. Therefore, we postulate a
default secretory pathway in which APP carrying the R672A mutation is
cleaved by
-secretase as well as by
-secretase (Fig. 8).

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Fig. 8.
Putative secretory pathways of intracellular
APP metabolism. Cells are postulated to have at least two protein
secretory pathways, the normal (upper) and default
(lower) secretory pathways of APP secretion. In healthy
(normal) cells, APP is generally directed into the normal secretory
pathway, in which APP is cleaved preferentially at the -site. An FAD
mutation such as the Swedish mutation (**) enhances the
amyloidogenic metabolism of APP, the cleavage of APP at the -site,
in the normal secretory pathway. Then, CTF / is always cleaved by
-secretase, and the resulting A and p3 fragment are secreted from
the cell. sAPP / is also secreted. On the other hand, the APP770
R747A/APP695 R672A mutation is thought to direct APP into the default
secretory pathway due to inefficient function of putative cytoplasmic
factor(s). APP in the default secretory pathway is cleaved at the
-site as well as the -site. The resulting sAPP / is not
always secreted, and CTF / is not always subject to subsequent
cleavage by -secretase. Some sAPP / and CTF / is degraded
intracellularly prior to secretion. When the regulatory steps of
-secretase in the default secretory pathway become aberrant for some
reason such as aging, the relatively large amount of CTF will be
further cleaved by -secretase prior to degradation, and an increased
amount of A will be generated. This may explain how the pathogenic
state of sporadic AD continues to develop.
|
|
The present observation also showed that CTF
does not always
generate A
and that sAPP is not always secreted, although the increased production of CTF
is thought to result in increased secretion of A
and sAPP in the case of APP carrying the FAD mutation (Fig. 8). The decreased secretion of sAPP derived from APP695 R672A may
be due to the intracellular degradation of sAPP in the default
secretory pathway. CTFs from the APP695 R672A mutation, as well as CTFs
from APP695wt, do not accumulate in the cell and are degraded. This may
be the reason why CTF
does not generate A
in the case of the
APP695 R672A mutation. These facts may be relevant to A
production
in the case of the sporadic type of AD. The molecular mechanism that
results in APP695 migration into a default secretory pathway that would
be enriched in
-secretase in the R672A mutant has not been
elucidated. Further analyses of the intracellular metabolism of APP695
R672A will be important for our understanding of what regulates
-secretase processing of CTF
and determines
-secretase
activation in the default secretory pathway.
APP695 R672A may be directed into a default secretory pathway by a
putative cytoplasmic factor that can associate with the APP cytoplasmic
domain sequence around Arg747 of APP770 and
Arg672 of APP695. The protein may play a role in the
direction of APP into the normal secretory pathway (Fig. 8). The effect
of the APP770 R747A (APP695 R672A) mutation on CTF
production
probably results from interference with the binding of this
hypothetical factor to the APP cytoplasmic domain. One recent report,
which indicates that transgenic mice expressing CTF
develop
extracellular A
with age (41), may be in support of this idea. Aging
may induce a loss of function of the cytoplasmic factor and activate
-secretase activity in a default secretory pathway. Another recent report, which indicates that knockout mice for presenilin-1 have increased levels of CTF
, but decreased secretion of A
(42), may
also be relevant to our present results. The basic amino acid (Arg672) of APP695 may play an important role in the APP
secretory pathway related to presenilin-1.
Furthermore, when compared with the CTF
production in cells
expressing APP695 R672A, secretion of A
into the medium
was reduced slightly (A
40) or did not change (A
42). This result is not necessarily cell-type specific, at least in non-neuronal cells,
because identical results, i.e. increased CTF
but
unchanged secretion of A
, were obtained when APP695 R672A was stably
expressed in Chinese hamster ovary cells (data not shown). Double
mutants of APP695 R672A and FAD also suppressed the secretion of
A
40, but not A
42, when compared with the FAD-only mutant,
indicating that the mechanism of A
42 production is different from
that of A
40 production. It has been shown clearly that the FAD
mutation increases the secretion of both A
40 and A
42, which are
thought to be generated in the normal secretory pathway (30, 34, 36, 37). Therefore, these results suggest that the cleavage of APP695 R672A
by
-secretase occurs in a compartment different from that used for
the cleavage of APPwt and APP carrying the FAD mutation at their
-sites (Fig. 8). Further analysis of the metabolism of APP695 R672A
in this default secretory pathway should contribute to our
understanding of the molecular mechanism of APP processing in the
sporadic type of AD pathogenesis.
We thank Drs. M. Oishi (Montifore Medical
Center, Hartsdale, New York) and S. Takeda (University of Tokyo) for
critical reading and comments on the manuscript, Drs. K. Yamamoto
(University of Tokyo) and T. Ozaki (Chiba Cancer Center, Chiba, Japan)
for technical advice, and S. Oguchi for technical assistance.