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INTRODUCTION |
ErbB-4 is a type I receptor tyrosine kinase that binds heregulin
(HRG)1 (1). HRG also binds to
ErbB-3. In addition some ErbB-1 ligands (heparin-binding epidermal
growth factor, betacellulin, and epiregulin) are reported to activate
ErbB-4.
Recently, ErbB-4 was shown to be processed by a novel proteolytic
pathway (2), analogous to that which generates cell fate determination
responses after activation of the Notch nontyrosine kinase receptor
(3). The amyloid precursor protein (APP) is also processed in a similar
manner (4). The first step in this pathway is cleavage within the
ectodomain close to the plasma membrane. In the case of ErbB-4, this
cleavage is stimulatable by TPA (5) and, in some cell lines, by HRG
(6). This proteolytic event results in the release of a 120-kDa ErbB-4
fragment into the extracellular milieu and the cellular retention
of an 80-kDa membrane-associated fragment. Ectodomain cleavage
requires the transmembrane metalloprotease tumor necrosis
factor-
-converting enzyme (7).
After ectodomain cleavage of ErbB-4, the cell-associated 80-kDa
fragment containing the transmembrane and cytoplasmic domains is
cleaved by a
-secretase activity releasing the cytoplasmic domain
into the cytosol (2, 8). Subsequently, the cytoplasmic domain fragment
of ErbB-4 translocates into the nucleus. The function of the 80-kDa
fragment in the nucleus is not known, although the carboxyl-terminal
region of this fragment does display weak activity in the GAL4
transcription activation assay (2). Importantly,
-secretase
inhibitors block the induction of cell death by HRG when T47D carcinoma
cells are placed in serum-free media (2). The nuclear localization of
other receptor tyrosine kinases, including ErbB-1 (9), ErbB-3 (10), and
fibroblast growth factor receptor 1 (11), have been described, but
these do not appear to involve proteolytic processing of the receptors.
At this time relatively little is known about how transmembrane
metalloprotease tumor necrosis factor-
-converting enzyme and
-secretase recognize their substrates. In this study, evidence is
provided that ErbB-4 PDZ domain recognition motif is necessary for
-secretase cleavage.
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EXPERIMENTAL PROCEDURES |
Materials--
HRG
1 was purchased from R&D Systems. The
cDNA for human ErbB-4 and rabbit antiserum to a carboxyl-terminal
sequence (residues 1108-1264) of human ErbB-4 was generously provided
by Dr. Matthias Kraus (University of Alabama, Birmingham, AL) and used
for Western blotting. Polyclonal antibodies C-18 to an epitope in the
carboxyl terminus of ErbB-4 and H-70 to an epitope in the amino
terminus of presenilin 1 were obtained from Santa Cruz Biotechnology.
Monoclonal anti-phosphotyrosine antibody (13-5900) and horseradish
peroxidase-conjugated Protein A were purchased from Zymed
Laboratories Inc. Protein A-Sepharose CL-4B, enhanced
chemiluminescence (ECL) reagents, and TPA were obtained from Sigma. The
-secretase inhibitor Compound E (12) was provided by Dr. Todd Golde
(Mayo Clinic, Jacksonville, FL). Nuclear export signal inhibitor
leptomycin B was provided by Dr. Minoru Yoshida (University of Tokyo,
Japan). G418 sulfate was obtained from Mediatech Inc. All other
chemicals were reagent grade from Sigma.
Cell Culture--
COS7 and NIH3T3 cell lines were obtained from
American Tissue Culture Collection. These cells and all NIH3T3-derived
cells, described below, were routinely grown in 5% CO2 at
37 °C in Dulbecco's modified Eagle's medium containing 10% fetal
bovine serum (FBS) and penicillin/streptomycin (Mediatech Inc.).
Experimental cultures were generally grown in 100-mm culture dishes
until 90% confluence unless otherwise noted in the text.
Gene Transfection and Establishment of ErbB-4 Wild-type
(WT) and
TVV Cell Lines--
LTR-W.T.ErbB4 is an ErbB-4
expression vector under the control of a LTR promoter and is described
elsewhere (13). It contains a neomycin selection marker. To produce a
cDNA construct to express ErbB-4 with the carboxyl-terminal three
residues TVV truncated, an 18-mer upstream primer,
5'-GACTACCTGCAGGAGTAC-3', was synthesized to match a sequence within
ErbB-4 cDNA containing a BamHI site. Also, a 31-mer
downstream primer 5'-ACGCGTCGACTTAATTCCGGTGTCTGTAAGG-3', was made
containing a SalI site next to stop code TAA, which
terminates translation 9 bp before the wild-type ErbB-4 stop sequence.
PCR amplification of LTR-W.T.ErbB4 cDNA with the above primers
produced a 160-bp fragment that was then cut with BamHI and
SalI to generate cohesive ends. The LTR-W.T.ErbB4 construct
was processed to remove the small fragment between BamHI and
SalI (next to the stop codon), and the PCR product was
inserted into the exact site. The resulting construct, called
LTR-
TVV, contains ErbB-4 cDNA with 9 bp corresponding to the
carboxyl-terminal three residues, i.e. TVV, deleted. The mutant construct was verified by enzymatic digestion and DNA sequencing.
For gene transfection, COS7 and NIH3T3 cells were transfected with
LTR-W.T.ErbB4 or LTR-
TVV using Effectene reagent (Qiagen) according
to the manufacturer's protocol. Typically, 2.0-2.5 µg of DNA was
used to transfect cells in a 100-mm dish. COS7 cells were assayed for
ErbB-4 receptor function 36-48 h after transfection. Stably
transfected NIH3T3 cell lines were obtained after selection in G418
sulfate (400 µg/ml) for 2 months. Each stably transfected cell line,
either WT ErbB-4 or
TVV mutant, represents a pool of several
individual colonies. For each receptor species, two stable cell lines
from independent transfections were established and assayed.
Cell Treatment and Lysis and Fractionation--
Cell lysates
were obtained essentially as described previously (5) with some
modifications. Briefly, 90% confluent cell monolayers in 100 mm dishes
were incubated overnight in DMEM containing 0.2% FBS (or 1% FBS, as
indicated in the text). Cells were then incubated at 37 °C with the
indicated additions for the indicated times. Next, the cells were
washed three times with Ca2+/Mg2+-free
phosphate-buffered saline (Ca2+/Mg2+-free PBS)
and lysed in 1 ml of cold TGH lysis buffer (1% Triton X-100, 10%
glycerol, 20 mM Hepes (pH 7.2), 100 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM Na3VO4, and
50 mM NaF) or mild TGH lysis buffer, having reduced levels of Triton X-100 (0.2%) and NaCl (20 mM). The lysed cells
were scraped from the dish and placed in a 1.5-ml
microcentrifuge tube. The lysates were incubated on ice for 30 min and then centrifuged (14,000 × g, 10 min) at
4 °C, and the supernatants were saved for Western blotting or
immunoprecipitation. Preparation of membrane and cytosol fractions was
described previously (5).
Immunoprecipitation and Western Blotting--
To analyze
ErbB-4 in total cell lysates, ~1-2 mg of cell lysate were
immunoprecipitated and underwent Western blotting or ~50 µg
underwent blotting without immunoprecipitation. To assess ErbB-4 in
cytosol and membrane fractions, equal volumes of each fraction
representing equal numbers of cells were immunoprecipitated and then blotted.
To precipitate proteins, ~4 µg of ErbB-4 or presenilin 1 antibody
was incubated (2 h at 4 °C) with lysates followed by a 1-h incubation with Protein A-Sepharose CL-4B. Immune complexes were washed
three times with the same lysis buffer (TGH or mild TGH), resuspended
in 1× Laemmli buffer, and boiled for 5 min. Subsequently, samples were
electrophoresed in a 7.5% SDS-polyacrylamide gel and transferred to
nitrocellulose membranes for Western blotting. Membranes were blocked
by a 1-h incubation at room temperature in TBST buffer (0.05% Tween
20, 150 mM NaCl, 50 mM Tris, pH 7.4) containing
3% bovine serum albumin (for tyrosine phosphorylation detection) or
5% nonfat dry milk (for all others). Subsequently, ErbB-4 antiserum or
phosphotyrosine antibody was added for 2 h at room temperature
with shaking. Membranes were then washed thrice with TBST buffer,
incubated with horseradish peroxidase-conjugated Protein A for 1 h, and after being washed with TBST buffer, visualized by ECL.
Immunofluorescence Staining in Fixed Cells--
Cells grown on
coverslips were fixed with methanol at
20 °C for 5 min. The cells
were then rehydrated with PBS for 10 min and, after a 45-min blocking
period using 10% goat serum (Sigma) in PBS, were incubated with C-18
ErbB-4 antibody for 45 min. After thorough washing (three times in
PBS), secondary antibody Alexa FluorTM 546 goat anti-rabbit
IgG (H+L) conjugate (Molecular Probes) was added for 30 min. The cells
were then mounted in Aqua Poly/Mount (Polysciences, Inc.). A
Zeiss LSM 410 confocal microscope equipped with ×40 (1.3 numerical
aperture) oil immersion objective lens, and the single fluorochrome
filter set for Alexa 546 (Molecular Probes) was used for visualization
and recording images.
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RESULTS |
Ligand Responsiveness of the ErbB-4
TVV Mutation--
The
carboxyl terminus of ErbB-4 encodes a PDZ domain recognition motif that
has been demonstrated to facilitate interaction with PSD95, a multi-PDZ
domain-containing protein that is highly expressed in neuronal cells
(14, 15). However, the biological significance of ErbB-4 interaction
with PDZ domains is not known.
To test the possible functional role of the PDZ domain binding motif in
ErbB-4, the three carboxyl-terminal residues that define this motif
(16), were deleted to create the ErbB-4 mutant designated
TVV. We
first tested by transient expression in COS7 cells the capacity of HRG
to stimulate tyrosine phosphorylation of both WT ErbB-4 and the
TVV mutant. The results
(Fig.1A) demonstrate that both
forms of ErbB-4 are activated by exogenous HRG. Subsequently, permanent
cell lines expressing WT ErbB-4 or the
TVV mutant were obtained by
transfection of NIH3T3 cells, which do not endogenously express any
ErbB family members. In these cells, HRG also activated both receptor
isoforms to approximately equivalent extents (Fig. 1B).
Consistent with growth factor-dependent activation of both ErbB-4 species, HRG also activated Erk1 and Erk2 protein kinases when
added to cells expressing WT ErbB-4 or the
TVV mutant (data not
shown).

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Fig. 1.
Ligand activation of WT ErbB-4 and
TVV mutant. A, Cos7 cells
transiently expressing WT ErbB-4 or TVV mutant ErbB-4 were treated
with HRG (20 ng/ml) for 10 min. The cells were then immediately washed
three times with cold Ca2+/Mg2+-free PBS and
then ysed in TGH buffer. After clarification, the lysate (0.8 mg) was
immunoprecipitated with C-18 ErbB-4 antibody. Western blots were
carried out with anti-phosphotyrosine followed by membrane stripping
and reblotting with ErbB-4 antibody. B, NIH3T3 cells stably
expressing WT ErbB-4 or the TVV mutant ErbB-4 were incubated
overnight in DMEM medium with 1% FBS, treated with HRG (20 ng/ml, 10 min), and lysed for immunoprecipitation and Western blotting as
described in the legend for A.
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The above results show that loss of the PDZ domain recognition motif
does not abrogate ligand-dependent receptor activation and
by inference cell surface expression. This result is slightly in
contrast to a previous report which concluded that the ErbB-4 PDZ
domain recognition motif was required for maximal growth factor activation of this receptor (15).
Influence of the
TVV Mutation on ErbB-4 Proteolytic
Processing--
The
TVV mutation of ErbB-4 was evaluated to assess
the potential role of the PDZ domain recognition motif in the
proteolytic processing pathway, which requires the participation of at
least two integral membrane proteases. The initial step in this pathway is the metalloprotease cleavage of ErbB-4, which produces an 80-kDa membrane-associated fragment (5). When cells expressing WT ErbB-4 or
the
TVV mutant were treated with TPA, the 80-kDa fragment was
produced at approximately the same rate from both receptor species for
the first 60 min, as shown in Fig. 2.
However, at a later time (3 h) the 80-kDa fragment generated in cells
expressing the ErbB-4
TVV mutant remained at a significant level,
whereas the level of the fragment generated in cells expressing the
wild-type receptor had significantly declined. These results indicate
that whereas the
TVV mutation did not influence the metalloprotease cleavage of ErbB-4, the cell-associated fragment generated from the
mutant receptor was significantly more metabolically stable.

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Fig. 2.
Influence of TVV
mutation on ErbB-4 ectodomain cleavage. A, NIH3T3 cells
expressing WT ErbB-4 or TVV were cultured until 90% confluent,
starved overnight in DMEM medium with 1% FBS, and then treated with
TPA (100 ng/ml) for the times indicated. TGH lysates were prepared for
Western blotting with ErbB-4 antibody to detect the full-length
(180-kDa) ErbB-4 molecule and the 80-kDa cell-associated fragment.
B, the bands were quantitated by scanning densitometry. To
compare the value of the 80-kDa band in the two cell lines, the amount
of 80-kDa fragment was normalized to the amount of 180-kDa ErbB-4
present at time 0 h.
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One means by which the membrane-associated 80-kDa ErbB-4 fragment is
metabolized is by
-secretase cleavage. This cleavage converts the
membrane-associated fragment, designated m80, to a cytosolic
fragment, designated s80 (2). Therefore, the TPA-dependent formation of the m80 and s80 ErbB-4 fragments from the WT and
TVV
ErbB-4 receptors were examined 30 min after the addition of the
agonist. Detection of anti-ErbB-4 reactive bands in the membrane
fraction (Fig. 3) shows that the
full-length (180-kDa) WT ErbB-4 and
TVV mutant proteins are degraded
in the presence of TPA and that the m80 fragment is generated from
each. However, the s80 ErbB-4 fragment is readily detected only in
cells that express the WT receptor. Only a very low level of s80 is
produced in cells expressing the
TVV mutant. After quantitating the
s80 bands, it is estimated that production of this fragment is reduced by 90-95% in the
TVV mutant.

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Fig. 3.
Influence of TVV
mutant on -secretase cleavage of ErbB-4.
NIH3T3 cells expressing WT ErbB-4 or TVV were cultured
until 90% confluent. The cells were then starved overnight in DMEM
with 0.2% FBS and treated for 30 min with TPA (100 ng/ml) or vehicle
Me2SO. The cells were then disrupted by homogenization, and
the membrane and cytosolic fractions were prepared as described under
"Experimental Procedures." Aliquots of membrane and cytosolic
fractions were then immunoprecipitated with ErbB-4 antibody (C-18) and
blotted with another ErbB-4 antibody.
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The above results indicate that introduction of the
TVV mutation
selectively abrogates conversion by
-secretase of the mutant m80
ErbB-4 fragment to the s80 fragment.
-Secretase catalytic activity
is generally considered to be a property of presenilin proteins;
however, this point remains unclear (17). Other substrates of
-secretase, such as APP and Notch, have been found to co-precipitate with presenilins (PS1, PS2) (18-22).
A co-precipitation strategy was employed to ascertain whether
presenilin associates with ErbB-4 and whether this is influenced by the
receptor's PDZ domain recognition motif. The results, as shown in Fig.
4 (left panel), demonstrate
that when PS1 is immunoprecipitated from untreated cells expressing the
WT ErbB-4 receptor, anti-ErbB-4 reactive proteins are detected
representing the full-length ErbB-4 (180-kDa) and the 80-kDa ErbB-4
fragment. When TPA is added to these cells, however, the level of
80-kDa ErbB-4 fragment detected in the PS1 immunoprecipitate is greatly
increased, as is the amount of 80-kDa fragment in these cells.
Comparison of the amount of full-length ErbB-4 in untreated cells and
80-kDa fragment in TPA-treated cells suggests that PS1 associates
preferentially with the 80-kDa fragment, which mostly represents the
m80
-secretase substrate. In contrast, an almost undetectable level
of ErbB-4 reactivity is found in PS1 immunoprecipitates from cells
expressing the
TVV mutant. The data in the right panel
show that in both cell types equal levels of full-length ErbB-4 are
present and that in the presence of TPA the 80-kDa fragment is also
detected at equivalent levels in both cell types. The results suggest
that s80 is not generated from the m80
TVV ErbB-4 mutant, because
the latter fragment is unable to associate with PS1. The presence of
PS2 in these cells was not detectable.

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Fig. 4.
Association of presenilin with ErbB-4 and the
80-kDa ErbB-4 fragment. NIH3T3 cells expressing WT
ErbB-4 or TVV were cultured until 90% confluent and then incubated
overnight in DMEM with 0.2% FBS. Subsequently, the cells were treated
with TPA (100 ng/ml) or vehicle Me2SO as a control for 30 min. The cells were lysed in mild TGH lysis buffer containing reduced
levels of Triton X-100 (0.2%) and NaCl (20 mM). Aliquots
of the lysates were then immunoprecipitated as indicated with anti-PS1
or anti-ErbB-4, and the precipitates (IP) were
blotted with anti-ErbB-4.
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If the absence of a PDZ domain recognition motif prevents efficient
-secretase cleavage of ErbB-4, then it would be expected that the
TVV mutant receptor would fail to be translocated to the nucleus
after the addition of TPA. The data in Fig.
5 show that whereas TPA addition to cells
expressing the WT ErbB-4 receptor does provoke nuclear localization of
ErbB-4 immunoreactivity, similar treatment of cells expressing the
TVV mutant reveals no nuclear ErbB-4 signal even if leptomycin B
(23) is added to prevent nuclear export. The data additionally
demonstrate that if leptomycin B is added but the cells are not
stimulated with TPA, ErbB-4 reactive material is present in the nucleus
of cells expressing the WT receptor but not in cells that express the
TVV mutant. This basal level of nuclear localized ErbB-4 is
prevented in the cells expressing WT receptor by addition of the
-secretase inhibitor Compound E (12).

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Fig. 5.
PDZ domain recognition
motif-dependent ErbB-4 nuclear localization. NIH3T3
cells expressing WT ErbB-4 or TVV were cultured on
coverslips until 80% confluent and then incubated overnight in DMEM
with 0.2% FBS (a, b, e,
f), or with leptomycin B (10 ng/ml) (c,
g), or with leptomycin B (LMB, 10 ng/ml) and
compound E (Cmpd E, 10 nM) (d,
h) as indicated. TPA (100 ng/ml) was then added
(b, f) for 90 min. All cells were then fixed and
immunostained with C-18 ErbB-4 antibody for confocal microscopy as
described under "Experimental Procedures." Bar, 25 µm,
applies to each image.
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Influence of
TVV Mutation on Cell Growth--
When T47D mammary
carcinoma cells are placed in serum-free medium, HRG-induced
ErbB-4 cleavage leads to cell death in a manner that requires
-secretase activity (2). Also, overexpression of the ErbB-4
cytoplasmic domain in COS cells results in cell death (8). Although a
basal level of ErbB-4 cleavage exists in all cell systems, this was not
sufficient to influence survival of the T47D cells. To investigate
further the potential biological significance of basal ErbB-4 cleavage,
the survival of the transfected NIH3T3 cells used in the experiments
described herein was evaluated.
The data in Fig. 6 show that when cells
expressing WT ErbB-4 or the
TVV mutant are placed in serum-free
medium, the WT ErbB-4 cells initially survived and slightly increased
in cell number, but by day 3 nearly all had died. In contrast, cells
that express the
TVV mutant ErbB-4 survive under the same culture
conditions for at least 4 days. This result indicates that cells
expressing the
-secretase cleavable WT ErbB-4 are sensitive to cell
death under these conditions, but the
TVV mutant does not produce a signal that provokes cell death. This also suggests that in these NIH3T3 cells, basal ErbB-4 cleavage is sufficient to provoke a cell
response.

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Fig. 6.
Influence of TVV
mutation on cell death. NIH 3T3 cells expressing WT
ErbB-4 ( ) or the TVV mutant ( ) were plated (~2 × 105 cells/well in 12-well dishes. On the next day (day 0),
the cells were washed with DMEM twice and cultured in serum-free DMEM.
Cell numbers were determined at 24-h intervals thereafter, using a
Coulter counter.
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To confirm that
-secretase activity is necessary for this growth
response, the experiment was repeated in the presence of Compound E, a
potent
-secretase inhibitor (12). The cells were plated as described
in Fig. 6, and cell numbers were determined 3 days later (Table
I). The results demonstrate that only
14% of the cells expressing the WT ErbB-4 survived under these
conditions, whereas 88% of the cells expressing the
TVV mutant
survived. The addition of Compound E almost completely prevented cell
death in cells expressing the WT ErbB-4 receptor but did not
significantly alter the response of cells that express the
TVV
mutant.
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Table I
Influence of -secretase inhibitor on cell growth
Approximately 2 × 105 cells expressing WT ErbB-4 or the
TVV mutant were plated in complete medium. On the following day the
medium was removed and replaced with serum-free medium. Compound E (10 nM) was added as indicated. Three days later cell numbers
were determined. Each cell count represents triplicate determination.
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DISCUSSION |
The major conclusion of the experimental results presented in the
manuscript is that PDZ domains have a central role in the mechanism by
which ErbB-4 is recognized and processed by
-secretase activity.
This proteolytic step seems to involve intramembranous cleavage of
ErbB-4 and results in the release of the receptor's cytoplasmic domain
(s80) into the cytosol and nucleus (2).
The immediate substrate for
-secretase activity is not the
full-length ErbB-4 but rather a membrane-associated fragment (m80) that
is derived from the native receptor by metalloprotease-mediated cleavage of the receptor ectodomain (2). The steps in this proteolytic
pathway for ErbB-4 are analogous to those described for the proteolytic
processing of Notch, a nontyrosine kinase receptor, and APP, a
nonreceptor transmembrane molecule. The intramembranous cleavage of all
three substrates requires
-secretase activity. However, the cleavage
sites in Notch and APP are dissimilar in sequence and relative location
within the transmembrane domain (17). Hence,
-secretase recognition
is not thought to depend on substrate primary sequence information, as
is the case with many other proteases. The transmembrane sequence of
ErbB-4 is unlike that of Notch or APP.
The data presented herein show that the carboxyl-terminal PDZ domain
recognition motif of ErbB-4 is required for
-secretase cleavage of
the ErbB-4 m80 fragment. This motif is also necessary for association
of PS1 with the m80 ErbB-4 fragment. These results imply that the
target of this ErbB-4 motif plays an important role in mediating
intramembranous cleavage of ErbB-4. Others have identified PSD95 as one
target of the ErbB-4 carboxyl-terminal sequence (14, 15). PSD95, which
is highly expressed in neuronal cells, contains three PDZ domains, two
of which recognize ErbB-4. In neuronal tissue PSD95 functions to
organize specialized complexes of transmembrane proteins (16). In the
non-neuronal cells used in the experiments described herein, we have
been able to detect PSD95 family members in ErbB-4, but not PS1,
immunoprecipitates (data not shown).
Interestingly, the carboxyl terminus of the Cooh-terminal fragment of
PS1 also contains a PDZ domain recognition sequence, and PDZ domain
containing-proteins that recognize PS1 through this motif have been
reported (22, 24). Hence, it seems plausible to suggest that ErbB-4
association with
-secretase involves PDZ domain-containing proteins
acting as a scaffold to bridge the two molecules. Both APP and Notch
have also been shown to associate with PS1 using co-precipitation
methodology. However, the associations are not known to be direct and
may involve other molecules. In the APP/PS1 association, evidence
exists that a PDZ domain-containing protein interacts by a PDZ domain
with PS1 and by another domain with APP (22). Because PDZ
domain-containing proteins often multimerize, it is possible that
multiple proteins having PDZ domains could facilitate ErbB-4
association with
-secretase activity. Alternatively, it is possible
that mutation of the ErbB-4 PDZ domain recognition motif alters the
cell surface trafficking of ErbB-4 and prevents
-secretase cleavage.