From the Department of Neurosciences, The Lerner
Research Institute, Cleveland Clinic Foundation,
Cleveland, Ohio 44195 and the ¶ Department of Molecular
Pharmacology, Albert Einstein College of Medicine,
Bronx, New York 10461
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ABSTRACT |
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Caveolae are plasma membrane invaginations where
key signaling elements are concentrated. In this report, both
biochemical and histochemical analyses demonstrate that the amyloid
precursor protein (APP), a source of A amyloid peptide, is enriched
within caveolae. Caveolin-1, a principal component of caveolae, is
physically associated with APP, and the cytoplasmic domain of APP
directly participates in this binding. The characteristic C-terminal
fragment that results from APP processing by
-secretase, an as yet
unidentified enzyme that cleaves APP within the A
amyloid sequence,
was also localized within these caveolae-enriched fractions. Further
analysis by cell surface biotinylation revealed that this cleavage
event occurs at the cell surface. Importantly,
-secretase processing was significantly promoted by recombinant overexpression of caveolin in
intact cells, resulting in increased secretion of the soluble extracellular domain of APP. Conversely, caveolin depletion using antisense oligonucletotides prevented this cleavage event. Our current
results indicate that caveolae and caveolins may play a pivotal role in
the
-secretase-mediated proteolysis of APP in vivo.
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INTRODUCTION |
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Senile plaques and paired helical filaments are the hallmarks of
the brain pathology of Alzheimer's disease (1). The principal component of the senile plaque is the A amyloid peptide, which is
composed of 39-43 amino acid residues. The A
amyloid peptide is
derived from a full-length precursor protein, termed
APP1 (amyloid precursor
protein) (2). Alternate splicing of the APP gene generates at least 10 distinct isoforms; APP695 is the brain-specific isoform.
The A
amyloid peptide is generated by the processing of APP with
- and
-secretases (3). Alternatively, APP is processed by
-secretase, which cleaves APP within the A
sequence, thereby
precluding the formation of A
(4). The identities of these
secretases remain unknown.
In some inherited forms of Alzheimer's disease, point mutations have been identified within the coding sequence of the APP gene (5). These mutations co-segregate with the disease phenotype and cause Alzheimer's disease. Understanding the molecular function and processing of APP is therefore critical to unraveling the molecular basis of Alzheimer's disease.
One approach to elucidate the function of APP is to identify APP-interacting proteins. At least five distinct classes of molecules have been identified as APP binding partners as follows: Go (6), Fe65 (7), X11 protein (8), Fe65-like protein (9), and APP-BP1 (10). The APP domain that interacts with Go has been localized to residues His657-Lys676 within the cytoplasmic domain of APP695. Go is a brain-specific member of heterotrimeric GTP-binding protein (G-protein) family. The in vivo interaction between APP695 and Go results in apoptotic cell death (11) and inhibition of cAMP response element trans-activation (12). In contrast, functional consequences of interactions between APP and other binding partners have not yet been described. However, it is likely that these APP-interacting protein molecules directly participate in APP-mediated cell signaling events.
A recent report described APP enrichment within caveolae-like domains in neurons (13). Caveolae are plasmalemmal microdomains where multiple signaling molecules are concentrated. Growth factor-mediated and G-protein-mediated signaling cascades have been shown to be initiated within these microdomains (14-16).
A major protein component of caveolae is caveolin. Molecular cloning
has recently identified a caveolin gene family. Three distinct caveolin
genes have been identified, cav-1, -2, and -3 (17-19). In addition, two isoforms of caveolin-1 (Cav-1 and
Cav-1
) are derived from a single transcript from alternate
translation initiation sites. Caveolins-1 and -2 are most abundantly
expressed in adipocytes, endothelial cells, and fibroblastic cell
types, whereas the expression of caveolin-3 is muscle-specific.
Caveolin proteins form homo-oligomers that bind cholesterol and
glycosphingolipids. These protein-protein and protein-lipid
interactions are thought to be essential for caveolae formation
(20).
Evidence is accumulating that caveolins can sequester molecules
involved in G-protein-coupled signaling within caveolae. Heterotrimeric G-proteins are concentrated within caveolae and interact directly with
caveolin via the G subunit (21). In addition, several G-protein-coupled receptors are sequestered within caveolae in a
ligand-independent (endothelin receptor) (22) and
ligand-dependent fashion (muscarinic acetylcholine,
-adrenergic, and bradykinin receptor) (23-26).
The molecular mechanism that underlies the recognition of signaling molecules by caveolin has recently been elucidated (27). Like other scaffolding proteins involved in signal transduction, caveolin contains a modular protein domain, termed the caveolin scaffolding domain. The caveolin scaffolding domain, in turn, recognizes a specific consensus motif within its interacting partners (28). As APP can function as a Go-coupled "receptor" (29) and contains a caveolin binding motif within its cytoplasmic domain, caveolin could provide a means for sequestering APP with caveolae membrane domains.
As APP is expressed in many non-neuronal cell types where caveolin-1 is abundantly expressed, we initiated this study to determine whether APP localizes within caveolae and if caveolin plays a role in this sequestration event. Here, we show that (i) endogenous APP co-fractionates with caveolin-1 in three distinct cell lines (COS-7, HEK293, and MDCK); (ii) recombinantly expressed APP co-immunoprecipitates with caveolin-1 when expressed in COS-7 cells; (iii) a GST fusion protein encoding the cytoplasmic domain of APP interacts directly with purified recombinant caveolin-1 protein. These three independent lines of evidence support the hypothesis that APP physically associates with caveolin.
Functionally, we show that the -secretase cleavage product of APP is
enriched within caveolae and that this cleavage event occurs at the
cell surface. Specifically, we find that recombinant overexpression of
caveolin-1 promoted
-secretase-mediated proteolysis of APP. The
production of the
-secretase cleavage product of APP was abrogated
by caveolin-1-based antisense oligonucleotides that effectively block
caveolin-1 expression. These in vivo functional data clearly
indicate that caveolae and caveolin proteins play a pivotal role in
-secretase-mediated processing of APP on the plasma membrane.
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EXPERIMENTAL PROCEDURES |
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Materials-- The cDNAs for canine caveolin-1 were as we described previously (30). Ni-NTA-agarose for purification of polyhistidine-tagged proteins was from Qiagen. Antibodies and their sources were as follows: anti-caveolin IgG polyclonal and monoclonal (2234) (gifts of Dr. John R. Glenney, Transduction Labs), anti-myc epitope IgG (monoclonal, 9E10; Santa Cruz Biotech), anti-HA epitope IgG (rat and mouse (12CA5) monoclonal, Boehringer Mannheim). A variety of other reagents were purchased commercially as follows: fetal bovine serum (JRH Biosciences), purified myelin basic protein (Sigma), and pre-stained protein markers (Bio-Rad and NOVEX). The anti-GST antibody was from Santa Cruz Biotechnology, and anti-myelin basic protein antibody was from Dako.
Antibodies against APP are well characterized and include the following: (i) monoclonal antibody: 22C11 specific for the extracellular domain of APP (Boehringer Mannheim), 4G8 specific for ACell Culture and cDNA Transfection-- COS-7, HEK293, and MDCK cells (all from ATCC) were grown in DMEM plus 10% fetal bovine serum supplemented with penicillin and streptomycin (complete DMEM). cDNA transfections were conducted as described previously (36). Briefly, COS-7 cells were seeded at 1 × 106 cells per 100-mm dish. Twenty-four hours later, DNA (6 µg) of interest was transfected with 30 µl of LipofectAMINE (Life Technologies, Inc.) in 5 ml of DMEM. 24 h post-transfection, the media were changed to complete DMEM. 48 h post-transfection, media or cells were harvested and subjected to further analyses.
cDNA Constructions-- For construction of the cDNA encoding HA-inserted APP, the APP695 cDNA in pCDNA-1 (37) was digested with XmaI and dephosphorylated with calf intestine alkaline phosphatase (New England Biolabs). The oligonucleotides that encode HA sequence possessing XmaI restriction sites at both ends were phosphorylated, annealed, and ligated with the dephosphorylated APP cDNA. The orientation and sequence of the inserted fragment was confirmed by DNA sequencing. The HA peptide sequence was inserted between residues Pro307 and Gly308 of APP695. For construction of the GST fusion protein cDNA encoding the cytoplasmic domain of APP (Lys649-Asn695), this domain was amplified as a fragment with additional restriction cleavage sites at both ends (EcoRI and BamHI sites). After PCR, the amplified fragment was digested with EcoRI and BamHI and subcloned in frame into the vector pGEX4T-1 (Amersham Pharmacia Biotech).
Detergent-free Purification of Caveolin-rich Membrane Domains-- Cultured cells were grown to confluence in 150-mm dishes and used to prepare caveolin-enriched membrane fractions, as described previously (38). After two washes with ice-cold phosphate-buffered saline, cultured cells (two confluent 150-mm dishes) were scraped into 2 ml of 500 mM sodium carbonate, pH 11.0, and homogenized sequentially with a loose-fitting Dounce homogenizer (10 strokes), a Polytron tissue grinder (three 10-s bursts; Kinematica GmbH, Brinkmann Instruments), and a sonicator (three 20-s bursts; Branson Sonifier 250, Branson Ultrasonic Corp.). The homogenate was then adjusted to 45% sucrose by the addition of 2 ml of 90% sucrose in MBS (25 mM Mes, pH 6.5, 0.15 M NaCl) and placed at the bottom of an ultracentrifuge tube. A 5-35% discontinuous sucrose gradient was formed above (4 ml of 5% sucrose, 4 ml of 35% sucrose; both in MBS containing 250 mM sodium carbonate) and centrifuged at 39,000 rpm for 16-20 h in an SW41 rotor (Beckman Instruments). A light-scattering band confined to the 5-35% sucrose interface contained caveolin but excluded most other cellular proteins. The caveolae-rich fractions were diluted 3-fold with MBS and centrifuged at 15,000 rpm for 20 min at 4 °C. The pellets were used as "purified caveolae-enriched membranes." This protocol separated caveolin from the bulk of cellular membranes and cytosolic proteins. This fractionation scheme is based on the specific buoyant density of caveolin-rich membrane domains and their resistance to solubilization by sodium carbonate. By using this scheme, endogenous MDCK (38) and COS-7 caveolin (39) are purified 2,000-fold relative to total cell lysates; approximately 90-95% of caveolin is recovered in fractions 4 and 5 of these sucrose density gradients while excluding greater than 99.95% of total cellular proteins. By using this same fractionation scheme, cav-myc and cav-myc-H7 co-fractionated with endogenous caveolin, an indication that the myc and polyhistidine tags do not interfere with the correct targeting of caveolin (38). Sodium carbonate extraction is routinely used to determine if proteins are firmly attached to membranes, and caveolin is not solubilized by sodium carbonate. By using this scheme, endogenous caveolin and cav-myc-H7 were not only recovered almost quantitatively in fractions 4 and 5, while excluding most cellular proteins, but also were separated from the glycosylphosphatidylinositol-linked plasma membrane marker, carbonic anhydrase IV (38). This is consistent with recent observations that glycosylphosphatidylinositol-linked proteins are not concentrated directly within caveolae but may reside in close proximity to the "neck regions" of caveolae within intact cells (40).
As an alternative approach to purify caveolae, a protocol developed by Smart et al. (41) was employed. A plasma membrane fraction was prepared from 10 100-mm dishes of confluent tissue culture cells. Each dish was washed twice with 5 ml of buffer A (0.25 M sucrose, 1 mM EDTA, 20 mM Tricine, pH 7.8). Cells were collected by centrifugation at 1,400 × g for 5 min (Sorval RT6000: 3000 rpm) and resuspended in 1 ml of buffer A and homogenized 15 times with Teflon glass homogenizer. Homogenized cells were centrifuged at 1,000 × g for 10 min (Sorval RT6000: 2500 rpm), and the supernatant was subjected to Percoll gradient centrifugation. The sample was overlaid on top of 23 ml of 30% Percoll solution in buffer A and centrifuged at 83,000 × g (30,000 rpm) for 30 min in a Beckman Ti-60 rotor. A plasma membrane fraction was collected, and the volume was adjusted to 2 ml in buffer A. 1.84 ml of 50% Optiprep in buffer B (0.25 M sucrose, 6 mM EDTA, 120 mM Tricine, pH 7.8) was added to 0.16 ml of buffer A (23% Optiprep solution), which was mixed with the sonicated plasma membrane fraction. The entire solution was placed at the bottom of a Beckman SW41 rotor tube and overlaid onto a linear 10-20% Optiprep gradient (prepared by diluting 50% Optiprep in buffer A and B) and centrifuged at 52,000 × g (18000 rpm) for 90 min using SW41 rotor (Beckman). The bottom fraction was collected (non-caveolae membrane). The top 5 ml of the gradient (fractions 1-6) was collected and mixed with 50% Optiprep in buffer B, which was then placed on the bottom of SW41 rotor tube and overlaid by 2 ml of 5% Optiprep in buffer A. The membrane fractions were centrifuged at 52,000 × g for 90 min. An opaque band located just above the 5% interface was designated the caveolae fraction (41).Immunoblotting of Gradient Fractions-- From the top of each gradient, 1-ml gradient fractions were collected to yield a total of 12 fractions. Caveolin migrates mainly in fractions 4 and 5 of these sucrose density gradients (38). Gradient fractions were separated by SDS-PAGE and transferred to Immobilon-PSQ sheets (Millipore). After transfer, sheets were stained with Ponceau S to visualize protein bands and subjected to immunoblotting. For immunoblotting using ECL, incubation conditions were as described by the manufacturer (Amersham Pharmacia Biotech), except we supplemented our blocking solution with both 1% bovine serum albumin, 3% nonfat dry milk, and 0.02% sodium azide.
Chemical Cross-linking Studies--
After transfection of
cDNAs, cells were incubated with DMEM plus 10% Me2SO
containing 48 µg/ml bis-[-(4-azidosalicylamindo)ethyl] disulfide
(Pierce) for 10 min at RT. Media were discarded, and cells were exposed
to ultraviolet light (wavelength 365 nm) (model LM20E, VWR Scientific)
for 15 min at 4 °C. Cells were solubilized in a lysis buffer A (10 mM Tris/HCl, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, 60 mM octylglycoside, 1%
Triton X-100, 1 mM aprotinin, 1 mM
phenylmethylsulfonyl fluoride) with sonication (three 20-s bursts,
Branson Sonifier 250, Branson Ultrasonic Corp). After centrifugation at
15,000 rpm for 20 min at 4 °C, supernatants were collected and
subjected to immunoprecipitation/immunoblot analysis. Cross-linking
reagents were cleaved by boiling samples at 95 °C for 5 min in the
presence of 5%
-mercaptoethanol, and the samples were subjected to
SDS-PAGE.
Immunoprecipitation of Caveolae-enriched Fractions-- Caveolae-enriched fractions were diluted with Mes-buffered saline (MBS) and solubilized in buffer A without Triton X-100. After dialysis against buffer A without detergent, immunoprecipitation was conducted with anti-HA (Boehringer Mannheim) or anti-myc antibodies (Santa Cruz Biotechnology) overnight at 4 °C. Fifty µl of a ~50% slurry protein G-Sepharose (Amersham Pharmacia Biotech) was added and further incubated for 2 h. The beads were washed 3 times with Tris-buffered saline (TBS, 10 mM Tris/HCl, pH 8.0, 0.15 M NaCl), once with TE buffer (0.1 M Tris/HCl, pH 6.8, 1 mM EDTA), and subjected to SDS-PAGE/immunoblotting.
Affinity Purification of Caveolin-myc-H7 Expressed in Insect Cells Using Ni-NTA-Agarose-- The Sf21 cells overproducing His-tagged caveolin-1 (76) were collected and solubilized in buffer A. Ni-NTA-agarose (200 µl) was then pre-equilibrated with TBS. Solubilized caveolin-containing cell extracts were then added to the resin and incubated for 6 h at 4 °C on a rotating platform. After binding, the beads were allowed to gently settle by gravity (5 min on ice) and washed extensively (four times, 5 min each; twice with TBS and twice with TBS plus 30 mM imidazole). After washing, bound proteins were eluted with TBS containing 200 mM imidazole, as per the manufacturer's instructions (Qiagen).
Purification of GST-APP Cytoplasmic Region-- An overnight culture of Escherichia coli harboring plasmids encoding either GST-APP or GST alone was purified as described previously (42).
Interaction of Caveolin-myc-H7 with GST-APP
Cytoplasmic Region Proteins--
The interaction of GST-APP fusion
proteins with caveolin-1-myc-H7 was evaluated as
described for the interaction of caveolin with baculovirus-expressed
heterotrimeric G-protein subunits (21). Briefly, GST or purified
GST-APP cytoplasmic fusion proteins bound to glutathione-agarose beads
were extensively prewashed with phosphate-buffered saline and lysis
buffer containing protease inhibitors. These beads contained ~100
pmol of a given fusion protein per 100 µl of packed volume.
Approximately 100 µl of this material was incubated with 1 µg of
caveolin-1-myc-H7 on a rotating platform
overnight at 4 °C. After binding, the beads were washed extensively
(6-8 times) with buffer B (50 mM HEPES/NaOH, pH 7.5, 120 mM NaCl, 1 mM EDTA, 0.5% CHAPS, and protease
inhibitors). Finally, bound proteins were eluted with 100 µl of
elution buffer containing 50 mM Tris, pH 8.0, 1 mM EDTA, 1% Triton X-100, 10 mM reduced glutathione, and protease inhibitors. The eluate was mixed 1:1 with 2×
sample buffer and subjected to SDS-PAGE (10% acrylamide) and Western
blot analysis with anti-caveolin polyclonal antibody (1:1000 dilution,
Transduction Laboratories). Horseradish peroxidase-conjugated secondary
antibodies (1:10000 dilution, Bio-Rad) were used to visualize bound
primary antibodies by ECL (Amersham Pharmacia Biotech). In place of
caveolin-myc-H7, purified myelin basic protein (a negative
control) and FE65 (a positive control) were also used for
binding experiments. For detection of myelin basic protein, anti-myelin
basic protein antibody was used. For the binding study of
FE65 and APP, the gene of human FE65 PTB2
domain (484-612 amino acids) was first cloned (43) by PCR with a set
of 5' and 3' primers (5' primer, ACGCGTCGACTCACAGGAAGGAGAGGAAACGCAC; 3'
primer, GCGGATCCTGTGAGGCACCTGCCAAGAAC) using human hippocampus cDNA
library (CLONTECH) as a template. PCR product was
digested with BamHI-SalI and ligated into
pCAL-n-EK vector (Stratagene) for generating a fusion protein cDNA
which contains calmodulin binding peptide and FE65-PTB2
fusion domains. Expression of calmodulin binding peptide-FE65-PTB2 protein was induced by
isopropyl-1-thio--D-galactopyranoside and was purified
using calmodulin affinity resin (Stratagene) as described previously
(44). The purified fusion protein (1 µg) was used for binding
analysis as described above. After transfer of eluted samples separated
in SDS-PAGE, the membrane was blocked with 2% skim milk and 2% bovine
serum albumin in PBS, 0.02% sodium azide overnight at 4 °C. The
membrane was incubated with 5 µg/ml biotinylated calmodulin
(Calbiochem) in washing buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 mM CaCl2) for 30 min at
RT, washed 3 times with the same washing buffer (10 min each),
incubated with 1/20,000 diluted streptavidin-horseradish
peroxidase-conjugated (Pierce) for 30 min at RT, and washed 3 times
with the same washing buffer. Finally bands were visualized by
diaminobenzidine staining (Vector).
Immunocytochemistry-- Transfected cells (50,000 cells) were split onto 4-well chamber slides (Becton Dickinson). Twenty-four hours later, the cells were incubated for 15 min at 8 °C with anti-HA rat monoclonal antibody (5 µg/ml, Boehringer Mannheim) and cholera toxin B subunit conjugated with peroxidase (1 µg/ml, Sigma) in complete DMEM (45). Cells were washed with PBS (4 × 5 min) and fixed in PBS containing 4% paraformaldehyde. After washing with PBS (5 × 5 min), nonspecific binding of antibodies was blocked with 3% normal goat serum (Life Technologies, Inc.) in PBS for 30 min at RT. Cells were then incubated with anti-cholera toxin rabbit antibody (Calbiochem) in PBS containing 3% normal goat serum for 1 h at RT. The chamber slides were washed with PBS (4 × 5 min) and incubated for 60 min at RT with secondary antibodies (Texas Red-conjugated goat anti-rabbit IgG (preabsorbed with rat and mouse IgG), 1:300 dilution, Jackson ImmunoResearch, and biotin-conjugated goat anti-rat IgG, 1:300 dilution, Boehringer Mannheim) and washed in PBS and in 0.1 M NaHCO3, pH 8.2 (4 × for 5 min). Cells were incubated with avidin-fluorescein isothiocyanate conjugate (1/300 dilution, Vector) in NaHCO3 buffer for 1 h at RT. Slides were mounted with Vectashield (Vector) and analyzed with a Leica TCS NT confocal microscope.
Immunoprecipitation of APPs--
Forty-eight hours
post-transfection, 1 ml of media was mixed with 1 ml of 2× RIPA (150 mM NaCl, 1.0% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS,
50 mM Tris/HCl, pH 8.0) and incubated with 2 µg each of
antibodies (anti-A-(1-28), 1153, CT-15, 6E10, 4G8, normal rabbit
serum (NRG) or normal mouse serum (NMG)) overnight at 4 °C. 50 µl
of ~50% slurry protein G-Sepharose was added and incubated for
2 h at 4 °C. Beads were washed 3 times with RIPA buffer and once with TE buffer. Final samples were incubated with 60 µl of 2 × sampling buffer containing
-mercaptoethanol and boiled at 95 °C for 3 min. Fifteen µl of each sample was subjected to
SDS-PAGE. As a critical control, 2 µg of anti-A
-(1-28) polyclonal
antibody was preincubated with 20 µg of the A
-(1-16) synthetic
peptide (Bachem) overnight at 4 °C and used for immunoprecipitation.
For immunoblotting, immunostained bands were visualized with ECL. In
Fig. 6C, we used ECL-Plus (Amersham Pharmacia Biotech).
Cell Surface Biotinylation--
Cells were washed twice with
ice-cold PBS and incubated with PBS containing 0.5 mg/ml
sulfo-NHS-biotin (Pierce) for 30 min at 4 °C (46), washed three
times with ice-cold PBS, and placed in complete DMEM. Four hours after
incubation at 37 °C in a CO2 incubator, media were
harvested and mixed with the same volume of 2× RIPA buffer, followed
by incubation with Neutravidin beads (Pierce). After overnight
incubation at 4 °C, the Neutravidin beads were washed three times
with RIPA buffer and once with TE buffer. Final samples were mixed with
sampling buffer containing -mercaptoethanol, boiled for 3 min, and
subjected to immunoblot analysis with 22C11.
Antisense Oligonucleotide Introduction-- COS-7 cells were seeded at a density of 1 × 106 cells per 100-mm dish. Twenty-four hours later, 3 µg each of antisense caveolin-1 oligonucleotide (TTTGCCCCCAGACAT; complementary to the 15-base initiation sequence of human caveolin-1) and the HA-APP695 cDNA were co-transfected into COS-7 cells using 30 µl of LipofectAMINE. Twelve hours later, the media were discarded and replaced with 8 ml of complete DMEM. Forty-eight hours post-transfection, media were harvested and mixed with 2× RIPA buffer. As a control, a sense-caveolin-1 oligonucleotide (ATGTCTGGGGGCAAA) which corresponded to the 15 bases of the initiation sequence was used. The media and cells were subjected to further analyses.
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RESULTS |
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Interaction of APP with Caveolin-1: A Requirement for the Caveolin Scaffolding Domain-- To investigate whether endogenous APP is associated with caveolae membranes, caveolae-enriched membrane domains from COS-7 cells were purified by a detergent-free procedure and were ultracentrifuged in a sucrose density gradient. Immunoblot analysis of the resulting gradient fractions with APP antibody (22C11) detected the vast majority of APP in fractions 4 and 5 (Fig. 1A, upper panel). Similarly, caveolin-1 was confined to the same region of the gradient (fractions 4 and 5), indicating that caveolae are contained within these fractions (Fig. 1A, lower panel). Densitometric analysis revealed that greater than 90% of the total APP-reactive material was recovered in fractions 4 and 5. These results demonstrate that molecules containing the 22C11 epitope are primarily enriched with caveolae microdomains in COS-7 cells.
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A C-terminal Degradation Product of APP Is Localized within
Caveolae-rich Fractions, and the Generation of This APP Product Is
Specifically Dependent upon the Expression of Caveolin-1--
The
-secretase-generated degradation product of APP can be detected in
COS-7 cells by immunoblotting with C-terminal APP antibodies (51). To
determine if
-secretase-mediated APP degradation occurred in our
experimental system, COS-7 cells were transiently transfected with
HA-tagged APP and subjected to detergent-free subcellular
fractionation. These fractions were then analyzed by immunoblotting
with C-terminal APP antibodies (AC-1). As shown in Fig.
4, two bands confined to caveolin-rich
fraction 4 were detected; the high molecular weight band represents
intact APP695, whereas the low molecular weight band is a
short fragment of APP that contains a C-terminal epitope recognized by
the AC-1 antibody. As this low molecular weight band was not detected
by immunoblotting with an antibody directed against the extracellular
domain of APP (22C11) (Fig. 2), these results indicate that this short
fragment represents a C-terminally derived APP degradation
product. To obtain a more precise estimate of the molecular mass
of this C-terminal APP fragment, the same sample was subjected to
Tris/Tricine SDS-PAGE and immunoblotting with AC-1. A 10-kDa band
was detected, supporting the hypothesis that it is derived from
APP
-secretase processing (51).
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DISCUSSION |
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We have presented several independent lines of evidence that APP
localizes within caveolae membranes and interacts directly with
caveolin-1, a principal structural component of caveolae. More
specifically, we have shown that (i) APP and caveolin-1 co-fractionate when cells are subjected to two independent detergent-free subcellular fractionation protocols previously used to purify caveolae from cultured cells; (ii) APP and caveolin-1 can form a physical complex in
intact cells as shown by chemical cross-linking and
co-immunoprecipitation experiments; (iii) association of APP with
caveolin-1 occurs via a direct interaction between the C-terminal
cytoplasmic domain of APP and the caveolin-1 scaffolding domain; (iv)
cell surface caveolae contain APP; (v) the characteristic C-terminal
fragment that results from APP processing by -secretase was also
localized within caveolae; (vi)
-secretase processing was
significantly promoted by recombinant overexpression of caveolin-1 in
intact cells, resulting in increased secretion of the soluble
extracellular domain of APP; and (vii) depletion of endogenous
caveolin-1 using antisense oligonucleotides prevented this cleavage
event. It is therefore likely that a physical interaction between APP
and caveolin-1 functionally sequesters APP within caveolae membranes
and thereby compartmentalizes the
-secretase-mediated proteolysis of
APP.
The caveolin-1 region that interacts with APP includes the caveolin scaffolding domain, a short stretch of 20 amino acids that directly participate in the recognition of multiple classes of signaling molecules. Recent evidence suggests that the caveolin scaffolding domain functions as a modular protein domain that interacts with peptide and protein ligands that contain a specific caveolin binding motif (28). APP contains a predicted caveolin binding motif within its C-terminal cytoplasmic tail. It is likely that this sequence is recognized by the caveolin scaffolding domain and functions to sequester APP within caveolae microdomains.
Cholesterol and sphingolipids are highly concentrated with caveolae
domains. Recent findings indicate that transport of newly synthesized
cholesterol from the endoplasmic reticulum to the plasma membrane is
mediated via caveolin proteins (52, 53). In addition, caveolin-1
directly binds cholesterol, and cholesterol is required for the
insertion of recombinant caveolin-1 into model lipid membranes (54,
55). There are several links between cholesterol metabolism and the
pathogenesis of Alzheimer's disease. Cholesterol affects APP
processing by interfering with the activity of -secretase (56). An
isoform of a major cholesterol carrier protein, apoE, is a genetic risk
factor for Alzheimer's disease (57). Thus, these links to cholesterol
metabolism may simply reflect the caveolar localization of APP.
The processing of APP has been intensively studied since the APP
molecule was first identified. Normal processing of APP occurs at the
so-called -site by
-secretase, which generates a
non-amyloidogenic extracellular domain of APP (APPs) (3, 58). This
processing event makes the extracellular domain of APP containing a
Kunitz-type protease inhibitor available in the extracellular space.
This protease inhibitor activity is thought to prevent thrombin from injuring neurons (59, 60). In addition,
-secretase is a target for
the development of potential agents for the treatment of Alzheimer's disease, as
-secretase may cleave and solubilize A
amyloid
peptide, a major insoluble component of the senile plaque. Recently, it has been reported that APPs activate microglial cells and thereby enhance the local release of neurotoxic agents (61). Therefore, APPs
possess a broad range of biological effects. As our current evidence
suggests that APP is processed at its
-site within caveolae membranes, this may help in the identification and cloning of the
protein or proteins that possess
-secretase activity.
Several previous reports support the view that -secretase-mediated
cleavage of APP occurs on the cell surface (62-65). These observations
fit well with our current findings that APP is processed at its
-site within caveolae, as caveolae are specialized microdomains of
plasma membrane. APP requires membrane anchoring for its cleavage by
-secretase suggesting that the protease itself is a
membrane-anchored protein (64). Interestingly, caveolae have been
reported to contain a protease activity (66). The cleavage mechanism
for APP may be similar to that of other proteolytic activities known to
cleave a number of single membrane-spanning precursors (tumor necrosis
factor and notch) at the cell surface to release their ectodomains to
the media (67).
-Secretase cleavage can also occur intracellularly (68-70).
Therefore, a second mechanism should exist that involves an
intracellular compartment that may be independent of plasma membrane
caveolae. Alternatively, this phenomenon may be explained by the
presence of intracellular forms of caveolae, i.e.
plasmalemmal vesicles. In our experiments, we could not detect a cell
surface cleavage product of APP in cells that were singly transfected
with APP (Fig. 5D); in contrast, in cells co-transfected
with APP and caveolin-1, APPs were produced at the cell surface.
Overall cleavage of APP by
-secretase may be modulated by the cycle
of caveolae internalization and recycling (APP could cycle between cell
surface and intracellular populations of caveolae). The
-secretase
cleavage occurs not only at Leu17 but also at
Val18 in the amyloid peptide (3), suggesting the existence
of multiple
-secretases. Thus, caveolae and an undefined
intracellular compartment may possess different
-secretases that
cleave APP at distinct
-sites.
Protein kinase C activation promotes the -secretase-mediated
processing of APP (71), by an unknown molecular mechanism. Since this
PKC
is a well established component of caveolae (72) and PKC
activators inhibit caveolae internalization (potocytosis) (73), it is
possible that PKC activation prolongs the amount of time APP spends in
cell surface caveolae and therefore in close contact with
-secretase
activity.
APP is known to transit through clathrin-coated pits and vesicles on
its way to endosomes and lysosomes (74). Recent findings suggest a
number of signaling receptors that are cleared from the cell surface
via clathrin-coated pits are first present within caveolae microdomains
(15, 21, 75). In fact, internalized caveolae microdomains and
clathrin-coated vesicles may be targeted to a common endosomal pool
(77). This scenario would provide two distinct mechanisms for clearing
signaling receptors from the cell surface. In fact, APPct was
reported to be enriched in lysosomes (62). It therefore is likely that
APP is first present within caveolae where
-secretase processing
takes place, whereas the remaining intact APP may be cleared from the
cell surface via clathrin-coated pits and targeted to endosomes and lysosomes for proteolytic processing.
Caveolae-like membrane domains exist in neuronal cells and have been
characterized by a number of independent laboratories (13, 75, 78). In
addition, Allinquant and co-workers (13) provided preliminary evidence
that axonal APP is concentrated within caveolae-like structures in
neurons. Thus, it is likely that a neuron-specific form of caveolin may
exist that promotes caveolae formation in neuronal cells and interacts
with neuronally expressed APP. In Alzheimer's disease, caveolae
dysfunction may cause reduced activity of -secretase and
accumulation of toxic A
amyloid peptide.
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ACKNOWLEDGEMENTS |
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We thank Y. Sakaki for the gift of APP cDNAs and Sangram S. Sisodia, Samuel E. Gandy, Richard W. Scott, Kazuaki Yoshikawa for generously donating APP antibodies.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported in part by United States Public Health Service Grant MH56036 (to T. O.).
§ Fellow of Japan Society for the Promotion of Science for Research Abroad.
Supported by National Institutes of Health FIRST Award
GM-50443 (to M. P. L.), a grant from the G. Harold and Leila Y. Mathers Charitable Foundation (to M. P. L.), and Scholarship in the
Medical Sciences from the Charles E. Culpeper Foundation (to
M. P. L.).
** To whom correspondence should be addressed: NC30, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-444-3592; Fax: 216-444-7927; E-mail: okamott{at}cesmtp.ccf.org.
1 The abbreviations used are: APP, amyloid precursor protein; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; RT, room temperature; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; HA, hemagglutinin; Mes, 4-morpholineethanesulfonic acid; MBS, Mes-buffered saline; MDCK, Madin-Darby canine kidney cells; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; TBS, Tris-buffered saline; NTA, nitrilotriacetic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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REFERENCES |
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