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J. Biol. Chem., Vol. 278, Issue 49, 49448-49458, December 5, 2003

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Novel Cadherin-related Membrane Proteins, Alcadeins, Enhance the X11-like Protein-mediated Stabilization of Amyloid -Protein Precursor Metabolism^*

Yoichi Araki¶, Susumu Tomita¶||, Haruyasu Yamaguchi**, Naomi Miyagi, Akio Sumioka, Yutaka Kirino, and Toshiharu Suzuki

From the Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-ku Kita-12 Nishi-6, Sapporo 060-0812, the Laboratory of Neurobiophysics, School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku Hongo 7-3-1, Tokyo 113-0033, and the ^**College of Medical Care and Technology, School of Medicine, Gunma University, Showa-machi 3-39-15, Maebashi 371-8514, Japan

Received for publication, June 9, 2003 , and in revised form, September 10, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Previously we found that X11-like protein (X11L) associates with amyloid -protein precursor (APP). X11L stabilizes APP metabolism and suppresses the secretion of the amyloid -protein (A) that are the pathogenic agents of Alzheimer's disease (AD). Here we found that Alcadein (Alc), a novel membrane protein family that contains cadherin motifs and originally reported as calsyntenins, also interacted with X11L. Alc was abundant in the brain and occurred in the same areas of the brain as X11L. X11L could simultaneously associate with APP and Alc, resulting in the formation of a tripartite complex in brain. The tripartite complex stabilized intracellular APP metabolism and enhanced the X11L-mediated suppression of A secretion that is due to the retardation of intracellular APP maturation. X11L and Alc also formed another complex with C99, a carboxyl-terminal fragment of APP cleaved at the -site (CTF). The formation of the Alc·X11L·C99 complex inhibited the interaction of C99 with presenilin, which strongly suppressed the -cleavage of C99. In AD patient brains, Alc and APP were particularly colocalized in dystrophic neurites in senile plaques. Deficiencies in the X11L-mediated interaction between Alc and APP and/or CTF enhanced the production of A, which may be related to the development or progression of AD.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The production, aggregation, and accumulation of amyloid -protein (A)¹ in the brain are initial steps in the pathogenesis of Alzheimer's disease (AD). A is generated by the intracellular processing of amyloid -protein precursor (APP). Major proteolytic processing of APP generates a large extracellular amino-terminal domain (sAPP) and a truncated carboxyl-terminal fragment (CTF) by the digestion of -secretase, which cleaves APP at the -site within the A domain. Another form of proteolytic processing occurring at the -site, by -secretase (or BACE), gives rise to low levels of sAPP and a carboxyl-terminal fragment (CTF) including the entire A domain. Both CTF and CTF are further cleaved by -secretase (presenilin (PS) complex) and generate the p3 peptide and A, respectively (1, 2).

A minority of AD cases fall into the familial category in which the overproduction of A appears to be due to mutations in genes encoding APP or PS (3). However, most AD cases are of the sporadic type (SAD) in which mutations in APP- or PS-encoding genes do not occur. The mechanisms that lead to the overproduction of A in SAD cases must therefore involve alternative mechanisms that cause the production, accumulation, and degradation of A (4-6). These mechanisms are currently being investigated intensively.

APP is a type I membrane protein (7). Immature APP (N-glycosylated form) is localized in the endoplasmic reticulum and cis-Golgi, whereas mature APP (N- and O-glycosylated form) is localized to compartments following trans-Golgi and on the plasma membrane. The cytoplasmic domain of APP (APPcyt), through its interactions with cytoplasmic proteins and/or its conformational changes due to phosphorylation (8-13), plays an important role in the regulation of APP metabolism. X11-like protein (X11L) was originally isolated as a factor that interacts with APPcyt, and the interaction results in the suppression of A production (14). X11L is a member of the X11 protein family. The X11 was initially identified on the basis of its possible link to Friedreich ataxia (15) and was originally denoted as Mint (16, 17). X11 and X11L are thought to be homologues of LIN-10 in Caenorhabditis elegans (18, 19) and dX11L in Drosophila melanogaster (20). They are neuron-specific adaptor proteins composed of a large amino-terminal region, a central phosphotyrosine interaction (PI) domain, and two carboxyl-terminal PDZ domains. The ⁶⁸¹GYENPTY⁶⁸⁷ motif of APPcyt (numbering is based on the APP695 isoform) interacts with the PI domain of X11L (14).

To understand how APP metabolism is regulated, including A generation, it is of interest to isolate other proteins that interact with X11L. This will help elucidate the molecular mechanism by which X11L regulates APP metabolism.

In the present study, we isolated novel human cDNAs encoding Alcadeins (Alcs), which are type I transmembrane proteins that interact with the PI domain of X11L. These genes are conserved in a wide variety of species, including D. melanogaster and C. elegans. We found that the cytoplasmic domain of Alc bound to the PI domain of X11L and initiated the formation of a tripartite complex comprised of Alc, X11L, and APP. This complex stabilized intracellular APP metabolism and significantly suppressed A production by slowing APP maturation. We found that Alc and X11L also formed a tripartite complex with C99, a CTF. This complex inhibited the interaction of C99 with PS1, a component of -secretase that generates A from C99. We could recover endogenous X11L·APP·Alc tripartite complexes from normal mouse brains, and normal mouse neurons displayed remarkable colocalization of these proteins. In AD brains, APP was found to colocalize with Alc in the dystrophic neurites of senile plaques. These observations indicate that APP exists in protein complexes composed of X11L and Alc that regulate APP metabolism, including A production in neurons, and that this regulatory mechanism may be perturbed in AD.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
cDNA Cloning of Human Alc1 and Plasmid Construction of Alc Family cDNAs—The yeast two-hybrid system used in this study has been described previously (14). Briefly, the human brain MATCHMAKER cDNA library (Clontech) was screened with bait composed of cDNA encoding part of the human X11L (hX11L) protein (amino acids 129-555). This yielded a cDNA clone (X11L-binding protein clone number 31 (XB31)) that contained a partial open reading frame of Alc1. Using this clone, a clone encoding the full-length Alc1 protein was isolated from a human brain gt11 cDNA library (Clontech). This cDNA was designated as human Alc1 (GenBank^TM accession number AF438482 [GenBank] , registered as XB31). The full-length hAlc1 open reading frame was subcloned into the HindIII and XbaI sites of pcDNA3 (Invitrogen). We identified two similar cDNA clones in the HUGE (Human Unidentified Gene-Encoded Large Proteins data base analyzed by the KAZUSA cDNA Project) Protein Database (21). The HUGE cDNA clones were denoted as human Alc2 (accession number KIAA0911) and Alc (accession number KIAA0726). We also found human Alc (accession number NM022131) in the GenBank^TM/EBI Data Bank. Alc cDNA, a generous gift from Dr. Nagase (KAZUSA DNA Research Institute, Chiba, Japan), was recloned into pcDNA3 at HindIII and XbaI sites to produce pcDNA3-hAlc. The human Alc1 and Alc cDNAs were also recloned via an XbaI site into pcDNA3 with a FLAG tag on the amino-terminal end of the insert so that pcDNA3-FLAG-hAlc1 and pcDNA3-FLAG-hAlc could be produced.

Protein Interaction Assays in Yeast—The MATCHMAKER two-hybrid system (Clontech) with pGBT9 and pGAD424 was used as described previously (22). -Galactosidase activity in the yeast two-hybrid system was measured in a liquid assay using o-nitrophenyl galactopyranoside and was expressed in Miller units.

Antibodies—The anti-Alc polyclonal rabbit antibody UT83 was raised against a peptide composed of Cys plus the sequence between positions 954 and 971 of human Alc1. The anti-Alc polyclonal rabbit antibody BS7 was raised against a peptide composed of Cys plus the sequence between positions 954 and 968 of human Alc. The anti-hX11L polyclonal antibodies, UT29 and UT30, were obtained as described previously (14), and the anti-X11L monoclonal antibody mint2 was purchased from BD Biosciences. Anti-FLAG (M2, Sigma) and anti-HA (12CA5, Roche Diagnostics) monoclonal antibodies were purchased. The anti-APP cytoplasmic domain polyclonal antiserum, G369, has been described previously (23). The anti-APP extracellular domain monoclonal (22C11, Roche Diagnostics, and LN27, Zymed Laboratories Inc.) and polyclonal (Sigma) antibodies were purchased. The 4G8 monoclonal anti-A antibody raised against the A-(17-24) peptide was purchased from Signet Laboratories (Dedham, MA). All polyclonal antibodies except G369 were affinity-purified before use. Monoclonal antibodies specific for protein-disulfide isomerase (1D3, Stressgen Biotechnologies, Victoria, British Columbia, Canada), 130-kDa Golgi matrix protein (GM-130) (clone no. 35, BD Biosciences), synaptotagmin (SYT) (clone no. 41, BD Biosciences), mouse kinesin heavy chain (KHC) (H2, Chemicon International; Temecula, CA), PS1 carboxyl-terminal fragment (PS1-CTF; Chemicon International), and PS1 amino-terminal fragment (PS1-NTF, Chemicon International) were purchased.

Coimmunoprecipitation and Western Blot Assays—COS7 and human embryonic kidney 293 (HEK293) cells (1 x 10⁷ cells) were transfected as described previously (14) with the indicated amounts of various combinations of the pcDNA3-hX11L, pcDNA3-hAlc1, pcDNA3-hAlc, pcDNA3APP695, and pcDNA3-PS1 plasmids. In another experiment, pcDNA3.1-HA-hX11, in which human X11 cDNA with an attached HA tag at the 5'-end was inserted into the NheI and EcoRV sites of pcDNA3.1, was transfected instead of pcDNA3-hX11L. pcDNA3-FLAG-hX11L and pcDNA3-FLAG-hX11, in which human X11L and X11 cDNA with an attached FLAG tag at the 5'-end, were also used in coimmunoprecipitation assay. Cells were harvested and lysed for 1 h on ice in CHAPS buffer, which consists of phosphate-buffered saline (PBS; 140 mM NaCl and 10 mM sodium phosphate (pH 7.4)) containing 10 mM CHAPS, 5 µg/ml chymostatin, 5 µg/ml leupeptin, 5 µg/ml pepstatin A, 1 mM Na₃VO₄, and 1 mM NaF. After centrifugation at 12,000 x g for 10 min at 4 °C, affinity-purified antibodies (indicated IgG amounts) or serum was added to the CHAPS lysate supernatant. The immunoprecipitates were then subjected to Western blot analysis using specific antibodies.

Subcellular Fractionation of Mouse Brain Proteins—The subcellular fractionation of mouse brains was performed as described previously (24, 25). Briefly, five brains of 8-week-old male C57BL/6 mice were homogenized on ice in 5 volumes of buffer (10 mM HEPES (pH 7.4), 0.32 M sucrose, 5 µg/ml chymostatin, 5 µg/ml leupeptin, and 5 µg/ml pepstatin) by 10 strokes of a loose-fitting (0.12-µm clearance) Teflon homogenizer. The postnuclei supernatants were further centrifuged at 100,000 x g for 1 h. The resulting precipitate (membrane fraction) was resuspended and separated by centrifugation for 115 min at 41,000 rpm in a Beckman SW41 rotor at 4 °C on a 0-28% (w/v) iodixanol density gradient. The supernatants were then fractionated into 13 tubes (0.8 ml/tube), and the fractions (20 µl) were analyzed by Western blot analysis with the indicated antibodies. Fraction 8 was solubilized by adding an equal amount of 2x CHAPS buffer consisting of 2x PBS (280 mM NaCl and 20 mM sodium phosphate (pH 7.4)) plus 20 mM CHAPS. The mixture was allowed to rotate for 30 min at 4 °C and then centrifuged at 100,000 x g for 60 min. The resulting supernatant was used in the coimmunoprecipitation analysis of endogenous proteins.

Pulse-Chase Study—Pulse-chase labeling of cells was performed with L-[³⁵S] in vitro Cell Labeling Mix (0.4 mCi/ml; AGQ 0080, Amersham Biosciences). HEK293 cells were transfected with the indicated combinations of the pcDNA3APP695, pcDNA3-hX11L, pcDNA3-FLAG-hAlc1, and pcDNA3 vector plasmids (transfection was performed with 9 µg of plasmids in total). After a 48-h transfection, the cells were metabolically labeled for 15 min. This was followed by a 0-8-h chase period, which was initiated by replacing the labeling medium containing excess amounts of unlabeled methionine. The cells were then lysed and subjected to immunoprecipitation with G369 (anti-APP cytoplasmic domain antibody) and protein G-Sepharose. The recovered APP was separated by SDS-PAGE (6% (w/v) polyacrylamide) and analyzed by Fuji BAS 2000 imaging analyzer.

Quantification of -Amyloid—C99 protein was used as an intracellular -secretase substrate (26). The construct containing Kozak sequence, first methionine, signal peptide sequence of APP, and CTF coding region was cloned into pcDNA3.1(+) at NheI and ApaI sites and designated as pcDNA3.1APP-C99. The C99 protein is expressed as an artificial CTF once its signal sequence is cleaved off by signal peptidase (26).

Neuro-2a cells (2 x 10⁶ cells) were transiently transfected with the indicated combinations of pcDNA3APP695 or pcDNA3.1APP-C99, pcDNA3-hX11L, pcDNA3-FLAG-hAlc1, and pcDNA3 vector plasmids (transfection was performed with 9 µg of plasmids in total). Cells were supplied with fresh growth medium 5 h after the start of transfection, and conditioned medium was collected 48 h after the medium replacement. A40 and A42 were quantified with sandwich ELISA by using three types of A-specific monoclonal antibodies (27). Intracellular A40 and A42 were also extracted (28) and quantified by the sandwich ELISA. Briefly, the cells were lysed by sonication in 40 µl of PBS containing 6 M guanidine chloride and centrifuged at 20,000 x g for 15 min at 4 °C. The resulting supernatant was diluted up to 12-fold by adding PBS and used in the ELISA. The single factor analysis of variance test followed by Tukey multiple comparisons was used to analyze differences among groups of data. Data were presented as means ± S.E.

Immunological Staining of Mouse Brain Sections—Experimental procedures were conducted in compliance with the guidelines of the Animal Studies Committee of Hokkaido University. Adult C57BL/6 mice (6 weeks, male) were perfused at 4 °C for 20 min with 4% (w/v) paraformaldehyde in 0.2 M sodium phosphate buffer, pH 7.5. The brains were excised and postfixed with the same fixative at 4 °C overnight followed by treatment with 30% (w/v) sucrose in PBS for 2-3 days at 4 °C. Brains were embedded into OCT compound (Miles Scientific), and frozen sagittal sections (20 µm) were prepared. The sections were incubated with 0.1% (v/v) Triton X-100 in PBS for 5 min at room temperature followed by treatment with 0.3% (v/v) H₂O₂ in PBS for 5 min at room temperature to quench endogenous peroxidase activity. The sections were blocked with 3% (w/v) bovine serum albumin in PBS for 10 min at room temperature and incubated for 3 h at 4 °C with antibodies in 1% (w/v) bovine serum albumin in PBS. After washing, the sections were further incubated with goat anti-mouse IgG coupled with Alexa Fluor 488 or goat anti-rabbit IgG coupled with Alexa Fluor 568 in 1% (w/v) bovine serum albumin for 1 h at room temperature. Sections were viewed using the confocal laser scanning microscope LSM510 (Carl Zeiss).

Immunological Staining of AD Brains—We used paraffin sections of frontal and temporal cortices from five AD subjects. Tissues were fixed with Kryofix (a mixture of ethanol, polyethylene glycol, and water; Merck) for 1-7 days and embedded in paraffin. Dewaxed serial tissue sections (cut into 4-µm sections) were immunostained with the ABC elite kit (Vector Laboratories, Burlingame, CA). Some sections were pretreated with a microwave antigen retrieval method for 10 min in 10 mM citrate buffer, pH 6.0 (29). Sections were incubated with antibodies specific for Alc (UT83, 0.8 µg/ml), APP (22C11, 0.5 µg/ml), and A (4G8, 1:1000 dilution). The peroxidase activity was visualized with diaminobenzidine-H₂O₂ solution. For control analyses, tissue sections were incubated with anti-Alc antibody UT83 in the presence of the antigen peptide (40 nM) or non-immune rabbit IgG (0.8 µg/ml).

For double immunofluorescence analyses, dewaxed sections were incubated with a mixture of UT83 and 22C11 or of UT83 and 4G8 at the same dilutions as above followed by incubation with a mixture of fluorescein isothiocyanate-tagged goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA; 1:30 dilution) and Cy3-tagged goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, 1:50 dilution). Prior to immunolabeling, the autofluorescence of the lipofuscin granules was blocked with Sudan black B staining (30).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Isolation, Identification, and Characterization of Novel Cadherin-related Membrane Protein Genes—Yeast two-hybrid screening of a human brain cDNA library with cDNA encoding a part of the hX11L protein resulted in the isolation of a novel cDNA denoted XB31 (GenBank^TM accession number AF438482 [GenBank] ). The cDNA encoded a type I transmembrane protein composed of 971 amino acids. In this paper, this protein has been entitled human Alcadein (Alzheimer-related cadherin-like protein) 1 or Alc1 (Fig. 1A). A thorough search of the human cDNA data base and genome data bases revealed three similar genes. These encode 981-, 968-, and 955-amino acid proteins, and we denoted these proteins as Alc2, Alc, and Alc, respectively (Fig. 1A). Alc2 is identical to human Alc1 except for 10 additional amino acids between the Alc1 residues at positions 71 and 72 (Fig. 1A, pink box). This extra sequence in Alc2 is derived from one exon, and both proteins are spliced variants of the XB31 (Alc) gene. The Alc and Alc proteins are both 50% homologous to Alc. They are encoded by different genes and belong to the same gene family to which Alc belongs. Further data base examinations revealed that there are Alc-like genes in D. melanogaster (GenBank^TM accession number AAF59384 [GenBank] ) and C. elegans (GenBank^TM accession number NP495189) as well.

View larger version (33K): [in this window] [in a new window]   FIG. 1. Comparison of structures and cytoplasmic amino acid sequences of the Alc family proteins and protein expression of Alc and X11L. A, schematic representation of structural features of the human Alc proteins. The pink box in the hAlc2 sequence indicates the amino acids derived from an exon of the hAlc gene. Yellow boxes include the predicted cadherin motif, while the black box within the human Alc sequence represents the leucine zipper motif. The green box includes a highly conserved region containing the X11L-binding sequence. The red box indicates an acidic region. The plasma membrane is indicated as a purple ladder-back structure. The underlines indicate the regions recognized by the specific antibodies UT83 and BS7. The numbers indicate amino acid (a.a.) residues. B, amino acid sequences of the cytoplasmic domains of Alc family proteins. The arrow reveals the NP motif that is the X11L-binding site (blue). Gaps produced by the alignment are indicated by a hyphen in the sequence. The numbers indicate amino acid position. C, detection of Alc proteins in the mouse brain by the UT83 Alc antibody. Mouse brain lysates (50 µg of protein) were subjected to SDS-PAGE (6% (w/v) polyacrylamide), transferred to a membrane, and probed with UT83 (0.16 µg/ml IgG) in the presence (+) or absence (-) of the antigen peptide used to raise it (40 nM). UT83 specifically detected two Alc proteins. The numbers refer to the molecular masses (kDa) of the protein standards. D, analysis of the distribution of Alc and X11L in various murine tissues and brain regions by Western blot analysis. Mouse lysates (50 µg of protein) of tissues (upper panel) and brain regions (lower panel) were subjected to SDS-PAGE (6% (w/v) polyacrylamide), transferred to the membrane, and probed with the anti-Alc (UT83) and anti-X11L (UT30) antibodies. Br, brain; Ht, heart; Lu, lung; Li, liver; Kid, kidney; Mus, muscle; OB, olfactory bulb, CC, cerebral cortex; ST, striatum; Hip, hippocampus; Ce, cerebellum; Mid, mid brain; Th, thalamus; Sci, sciatic nerve.

View larger version (37K): [in this window] [in a new window]   FIG. 2. Determination of the X11L-binding site of Alc. Various protein constructs based on the cytoplasmic domain of Alc (shown schematically in the upper panel) were expressed in yeast and examined by the yeast two-hybrid system for their ability to bind X11L1-125. The first 125 amino acids in the amino-terminal domain of X11L were deleted as they generate a nonspecific and constitutive positive signal in this assay system (14). The following Alc deletion and point mutant constructs were tested: wild type (WT), the cytoplasmic domain of Alc (amino acids 871-971 of Alc1); CTF1, amino acids 903-971; CTF2, amino acids 909-971; CTF3, amino acids 871-902; CTF4, amino acids 871-908; NP-AA, Ala-Ala substitution for Asn-Pro in NPMETY sequence; Y-A, Ala substitution for Tyr in NPMETY. The ability to grow in selective medium was examined, and -galactosidase activity was quantified by a liquid assay and calculated in Miller units ± S.D. (lower panel, n = 3). The plasmid alone was used as a control. GAL4AD represents the yeast Gal4 activator domain.

Expression and Distribution of Alc—

Expression of Alc protein in mouse brain tissue was examined by Western blot analysis. The anti-Alc antibody UT83 recognizes two endogenous proteins in mouse brain lysates (Fig. 1C) as well as recombinant human Alc1 but not recombinant Alc (data not shown, please see Supplemental Fig. 2). The antigen peptide to which UT83 was raised competed with the endogenous proteins for binding to UT83 (Fig. 1C, +). Thus, the UT83 antibody specifically recognizes two endogenous Alc proteins in the mouse brain. When UT83 was used to analyze Alc expression in a variety of murine tissues, the highest levels were found in the brain, and small amounts were found in the lung (Fig. 1D, upper panel). Assessment of X11L expression with the UT30 X11L-specific antibody confirmed its brain-specific distribution (Fig. 1D, upper panel). These observations suggest that, like X11L, Alc is abundant in neurons. This colocalization in the brain supports the notion that X11L and Alc interact physiologically. This idea was further supported when we examined Alc and X11L expression in various parts of the brain by dissecting adult mouse brains into various component regions and blotting them with UT83 and UT30. Relatively high levels of Alc were detected in the cerebral cortex, striatum, hippocampus, and thalamus; moderate levels were found in the cerebellum; and low levels were observed in the olfactory bulb, midbrain, and pons (Fig. 1D, lower panel). Expression in the sciatic nerve fiber was not observed. X11L was distributed in a very similar pattern (Fig. 1D, lower panel). Thus, X11L and Alc are both largely expressed in neural tissues apart from peripheral nervous system.

Alc and X11L Interaction—We investigated the interactions between Alc and X11L by coimmunoprecipitation assays from COS7 cells expressing X11L together with Alc1 or Alc followed by Western blot analysis. The immunoprecipitates resulting from incubating the lysates with anti-Alc (UT83) or anti-Alc (BS7) antibodies included X11L as detected by the X11L antibody (UT30) (data not shown, please see Supplemental Fig. 3). Conversely the immunoprecipitates generated by the anti-X11L antibody contained Alc1 and Alc. In contrast, non-immune antibody (control IgG) immunoprecipitates did not contain these proteins. Thus, both the Alc and Alc proteins bind to X11L within cells.

We identified the Alc- and Alc-binding domain of X11L by assessing the in vitro binding of various X11L domain constructs to the putative cytoplasmic domains of Alc and Alc. Cell lysates containing X11L protein constructs were incubated with beads coupled to the cytoplasmic domains of human Alc or Alc fused to GST. GST alone was used as a negative control. The pull-downs were subjected to Western blot analysis with the anti-X11L amino-terminal antibody UT29. Full-length X11L and its construct encoding the X11L amino-terminal domain attached to the PI domain (N + PI) bound the GST-Alc and GST-Alc fusion proteins (data not shown, please see Supplemental Fig. 4). However, the amino-terminal domain alone did not bind either protein. GST alone also did not bind to any of the X11L constructs. Thus, as with APP (14), the cytoplasmic domains of Alc and Alc bind the PI domain of X11L.

We also identified the region in the cytoplasmic domain of Alc (AlcCTF) that is required for the interaction with X11L by yeast two-hybrid analysis. Truncated Alc1CTF constructs lacking the amino-terminal regions (CTF1, 871-902; CTF2, 871-908) or carboxyl-terminal regions (CTF3, 903-971; CTF4, 909-971) were examined for interaction with the N + PI domain (amino acids 129-555) of X11L. -Galactosidase activity was measured in a liquid assay and calculated as Miller units. The CTF1 and CTF4 constructs, which have the conserved ⁹⁰⁴NPMETY⁹⁰⁹ (numbering is based on the hAlc1 isoform) sequence in common, bound X11L, but CTF2 and CTF3, which both lack the NPMETY sequence, did not interact with X11L (Fig. 2). The NPXXXY sequence is a modified version of the NPXY motif of the cytoplasmic domain of APP that binds X11L (14). When we introduced mutations into the first two amino acids of the NPMETY sequence generating AAMETY, denoted as NP-AA, AlcCTF did not interact with X11L. However, modification of NPMETY to NPMETA (Y-A) had no effect on the ability of the protein to bind to X11L (Fig. 2). Thus, it appears that the first two amino acids of the NPMETY motif are essential for the association of Alc with X11L. This is supported by the mutational analyses of the site used by APP to bind X11 as it was shown that the end tyrosine residue of the NPTY motif of APP is not important for binding to X11 (31). Moreover, the first two Asp and Pro amino acids are conserved in all the Alc proteins, whereas the end Tyr residue is not conserved in Alc or the D. melanogaster and C. elegans Alc proteins (Fig. 1B).

Alc, X11s, and APP Form a Tripartite Complex—The cytoplasmic domains of Alc and APP both bind to the PI domain of X11L. We thus investigated whether Alc and APP compete for binding to X11L or act cooperatively. APP695 (the human APP isoform composed of 695 amino acids) and X11L were expressed together in HEK293 cells in the presence or absence of hAlc1 or FLAG-tagged hAlc, and APP was immunoprecipitated from cell lysates with the anti-APP cytoplasmic domain antibody G369. The immunoprecipitates were analyzed by Western blot analysis with anti-APP (G369), anti-X11L (UT29), anti-Alc (UT83), and anti-FLAG (M2) antibodies. As reported previously (14), X11L was coimmunoprecipitated with the anti-APP antibody in the absence of Alc. Surprisingly, in the presence of Alc1 or FLAG-Alc, higher levels of X11L were recovered in the APP immunoprecipitates (Fig. 3A), and the two Alc proteins were found to be bound to X11L·APP complexes (Fig. 3A). However, when APP and FLAG-Alc1 were coexpressed in the absence of X11L, the G369 antibody did not coimmunoprecipitate Alc. The identical result was obtained when 22C11 anti-APP extracellular domain antibody was used (data not shown, please see Supplemental Fig. 5). Moreover M2 antibody did not coimmunoprecipitate APP (Fig. 3B). Notably, in the cells that coexpress X11L and APP, the levels of immature APP (lower band of APP, N-glycosylated form) were increased (Fig. 3, A and B). This effect is related to the fact that X11L stabilizes APP metabolism (see Fig. 5). These findings clearly indicate that APP and Alc form a complex by interacting with X11L and that the binding of APP and X11L is greatly stabilized by Alc.

View larger version (26K): [in this window] [in a new window]   FIG. 3. Alc enhances APP-X11L binding, and the three proteins form a tripartite complex. A, role of Alc in the binding of X11L to APP. HEK293 cells (1 x 107 cells) were transiently cotransfected with pcDNA3APP695 (2 µg) and pcDNA3-hX11L (0.25 µg) with or without pcDNA3-hAlc1 (6.75 µg) or pcDNA3FLAG-hAlc (6.75 µg) as indicated. To standardize the plasmid concentrations, the pcDNA3 vector (-) was added (to yield 9 µg of plasmid in total). The cells were lysed, and APP in the cell lysate was recovered by immunoprecipitation with the anti-APP cytoplasmic domain antibody G369. The immunoprecipitates were then analyzed by Western blotting with anti-APP (G369), anti-X11L (UT29), anti-Alc (UT83), and anti-FLAG (M2) antibodies. B, role of X11L in the formation of the APP·X11L·Alc tripartite complex. HEK293 cells (1 x 107 cells) were transiently cotransfected with pcDNA3APP695 (3 µg) and pcDNA3-FLAG-hAlc1 (3 µg) in the presence or absence of pcDNA3-hX11L (3 µg). To standardize the plasmid amounts, 3 µg of pcDNA3 vector was added in the absence of pcDNA3-hX11L (-). The cell lysates were immunoprecipitated with the anti-APP antibody G369 (upper three panels) or with the anti-FLAG antibody M2 (lower three panels). The immunoprecipitates were analyzed by Western blot analysis with anti-APP (G369), anti-X11L (UT29), and anti-FLAG (M2) antibodies. C, X11 can complex with APP and Alc1. COS7 cells (1 x 107 cells) were cotransfected with pcDNA3APP695 (2 µg), pcDNA3-HA-hX11 (0.25 µg), and pcDNA3-hAlc1 (6.75 µg) in various combinations. To standardize the plasmid concentration, adequate amounts of pcDNA3 vector (-) were added (to yield 9 µg of plasmid in total). The cell lysates were immunoprecipitated with anti-Alc antibody (UT-83, upper panel) or anti-APP antibody (22C11, middle panel). The crude lysate (Lysate, lower panel) and immunoprecipitates (IP) were then analyzed by Western blotting with anti-Alc (UT83), anti-HA (12CA5), and anti-APP (G369) antibodies. D, Alc stabilizes APP binding to X11L and X11. COS7 cells (1 x 107 cells) were cotransfected with pcDNA3APP695 (2 µg), pcDNA3-Alc1 (6.75 µg), and pcDNA3-FLAG-hX11L or pcDNA3-FLAG-hX11 (0.25 µg) in various combinations. To standardize the plasmid concentration, adequate amounts of pcDNA3 vector (-) were added (to yield 9 µg of plasmid in total). The cell lysates were immunoprecipitated with anti-FLAG antibody (M2). The immunoprecipitates were analyzed by Western blotting with anti-FLAG (M2, first panel), anti-APP (G369, second and third panels), and anti-Alc (UT83, fourth panel) antibodies. The third panel shows a longer exposure (exp) of film than that of the second panel.

View larger version (26K): [in this window] [in a new window]   FIG. 5. Alc enhances the X11L-mediated stabilization of APP metabolism and suppression of A and sAPP secretion. A and B, effect of coexpressing Alc1 and X11L on APP metabolism. HEK293 cells (1 x 107 cells) were transiently transfected with pcDNA3APP695 (2 µg) in the presence or absence of pcDNA3-hX11L (0.25 µg) and with or without pcDNA3FLAG-hAlc1 (6.75 µg). To standardize the plasmid amounts, pcDNA3 vector was added (to yield 9 µg of plasmid in total). After a 48-h transfection, the cells were metabolically pulse-labeled with [35S]methionine for 15 min and chased for the indicated times. APP was immunoprecipitated from cell lysates with the anti-APP cytoplasmic domain antibody G369, separated by SDS-PAGE (6% (w/v) polyacrylamide), detected by autoradiography (A), and quantified using a Fuji BAS 2000 analyzer. The relative ratios of the levels of immature APP to the maximum level of immature APP at 0 h (the latter level was assigned a reference value of 1.0) were calculated (B). mAPP, mature APP695; imAPP, immature APP695. C, effect of coexpressing Alc1 and X11L on A secretion. Neuro-2a cells (1 x 107 cells) were transiently transfected with pcDNA3APP695 (3 µg) with or without pcDNA3-hX11L (0.3 µg) and in the presence or absence of pcDNA3-FLAG-hAlc1 (5.7 µg). To standardize the plasmid amounts, pcDNA3 vector was added (to yield 9 µg of plasmid in total). The culture medium was collected and assessed for A40 and A42 levels using a sandwich ELISA. The concentrations of A40 and A42 are presented as means with S.E. (n = 6). The data were analyzed by one-way analysis of variance followed by the Tukey test (**, p < 0.01; ***, p < 0.001). D, effect of coexpressing Alc1 and X11L on sAPP secretion. Neuro-2a cells (1 x 107 cells) were transiently transfected with pcDNA3APP695 (3 µg) with or without pcDNA3-hX11L (0.3 µg) and in the presence or absence of pcDNA3-FLAG-hAlc1 (5.7 µg). To standardize the plasmid amounts, pcDNA3 vector (-) was added (to yield 9 µg of plasmid in total). The culture medium (2 ml) was collected, and sAPP was recovered by immunoprecipitation with anti-APP extracellular domain antibody (22C11) as described previously (4). The immunoprecipitates (Medium) and cell lysates (Cell, 50 µg of protein) were analyzed by Western blotting with 22C11. The levels of sAPP (a relative ratio) were normalized to the amount of intracellular full-length mature and immature APP (APPFL) and indicated relative to the level of lane 1, which was assigned a reference value of 1.0. Results are the average of duplicate assays, and error bars are indicated.

It is usually documented that the PI domain does not interact at the same time with two proteins possessing a similar interaction motif (32). Nevertheless the stoichiometry of X11L bound to APP seems to be increased by addition of Alc (Fig. 3A), but is the APP·X11s·Alc complex stoichiometry 1:1:1 or 1:2 (or more):1? Thus, we performed coimmunoprecipitation of APP and Alc with the M2 antibody from cells expressing APP, Alc1, and FLAG-X11L or FLAG-X11 (Fig. 3D). If the stoichiometry of X11L bound to APP is increased by Alc, the amount of APP recovered by coimmunoprecipitation with anti-FLAG antibody is expected to decrease in the presence of Alc1. However, FLAG-X11L and FLAG-X11 coprecipitated more APP in the presence of Alc1 than in the absence of Alc1. This result suggests that the stoichiometry of X11s bound to APP does not increase by the addition of Alc. The most simple explanation is that the population of X11s bound to APP is increased in cells by the expression of Alc. Alc may increase the chance that APP will interact with X11L or decrease the dissociation constant of the APP·X11L complex. Furthermore our preliminary result derived from studies to determine APP- and Alc-binding sites by introducing amino acid substitution in the PI domain of X11L supports the interaction of APP and Alc with different sites of the single PI domain of X11L.² However, we cannot rule out the possibility that more than one X11L molecule is bound to APP because both X11s could form homo- and/or heterodimers when the respective proteins were overexpressed in cells.³

We examined whether APP, Alc, and X11L form similar tripartite complexes in the brain. When we separated the post-nuclear fraction from the brains of five mice into cytoplasmic and membrane fractions and subjected both fractions to immunoprecipitation assays, we found that APP and Alc (which are both membrane proteins) were not recovered in the cytoplasmic fraction (data not shown). However, the majority of X11L was recovered in the cytoplasmic fraction, although a moderate amount of X11L was also recovered in the membrane fraction (data not shown). To examine formation of these proteins into tripartite complexes in the brain, the membrane fraction was further fractionated by iodixanol density gradient centrifugation, and these fractions were subjected to Western blot analysis with antibodies specific for APP, X11L, Alc, the endoplasmic reticulum protein protein-disulfide isomerase, the Golgi-resident protein GM-130, the synaptic vesicle protein SYT, the vesicle transport motor protein KHC, and PS1. The vesicles bearing APP, APPCTF, X11L, and Alc all sedimented in the same medium density membranous protein fractions 7-9, which were a bit heavier than the SYT-containing vesicles in fractions 9 and 10 (Fig. 4A). APP, KHC, and PS1 were observed in the same fractions, confirming earlier observations (33, 34). These data suggest that APP, X11L, and Alc could form tripartite complexes on organelles composed of medium density membranes. To test this, the proteins in fraction 8 were solubilized with CHAPS buffer, immunoprecipitated with anti-APP (G369) or non-immune control antibodies, and subjected to Western blot analysis with antibodies specific for APP, X11L, Alc, SYT, and rabbit IgG antibodies. Alc and X11L were coimmunoprecipitated by anti-APP but not by non-immune antibody (Fig. 4B). Also SYT was not coimmunoprecipitated by anti-APP antibody. Thus, APP and/or APPCTF, Alc, and X11L form a tripartite complex in vivo, probably on medium density membrane organelles that contain cargo proteins including PS and that differ from the synaptic vesicles that contain SYT.

View larger version (68K): [in this window] [in a new window]   FIG. 4. APP, X11L, and Alc form a tripartite complex in the neurons of mouse brains. A, colocalization of APP, X11L, and Alc in the same brain membrane fractions. The membrane fraction of five mouse brains was subjected to further fractionation by iodixanol density gradient centrifugation. The density is indicated (upper panel). The fractions were analyzed by Western blotting with antibodies to APP and APPCTF (G369), X11L (mint2), Alc (UT83), protein-disulfide isomerase (PDI)(ID3), GM-130 (clone no. 35), SYT (clone no. 41), KHC (H2), carboxyl-terminal half of PS1 (PS1-CTF) (lower panel). B, presence of APP·X11L·Alc tripartite complexes in the brain. Fraction 8 in A was lysed, and APP was recovered by immunoprecipitation with anti-APP (G369) antibody. As a control, non-immune antibody was used instead of G369. Crude lysate (Lysate) and the immunoprecipitates (IP) were subjected to Western blot analysis with antibodies specific for APP and APPCTF (G369), X11L (mint2), Alc (UT83), SYT (clone no. 41), and rabbit IgG antibodies. (H) indicates heavy chain of IgG. C, APP, Alc, and X11L colocalize in mouse neurons. Sagittal sections of an adult mouse brain were double stained with antibodies against APP (22C11, red) and X11L (mint2, green)(panels 1-3), Alc (UT83, red) and X11L (mint2, green) (panels 4-6), or APP (LN27, red) and Alc (UT83, green) (panels 7-9). The merging of the signals (yellow) indicates the colocalization of the two proteins being assessed (panels 3, 6, and 9). CA3 region of the hippocampus is shown as are high power views of the cell bodies of the large pyramidal neurons of the cerebral cortex. The scale bar indicates 10 µm.

Alc Enhances the X11L-mediated Stabilization of APP Metabolism and Suppression of A and sAPP Secretion—X11 stabilizes intracellular APP metabolism (35, 36), and we previously reported that X11L suppresses the secretion of A40 (14), indicating that X11L also stabilizes APP metabolism. Since Alc and APP form a complex with X11L through cytoplasmic interactions, and this complex formation strengthens the interaction between APP and X11L (Fig. 3), we speculated that Alc may enhance the X11L-mediated stabilization of APP metabolism and thereby further suppress A and sAPP production. To test this, we assessed the intracellular APP metabolism in HEK293 cells that express APP with or without X11L in the presence or absence of Alc1 by pulse-chase assay (Fig. 5). The metabolically radiolabeled APP was recovered by immunoprecipitation at the indicated chase periods and separated by SDS-PAGE. The levels of immature APP were quantified by autoradiography and calculated with respect to the 0 h levels (1.0) (Fig. 5B). When APP was expressed alone, the immature APP levels decreased gradually with time due to the maturation of APP and the secretion of large extracellular domain truncated at - or -site (sAPP) during the chase period. As expected, X11L coexpression slightly delayed this decrease in immature APP levels, indicating that X11L stabilizes APP metabolism. When Alc1 was also coexpressed, the effect of X11L was greatly enhanced. However, the increased stabilization of APP metabolism due to Alc1 was not observed if X11L was not coexpressed.

We next investigated the production of A and sAPP in Neuro-2a cells expressing APP with or without X11L in the presence or absence of Alc1. The amount of A in medium secreted from the cells was quantified using sandwich ELISA. As we previously demonstrated (14), coexpression of X11L suppresses the secretion of A40 (Fig. 5C). X11L did tend to suppress A42 secretion, but it was not significant statistically. When X11L and Alc1 were coexpressed, the effect of X11L was remarkably enhanced. However, enhanced suppression of A40 secretion was not observed if only Alc1 was expressed. Alc1 and X11L coexpression did not significantly effect A42 secretion. Thus, Alc clearly enhances the X11L-mediated inhibition of A, at least A40, secretion from APP, which correlates with the finding that Alc stabilizes the interaction between X11L and APP (Fig. 3A). The amount of sAPP in the medium secreted from the cells was detected by Western blot analysis with anti-APP extracellular domain antibody 22C11 (Fig. 5D). X11L suppressed sAPP secretion, and Alc1 enhanced this effect. It is likely that the decreased A40 and sAPP secretion by cells expressing APP, X11L, and Alc is due to the slow-down of intracellular APP maturation caused by their tripartite complex formation. Because we could not quantify the A levels in the cell lysates, we performed another study with cells expressing C99 protein (see Fig. 6).

View larger version (33K): [in this window] [in a new window]   FIG. 6. The Alc·X11L·C99 tripartite complex inhibits A generation from C99 and blocks the association between C99 and PS1. A, effect of X11L and Alc on C99 cleavage. Neuro-2a cells (1 x 107 cells) were transiently transfected with pcDNA3-APPC99 (3 µg) in the presence or absence of pcDNA3-hX11L (0.3 µg) and with and without pcDNA3-hAlc1 (5.7 µg). To standardize the plasmid concentrations, pcDNA3 vector (-) was added (to yield 9 µg of plasmid in total). The cells were lysed, and C99 was analyzed by Western blotting with anti-APP (G369) antibody. The lower panel indicates a shorter exposure (exp) of the film. B and C, effect of X11L and Alc on the generation of A from C99. A40 (left panel) and A42 (right panel) in the medium (B) and the lysate (C) of the cells in A were quantified by a sandwich ELISA. The A40 and A42 concentrations are presented as means with S.E. (n = 6). The data were analyzed by one-way analysis of variance followed by the Tukey test (*, p < 0.05; **, p < 0.01; ***, p < 0.001). D, effect of X11L and Alc1 on the interaction between C99 and PS1. HEK293 cells (1 x 107 cells) were transiently transfected with pcDNA3-APPC99 (2 µg) and pcDNA3-PS1 (1 µg) in the presence or absence of pcDNA3-hX11L (2 µg) and with or without pcDNA3-hAlc1 (4 µg). To standardize the plasmid amounts, pcDNA3 vector (-) was added (to yield 9 µg of plasmid in total). The cells were cultured for 24 h in the presence (+) or absence (-) of N-acetyl-leucyl-norleucinal (LLnL) (10 µM) and then lysed. C99 in the cell lysate was recovered by immunoprecipitation with the G369 anti-APP antibody. The immunoprecipitates were analyzed by Western blotting with antibodies specific for APP (G369), the full-length PS1 (PS1-NTF), X11L (mint2), and Alc (UT83). E, schematic diagram showing how the tripartite complex composed of CTF, X11s (X11L and X11), and Alc can block the -cleavage of CTF by PS. EC1 and EC2 in Alcadein indicate cadherin motifs 1 and 2, respectively. Although the stoichiometry in the complex APP·X11L/X11·Alc is drawn as 1:1:1 for the sake of convenience, further analysis is needed to reveal the substance of the complex.

X11L and Alc Form a Tripartite Complex with C99 and Suppress the -Cleavage of C99 —

When the A in the medium from the transfected Neuro-2a cells described above was quantified, it was found that X11L coexpression with C99 suppresses the secretion of both A40 and A42 into medium (Fig. 6B). Moreover, when Alc1 was also coexpressed, this effect was remarkably enhanced (Fig. 6B). We could not quantify the level of intracellular A derived from full-length APP. Thus, we quantified the A in cells expressing C99. When the intracellular levels of A in the transfected Neuro-2a cells were examined, the same effects of X11L and Alc1 expression were observed for intracellular A40 generation (Fig. 6C). However, expression of X11L and Alc1 did not have a significant effect on the intracellular A42 levels, although the expression of both X11L and Alc1 did tend to suppress the amount of intracellular A42 (Fig. 6C). Thus, we concluded that X11L could suppress the -cleavage of CTF, and Alc enhances this effect.

PS is essential for -secretase activity (37). We investigated whether X11L inhibits the interaction of CTF with PS1 and whether Alc enhances this inhibitory activity. HEK293 cells were cotransfected with C99 and PS1 in the presence or absence of X11L and with or without Alc1. These cells were subjected to immunoprecipitation assays with the anti-APP antibody G369, which recognizes C99, followed by Western blotting to determine the interaction of C99 with PS1. A previous report showed that the interaction between CTF and PS is detectable in the presence of an aspartic protease inhibitor N-acetyl-leucyl-norleucinal (38), and thus we cultured the transfected cells in the presence or absence of N-acetyl-leucylnorleucinal. We observed that, in the presence of N-acetylleucyl-norleucinal, the anti-APP antibody coprecipitated PS1, confirming that C99 can interact with PS1 as expected (Fig. 6D). In the presence of X11L, the anti-APP antibody recovered a smaller amount of PS1, indicating that X11L suppresses the interaction between C99 and PS1. Surprisingly coexpression of Alc1 abolished the interaction between C99 and PS1. Thus, it appears that X11L blocks the association of CTF with PS1 and inhibits the -cleavage of CTF, thereby suppressing A generation. Furthermore Alc enhances this effect of X11L by forming a stable complex comprised of CTF, X11L, and Alc. The mechanism by which CTF processing is regulated through its interactions with X11L and Alc is depicted schematically in Fig. 6E.

Localization of Alc in the AD Brain—To investigate the localization of Alc in AD patient brains, we immunolabeled serial paraffin-embedded tissue sections with antibody specific for Alc. In ethanol (Kryofix)-fixed sections (Fig. 7, panel 1) that had not been subjected to the antigen retrieval method, the anti-Alc antibody consistently detected intense Alc immunolabeling of dystrophic neurites in the neuritic plaques together with faint neuronal labeling. The APP labeling pattern was similar (Fig. 7, panel 2). Labeling was not observed in sections stained with antigen-preabsorbed UT83 (data not shown) or non-immune rabbit IgG used at the same concentration as UT83 (Fig. 7, panel 3). To confirm the colocalization of Alc and APP, paraffin-embedded tissue sections from AD brains were double labeled for Alc and APP (Fig. 7, panels 4-6). In a high power view of a neuritic plaque, most of the Alc immunofluorescence (panel 4) colocalized with that of APP (panel 5). The merged view is shown in panel 6. Furthermore double labeling of Alc and A revealed Alc-positive neurites around the amyloid core of plaques (Fig. 7, panel 7). Unfortunately antibody specific for X11L (mint2) did not work on the paraffin-embedded tissue sections (data not shown). These observations suggest that in AD, Alc and APP accumulate in dystrophic neurites around the amyloid core of plaques.

View larger version (98K): [in this window] [in a new window]   FIG. 7. Localization of Alc, APP, and A in AD brain. Panels 1-3, staining with single antibodies. Alc (panel 1, UT83) localized in the dystrophic neurites of disseminated senile plaques in a Kryofix-paraffin section of an AD brain. An adjacent section shows a similar pattern of APP labeling (panel 2, 22C11). This labeling was not observed in a control AD section stained with non-immune rabbit IgG (panel 3). Panels 4-6, double immunofluorescence staining for Alc (panel 4, UT83, green) and APP (panel 5, 22C11, red) demonstrated the similar localization of the two proteins in a neuritic plaque of an AD brain. A merged image is shown in panel 6 (yellow). Panel 7, double immunofluorescence staining for Alc (UT83, green) and A (4G8, red) demonstrated accumulation of Alc-positive dystrophic neurites around the plaque core. Scale bar, 20 µm (white) and 100 µm (black).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The first member of the Alc family we isolated was human Alc1. On the basis of its sequence, we also identified the cDNAs for human Alc2, Alc, and Alc in the cDNA and genome data bases. Homology searching also revealed Alc-like genes in D. melanogaster and C. elegans. These proteins share two cadherin motifs and a putative Ca²⁺-binding site in the amino-terminal ectodomain, a laminin G domain in the middle ectodomain, and an X11L-binding site and an acidic region in the cytoplasmic domain. It is likely that these proteins belong to the same family and play identical roles in neural function. However, the roles played by these putative Alc domains (apart from the X11L-binding site) in the metabolism and/or function of APP require further investigation. Supporting the notion that the Alc proteins participate in neural function(s) is that a chicken protein that is homologous to Alc has been reported recently to be a postsynaptic membrane protein that may play a role in postsynaptic Ca²⁺ signaling (47). It is possible that Alc may transmit unidentified extracellular information through an as yet unknown mechanism or that it may serve as a receptor, together with APP, of cargo proteins in membrane transport vesicles (39). Supporting the first possibility, we demonstrated in the present study that Alc couples with APP through cytoplasmic interactions bridged by X11L or X11. APPcyt is thought to transmit some extracellular signals into nucleus by the mechanism of regulated intracellular proteolysis by coupling with adaptor proteins such as FE65 (48). Alc and X11s may moderate this signaling process by regulating APP processing by -secretase complex including PS. Supporting the possible cargo protein receptor function of Alc is our demonstration that APP, X11L, and Alc were recovered together with KHC in identical subcellular fractions. However, there is no direct evidence that APP and Alc operate as receptors of cargo proteins in membrane transport vesicles (39).

We found that the coupling of APP with Alc through X11L significantly stabilized APP metabolism and enhanced the suppression of A production. This effect was due to the suppression of APP maturation, resulting in the suppression of the first cleavage of APP at the - and -sites. Normally the majority of APP is subjected to non-amyloidogenic processing by - and -secretases that does not generate A (2), although a small proportion of APP is processed into A by an intracellular amyloidogenic pathway in which the protein is cleaved by - and -secretases. We found that X11L could also associate with the carboxyl-terminal fragments of APP that are metabolic products of APP cleavage at the -or -site. Thus, we examined whether X11L and Alc could also suppress the -cleavage of C99/CTF. We found that the X11L-mediated association of Alc with C99 enhanced the suppressive effects of X11L on A generation from C99. Moreover, since a recent report indicates that PS is an essential component of -secretase (35), we examined whether the interaction between C99/CTF and PS1 is inhibited by formation of the C99·X11L·Alc complex. We found that X11L blocked PS from interacting with C99/CTF and that Alc enhanced this effect. The stable tripartite complex formed by C99/CTF, X11L, and Alc may block the access of the -secretase complex to CTF. These observations suggest that the development of drugs that up-regulate X11L and Alc function in AD patients and thereby down-regulate CTF cleavage may be useful in the treatment of AD.

Both APP and Alc recognized the PI domain of X11L, but the binding of these two proteins to PI was cooperative, not competitive. The association of Alc with X11L may induce some conformational change of the PI domain of X11L and result in the stable inter