Interaction of a Neuron-specific Protein Containing PDZ Domains with Alzheimer's Amyloid Precursor Protein*

Susumu TomitaDagger §, Toshinori Ozaki, Hidenori TaruDagger , Shinobu OguchiDagger , Shizu TakedaDagger parallel , Yoshimasa YagiDagger parallel , Shigeru Sakiyama, Yutaka KirinoDagger , and Toshiharu SuzukiDagger **

From the Dagger  Laboratory of Neurobiophysics, School of Pharmaceutical Sciences, the University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033 Japan, the  Division of Biochemistry, Chiba Cancer Center Research Institute, 666-2, Nitona, Chuou-ku, Chiba 260-0801, Japan, and parallel  Bio-oriented Technology Research Advancement Institution, 3-18-19 Toranomon, Minato-ku, Tokyo 105-0001, Japan

    ABSTRACT
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Abstract
Introduction
References

A novel protein, human X11-like (human X11L), contains a phosphotyrosine interaction (PI) domain and two PDZ domains and displays 55.2% amino acid homology with the human X11 (human X11). The PI domain of human X11L interacts with a sequence containing the NPXY motif found in the cytoplasmic domain of Alzheimer's amyloid precursor protein. A construct lacking the carboxyl-terminal domain, which comprises two PDZ domains (N + PI), enhances PI binding to APP, whereas another construct lacking an amino-terminal domain relative to PI domain (PI + C) suppresses PI binding to APP. Overexpression of full-length human X11L (N + PI + C) in cells that express APP695 stably decreased the secretion of Abeta 40 but not that of Abeta 42. However, overexpression of the PI domain alone and the N + PI construct in cells did not affect the secretion of Abeta despite their ability to bind to the cytoplasmic domain of Alzheimer's amyloid precursor protein. These observations suggest that the amino-terminal domain regulates PI binding to APP and that the carboxyl-terminal domain containing PDZ motifs is essential to modulate APP processing. Because expression of the human X11L gene is specific to brain, the present observations should contribute to shedding light on the molecular mechanism of APP processing in Alzheimer's disease.

    INTRODUCTION
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Abstract
Introduction
References

Alzheimer's amyloid precursor protein (APP)1 is an integral membrane protein with a receptor-like structure (1). A principal component of parenchymal amyloid deposits in Alzheimer's disease (AD) is beta -amyloid (Abeta ) (2-4), which is derived from APP by proteolytic cleavage (1, 5-11). Abeta is thought to be generated through an intracellular protein secretory pathway of APP (for a review, see Ref. 12). The short APP cytoplasmic domain consisting of 47 amino acid residues is thought to be responsible for determination of APP metabolism (13-15) and possible signal transduction from a putative extracellular ligand that has yet to be identified (for a review, see Ref. 16). Because APP is a candidate pathogenic factor of AD, elucidation of the physiological function of APP as well as the determination of the metabolic mechanism of Abeta production should increase our understanding of the pathogenesis of AD. Using a yeast two-hybrid system, we isolated cDNA of proteins that interact with the cytoplasmic domain of APP (APPCOOH) in order to elucidate the molecular mechanisms of APP metabolism and function of APP. Previous efforts to identify and isolate proteins associating with APPCOOH, utilizing the yeast two-hybrid system, have resulted in the isolation of proteins carrying phosphotyrosine binding/phosphotyrosine interaction (PI) domains such as Fe65 (17), Fe65-like (18, 19), and X11 (20).

The X11 gene, which is located on chromosome 9, was originally isolated as a gene candidate for Friedreich ataxia (21) and its partial cDNA was identified as a clone encoding a protein that associates with APPCOOH (22). The PI domain of X11 interacts with the YENPTY motif of APPCOOH (20). In the present study, we isolated a complete cDNA encoding an X11-like protein (X11L) from a human adult brain cDNA library, utilizing the yeast two-hybrid system and APPCOOH as a bait. A partial short cDNA encoding approximately 190 amino acids in the PI domain of X11L has already been isolated using a similar procedure (22). However, detailed characterization of X11L binding to APP and identification of the role X11L plays in the physiological function of APP have not been performed. We found that human X11L requires a sequence containing the NPXY motif of APPCOOH for APP binding and that association of the PI domain with APP was suppressed by a deletion of a amino-terminal domain fused to the PI domain (PI + C construct) but enhanced by a deletion of a carboxyl-terminal domain fused to the PI domain (N + PI construct). Co-transfection of full-length human X11L into cells that express stably transfected human APP695 cDNA resulted in decreased secretion of Abeta 40 but not Abeta 42. However, co-transfection into cells of the cDNA lacking the carboxyl-terminal domain (N + PI construct) or a cDNA encoding only the PI domain (PI construct), whose protein products preserve the ability to bind to APPCOOH, did not present the ability to modulate Abeta production. The present results suggest that the amino-terminal region of the PI domain is needed to regulate binding affinity to APPCOOH and that the carboxyl-terminal region of the PI domain is essential for modulation of APP metabolism. Furthermore, Northern blot and Western blot analyses demonstrated strong specific expression of the X11L gene in brain tissues. Together with the present results, this suggests that X11L may play an important role in the physiological function and metabolism of APP in neuronal tissues.

    EXPERIMENTAL PROCEDURES

Yeast Two-hybrid System, cDNA Cloning of Human and Mouse X11-like Protein, and Plasmid Construction-- The yeast two-hybrid system, MATCH MAKER Two-Hybrid System, was purchased from CLONTECH Laboratories, Inc. (Palo Alto, CA). The cDNA encoding APPCOOH, located between amino acids 649 and 695 (numbering as for the APP695 isoform), was amplified by PCR using the APP695 cDNA present in pcDNA3 (23). The PCR product, which was produced by additional EcoRI sequence in 5' upstream and BamHI sequence in 3' downstream, was digested with EcoRI plus BamHI, and the restriction fragment was inserted into pGBT9 at identical restriction sites in-frame with the GAL4 encoding sequence present on this vector. The resulting pGBT9APPCOOH "bait" plasmid was used to isolate the cDNA of the APP-binding protein from a human adult brain cDNA library cloned into the vector pGAD424, which contains the GAL4 transactivation domain. Both plasmids were co-transfected into the yeast HF7c strain (MATa, ura3-52, his3-200, ade2-101, lys2-801, trp1-901, leu2-3, 112, gal4-542, gal80-538, LYS::GAL1-HIS3, URA:: (GAL4 17-mer)3-CYC1-LacZ) (24) using the lithium acetate/heat shock procedure according to the manufacturer's product protocol (catalog no. PT1265-1, CLONTECH Laboratories). Because the HF7c strain harbors the reporter genes lacZ and HIS3, co-transformants were streaked first on selection medium lacking tryptophan, leucine, and histidine to assay for activation of the HIS3 reporter gene. Positive colonies were then picked and assayed for activation of the lacZ reporter gene as evidenced by their blue color. Plasmids from colonies positive for both reporter genes were rescued into Escherichia coli HB101. The rescued plasmids were reassayed for their binding activity to the cytoplasmic domain of APP by transfection into HF7c harboring the "bait" plasmid. Finally, the nucleotide sequences of the positive clones were determined by the dideoxy terminator cycle sequencing using a Prism 377 DNA Sequencer (Perkin-Elmer), and the homology of the deduced amino acid sequence to existing sequences in the data bases was investigated using the BLAST program.

The 5' nucleotide sequence of one cDNA, pGAD424#104, isolated by the yeast two-hybrid system encoded the 624-amino acid sequence of human X11L protein. The deleted amino-terminal region consisting of 125 amino acids was determined by the RACE procedure (Life Technologies, Inc.; 5'-RACE system version 2) using human whole brain poly(A)+ RNA (CLONTECH Laboratories; catalog no. 6516-1) and primers corresponding to the amino acid sequence indicated by the underline in Fig. 1a. The 5'-RACE product was inserted into the pCRII TA cloning vector (Invitrogen), pCRII5'hX11L. Mouse X11L (previously referred to as mouse X11 in Refs. 22 and 25) cDNA was isolated by reverse transcription-PCR using two sets of primers. The primer sequence was designed from the known partial nucleotide sequence of mouse X11 (DDBJ/EMBL/GenBankTM accession no. L34676). A combination of primers consisting of primer 1 corresponding to nucleotide positions 5-22 (forward, numbering for L34676) and primer 2 corresponding to nucleotide positions 839-856 (reverse, numbering for L34676) and another combination of primers consisting of primer 3 corresponding to nucleotide positions 839-856 (forward, numbering for L34676) and primer 4 corresponding to nucleotide positions 2041-2059 (reverse, numbering as for L34676) were used to amplify whole mouse brain poly(A)+ RNA using Pwo DNA polymerase (Boehringer Mannheim). The resulting 0.8-kb (primers 1 and 2) and 1.2-kb PCR (primers 3 and 4) products were cloned separately into pCRII to give, respectively, pCRIImX11L-1, which contains the nucleotide sequence encoding the amino acid sequence between positions 70 and 353 (numbering as for Fig. 1a), and pCRIImX11L-2, which contains the nucleotide sequence encoding the amino acid sequence between positions 348 and 748 (numbering as for Fig. 1a). The 5' unknown nucleotide sequence was also determined by the RACE procedure using the mouse brain poly(A)+ RNA and primers corresponding to the amino acid sequences indicated by underlines in Fig. 1. The 5'-RACE product, corresponding to amino acid positions 1-69 (numbering for Fig. 1a), was inserted into pCRII to give pCRIITA5'mX11L.

The insert from pGAD424#104 and the cDNA encoding the 5' region of human X11L, pcRIITA5'hX11L, were ligated. The resulting entire cDNA encoding the entire human X11L protein was inserted into pcDNA3 (Invitogen, Carlsbad, CA) with (pcDNA3-FLAG-hX11L) or without (pcDNA3-hX11L) the addition of a synthetic linker encoding the FLAG epitope on the carboxyl-terminal side of the inserts. The amino acid sequence of human X11L, consisting of 749 amino acids, and that of mouse X11L, consisting of 748 amino acids, were deduced from the nucleotide sequence of each cDNA and are indicated in Fig. 1.

The cDNA clones encoding the partial amino-terminal domain (pGAD424#6; amino acids 121-367), PI domain (pGAD424#PI; 368-555), carboxyl-terminal domain (pGAD424#7; 556-749), amino-terminal domain attached to the PI domain (pGAD424#1; 121-555), PI domain attached with the carboxyl-terminal domain (pGAD424#4; 368-749), and almost the entire sequence of human X11L (pGAD424#104; 121-749) were subcloned into pGAD424 harboring the GAL4 transactivation domain. These plasmids were used for the APP binding assay in co-transfections with pGBT9APPCOOH using the yeast two-hybrid system. The cDNA encoding APPCOOH was truncated internally at regions 681-690 (pGBT9APP695Delta 681-690) and 669-680 (pGBT9APP695Delta 669-680). The cDNA encoding the cytoplasmic domain of amyloid precursor-like protein 2 (APLP2) was cloned from an adult human brain cDNA library by the reverse yeast two-hybrid system with the "bait vector," pGBT9#104, and the cDNA encoding the cytoplasmic domain of human amyloid precursor-like protein 1 (APLP1) was cloned by reverse transcription-PCR using human brain poly(A)+ RNA and primers with nucleotide sequences corresponding to positions 605-611 (forward) and 644-650 (reverse) (numbering as for the human APLP1 genome sequence, DDBJ/EMBL/GenBankTM accession no. AD000864). These cDNAs encoding 53 and 47 amino acid residues of the cytoplasmic domain of APLP1 and APLP2, respectively, were inserted into pGBT9 to produce pGBT9hAPLP2COOH and pGBT9hAPLP1COOH. These constructs in pGBT9 vectors were used to examine the binding ability for human X11L (pGAD424#104) in the yeast two-hybrid system.

beta -Galactosidase activity in the yeast two-hybrid system was measured in a liquid assay using o-nitrophenylgalactopyranoside according to the manufacturer's protocol (CLONTECH Laboratories; catalog no. PT1265-1) and was expressed in Miller units.

Antibodies-- The polyclonal anti-X11L amino-terminal domain antibody UT-29 was raised against a peptide, "Cys" plus containing the amino acid sequence between positions 207 and 223 of human X11L. Another polyclonal anti-X11L carboxyl-terminal domain antibody, UT-30, was raised against the peptide "Cys" plus containing the amino acid sequence between positions 735 and 749 of human X11L. These peptides were synthesized at Quality Controlled Biochemicals Inc. (Hopkinton, MA). UT-29 and UT-30 are specific to X11L (see "Results") and appear not to react with X11. These antibodies were affinity-purified using antigen coupled to SulfoLink gel (Pierce). Anti-APP polyclonal antibody UT-18 was prepared against APP695-(676-695). The specificity was identical to that of another anti-APP polyclonal antibody, UT-421, which has been fully characterized (15, 23). Anti-Abeta monoclonal antibody, 2D1, recognizes a human-specific epitope FRH-(600-602), which lies between the beta - and alpha -secretase sites (23); 25C was prepared against human APP695-(621-631) (Abeta -(25-35)). Anti-FLAG monoclonal antibody M2 (Eastman Kodak Co.) and anti-APP extracellular domain monoclonal antibody LN27 (Zymed Laboratories Inc., San Francisco, CA) were purchased.

Preparation of Proteins and in Vitro Interaction of Human X11L with APP-- The cDNA clones encoding human X11L, the domain structures of human X11L, and the cytoplasmic domain of human APP695 were subcloned into pGEX-4T-1 (Amersham Pharmacia Biotech) to produce GST fusion protein. The recombinant plasmids were introduced into E. coli JM109. Production of the fusion protein was induced by culturing the recombinant E. coli strains in the presence of 1 mM isopropyl-beta -D-thiogalactopyranoside. Cells were lysed in phosphate-buffered saline (PBS; 140 mM NaCl and 10 mM sodium phosphate (pH 7.4)) by sonication, and the lysate was centrifuged at 10,000 × g for 20 min at 4 °C. The resulting supernatant was applied to a glutathione-Sepharose 4B (Amersham Pharmacia Biotech) column (1-ml bed volume), and proteins bound to the beads were recovered with an elution buffer consisting of 5 mM glutathione and 50 mM Tris-HCl (pH 7.4). Affinity-purified GST fusion proteins were dialyzed against a buffer consisting of 20 mM Tris-HCl (pH 7.5)/2 mM EDTA/1 mM dithiothreitol, and the purity of the protein was examined by staining with Coomassie Brilliant Blue R-250 following SDS-PAGE. To prepare the affinity beads coupled to the GST fusion protein, 2-4 nmol of GST fusion proteins were incubated with 100 µl of glutathione-Sepharose 4B for 1 h at 4 °C with rotation. The efficiency of coupling, almost greater than 95% under these conditions, was determined by examining the protein content of the uncoupled protein.

Whole cell extracts of 293 cells that expressed stably transfected APP695 cDNA (pcDNA3APP695) (23), human X11L cDNA (pcDNA3-hX11L), and cDNA encoding the domain structures of human X11L were prepared by lysing cells in lysis buffer (PBS containing 10 mM CHAPS, 5 µg/ml chymostatin, 5 µg/ml leupeptin, 5 µg/ml pepstatin A, 1 mM Na3VO4, and 1 mM NaF) for 1 h on ice, followed by centrifugation at 12,000 × g for 10 min at 4 °C. The resulting supernatant containing APP695 and protein constructs from human X11L were used for the binding assay.

The protein samples, which were adjusted to contain approximately 2-4 nmol of protein, were applied to a column (100-µl bed volume) of glutathione-Sepharose beads bearing GST fusion protein or GST alone (control). The beads were washed with binding buffer three times, and the protein complex was eluted with elution buffer. Following SDS-PAGE, the proteins were analyzed by immunoblotting with the indicated antibodies and 125I-Protein A (Amersham Pharmacia Biotech). Protein concentration was determined by the micro-Lowry method with bovine serum albumin as a standard (26).

Co-immunoprecipitation-- COS7 cells (~1 × 107 cells) were doubly transfected with 2 µg of pcDNA3APP695 and 2 µg of pcDNA3-hX11L in Lipofectamine (Life Technologies) for 18 h. After changing the medium, the cells were cultured for 48 h in Dulbecco's modified Eagle's medium containing 10% (v/v) heat-inactivated fetal bovine serum. The cells were harvested and lysed in 1 ml of lysis buffer (PBS containing 10 mM CHAPS, 5 µg/ml chymostatin, 5 µg/ml leupeptin, 5 µg/ml pepstatin A, 1 mM Na3VO4, and 1 mM NaF) for 1 h on ice and centrifuged at 12,000 × g for 10 min at 4 °C. The affinity-purified polyclonal antibodies (50 µg) UT-18 and UT-30 and monoclonal antibodies (50 µg) 2D1 and 25C were added to the supernatant and incubated on ice for 1 h. Protein G-Sepharose beads were then added and incubated for 1 h at 4 °C, and the samples were centrifuged. The pellets were washed with the lysis buffer three times, and proteins were eluted by boiling the pellet in SDS-sample buffer (23). Proteins were then analyzed by SDS-PAGE (6% (w/v) polyacrylamide) and immunoblotted with the indicated antibodies, and immunocomplexes were revealed by ECL (Amersham Pharmacia Biotech).

Immunocytochemistry-- 293 cells that express stably transfected pcDNA3-hX11L were fixed for 20 min with 4% (w/v) paraformaldehyde in PBS (pH 7.4) containing 4% (w/v) sucrose, permeabilized with 0.2% (v/v) Triton X-100 for 5 min and blocked for 1 h in PBS containing 0.2% (v/v) goat serum. The same cells were double-stained with rhodamine-conjugated Con A (Vector Laboratories, Inc., Burlingame, CA), which binds with high affinity to glycoproteins in the ER and cis-Golgi, or with rhodamine-conjugated WGA (Vector Laboratories), which binds with high affinity to glycoproteins in medial and trans-Golgi (27, 28). In a separate study, 293 cells that express stably transfected pcDNA3-APP695 were further transfected with pcDNA3-hX11L transiently. The cells were incubated with anti-human X11L antibody UT-30 and anti-APP monoclonal antibody LN27 and then with fluorescein isothiocyanate- or rhodamine isothiocyanate-conjugated secondary antibody (Zymed Laboratories Inc., San Francisco, CA). The coverslips were mounted in immersion oil type B (R. P. Cargille Laboratories Inc., Cedar Grove, NJ), and the cells were viewed using a confocal laser scanning microscope, Bio-Rad MRC 600.

Western Blot Analysis of Mouse X11L from Adult Tissues-- Tissues from adult ddY mice (4-5 weeks) were homogenized and sonicated in a solution containing 50 mM Tris-HCl (pH 7.4), 1% (w/v) SDS, 2.7 M urea, 25 µg/ml pepstatin A, 25 µg/ml leupeptin, and 25 µg/ml chymostatin. Samples were centrifuged (10,000 × g, 10 min), the resulting supernatant (150 µg of protein) was subjected to SDS-PAGE (7.5% (w/v) polyacrylamide), and proteins were electrophoretically transferred to a nitrocellulose membrane. The membrane was probed with UT-30 and 125I-Protein A (Amersham Pharmacia Biotech). Radioactivity was analyzed by autoradiography.

Comprehensive Gene Mapping-- The somatic cell hybrid Southern DNA membranes, BIOSMAP, were purchased from BIOS Laboratories (New Haven, CT). Southern hybridization was performed with a probe prepared from a template containing the insert from pcDNA3-hX11L with random oligonucleotides (High Prime DNA labeling kit; Boehringer Mannheim) and [alpha -32P]dCTP (3000 Ci/mmol; NEN Life Science Products) according to the user guidelines.

Northern Analysis of Human X11L-- Human multiple tissue Northern blots (catalog no. 7760-1) were purchased from CLONTECH Laboratories. Hybridizations were carried out using the standard procedure (29). Radioactive probes with a specific activity equal to, or higher than, 1 × 108 cpm/µg were prepared from a template of human X11L cDNA coding for the amino acid sequence between positions 126 and 749 using [alpha -32P]dCTP (3000 Ci/mmol, NEN Life Science Products) and random oligonucleotides (High Prime DNA labeling kit; Boehringer Mannheim).

ELISA Analysis-- The 293 cell line that expresses stably transfected pcDNA3APP695 (15, 23) was co-transfected with 10 µg of pcDNA3-hX11L, which contained the cDNA sequence encoding the full-length form of human X11L (amino acid sequence 1-749), pcDNA3-N + PI, which contained the cDNA encoding the amino-terminal region attached to the PI domains (amino acid sequence 1-555), pcDNA3-PI, which contained the cDNA encoding the PI domain (amino acid sequence 368-555), or the control plasmid pcDNA3, which lacked a cDNA sequence (Invitrogen, Carlsbad, CA). The cells were supplied with fresh growth media 12 h after the start of transfection, and conditioned medium from cells (2 × 10 6 cells) was collected 72 h after the medium change. Abeta 40 and Abeta 42 were quantified with sandwich ELISA using three types of Abeta -specific monoclonal antibodies as described previously (15, 23). Briefly, wells were coated with the Abeta 40 (4D1) or Abeta 42 (4D8) end-specific monoclonal antibodies (0.3 µg of IgG in PBS), washed with PBS containing 0.05% (v/v) Tween 20 (washing buffer), blocked with bovine serum albumin (3% (w/v) in PBS), and washed with washing buffer, and then a sample (100 µl) diluted suitably with washing buffer containing 1% (w/v) bovine serum albumin (dilution buffer) was incubated together with a standard dose of synthetic Abeta -(1-40) and Abeta -(1-42) peptides (synthesized using solid phase t-BOC (N-tert-butyloxycarbonyl)-chemistry by Dr. James I. Elliott at the W. M. Keck Foundation Biotechnology Resource Laboratory, Yale University). After washing, wells were treated with biotinized 2D1 (12. 5 ng in dilution buffer), washed, and incubated with 100 µl of a streptavidin-horseradish peroxidase complex (1:2000 dilution; Amersham Pharmacia Biotech catalog no. RPN1051). The plates (96 wells) were washed further, 100 µl of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid solution (KPL 5062-01, Kirkegaard & Perry Laboratories, Gaithersburg MD) was added to the wells, and incubation was continued at room temperature. The reaction was stopped by adding 100 µl of 1% (w/v) SDS, and the absorbance at 405 nm was measured.

    RESULTS

Molecular Cloning of a cDNA Encoding Human X11L Protein: Primary Structure and Chromosome Location-- The short cytoplasmic domain of APP is the sole region exposed to the cytoplasm and contains several functional amino acid motifs that regulate the metabolism of APP (13-15, 30, 31). Therefore, the isolation and characterization of proteins which interact with APPCOOH are important for our understanding the physiological function and metabolism of APP. Approximately 106 independent clones from an adult human brain cDNA library were screened by the yeast two-hybrid system with a "bait" vector containing the cDNA encoding the 47-amino acid APPCOOH region, pGBT9APPCOOH. Ultimately, 125 clones were isolated as positive for nutrient (His) selection and beta -galactosidase activity. The nucleotide sequences of all the positive candidates were determined. Out of the 125 clones, 88 encoded the amino acid sequence of Fe65 (17) or Fe65-like (18) proteins, which have previously been reported to be APP-binding proteins. Nucleotide sequences of the other four clones revealed a novel protein sequence with higher homology to mouse X11 than to human X11 (25). Other sequences were nonsense clones, which were not thought to encode significant proteins. One of the four cDNA clones, pGAD424#104, contained an open reading frame coding for a potentially novel protein of 624 amino acids, as shown in amino acid positions 126-749 of human X11L (Fig. 1a), and contained a 3'-untranslated and poly(A)+ sequence. To identify the 5' nucleotide sequence of this clone, we performed the RACE procedure using poly(A)+ RNA from human adult brain and the indicated primer (underlined in Fig. 1a), which corresponds to amino acid positions 130-135. An approximately 620-nucleotide sequence including the missing 260 base pairs of the 5'-untranslated region was determined, and the whole amino acid sequence of this clone was deduced (Fig. 1a). This protein showed high homology to the partial amino acid sequence of mouse X11 rather than to human X11. Because the amino-terminal sequence of mouse X11 has not been reported (25), we also determined the 5' nucleotide sequence of mouse X11 by the RACE procedure using poly(A)+ RNA from mouse adult brain and the indicated primer (underlined in Fig. 1a), which corresponds to amino acids 80-85. The entire amino acid sequence of the amino-terminal part of mouse X11 was deduced, and the sequence was used to look for homology between the three related proteins (Fig. 1).


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Fig. 1.   Comparison of amino acid sequences and homology between the human X11-like, mouse X11-like, and human X11 proteins. a, alignment of human X11L (hX11L), mouse X11L (mX11L), and human X11 (hX11) protein amino acid sequences. Gaps produced by the alignment are indicated by a hyphen in the sequence. Predicted phosphotyrosine interaction (PI) domain (yellow box) and PDZ domains (blue boxes) are indicated. The first methionine residue was chosen as the methionine after the termination codon. The nucleotide sequence surrounding the methionine seems to match the Kozak consensus sequence (55). Although mouse X11-like was previously reported as a partial amino acid sequence of mouse X11 (25), we designated this mouse X11 as mouse X11L (see "Experimental Procedures"). The positions of amino acids are relative to those of human X11L. b, general characteristics and homology between the three proteins. Amino acid homologies between human X11L and other proteins are indicated. PI, phosphotyrosine interaction domain (yellow); PDZ, PDZ domain (blue) predicted from the consensus sequence of other PDZ domains (50). Numbers indicate amino acid positions of human X11L. Amino acid homology (percentage of human X11L) of mouse X11L and human X11 to human X11L is indicated.

The human X11L cDNA encoded a protein consisting of 749 amino acids and presented 55.2% amino acid homology to the partially known amino acid sequence of human X11 and 90.2% homology to the complete sequence of mouse X11, which consists of 748 amino acids. Thus, we designated this protein as human X11-like protein (human X11L), whose partial sequence consisting of 188 amino acids comprising the PI domain has already been reported (22). Furthermore, mouse X11 (25) is thought to be a homologue of human X11L because it possesses higher homology to human X11L than to human X11. Thus, we surmise that the mouse counterpart of the human X11 gene has not yet been identified. Therefore, we have referred to mouse X11 as mouse X11L in the present paper (Fig. 1a).

The human X11L protein contains a PI domain (Fig. 1a, yellow box) and two PDZ domains (Fig. 1a, blue boxes) (the repeated sequences of the brain-specific protein PSD-95, Drosophila septate junction protein disks-large, and the epithelial tight-junction protein ZO1) (32) in its carboxyl-terminal domain, as does human X11 (Fig. 1b). The PI domain (amino acid positions 368-555, 82.4% homology) as well as the region that lies on the carboxyl-terminal side (amino acid positions 556-749, 85.6% homology) of the PI domain, but not the amino-terminal domain (amino acid positions 1-367, 20.6% homology), are highly conserved between the human X11L and X11 proteins (Fig. 1).

The chromosomal location of the human X11 gene has been fully characterized (21). The gene is located on 9q13-q21 within 0.7 centimorgans of the D9S5 and D9S15 loci. We identified the chromosome on which human X11L gene is located using a comprehensive gene mapping procedure with the somatic cell hybrid Southern DNA membrane as described under "Experimental Procedures". Southern hybridization with a probe of human X11L whole cDNA, pcDNA3-hX11L, indicated that the human X11L gene is located on human chromosome 15 (data not shown), a location that is distinct from that of the X11 gene located on chromosome 9.

Binding of Human X11L Protein to APP-- X11 and Fe65 have been shown to interact with the YENPTY (APP-(682-687) of APP695 isoform) motif of APPCOOH through their PI domains. In contrast to the interaction involving the Shc PI domain, the interaction is independent of APP tyrosine phosphorylation (20, 33). To characterize the binding of X11L to APP, we performed a yeast two-hybrid study using a combination of the cDNA encoding the domain sequence derived from human X11L in pGAD424 and the cDNA of APPCOOH, pGBT9APPCOOH (Fig. 2), and another combination of the cDNA coding for almost the whole length of human X11L, pGAD424#104, and the cDNA of APPCOOH truncated internally in pGBT9 (Fig. 3a). The beta -galactosidase activities (Miller units) of both combinations were measured and compared (Figs. 2 and 3a). The PI domain (pGAD424#PI, containing the cDNA coding for amino acids 368-555) was essential for binding to APP and gave almost the same activity as that of the plasmid coding for the full-length human X11L, pGAD424#104. We found that when the PI domain was attached to the amino-terminal domain (pGAD424#1, containing the cDNA coding for amino acids 126-555) the activity was enhanced (2-fold). However, when the PI domain was attached to the carboxyl-terminal domain (pGAD424#4, containing the cDNA coding for amino acids 368-749) APP binding ability was markedly suppressed (Fig. 2). The amino-terminal (pGAD424#6 containing cDNA coding for amino acids 126-367) and carboxyl-terminal (pGAD424#7 containing cDNA coding for amino acids 556-749) domains alone were negative for both beta -galactosidase activity and His nutrient selection and had no ability by themselves to bind to APPCOOH. A cDNA construct coding for amino acids 1-125 gave a nonspecific and constitutive positive signal in the nutrient and beta -galactosidase assays of the yeast two-hybrid system, which indicates that this region contains an artificial trans-activator sequence of Gal4-mediated transcription. Therefore, we deleted this region from the cDNA constructs for the two-hybrid assays.


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Fig. 2.   Binding of the human X11-like protein constructs to the cytoplasmic domain of APP in yeast. a, binding between constructs derived from human X11L and APPCOOH was quantified by the liquid beta -galactosidase assay. Results are an average of six independent studies (n = 6), and the error bar indicates S.D. Control indicates a study with the plasmid alone. beta -Galactosidase activity is indicated in Miller units. b, schematic structures of human X11L and the constructs derived from human X11L used in this study. PI, phosphotyrosine interaction domain; PDZ, PDZ domain. Numbers indicate amino acid position.


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Fig. 3.   Binding of human X11-like protein to protein constructs derived from the cytoplasmic domain of APP and to the cytoplasmic domain of APP family proteins in yeast. a, binding between human X11L (hX11L) and the protein constructs derived from APPCOOH was quantified by the liquid beta -galactosidase assay. Results are the average of six independent studies (n = 6), and the error bar indicates S.D. wt, the cytoplasmic domain of APP695; Delta 669-680, the cytoplasmic domain of APP695 lacking the amino acid sequence of APP695-(669-680); Delta 681-690, the cytoplasmic domain of APP695 lacking the amino acid sequence of APP695-(681-690). The amino acid sequences of the protein constructs are also indicated. b, binding activity of human X11L to the cytoplasmic domain of APP gene family, APP695, APLP1650, and APLP2763. Results are the average of six independent studies (n = 6), and the error bar indicates S.D. The amino acid sequence of the cytoplasmic domain of the APP gene family proteins is also indicated. Control indicates a study with the pGBT9 plasmid alone. beta -Galactosidase activity is indicated in Miller units.

When the binding site of the cytoplasmic domain of APP was examined (Fig. 3a), we found that a cDNA, pGBT9APP695Delta 681-690, encoding the cytoplasmic domain with a deletion of amino acids 681-690 containing the NPTY sequence had lost binding ability for human X11L. Another construct, pGBT9APP695Delta 669-680, with a deletion of the amino-terminal 15 amino acid residues to the NPTY motif retained binding ability, but the binding was slightly weaker compared with that of the whole sequence (wild type). The results indicate that the sequence containing the NPTY motif of APP is essential for association with human X11L as well as with X11 and Fe65/Fe65-like (17-20).

APP is a member of a gene family that includes APLP1 (34) and APLP2 (35). The amino acid sequences of these proteins are highly conserved, especially within their cytoplasmic domains. The binding ability of human X11L to the cytoplasmic domain of APLPs was examined using the yeast two-hybrid system (Fig. 3b). When pGAD424#104 was co-transfected with pGBT9APLP2COOH consisting of the cDNA encoding the 47-amino acid human APLP2 cytoplasmic domain (Fig. 3b, APLP2), or pGBT9APLP1COOH consisting of the cDNA encoding the 46-amino acid human APLP1 cytoplasmic domain (Fig. 3b, APLP1) in yeast, equal or greater beta -galactosidase activities were observed than for APPCOOH (Fig. 3b, APP). These results indicate that human X11L associates with APP family proteins, APP, APLP1, and APLP2, all of which contain NPXY motifs. The control vector, pGBT9, which did not contain cDNA gave no beta -galactosidase activity (Fig. 3b, control).

Interaction of APP with Human X11L Using GST Fusion Proteins in Vitro-- The protein-protein interaction of human X11L with APP was examined in vitro using GST-human X11-like fusion protein (GST-hX11L) and human APP695 (Fig. 4, a and b). Glutathione-Sepharose beads bearing GST fusion proteins, whose constructs are shown in Fig. 4d, were incubated with the lysate of 293 cells that were stably transfected with pcDNA3APP695 containing human APP695 cDNA. The beads were washed with binding buffer three times, and protein complexes were eluted with PBS containing 5 mM reduced glutathione. Following SDS-PAGE, the proteins were analyzed by immunoblotting with anti-APP antibody UT-18 plus 125I-Protein A (arrows in Fig. 4, a and b (longer exposure of a), indicate mature (m, upper) and immature (im, lower) APP695 isoforms). APP was found to associate with the PI domain (PI, amino acid positions 368-555) as well as with full-length human X11L (human X11L). The PI domain attached to the amino-terminal domain (N + PI; amino acid positions 1-555) enhanced binding to APP, and the PI domain attached to the carboxyl-terminal domain (PI + C, amino acid positions 368-749) suppressed binding to APP. The amino-terminal (N, amino acid positions 1-367) and carboxyl-terminal (C, amino acid positions 556-749) domains alone did not bind to APP. The results of these assays corroborate the results obtained using the yeast two-hybrid assay (Fig. 2). In addition, the results suggested that the amino- and/or carboxyl-terminal domains may be regulatory element(s) of PI domain binding to APP.


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Fig. 4.   Interaction of APP695 and the cytoplasmic domain of APP695 with protein constructs derived from human X11-like protein in vitro. a, binding of APP695 to protein constructs derived from human X11L. Whole cell extracts (approximately 1 mg of protein) of 293 cells stably transfected with APP695 cDNA were applied to columns of glutathione beads bearing the different GST fusion protein constructs as indicated in d. The bound APP695 protein was eluted together with the protein constructs of human X11L, separated by SDS-PAGE (10% (w/v) polyacrylamide), transferred to a nitrocellulose membrane, and detected by anti-APP antibody UT-18. Arrows indicate APP695 (m, mature APP; im, immature APP). hX11L, full-length human X11L; N + PI, amino-terminal domain attached to the PI domain; N, amino-terminal domain alone; PI + C, PI domain attached to the carboxyl-terminal domain; PI, PI domain alone; C, carboxyl-terminal domain alone. b, longer exposure of the autoradiogram of a. c, whole cell extract (approximately 1 mg of protein) of 293 cells stably transfected with cDNAs encoding constructs derived from human X11L was applied to a column of glutathione beads bearing the GST-APPCOOH fusion protein (APPCOOH) and GST protein alone (GST). The protein constructs from the human X11L-bound fraction of the GST-APPCOOH beads were recovered, separated by SDS-PAGE (7.5% (w/v) polyacrylamide), transferred to a nitrocellulose membrane, and detected by immunoblotting with UT-29 antibody. Crude indicates the result of Western blot analysis with cell lysates (10 µg of protein) containing the full-length human X11L protein (hX11L), the amino-terminal domain attached with the PI domain (N + PI) or the amino-terminal domain alone (N). d, schematic structure of the human X11L construct used in this study. N, amino-terminal region of the PI domain; PI, phosphotyrosine interaction domain; C, carboxyl-terminal region of the PI domain; PDZ, PDZ domain. Amino acid numbers of human X11L are indicated. 170, 116, and 76 refer to the molecular masses (kDa) of the protein standards.

In order to confirm the protein-protein interaction of human X11L with APPCOOH, the studies described in Fig. 4, a and b, were performed again but with the difference that glutathione-Sepharose beads bearing GST- APPCOOH fusion protein (APPCOOH) were incubated with the cell lysates from 293 cells that expressed stably transfected pcDNA3-hX11L (human X11L), pcDNA3-N + PI (N + PI), or pcDNA3-N (N) (Fig. 4c). These cell lysates contain the respective protein constructs as shown in "crude" of Fig. 4c. The beads were washed with binding buffer three times, and protein complexes were eluted with PBS containing 5 mM reduced glutathione. Following SDS-PAGE, the proteins were analyzed by immunoblotting with anti-X11L antibody UT-29 plus 125I-Protein A. Full-length human X11L was found to associate with GST-APPCOOH fusion protein on the beads (human X11L). The PI domain fused to the amino-terminal domain (amino acid positions 1-555) associated strongly with the GST-APPCOOH fusion protein on the beads (N + PI). However, the amino-terminal domain (amino acid positions 1-367) alone did not associate with GSTAPPCOOH fusion protein (N). The beads carrying GST peptide alone (GST) did not present any appreciable ability to associate with human X11L or its domain structure proteins. We have not studied binding of the PI domain (PI) alone, nor the PI domain fused to the carboxyl-terminal domain (PI + C), nor the carboxyl-terminal domain alone (C), because we had no PI-specific antibody and could not establish 293 cell lines stably transfected with pcDNA3-PI + C or pcDNA3-C. However, the results broadly confirmed the data presented in Fig. 4, a and b, and, especially, proved that deletion of the human X11L carboxyl-terminal domain enhances the binding of human X11L to APPCOOH.

Specificity of the Anti-X11L Antibody and Brain-specific Expression of X11L-- Antibodies raised against sequences in the amino-terminal (UT-29) and the carboxyl-terminal (UT-30) regions of the PI domain were examined for their specificity. Human X11L tagged with the FLAG peptide on its carboxyl-terminal side (FLAG-hX11L) was prepared. From the lysates of 293 cells, which were stably transfected with FLAG-hX11L cDNA (pcDNA3-FLAG-hX11L), proteins were immunoprecipitated (IP) with anti-FLAG (M2), UT-29, or UT-30 antibodies, separated by SDS-PAGE, transferred to nitrocellulose membrane, and probed (WB) with the indicated antibodies (Fig. 5a). Because a ~135-kDa protein (arrow) was detected with all three types of antibodies, we designated the ~135-kDa protein as FLAG-hX11L. When crude cell lysates were probed with these three antibodies, the ~135-kDa protein was also specifically detected (Fig. 5a, crude). Preincubation of antibodies with the corresponding antigen peptides (10 µM) resulted in the failure of these antibodies to recognize the ~135-kDa protein (data not shown). The results demonstrate that both UT-29 and UT-30 antibodies are highly specific for human X11L and that they are potentially useful for detecting endogenous X11L. Endogenous X11L was also analyzed (Fig. 5b); mouse X11L was immunoprecipitated (IP) from mouse brain lysate with UT-29 or UT-30. The immunoprecipitate was analyzed by Western blot (WB) with UT-29 or UT-30. When crude brain lysate (Fig. 5b, crude) was probed directly, the ~135-kDa protein (arrow) was also detected by both UT-29 and UT-30. The efficiency of immunoprecipitation of mouse X11L by UT-30 is much higher than that of UT-29 (compare left part with middle part of Fig. 5b), although UT-29 and UT-30 immunoprecipitate human X11L equally well as shown in Fig. 5a. The amino acid sequence of the region used to raise the UT-29 antibody was not identical between human and mouse (15/17 amino acid identity) X11L, although that used to raise UT-30 was identical (15/15 amino acid identity). Since UT-29 was raised against the human sequence, the antibody may not be as effective in recognizing the mouse protein, especially in immunoprecipitation. The molecular size of X11L deduced from the cDNA is 82.5 kDa, which is much smaller than 135 kDa, the value determined by SDS-PAGE. One explanation for this discrepancy could be that X11L is subject to modifications such as glycosylation. However, this does not seem plausible, because the GST-hX11L fusion synthesized in E. coli also migrated as a 160-kDa protein during SDS-PAGE, and a ~135-kDa protein after the removal of the GST peptide by digestion with thrombin (data not shown). The most plausible explanation is that X11L adopts a secondary structure that results in anomalous migration during SDS-PAGE.


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Fig. 5.   Specificity of antibodies and identification of the X11-like protein. a, crude lysate (crude, 100 µg of protein) from 293 cells stably transfected with pcDNA3-FLAG-hX11L was subjected to immunoprecipitation (IP) using anti-FLAG (M2), anti-X11L amino-terminal domain (UT-29), or anti-X11L carboxyl-terminal domain (UT-30) antibodies. Immunoprecipitates from the lysate (approximately 1 mg of protein) were subjected to SDS-PAGE (6% (w/v) polyacrylamide), proteins were transferred to a nitrocellulose membrane, and the membrane was probed (WB) with M2, UT-29, or UT-30 antibodies. Immunocomplexes were detected by incubation with anti-mouse IgG peroxidase or anti-rabbit IgG peroxidase followed by ECL. The ~135-kDa protein indicated with an arrow is FLAG-hX11L. b, mouse brain lysate (crude, 100 µg of protein) was subject to immunoprecipitation (IP) using UT-29 and UT-30 antibodies. Immunoprecipitates from the lysate (approximately 1 mg of protein) were probed (WB) with UT-29 and UT-30 antibodies, and immunocomplexes were detected by ECL with anti-rabbit IgG following SDS-PAGE (6% (w/v) polyacrylamide) and transfer of proteins to a nitrocellulose membrane. PC, sample was subjected to preclearing with Protein A-Sepharose prior to the addition of the antibody; w/o PC, sample was immunoprecipitated without preclearing; Comp, sample was immunoprecipitated with indicated antibodies in the presence of antigen peptide (10 µM). The ~135-kDa protein indicated with an arrow is mouse X11L. UT-29 does not react with mouse X11L in immunoprecipitation (see text). Numbers are the molecular masses (kDa) of the protein standards. c, mouse tissues lysate (150 µg of protein) was subject to SDS-PAGE (6% (w/v) polyacrylamide), and proteins were transferred to a nitrocellulose membrane. The membrane was probed with UT-30 and 125I-Protein A and then analyzed by autoradiography. An arrow indicates the position of mouse X11L.

It has been reported that human X11 is expressed dominantly (21) but not specifically2 in brain tissues. However, the expression patterns of human and mouse X11L have not been examined, although both X11 and X11L proteins are similar with respect to structure and APP binding. The expression of mouse X11L in various tissues was examined (Fig. 5c). Protein extracts (150 µg of protein) from various adult mouse tissues were analyzed by Western blot with UT-30 antibody. A ~135-kDa mouse X11L protein (Fig. 5c, arrow) was detected specifically in brain but not in other nonneuronal tissues such as heart, lung, liver, testis, spleen, thymus, or kidney. The results indicated brain-specific expression of X11L protein.

We further performed Northern blot analysis using a probe derived from human X11L cDNA as described under "Experimental Procedures." This Northern blot analysis showed a strong 4.4-kb and a comparatively weak 4.0-kb message in brain tissue but not in the other nonneuronal tissues examined (Fig. 6a). The actin gene was found to exist in all tissues (Fig. 6b). The size of the mRNA is in good agreement with the size of human X11L cDNA and clearly differs from the mRNA size of human X11 (21). Thus, we concluded that the major 4.4-kb product is the mRNA that encodes human X11L. The minor 4.0-kb product may be a truncated mRNA or a minor product derived from alternative splicing of human X11L gene transcripts. These results demonstrate that X11L gene is specifically expressed in neuronal tissue and suggests that the protein product may play a role in neuron-specific function(s) through association with APP.


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Fig. 6.   Northern blot analysis with probes for human X11-like (hX11L) and beta -actin genes. Human multiple tissue Northern blots containing 2 µg of poly(A)+ RNA were hybridized with the probe, which was radiolabeled by random priming of human X11L cDNA coding for amino acids 126-749 (a) and with a radioactive probe derived from the cDNA of human beta -actin (b). The 4.4- and 4.0-kb bands in brain hybridized with the human X11L probe (a). The 2-kb band was hybridized ubiquitously, and an additional 1.8-kb band was hybridized in heart and skeletal muscle with beta -actin probe (b).

In Vivo Interaction of Human X11L with APP-- To examine whether human X11L and APP interacted in cells, co-immunoprecipitation experiments were performed. COS7 cells that transiently express human X11L and APP695 were lysed in the presence of 10 mM CHAPS. The detergent-soluble fraction was subjected to immunoprecipitation (IP) using affinity-purified UT-30 or anti-APP extracellular domain monoclonal antibodies 2D1 and 25C (Fig. 7). The same quantity of normal rabbit or mouse IgG was used as a control. The immunoprecipitates were analyzed by Western blot (WB) using UT-18 (Fig. 7a) or UT-30 (Fig. 7b) antibody. APP was detected in the sample immunoprecipitated with UT-30 (Fig. 7a). Human X11L was detected in the sample immunoprecipitated with 2D1 and 25C (Fig. 7b). APP and human X11L could not be detected in the sample immunoprecipitated using normal rabbit or mouse IgG, indicating that the co-immunoprecipitation of APP and human X11L was specific. The result suggests that APP and X11L interact in mammalian cells.


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Fig. 7.   Co-immunoprecipitation of the human X11-like protein and APP. COS7 cells transiently expressing human X11L and APP were lysed in PBS containing 10 mM CHAPS, and the lysate (crude) was used for immunoprecipitation (IP) with the indicated antibodies. The immunoprecipitates were analyzed by Western blot (WB) with antibodies UT-18 (a) and UT-30 (b) following SDS-PAGE (6% (w/v) polyacrylamide) and transfer to a nitrocellulose membrane. Mature (m) and immature (im) APP695 and immature endogenous APP (imAPP endogenous) were co-immunoprecipitated with human X11L using anti-X11L antibody UT-30 (a). The human X11L protein was also co-immunoprecipitated with APP using anti-APP extracellular domain antibodies 2D1 and 25C (b). Rabbit IgG, normal rabbit IgG; mouse IgG, normal mouse IgG; 2D1 and 25C, anti-Abeta monoclonal antibodies; UT-30, anti-human X11L polyclonal antibody.

Intracellular Distribution of Human X11L and APP-- The intracellular distribution of human X11L was examined by an immunocytochemical study using 293 cells stably expressing human X11L protein. The 293 cells that express stably transfected pcDNA3-human X11L were double-stained with UT-30 and WGA (medial- plus trans-Golgi marker) or ConA (ER plus cis-Golgi marker) (23, 27, 28) to analyze the intracellular distribution of human X11L (Fig. 8, a and b). In a separate study, the 293 cells stably transfected with pcDNA3-APP695 and transiently transfected with pcDNA3-hX11L were double-stained with UT-30 and LN-27 antibodies to analyze for co-distribution of human X11L and APP (Fig. 8c). The cells were then observed under a confocal laser scanning microscope. We found that the staining of human X11L with UT-30 (green in Fig. 8, a and b) partially co-localized with the staining obtained using WGA (red in Fig. 8a) and ConA (red in Fig. 8b). It is also clear that UT-30 did not stain the plasma membrane. These results show that X11L protein is present in the cytoplasm and to a lesser extent in the ER and Golgi apparatus as shown by the yellow color (hX11L/WGA in Fig. 8a and hX11L/ConA in Fig. 8b). LN27 stained the Golgi apparatus strongly (red in Fig. 8c), which agrees with a previous report on the cellular distribution of APP (23). Taken together, these observations indicate that APP (red) may be colocalized with human X11L (green) mostly in the Golgi apparatus (hX11L/APP in Fig. 8c), although the majority of human XIIL was localized in the cytoplasm. The antibody that had been preadsorbed to antigen did not show any staining (data not shown).


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Fig. 8.   Localization of the human X11-like protein in 293 cells. Intracellular localization of human X11L is shown as follows (left parts of a-c): medial- and trans-Golgi (a), ER and cis-Golgi (b), and APP (c). 293 cells that express stably transfected pcDNA3-hX11L were stained with UT-30 antibody (a and b, green) and observed under a confocal laser scanning microscope. The cells were also double-stained with rhodamine-conjugated WGA (a, red) to identify the location of medial- and trans-Golgi and rhodamine-conjugated ConA (b, red) to identify the location of ER and cis-Golgi. c, 293 cells stably transfected with pcDNA3-APP695 were further transiently transfected with pcDNA3-hX11L. The cells were double-stained with UT-30 (green) and LN27 (red) antibodies. Co-localization of human X11L and WGA (hX11L/WGA), human X11L and ConA (hX11L/ConA), and human X11L and APP (hX11L/APP) are indicated by the yellow. Scale bar, 20 µm.

The distribution of X11L was confirmed by a biochemical study. Mouse brain homogenate was fractionated into nuclear, membrane, and cytosolic fractions and then analyzed by immunoblot analysis with the UT-30 antibody. Most of the mouse X11L protein was detected in the cytosolic fraction with a smaller quantity of mouse X11L being also detected in crude membrane fractions consisting of the plasma membrane, ER, and Golgi apparatus membranes. This indicates that X11L may be attached to these membranes through association with APP (data not shown). The results of the immunocytochemical (Fig. 8) and biochemical studies indicate that X11L is most likely a cytosolic protein with a tendency to co-distribute with APP.

Role of X11L Protein in APP Processing: Generation of beta -Amyloid Peptides-- To examine whether X11L regulates APP processing through its interaction with APPCOOH, the effect of X11L on the generation of both Abeta 40 and Abeta 42 peptides was examined (Fig. 9). The 293 cells that stably expressed human APP695 (15, 23) were co-transfected transiently with the cDNA encoding full-length human X11L, N + PI (amino acid positions 1-555), PI (amino acid positions 369-555), or vector alone (control) (see Fig. 4 for protein construction). We have already demonstrated that these three constructs, full-length human X11L, N + PI, and PI, can associate with APPCOOH (Figs. 2 and 4). At 72 h after supplementation of the cells with fresh medium following transfection, the conditioned media were collected, and the levels of Abeta 40 and Abeta 42 were quantified by sandwich ELISA (15, 23). The relative ratios of the levels of Abeta to the control level (reference value set to 1.0) were determined. The full-length human X11L construct decreased the level of Abeta 40 secretion but not that of Abeta 42. The levels of Abeta 40 produced from cells expressing N + PI or PI domains did not change compared with the levels produced by control cells, despite the fact that the N + PI and PI domain have binding ability for APPCOOH (Fig. 4). The secretion level of Abeta 42 was not altered remarkably by co-transfection with human X11L or its domain structures (Fig. 9b). These results suggest that human X11L functions to modulate APP processing. The constructs N + PI and PI did not have this modulator function, indicating that the carboxyl-terminal region is important for APP processing. The study indicates that the PDZ domains in the carboxyl-terminal domain of human X11L play an important role in APP processing. Furthermore, the results suggest that regulation of Abeta 42 production may differ from that of Abeta 40.


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Fig. 9.   Quantification of Abeta in the medium of cells expressing human X11-like protein and derived constructs. The 293 cells stably transfected with pcDNA3APP695 (15, 23) were co-transfected transiently with human X11L and derived constructs. Fresh medium was supplied 12 h after transfection, conditioned media were collected 72 h after the medium change, and the concentrations of Abeta 40 (a) and Abeta 42 (b) in the medium were measured using sandwich ELISA as described under "Experimental Procedures." The quantity of Abeta is indicated as the relative ratio of the levels of Abeta observed using cells transfected with "control" or the pcDNA3 vector alone (a reference value of 1.0). hX11L, full-length hX11L; N + PI, amino-terminal domain attached to the PI domain; PI, PI domain. Results are the average of 12-16 independent assays. The error bar indicates S.D. (**, p = 0.005).


    DISCUSSION

APP was identified as a causative factor in the pathogenesis of AD (1, 5-11). Several mutations of APP and other AD-related genes have been found in familial AD, and it has been reported that these mutations contribute to the pathogenesis of familial AD (reviewed in Refs. 36 and 37). However, familial AD represents only a small minority of AD cases. The majority of AD cases are of the sporadic type, whose patients are not thought to carry the mutation. Therefore, elucidation of the physiological function(s) and metabolism of APP is critical for our understanding of the pathogenesis of sporadic type AD. The cytoplasmic domain of APP, or APPCOOH, which is highly conserved phylogenetically in vertebrates (38), contains signals for APP metabolism (13-15) and is thought to contain the amino acid sequence essential for the transduction of signals from an extracellular ligand that remains to be identified (reviewed in Ref. 16). Consequently, research based on the identification and isolation of factors that interact with APPCOOH has been pursued (17-20, 39, 40). In the present paper, we isolated a cDNA encoding a novel protein, human X11L, as a protein that binds to APPCOOH. This human X11L possesses 55.2% homology to human X11, which has been identified as a gene in the region of the Friedreich ataxia locus (21), a degenerative disorder involving both the central and peripheral nervous system (41). Although a partial amino acid sequence consisting of 165 amino acids in the PI domain of X11L has been identified as the APP binding motif (22) and the interaction between the PI domain of X11 and a sequence containing the NPTY motif of APPCOOH has been fully analyzed (20, 33), the biological function of these proteins has not been determined. Because the amino acid homology between the PI domains from human X11 and human X11L is very high (82.9%), the architecture and APPCOOH recognition mechanism of the PI domains from both proteins may be similar. In the present study, we demonstrate that the interaction of human X11L with APPCOOH is enhanced by deletion of the carboxyl-terminal domain and suppressed by deletion of the amino-terminal domain (Figs. 2 and 4). One possible explanation for these results is that both domains function as regulatory elements of PI domain binding to APPCOOH, possibly indicating that the amino-terminal domain contains an "enhancer" sequence and the carboxyl-terminal domain contains a "suppressor" sequence for PI domain binding to APPCOOH. The deletion of the amino- or carboxyl-terminal domain may alter the conformation of the PI domain, and this may affect the binding affinity to APPCOOH. Although these deleted constructs of human X11L are not physiological products, in vivo, proteins that can interact with the amino- and/or the carboxyl-terminal domains of X11L may regulate APP binding to the PI domain by inducing conformational changes in human X11L. We propose a possible hypothesis for the regulation of X11L binding to APPCOOH (Fig. 10). In cells, X11L exists in a free form and shows a weak affinity for APPCOOH (Fig. 10a). When an unidentified "factor X" binds to the amino-terminal domain of X11L, the PI domain adopts an altered conformation and acquires strong binding affinity for APPCOOH (Fig. 10b). The construct deleted for the carboxyl-terminal domain (N + PI construct) may mimic this strong binding state of X11L (Fig. 10d). Then the PDZ domain(s) of the stable X11L·APP complex, associate with another factor, "factor Y," resulting in the suppression of Abeta 40 production (Fig. 10c). We have already isolated several novel proteins that bind to the amino-terminal domain.3 One of these proteins may be "factor X."


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Fig. 10.   Possible role of X11L in APP processing. a, the X11L protein binds to the APPCOOH domain weakly via its PI domain ("weak associated state"). b, when factor X binds to the amino-terminal domain, the conformation of X11L changes and the X11L·factor X complex binds to APPCOOH with high affinity ("strong associated state"). c, when the PDZ domain(s) in the carboxyl-terminal domain of the X11L are in a strong associated state, another "factor Y" binds. This binding may be essential for suppression of Abeta 40 secretion. d, A construct with a deletion of the carboxyl-terminal domain (N + PI) can bind to APPCOOH with high affinity. In this case, the conformation of N + PI is thought to mimic the strong associated state. This construct does not suppress the secretion of Abeta 40, because it lacks the carboxyl-terminal domain. e, the construct containing the PI domain alone can bind to APPCOOH perhaps without any regulation. f, the construct with a deletion of the amino-terminal domain (PI + C) cannot bind to APPCOOH because of the suppressor effect of the carboxyl-terminal domain and the missing amino-terminal domain. M, membrane; Abeta , beta -amyloid; N, amino-terminal domain of X11L; PI, PI domain of X11L; C, carboxyl-terminal domain of X11L; X, a putative factor X, which binds to the amino-terminal domain of X11L; Y, a putative factor Y, which binds to the carboxyl-terminal domain of X11L.

When the ability of full-length human X11L, N + PI, or PI constructs on Abeta production was examined in cells, only full-length human X11L reduced the secretion of Abeta 40. This result agrees well with our hypothesis. The constructs deleted for the carboxyl-terminal domain (N +PI construct), which preserves human X11L binding ability for APPCOOH, did not have this modulator activity to regulate Abeta 40 secretion. This observation would also appear to support our hypothesis. Neither the PI domain alone nor the N + PI domain is sufficient to regulate APP metabolism if it binds to APPCOOH with higher affinity (Fig. 10, d and e). The human X11L construct lacking the amino-terminal domain (PI + C) loses its binding affinity for APPCOOH (Fig. 10f). The carboxyl-terminal domain containing the PDZ domains may be essential for the regulation of Abeta 40 production. We have not examined how the PDZ domains function to regulate Abeta 40 production in detail. However, it has been well established that the PDZ domain interacts with other proteins (32, 42, 43). Fig. 10 proposes that an association of an unidentified "factor Y" to the PDZ domain(s) may be necessary to regulate APP processing.

APPCOOH is not subject to tyrosine phosphorylation,4 and it is believed that the mode of binding of X11 and X11L to a sequence containing the NPXY motif in APPCOOH is a phosphotyrosine-independent interaction such as in the case of Shc phosphotyrosine binding/PI (44). However, APPCOOH contains other phosphorylation sites (45-47). One of these, Thr668, is located at position 16 in the amino-terminal region of the NPTY motif and is phosphorylated in brain tissue (48). Furthermore, we have found that Thr668 phosphorylation of mature APP (mAPP, N- and O-glycosylated form) is neuron-specific,5 although Thr668 phosphorylation of immature APP (N-glycosylated form) is observed in nonneuronal cells during the mitotic phase of cell division (47-49). Therefore, we examined the effect of phosphorylation of APP at Thr668 on the interaction between X11L and APPCOOH. A single amino acid mutation that changed this threonine to alanine or glutamic acid did not alter the binding of APP to the PI domain of human X11L.2 This result indicates that the association of X11L with APPCOOH is not dependent on the phosphorylation of APP.

Because proteins containing the PDZ domain are known to play a role in the clustering of membrane receptors and channels (50) and because the expression of X11L is brain-specific, X11L may also play an important role in neuronal function(s) through its association with APP. While we were preparing this manuscript, we found that a cDNA similar to human X11L could be isolated from rat as a protein interacting with Munc 18, Mint (51). Munc 18 is thought to be a protein required for synaptic vesicle exocytosis. Mint-1 and Mint-2 seem to be rat homologues of human X11 and X11L, respectively. The Munc 18 binding domain is located in the amino-terminal region of the PI domain of X11L/Mint-2, and a complex consisting of Mint and Munc18 is thought to play a role in the docking of synaptic vesicles to the active zone (51). This observation suggests that APP may play an important role in neurotransmitter release at nerve terminals or that Mint-2 may regulate the binding ability of X11L to APP. Since we have demonstrated that mAPP localizes in the nerve terminals of cholinergic neurons (38), one may hypothesize that APP interacts with the exocytotic machinery proteins of synaptic vesicles via X11L/Mint-2. Since cholinergic neurotransmission is thought to be involved in learning and memory processes (52) and loss of function in cholinergic neurotransmission has been observed in senile dementia of AD (53), further characterization of X11L should increase our understanding of the physiological function of APP and the pathogenesis of sporadic type AD.

Furthermore, while this manuscript was being reviewed, a report appeared showing that X11 has an inhibitory effect on both Abeta 40 and Abeta 42 secretion using a double transient expression system with APP and X11 cDNAs (54). The X11 and X11L proteins may regulate APP metabolism in a different fashion due to differences in their amino-terminal domain structures.

    ACKNOWLEDGEMENTS

We thank Drs. M. Oishi (Montifore Medical Center, New York), T. Seki (Juntendo University School of Medicine, Tokyo, Japan), and S. Itohara (RIKEN: The institute of Physical and Chemical Research, Wako, Japan) for critical reading and comments on the manuscript and Y. Morimoto and N. Kobayashi for technical assistance

    FOOTNOTES

* This work was supported in part by a Grant from Program for Promotion of Basic Research Activities for Innovative Biosciences.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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB014719 (human X11-like) and AB014720 (mouse X11-like).

§ Recipient of Japan Society for the Promotion of Science Research Fellowships for Young Scientists.

** To whom correspondence should be addressed. Tel./Fax: 81-3-3814-6937; E-mail: tsuzuki{at}mayqueen.f.u-tokyo.ac.jp.

The abbreviations used are: APP, beta -amyloid precursor protein; Abeta , beta -amyloid; AD, Alzheimer's disease; APPCOOH, the cytoplasmic domain of APP; CHAPS, 3-[(3-cholamidpropyl)dimethylammonio]-1-propanesulfonic acid; ConA, concanavalin A; COS7 cells, SV40-transformed kidney cells from African green monkey; ELISA, enzyme-linked immunosorbent assay; ER, endoplasmic reticulum; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PDZ, repeated sequences in the brain-specific protein PSD-95, the Drosophila septate junction protein disks-large, and the epithelial tight junction protein ZO-1; PI, phosphotyrosine interaction; RACE, rapid amplification of cDNA ends; WGA, wheat germ agglutinin; kb, kilobase pair(s); X11, a gene in the region of the Friedreich ataxia locus; X11L, X11-like.

2 S. Tomita and T. Suzuki, unpublished observation.

3 S. Tomita, D.-S. Lee, and T. Suzuki, unpublished observations.

4 T. Suzuki, unpublished observation.

5 Iijima, K., Satoh, Y., Ando, K., Seki, T., Arai, Y., Greengard, P., Nairn, A. C., Kirino, Y., and Suzuki, T., manuscript in preparation.

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Abstract
Introduction
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