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The Journal of Neuroscience, September 1, 2002, 22(17):7340-7351
Regulation of APP-Dependent Transcription Complexes by
Mint/X11s: Differential Functions of Mint Isoforms
Thomas
Biederer,
Xinwei
Cao,
Thomas C.
Südhof, and
Xinran
Liu
The Center for Basic Neuroscience, Department of Molecular
Genetics, and Howard Hughes Medical Institute, The University of Texas
Southwestern Medical Center, Dallas Texas 75390-9111
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ABSTRACT |
Mints/X11s are neuron-specific (Mints 1 and 2) and ubiquitous
(Mint 3) adaptor proteins composed of isoform-specific
N-terminal sequences and common C-terminal
phosphotyrosine-binding (PTB) and PDZ domains. We now show
that all three Mints bind to the cytoplasmic tail of amyloid-
precursor protein (APP) and presenilins and strongly increase the
levels of cellular APP in transfected cells. Immunocytochemistry
revealed that in neurons, Mints 1 and 2 were colocalized with APP in
the trans-Golgi network, with lower levels throughout
the cell body and neurites. Using an APP-dependent transactivation
assay that uses a fusion protein of APP coupled to the potent
transcription factor Gal4/VP16, we examined the effects of Mints on the
proteolytic processing and putative transcriptional function of APP.
Although all Mints were biochemically similar, only Mints 1 and 2 but
not Mint 3 strongly inhibited transactivation by APP-Gal4/VP16.
Inhibition was enhanced by a mutation of the first PDZ domain and by
deletion of the PDZ domains or the N-terminal sequences but abolished
by inactivation of the PTB- and PDZ domains. Mint 1 also inhibited
transactivation by the "precleaved" cytoplasmic tail of APP fused
to Gal4/VP16, whereas Fe65 (which binds to APP as strongly as Mints)
enhanced transactivation. Our data suggest that Mints 1 and 2 but not
Mint 3 have a specific effect on APP function that cannot be explained
simply by their interaction with presenilins and occurs at least partly
after cleavage of APP. In view of their biochemical similarity, the
functional differences among Mints are unexpected, suggesting that
Mints 1 and 2 have a brain-specific function related to APP that is not
executed by the ubiquitous Mint 3.
Key words:
APP; Alzheimer's disease; synapse; Mint; X11; Lin-10; Fe65; CASK; Munc18-1
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INTRODUCTION |
Amyloid- precursor protein (APP)
of Alzheimer's disease is a ubiquitous type 1 membrane protein (Kang
et al., 1987 ; Kitaguchi et al., 1988; Tanzi et al., 1988). APP is
physiologically processed by proteolytic cleavage (for review, see
Selkoe, 1998; Bayer et al., 1999; Haass and De Strooper, 1999; Wolfe
and Haass, 2001). First, cleavage by  - or -secretases releases
the large extracellular part of APP. Subsequently, the remaining
sequences of APP composed of a small extracellular stub, the
transmembrane region (TMR), and the cytoplasmic tail are digested by
 -secretase at multiple positions (Sastre et al., 2001; Yu et al.,
2001). -Cleavage liberates an intracellular cytoplasmic fragment
that may be translocated to the nucleus (Cupers et al., 2001; Kimberly
et al., 2001) and function as a transcriptional activator (Cao and
Südhof, 2001; Gao and Pimplikar, 2001), In addition, -cleavage
generates small peptides derived from the TMR and adjacent
extracellular sequences that include A40 and A42, which form the
amyloid fibrils in Alzheimer's disease (Glenner and Wong, 1984;
Masters et al., 1985) (for review, see Selkoe, 1998; Haass and De
Strooper, 1999).
The short cytoplasmic tail of APP contains an NPTY sequence that
binds to phosphotyrosine-binding (PTB) domains in multiple proteins, including Fe65 and Mints/X11s (Fiore et al., 1995; Borg et
al., 1996; Guenette et al., 1996; McLoughlin and Miller, 1996; Zhang et
al., 1997). Fe65 is an adaptor protein that forms a transcriptionally active complex with the released APP tail and a nuclear histone acetyltransferase, Tip60 (Cao and Südhof, 2001). The genes for Mints 1 and 2 were identified as candidates for Friedreich's ataxia and, on the basis of partial sequences, were thought to be orthologs (Duclos and Koenig, 1995). However, sequencing full-length cDNAs showed
that these proteins were products of distinct genes (Okamoto et al.,
1997). To prevent confusion among different types of X11s, we
called these proteins Mints 1 and 2, and we named a third isoform Mint
3 (Okamoto et al., 1997; Okamoto and Südhof, 1998). Subsequent recloning of the same proteins led to further renaming, and they are
now also variably referred to as X11//, mLin-10s,
X11a/b/c, or X11L1/L2.
Mints/X11s are composed of a long isoform-specific N-terminal sequence,
a central PTB domain, and two C-terminal PDZ domains. Mints
interact with several other proteins in addition to APP. Mint 1 (but
not Mints 2 and 3) binds to CASK (Butz et al., 1998), another
adaptor protein (Hata et al., 1996). In Caenorhabditis elegans, CASK and Mint 1 homologs are encoded by the Lin-2 and Lin-10 genes whose mutation causes similar vulvaless phenotypes, suggesting that the Mint 1/CASK complex is evolutionarily conserved (Borg et al., 1998b, 1999; Butz et al., 1998; Kaech et al., 1998;). Mints 1 and 2 also bind to Munc18-1, an essential fusion protein at
the synapse (Okamoto et al., 1997; Biederer and Südhof, 2000; Verhage et al., 2000), and to presenilins, which are intrinsic components of the -secretase (Lau et al., 2000).
The functions of Mints remain obscure. In C. elegans, Lin-10
(Mint 1) mediates the correct targeting of EGF-like receptors to
the basolateral membrane of vulval precursor cells (Whitfield et al.,
1999) and is necessary for delivery of AMPA-like glutamate receptors to
synapses (Rongo et al., 1998). These data suggest that Lin-10/Mint 1 functions in membrane traffic of proteins to specific plasma membrane
domains. In vertebrates, however, various somewhat contradictory
functions for Mints have been proposed. Transfection experiments
revealed that Mints alter production of A peptides, indicating a
role in APP cleavage (Borg et al., 1998a; Sastre et al., 1998; Mueller
et al., 2000). In contrast, an interaction of Mint 1 with KIF17
in vitro led to the proposal that Mint 1 functions in
trafficking neuronal NMDA- but not AMPA-type glutamate receptors in
vertebrates (Setou et al., 2000). This study renamed Mint 1 "mLin-10" in analogy to the C. elegans gene but did not
reference the previous finding that in nematodes Lin-10 affects only
AMPA receptors and not NMDA receptors (Rongo et al., 1998).
In the present study, we compared all three Mint isoforms in the same
experiments to test their functional relation to APP. APP was chosen
because in vertebrates this appears to be the functionally best
validated interaction (Borg et al., 1998a; Sastre et al., 1998),
because the relation of APP to Alzheimer's disease makes understanding
its biology imperative and because recent insights into a possible
transcriptional function of APP have opened new avenues to address this
question. Our data reveal that various Mints have similar biochemical
properties but different functional effects and that these effects
cannot be explained only by regulating APP cleavage.
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MATERIALS AND METHODS |
Plasmid construction
Gal4-containing transactivation plasmids. Most of the
plasmids used for the transactivation experiments described here were reported previously (Cao and Südhof, 2001). All eukaryotic
expression vectors containing Gal4 or Gal4/VP16 were based on
pMst (Gal4) and pMst-GV (Gal4/VP16), which are derived from the
SV40 promoter-based mammalian expression vector pM (Clontech) (Cao and
Südhof, 2001). In addition to the previously described vectors,
the following vectors were constructed:
pMst-GV-APPICF
(APPICF-Gal4/VP16), generated by cloning the
intracellular fragment of human APP695
(APPICF, residues 652-695) into the
BamHI-SalI sites of pMst-GV; pMst-GV-APP (APP-Gal4/VP16), by cloning the extracellular and TMR fragments of
human APP695 (APPe, residues 1-651) into the
NheI site of pMst-GV-APPICF; pMst-GV-APP (APP-Gal4/VP16), by cloning residues 639-651 of human APP695 preceded by a methionine into the
BglII-NheI sites of
pMst-GV-APPICF.
pMst-GV-APPICF*
(APPICF*-Gal4/VP16), pMst-GV-APP* (APP*-Gal4/VP16), and pMst-GV-APP* (APP*-Gal4/VP16) were
generated from their respective parent plasmid by site-specific
mutagenesis replacing NPTY(684-687) with
NATA(684-687) using the QuikChange kit
(Stratagene, La Jolla, CA). pMst-GV-NRX (NRX-Gal4/VP16) was generated
by cloning the intracellular fragment of rat Neurexin 1
(NRXICF, residues 414-468) into the
BamHI-SalI sites of pMst-GV, followed by cloning
the extracellular and TMR fragments (NRXe, residues 1-417) into the
NheI site. pMst-GV-NA
(NRXe-Gal4/VP16-APPICF) was generated by cloning
NRXe into the NheI site of
pMst-GV-APPICF, and pMst-GV-AN
(APPe-Gal4/VP16-NRXICF) was generated by cloning the intracellular fragment of Neurexin 1
(NRXICF) into the
BamHI-SalI sites of pMst-GV, followed by cloning
of APPe into the NheI site.
Mint plasmids. The eukaryotic pCMV5 expression vectors for
full-length rat Mints 1, 2, and 3 have been described previously (Okamoto and Südhof, 1997, 1998). Mutations were inserted in these parent vectors by site-specific mutagenesis as described above.
pCMV-Mint 1-PDZ1* was constructed by mutating
GV(670,671) to AA(670,671)
in the carboxylate binding loop of the first PDZ domain, and pCMV-Mint
1-PDZ2* was created by mutating GF(762,763) to
AA(762,763) in the carboxylate binding loop of
the second PDZ domain. pCMV-Mint 1- PDZ was generated by introducing
a stop codon after residue 659 in pCMV-Mint 1. A hydrophobic pocket of
the PTB domain of Mint 1 was altered at positions
YQEF(613-616) to
SQES(613-616) to generate the pCMV-Mint 1-PTB*
construct. pCMVmyc-Mint 1 was generated by cloning the full-length
Mint 1 coding sequence from pEGFP-Mint 1 (a gift from Dr. Anton
Maximov, University of Texas Southwestern, Dallas, TX) into the
EcoRI-KpnI sites of pCMVmyc. To construct
pCMVmyc-Mint 1-Nterm, the C-terminal part of Mint 1 encoding
residues 451-839 was cloned into the KpnI-XbaI sites of pCMVmyc. pCMV-Mint 2-PDZ was generated by introducing a
stop codon after residue 570 in pCMV-Mint 2, and pCMV-Mint 3-PDZ was
generated by introducing a stop codon after residue 391 in pCMV-Mint 3.
Other plasmids. The eukaryotic expression vectors for CASK
(Hata et al., 1996) and Fe65 (Cao and Südhof, 2001) were
described previously.
Antibodies
Most antibodies have been described previously (Biederer and
Südhof, 2000, 2001; Cao and Südhof, 2001). The APP antibody used was a polyclonal rabbit serum (U955) raised against the cytosolic, extreme C-terminal 15 residues of APP coupled to keyhole-limpet hemocyanin. Monoclonal antibodies to Mints 1, 2, and 3, TGN 38, and
early endosome antigen (EEA1) were from Transduction
Laboratories, antibodies to calnexin were from Chemicon, and antibodies
to Golgi 58K protein were from Sigma. Polyclonal Mint 1 antibodies have been described previously (P932) (Okamoto and Südhof, 1997), as
were antibodies against Velis (T813) (Butz et al., 1998). Polyclonal anti-myc antibodies were from Santa Cruz Biotechnology (Santa Cruz,
CA), and polyclonal antibodies directed against Mint 3 were from
Affinity Bioreagents (Denver, CO). For all Mint antibodies, specificity
was confirmed using preparations from COS cells transfected with
expression vectors for Mints 1-3 (see Fig. 1A and
data not shown).
Biochemical preparations
All steps were performed on ice or at 4°C. Rat forebrains (Pel
Freez, Rogers, AR) were homogenized in a pestle tissue grinder using a
slow-speed stirrer at a tissue to buffer ratio of 10% (w/v) in buffer
RMP (20 mM HEPES-KOH, pH 7.4, 125 mM
K-acetate, 5 mM MgCl2, 320 mM sucrose) adjusted to 1.0% Triton X-100 in the presence
of protease inhibitors. For preparation of membrane proteins, rat
forebrains were homogenized in buffer RMP, the samples were centrifuged
in an Eppendorf microcentrifuge at 600 × g for 10 min
to obtain the postnuclear supernatant, and membranes were pelleted in a
Sorvall S80-AT3 rotor at 280,000 × g for 20 min. The
membrane pellet was extracted in buffer RMP adjusted to 1.0% Triton
X-100 using a pestle tissue grinder and centrifuged again at
280,000 × g for 20 min to yield the solubilized
membrane proteins.
Peptide bead affinity chromatography
Peptides were synthesized on an ABI synthesizer with an added
N-terminal cysteine for coupling to SulfoLink Beads (Pierce, Rockford,
IL) according to the manufacturer's instructions at 1.0 mg peptide per
1 ml beads. For binding to the APP NPTY motif, a peptide corresponding
to the APP-derived sequence CGYENPTYKFFEQMQN (human APP, residues
398-412) was immobilized on SulfoLink beads. For binding to
presenilin C-terminal sequences, peptides corresponding to the
extreme C termini of human Presenilin 1 (sequence CMDQLAFHQFYI), human
Presenilin 2 (sequence CMDTLASHQLYI), or Drosophila
Presenilin (sequence CMEDLSAKQVFI) were immobilized. As negative
controls, peptides corresponding to the extreme C terminus of human
HPV2 (poliovirus receptor-related protein 2; sequence CGSLISRRAVYV) and
a peptide derived from the gp41 glycoprotein (sequence CWFSITNWLWYI) were used. Extracts of transfected eukaryotic cells or proteins solubilized from rat brain were incubated with 20 µg peptide
immobilized on SulfoLink beads for 12-16 hr at 4°C under mild
agitation. Binding was performed in buffer RMP adjusted to 1.0% Triton
X-100 and 600 mM potassium acetate. For APP
competition experiments, the soluble peptide QNGYENPTYKFFEQ or
QNGYENATAKFFEQ, corresponding to the native or mutated APP NPTY motif,
was added during the binding incubation at the concentrations of 0.1 mg/ml and 1.0 mg/ml. Bound proteins were eluted with 2% SDS.
Immunoprecipitations
Human embryonic kidney (HEK) 293 cells were cotransfected for
APP and the individual Mints 1, 2, or 3, respectively, and after 2 d they were collected in IP buffer [25 mM HEPES-KOH, pH
7.4, 125 mM K-acetate, 5 mM
MgCl2, 1.0% IGEPAL CA-630 (Sigma), 10% glycerol] in the presence of protease inhibitors. After the cell suspension was passed through a 28 gauge syringe, the lysate was centrifuged in an Eppendorf microcentrifuge at 21,000 × g for 20 min, and the detergent-extracted material was
subjected for 2 hr to immunoprecipitation using antibodies directed
against APP (U955) or the respective preimmune serum.
Miscellaneous biochemical procedures
SDS-PAGE and immunoblotting were performed as described
(Laemmli, 1970; Towbin et al., 1979). For standard
immunodetection on Western blots, enhanced chemiluminescence (ECL;
Amersham) was applied. Quantitative immunoblotting was performed using
radiolabeled 125I secondary antibodies
(Amersham), and the signals were quantitated on a PhosphorImager
(Molecular Dynamics) using ImageQuant software. To determine levels of
expressed Mint 1 protein, signals of cell lysates were compared with
those from known amounts of purified glutathione
S-transferase-Mint 1. Protein concentrations were determined using the BCA protein assay (Pierce).
Immunocytochemistry
Adult mice were perfusion-fixed in 4% paraformaldehyde in PBS,
pH 7.4. Vibratome sections (35 µm) from brain were blocked for 1 hr
in 10% normal goat serum containing 0.1% Triton X-100 and incubated
with the various primary antibodies overnight at 4°C and with the
biotinylated secondary antibody for 1 hr. Sections were processed using
a VectaStain ABC Elite Kit (Vector, Burlingame, CA) according to the
manufacturer's instruction. The final immunosignal was developed using
3'3-diaminobenzidine tetrahydrochloride. For immunofluorescence
labeling, primary hippocampal cells on coverslips were fixed in
situ for 10 min with absolute methanol at  20°C, permeabilized
in 0.1% saponin/PBS, and blocked in 3% milk/PBS. The primary
incubation was performed in blocking buffer for 1 hr at room
temperature. After washing with PBS, the cells were incubated with goat
anti-rabbit or goat anti-mouse secondary antibodies that were coupled
with Alexa Fluor 488 and Alexa Fluor 546 (Molecular Probes). Labeled
cells were viewed with a Leica TCS SP2 confocal microscope or a Zeiss
fluorescent microscope with a Hamamatsu ORCA-100 digital camera. Final
images were processed by MetaMorph (Universal Imaging) and Adobe Photoshop.
Transactivation assays
PC12, COS, HeLa, and HEK293 cells were cotransfected with
three or four plasmids: (1) pG5E1B-luc (HEK293 cells, HeLa cells, and
COS cells = 0.2-0.5 µg DNA; PC12 cells = 1.0 µg); (2)
pCMV-LacZ (HEK293 cells, HeLa cells, and COS cells = 0.05 µg
DNA; PC12 cells = 0.5 µg DNA); (3) pMst (Gal4), pMst-GV-APP
(APP-GV), pMst-GV (GV), pMst-GV-APPICF
(APPICF-GV), pMst-APPICF
(APPICF-Gal4), pMst-GV-APP* (APP*-GV),
pMst-GV-APPICF*
(APPICF*-GV), pMst-APPICF*
(APPICF*-Gal4), pMst-GV-APP (APP-GV),
pMst-GV-NRX (NRX-GV), pMst-GV-NA (NRXe-GV-APPc), pMst-GV-AN
(APPe-GV-NRXc)(HEK293, HeLa, and COS cells = 0.1-0.3 µg DNA;
PC12 cells = 1.0 µg DNA). Where indicated, a fourth plasmid was
cotransfected: pcDNA3.1-PS2D366A (kind gift of Dr. C. Haass, Ludwig-Maximilians-Universität, Munich); pCMV-Mint1;
pCMV-Mint 1-PDZ1*; pCMV-Mint 1-PDZ2*; pCMV-Mint 1-PTB*;
pCMVmyc-Mint 1; pCMVmyc-Mint 1-Nterm; pCMV-Mint 1-PDZ; pCMV-Mint
2; pCMV-Mint 2-PDZ; pCMV-Mint 3; pCMV-Mint 3-PDZ; pCMV5-Fe65;
pCMV-CASK (HEK293, HeLa, and COS cells = 0.1-0.3 µg DNA or as
described in the Figure Legends; PC12 cells = 0.5 µg DNA). For
negative controls, the expression vector pCMV5 was used without insert.
Cells were harvested 48 hr after transfection in 0.2 ml per well
reporter lysis buffer (Promega), and their luciferase and
-galactosidase activities were determined with the Promega
luciferase assay kit using a chemiluminescence reader (Lucy2, Anthos
Labtec, Salzburg, Austria) and the standard
O-nitrophenyl-D-galacto-pyranoside
(Sigma) method, respectively. The luciferase activity was standardized
by the -galactosidase activity to control for transfection
efficiency and general effects on transcription and further normalized
for the transactivation observed in cells expressing Gal4 alone where indicated. Values shown are averages of transactivation assays performed in duplicate and repeated at least three times for each cell
type and constructs. All constructs were assayed in three or four cell
lines, but usually only representative results for one cell line are shown.
Transfections were performed at 50-80% confluency in six-well plates
using Fugene6 (Roche).
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RESULTS |
Comparison of the binding of Mints/X11 1, 2, and 3 to APP
Previous results have separately examined the ability of Mints to
bind to APP or presenilins and to stabilize APP in cotransfected cultured cells (Borg et al., 1996, 1998; McLoughlin and Miller, 1996;
Zhang et al., 1997; Sastre et al., 1998; Lau et al., 2000; Okamoto et
al., 2001). These experiments established a potentially important connection between Mints, presenilins, and APP, but the
relative activities of the three Mints and the generality of these
putative targets were not analyzed. As an initial step toward
understanding the common versus unique properties of Mint isoforms, we
compared the binding of different Mints to APP and presenilin and their
effect on APP in transfected cells. Because these and subsequent
experiments critically depended on the specificity of the Mint
antibodies used, we first validated the specificity of these antibodies
using transfected COS cells that express individual Mints.
Immunoblotting confirmed the monospecificity of the antibodies (Fig.
1A, lanes
1-4), which could thus be applied to detect each Mint
isoform separately in complex solutions containing multiple Mints, such
as brain homogenates.
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Figure 1.
Similar tight binding of Mints 1, 2, and 3 to the
cytoplasmic tail of APP. A, Immunoblot analysis of Mint
binding to the immobilized cytoplasmic tail of APP. Lanes
1-4 were loaded with proteins extracted from COS cells
transfected with a control vector (lane 1) or Mint 1-3
expression vectors (lanes 2-4) to demonstrate
specificity of the antibodies used for the respective Mint isoforms.
Proteins from rat forebrain homogenates (lane 5) were
bound to an immobilized cytoplasmic peptide derived from APP in the
absence of added soluble peptide (lane 6) or in
the presence of 0.1 or 1.0 mg/ml of a soluble peptide containing the
wild-type sequence of the APP cytoplasmic tail (lanes 7,
8), or of this soluble APP tail peptide point-mutated in
the NPTY binding sequence for Mints (lane 9). As a
further control, binding of Mints to a control column with an
immobilized peptide derived from gp41 was examined (lane
10). Binding was performed at 600 mM salt. The
samples tested in lanes 6-10 are proteins bound to
beads after the respective binding and peptide competition experiments.
Immunoblotting was performed using monoclonal antibodies against Mints
1 and 2 and polyclonal antibodies against Mint 3. Fractions were also
analyzed by immunoblotting for the negative control proteins Rab
GDP-dissociation inhibitor protein (GDI), a
soluble protein, and for synaptophysin 1 (Syp), a
membrane protein of synaptic vesicles (bottom panels).
B, Quantitation of the amount of Mints bound to the
immobilized cytoplasmic tail of APP as percentage of Mints in the
starting brain extract. Note that all Mints specifically bind only to
the immobilized APP but not to the control column and that their
binding can be inhibited only by high concentrations of wild-type APP
peptide but not by mutant peptide.
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We then used affinity chromatography of rat brain proteins on the
immobilized cytoplasmic tail of APP to examine the binding of
endogenous Mints to APP. Proteins bound to a peptide corresponding to
the cytoplasmic C-terminal tail of APP were analyzed by immunoblotting. Recovery of the three Mints from the brain homogenates was quantitated with 125I-labeled secondary antibodies
(Fig. 1A, lanes 5-10). All three Mints
tightly bound to APP (lane 6), and binding was
inhibited by high concentrations of the corresponding wild-type but not mutant APP tail peptide (Fig. 1A, lanes
7-9). Quantitation revealed that Mints from the brain homogenate
were recovered efficiently on the affinity column with yields of
25-90% (Fig. 1B); for example, the immobilized APP
tail extracted almost all of the brain Mint 3, and even 1 mg/ml of
competing peptide was unable to completely block it from binding to the
column. In contrast, only 25% of brain Mint 1 was bound. No Mint
binding to a control peptide was observed (Fig. 1A,
lane 10).
To test whether Mints interact with APP in vivo, we measured
the levels of transfected APP by quantitative immunoblotting using
125I-labeled secondary antibodies (Fig.
2A,B).
As a control, we cotransfected Fe65, which also binds to the
cytoplasmic tail of APP (Fiore et al., 1995). Cotransfection of all
three Mints dramatically increased APP levels (up to threefold) (Fig.
2, lanes 3-5), extending previous observations that Mint 1 stabilizes transfected APP and increases its steady-state levels (Borg
et al., 1998a; Sastre et al., 1998). As a control, Fe65 (which also
binds to APP) slightly decreased the steady-state levels of transfected
APP, although this was not necessarily a specific effect as
cotransfection of any protein usually decreases expression
because it dilutes the transcription/translation machinery. The
stabilization of APP in the transfected cells could be attributable to
a direct or indirect interaction of Mints with APP. Although the
affinity chromatography experiments already suggested a direct
interaction (Fig. 1), we examined this question further by testing
whether Mints were coimmunoprecipitated with APP from the transfected
cells (Fig. 2C). Indeed, Mints 1, 2, and 3 were
coimmunoprecipitated with antibodies to APP, but not with control
antibodies (Fig. 2C), supporting the notion that Mints
directly bind to APP.
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Figure 2.
Cotransfection of Mints 1, 2, or 3 increases the
steady-state levels of APP. A, Immunoblot analysis of
HEK293 cells cotransfected with expression plasmids encoding APP695,
Mints 1-3, and Fe65 as indicated. Under control conditions
(lane 1), little APP695 is expressed in the cells
because the predominant splice variants in peripheral tissues include
the Kunitz domain (Kitaguchi et al., 1988; Tanzi et al., 1988). The
migration position of the endogenous APP containing the Kunitz domain
and the transfected APP695 lacking this domain are indicated on the
right. The quantities of loaded lysates were normalized
to equal amounts of transfected cells based on the activity of
cotransfected -galactosidase. B, Quantitation of the
levels of APP695 in transfected HEK293 cells shown in A.
Note that Fe65 slightly decreases APP695 levels, whereas all Mints
increase it several fold. The levels of endogenous APP do not change
significantly because most of the cells are not transfected and thus
are not exposed to the transfected Mints. C,
Immunoprecipitation assay of Mint binding to APP. APP695 and Mints 1, 2, or 3 were coexpressed in transfected HEK293 cells
(Start, lane 1). Cellular proteins were
then immunoprecipitated with an antibody to the C terminus of APP
(IP APP, lane 2) or a control antibody
(IP Control, lane 3). Samples were
analyzed by immunoblotting with monoclonal antibodies to the indicated
proteins. The double band for Mint 2 is probably caused by a
hypersensitive proteolytic site in the N terminus of Mint 2. For
detection of APP, the quantity loaded in lanes 2 and
3 was fivefold less than for detection of Mints.
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Binding of Mints to presenilins
We next examined the potential interaction of Mints with
presenilins using the same affinity chromatography approach used for
APP binding. Immunoblotting showed that all three Mints bound to the
cytoplasmic C-terminal sequence of presenilin 1 in agreement with
previous observations (Fig.
3A) (Lau et al., 2000).
Binding was specific because Mints were not bound to control beads, and control proteins such as dissociation inhibitor protein (GDI) and
synaptophysin were not retained on the presenilin 1 column. However,
quantitations revealed that presenilin binding differed among Mints
(Fig. 3B) (and data not shown). Of endogenous Mints 1 and 3 from brain, 14-16% were recovered on the presenilin column, but only
3% of Mint 2 was bound. Mints bind to the cytoplasmic tail of APP via
an interaction of the Mint PTB domain with the NPTY sequence in APP
(Borg et al., 1996; McLoughlin and Miller, 1996; Zhang et al., 1997).
Presenilin 1 does not include an NPxY sequence but features a
C-terminal sequence that resembles that of neurexins, which bind to the
PDZ domains of Mints (Biederer and Südhof, 2000), suggesting that
the binding of Mints to presenilin 1 may be mediated by one or both
Mint PDZ domains. To test whether other PDZ domain proteins also bind
to the C-terminal sequence of presenilin 1, we examined the presenilin
binding of PSD-95, an abundant component of the postsynaptic density
that contains three PDZ domains (Cho et al., 1992; Kistner et al.,
1993), and of Velis, a family of three proteins that contain a single
PDZ domain (Butz et al., 1998). Both PDZ domain proteins were bound; although binding of PSD-95 was weak, Velis were captured to the same
extent as Mints 1 and 3 (Fig.
3A,B).
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Figure 3.
Binding of both PDZ domains of Mints to the
cytoplasmic C-terminal sequence of presenilins. A,
Proteins solubilized from a rat forebrain membrane preparation
(Start, lane 1) were bound to an affinity
column containing a peptide corresponding to the C terminus of
presenilin 1 (Pres1, lane 2) or a control
peptide derived from gp41 (Control, lane
3). Bound proteins were analyzed by immunoblotting with
antibodies to the indicated proteins, which included the negative
control proteins GDI and synaptophysin (Syp). Note that
the antibody to Velis that was used (Butz et al., 1998) recognizes all
three Veli isoforms, which migrate as two distinct bands.
B, Percentage of Mints solubilized from a rat forebrain
membrane preparation that bound to an affinity column containing a
peptide corresponding to the C terminus of presenilin 1 or a control
peptide derived from gp41 (n = 3).
C, Binding of Mint 1 solubilized from a rat forebrain
preparation to the immobilized C-terminal sequences of human
presenilins 1 and 2, Drosophila presenilin, and control
peptides derived from HPV2 and gp41 (n = 3).
D, Binding of various Mint 1 mutants prepared from
transfected COS cells to the immobilized cytoplasmic C-terminal
sequence of presenilin 1. Mint 1 mutants containing inactivating point
mutations in either the first (Mint 1 PDZ1*), the second
(Mint 1 PDZ2*), or both PDZ domains (Mint 1 PDZ1*/2*), or a truncation mutant of Mint 1 lacking the two PDZ domains (Mint 1
PDZ) were analyzed by affinity chromatography on
immobilized presenilin 1 or gp41 control peptides.
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The fact that multiple unrelated PDZ domain proteins bind to the
C-terminal sequence of presenilin 1 but various Mints exhibit large
differences in binding raises concerns about the specificity of the
interaction of Mints with presenilins, prompting us to examine it
further. The C-terminal presenilin 1 sequence is conserved in
vertebrate presenilin 2 and in Drosophila presenilin.
C-terminal peptides from all of these presenilins captured Mint 1, whereas control peptides did not, suggesting that all of these
presenilins potentially interact with PDZ domain proteins (Fig.
3C). To identify which of the two PDZ domains in Mints binds
to presenilins, we mutated the PDZ1 and PDZ2 domains of Mint 1 separately or together, or deleted them both. Presenilin binding assays
with wild-type and mutant Mint proteins produced in transfected COS
cells revealed that mutations in each of the two Mint 1 PDZ domains
resulted in an approximately twofold decrease in binding (Fig.
3D). Mutations in both PDZ domains or deletion of both PDZ
domains almost completely abolished binding. Together these data
suggest that each of the two Mint PDZ domains individually binds to
presenilins in vitro.
Localization of endogenous Mints in neurons
In vertebrates, Mints 1 and 2 are detectable only in brain,
whereas Mint 3 is expressed ubiquitously (Okamoto et al., 1997; Okamoto
and Südhof, 1998). To study the localization of Mints, we stained
rat brain sections with antibodies to Mints 1 and 2. Mint 3 could not
be investigated because the available antibodies were not suitable for
immunocytochemistry (data not shown). Abundant labeling of the neuronal
cell bodies, with less staining of the dendrites, was observed in the
hippocampus (Fig.
4a,b,d,e)
[see also McLoughlin et al. (1999)]. Mint 1 was particularly strongly expressed in interneurons. APP exhibited a very similar distribution to
Mints 1 and 2 (Fig. 4c,f). Notably, nuclei
were not labeled. Staining throughout the neuropil was detected that
was weaker than the cell body staining, indicating that low levels of
Mints may be present at synapses as suggested by Okamoto et al.
(2000).
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Figure 4.
Immunoreactivity of Mint 1, Mint 2, and APP in
mouse hippocampus. Vibratome sections (35 µm) from adult mouse
hippocampus were stained for Mint 1 (a,
d), Mint 2 (b, e), and APP
(c, f) using the avidin-biotin
peroxidase method. d, e, and
f are enlarged images of CA3 regions from
a, b, and c and show that
the immunostaining is clearly present in both cell soma and proximal
apical dendrites. Antibodies against Mint 1 were polyclonal and
monoclonal against Mint 2. The polyclonal APP antibody that was used is
directed against a C-terminal epitope in the cytoplasmic APP tail that
is liberated after -cleavage. Scale bars (shown in c
for a-c): 0.5 mm; (shown in
f for d-f): 0.1 mm.
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To examine the subcellular distribution of Mints, we
performed immunofluorescence staining of
cultured hippocampal neurons (Figs. 5,
6). Mints 1 and 2 were predominantly
localized in a perinuclear compartment where they overlapped almost
completely (Fig. 5a-c). Double labeling with
antibodies to synapsins as a presynaptic marker failed to detect high
levels of Mints in synapses (Fig. 5d-f)
(and data not shown). APP was mostly colocalized with Mints
(Fig. 5g-l). Compared with Mints, more
APP appeared to be present in neurites, suggesting that at steady
state, Mints are more concentrated in the perinuclear compartments than
APP.
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Figure 5.
Immunofluorescence localization of Mints 1 and 2 in cultured hippocampal neurons. The left
(red) and center
(green) pictures show the separate
fluorescence channels from double-immunofluorescence labeling
experiments, whereas the right pictures show the merged
images. Neurons were labeled with the following antibodies:
a-c, antibodies to Mint 1 (a) and Mint 2 (b). Note
that the pictures show a low-magnification overview with a
high-magnification inset.
d-f, antibodies to synapsins
(d) and Mint 2 (e).
g-i, Antibodies to APP
(g) and Mint 1 (h).
j-l, Antibodies to APP
(j) and Mint 2 (k). Scale
bars (shown in a for low-magnification views in
d-l): 50 µm; (shown in l for
insets in a-c and the
full images in a-l): 20 µm.
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Figure 6.
Mints 1 and 2 are concentrated in the
trans-Golgi complex. Subcellular distribution of Mints
has been investigated further with a group of well characterized
markers to Golgi apparatus, endoplasmic reticulum, and early endosome.
Cultured hippocampal neurons were double labeled with antibodies to the
following proteins: a-c, Mint 2 (a), TGN38 (b), and merged
images (c); d-f,
Mint 1 (d), the Golgi 58K protein
(e), and merged images (f);
g-i, Mint 2 (g),
calnexin (h), and merged images
(i); j-l, Mint 2 (j), EEA1 (k), and merged
images (l). Scale bar (shown in l
for a-l): 15 µm.
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In the next set of experiments, we double labeled cultured hippocampal
neurons with antibodies to marker proteins to identify the perinuclear
compartment containing Mints (Fig. 6). We found that Mints colocalize
best with TGN38 (Fig. 6a-c) (and data not shown), a marker of the trans-Golgi complex (Luzio et al.,
1990). A similar localization, but not quite as precise, was observed with the 58K Golgi protein (Fig. 6d-f)
(and data not shown), a peripheral membrane protein that is enriched
in, but also found outside of, the trans-Golgi complex
(Bloom and Brashear, 1989). By contrast, double labeling of neurons
with antibodies to Mint 1 or 2 and the endoplasmic reticulum protein
calnexin (Wada et al., 1991) failed to detect an overlap in
localization (Fig. 6g-i). Similarly, the early
endosomal protein EEA1 (Mu et al., 1995) exhibited a different staining
pattern (Fig. 6j-l). Together these data
support the conclusion that in mature neurons in situ (Fig. 4) and in culture (Figs. 5, 6), Mints 1 and 2 are colocalized in the
trans-Golgi complex, a localization consistent with a role in APP trafficking (Borg et al., 1998a; Sastre et al., 1998) and in
directing proteins out of the trans-Golgi apparatus toward defined plasma membrane domains (Rongo et al., 1998; Whitfield et al.,
1999). Thus at steady state, Mints exhibit a localization that is
similar to that of APP but notably distinct from that of either CASK or
Munc18-1.
A transactivation assay of APP cleavage
We recently described a function for the cytoplasmic tail of APP
in activating transcription by forming a protein complex with Fe65 (Cao
and Südhof, 2001). The transcriptional activation observed in
these assays could potentially be used to measure APP cleavage but
depends on Fe65, which binds to the same NPTY sequence of APP as Mints
[(which, however, do not activate transcription (Cao and Südhof,
2001)]. To test the potential function of Mints in APP cleavage and
signaling, we used a variant of our assay that allows monitoring of APP
cleavage independent of Fe65 binding. For this purpose we introduced
both Gal4 and VP16 into the cytoplasmic tail of
APP695 at the cytoplasmic boundary of the TMR.
This assay differs from the original assay (Cao and Südhof, 2001)
in that the powerful viral transcriptional activator VP16 (Sadowski et al., 1988) is introduced together with the yeast DNA binding protein Gal4 into APP. Thus transactivation by APP-Gal4/VP16 is independent of
the binding of cellular transcriptional activators but can occur only
after APP is cleaved by -secretase and the released cytoplasmic tail
fragment moves into the nucleus. We transfected the APP-Gal4/VP16
fusion protein into PC12, HEK293, COS, or HeLa cells and measured
transactivation of transcription from a cotransfected Gal4-dependent
reporter plasmid encoding luciferase. As a negative control, we used
Gal4 alone without VP16 or APP (Cao and Südhof, 2001), and as a
positive control, we used Gal4/VP16 without APP. In all
experiments, cells were cotransfected with a constitutive -galactosidase expression vector to control for transfection efficiency and to rule out direct effects of transfected proteins on
transcription. Furthermore, in all cases, expression of transfected proteins was verified by immunoblotting.
In all cell types tested, full-length APP-Gal4/VP16 (APP-GV)
transactivated Gal4-dependent transcription almost as strongly as
Gal4/VP16 alone (~500- to 2000-fold activation over Gal4 alone, depending on cell type), suggesting that cleavage of APP to release the
intracellular fragment was not the major rate-limiting step (Fig.
7, columns 1-3). A
chimeric protein in which Gal4/VP16 was fused to the isolated
cytoplasmic tail of APP (APPICF-GV) was an even
more potent transactivator than Gal4/VP16 alone or full-length APP-Gal4/VP16 (~4000 vs ~500- to 2000-fold activation) (Fig. 7, column 5). Addition of the 12 hydrophobic residues from the
TMR that are present in the initial -cleavage product had no
inhibitory effect on transactivation but induced a moderate stimulation
(Fig. 7, column 6). In contrast, the cytoplasmic APP
tail containing only Gal4 without VP16
(APPICF-Gal4) was inactive when Fe65 was not
cotransfected (less than fivefold activation) (Fig. 7, column 7) (Cao and Südhof, 2001). Cleavage of APP does not
appear to require binding of endogenous cellular proteins to the NPTY
tail sequence (residues 684-687 of APP695)
because mutation of NPTY to NATA had no effect on transactivation.
Specifically, no effect of this mutation was observed with either
full-length APP or the cytoplasmic APP tail fused to Gal4/VP16 (Fig. 7,
columns 4 and 7).
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Figure 7.
Use of APP-Gal4/VP16 fusion proteins to measure
-cleavage of APP. The bar diagram on
top shows results from Gal4-transactivation assays in PC12
cells that were cotransfected with a Gal4-dependent luciferase reporter
plasmid (to measure transactivation using luciferase expression), a
-galactosidase control plasmid (to normalize for transfection
efficiency), and the test plasmids identified by numbers
below the bars. The domain structures of the proteins
encoded by the test plasmids are shown schematically below the
bar diagram (APPe,
extracellular sequences of APP; APPICF,
cytoplasmic sequences of APP). Constructs marked with an
asterisk (APP*-GV,
APPICF*-GV, and
APPICF*-Gal4) contain a
point mutation in which the NPTY sequence in the cytoplasmic tail of
APP is replaced by NATA. Transfected cells were harvested 2 d
after transfection, luciferase, and -galactosidase activities were
determined, and the luciferase activity was normalized for the
-galactosidase activity to control for transfection efficiency as
described in Materials and Methods. The -galactosidase-normalized
luciferase activity is expressed in relation to the activity of cells
cotransfected with Gal4 alone. Data shown are from a representative
experiment repeated multiple times in PC12 cells and in COS, HEK293,
and HeLa cells with similar results. GV, Gal4/VP16 module;
APPICF, intracellular fragment of APP;
APP , -secretase cleavage product
of APP.
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APP sequences required for cleavage
The assay described above uses transfected cells that overexpress
the respective test proteins, raising the concern that transactivation by APP-Gal4/VP16 may be caused by nonspecific proteolysis of the APP-Gal4/VP16 fusion protein instead of specific -/- and
-cleavage. To address this concern, we tested whether specific
sequences of APP were required for transactivation by the embedded
Gal4/VP16 module (Fig. 8).
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Figure 8.
Sequence requirements of APP -cleavage measured
by Gal4/VP16-dependent transactivation. The bar
diagrams on top show results of Gal4/VP16
transactivation assays obtained with the constructs that are
schematically displayed and identified by numbers below the diagrams.
A, Requirements of extracellular and intracellular APP
sequences for transactivation. APP-Gal4/VP16 proteins that contain all
APP sequences (construct 3) or lack the intracellular
(construct 4) or extracellular and transmembrane
sequences (construct 5) were analyzed as described in the
legend to Figure 7. Gal4 (construct 1) and Gal4/VP16
(construct 2) were used as standardization controls to
establish the background and maximal response, respectively.
B, Test of the specific functions of extracellular versus
intracellular APP sequences in -cleavage and of the effect of a
dominant negative mutant of presenilin 2 (PS2DA) (Steiner et
al., 1999). Transactivation was measured by constructs in which
Gal4/VP16 is placed in all possible combinations into the context of
the extracellular and intracellular sequences of APP
(APPe and
APPICF = extracellular and
cytoplasmic sequences of APP, respectively) or neurexin
1 (NRXe and
NRXICF = extracellular and
cytoplasmic sequences of neurexin, respectively). Gal4/VP16 constructs
were transfected without () and with (+) the dominant negative
presenilin 2 expression vector. All bar diagrams exhibit
representative experiments in the cell types identified on the
top. Experiments were performed with test plasmids
cotransfected with a Gal4 luciferase reporter plasmid and a
-galactosidase control plasmid as described in the legend to Figure
7. In A, transactivation as measured by
-galactosidase-normalized luciferase activity is expressed relative
to the activity of Gal4 alone, whereas in B, transactivation
as measured by luciferase activity is shown in arbitrary units only
normalized to -galactosidase activities.
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We first removed the cytoplasmic tail of APP and generated a
"tailless" APP-Gal4/VP16 fusion protein in which the extracellular sequences and the TMR of APP were linked to intracellular Gal4/VP16 followed by a stop codon (APPe-GV). The tailless
APP-Gal4/VP16 protein was almost completely inactive in transactivation
assays compared with Gal4/VP16 alone or APP-Gal4/VP16 or APP-GV,
which represents the initial -secretase cleavage product (Fig.
8A, columns 2-5). This suggests that
Gal4/VP16 is not released from APP-Gal4/VP16 by nonspecific degradation
and that the tail of APP is required either for recognition by the APP
cleavage enzymes or for trafficking of APP to the cleavage compartments.
To differentiate between these two possibilities, we inserted Gal4/VP16
into the cytoplasmic tail of neurexin 1 (NRX-GV). Neurexin 1 is
expressed on the neuronal cell surface similar to APP but is not known
to be processed by proteolytic cleavage (Ushkaryov et al., 1992).
Neurexin 1-Gal4/VP16 was nearly inactive in transactivation assays
in contrast to APP-Gal4/VP16 (Fig. 8B, column
3 vs 6), suggesting that Gal4/VP16-mediated
transactivation requires specific sequences in the APP molecule. To
identify these sequences, we constructed chimeric Gal4/VP16-fusion
proteins containing either extracellular APP sequences with the
intracellular neurexin 1 region, or extracellular neurexin 1
sequences with the intracellular APP region. A fusion protein
composed of the extracellular sequences and TMR of APP coupled to
intracellular Gal4/VP16 and the cytoplasmic tail of neurexin 1 (APPe-GV-NRXICF) strongly
transactivated transcription (Fig. 8B, column
8). In contrast, the reverse fusion protein of the extracellular
neurexin sequences and the neurexin TMR with the cytoplasmic APP
sequences (NRXe-GV-APPICF)
was inactive (Fig. 8B, column 7).
These experiments demonstrate that the extracellular sequences of APP
are essential for proper cleavage, in agreement with results of Struhl
and Adachi (2000). Although the intracellular sequences of APP
are also essential for its processing (see tailless mutant
APPeGV, Fig. 8), they can be functionally
replaced by the intracellular fragment of a plasma membrane protein
like neurexin that exhibits no sequence similarity with APP, and in
particular does not contain an NPxY sequence (Ushkaryov et al., 1992).
This may indicate a role of the APP tail in trafficking to a cleavage compartment at the plasma membrane or derived from the plasma membrane.
However, it should be noted that because APP is fused to a DNA-binding
domain (Gal4) and a transactivation domain (VP16) in the present assay,
this assay does not allow conclusions about the physiological function
of the NPxY sequence in the transactivation process mediated by APP as
described previously (Cao and Südhof, 2001).
Finally, to test whether presenilins are involved in transactivation by
APP-Gal4/VP16, we cotransfected a dominant negative mutant of
presenilin 2 (Steiner et al., 1999) with APP-Gal4/VP16 (Fig.
8B). Transactivation of Gal4-dependent transcription
by full-length APP-Gal4/VP16 was inhibited by the presenilin 2 mutant, whereas the small amount of residual transactivation observed with
neurexin 1-Gal4/VP16 was insensitive to presenilin 2 (Fig. 8B, column 3 vs 6). As
expected, mutant presenilin 2 also potently inhibited transactivation
by the fusion protein of the extracellular domain of APP with
intracellular neurexin sequences (Fig. 8B, column 8) but had no effect on the residual transactivation
observed with the reverse fusion protein containing the extracellular
domain of neurexin 1 coupled to the cytoplasmic tail of APP (Fig.
8B, column 7).
Mint 1 inhibits transactivation mediated by APP-Gal4/VP16
We used the transactivation assay to study the effects of Mints on
APP. Cotransfection of Mint 1 strongly inhibited transactivation mediated by APP-Gal4/VP16 (Fig.
9A). This inhibition was
abolished by mutation of the NPTY sequence in the cytoplasmic tail of
APP-Gal4/VP16, consistent with the notion that direct binding of Mint 1 to APP is required. When we compared the activity of the three Mint
isoforms in the transactivation assay, Mints 1 and 2 potently inhibited transactivation by APP-Gal4/VP16, whereas Mint 3 had no significant effect (Fig. 9B). Quantitative immunoblotting confirmed that
all three Mints were expressed at high levels in the cotransfected cells (Fig. 9C).
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Figure 9.
Mints 1 and 2 but not Mint 3 inhibit
transactivation by APP-Gal4/VP16 fusion proteins. A,
Dose-dependent inhibition by Mint 1 of wild-type APP-Gal4/VP16
transactivation but not of APP-Gal4/VP16 carrying a point mutation in
the cytoplasmic NPTY binding sequence for Mints
(APP*-GV). A constant amount of
wild-type or mutant APP-Gal4/VP16 plasmid (100 ng DNA per well) was
cotransfected with the indicated amounts of Mint 1 expression vector
into HEK293 cells. Transactivation and Mints levels in the cells were
quantified in the same samples as described in Materials and Methods.
B, Effects of Mints 1, 2, and 3 on
APP-Gal4/VP16-dependent transactivation. Mint amounts in
transactivation assay samples were quantified on immunoblots and are
expressed as a fraction of the amount observed with the maximal amount
of DNA transfected to control for the nonlinearity of the relation
between transfected DNA and expressed protein. C,
Quantitated immunoblot analysis of the Mints expressed in the
experiment shown in B.
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Because all three Mints bind to APP in vitro and in
transfected cells (Figs. 1, 2), the selective inability of Mint 3 to
inhibit transactivation was surprising. To gain insight into the
mechanism by which Mint 1 inhibits APP-Gal4/VP16-mediated
transactivation and to understand why Mint 3 has no effect, we examined
a series of Mint mutants. We found that the inactivating point mutation in the first PDZ domain of Mint 1 or deletion of both PDZ domains that
were studied above in the presenilin-binding experiments (Fig. 3)
dramatically increased the inhibition of transactivation by Mint 1 (Fig. 10A). In
contrast, the second PDZ domain point mutation did not alter the
inhibitory effect of Mint 1. Because the various Mint 1 mutants
exhibited different expression levels, the relative amounts of
expressed protein were quantified using 125I-labeled secondary antibodies. These
quantitations showed that the PDZ domain deletion mutant was ~10
times more potent than wild-type Mint 1 (Fig. 10A).
We also tested a Mint 3 mutant with a PDZ domain deletion, which was
completely inactive in the assay, similar to wild-type Mint 3 (Fig.
10B,C). Control transfections showed that the various Mint 1 proteins did not inhibit general transcription but specifically impaired transactivation by
APP-Gal4/VP16 (data not shown).
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Figure 10.
Structure-function analysis of Mint 1: role of
PDZ domains in inhibiting APP-Gal4/VP16-dependent transactivation.
A, HEK293 cells were cotransfected with a constant
amount of APP-Gal4/VP16 expression vector and reporter plasmids and
increasing amounts of expression vectors expressing wild-type Mint 1 or
mutants of Mint 1 carrying point mutations in the first or second PDZ
domains (Mint 1 PDZ1* or PDZ2*,
respectively) or lacking both C-terminal PDZ domains (Mint
1 PDZ). Transactivation and Mint 1 amounts in
the cells were then quantified in the same samples as described in
Materials and Methods. B, Transactivation observed in
HEK293 cells cotransfected with APP-Gal4/VP16 and a control plasmid, or
wild-type Mint 1, a Mint 1 mutant lacking the PDZ domains, wild-type
Mint 3, or a Mint 3 mutant lacking both PDZ domains. All
transactivation levels in A and B are
normalized for the amount of transactivation observed under control
conditions. C, Immunoblot analysis of the Mint mutants
analyzed in B to control expression of the constructs in
the experiments shown.
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The results of Figure 10 suggest that Mint 1 binding to APP may couple
it to another protein that binds to the first PDZ domain, implying that the two PDZ domains of Mint 1 are not equivalent. To examine this further, we measured the effect of mutating the PTB
domain of Mint 1 on its inhibitory activity. On the basis of available
structural information (Zhang et al., 1997), a mutant Mint 1 PTB* was
designed in which critical residues involved in binding to the NPTY
sequence of APP were altered. The PTB domain mutation decreased
inhibition of transactivation but did not abolish it (Fig.
11A), possibly
because the mutant PTB domain retained residual binding activity for
APP. Only the combination of the PTB domain mutation with the point
mutation in the first PDZ domain of Mint 1 or with the deletion of both
PDZ domains abolished its inhibitory effect on transactivation (Fig.
11A) (and data not shown). Conversely, after deletion
of the isoform-specific N-terminal residues of Mint 1 (the sequences
that are N terminal to the PTB- and PDZ domains and account for 451 of
the 839 residues of Mint 1) (Okamoto et al., 1997), the specific
inhibitory activity of Mint 1 was also increased significantly (Fig.
11B). The expression level of the N-terminal Mint 1 deletion mutant was low, suggesting that it may be partially cytotoxic
(Fig. 11C).
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Figure 11.
The PTB and PDZ domains of Mint 1 cooperate in
inhibiting transactivation by APP-Gal4/VP16. A, Effect
of cotransfecting Mint 1 and various Mint 1 point mutants in the PTB
domain and the first PDZ domain with APP-Gal4/VP16. The same DNA amount
of control vector or Mint 1 vector or the vectors encoding the
indicated Mint 1 mutants was cotransfected into HEK293 cells
with the APP-Gal4/VP16 and reporter plasmids, and transactivation was
determined as described in Materials and Methods. B,
Effect of deleting the N-terminal isoform-specific Mint 1 sequences on
inhibition of transactivation. Same amounts of myc-tagged full-length
Mint 1 or an N-terminally truncated Mint 1 mutant containing only the
PTB and PDZ domains were cotransfected with APP-Gal4/VP16 and reporter
constructs into HEK293 cells, and their specific inhibitory activity on
transactivation was determined. To exclude an effect of the
myc-epitope, the specific activity of wild-type and myc-tagged Mint 1 was compared. C, Quantitated immunoblot analysis of Mint
expression in the experiment shown in B. Expressed
proteins were detected using antibodies against the myc epitope.
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Effect of Mints on transactivation by the intracellular fragment
of APP
A possible explanation for the effects of Mints on transactivation
by APP-Gal4/VP16, on the basis of the binding of Mints to APP via their
PTB domain and to presenilins via their PDZ domains (Fig. 1-3), would
be that Mints 1 and 2 interfere with -cleavage of APP. However, the
observations that Mint 3 also binds to APP and presenilins better than
Mint 2 but does not inhibit transactivation, and that both Mint 1 PDZ
domains bind to presenilin 1 in vitro but only the first PDZ
domain is involved in the inhibition of transactivation, argue
against this hypothesis. An alternative explanation for the
transactivational inhibition is that Mints 1 and 2 act on the APP
cytoplasmic tail after it has been released by -cleavage. To
differentiate between these explanations, we measured the effect of
Mints on transactivation by the "precleaved" cytoplasmic tail of
APP fused to Gal4/VP16 (APPICF-GV). To
distinguish specific, i.e., binding-dependent effects from nonspecific
effects, we also analyzed in the same experiments the mutant
cytoplasmic tail of APP that is unable to bind to Mints
(APPICF*-GV). In addition, Mints were compared
with Fe65 as another APP-binding protein and with CASK as an unrelated
control (Fig. 12).
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Figure 12.
Binding-dependent effect of Mints on
transactivation mediated by a fusion protein of the intracellular
fragment of APP with Gal4/VP16. Constant amounts of plasmids encoding
APPICF-GV or the NPTY (684-687) to NATA (684-687) mutant APPICF*-GV
were cotransfected into HEK293 cells together with reporter plasmids
and expression vectors for the indicated proteins. Note that Fe65
actually further enhances transcription even when Gal4 is fused to the
cytoplasmic tail of APP together with the potent transcriptional
activator VP16.
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Mint 1 expression significantly inhibited transactivation mediated by
the cytoplasmic tail of APP fused to Gal4/VP16; this inhibition was
observed only when the APP tail contained a normal Mint binding
sequence (Fig. 12). A similar inhibition was detected with Mint 2 (data
not shown). Mint 3, by contrast, had no significant effect, in
agreement with the results obtained with full-length APP-Gal4/VP16
(Figs. 9B, 10B). Fe65 enhanced
transactivation, again only when the NPTY sequence in the cytoplasmic
tail was intact (Fig. 12), consistent with the overall function of Fe65
in stimulating transcription (Cao and Südhof, 2001). CASK used as
a negative control had no effect on transactivation. Identical effects
of both Mints 1 and 3 and Fe65 were observed when transactivation was
assayed by a construct that mimicked the initial -cleavage product
of APP, i.e., that contained 12 hydrophobic residues from the TMR
preceding the cytoplasmic tail of APP (data not shown). These results
demonstrate that Mint 1 inhibits transactivation downstream of APP
cleavage and that this effect is highly specific for neuronal versus
ubiquitous Mints.
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DISCUSSION |
Mints, also known as X11 and mLin-10 proteins, are a family of
adaptor proteins that have been associated with several localizations and implicated in many potential functions. In adult mammals, Mints 1 and 2 are specific for brain, whereas Mint 3 is expressed ubiquitously
(Okamoto and Südhof, 1997, 1998). Mints are composed of
N-terminal sequences that lack easily identifiable domains and differ
between isoforms and C-terminal sequences that encode similar PTB and
PDZ domains in all Mints. The combination of distinct and shared
domains suggests that the isoform-specific N-terminal sequences of
Mints may confer distinct functions onto the activities of their common
C-terminal domains. However, what do Mints actually do and how similar
or dissimilar are their biological roles? As a first step toward
addressing these questions, we have focused on the best characterized
interaction of Mints, their binding to APP (Borg et al., 1996;
McLoughlin and Miller, 1996; Zhang et al., 1997), and related the
biochemical properties and localizations of Mints to their functional
effects on APP in a transcriptional transactivation assay.
We first showed that all Mints bind to APP and presenilins in
vitro and increase the steady-state levels of APP in transfected cells (Figs. 1-3). These observations agree with and extend previous data (Borg et al., 1996, 1998; McLoughlin and Miller, 1996; Zhang et
al., 1997; Sastre et al., 1998; Lau et al., 2000). Although the
strength of APP binding by Mints and the stabilization of APP by Mints
in transfected cells suggest that the APP-Mint interaction is
physiologically important, the presenilin binding is questionable. The
differences among Mint isoforms, the fact that both PDZ domains of Mint
1 bind equally, and the promiscuous binding properties of the PDZ
domains involved give rise to caution. Not surprisingly, a large number
of interactions mediated by these domains have been reported, the
biological significance of which has been difficult to assess.
As one approach to evaluate the significance of the interaction of
Mints with APP, we next examined the localization of endogenous Mints
in neurons (Figs. 4-6). The observed predominant localization of Mints
in the trans-Golgi network rules out the possibility that
the majority of Mint 1 is part of a permanent presynaptic PDZ domain
complex that could provide a scaffold for synaptic molecules as
originally envisioned (Okamoto et al., 1997; Butz et al., 1998).
Instead, in conjunction with the C. elegans data, these
results indicate that the PDZ domain complexes formed by Mints may be
transient, highlighting a potential role for Mints as escort proteins
that mediate one particular phase in the life of other proteins, for
example protein targeting to a specific part of the cell (Rongo et al.,
1998; Whitfield et al., 1999). However, these results do not resolve
the question of whether the in vitro interactions of Mints
with CASK (Mint 1 only) or Munc18-1 (Mints 1 and 2) are
physiologically relevant. In fact, one possibility is that Mints
participate in trafficking proteins such as Munc18-1 and CASK to
synapses, because we observed a steady-state localization and not a
dynamic view.
Using a transactivation assay that depends on APP cleavage but is
independent of transcriptional activators (Figs. 7, 8), we demonstrated
that Mints 1 and 2 but not Mint 3 dramatically inhibit transactivation
by APP-Gal4/VP16 (Figs. 9-12). Three principal findings were made.
First, compared with the rather moderate effects of Mints on A
production and secretion (Borg et al., 1998a; Sastre et al., 1998), the
inhibition that we observed was nearly complete at higher expression
levels of Mints 1 and 2. This inhibition was specific because two other
proteins that bind to the cytoplasmic tail of APP, Mint 3 and Fe65,
were without inhibitory effect. Given the similarity between the
biochemical properties of Mints 1 and 3 in binding to APP and
presenilin (Figs. 1, 3) and in raising the steady-state levels of APP
in transfected cells (Fig. 2), the lack of inhibition by Mint 3 is
surprising. This observation suggests a functional differentiation of
neuronal versus ubiquitous Mints. It also indicates that the inhibition
cannot be explained simply by binding of Mints to APP and presenilin,
because the inhibitory Mints 1 and 2 and the noninhibitory Mint 3 bind
to APP comparably well (Figs. 1, 2), and the two inhibitory Mints 1 and
2 interact with presenilin with different strength. Nevertheless, inhibition appeared to involve direct binding of Mints 1 and 2 to APP
because it required the Mint-binding site in the cytoplasmic tail of APP.
Second, we found that deletion of the two PDZ domains or of the
isoform-specific N-terminal sequences of Mint 1 greatly potentiated its
inhibitory activity, although the expression level of the N-terminally
truncated mutant dropped precipitously, presumably because it is
cytotoxic. This indicated that the inhibition may be caused by
interference with a normal adaptor function of an endogenous protein
and that this interference is enhanced by deletion of one of the
domains. Interestingly, the PDZ domain mutations showed that the two
PDZ domains are not equivalent because only the mutation in the first
but not the second PDZ domain increased inhibition (Fig. 10). Both
mutations were effective in disrupting PDZ domain function; both
abolished ~50% of presenilin binding, which was eliminated by the
double PDZ domain mutation (Fig. 3C) (data not shown). Thus
the inh |
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ABSTRACT
INTRODUCTION
