Densin-180 Interacts with δ-Catenin/Neural Plakophilin-related Armadillo Repeat Protein at Synapses*

Densin-180, a protein purified from the postsynaptic density fraction of the rat forebrain, is the founding member of a newly described family of proteins termed the LAP (leucine-rich repeats and PSD-95/Dlg-A/ZO-1 (PDZ) domains) family that plays essential roles in establishment of cell polarity. To identify Densin-180-binding proteins, we screened a yeast two-hybrid library using the carboxyl-terminal fragment of Densin-180 containing PDZ domain as bait, and we isolated δ-catenin/neural plakophilin-related armadillo repeat protein (NPRAP) as a Densin-180-interacting protein. δ-catenin/NPRAP, a member of the armadillo repeat family, is a nervous system-specific adherens junction protein originally discovered as an interactor with presenilin-1, a protein involved in Alzheimer's disease. Densin-180 PDZ domain binds the COOH terminus of δ-catenin/NPRAP containing the PDZ domain-binding sequence. Endogenous Densin-180 was co-immunoprecipitated with δ-catenin/NPRAP and N-cadherin. Although Densin-180 was reported to be a transmembrane protein, Densin-180 was not accessible to surface biotinylation in dissociated hippocampal neurons; hence Densin-180 may be a cytosolic protein. Densin-180 co-localized with δ-catenin/NPRAP at synapses in dissociated hippocampal neurons. We propose that Densin-180 is associated in vivo with δ-catenin/NPRAP and may be involved in organization of the synaptic cell-cell junction through interaction with the δ-catenin/NPRAP-N-cadherin complex.

Neurotransmission takes place at synapses that are highly specialized sites of cell-cell contact between neurons (1). When viewed electron-microscopically, excitatory synapses have an electron-dense thickening of postsynaptic membrane termed postsynaptic density (PSD) 1 (2,3). PSD has specific receptors for glutamate as well as numerous receptor-associated proteins; hence PSD can be regarded as a proteinaceus organelle specialized for postsynaptic signal transduction (4).
Densin-180 was identified as a transmembrane protein tightly associated with PSD in central nervous system neurons and was postulated to function as a synaptic adhesion molecule (5,6). The sequence of Densin-180 contains 16 leucine-rich repeats, a putative transmembrane domain, and a PSD-95/Dlg-A/ZO-1 (PDZ) domain. Four splice variants (A-D) of the putative cytosolic tail of Densin-180 are differentially expressed during development of the rat brain (7). Densin-180 is phosphorylated by Ca 2ϩ /calmodulin-dependent kinase II (CaMKII) (5) and binds CaMKII (6,7), thereby suggesting a possible role for Densin-180 in the localization of CaMKII at PSD (6,7). It was also reported that Densin-180 forms a ternary complex with the ␣-subunit of CaMKII and ␣-actinin (6). Densin-180 belongs to a recently identified family of PDZ domain-containing proteins. These proteins include Scribble, a Drosophila protein essential for epithelial integrity (8) with tumor suppressive function (9); ERBIN, an ErbB2-interacting protein (10); LET-413, an ERBIN ortholog in Caenorhabditis elegans (11). In addition to the PDZ domain in the COOH termini, members of this family have 16 leucine-rich repeats in the NH 2 termini and have thus been named LAP (for leucine-rich repeats and PDZ) proteins (10).
To identify proteins interacting with Densin-180, we screened a human brain cDNA library using the yeast twohybrid procedure and the putative intracellular domain of Densin-180 as bait. We isolated ␦-catenin/neural plakophilin-related armadillo repeat protein (NPRAP) (12,13) as a potential binding partner for Densin-180. ␦-catenin was identified based on its ability to bind to the loop region of presenilin-1, which is encoded by the gene most commonly mutated in familial Alzheimer's disease (12). Independently, the same gene was identified as a protein homologous to plakophilin 1 and was termed NPRAP (13). ␦-catenin/NPRAP is a member of the p120 catenin subfamily, defined as proteins with 10 armadillo repeats (in contrast to the 13 armadillo repeats of ␤-catenin) in characteristic spacing and often with quite diverse NH 2 -and COOHterminal sequences that flank the repeats (14). ␦-catenin is reported to co-localize and interact with N-cadherin in the mouse brain (15) and to undergo dynamic relocalization during brain development (16). It was recently reported that ␦-catenin/ NPRAP binds to PDZ domains of the synaptic scaffolding molecule (S-SCAM) (17) and PAPIN (18). We have now obtained evidence for the in vivo association of Densin-180 with ␦-catenin/NPRAP. We propose that Densin-180 may possibly be involved in the organization of synaptic cell-cell adhesion.

EXPERIMENTAL PROCEDURES
Cloning of Full-length Human Densin-180 -We carried out BLAST searches to identify cDNAs encoding human Densin-180 using the rat Densin-180 cDNA sequence (GenBank accession number U66707). KIAA1365 cDNA encoding COOH-terminal sequences of human Densin-180 cDNA was identified (kindly provided by Drs. T. Nagase and N. Kusuhara from Kazusa DNA Research Institute, Chiba, Japan). We also found a genomic DNA clone (EMBL accession number AL359412) harboring DNA sequences that are highly homologous to rat Densin-180 cDNA and thought to be a human Densin-180. An NH 2 -terminal half of cDNA of human Densin-180 was amplified by polymerase chain reaction using a human brain cDNA library (CLONTECH, Palo Alto, CA) and PyroBest polymerase (Takara, Tokyo, Japan) with a set of primers designed according to the genomic sequence. A human cDNA insert of splice variant D reported in the putative intracellular domain of rat Densin-180 (7) was also cloned using polymerase chain reaction. These DNAs were sequenced using an ABI 310 genetic analyzer (Applied Biosystems, Foster City, CA).
Yeast Two-hybrid Screening-cDNA encoding the putative intracellular domain of human Densin-180 (amino acid residues 1242-1537) (Densin-C) was cloned into the yeast GAL4 DNA-binding domain vector (pGBD-C1), which was kindly provided by P. James (University of Wisconsin, Madison, WI). The resulting plasmid, pGBD-Densin-C was used in the two-hybrid screen of a human brain cDNA library fused to the pACT2 vector (CLONTECH), following the Matchmaker two-hybrid system protocol (CLONTECH). Positive clones were screened for the potential to grow on selective medium and for the expression of ␤-galactosidase. Subsequent two-hybrid interaction analyses were carried out by co-transformation of plasmids, containing the GAL4 DNA-binding (pGBD-C1) and -activation (pGAD-C1) domains into Saccharomyces cerevisiae strain Y190, as described (19). Full-length Densin-180 and the putative intracellular domain lacking the PDZ domain (amino acid residues 1242-1442) of Densin-180 were constructed into the pGBD-C1 and pGAD-C1 vectors by means of enzyme digestion and use of polymerase chain reaction techniques. The cDNAs encoding amino acid residues 1014 -1225 (C-SWV (wild-type)) and 1014 -1222 (C-⌬SWV) of human ␦-catenin/NPRAP were inserted into the yeast prey vector (pGAD-C1). The C-SWA fragment, which contains a mutation of the COOHterminal valine to alanine in C-SWV, was also cloned into the pGAD-C1 vector. A cDNA fragment of the intracellular domain of human Ncadherin was constructed into pGBD-C1 and pGAD-C1 vectors.
Generation of Anti-Densin-180 Antibody and Immunoblotting-Anti-Densin-Ext antibody was produced in rabbits injected with recombinant His 6 -Densin-Ext protein and affinity-purified with recombinant GST-Densin-Ext fusion protein. Lysates prepared from rat brain homogenate were separated by SDS-PAGE and transferred onto a polyvinylidine difluoride membrane (Atto, Tokyo, Japan). The blots were incubated with anti-Densin-Ext antibody and horseradish-peroxidaseconjugated second antibody, and immunoreactive bands were visualized using chemiluminescence detection reagents (Renaissance; PerkinElmer Life Sciences). To detect the Myc-tag, the membranes were incubated with anti-Myc monoclonal antibody (9E10).
In Vitro Translation, Pull-down Assays, and Immunoprecipitation-The cDNAs encoding amino acid residues 1014 -1225 (C-SWV (wildtype)) and 1014 -1222 (C-⌬SWV) of human ␦-catenin/NPRAP were inserted into the pCMV-Tag3B (Myc) expression vector (Stratagene, La Jolla, CA). The C-SWA fragment, which contains a mutation of the COOH-terminal valine to alanine in C-SWV, was also cloned into the pCMV-Tag3B plasmid. These pCMV-Tag3B vectors harboring various constructs of ␦-catenin/NPRAP COOH termini were used for coupled in vitro transcription/translation in rabbit reticulocyte lysates using the TNT kit (Promega, Madison, WI). Labeled ␦-catenin/NPRAP C-SWV, C-SWA, and C-⌬SWV polypeptides in lysates were detected using Transcend non-radioactive translation detection systems (Promega). Crude synaptosomes were prepared as described by Hirao et al. (22). In brief, one adult rat brain was homogenized in 8 ml of 0.32 M sucrose containing 4 mM Hepes/NaOH at pH 7.4 and centrifuged at 800 ϫ g for 10 min at 4°C. The supernatant (S1) was centrifuged at 9,200 ϫ g for 15 min at 4°C to collect the pellet. The pellet was resuspended in 8 ml of 0.32 M sucrose containing 4 mM Hepes/NaOH at pH 7.4 and centrifuged 10,200 ϫ g for 15 min at 4°C. The pellet (P2; the crude synaptosomal fraction) was resuspended with 8 ml of a buffer containing 20 mM Hepes/NaOH (pH 8.0), 100 mM NaCl, 5 mM EDTA, and 1% (w/v) Triton X-100 and centrifuged at 100,000 ϫ g for 30 min at 4°C. The supernatant was used in the pull-down assay and immunoprecipitation.
Equal amounts of GST or GST-Densin-C fusion protein beads (ϳ50 g of protein) were incubated with in vitro-translated products, lysates from COS7 cells overexpressing Myc-␦-catenin or 1% Triton X-100 soluble lysates of crude synaptosomal fractions, for 4 h at 4°C on a rotating platform. After centrifugation, beads were washed four times with wash buffer (20 mM Tris-HCl, pH 7.5, 1% Triton X-100, and 150 mM NaCl). Bound proteins were eluted with SDS sample buffer and subjected to SDS-PAGE and immunoblotting. 1% Triton X-100 soluble lysates of crude synaptosomal fractions were incubated overnight at 4°C with anti-Densin-Ext polyclonal antibody, anti-␦-catenin monoclonal antibody (BD Transduction Laboratory, Lexington, KY), anti-N-cadherin monoclonal antibody (BD Transduction Laboratory), control rabbit IgG, or control mouse IgG, and immunocomplexes were immobilized on protein G-agarose beads (Sigma). Immunoprecipitates were analyzed by immunoblotting with anti-Densin-Ext polyclonal antibody, anti-␦-catenin monoclonal antibody (BD Transduction Laboratory), or anti-N-cadherin monoclonal antibody (BD Transduction Laboratory).
Labeling of Surface Proteins-To label surface proteins, rat hippocampal neurons were cultured on a 10-cm dish for 4 days, washed with cold phosphate-buffered saline containing 1 mM MgCl 2 and 0.1 mM CaCl 2 , and incubated with 0.5 mg/ml sulfo-NHS-LC-biotin (Pierce) in the same buffer at room temperature for 30 min. The labeling reaction was quenched by incubation with 100 mM glycine for 10 min at room temperature. Cells were then lysed in a buffer containing 1% Triton X-100 and 1% deoxycholate. Lysates were clarified by centrifugation at 15 rpm for 30 min, and the supernatants were incubated with streptavidin-agarose beads overnight at 4°C. Bound proteins were subjected to SDS-PAGE and immunoblotting.
Immunofluorescence-Rat hippocampal neurons dissociated at E18 were grown in culture on coverslips for 14 days and fixed by incubation 4% paraformaldehyde in phosphate-buffered saline for 10 min, followed by treatment with Ϫ20°C methanol for 10 min. For double staining of Densin-180 and either ␦-catenin or N-cadherin, cells were first incubated with anti-Densin-Ext polyclonal antibody followed by Alexa 488labeled anti-rabbit antibody (Molecular Probes, Eugene, OR). Next the cells were incubated with anti-␦-catenin antibody (BD Transduction Laboratory) or anti-N-cadherin antibody (BD Transduction Laboratory) followed by FluoroLink Cy3-linked anti-mouse antibody (Amersham Biosciences). An Olympus LSM-GB200 microscope was used to examine the coverslips.

RESULTS
Identification of ␦-catenin/NPRAP As a Densin-180-interacting Protein-We first obtained the full-length cDNA for human Densin-180 (AF434715), as described under "Experimental Procedures." To identify proteins that bind Densin-180 we screened a human brain cDNA library, using the yeast twohybrid technique and the putative cytosolic domain of human Densin-180 as bait. From 1.7 ϫ 10 7 transformants, 30 positive clones were obtained. Among them, eight positive clones encoded human ␦-catenin/NPRAP (12, 13) (Fig. 1A), and two clones were p0071 (23). In addition, the positive clones con-tained one clone of the ␣-subunit of CaMKII and one clone of ␣-actinin reported to be Densin-180-binding proteins (6, 7). ␦-catenin/NPRAP has a structure similar to that of p0071 and is considered to be a neural isoform of p0071, which is expressed ubiquitously (18). Because Densin-180 has been detected only in the brain, we further examined the interaction of ␦-catenin/NPRAP with Densin-180.
Densin-180 PDZ Domain Binds to the Carboxyl Terminus of ␦-catenin/NPRAP-PDZ domains of mammalian LAP proteins belong to class I domains that interact with peptides containing a carboxyl-terminal (S/T)XV (in single letter amino acid code, where X is any residue) motif (24,25). ␦-catenin/NPRAP contains a COOH-terminal SWV motif matching a canonical class I PDZ domain binding site. Therefore, the PDZ domain of Densin-180 was speculated to bind the COOH-terminal PDZbinding motif of ␦-catenin/NPRAP. We next examined interactions between several constructs of Densin-180 and ␦-catenin/ NPRAP, using yeast two-hybrid methods (Fig. 1, B and C). The Densin-180 intracellular domain lacking the PDZ domain (C-⌬PDZ) did not bind wild-type ␦-catenin/NPRAP COOH terminus (C-SWV). Conversely, mutation of the carboxyl-terminal valine to alanine in ␦-catenin/NPRAP (C-SWA) or deletion of the COOH-terminal three amino acids in ␦-catenin/NPRAP (C-⌬SWV) abrogated the interaction (Fig. 1C). These results show that Densin-180 interacts with the carboxyl terminus of ␦-catenin/NPRAP via its PDZ domain.
Unlike other LAP proteins, which are cytosolic membraneassociated proteins, Densin-180 was originally reported to be a transmembrane glycoprotein (5). ERBIN, a LAP family protein showing high homology to Densin-180, was shown to be a cytosolic protein (determined using surface biotinylation) (26). We biotinylated surface proteins in dissociated hippocampal neurons, which were then isolated with streptavidin-agarose beads (Fig. 3C). As described above, Densin-180 co-immunoprecipitated with N-cadherin from a 1% Triton X-100 soluble fraction of crude synaptosomal fractions of the rat brain. To exclude the possibility that Densin-180 may be precipitated with streptavidin-agarose beads through association with biotinylated N-cadherin, cells were solubilized with 1% Triton X-100 and 1% deoxycholate, the detergent condition in which Densin-180 did not co-immunoprecipitate with N-cadherin (data not shown). As shown in Fig. 3C, biotin markedly labeled N-cadherin, a protein with a transmembrane domain. In contrast, Densin-180 in the pull-down complex was barely detectable. These observations suggest that Densin-180 may be a cytosolic protein.
As described above, Densin-180 forms a ternary complex with ␦-catenin/NPRAP and N-cadherin, possibly as a cytosolic protein. To address more precisely the nature of the interaction, we checked the interaction of Densin-180 and N-cadherin, using the two-hybrid method (Fig. 3D). With this system, the intracellular domain of N-cadherin interacted with ␦-catenin/ NPRAP but not with full-length Densin-180. Our findings suggest that Densin-180 forms a complex with N-cadherin through ␦-catenin/NPRAP (Fig. 3D).
Densin-180 Co-localizes with ␦-catenin/NPRAP in Dissociated Hippocampal Neurons-To examine the intracellular distribution of Densin-180 and to determine whether Densin-180 co-localizes with ␦-catenin/NPRAP, an anti-Densin-Ext antibody was used for immunocytochemical staining of dissociated rat brain hippocampal cell cultures (Fig. 4). Hippocampal neurons plated at E18 were grown in culture for 2 weeks. The staining for Densin-180 was membrane-associated and punc-tate along dendrites with little cytoplasmic staining, as described (5). We also observed co-localization of Densin-180 and PSD-95 (data not shown) as reported (5), thus indicating that Densin-180 is located at synapses. Double staining with anti-Densin-Ext antibody and anti-␦-catenin antibody revealed colocalization of Densin-180 and ␦-catenin/NPRAP (Fig. 4, a-f). The staining pattern of ␦-catenin/NPRAP was reported to be similar to that of N-methyl D-aspartate receptor 1 in rat hippocampal culture, suggesting the localization of ␦-catenin/ NPRAP at synapses (17). There was also an overlap between Densin-180 and N-cadherin (Fig. 4, g-i). In light of all of this evidence, we propose that Densin-180 is associated with ␦-catenin/NPRAP and N-cadherin at synapses. DISCUSSION In the present study, we attempted to identify new Densin-180 binding partners and characterize protein complexes important for function. Using the two-hybrid system, GST pulldown assay, co-immunoprecipitation analysis, and immunocytochemical staining of rat hippocampal neurons, we found that Densin-180 interacts with ␦-catenin/NPRAP. The Densin-180 PDZ domain binds to the SWV motif present in the carboxyl terminus of ␦-catenin/NPRAP. Densin-180 was immunoprecipitated not only with ␦-catenin/NPRAP but also with N-cadherin. Although Densin-180 was reported to be an integral protein (5), Densin-180 was not accessible to surface biotinylation in dissociated hippocampal neurons, which suggests that Densin-180 may be a cytosolic protein (Fig. 5). In rat hippocampal neurons, Densin-180 co-localizes with ␦-catenin/NPRAP and N-cadherin at synapses. These results suggest that Densin-180 may be involved in organization of the synaptic cell-cell junction through association with ␦-catenin/NPRAP and N-cadherin. At synapses, cadherin-catenin complexes show both presynaptic and postsynaptic locations (27). In contrast, Densin-180 was originally reported to be enriched in PSD, although it has not yet been determined whether Densin-180 is presynaptic, postsynaptic, or both. Therefore, it is yet unclear if Densin-180 interacts with ␦-catenin/NPRAP and N-cadherin at presynapses.
By way of summary, we provide evidence that Densin-180 is associated in vivo with an armadillo protein, ␦-catenin/NPRAP, and we suggest that Densin-180 may be involved in the organization of synaptic cell-cell contacts. As other LAP proteins regulate cell polarity in epithelial cells, it may be that brainspecific Densin-180 can affect cell polarity in neurons through interactions with an armadillo protein. Identification of proteins binding to other domains (e.g. leucine-rich repeats) of Densin-180 will be necessary to unravel the entire function of Densin-180. FIG. 5. Working model for Densin-180-␦-catenin/NPRAP interaction at synapses. Densin-180 was reported to be a transmembrane protein (5). In this case, Densin-180 is associated with ␦-catenin/ NPRAP and N-cadherin as shown in A. In the present study we noted a possibility that Densin-180 may be a cytosolic protein to form a complex with ␦-catenin/NPRAP and N-cadherin as shown in B.