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INTRODUCTION |
Mutations in the two presenilin genes (PS1 and
PS2) are the most common cause of early-onset familial
Alzheimer's disease (for review, see Ref. 1). Encoded by genes on
chromosomes 14 and 1, respectively, PS1 and PS2 are highly homologous
proteins predicted to contain eight transmembrane helices (2, 3). Immunocytochemical analysis has shown that PS1 is localized mainly in
the endoplasmic reticulum and, to a lesser extent, in the Golgi compartment (4, 5). More than 40 missense mutations have been
identified in PS1 (6, 7), and these mutations account for up to 60% of
early-onset cases of familial Alzheimer's disease (8). Many of these
mutations occur within transmembrane domains or immediately adjacent to
the predicted hydrophilic loop between transmembrane domains 6 and 7 (9). A large family from Colombia has a E280A mutation in the
hydrophilic loop of PS1; affected individuals show massive deposits of
A
42 peptide in many brain regions (10). The mutant gene products of
PS1 and PS2 cause dysfunction and/or death of vulnerable populations of
nerve cells, with the resulting clinical syndrome of progressive dementia.
To examine the in vivo role of PS1 in mammalian development,
mice with a targeted disruption of the PS1 gene were
generated (11, 12). Homozygous mutant mice failed to survive beyond the
first 10 min after birth. The most striking phenotype observed in
PS1 null embryos was a severe perturbation in the
development of the axial skeleton and ribs. The failed development of
the axial skeleton in PS1 null mutants was traced to defects
in somitogenesis; at embryonic days 8.5 and 9.5, somites were
irregularly shaped and misaligned along the entire length of the neural
tube and largely absent in the most caudal regions. The abnormal somite patterns in PS1 null embryos are highly reminiscent of
somite segmentation defects in mice with functionally inactivated
notch-1 and dll-1 genes (13, 14). Indeed, the
expression of mRNA that encodes Notch-1 and Dll-1 is strongly
reduced in the presomitic mesoderm of PS1 null mice
(12).
This raises the possibility that the presenilins play an important role
in Notch signaling during embryonic development and/or cellular
differentiation. In support of this notion, Sel-12, the presenilin
homolog in Caenorhabditis elegans, functions as a
co-receptor for the nematode notch receptor, Lin-12 (15), and a
misfunction of the sel-12 gene causes constitutive
activation of lin-12, resulting in an egg-lying defect (15).
How these functions contribute to the pathological properties of mutant
presenilins in Alzheimer's disease remains to be determined.
To elucidate the normal biological role of the presenilins, efforts
have been made to identify molecules that interact with the
presenilins. In three recent reports (16-18), PS1 was shown to
interact with different members of the catenin family of proteins, termed
- and
-catenin. Pathogenic mutations in the PS1
gene reduced the ability of PS1 to stabilize
-catenin and caused an increased degradation of
-catenin in the brains of transgenic mice
(17).
The catenins are mammalian homologs of the Drosophila
armadillo protein predicted to participate in the Wingless signaling pathway. The Notch and Wingless signaling pathways have been described to be mutually inhibitory and are functionally associated with the
protein called Dishevelled (19). Interestingly, the human homolog of
Dishevelled has been mapped to a region of chromosome 3 that has
recently been linked to late-onset Alzheimer's disease (6, 20).
We have now used yeast two-hybrid screens to identify molecules that
bind to the hydrophilic loop of PS1. A single interacting protein was
discovered, termed p0071, identical to a recently reported novel member
of the armadillo family (21). The p0071 sequence covers three distinct
domains: the N- and C-terminal domains lack significant homology to
other known proteins, whereas the central domain represents a stretch
of 10 armadillo repeat domains with strong homology to
-catenin,
p120, and B6P/plakophilin-1 (18, 21). The interaction of PS1 with p0071
was confirmed by three independent methods, i.e. yeast
two-hybrid retransformation assays, binding to
GST1 fusion proteins, and
immunoprecipitation from brain and cotransfected COS cells. The
interaction is mediated by the C-terminal fragment of PS1
constitutively formed by proteolytic processing under in vivo conditions (22). Neither the hydrophilic N or C terminus of
PS1 nor the loop of PS2 shows an interaction with p0071. Nine out of 10 armadillo repeat domains in p0071 are essential for mediating this
interaction, consistent with the paradigm that individual motifs or
couples of these motifs function as protein modules in signaling
cascades establishing protein-protein interactions (23).
Our studies provide compelling evidence that a novel member of the
armadillo family, termed p0071, interacts in a specific manner with
PS1. In contrast to the recently reported armadillo protein
-catenin, which interacts with PS1 and shows a brain-specific expression pattern (18), p0071 and
-catenin are broadly expressed in
a variety of tissues (1, 21), fitting well with the widespread expression of PS1 (24). Thus, it seems that differential interactions modulated in a tissue- or developmental stage-specific manner may be
the molecular basis for the different functions of the normal and
mutant forms of the presenilins.
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MATERIALS AND METHODS |
cDNA Cloning, Construction of Yeast Bait and Prey Vectors,
Construction of Bacterial and Eukaryotic Expression Vectors, COS Cell
Expression, and Sequencing--
Full-length cDNAs for rat PS1 and
PS2 were generated by polymerase chain reaction (PCR) using primers
specific for the respective 5'- and 3'-ends of the presenilin coding
regions and rat brain cDNA (CLONTECH) as
template. For amplification by PCR, Pfu polymerase (Stratagene) was used. Subcloning into pBluescript SK II and sequencing confirmed the identity of PS1 and PS2. These presenilin constructs served as templates to amplify DNA fragments with specific primers containing the restriction enzyme sites BamHI at the 5'-end
and EcoRI at the 3'-end, respectively, for subcloning into
the yeast bait vector pLexN (25).
Yeast prey vector constructs were obtained by subcloning of p0071 DNA
fragments into pVP16 (25) using BamHI and NotI as restriction sites. p0071 fragments were generated by PCR as described above. A partial p0071 clone obtained by a yeast two-hybrid screen of a
rat brain library was used as template.
A PS1 construct with its hydrophilic loop fused to GST was generated by
subcloning an EcoRI-SalI fragment into the vector pGEX-5X-1 (Amersham Pharmacia Biotech). It was expressed in
Escherichia coli (XL1-Blue) and purified as described
(26).
His6-tagged p0071 was created by cloning a 2.5-kilobase
pair BamHI-HindIII fragment into pQE-9 (Amersham
Pharmacia Biotech). The final construct was obtained by PCR
amplification introducing a Kozak sequence (CCACCATGA) and subsequent
cloning into the pCMV-based vector pcDNA3 (Invitrogen). Full-length
PS1 was amplified by PCR using a sense primer with a Kozak sequence and
a PS1 construct as template (see above). Subsequently, a 1.4-kilobase
PCR fragment was cloned into the BamHI-EcoRI site
of pcDNA3. These two pcDNA3 constructs were used for
transfection of COS cells. Plasmid DNA was transfected into COS cells
using DEAE-dextran, and transfected COS cells were harvested 48 h
after transfection (27).
The following primers were used for amplification by PCR: for
PS1, 5'-ACCGGATCCGCTGCTCCAATGACAGAGATACCTGCAC (sense, starting at aa
1), 5'-GGAGAATTCTTAGATATAAAATTGATGGAAT (antisense, ending at aa 468),
5'-GGAGAATTCTGCCCCAAAGGTCCACTTCG (sense, starting at aa 263),
5'-GGAGAATTCATGGTGTGGTTGGTGAATATG (sense, starting at aa 292),
5'-GGAGAATTCGCTGAAGGAGACCCAGAAGC (sense, starting at aa 299),
5'-GGAGGATCCTTATGTGTTCCAGTCCCCACTGGC (antisense, ending at aa 407),
5'-GGAGGATCCTTAGACAGCAGCTCTTGACTCAG (antisense, ending at aa 362),
5'-GGAGGATCCTTAGCCAGTGTCCTGTGTCTCTTC (antisense, ending at aa
329), 5'-GCTGAATTCATGACAGAGATACCTGCAC (sense, starting at aa 1),
5'-CGGGGATCCTTAGACGTGCTTGGCTCCATAT (antisense, ending at aa 82),
5'-GCTGAATTCTTCAAGAAAGCGTTGCCGG (sense, starting at aa 429), and
5'-CGGGGATCCCTAGATATAAAATTGATGGA (antisense, ending at aa 468); for
PS2, 5'-ACCGGATCCATGCTCACATTCATGGCCTC (sense, starting at aa 1),
5'-GGAGAATTCTCAGATGTAGAGCTGGTGGG (antisense, ending at aa 448),
5'-GCTGAATTCTGCCCCAAAGGGCCACTGAG (sense, starting at aa 269), and
5'-CGGGGATCCTTAAGTGTTCCAGTCTCCGT(antisense, ending at aa 387); and for
p0071, 5'-CCGGATATCCCACCATGAGAGGATCGCATCACC (sense, starting at aa
104), 5'-GCATGCGGCCGCCCAAGCTCAGCTAATTAAGC (antisense, ending at aa
963), 5'-CTTGGATCCACGACACTGTTCATCCCAAC (sense, starting at aa 107),
5'-ATTGGATCCGATCCCCATCAATAGACAG (sense, starting at aa 510),
5'-ATTGGATCCGACTGCAGCACTTGTGTTTTGG (sense, starting at aa 552),
5'-ATTGGATCCGACTGCGAAACCTTGTGTTTGG (sense, starting at aa 594),
5'-ATTGGATCCGACTTTGGAATTTATCCTCATG (sense, starting at aa 639),
5'-TCAGCGGCCGCTTATCTTGTGGTGCCATCATCAG (antisense, ending at aa 509),
5'-TCAGCGGCCGCTTAACAGCAGATGGCTGCCACAG (antisense, ending at aa 940),
and 5'-TTTATAGCGGCCGCTTACTCTATACCTCCCGAGTCAG (antisense, ending
at aa 963).
The identity of most DNA constructs was confirmed by sequencing. DNA
sequencing was performed by the dideoxy nucleotide chain termination
method using fluorescently labeled primers and an ABI370A DNA sequencer.
Yeast Two-hybrid Screens and Interaction Assays--
The yeast
strain L40 (25) was sequentially transfected with the bait vector
(hydrophilic loop of PS1 cloned into pLexN) and a rat brain cDNA
library (25) using lithium acetate (28). Transformants were selected on
plates lacking histidine, uracil, tryptophan, lysine, and leucine.
Positive clones were picked after 4 days at 30 °C, and the
-galactosidase activity of the clones was assayed by the filter
method. Extrachromosomal DNA was isolated from clones that grew in the
absence of histidine and were
-galactosidase-positive using the
glass bead method (29). Prey plasmids were rescued in E. coli HB101 cells by electroporation and selection on M9 plates
containing 50 mg/liter proline and 0.1 g/liter ampicillin. For
quantitation of
-galactosidase activity, yeast strains transfected with bait and prey vectors were grown in supplemented minimal medium
lacking uracil, tryptophan, and leucine in a shaking incubator at
30 °C for 48 h.
-Galactosidase assays were performed on
yeast extracts with protein concentrations of 0.5 mg/ml/assay as
described previously (30).
Binding of Recombinant p0071 to Immobilized GST Fusion
Proteins--
COS cells transfected with His6-p0071 in
pcDNA3 were solubilized in phosphate-buffered saline containing 1%
Triton X-100 and 1 mM phenylmethylsulfonyl fluoride and
centrifuged at high speed (10,000 × g) to remove
insoluble material. The hydrophilic loop of PS1 fused to GST was
expressed in E. coli and purified according to standard
procedures (Amersham Pharmacia Biotech). Glutathione beads with
immobilized GST fusion protein were incubated with solubilized
His6-p0071-transfected COS cells at 4 °C for 1 h. After four washing steps with phosphate-buffered saline containing 1%
Triton X-100 and 1 mM phenylmethylsulfonyl fluoride, SDS
sample buffer was added to the beads. Proteins eluted from these beads were analyzed by SDS-PAGE and immunoblotting using the enhanced chemiluminescence method as the detection system (Amersham Pharmacia Biotech).
Antibodies--
Antibodies were raised in rabbits immunized with
the N terminus of rat PS1 encompassing residues 1-80. These antibodies
were affinity-purified on GST-PS1 (aa 1-80) immobilized on a
polyvinylidene difluoride membrane. The monoclonal antibody APS 18 (31)
was kindly provided by K. Beyreuther (Center for Molecular Biology, 69120 Heidelberg, Germany). It was raised against a peptide
corresponding to amino acids 314-334 of the large loop of PS1. A
rabbit antiserum directed against human p0071 (21) was a gift from C. Nachtsheim (Max Planck Institute for Physical Chemistry, 37077 Göttingen, Germany) and M. Hatzfeld (Zentrale Abteilung der
nedizinischen Fakultät Halle, Halle, Germany). A monoclonal
antibody directed against the His6 epitope was purchased
from QIAGEN Inc.
Miscellaneous Procedures--
SDS-PAGE, immunoblotting, and
immunoprecipitations were performed using standard procedures as
described previously (32). Protein loads were equalized based on
Coomassie Blue staining of the gels and on protein assays. Protein
concentrations were determined according to Bradford (33) using bovine
serum albumin as a standard.
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RESULTS |
Yeast Two-hybrid Screen for PS1-interacting Proteins--
Many of
the mutations causing an early onset of familial Alzheimer's disease
are located in the proximal part of the hydrophilic loop in PS1 (1),
implying an important cellular function for this domain. Therefore, the
loop domain was chosen as bait for a yeast two-hybrid screen. Yeast
transformants (85 × 106) with a rat brain cDNA
library were screened for proteins interacting with the hydrophilic
loop of PS1 encompassing residues 263-407. Only a single positive
clone was identified from this screen, termed pPrey D1.
Retransformations of yeast with a combination of pPrey D1 and a set of
different bait plasmids demonstrated that
-galactosidase activation
occurred only when pPrey D1 was cotransfected with a bait construct
containing the hydrophilic loop of PS1 (Fig.
1). Other constructs of PS1, comprising
the hydrophilic parts of the N or C terminus, did not show any
detectable interaction, suggesting that the interaction observed was
specific. Sequencing revealed that pPrey D1 represented a rat homolog
of the human armadillo protein p0071 spanning residues 104-963
(21).

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Fig. 1.
PS1 interacts directly with armadillo protein
p0071. A yeast-two hybrid screen with a bait plasmid encoding the
hydrophilic loop of PS1 and a prey cDNA library from rat brain was
performed. As prey, a single positive clone encoding p0071 was
identified. The column diagram shows -galactosidase activities of
yeast clones harboring the respective bait and prey plasmids. The
interaction with p0071 was specific for the hydrophilic loop of PS1
(second column). -Galactosidase activity depended on both
of them (second column). There was no detectable
-galactosidase activity when one of these proteins was missing
(first and third columns). Neither the N terminus
of PS1 (fourth column) nor its C terminus (fifth
column) showed any interaction with p0071.
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Confirmation of the Interaction between PS1 and p0071--
To
confirm the validity of the interaction, three additional independent
sets of experiments were performed. The binding of p0071 to an
immobilized GST fusion protein containing the hydrophilic loop of PS1
was analyzed by SDS-PAGE and subsequent immunoblotting (Fig.
2). For this purpose, a His6
tag at the N terminus of p0071 was used as a recognition site for a
commercially available monoclonal antibody. His6-tagged
p0071 interacted specifically with the hydrophilic loop of PS1 fused to
GST (Fig. 2A, second lane). No binding to GST was
observed (Fig. 2A, first lane), demonstrating the
specificity of the interaction.

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Fig. 2.
The immobilized hydrophilic loop of PS1 binds
to p0071. A, a GST fusion protein containing the
hydrophilic loop of PS1 was immobilized on glutathione beads. These
beads were incubated with a Triton X-100 extract from COS cells
transfected with His6-p0071. Proteins bound to these beads
were analyzed by SDS-PAGE and immunoblotting for the His6
epitope. The hydrophilic loop of PS1 interacted specifically with
His6-p0071 (second lane). It did not interact
with endogenous COS cell proteins (third lane). GST alone
did not bind to His6-p0071 (first lane).
B, electrophoretically separated GST and GST-PS1
encompassing residues 263-407 were stained with Coomassie Blue. Equal
amounts of both proteins were used for binding assays.
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We conclude from this experiment that p0071 interacts in
vitro with the hydrophilic loop of PS1. To demonstrate an
interaction in vivo, we used COS cells and brain tissue for
co-immunoprecipitation experiments. COS cells were transfected
with His6-tagged p0071, full-length PS1, or a combination
of both. A polyclonal antibody raised against the N terminus of PS1
specifically recognized a 43-kDa protein in transfected COS cells and a
28-kDa protein in both transfected and untransfected cells (Fig.
3A). The relative molecular
masses of these proteins perfectly matched the expected sizes of
full-length PS1 and the N-terminal fragment of PS1, respectively (22).

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Fig. 3.
PS1 and p0071 co-immunoprecipitate from
transfected COS cells and human brain tissue. A,
extracts from COS cells transfected with full-length PS1 were
immunostained with a polyclonal antibody raised against the N terminus
of PS1. The N-terminal (N-term.) fragment of PS1 was
detected in both untransfected and transfected cells (marked by an
asterisk), whereas full-length PS1 was found only in
transfected cells (second lane, marked by an
arrow). B, COS cells were transfected with PS1
(first lane), His6-p0071 (second
lane), or a combination of both proteins (third lane).
PS1 was immunoprecipitated from solubilized COS cells with a specific
PS1 antibody (specificity of this antibody is shown in A).
From these cells, immunoprecipitates were analyzed by SDS-PAGE and
immunoblotting for the His6 epitope. Immunoprecipitates
from cotransfected COS cells contained His6-p0071
(third lane). His6-p0071 was not found in
immunoprecipitates from COS cells transfected with either
His6-p0071 (second lane) or PS1 (first
lane), demonstrating that PS1 and p0071 interact specifically in
cotransfected cells. The arrow and the asterisk
indicate the positions of His6-p0071 and the heavy chain of
the antibody used for immunoprecipitation, respectively. C,
PS1 was immunoprecipitated from solubilized human brain tissue (cortex)
with the monoclonal antibody APS 18, specific for the C-terminal
fragment of human PS1. Analysis of these immunoprecipitates with a
polyclonal antibody raised against p0071 revealed a strong band with a
molecular mass of 130-140 kDa (second lane, marked by an
arrow). This band was absent when a monoclonal antibody
directed against the synaptic vesicle protein synaptophysin was used
for immunoprecipitation (first lane, control). Please note
that endogenous p0071 (C) is bigger than recombinant p0071
(B) because a partial cDNA clone (aa 104-963) was used
for expression in COS cells. D, p0071 was precipitated from
solubilized brain tissue using the polyclonal antibody mentioned above.
These immunoprecipitates contained the C-terminal fragment
(C-term.) of PS1 (second lane, marked by an
arrow). Full-length PS1 did not co-immunoprecipitate. The
C-terminal fragment of PS1 was not detectable using the preimmune serum
instead of the p0071 antiserum (first lane, control).
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This antibody was used to immunoprecipitate solubilized PS1 from
transfected COS cells. The immunoprecipitates were analyzed by SDS-PAGE
and immunoblotting for His6-p0071. His6-tagged
p0071 and PS1 co-immunoprecipitated from cotransfected COS cells (Fig. 3B, third lane), demonstrating an interaction of
both proteins in vivo. No interaction was observed in single
His6-p0071-transfected cells (Fig. 3B,
second lane), although COS cells express endogenous PS1
(22). Most likely, endogenous PS1 was cleaved by a constitutively active protease, forming an N- and a C-terminal fragment (22). Since
the antibody used for immunoprecipitation is directed against the
N-terminal part of PS1, binding of p0071 to the C-terminal part would
not be detectable. However, in transfected cells overexpressing PS1,
full-length PS1 is the predominant form (Fig. 3A) (22).
The question now arises whether PS1 and p0071 interact in a human
tissue, e.g. in human brain. To address this question, PS1 was immunoprecipitated from solubilized human brain tissue (cortex) using the monoclonal antibody APS 18 (31). APS 18 was reported to be
specific for the C-terminal fragment of human PS1 (31). The
immunoprecipitates obtained with the PS1 and p0071 antibodies were
analyzed by gel electrophoresis and immunoblotting (Fig. 3,
C and D). Indeed, p0071 and PS1
co-immunoprecipitated, strongly supporting the notion that both
proteins form a stable complex in the brain. Taken together, we
conclude from these experiments that PS1 and the armadillo protein
p0071 specifically interact in vitro and in
vivo.
Determination of the Binding Domains in p0071 and PS1--
To
determine the domains mediating the interaction between p0071 and PS1,
we made a series of bait and prey vector constructs with inserts
shortened at their respective 5'- and 3'-ends. We then measured the
-galactosidase activities for various combinations of these
constructs by the yeast two-hybrid method.
p0071 is composed of three distinct domains: unique N- and C-terminal
parts encompassing residues 1-509 and 988-1211, respectively, and a
highly conserved middle part comprising a set of 10 armadillo repeat
domains (21). As depicted in Fig. 4,
armadillo repeat domains 2-10 are necessary and sufficient to mediate
the interaction with the hydrophilic loop of PS1. The unique N- and
C-terminal parts of p0071 are not necessary for interaction, although
it seems that these parts might strengthen the interaction (for
comparison, see constructs A and E). Since the
highly conserved armadillo repeats mediate the interaction, it is not
surprising that other members of the armadillo family, like
- and
-catenin, can also bind to PS1 (16-18).

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Fig. 4.
The armadillo repeats of p0071 mediate
interaction with PS1. N- and C-terminal deletion mutants of p0071
were coexpressed in yeast together with the hydrophilic loop of PS1.
A, the numbers on both sides of the p0071
constructs represent the N- and C-terminal amino acids of the various
constructs, respectively. The armadillo repeats of p0071 are depicted
as black boxes. B, the interaction of various
p0071 constructs with the hydrophilic loop of PS1 was determined by
measuring the specific activity of the reporter gene -galactosidase.
-Galactosidase activity was measured in triplicates and is shown as
the mean ± S.D. The shortest fragment of p0071 that could
interact with PS1 encompassed residues 552-963 (construct
E), showing that 8.5 out of 10 armadillo repeats are necessary and
sufficient to mediate the interaction.
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Under in vivo conditions, PS1 is specifically cleaved by a
protease, leading to the formation of stable N- and C-terminal fragments (22). These two fragments stay together and form a tight
complex with an unknown biological function (31). The question now
arises as to which part of the complex interacts with p0071. Therefore,
we generated multiple bait vector constructs. Two of these constructs
start with the residues reported recently (34) as the major
physiological cleavage sites in PS1. As shown in Fig.
5 (constructs D and
E), the C-terminal fragment of PS1 mediates the interaction
with p0071. Interestingly, the strength of the interaction was enhanced
by shortening the loop fragments (for comparison, see constructs
A, B, D, and E). The N-terminal presenilin fragment did not bind to p0071 (construct C).
Thus, only one part of the complex consisting of the presenilin
fragments is responsible for the interaction with p0071.

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Fig. 5.
The C-terminal part of PS1 binds specifically
to p0071. N- and C-terminal deletion mutants for the hydrophilic
loop of PS1 were generated and coexpressed in yeast together with
p0071. A, the numbers on both sides of the PS1
constructs specify the N- and C-terminal amino acids of individual
constructs, respectively. The arrows point to two major
physiological cleavage sides of PS1. B, the interaction of
these PS1 constructs with p0071 was determined by measuring the
specific activity of the reporter gene -galactosidase.
-Galactosidase activity was measured in triplicates and is shown as
the mean ± S.D. The N-terminal part of the PS1 loop failed to
bind to p0071 (construct C), whereas the C-terminal part
bound specifically to p0071 (construct E). A prey construct
devoid of p0071 (construct F) did not show any interaction
with the C-terminal part of PS1.
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Comparison of PS1 and PS2 with Regard to p0071 Binding--
As
mentioned above, the presenilins exist in two distinct isoforms, PS1
and PS2. The amino acid sequences of both isoforms are very similar,
implying that they are derived from a common ancient precursor. The
presenilin isoforms seem to function in a common and distinct manner
(24). Therefore, we asked whether the interaction with p0071 is a
common function of both presenilin isoforms.
The presenilin loop sequences from different species are depicted in
Fig. 6A using the CLUSTAL
method for alignment. Residues shared by at least three of the four
presenilins are shown on a black background. The sequences
at both ends of the loop are highly conserved (Fig. 6A). In
contrast, in the middle part of the loop, the sequences of rat PS1 and
PS2 are very divergent. This is exactly the region to which p0071 binds
(see Fig. 5 and the line in Fig. 6A). The high
degree of PS1-PS2 diversity in the middle of the loop predicts that
PS1, but not PS2, can interact with p0071. Indeed, the loop of PS2 did
not bind to p0071 (Fig. 6B, second column).

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Fig. 6.
PS2 does not interact with p0071.
A, the amino acid sequences of the hydrophilic loops of rat
PS1 and PS2 and mouse and human PS1 are aligned (9, 39, 40). The
sequences of rat PS1 and PS2 are highly conserved in the vicinity of
their N and C termini, but are very divergent in the middle part. The
arrows mark the positions of two major physiological
cleavage sites in PS1. The line indicates the binding domain
of rat PS1 to p0071 (see Fig. 5). In this region, rat PS1 and PS2 are
very divergent, explaining the inability of PS2 to bind to p0071 (see
B). In contrast, rat, mouse, and human PS1 are highly
conserved, predicting an interaction of these PS1 forms with rat p0071.
B, the hydrophilic loops of PS1 and PS2, respectively, were
coexpressed with p0071 in yeast. The interaction was determined by
measuring the specific activity of the reporter gene -galactosidase.
In contrast to PS1 (third column), PS2 did not bind to p0071
(second column).
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We conclude from this experiment that the presenilin-p0071 interaction
is confined to PS1, suggesting that the presenilin isoforms differ in
their functions. Indeed, quantitative reverse transcription-PCR studies
revealed that PS1 and PS2 mRNAs are expressed
at significantly different levels among tissues and in brain
development (24). Furthermore, the functional inactivation of the mouse
PS1 gene leading to a lethal phenotype could not be
compensated by the PS2 gene (11, 12), implying that the presenilin isoforms have common and distinct functions.
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DISCUSSION |
The presenilins PS1 and PS2 are the major
genes responsible for early-onset familial Alzheimer's disease (1).
The aim of this study was to find interacting proteins for PS1 because
most cellular functions of proteins are mediated by protein-protein interactions. Thus, the identification of interacting molecules helps
us to understand the biological function of a protein. Therefore, we
performed a yeast two-hybrid screen for the hydrophilic loop of PS1,
located between transmembrane domains 6 and 7. In this screen, we
identified a recently discovered novel member of the armadillo family,
termed p0071 (21), that interacts in a specific manner with PS1. We
confirmed the interaction by three independent methods, i.e.
yeast two-hybrid retransformation assays, binding to GST fusion
proteins, and immunoprecipitations from human brain and transfected COS cells.
As mentioned, p0071 belongs to a protein family named after the
Drosophila segment polarity gene armadillo, which
is involved in inductive signaling events during development (35). The
members of the armadillo family are characterized by a series of
imperfect 42-amino acid repeats (36). Based on the modular composition of the armadillo family members, it was suggested that individual motifs or couples of these motifs function in signaling cascades establishing protein-protein interactions (23). Therefore, we were
interested in determining the binding motif in p0071.
Using the yeast two-hybrid method, we could demonstrate that armadillo
repeats 2-10 are necessary and sufficient to mediate the interaction
with PS1 (Fig. 4). As recently reported,
-catenin interacts with
conductin via its armadillo repeats 3-7 (37). Thus, the interactions
mediated by p0071 or
-catenin support the above-mentioned paradigm
of how armadillo proteins mediate protein-protein interactions.
The armadillo family members
/
-catenin and p0071 show a high
degree of amino acid identity in the armadillo repeat domain located in
the middle of the respective molecules. For instance, p0071 and
-catenin are 69.2% identical in their armadillo repeat domains.
This high degree of identity in the domains mediating protein-protein
interactions predicts that the respective binding partners are similar
or even identical. Thus, it is not surprising that
/
-catenin can
also interact with PS1 (16-18).
The question now arises as to what the biological significance is of
multiple interacting molecules. Most likely, the interactions of PS1
with different members of the armadillo family may be a function of the
cell type or the developmental stage of an organism. In support of this
hypothesis,
-catenin shows an almost brain-specific expression
pattern (18), whereas p0071 and
-catenin are widely expressed in all
tissues (1, 21), reminiscent of the PS1 expression pattern (24).
A direct interaction of PS1 with members of the armadillo family has a
number of implications.
-Catenin and its Drosophila homolog armadillo are multifunctional proteins: both are key components of cell-cell adhesive junctions and also participate in transduction of
Wingless-Wnt cell-cell signals. Wingless-Wnt signals direct many key
developmental decisions, regulating anterior-posterior pattern in flies
and vertebrates.
-Catenin/armadillo is a key effector of signal
transduction. In the absence of signals, levels of free
-catenin/armadillo are low: the Wingless-Wnt signal stabilizes free
-catenin/armadillo. Stabilized
-catenin binds to a transcription factor of the Tcf-Lef family and enters the nucleus, inducing expression of genes involved in proliferation or apoptosis (38).
The high degree of similarity between the armadillo family members
implies that p0071 acts in a similar manner. Indeed, p0071 contains a
nuclear targeting signal (KKKKKKKR) within its armadillo repeat domain.
Thus, it is tempting to speculate that p0071, in concert with a
transcription factor, enters the nucleus and alters gene expression.
The function of PS1 might be to form a complex with p0071 in the
endoplasmic reticulum or Golgi compartment and to control its targeting
and/or proteolytic degradation. The targeted disruption of the
PS1 gene in mice strongly reduces the expression of Notch-1
in the presomitic mesoderm (12). In addition, the abnormal somite
patterns in PS1 null embryos are highly reminiscent of
somite segmentation defects described in mice with a functionally inactivated notch-1 gene (13). Thus, the members of the
armadillo family might be mediators of PS1 function in signaling
cascades. Further experiments will be needed to elucidate the precise
function of the presenilins and armadillo proteins in the Wingless and Notch signaling pathways.