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- 10.1074/jbc.273.17.10485
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Abstract
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 17, Issue of April 24, pp. 10485–10495, 1998 © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Caveolae, Plasma Membrane Microdomains for a-Secretase- mediated Processing of the Amyloid Precursor Protein* (Received for publication, November 4, 1997, and in revised form, January 26, 1998) Tsuneya Ikezu‡§, Bruce D. Trapp‡, Kenneth S. Song¶i, Amnon Schlegel¶i, Michael P. Lisanti¶i, and Takashi Okamoto‡** 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 Caveolae are plasma membrane invaginations where isoforms; APP is the brain-specific isoform. The Ab amyloid key signaling elements are concentrated. In this report, peptide is generated by the processing of APP with b- and both biochemical and histochemical analyses demon- g-secretases (3). Alternatively, APP is processed by a-secretase, strate that the amyloid precursor protein (APP), a which cleaves APP within the Ab sequence, thereby precluding b amyloid peptide, is enriched within caveo- source of A the formation of Ab (4). The identities of these secretases lae. Caveolin-1, a principal component of caveolae, is remain unknown. physically associated with APP, and the cytoplasmic do- In some inherited forms of Alzheimer’s disease, point muta- main of APP directly participates in this binding. The tions have been identified within the coding sequence of the characteristic C-terminal fragment that results from APP gene (5). These mutations co-segregate with the disease a-secretase, an as yet unidentified APP processing by phenotype and cause Alzheimer’s disease. Understanding the b amyloid se- enzyme that cleaves APP within the A molecular function and processing of APP is therefore critical quence, was also localized within these caveolae-en- to unraveling the molecular basis of Alzheimer’s disease. riched fractions. Further analysis by cell surface bioti- One approach to elucidate the function of APP is to identify nylation revealed that this cleavage event occurs at the APP-interacting proteins. At least five distinct classes of mol- a-secretase processing was cell surface. Importantly, ecules have been identified as APP binding partners as follows: significantly promoted by recombinant overexpression of caveolin in intact cells, resulting in increased secre- G (6), Fe (7), X11 protein (8), Fe -like protein (9), and o 65 65 tion of the soluble extracellular domain of APP. Con- APP-BP1 (10). The APP domain that interacts with G has 657 676 versely, caveolin depletion using antisense oligonucle- been localized to residues His -Lys within the cytoplasmic totides prevented this cleavage event. Our current domain of APP .G is a brain-specific member of heterotri- 695 o results indicate that caveolae and caveolins may play a meric GTP-binding protein (G-protein) family. The in vivo in- a-secretase-mediated proteolysis of pivotal role in the teraction between APP and G results in apoptotic cell death 695 o APP in vivo. (11) and inhibition of cAMP response element trans-activation (12). In contrast, functional consequences of interactions be- tween APP and other binding partners have not yet been de- Senile plaques and paired helical filaments are the hall- scribed. However, it is likely that these APP-interacting pro- marks of the brain pathology of Alzheimer’s disease (1). The tein molecules directly participate in APP-mediated cell principal component of the senile plaque is the Ab amyloid signaling events. peptide, which is composed of 39 – 43 amino acid residues. The A recent report described APP enrichment within caveolae- Ab amyloid peptide is derived from a full-length precursor like domains in neurons (13). Caveolae are plasmalemmal mi- protein, termed APP (amyloid precursor protein) (2). Alter- crodomains where multiple signaling molecules are concen- nate splicing of the APP gene generates at least 10 distinct trated. Growth factor-mediated and G-protein-mediated signaling cascades have been shown to be initiated within these microdomains (14 –16). * The costs of publication of this article were defrayed in part by the A major protein component of caveolae is caveolin. Molecular payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to cloning has recently identified a caveolin gene family. Three indicate this fact. This work was supported in part by United States distinct caveolin genes have been identified, cav-1, -2, and -3 Public Health Service Grant MH56036 (to T. O.). (17–19). In addition, two isoforms of caveolin-1 (Cav-1a and § Fellow of Japan Society for the Promotion of Science for Research Cav-1b) are derived from a single transcript from alternate Abroad. i Supported by National Institutes of Health FIRST Award GM- translation initiation sites. Caveolins-1 and -2 are most abun- 50443 (to M. P. L.), a grant from the G. Harold and Leila Y. Mathers dantly expressed in adipocytes, endothelial cells, and fibroblas- Charitable Foundation (to M. P. L.), and Scholarship in the Medical tic cell types, whereas the expression of caveolin-3 is muscle- Sciences from the Charles E. Culpeper Foundation (to M. P. L.). ** To whom correspondence should be addressed: NC30, Cleveland specific. Caveolin proteins form homo-oligomers that bind Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216- cholesterol and glycosphingolipids. These protein-protein and 444-3592; Fax: 216-444-7927; E-mail: [email protected]. protein-lipid interactions are thought to be essential for caveo- The abbreviations used are: APP, amyloid precursor protein; GST, glu- lae formation (20). tathione S-transferase; PAGE, polyacrylamide gel electrophoresis; RT, room temperature; PBS, phosphate-buffered saline; DMEM, Dulbecco’s modified Evidence is accumulating that caveolins can sequester mol- Eagle’s medium; HA, hemagglutinin; Mes, 4-morpholineethanesulfonic acid; ecules involved in G-protein-coupled signaling within caveolae. MBS, Mes-buffered saline; MDCK, Madin-Darby canine kidney cells; Heterotrimeric G-proteins are concentrated within caveolae Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; TBS, Tris-buff- and interact directly with caveolin via the Ga subunit (21). In ered saline; NTA, nitrilotriacetic acid; CHAPS, 3-[(3-cholamidopropyl)di- methylammonio]-1-propanesulfonic acid. addition, several G-protein-coupled receptors are sequestered This paper is available on line at http://www.jbc.org 10485 This is an Open Access article under the CC BY license. 10486 a-Secretase Processing of APP within Caveolae possessing XmaI restriction sites at both ends were phosphorylated, within caveolae in a ligand-independent (endothelin receptor) annealed, and ligated with the dephosphorylated APP cDNA. The ori- (22) and ligand-dependent fashion (muscarinic acetylcholine, entation and sequence of the inserted fragment was confirmed by DNA b-adrenergic, and bradykinin receptor) (23–26). sequencing. The HA peptide sequence was inserted between residues The molecular mechanism that underlies the recognition of 307 308 Pro and Gly of APP . For construction of the GST fusion protein 649 695 signaling molecules by caveolin has recently been elucidated cDNA encoding the cytoplasmic domain of APP (Lys -Asn ), this (27). Like other scaffolding proteins involved in signal trans- domain was amplified as a fragment with additional restriction cleav- age sites at both ends (EcoRI and BamHI sites). After PCR, the ampli- duction, caveolin contains a modular protein domain, termed fied fragment was digested with EcoRI and BamHI and subcloned in the caveolin scaffolding domain. The caveolin scaffolding do- frame into the vector pGEX4T-1 (Amersham Pharmacia Biotech). main, in turn, recognizes a specific consensus motif within its Detergent-free Purification of Caveolin-rich Membrane Domains— interacting partners (28). As APP can function as a G -coupled Cultured cells were grown to confluence in 150-mm dishes and used to “receptor” (29) and contains a caveolin binding motif within its prepare caveolin-enriched membrane fractions, as described previously cytoplasmic domain, caveolin could provide a means for seques- (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 tering APP with caveolae membrane domains. sodium carbonate, pH 11.0, and homogenized sequentially with a loose- As APP is expressed in many non-neuronal cell types where fitting Dounce homogenizer (10 strokes), a Polytron tissue grinder caveolin-1 is abundantly expressed, we initiated this study to (three 10-s bursts; Kinematica GmbH, Brinkmann Instruments), and a determine whether APP localizes within caveolae and if caveo- sonicator (three 20-s bursts; Branson Sonifier 250, Branson Ultrasonic lin plays a role in this sequestration event. Here, we show that Corp.). The homogenate was then adjusted to 45% sucrose by the (i) endogenous APP co-fractionates with caveolin-1 in three 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% distinct cell lines (COS-7, HEK293, and MDCK); (ii) recombi- discontinuous sucrose gradient was formed above (4 ml of 5% sucrose, 4 nantly expressed APP co-immunoprecipitates with caveolin-1 ml of 35% sucrose; both in MBS containing 250 mM sodium carbonate) when expressed in COS-7 cells; (iii) a GST fusion protein en- and centrifuged at 39,000 rpm for 16 –20 h in an SW41 rotor (Beckman coding the cytoplasmic domain of APP interacts directly with Instruments). A light-scattering band confined to the 5–35% sucrose purified recombinant caveolin-1 protein. These three independ- interface contained caveolin but excluded most other cellular proteins. ent lines of evidence support the hypothesis that APP physi- The caveolae-rich fractions were diluted 3-fold with MBS and centri- fuged at 15,000 rpm for 20 min at 4 °C. The pellets were used as cally associates with caveolin. “purified caveolae-enriched membranes.” This protocol separated Functionally, we show that the a-secretase cleavage product caveolin from the bulk of cellular membranes and cytosolic proteins. of APP is enriched within caveolae and that this cleavage event This fractionation scheme is based on the specific buoyant density of occurs at the cell surface. Specifically, we find that recombi- caveolin-rich membrane domains and their resistance to solubilization nant overexpression of caveolin-1 promoted a-secretase-medi- by sodium carbonate. By using this scheme, endogenous MDCK (38) ated proteolysis of APP. The production of the a-secretase 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 cleavage product of APP was abrogated by caveolin-1-based and 5 of these sucrose density gradients while excluding greater than antisense oligonucleotides that effectively block caveolin-1 ex- 99.95% of total cellular proteins. By using this same fractionation pression. These in vivo functional data clearly indicate that scheme, cav-myc and cav-myc-H co-fractionated with endogenous caveolae and caveolin proteins play a pivotal role in a-secre- caveolin, an indication that the myc and polyhistidine tags do not tase-mediated processing of APP on the plasma membrane. interfere with the correct targeting of caveolin (38). Sodium carbonate extraction is routinely used to determine if proteins are firmly attached EXPERIMENTAL PROCEDURES to membranes, and caveolin is not solubilized by sodium carbonate. By Materials—The cDNAs for canine caveolin-1 were as we described using this scheme, endogenous caveolin and cav-myc-H were not only previously (30). Ni-NTA-agarose for purification of polyhistidine-tagged recovered almost quantitatively in fractions 4 and 5, while excluding proteins was from Qiagen. Antibodies and their sources were as follows: most cellular proteins, but also were separated from the glycosylphos- anti-caveolin IgG polyclonal and monoclonal (2234) (gifts of Dr. John R. phatidylinositol-linked plasma membrane marker, carbonic anhydrase Glenney, Transduction Labs), anti-myc epitope IgG (monoclonal, 9E10; IV (38). This is consistent with recent observations that glycosylphos- Santa Cruz Biotech), anti-HA epitope IgG (rat and mouse (12CA5) phatidylinositol-linked proteins are not concentrated directly within monoclonal, Boehringer Mannheim). A variety of other reagents were caveolae but may reside in close proximity to the “neck regions” of purchased commercially as follows: fetal bovine serum (JRH Bio- caveolae within intact cells (40). sciences), purified myelin basic protein (Sigma), and pre-stained pro- As an alternative approach to purify caveolae, a protocol developed tein markers (Bio-Rad and NOVEX). The anti-GST antibody was from by Smart et al. (41) was employed. A plasma membrane fraction was Santa Cruz Biotechnology, and anti-myelin basic protein antibody was prepared from 10 100-mm dishes of confluent tissue culture cells. Each from Dako. dish was washed twice with 5 ml of buffer A (0.25 M sucrose, 1 mM Antibodies against APP are well characterized and include the fol- EDTA, 20 mM Tricine, pH 7.8). Cells were collected by centrifugation at lowing: (i) monoclonal antibody: 22C11 specific for the extracellular 1,400 3 g for 5 min (Sorval RT6000: 3000 rpm) and resuspended in 1 ml domain of APP (Boehringer Mannheim), 4G8 specific for Ab residues of buffer A and homogenized 15 times with Teflon glass homogenizer. 18 –24, and 6E10 specific for Ab residues 1–17 (Senetek) (31); and (ii) Homogenized cells were centrifuged at 1,000 3 g for 10 min (Sorval polyclonal antibody: Ab-(1–28) (Zymed), 1153 specific for Ab-(1–28) RT6000: 2500 rpm), and the supernatant was subjected to Percoll (kindly provided by Richard Scott) (32), 369 (kindly provided by Samuel gradient centrifugation. The sample was overlaid on top of 23 ml of 30% E. Gandy) (33), AC-1 (kindly provided by Kazuaki Yoshikawa) (34), both Percoll solution in buffer A and centrifuged at 83,000 3 g (30,000 rpm) specific for the cytoplasmic region, and CT-15 specific for the C-terminal for 30 min in a Beckman Ti-60 rotor. A plasma membrane fraction was 15 amino acids (kindly provided by Sangram S. Sisodia) (35). collected, and the volume was adjusted to 2 ml in buffer A. 1.84 ml of Cell Culture and cDNA Transfection—COS-7, HEK293, and MDCK 50% Optiprep in buffer B (0.25 M sucrose, 6 mM EDTA, 120 mM Tricine, cells (all from ATCC) were grown in DMEM plus 10% fetal bovine pH 7.8) was added to 0.16 ml of buffer A (23% Optiprep solution), which serum supplemented with penicillin and streptomycin (complete was mixed with the sonicated plasma membrane fraction. The entire DMEM). cDNA transfections were conducted as described previously solution was placed at the bottom of a Beckman SW41 rotor tube and (36). Briefly, COS-7 cells were seeded at 1 3 10 cells per 100-mm dish. overlaid onto a linear 10 –20% Optiprep gradient (prepared by diluting Twenty-four hours later, DNA (6 mg) of interest was transfected with 30 50% Optiprep in buffer A and B) and centrifuged at 52,000 3 g (18000 ml of LipofectAMINE (Life Technologies, Inc.) in 5 ml of DMEM. 24 h rpm) for 90 min using SW41 rotor (Beckman). The bottom fraction was post-transfection, the media were changed to complete DMEM. 48 h collected (non-caveolae membrane). The top 5 ml of the gradient (frac- post-transfection, media or cells were harvested and subjected to fur- tions 1– 6) was collected and mixed with 50% Optiprep in buffer B, ther analyses. which was then placed on the bottom of SW41 rotor tube and overlaid cDNA Constructions—For construction of the cDNA encoding HA- by 2 ml of 5% Optiprep in buffer A. The membrane fractions were inserted APP, the APP cDNA in pCDNA-1 (37) was digested with centrifuged at 52,000 3 g for 90 min. An opaque band located just above XmaI and dephosphorylated with calf intestine alkaline phosphatase the 5% interface was designated the caveolae fraction (41). (New England Biolabs). The oligonucleotides that encode HA sequence Immunoblotting of Gradient Fractions—From the top of each gradi- a-Secretase Processing of APP within Caveolae 10487 ent, 1-ml gradient fractions were collected to yield a total of 12 fractions. which contains calmodulin binding peptide and FE -PTB2 fusion do- Caveolin migrates mainly in fractions 4 and 5 of these sucrose density mains. Expression of calmodulin binding peptide-FE -PTB2 protein gradients (38). Gradient fractions were separated by SDS-PAGE and was induced by isopropyl-1-thio-b-D-galactopyranoside and was puri- SQ transferred to Immobilon-P sheets (Millipore). After transfer, sheets fied using calmodulin affinity resin (Stratagene) as described previ- were stained with Ponceau S to visualize protein bands and subjected to ously (44). The purified fusion protein (1 mg) was used for binding immunoblotting. For immunoblotting using ECL, incubation conditions analysis as described above. After transfer of eluted samples separated were as described by the manufacturer (Amersham Pharmacia Bio- in SDS-PAGE, the membrane was blocked with 2% skim milk and 2% tech), except we supplemented our blocking solution with both 1% bovine serum albumin in PBS, 0.02% sodium azide overnight at 4 °C. bovine serum albumin, 3% nonfat dry milk, and 0.02% sodium azide. The membrane was incubated with 5 mg/ml biotinylated calmodulin Chemical Cross-linking Studies—After transfection of cDNAs, cells (Calbiochem) in washing buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, were incubated with DMEM plus 10% Me SO containing 48 mg/ml 1mM CaCl ) for 30 min at RT, washed 3 times with the same washing 2 2 bis-[b-(4-azidosalicylamindo)ethyl] disulfide (Pierce) for 10 min at RT. buffer (10 min each), incubated with 1/20,000 diluted streptavidin- Media were discarded, and cells were exposed to ultraviolet light (wave- horseradish peroxidase-conjugated (Pierce) for 30 min at RT, and length 365 nm) (model LM20E, VWR Scientific) for 15 min at 4 °C. Cells washed 3 times with the same washing buffer. Finally bands were were solubilized in a lysis buffer A (10 mM Tris/HCl, pH 8.0, 1 mM visualized by diaminobenzidine staining (Vector). EDTA, 1 mM dithiothreitol, 60 mM octylglycoside, 1% Triton X-100, 1 Immunocytochemistry—Transfected cells (50,000 cells) were split mM aprotinin, 1 mM phenylmethylsulfonyl fluoride) with sonication onto 4-well chamber slides (Becton Dickinson). Twenty-four hours later, (three 20-s bursts, Branson Sonifier 250, Branson Ultrasonic Corp). the cells were incubated for 15 min at 8 °C with anti-HA rat monoclonal After centrifugation at 15,000 rpm for 20 min at 4 °C, supernatants antibody (5 mg/ml, Boehringer Mannheim) and cholera toxin B subunit were collected and subjected to immunoprecipitation/immunoblot anal- conjugated with peroxidase (1 mg/ml, Sigma) in complete DMEM (45). ysis. Cross-linking reagents were cleaved by boiling samples at 95 °C Cells were washed with PBS (4 3 5 min) and fixed in PBS containing for 5 min in the presence of 5% b-mercaptoethanol, and the samples 4% paraformaldehyde. After washing with PBS (5 3 5 min), nonspecific were subjected to SDS-PAGE. binding of antibodies was blocked with 3% normal goat serum (Life Immunoprecipitation of Caveolae-enriched Fractions—Caveolae-en- Technologies, Inc.) in PBS for 30 min at RT. Cells were then incubated riched fractions were diluted with Mes-buffered saline (MBS) and sol- with anti-cholera toxin rabbit antibody (Calbiochem) in PBS containing ubilized in buffer A without Triton X-100. After dialysis against buffer 3% normal goat serum for1hatRT.The chamber slides were washed A without detergent, immunoprecipitation was conducted with anti-HA with PBS (4 3 5 min) and incubated for 60 min at RT with secondary (Boehringer Mannheim) or anti-myc antibodies (Santa Cruz Biotech- antibodies (Texas Red-conjugated goat anti-rabbit IgG (preabsorbed nology) overnight at 4 °C. Fifty mlofa ;50% slurry protein G-Sepharose with rat and mouse IgG), 1:300 dilution, Jackson ImmunoResearch, and (Amersham Pharmacia Biotech) was added and further incubated for biotin-conjugated goat anti-rat IgG, 1:300 dilution, Boehringer Mann- 2 h. The beads were washed 3 times with Tris-buffered saline (TBS, 10 heim) and washed in PBS and in 0.1 M NaHCO , pH 8.2 (4 3 for 5 min). mM Tris/HCl, pH 8.0, 0.15 M NaCl), once with TE buffer (0.1 M Tris/HCl, Cells were incubated with avidin-fluorescein isothiocyanate conjugate pH 6.8, 1 mM EDTA), and subjected to SDS-PAGE/immunoblotting. (1/300 dilution, Vector) in NaHCO buffer for1hatRT. Slides were Affinity Purification of Caveolin-myc-H Expressed in Insect Cells mounted with Vectashield (Vector) and analyzed with a Leica TCS NT Using Ni-NTA-Agarose—The Sf21 cells overproducing His-tagged confocal microscope. caveolin-1 (76) were collected and solubilized in buffer A. Ni-NTA- Immunoprecipitation of APPs—Forty-eight hours post-transfection, agarose (200 ml) was then pre-equilibrated with TBS. Solubilized caveo- 1 ml of media was mixed with 1 ml of 23 RIPA (150 mM NaCl, 1.0% lin-containing cell extracts were then added to the resin and incubated Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris/HCl, pH 8.0) for6hat4 °Cona rotating platform. After binding, the beads were and incubated with 2 mg each of antibodies (anti-Ab-(1–28), 1153, allowed to gently settle by gravity (5 min on ice) and washed extensively CT-15, 6E10, 4G8, normal rabbit serum (NRG) or normal mouse serum (four times, 5 min each; twice with TBS and twice with TBS plus 30 mM (NMG)) overnight at 4 °C. 50 mlof ;50% slurry protein G-Sepharose imidazole). After washing, bound proteins were eluted with TBS con- was added and incubated for2hat4 °C. Beads were washed 3 times taining 200 mM imidazole, as per the manufacturer’s instructions with RIPA buffer and once with TE buffer. Final samples were incu- (Qiagen). bated with 60 mlof2 3 sampling buffer containing b-mercaptoethanol Purification of GST-APP Cytoplasmic Region—An overnight culture and boiled at 95 °C for 3 min. Fifteen ml of each sample was subjected of Escherichia coli harboring plasmids encoding either GST-APP or to SDS-PAGE. As a critical control, 2 mg of anti-Ab-(1–28) polyclonal GST alone was purified as described previously (42). antibody was preincubated with 20 mg of the Ab-(1–16) synthetic pep- Interaction of Caveolin-myc-H with GST-APP Cytoplasmic Region tide (Bachem) overnight at 4 °C and used for immunoprecipitation. For Proteins—The interaction of GST-APP fusion proteins with caveolin-1- immunoblotting, immunostained bands were visualized with ECL. In myc-H was evaluated as described for the interaction of caveolin with Fig. 6C, we used ECL-Plus (Amersham Pharmacia Biotech). baculovirus-expressed heterotrimeric G-protein subunits (21). Briefly, Cell Surface Biotinylation—Cells were washed twice with ice-cold GST or purified GST-APP cytoplasmic fusion proteins bound to gluta- PBS and incubated with PBS containing 0.5 mg/ml sulfo-NHS-biotin thione-agarose beads were extensively prewashed with phosphate-buff- (Pierce) for 30 min at 4 °C (46), washed three times with ice-cold PBS, ered saline and lysis buffer containing protease inhibitors. These beads and placed in complete DMEM. Four hours after incubation at 37 °C in contained ;100 pmol of a given fusion protein per 100 ml of packed aCO incubator, media were harvested and mixed with the same volume. Approximately 100 ml of this material was incubated with 1 mg volume of 23 RIPA buffer, followed by incubation with Neutravidin of caveolin-1-myc-H on a rotating platform overnight at 4 °C. After beads (Pierce). After overnight incubation at 4 °C, the Neutravidin binding, the beads were washed extensively (6 – 8 times) with buffer B beads were washed three times with RIPA buffer and once with TE (50 mM HEPES/NaOH, pH 7.5, 120 mM NaCl, 1 mM EDTA, 0.5% buffer. Final samples were mixed with sampling buffer containing CHAPS, and protease inhibitors). Finally, bound proteins were eluted b-mercaptoethanol, boiled for 3 min, and subjected to immunoblot anal- with 100 ml of elution buffer containing 50 mM Tris, pH 8.0, 1 mM EDTA, ysis with 22C11. 1% Triton X-100, 10 mM reduced glutathione, and protease inhibitors. Antisense Oligonucleotide Introduction—COS-7 cells were seeded at The eluate was mixed 1:1 with 23 sample buffer and subjected to a density of 1 3 10 cells per 100-mm dish. Twenty-four hours later, 3 SDS-PAGE (10% acrylamide) and Western blot analysis with anti- mg each of antisense caveolin-1 oligonucleotide (TTTGCCCCCAGACAT; caveolin polyclonal antibody (1:1000 dilution, Transduction Laborato- complementary to the 15-base initiation sequence of human caveolin-1) ries). Horseradish peroxidase-conjugated secondary antibodies (1:10000 and the HA-APP cDNA were co-transfected into COS-7 cells using 30 dilution, Bio-Rad) were used to visualize bound primary antibodies by ml of LipofectAMINE. Twelve hours later, the media were discarded and ECL (Amersham Pharmacia Biotech). In place of caveolin-myc-H7, pu- replaced with 8 ml of complete DMEM. Forty-eight hours post-trans- rified myelin basic protein (a negative control) and FE (a positive fection, media were harvested and mixed with 23 RIPA buffer. As a control) were also used for binding experiments. For detection of myelin control, a sense-caveolin-1 oligonucleotide (ATGTCTGGGGGCAAA) basic protein, anti-myelin basic protein antibody was used. For the which corresponded to the 15 bases of the initiation sequence was used. binding study of FE and APP, the gene of human FE PTB2 domain The media and cells were subjected to further analyses. 65 65 (484 – 612 amino acids) was first cloned (43) by PCR with a set of 59 and 39 primers (59 primer, ACGCGTCGACTCACAGGAAGGAGAG- RESULTS GAAACGCAC; 39 primer, GCGGATCCTGTGAGGCACCTGCCAA- Interaction of APP with Caveolin-1: A Requirement for the GAAC) using human hippocampus cDNA library (CLONTECH) as a Caveolin Scaffolding Domain—To investigate whether endog- template. PCR product was digested with BamHI-SalI and ligated into pCAL-n-EK vector (Stratagene) for generating a fusion protein cDNA enous APP is associated with caveolae membranes, caveolae- 10488 a-Secretase Processing of APP within Caveolae FIG.1. Endogenous APP/APLP and caveolin-1 are co-localized within caveolae-enriched membrane frac- tions. COS-7 (A), HEK293 (B), and MDCK cells (C) grown to confluence in 150-mm dishes were used to prepare caveolin-enriched membrane domains, as described previously (38). After two washes with ice-cold phosphate-buffered saline, cells (two confluent 150-mm dishes) were scraped into 2 ml of 500 mM sodium carbonate, pH 11.0. Homogeniza- tion was carried out using a loose-fitting Dounce homogenizer, a Polytron tissue grinder, and a sonicator. The homogenate was adjusted to 45% sucrose 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 contain- ing 250 mM sodium carbonate) and centri- fuged at 39,000 rpm for 16 –20 h. From the top of each gradient, 1-ml gradient fractions were collected to yield a total of 12 fractions. Gradient fractions were sep- arated by SDS-PAGE (4 –20% linear gra- dient gel) and transferred to Immobilon- SQ P . After transfer, the sheets were subjected to immunoblotting with 22C11 (upper panel) and anti-caveolin-1 antibod- ies (lower panel). Arrows indicate APP/ APLP (upper panel) and caveolin-1 (lower panel). enriched membrane domains from COS-7 cells were purified by fractions. In addition, when the independent fractionation pro- a detergent-free procedure and were ultracentrifuged in a su- cedure involved homogenization in Triton X-100 containing crose density gradient. Immunoblot analysis of the resulting buffers, essentially identical results were obtained (not shown). gradient fractions with APP antibody (22C11) detected the vast Therefore, it is likely that the 22C11 immunoreactive proteins majority of APP in fractions 4 and 5 (Fig. 1A, upper panel). physically associated with specialized caveolin-enriched mem- Similarly, caveolin-1 was confined to the same region of the brane microdomains in these three distinct cell lines. gradient (fractions 4 and 5), indicating that caveolae are con- The antibody 22C11 recognizes both APP and other mole- tained within these fractions (Fig. 1A, lower panel). Densito- cules closely related to APP, termed APLPs (47). To determine metric analysis revealed that greater than 90% of the total if APP itself is confined to caveolae-enriched domains, we next APP-reactive material was recovered in fractions 4 and 5. employed a recombinant approach. COS-7 cells were co-trans- These results demonstrate that molecules containing the fected with epitope-tagged forms of APP and caveolin-1 (the HA 22C11 epitope are primarily enriched with caveolae microdo- epitope was inserted into the extracellular domain of APP mains in COS-7 cells. and the myc epitope was placed at the extreme C terminus of To extend these initial observations, the same fractionation caveolin-1). These transiently transfected cells were then sub- procedure was employed on HEK293 and MDCK cells. In jected to the same detergent-free fractionation procedure de- HEK293 cells, endogenous 22C11 immunoreactive bands were scribed above. HA-tagged APP and myc-tagged caveolin-1 again highly enriched in fractions 4 and 5 and precisely coin- were confined mainly to fraction 4 and were undetectable in the cided with the distribution of caveolin-1 (Fig. 1B). In MDCK remaining 11 fractions (Fig. 2A). These data indicate that the cells, three major bands were detected by immunoblot analysis majority of recombinant APP and caveolin-1 co-fractionate with 22C11 (Fig. 1C). Approximately 80% of the total 22C11 and are localized within caveolae-enriched membrane immunoreactive material was confined to the caveolin-rich domains. a-Secretase Processing of APP within Caveolae 10489 FIG.2. Recombinant APP and caveolin-1 form a physical complex. A, recombinant APP and recombinant caveolin-1 co-fractionate and are targeted to caveolae-enriched membranes. HA-APP and myc-caveolin-1 were transfected into COS-7 cells with LipofectAMINE. 48 h post- transfection, cells were harvested and subjected to detergent-free caveolae purification as in Fig. 1. Gradient fractions were separated by SDS-PAGE (4 –20% linear gradient gel) and subjected to immunoblotting with IgG directed against the epitope tags, HA (upper panel) and myc (lower panel). B, recombinant APP and caveolin-1 form a stable complex in vivo. After co-transfection of HA-APP with either vector alone (mock), caveolin-1 (cav-1), caveolin-1 lacking residues 61–100 (D61–100), or caveolin-1b (cav-1b), cells were incubated with DMEM plus Me SO containing bis-[b-(4-azidosalicylamindo)ethyl] disulfide and were exposed to ultraviolet light. Cells were solubilized in buffer A with the help of sonication. After centrifugation, supernatants were collected and subjected to immunoprecipitation with anti-myc antibody and immunoblotted with anti-HA (upper panel) or anti-myc antibodies (middle panel). Cell lysates without chemical cross-linking were also subjected to immunoblotting with anti-HA antibodies (lower panel). C, recombinant APP and caveolin-1 co-immunoprecipitate (IP). After transfection of HA-APP with either caveolin-1b (cav-1) or caveolin-1 lacking residues 61–100 (D61–100), cells were subjected to detergent-free caveolae purification as in Fig. 1. Caveolae fractions were diluted with Mes-buffered saline, collected by centrifugation, and solubilized in buffer A lacking Triton X-100. After centrifugation at 15,000 rpm for 30 min, supernatants were collected and subjected to dialysis against buffer A without detergent. Immunopre- cipitation was conducted with (i) anti-HA (HA) or normal rat IgG (NRG)(left panel), (ii) with anti-myc (myc) or normal mouse IgG antibody (NMG) (middle panel) and (iii) with anti-HA or anti-myc (right panel). The samples were subjected to immunoblotting with anti-myc, HA, or AC-1 antibodies. D, purified recombinant APP cytoplasmic domain interacts with purified recombinant caveolin-1. Recombinant caveolin-1 was affinity purified from the Sf21 cells overproducing His-tagged caveolin using the Ni-NTA-agarose. Recombinant GST-APP or GST was purified from E. coli harboring plasmids encoding either GST-APP or GST alone. GST-APP or GST alone were pre-bound to glutathione-agarose beads and incubated with caveolin-1-myc-H . Bound proteins were eluted with buffer containing 10 mM reduced glutathione and were subjected to SDS-PAGE (12% acrylamide) and Western blot analysis with anti-caveolin-1 or anti-GST antibodies. Under the same binding conditions, the binding capacity of myelin basic protein and FE PTB2 domain (484 – 612 amino acids) was also determined. Myelin basic protein was detected by anti-myelin basic protein antibody, whereas FE was detected by biotin conjugated-calmodulin binding, followed by diaminobenzidine staining. G-protein-coupled receptors (the endothelin receptor) and recover epitope-tagged caveolin. After immunoprecipitation, growth factor receptors can form physical complexes with samples were subjected to immunoblot analysis to visualize caveolin-1 (48, 49). In the case of epidermal growth factor HA-tagged APP. receptor, caveolin-1 interacts with the epidermal growth factor As shown in Fig. 2B, two major bands corresponding to receptor kinase domain via the caveolin scaffolding domain. As caveolin-1a and caveolin-1b were detected in these immuno- APP is physically and functionally coupled to the heterotri- precipitates using the myc antibody for Western blot analysis meric G-protein, G (29), APP may also interact directly with (Fig. 2B, lane 2). Anti-HA immunoblot analysis revealed that caveolin-1. Direct interaction of caveolin-1 with receptors and HA-tagged APP was also detected in these immunoprecipi- downstream signaling molecules is thought to facilitate seques- tates. In the mock-transfected cells, neither caveolin-1 nor APP tration of multiple signaling molecules within caveolae mem- immunoreactivity were detected by Western blotting with an- branes. To test this hypothesis, we employed a chemical cross- ti-HA or anti-myc (Fig. 2B, lane 1). The results establish that linking approach coupled with immunoprecipitation. caveolin-1 forms a physical complex with APP in intact COS-7 COS-7 cells were transiently co-transfected with epitope- cells. Under the same experimental conditions expect using no tagged APP and caveolin-1 as described above and subjected to chemical cross-linking reagent, the physical association of APP chemical cross-linking with the membrane-permeant photore- and caveolin was not observed, suggesting that treatment with active agent, 9-bis-[b-(4-azidosalicylamindo)ethyl] disulfide. chemical cross-linking reagent is necessary to observe suffi- After photolysis the cross-linked cells were solubilized with a cient binding of APP and caveolin in this transient expression buffer containing 1% Triton X-100 and 60 mM octylglycoside. system. Samples were then immunoprecipitated with anti-myc IgG to To investigate further the specificity of this interaction, the 10490 a-Secretase Processing of APP within Caveolae same experiment was performed using other forms of caveo- lin-1. We used myc-tagged caveolin-1b, the shorter isoform of caveolin-1 that lacks caveolin residues 1–31, and myc-tagged caveolin-1-(D 61–100), an internal deletion mutant that lacks the caveolin-1 scaffolding domain. Immunoprecipitation and Western blotting with anti-myc IgG detected both caveolin-1b and caveolin-1-(D 61–100) (Fig. 2B, lanes 3 and 4), as expected. However, immunoblot analysis with anti-HA antibody revealed that APP only co-immunoprecipitated with caveolin-1b but not with caveolin-1-(D 61–100). These results indicate that caveolin residues 1–31 are not required for interaction of APP with caveolin-1; in addition, they implicate caveolin residues 61–100 in this process, demonstrating a crucial role for the caveolin-1 scaffolding domain in the recognition of APP by caveolin-1 in vivo. In another independent approach, we employed detergent- free immunoprecipitation. COS-7 cells were transiently co- transfected with epitope-tagged forms of APP and caveolin-1 (caveolin-1b and caveolin-1-(D 61–100)) as described above. After detergent-free cell fractionation, caveolae containing fractions were collected and solubilized with 60 mM octylglyco- side. After dialysis to remove detergent, these caveolin-rich membranes were subjected to immunoprecipitation with either FIG.3. Localization of APP and cholera toxin B subunit bind- anti-myc or anti-HA antibodies. Immunoblot analysis revealed ing site within a single cell. COS-7 cells transfected with HA-APP that APP is associated with caveolin-1b but not caveolin-1-(D and myc-tagged caveolin-1 were incubated with cholera toxin B subunit 61–100) (Fig. 2C). Also, normal mouse IgG did not immunopre- and immunostained with anti-HA (A and C) and anti-cholera toxin (B cipitate either HA-tagged APP or the myc-tagged forms of and D) antibodies. Primary antibodies were detected using differen- tially tagged fluorescent secondary antibodies (fluorescein isothiocya- caveolin-1, confirming the specific nature of these interactions. nate-conjugated for HA and Texas Red-conjugated for cholera toxin B The results indicate that APP and caveolin form physical com- subunit). Both epitopes were visualized by laser confocal microscopy. plexes and they are consistent with chemical cross-linking Two single cells immunostained with anti-HA antibody (green), and studies of intact cells (Fig. 2B). with anti-cholera toxin B subunit antibody (red) are presented. Similar results were observed in at least 100 independent cells. Arrows point at Although the experiments described above indicate that APP co-localization of APP and cholera toxin B subunit binding site. and caveolin-1 form a physical complex in intact cells, it re- mains unknown whether this interaction is direct or occurs via support the hypothesis that APP is localized within cell surface another protein that may act as a bridge to link APP with caveolae in living cells. caveolin-1. To address this issue, the interaction between a In summary, we have shown here that (i) both endogenous purified GST fusion protein encoding the short cytoplasmic tail and recombinant APP co-fractionate with caveolin-1, (ii) APP of APP and purified recombinant caveolin-1 containing both forms a physical complex with caveolin, (iii) the scaffolding myc and His tags (caveolin-1-myc-His ) was investigated. GST domain of caveolin and the cytoplasmic domain of APP partic- alone or GST-APP cytoplasmic tail immobilized on glutathione- ipate in this reciprocal interaction, and (iv) cell surface caveo- agarose beads were incubated with purified recombinant lae contain APP. caveolin-1. After binding, the beads were extensively washed A C-terminal Degradation Product of APP Is Localized with buffers containing high salt and detergents, and the within Caveolae-rich Fractions, and the Generation of This APP bound proteins were then specifically eluted with buffer con- Product Is Specifically Dependent upon the Expression of taining excess reduced glutathione. These eluates were then Caveolin-1—The a-secretase-generated degradation product of subjected to immunoblot analysis with anti-caveolin-1 IgG. Fig. APP can be detected in COS-7 cells by immunoblotting with 2D shows that caveolin-1 specifically associated with GST-APP C-terminal APP antibodies (51). To determine if a-secretase- cytoplasmic tail but not with GST alone. mediated APP degradation occurred in our experimental sys- We also used FE APP binding domain as a positive control 65 tem, COS-7 cells were transiently transfected with HA-tagged and myelin basic protein as a negative control for these exper- APP and subjected to detergent-free subcellular fractionation. iments. Under our conditions, FE PTB2 domain (484 – 612 65 These fractions were then analyzed by immunoblotting with amino acids) but not myelin basic protein bound GST-APP C-terminal APP antibodies (AC-1). As shown in Fig. 4, two cytoplasmic tail (Fig. 2D), indicating that the employed exper- bands confined to caveolin-rich fraction 4 were detected; the imental conditions were appropriate to evaluate the direct in- high molecular weight band represents intact APP , whereas teraction between the APP cytoplasmic region and its associ- the low molecular weight band is a short fragment of APP that ated molecules. Taken together, these results clearly indicate contains a C-terminal epitope recognized by the AC-1 antibody. that APP interacts directly with caveolin-1 through a portion of As this low molecular weight band was not detected by immu- the cytoplasmic tail of APP. noblotting with an antibody directed against the extracellular Cell surface caveolae can be detected by cholera toxin B domain of APP (22C11) (Fig. 2), these results indicate that this subunit binding (45, 50). To investigate the possibility that cell short fragment represents a C-terminally derived APP degra- surface caveolae contain APP, we immunostained living COS-7 dation product. To obtain a more precise estimate of the cells with anti-HA and cholera toxin antibodies following incu- molecular mass of this C-terminal APP fragment, the same bation of the cells with cholera toxin B subunit after transfec- sample was subjected to Tris/Tricine SDS-PAGE and immu- tion of recombinant HA-APP and myc-caveolin cDNAs. As noblotting with AC-1. A 10-kDa band was detected, sup- shown in Fig. 3, HA (green) and cholera toxin B subunit stain- porting the hypothesis that it is derived from APP a-secretase ing (red) are co-localized on the cell surface. These results processing (51). a-Secretase Processing of APP within Caveolae 10491 FIG.4. A C-terminal fragment of APP is the product of a-secretase cleavage and is concentrated within caveolae-enriched membrane fractions. A, a well defined C-terminal fragment of APP is concentrated within caveolae-enriched membranes fractions. HA-APP was transfected into COS-7 cells with LipofectAMINE. 48 h post-transfection, cells were harvested and subjected to detergent-free caveolae purification as in Fig. 1. Gradient fractions were collected, separated by SDS-PAGE (4 –20% linear gradient gel), and subjected to immunoblotting with AC-1 antibody which is directed against the C terminus of APP. Inset, lysates from the transfected cells were also subjected to Tris/Tricine SDS-PAGE (10 –20% linear gradient gel) and to immunoblotting with AC-1. B, the C-terminal fragment is an a-secretase-cleaved product of APP. COS-7 cells transfected with HA-APP were subject to immunoprecipitation with various C-terminal APP antibodies (369, CT-15, and AC-1) followed by immunoblotting with anti-Ab antibodies (4G8 which is directed against residues 18 – 40 of Ab peptide and 6E10 which is directed against residues 1–17). C, a soluble form of the extracellular domain of APP (APPs) is shed into the culture media. The culture media of cells transfected with HA-APP were collected and subjected to immunoprecipitation with anti-Ab antibodies (4G8 and 6E10) followed by immunoblotting with 22C11. D, APP and its C-terminal fragment are concentrated in caveolae membrane fractions. The Optiprep method was employed to confirm further the observation that both APP and its C-terminal fragment are in caveolae. Plasma membrane and caveolae membrane fractions were obtained from COS-7 cells transfected with HA-APP by the method developed by Smart et al. (41), and 4 mg each of protein was loaded, separated by SDS-PAGE (4 –20% linear gradient gel), and subjected to immunoblotting with AC-1 antibody (upper panel) and caveolin antibody (middle panel). The protein concentration profile of fractionated samples is also presented (lower panel). To substantiate further these observations, we subjected the fragment represents an a-secretase processed product of APP samples to immunoprecipitation with three independent anti- (aAPPct). These results also establish that both intact APP and bodies directed against the C-terminal region of APP (termed aAPPct co-fractionate with caveolin-1 and are concentrated 369, CT15, and AC-1). These immunoprecipitates were then within caveolae-enriched fractions. immunoblotted with antibodies directed against the Ab amy- As a-secretase promotes secretion of the APP extracellular loid region of APP (termed 4G8 and 6E10). 4G8 recognizes domain (APPs), we investigated the possibility that APPs are residues 18 –24 of the amyloid peptide, whereas 6E10 recog- present in the media containing our transfected cells. The nizes residues 1–17 of the amyloid peptide. As the a-secretase media from cells transfected with HA-APP were collected and cleavage site is located at residue 17 or 18 of the amyloid subjected to immunoprecipitation with anti-Ab peptide anti- peptide, these antibodies should allow us to the precisely de- bodies followed by immunoblotting with 22C11. As shown in termine the origin of this C-terminal low molecular weight Fig. 4C, a band corresponding to APPs was detected by immu- product of APP. As shown in Fig. 4B, the short APP fragment noprecipitation with 6E10 but not with 4G8. Detection of a that was immunoprecipitated with antibodies directed against soluble fragment of the extracellular domain of APP that con- the C terminus of APP is immunoreactive with the 4G8 anti- tains residues 1–17 of the Ab amyloid peptide (the epitope of body but not with 6E10. These data indicate that the short 6E10) provides additional support for a-secretase cleavage of C-terminal APP fragment contains both the C terminus of APP APP in COS-7 cells. Taken together, these findings demon- and the 4G8 epitope, but it does not contain the 6E10 epitope. strate that APP is correctly processed at its a-secretase cleav- These studies provide additional evidence that this 10-kDa age site in COS-7 cells. 10492 a-Secretase Processing of APP within Caveolae As an alternative approach, we employed a method devel- cell surface. To investigate this possibility, we measured the amount of a-secretase cleavage product of APP that was shed oped by Smart et al. (41) to purify caveolae (Optiprep approach) to substantiate further our observation that intact full-length from the cell surface by exploiting the membrane-impermeant probe, sulfo-NHS-biotin. COS-7 cells were co-transfected with APP and its a-secretase-cleaved C-terminal fragment are local- HA-APP and myc-tagged caveolin-1, subjected to cell surface ized in caveolae. We first transiently transfected HA-APP and biotinylation, and placed in fresh media for 4 h. The media myc-caveolin-1 into COS-7 cells and then purified caveolae by were then collected, and cell surface biotinylated molecules the Optiprep method. In this approach, the bulk of protein were specifically recovered by incubating the media with Neu- which was recovered in fractions 7–13, termed “plasma mem- travidin beads. After extensively washing the beads with buffer brane fractions,” was successfully separated from the “caveolae containing high salt and detergents, these samples were im- membrane fraction” (fractions 1– 6) (Fig. 4D, lower panel), con- munoblotted with 22C11. As shown in Fig. 5D (right panel), sistent with the method originally described by Smart et al. biotinylated APPs were only detected in the medium of cells (41). By using this approach, full-length APP and its processed co-transfected with APP and caveolin-1; in contrast, no bioti- product were dramatically enriched within the caveolae mem- nylated APPs were detected in the medium of cells transfected brane fraction (Fig. 4D, upper panel). As reported previously, with APP alone (middle panel). Immunoblotting of these cell the plasma membrane fraction did not contain caveolin, which lysates confirmed that APP expression was similar in the pres- was specifically recovered in the caveolae membrane fraction ence or absence of caveolin transfection (left panel). These data (Fig. 4D, middle panel). Importantly, the full-length APP and indicate that APPs are created on the cell surface, and its its processed product were not detected in these plasma mem- production is dependent on caveolin expression, further sup- brane fractions (Fig. 4D, upper panel). These data clearly sup- porting the hypothesis that caveolae organelles and the caveo- port the hypothesis that not only full-length APP but also its lin-1 protein play a pivotal role in a-secretase-mediated APP short product processed by a-secretase are present within degradation. caveolae microdomains. If caveolin-1 overexpression greatly promoted a-secretase- These observations prompted us to investigate if caveolae mediated degradation of APP, loss of endogenous caveolin-1 play any role in the a-secretase processing of APP. One possi- expression should block the basal production of APPs. To ex- bility is that the relative amount of caveolin expression could amine if the expression of endogenous caveolin-1 is strictly modulate whether APP undergoes a-secretase processing. To required for this processing event, we introduced caveolin-1 test this hypothesis, we (i) co-transfected COS-7 cells with antisense oligonucleotides to deplete the cells of endogenous HA-tagged APP and myc-tagged caveolin-1 and (ii) assessed caveolin-1. The caveolin-1 antisense oligonucleotide included a-secretase activity by measuring the amounts of aAPPct in the first 15 bases of the 59-coding sequence of the caveolin-1 cell lysates and of APPs in the culture media. In addition, we gene. This antisense oligonucleotide and the corresponding performed detergent-free subcellular fractionation on these sense nucleotide were introduced into COS-7 cells by transient samples. As shown in Fig. 5A, two major bands corresponding transfection in combination with HA-tagged APP. The media to intact APP and aAPPct that were confined to fractions 4 and were collected, and the amount of APPs produced was detected 5 were detected by immunoblot analysis with C-terminal APP by immunoprecipitation with the anti-Ab-(1–28) antibody fol- antibodies. The ratio aAPPct to intact APP was increased sig- lowed by Western blotting with 22C11. Expression of endoge- nificantly by co-expression with caveolin-1 (as compared with nous caveolin-1 was virtually undetectable after transfection HA-APP expressed alone). Densitometric analysis revealed with the antisense oligonucleotide indicating that the synthesis that the ratio increased from 1:1 to 5:1. In contrast, overexpres- of endogenous caveolin-1 was efficiently abrogated; in contrast, sion of the caveolin mutant which lacks the APP binding do- the sense oligonucleotide had no effect on the expression of main (caveolin-1-(D 61–100)) did not increase aAPPct (Fig. 5A), endogenous caveolin-1 (Fig. 6B). Co-transfection with the an- indicating that caveolin binding to APP promotes APP tisense oligonucleotide decreased the amount of APPs produced degradation. and secreted to the media by more than 80% as compared with The culture medium was then collected from these cells, cells transfected with HA-APP alone (Fig. 6A). Accordingly, the immunoprecipitated with an anti-Ab-(1–28) antibody, and an- amount of aAPPct produced was dramatically inhibited by alyzed by Western blotting with 22C11. As shown in Fig. 5B, co-transfection of HA-APP with the caveolin-1 antisense oligo- APPs were detected in the media, and this immunoreactivity nucleotide, without affecting the production of intact APP (Fig. was abolished by preincubating the anti-Ab antibody with a 6B). Importantly, co-transfection with the sense oligonucleo- synthetic peptide containing Ab residues 1–16. In contrast, tide had little or no effect on the production of APPs or aAPPct non-immune rabbit IgG did not immunoprecipitate a band (Fig. 6, A and B). corresponding to APPs. These results clearly indicate the APPs DISCUSSION in the media are an end product of a-secretase-processed APP. The amount of APPs secreted into the media from cells We have presented several independent lines of evidence transiently transfected with HA-APP and myc-tagged forms of that APP localizes within caveolae membranes and interacts caveolin-1 was compared (Fig. 5C). Co-transfection of caveo- directly with caveolin-1, a principal structural component of lin-1 with APP significantly increased the amount of APPs caveolae. More specifically, we have shown that (i) APP and secreted into the media detected by 22C11 immunoblotting. In caveolin-1 co-fractionate when cells are subjected to two inde- contrast, co-transfection with an internal deletion mutant of pendent detergent-free subcellular fractionation protocols pre- caveolin lacking the APP binding domain (caveolin-1-(D 61– viously used to purify caveolae from cultured cells; (ii) APP and 100)) failed to increase the amount of APP secreted into the caveolin-1 can form a physical complex in intact cells as shown media. Immunoblots with 22C11 and myc antibodies showed by chemical cross-linking and co-immunoprecipitation experi- similar expression levels of HA-APP and caveolin among the ments; (iii) association of APP with caveolin-1 occurs via a different transfectants. These results establish that caveolin-1 direct interaction between the C-terminal cytoplasmic domain overexpression promotes a-secretase processing of APP and of APP and the caveolin-1 scaffolding domain; (iv) cell surface that the caveolin scaffolding domain is required for this effect. caveolae contain APP; (v) the characteristic C-terminal frag- As caveolae represent a subcompartment of the plasma ment that results from APP processing by a-secretase was also membrane, a-secretase processing should be observed on the localized within caveolae; (vi) a-secretase processing was sig- a-Secretase Processing of APP within Caveolae 10493 FIG.5. Overexpression of caveolin-1 promotes a-secretase processing of APP. A, production of the C-terminal fragment of APP is greatly enhanced by overexpression of caveolin (cav-1) but not by overexpression of caveolin-1 lacking residues 61–100 (cav-1D61–100). COS-7 cells were transfected with HA-APP and myc-caveolin-1 or myc-caveolin lacking residues 61–100. 48 h post-transfection, cells were harvested and subjected to detergent-free caveolae fractionation. Gradient fractions were collected, separated by SDS-PAGE (4 –20% linear gradient gel), and subjected to immunoblotting with AC-1 and anti-myc antibodies. The equivalent expression of recombinant caveolin-1 and mutant caveolin-1 was confirmed by immunoblotting with the anti-myc antibody (data not shown). B, media from cells co-transfected with HA-APP and myc-caveolin-1 were subjected to immunoprecipitation with anti-Ab-(1–28) antibody (anti-Ab)(lanes 1 and 2) or normal rabbit immunoglobulin (NRG)(lane 3) and analyzed by immunoblotting with 22C11. Prior to immunoprecipitation, the anti-Ab-(1–28) antibody was pre-absorbed with an Ab-(1–16) synthetic peptide and was then used for immunoprecipitation (lane 2). C, media from cells transfected with or without HA-APP (1APP or 2APP) together with either mock (2cav-1), caveolin (cav-1), or caveolin which lacks residues 61–100 (D61-100) were subjected to immunoprecipitation (IP) with an anti-Ab- (1–28) antibody or 1153 and immunoblotted with 22C11 (lower panel). Cell lysates (100 mg) were also subjected to immunoblotting with 22C11 (upper panel) or anti-myc antibodies (middle panel). D, APP degradation occurs on the cell surface. Left panels, cells transfected with HA-APP alone or in combination with myc-tagged caveolin-1 were surface-labeled with the cell-impermeant probe sulfo-NHS biotin. After incubation for 0 (0h) or4h(4h), the media were collected and subjected to precipitation with Neutravidin beads. Precipitated proteins were to immunoblotted with 22C11. Right panel, cells transfected with HA-APP alone or in combination with myc-tagged caveolin-1 were subjected to immunoprecipitation with CT-15, followed by immunoblotting with 22C11. nificantly promoted by recombinant overexpression of caveo- scaffolding domain functions as a modular protein domain that lin-1 in intact cells, resulting in increased secretion of the interacts with peptide and protein ligands that contain a spe- soluble extracellular domain of APP; and (vii) depletion of cific caveolin binding motif (28). APP contains a predicted endogenous caveolin-1 using antisense oligonucleotides pre- caveolin binding motif within its C-terminal cytoplasmic tail. It vented this cleavage event. It is therefore likely that a physical is likely that this sequence is recognized by the caveolin scaf- interaction between APP and caveolin-1 functionally seques- folding domain and functions to sequester APP within caveolae ters APP within caveolae membranes and thereby compart- microdomains. mentalizes the a-secretase-mediated proteolysis of APP. Cholesterol and sphingolipids are highly concentrated with The caveolin-1 region that interacts with APP includes the caveolae domains. Recent findings indicate that transport of caveolin scaffolding domain, a short stretch of 20 amino acids newly synthesized cholesterol from the endoplasmic reticulum that directly participate in the recognition of multiple classes of to the plasma membrane is mediated via caveolin proteins (52, signaling molecules. Recent evidence suggests that the caveolin 53). In addition, caveolin-1 directly binds cholesterol, and cho- 10494 a-Secretase Processing of APP within Caveolae FIG.6. Ablation of caveolin-1 expression leads to inhibition of a-secretase processing of APP. A, transfection with caveolin-1-derived antisense oligonucleotides decreases the amount of APPs that are shed into the media. COS-7 cells were transfected with 3 mg each of antisense caveolin-1 oligonucleotide and HA-APP cDNA using LipofectAMINE. Twelve hours later, the media were discarded and replaced with complete DMEM. 48 h post-transfection, the medium was harvested, mixed with 23 RIPA buffer, and subjected to immunoprecipitation with an anti-Ab polyclonal antibody or 1153. Each of the final samples was subjected to immunoblot analysis with 22C11, and positive bands were visualized with ECL-Plus. Lane 1, no oligonucleotide transfection; lane 2, sense oligonucleotide transfection; lane 3, antisense oligonucleotide transfection. B, transfection with caveolin-1-derived antisense oligonucleotides decreases the amount of C-terminal APP fragment within cell lysates. COS-7 cells were transfected with the following: lane 1, no oligonucleotides; lane 2, a caveolin-1 sense oligonucleotide; and lane 3, a caveolin-1 antisense oligonucleotide. Cell lysates were prepared and subjected to immunoblot analysis with anti-APP C-terminal antibody (AC-1)(left panel)or anti-caveolin monoclonal antibody (2234)(right panel). lesterol is required for the insertion of recombinant caveolin-1 similar to that of other proteolytic activities known to cleave a into model lipid membranes (54, 55). There are several links number of single membrane-spanning precursors (tumor necro- between cholesterol metabolism and the pathogenesis of Alz- sis factor and notch) at the cell surface to release their ectodo- heimer’s disease. Cholesterol affects APP processing by inter- mains to the media (67). fering with the activity of a-secretase (56). An isoform of a a-Secretase cleavage can also occur intracellularly (68 –70). major cholesterol carrier protein, apoE, is a genetic risk factor Therefore, a second mechanism should exist that involves an for Alzheimer’s disease (57). Thus, these links to cholesterol intracellular compartment that may be independent of plasma metabolism may simply reflect the caveolar localization of APP. membrane caveolae. Alternatively, this phenomenon may be The processing of APP has been intensively studied since the explained by the presence of intracellular forms of caveolae, i.e. APP molecule was first identified. Normal processing of APP plasmalemmal vesicles. In our experiments, we could not de- occurs at the so-called a-site by a-secretase, which generates a tect a cell surface cleavage product of APP in cells that were non-amyloidogenic extracellular domain of APP (APPs) (3, 58). singly transfected with APP (Fig. 5D); in contrast, in cells This processing event makes the extracellular domain of APP co-transfected with APP and caveolin-1, APPs were produced containing a Kunitz-type protease inhibitor available in the at the cell surface. Overall cleavage of APP by a-secretase may extracellular space. This protease inhibitor activity is thought be modulated by the cycle of caveolae internalization and re- to prevent thrombin from injuring neurons (59, 60). In addi- cycling (APP could cycle between cell surface and intracellular tion, a-secretase is a target for the development of potential populations of caveolae). The a-secretase cleavage occurs not 17 18 agents for the treatment of Alzheimer’s disease, as a-secretase only at Leu but also at Val in the amyloid peptide (3), may cleave and solubilize Ab amyloid peptide, a major insolu- suggesting the existence of multiple a-secretases. Thus, caveo- ble component of the senile plaque. Recently, it has been re- lae and an undefined intracellular compartment may possess ported that APPs activate microglial cells and thereby enhance different a-secretases that cleave APP at distinct a-sites. the local release of neurotoxic agents (61). Therefore, APPs Protein kinase C activation promotes the a-secretase-medi- possess a broad range of biological effects. As our current ated processing of APP (71), by an unknown molecular mech- evidence suggests that APP is processed at its a-site within anism. Since this PKCa is a well established component of caveolae membranes, this may help in the identification and caveolae (72) and PKC activators inhibit caveolae internaliza- cloning of the protein or proteins that possess a-secretase tion (potocytosis) (73), it is possible that PKC activation pro- activity. longs the amount of time APP spends in cell surface caveolae Several previous reports support the view that a-secretase- and therefore in close contact with a-secretase activity. mediated cleavage of APP occurs on the cell surface (62– 65). APP is known to transit through clathrin-coated pits and These observations fit well with our current findings that APP vesicles on its way to endosomes and lysosomes (74). Recent is processed at its a-site within caveolae, as caveolae are spe- findings suggest a number of signaling receptors that are cialized microdomains of plasma membrane. APP requires cleared from the cell surface via clathrin-coated pits are first membrane anchoring for its cleavage by a-secretase suggesting present within caveolae microdomains (15, 21, 75). In fact, that the protease itself is a membrane-anchored protein (64). internalized caveolae microdomains and clathrin-coated vesi- Interestingly, caveolae have been reported to contain a prote- cles may be targeted to a common endosomal pool (77). This ase activity (66). The cleavage mechanism for APP may be scenario would provide two distinct mechanisms for clearing a-Secretase Processing of APP within Caveolae 10495 Wisniewski, H. M. (1990) Neurosci. Res. Commun. 7, 118 –122 signaling receptors from the cell surface. In fact, aAPPct was 32. Reaume, A. G., Howland, D. S., Trusko, S. P., Savage, M. J., Lang, D. M., reported to be enriched in lysosomes (62). It therefore is likely Greenberg, B. D., Siman, R., and Scott, R. W. (1996) J. Biol. Chem. 271, 23380 –23388 that APP is first present within caveolae where a-secretase 33. Buxbaum J. D., Gandy, S. E., Cicchetti, P., Ehrlich, M. E., Czernk, A. J., processing takes place, whereas the remaining intact APP may Fracasso, R. P., Ramabhadran, T. V., Unterbeck, A. J., and Greengard, P. be cleared from the cell surface via clathrin-coated pits and (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6003– 6006 34. Hayashi, Y., Kashiwagi, K., and Yoshikawa, K. (1992) Biochem. Biophys. Res. targeted to endosomes and lysosomes for proteolytic Commun. 187, 1249 –1255 processing. 35. Sisodia, S. S., Koo, E. H., Hoffman, P. N., Perry, G., and Price, D. L. (1993) Caveolae-like membrane domains exist in neuronal cells and J. Neurosci. 13, 3136 –3142 36. Ikezu, T., Okamoto, T., Murayama, Y., Okamoto, T., Homma, Y., Ogata, E., have been characterized by a number of independent laborato- and Nishimoto, I. (1994) J. Biol. Chem. 269, 31955–31961 ries (13, 75, 78). In addition, Allinquant and co-workers (13) 37. Yamatsuji, T., Okamoto, T., Takeda, S., Murayama, Y., Tanaka, N., and Nishimoto, I. (1996) EMBO J. 15, 498 –509 provided preliminary evidence that axonal APP is concentrated 38. Song, K. S., Li, S., Okamoto, T., Quilliam, L., Sargiacomo, M., and Lisanti, within caveolae-like structures in neurons. Thus, it is likely M. P. (1996) J. Biol. Chem. 271, 9690 –9697 that a neuron-specific form of caveolin may exist that promotes 39. Tang, Z., Okamoto, T., Boontrakulpoontawee, P., Katada, T., Otsuka, A. J., and Lisanti, M. P. (1997) J. Biol. Chem. 272, 2437–2445 caveolae formation in neuronal cells and interacts with neuro- 40. Schnitzer, J., McIntosh, D., Dvorak, A. M., Liu, J., and Oh, P. (1995) Science nally expressed APP. In Alzheimer’s disease, caveolae dysfunc- 269, 1435–1439 tion may cause reduced activity of a-secretase and accumula- 41. Smart, E. J., Y. Ying, C. Mineo, and Anderson, R. G. W. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10104 –10108 tion of toxic Ab amyloid peptide. 42. Frangioni, J. V., and Neel, B. G. (1993) Anal. Biochem. 210, 179 –187 43. Ermekova, K. S., Zambrano, N., Linn, H., Minopoli, G., Gertler, F., Russo, T., Acknowledgments—We thank Y. Sakaki for the gift of APP cDNAs and Sudol, M. (1997) J. Biol. Chem. 272, 32869 –32877 and Sangram S. Sisodia, Samuel E. Gandy, Richard W. Scott, Kazuaki 44. Sharma, R. K., Taylor, W. A., and Wang, J. H. (1983) Methods Enzymol. 102, Yoshikawa for generously donating APP antibodies. 210 –219 45. Parton, R. G., Joggerst, B., and Simons, K. (1994) J. Cell Biol. 127, 1199 –1215 REFERENCES 46. Lisanti, M. P., Sargiacomo, M., Graeve, L., Saltiel, A. R., and Rodriguez- 1. Selkoe, D. J. (1989) Cell 58, 611– 612 Boulan, E. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 9557–9561 47. Slunt, H. H., Thinakaran, G., Von Koch, C., Lo, A. C., Tanzi, R. E., and Sisodia, 2. Kang, J., Lemaire, H. G., Unterbeck, A., Salbaum, J. M., Masters, C. L., Grzeschik, K. H., Multhaup, G., Beyreuther, K., and Muller-Hill, B. (1987) S. S. (1994) J. Biol. Chem. 269, 2637–2644 Nature 325, 733–736 48. Chun, M., Liyanage, U., Lisanti, M. P., and Lodish, H. F. (1994) Proc. Natl. 3. Selkoe, D. J. (1994) Annu. Rev. Cell Biol. 10, 373– 403 Acad. Sci. U. S. A. 91, 11728 –11732 4. Sisodia, S. S., Koo, E. H., Beyreuther, K., Unterbeck, A., and Price, D. L. (1990) 49. Couet, J., Sargiacomo, M., and Lisanti, M. P. (1997) J. Biol. Chem. 272, Science 248, 492– 495 30429 –30438 5. van Duijn, C. M., Hendriks, L., Cruts, M., Hardy, J. A., Hofman, A., and Van 50. Parton, R. G. (1994) J. Histochem. Cytochem. 42, 155–166 Broeckhoven, C. (1991) Lancet 337, 978 51. Wang, R., Meschia, J. F., Cotter, R. J., and Sisodia, S. S. (1991) J. Biol. Chem. 6. Nishimoto, I., Okamoto, T., Matsuura, Y., Okamoto, T., Murayama, Y., and 266, 16960 –16964 Ogata, E. (1993) Nature 362, 75–79 52. Fielding, C., Bist, A., and Fielding, P. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 7. Fiore, F., Zambrano, N., Minopoli, G., Donini, V., Duilio, A., and Russo, T. 3753–3758 (1995) J. Biol. Chem. 270, 30853–30856 53. Smart, E., Ying, Y., Donzell, W., and Anderson, R. (1996) J. Biol. Chem. 271, 8. Borg, J. P., Oo, I. J., Levy, E., and Margolis, B. (1996) Mol. Cell. Biol. 16, 29427–29435 6229 – 6241 54. Murata, M., Peranen, J., Schreiner, R., Weiland, F., Kurzchalia, T., and 9. Guenette, S. Y., Chen, J., Jondro, P. D., and Tanzi, R. E. (1996) Proc. Natl. Simons, K. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10339 –10343 Acad. Sci. U. S. A. 93, 10832–10837 55. Li, S., Song, K. S., and Lisanti, M. P. (1996) J. Biol. Chem. 271, 568 –573 10. Chow, N., Korenberg, J.R., Chen, X.-N., and Neve, R.L. (1996) J. Biol. Chem. 56. Bodovitz, S., and Klein, W. (1996) J. Biol. Chem. 271, 4436 – 4440 271, 11339 –11346 57. Czech, C., Forstl, H., Hentschel, F., Monning, U., Besthorn, C., Geiger- 11. Yamatsuji, T., Matsui, T., Okamoto, T., Komatsuzaki, K., Takeda, S., Fuku- Kabisch, C., Sattel, H., Masters, C., and Beyreuther, K. (1994) Eur. Arch. moto, H., Iwatsubo, T., Suzuki, N., Asami-Odaka, A., Ireland, S., Kinane, Psychiatry Clin. Neurosci. 243, 291–292 T. B., Giambarella, U., and Nishimoto, I. (1996) Science 272, 1349 –1352 58. Sisodia, S. S., Koo, E. H., Beyreuther, K., Unterbeck, A., and Price, D. L. (1990) 12. Ikezu, T., Okamoto, T., Komatsuzaki, K., Matsui, T., Martyn, J. A. J., and Science 248, 492– 495 Nishimoto, I. (1996) EMBO J. 15, 2468 –2475 59. Van Nostrand, W. E., Wagner, S. L., Suzuki, M., Choi, B. H., Farrow, J. S., 13. Bouillot, C., Prochiantz, A., Rougon, G., and Allinquant, B. (1996) J. Biol. Geddes, J. W., Cotman, C. W., and Cunningham, D. D. (1989) Nature 341, Chem. 271, 7640 –7644 546 –549 14. Mineo, C., James, G. L., Smart, E. J., and Anderson, R. G. W. (1996) J. Biol. 60. Oltersdorf, T., Fritz, L. C., Schenk, D. B., Lieberburg, I., Johnson-Wood, K. L., Chem. 271, 11930 –11935 Beattie, E. C., Ward, P. J., Blacher, R. W., Dovey, H. F., and Sinha, S. (1989) 15. Liu, P., Ying, Y., Ko, Y.-G., and Anderson, R. G. W. (1996) J. Biol. Chem. 271, Nature 341, 144 –147 10299 –10303 61. Barger, S. W., and Harmon, A. D. (1997) Nature 388, 878 – 881 16. Lisanti, M. P., Scherer, P., Tang, Z.-L., and Sargiacomo, M. (1994) Trends Cell 62. Haass, C., Koo, E. H., Mellon, A., Hung, A. Y., and Selkoe, D. J. (1992) Nature Biol. 4, 231–235 357, 500 –503 17. Glenney, J. R., and Soppet, D. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 63. Koo, E. H., Park, L., and Selkoe, D. J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10517–10521 4748 – 4752 18. Scherer, P. E., Tang, Z., Chun, M., Sargiacomo, M., Lodish, H. F., and Lisanti, 64. Sisodia, S. S. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6075– 6079 M. P. (1995) J. Biol. Chem. 270, 16395–16401 65. Arribas, J., Lopez-Casillas, F., and Massague, J. (1997) J. Biol. Chem. 272, 19. Tang, Z., Scherer, P. E., Okamoto, T., Song, K., Chu, C., Kohtz, D. S., Nishi- 17160 –17165 moto, I., Lodish, H. F., and Lisanti, M. P. (1996) J. Biol. Chem. 271, 66. Sevinsky, J. R., Rao, L. V., and Ruf, W. (1996) J. Cell Biol. 133, 293–304 2255–2261 67. Blobel, C. P. (1997) Cell 90, 589 –592 20. Sargiacomo, M., Scherer, P. E., Tang, Z., Kubler, E., Song, K. S., Sanders, 68. De Strooper, B., Umans, L., Van Leuven, F., and Van Den Berghe, H. (1993) M. C., and Lisanti, M. P. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, J. Cell Biol. 121, 295–304 9407–9411 69. Sambamurti, K., Refolo, L. M., Shioi, J., Pappolla, M. A., and Robakis, N. K. 21. Li, S., Okamoto, T., Chun, M., Sargiacomo, M., Casanova, J. E., Hansen, S. H., (1992) Ann. N. Y. Acad. Sci. 674, 118 –128 Nishimoto, I., and Lisanti, M. P. (1995) J. Biol. Chem. 270, 15693–15701 70. Haass, C., Koo, E. H., Capell, A., Teplow, D. B., and Selkoe, D. J. (1995) J. Cell 22. Chun, M., Liyanage, U. K., Lisanti, M. P., and Lodish, H. F. (1994) Proc. Natl. Biol. 128, 537–547 Acad. Sci. U. S. A. 91, 11728 –11732 71. Jacobsen, J. S., Spruyt, M. A., Brown, A. M., Sahasrabudhe, S. R., Blume, A. J., 23. Feron, O., Smith, T. W., Michel, T., and Kelly, R. A. (1997) J. Biol. Chem. 272, Vitek, M. P., Muenkel, H. A., and Sonnenberg-Reines, J. (1994) J. Biol. 17744 –17748 Chem. 269, 8376 – 8382 24. de Weerd, W. F., and Leeb-Lundberg, L. M. (1997) J. Biol. Chem. 272, 72. Lisanti, M. P., Scherer, P. E., Vidugiriene, J., Tang, Z.-L., Hermanoski-Vo- 17858 –17866 satka, A., Tu, Y.-H., Cook, R. F., and Sargiacomo, M. (1994) J. Cell Biol. 25. Raposo, G., Dunia, I., Marvelo, S., Andre, C., Guillet, J. G., Strosberg, A. D., 126, 111–126 Benedeth, E. L., and Hoebeke, J. (1987) Biol. Cell 60, 117–124 73. Smart, E. J., Foster, D. C., Ying, Y. S., Kamen, B. A., and Anderson, R. G. 26. Raposo, G., Dunia, I., Delavier-Klutchko, C., Kaveri, S., Stroberg, A. D., and (1994) J. Cell Biol. 124, 307–313 Benedeth, E. L. (1989) Eur. J. Cell Biol. 50, 340 –352 74. Nordstedt, C., Caporaso, G. L., Thyberg, J., Gandy, S. E., and Greengard, P. 27. Okamoto, T., Schlegel, A., Scherer, P. E., and Lisanti, M. P. (1998) J. Biol. (1993) J. Biol. Chem. 268, 608 – 612 Chem. 273, 5419 –5422 75. Wu, C., Butz, S., Ying, Y., and Anderson, R. G. W. (1997) J. Biol. Chem. 272, 28. Couet, J., Li, S., Okamoto, T., Ikezu, T., and Lisanti, M. P. (1997) J. Biol. 3554 –3559 Chem. 272, 6525– 6533 29. Okamoto, T., Takeda, S., Murayama, Y., Ogata, E., and Nishimoto, I. (1995) 76. Li, S., Song, K. S., Koh, S. S., Kikuchi, A. and Lisanti, M.P. (1996) J. Biol. Chem. 271, 28647–28654 J. Biol. Chem. 270, 4205– 4208 30. Song, K. S., Tang, Z., Li, S., and Lisanti, M. P. (1997) J. Biol. Chem. 272, 77. Simons, K., and Ikonen, E. (1997) Nature 387, 569 –572 78. Bickel, P. E., Scherer, P. E., Schnitzer, J. E., Oh, P., Lisanti, M. P., and Lodish, 4398 – 4403 31. Kim, K. S., Wen, G. Y., Bancher, C. M., Chen, J., Sapeenza, V. J., Hong, H., and H. F. (1997) J. Biol. Chem. 272, 13793–13802
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Journal of Biological Chemistry – Unpaywall
Published: Apr 1, 1998