Interaction with Protocadherin- (cid:1) Regulates the Cell Surface Expression of Protocadherin- (cid:2) *

The protocadherin- (cid:2) (CNR/Pcdh (cid:2) ) and protocad-herin- (cid:1) (Pcdh (cid:1) ) proteins, members of the cadherin superfamily, are putative cell recognition/adhesion molecules in the brain. Overexpressed cadherins are generally expressed on the cell surface and elicit cell adhesion activity in several cell lines, although hardly any overexpressed CNR/Pcdh (cid:2) proteins are expressed on the cell surface, except on HEK293T cells, which show low expression. We analyzed the expression of CNR/Pcdh (cid:2) and Pcdh (cid:1) in HEK293T cells and found that they formed a protein complex and that Pcdh (cid:1) enhanced the surface expression of CNR/Pcdh (cid:2) . This enhanced surface expression was confirmed by flow cy-tometry analysis and by marking cell surface proteins with biotin. The enhancement was observed using different combinations of CNR/Pcdh (cid:2) and Pcdh (cid:1) proteins. The surface expression activity was enhanced by the extracellular domains The biochemical and functional interactions of CNR/Pcdh (cid:1) and Pcdh (cid:3) , however, remain unknown. Here we show that CNR/Pcdh (cid:1) and Pcdh (cid:3) formed a protein complex in the brain and neuroblastoma. They bound each other at sites in both their extracellular and cytoplasmic domains. Formation of the complex enhanced the cell surface expression of the CNR/Pcdh (cid:1) proteins. Thus CNR/

Cell recognition molecules play significant roles in the building of neuronal networks in the central nervous system. The cadherin superfamily is a large family of calcium-dependent cell adhesion molecules, which have been implicated in the morphogenesis of nonneuronal and neuronal tissues (1). Classic cadherins have five tandemly repeated extracellular (EC) 1 domains that mediate calcium-dependent homophilic protein-protein interactions (2). Overexpressed classic cadherins are generally expressed on the cell surface and elicit cell adhesion activity in several cell lines. The cell adhesion activity of the classic cadherins plays an important role in neuronal development, including the formation and maintenance of neuronal connectivity and synaptic plasticity (3)(4)(5)(6)(7)(8).
The protocadherins (Pcdhs) and cadherin-related neuronal receptor (CNR) are putative trans-synaptic recognition molecules that have six EC domains and a cytoplasmic domain (9,10). Genes for the CNR/Pcdhs are organized into clusters (termed ␣ (CNR), ␤, and ␥ (11,12). Within the ␣ and ␥ clusters, three exons encode the cytoplasmic domain for each Pcdh, making these domains identical within a cluster. The extracellular regions of Pcdh␥ proteins have been shown to mediate weak, homophilic cell adhesion (13). The CNR/Pcdh␣ proteins appear not to possess homophilic-binding activity, whereas CNR/Pcdh␣-v4 becomes a calcium-dependent cell adhesion molecule upon interacting with integrins. The interacting site is an RGD motif in the EC1 region, which is highly conserved among mammalian CNR/Pcdh␣ members (14).
We previously transfected full-length CNR/Pcdh␣-v4 into several cell lines and did not detect the overexpressed protein on the cell surface of L1, Neuro2A, COS7, Chinese hamster ovary, CHP212, or MDCKII cells. In these cell lines, the CNR/ Pcdh␣-v4 was concentrated in the endoplasmic reticulum. However, in HEK293T cells, low levels of CNR/Pcdh␣-v4 were detected on the cell surface, although most remained in the endoplasmic reticulum. These results indicated that in the cell lines tested, almost no CNR/Pcdh␣ protein is transported to the cell surface, except for HEK293T cells, where there is a low level of surface expression (14). The immunolocalization of Pcdh␥s in hippocampal neurons showed it to be strongly expressed on a subpopulation of tubulovesicular structures and to be recruited to the synapse in Pcdh␥-positive neuron pairs (15). These results suggested that the CNR/Pcdh␣ and Pcdh␥ proteins have molecular features that enable the regulation of their expression on the surface of the synaptic plasma membrane from intracellular compartments.
CNR/Pcdh␣ and Pcdh␥ are widely expressed in the brain. Single cell analyses using reverse transcription-PCR and in situ hybridization have revealed that each neuronal cell expresses distinct sets of CNR/Pcdh␣ and Pcdh␥ proteins (10,16,17). CNR/Pcdh␣ and Pcdh␥ proteins are commonly concentrated in the post-synaptic density fraction. The biochemical and functional interactions of CNR/Pcdh␣ and Pcdh␥, however, remain unknown. Here we show that CNR/Pcdh␣ and Pcdh␥ formed a protein complex in the brain and neuroblastoma. They bound each other at sites in both their extracellular and cytoplasmic domains. Formation of the complex enhanced the cell surface expression of the CNR/Pcdh␣ proteins. Thus CNR/ Pcdh␣ and Pcdh␥ proteins form a hetero-protein complex, which plays a role in the regulation of protein localization to the plasma membrane.
Cell Culture and Transfection-HEK293T was maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. C1300 and NB2a cells were grown in RPMI1640 medium supplemented with 10% fetal bovine serum. Transfection of plasmid DNA into HEK293T cells was performed using LipofectAMINE 2000 according to the manufacturer's procedure (Invitrogen).
Immunoprecipitation-HEK293T cells were lysed with Buffer A (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.25% sodium deoxycholate) and incubated at 4°C for 20 min. The lysates were spun at 20,000 ϫ g for 20 min, and the supernatant was used for immunoprecipitation. Mouse P7 brains were homogenized in 0.32 M sucrose. The homogenate was spun at 800 ϫ g at 4°C for 10 min. The supernatant (S1) was spun at 20,000 ϫ g at 4°C for 30 min to obtain the pellet fraction (P2). The P2 fraction was lysed with buffer B (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100) containing 1 mM EDTA or 1 mM CaCl 2 and spun at 20,000 ϫ g at 4°C for 30 min. The supernatant (S3) was used for immunoprecipitation. For immunoprecipitation, the supernatant was incubated with 1 g of anti-CNRA, anti-CNR/Pcdh␣, anti-Pcdh␥-pep, or anti-Pcdh␥ at 4°C for 1 h. Protein A-or G-Sepharose beads (Amersham Biosciences) were added to the sample, and incubation was continued at 4°C for 1 h. After the beads were extensively washed with Buffer A or Buffer B, the bound proteins were eluted by boiling the beads in SDS sample buffer (60 mM Tris-Cl, pH 6.7, 2% SDS, 2% v/v 2-mercaptoethanol, and 5% glycerol) and subjected to SDS-PAGE followed by Western blot analysis.
Flow Cytometry-Transfected cells were detached from the plates with phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin and 1 mM EDTA. Detached cells (1 ϫ 10 6 ) were incubated with anti-Myc antibodies diluted in PBS containing 0.5% bovine serum albumin for 30 min. The cells were then washed with PBS and incubated with Alexa 488-conjugated goat anti-mouse IgG for 30 min at 4°C. After extensive washing, the surface expression of the tagged proteins was quantified using an EPICS Altra (Beckman Coulter).
Biotinylation of Cell Surface Proteins-Transfected HEK293T cells were washed with ice-cold PBS three times and incubated 30 min with 0.5 mg/ml EZ-link sulfo-hydroxysulfosuccinimide (NHS)-S-S-biotin (Pierce) in cold PBS. After washing with ice-cold PBS three times, the cells were lysed with radioimmune precipitation assay buffer (20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) and spun at 20,000 ϫ g at 4°C for 30 min. Streptavidin-agarose beads (Pierce) were added to the supernatants, and the mixture was incubated at 4°C for 1 h. After the beads were extensively washed with radioimmune precipitation assay buffer, the bound proteins were eluted by boiling the beads in SDS sample buffer and subjected to SDS-PAGE followed by Western blot analysis.
Subcellular Fractionation of Mouse Brain-Subcellular fractionation of the mouse brain was performed as described previously (19,20). Briefly, adult mouse brains were homogenized in an ice-cold solution containing 0.32 M sucrose, 1 mM HEPES-KOH, pH 7.4, 1 mM NaHCO 3 , 1 mM MgCl 2 , 0.1 mM phenylmethanesulfonyl fluoride, 10 g/ml aprotinin, 10 g/ml leupeptin, 1 mM NaF, and 1 mM Na 3 VO 4 , and spun at 1,000 ϫ g and then 13,800 ϫ g, each for 10 min. The resulting pellet (P2) was further fractionated by discontinuous sucrose density gradient centrifugation at 82,500 ϫ g for 2 h to obtain the crude synaptosome fraction. This fraction was lysed by osmotic shock, and the lysate was spun at 38,500 ϫ g for 20 min to yield a crude synaptic membrane fraction pellet. The pellet was solubilized with 0.5% Triton X-100 and spun at 201,800 ϫ g for 20 min to obtain the post-synaptic density fraction.
Immunohistochemistry-ICR mice (postnatal days 10 -14) were used for the immunohistochemical detection of CNR/Pcdh␣ and Pcdh␥ proteins. Mice were deeply anesthetized and decapitated. The brains were rapidly removed and immersed in embedding material (OCT compound, Miles). The brains were quickly frozen by isopentane cooled with dry ice. The fresh-frozen sections were then cut on a cryostat (Leica CM3050) and thaw-mounted on slides. Prior to the immunohistochemical procedure, the sections were fixed with cold methanol (Ϫ20°C) for 15 min. The sections were blocked with blocking reagent for 1 h and then incubated with the affinity-purified antibodies (1 g/ml) diluted in PBS containing 5% blocking reagent and 0.1% sodium azide, overnight at room temperature. After being washed with PBS, the sections were incubated with Alexa 488-conjugated goat anti-rabbit IgG and Alexa 594-conjugated goat anti-guinea pig IgG in PBS containing 5% blocking reagent for 1 h at room temperature. Finally, the sections were rinsed with PBS, coverslipped with glycerol-PBS containing p-phenylenediamine, and analyzed using a Zeiss confocal laser-scanning microscope, Axiovert 135. Hippocampal neurons were prepared as described (21). In brief, hippocampi were isolated from ICR mouse embryos (E16.5), dissociated, plated on 100 g/ml poly-L-lysine-coated glass coverslips, and cultured in Neurobasale medium (Invitrogen) with B27 supplement (Invitrogen). After 14 days in culture, neurons were fixed with 2% paraformaldehyde and in methanol at Ϫ20°C. HEK293T cells were fixed with 2% paraformaldehyde. For blocking, fixed cells were incubated in PBS containing 5% goat normal serum for 30 min in the presence or absence of 0.05% Triton X-100 with the primary antibody, followed by washing with PBS and incubation with Alexa-488-conjugated anti-rabbit IgG or Alexa-594-conjugated anti-guinea pig IgG for 1 h. After washing, the cells were mounted in 1% p-phenylenediamine and analyzed using a Zeiss confocal laser-scanning microscope, LSM 410, or an Olympus light microscope, Power BX51.

Localization of CNR/Pcdh␣ and Pcdh␥ Proteins-CNR/
Pcdh␣ family proteins are localized, in part, to synaptic regions (10), and the Pcdh␥ proteins are targeted to subsets of synapses and intracellular organelles in the hippocampal neurons (15). To address the protein localization of both CNR/Pcdh␣ and Pcdh␥ family proteins, we produced affinity-purified antibodies to the constant cytoplasmic domain of CNR/Pcdh␣ (anti-CNR/ Pcdh␣) and Pcdh␥ (anti-Pcdh␥). The variable exons are cisspliced to the common exon of the CNR/Pcdh␣ or Pcdh␥ transcript in each gene cluster; therefore, the antibodies should mark the regions in which any of the 14 CNR/Pcdh␣ or 25 Pcdh␥ proteins is localized, respectively. We tested the antibodies for specificity against lysates from HEK293T cells transfected with mouse CNR/Pcdh␣ and Pcdh␥ proteins. The anti-CNR/Pcdh␣ and anti-Pcdh␥ antibodies reacted specifically with the transfected CNR/Pcdh␣ and Pcdh␥ proteins (data not shown).
We next evaluated the distribution of the CNR/Pcdh␣ and Pcdh␥ proteins in cultured hippocampal neurons. The CNR/ Pcdh␣ and Pcdh␥ staining occurred as fine puncta, and their expression patterns were partially colocalized (Fig. 1A). A similar distribution of Pcdh␥ was reported for cultured hippocampal neurons (15). Using adult brains, subcellular fractionation analysis showed both CNR/Pcdh␣s and Pcdh␥s to be concentrated in the synaptic membrane and post-synaptic density fractions (Fig. 1B). These results suggested that CNR/Pcdh␣ and Pcdh␥ are colocalized in the brain. To elucidate their colocalization in the hippocampus, we performed immunostaining with the anti-CNR/Pcdh␣ and anti-Pcdh␥ antibodies. The CNR/ Pcdh␣ and Pcdh␥ proteins were both detected in the molecular layers of the dentate gyrus, CA1, and CA3 regions in the hippocampus (data not shown). At high magnification, the signals of the CNR/Pcdh␣ and Pcdh␥ proteins showed similar punctate patterns (Fig. 1C). Double staining analysis revealed some double-stained puncta, in addition to the single-stained puncta (Fig. 1B). That is, the patterns of the puncta stained for CNR/Pcdh␣ and Pcdh␥, respectively, were not identical, but partially overlapped.
The staining pattern and subcellular fractionation of the CNR/Pcdh␣ and Pcdh␥ proteins suggested that they might form a complex in vivo. To investigate this possibility, we performed immunoprecipitation analyses of the CNR/Pcdh␣ and Pcdh␥ proteins. Lysates from mouse brains at postnatal day 7 were subjected to immunoprecipitation with the antibody to CNR/Pcdh␣s (anti-CNRA) followed by immunoblotting with anti-Pcdh␥-pep, an antibody raised against the Pcdh␥-a12 peptide. As shown as in Fig. 2A, Pcdh␥ was identified as a 100-to 120-kDa band and coimmunoprecipitated with CNR/Pcdh␣s. Similarly, immunoprecipitation of the lysate with anti-Pcdh␥pep followed by immunoblotting with anti-CNRA revealed an association between CNR/Pcdh␣ and Pcdh␥. N-cadherin and Pcdh␥ proteins are reported to be detected in the same fractions of synaptic components and intracellular membranes (15). However, neither CNR/Pcdh␣ nor Pcdh␥ formed a protein complex with N-cadherin ( Fig. 2A). These results indicate that CNR/Pcdh␣ specifically associates with Pcdh␥ in vivo.
To further confirm the protein interaction between CNR/ Pcdh␣ and Pcdh␥ in vivo, we used two neuroblastoma cell lines, C1300/N1 and NB2a, which endogenously express both proteins. Fig. 2B shows that extracts immunoprecipitated with the anti-CNRA antibody contained CNR/Pcdh␣ and Pcdh␥ proteins. This result indicated that CNR/Pcdh␣ also specifically associated with Pcdh␥ in neuroblastoma cells. Both CNR/ Pcdh␣ and Pcdh␥ have putative calcium-binding motifs, which are conserved in the cadherin family. In the classic cadherins, the calcium-binding motifs are necessary for homophilic binding activity. To test the effect of calcium ions on the association between the CNR/Pcdh␣ and Pcdh␥ proteins, we performed the coimmunoprecipitation analysis in the presence of 1 mM of calcium ions or EDTA. Regardless of the presence of calcium ions or EDTA, CNR/Pcdh␣ associated with Pcdh␥, suggesting that the formation of the CNR/Pcdh␣ and Pcdh␥ complex was independent of calcium ions (Fig. 2C). were double-labeled with anti-CNR/ Pcdh␣ and anti-Pcdh␥ antibodies. The immunostaining patterns of CNR/Pcdh␣ (green) and Pcdh␥ (red) partially overlapped. Double-labeled dots are indicated in yellow (arrows). Red or green dominant dots were also observed (arrowheads). Scale bar, 20 m (upper panels); 5 m (lower panels). B, subcellular distribution of CNR/Pcdh␣ and Pcdh␥. P14 mouse brains were homogenized and fractionated by sucrose density centrifugation as described under "Experimental Procedures." CNR/Pcdh␣ were detected in the P2 (crude membrane fraction), synaptic membrane (synaptic membrane fraction), and post-synaptic density (PSD fraction). Pcdh␥ showed an identical subcellular distribution with CNR/Pcdh␣. C, the immunostaining patterns of CNR/Pcdh␣ (red) and Pcdh␥ (green) partially overlapped in the CA1 and CA3 regions. Double-labeled dots are indicated in yellow (arrow). Scale bar, 5 m.
CNR/Pcdh␣ Associated with Pcdh␥ at Regions in Their Cytoplasmic and Extracellular Domains-Using PCR amplification from full-length CNR/Pcdh␣ or Pcdh␥, we constructed deletion, chimeric, Myc-tagged, or His-tagged constructs and subcloned them into mammalian expression vectors or bacterial expression vectors, as described under "Experimental Procedures." These protein structures are shown in Fig. 4.
To identify the domain of CNR/Pcdh␣-v4 responsible for its interaction with Pcdh␥, we performed a GST pull-down assay with a bacterially produced GST and GST-CNR/Pcdh␣-v4CP, which contained only the cytoplasmic domain of CNR/Pcdh␣-v4, against full-length Pcdh␥-a12 or -b2 proteins, or these proteins with the cytoplasmic region deleted, Pcdh␥-a12delCP and -b2delCP. We could detect GST-CNR/Pcdh␣-v4CP binding to the full-length Pcdh␥-a12 and -b2 proteins, but not to Pcdh␥-a12delCP or -b2delCP (Fig. 5A). The control, GST protein alone, did not interact with any of the Pcdh␥ proteins used here. These results indicated that the cytoplasmic region of CNR/Pcdh␣ had binding activity with the cytoplasmic region of the Pcdh␥ proteins.
To further examine the interaction between CNR/Pcdh␣ with Pcdh␥, we constructed expression vectors, Pcdh␥-EC-His and CNR/Pcdh␣-EC-Myc, that contained only the extracellular domains of Pcdh␥ and CNR/Pcdh␣. The carboxyl terminus of Pcdh␥ was tagged with His 8 and that of CNR/Pcdh␣ was tagged with the Myc-epitope. The Pcdh␥-a12-EC-His or -b1-EC-His was then transfected into HEK293T cells along with the control vector or CNR/Pcdh␣-v4-EC-Myc. Forty-eight hours after transfection, the proteins tagged with His or Myc were affinitypurified from the culture medium using a nickel column or Protein G-Sepharose containing immobilized anti-Myc antibody. The purified proteins were then detected by Western blotting with an anti-His or anti-Myc antibody. All the tagged proteins were affinity-purified: the His-tagged and Myc-tagged  1 and 2) or Pcdh␥-a12 (lanes 4 and 5), respectively. B, HEK293T were cotransfected with CNR/Pcdh␣-v4 or -v7 and the empty vector or Pcdh␥-a1, -a3, -b2, or -b4. Transfected cells were immunoprecipitated with anti-Pcdh␥ antibodies, followed by immunoblotting with anti-CNR/Pcdh␣ or anti-Pcdh␥ antibodies. The different forms of CNR/Pcdh␣ and Pcdh␥ coimmunoprecipitated. proteins were pulled down by the nickel column and anti-Myc antibody, respectively. In addition, the association of CNR/ Pcdh␣-v4EC-Myc with Pcdh␥-a12-EC-His or with -b1-EC-His was also detected in the culture mediums from HEK293 cells coexpressing both CNR/Pcdh␣-EC-Myc and Pcdh␥-EC-His (Fig. 5B). Thus, the extracellular domains of CNR/Pcdh␣-EC and Pcdh␥-EC associated with each other. We also confirmed that the extracellular domain of CNR/Pcdh␣-v7 also binds to that of Pcdh␥-a12 and -b1 (data not shown).
The above results indicated that the extracellular and cytoplasmic regions of the CNR/Pcdh␣ protein could interact with those of the Pcdh␥ protein. To further confirm these interactions, we performed immunoprecipitation assays of the fulllength Pcdh␥ proteins (Pcdh␥-a12 and -b2) with extracellular domain-deleted CNR/Pcdh␣-v4 (Myc-CNR/Pcdh␣-v4delEC) or cytoplasmic region-deleted CNR/Pcdh␣-v4 (Myc-CNR/Pcdh␣-v4delCP). Transfected cells were lysed and immunoprecipitated with an anti-Myc antibody. Both the Pcdh␥-a12 and -b2 full-length proteins were detected in immunoprecipitates from cells transfected with both the extracellular-and cytoplasmic domain-deleted CNR/Pcdh␣-v4, using the anti-Myc antibody (Fig. 5C). These data indicated that both the extracellular and

FIG. 4. Deletion mutants and chimeras of CNR/Pcdh␣ or Pcdh␥ used in pull-down assay and transfection experiments.
Deletion and chimera constructs of CNR/Pcdh␣ and Pcdh␥ used in the transfection experiments. Myc and His 8 are epitope tags used for immunoprecipitation, immunocytochemistry, and Western blotting. For the pull-down assay, the cytoplasmic region of CNR/ Pcdh␣-v4 was fused with glutathione Stransferase (GST). Sig is the signal sequence; EC is the extracellular domain; TM is the transmembrane domain; and CP is the cytoplasmic domain.  3 and 6). Bound Pcdh␥s were detected by immunoblotting. Only Pcdh␥s containing the cytoplasmic region were able to bind GST-CNR/Pcdh␣-v4CP. Lanes 1 or 4 correspond to the lysates of cells expressing full-length Pcdh␥-a12 or -b1 (upper panel) or cytoplasmic region-deleted mutants (lower panel). Truncated proteins were found in the Pcdh␥-a12 lysates. B, interaction between CNR/Pcdh␣ and Pcdh␥ through their extracellular domains. CNR/Pcdh␣-v4EC-Myc, Pcdh␥-a12EC-His, Pcdh␥-b1EC-His, both CNR/Pcdh␣-v4EC-Myc and Pcdh␥-a12EC-His, or both CNR/Pcdh␣-v4EC-Myc and Pcdh␥-b1EC-His were affinity-purified from the culture medium with a nickel column or Protein G-Sepharose containing immobilized anti-Myc antibody. The purified proteins were resolved by SDS-PAGE and transferred to PVDF membranes. The blots were probed with an anti-His or anti-Myc antibody. C, CNR/Pcdh␣-v4 associated with both the extracellular and cytoplasmic domain of Pcdh␥s in HEK293T cells. HEK293T cells were transfected with Pcdh␥-a12 or -b2 along with Myc-CNR/ Pcdh␣-v4delEC, -delCP, or empty vector. After 24 h, Transfected cells were immunoprecipitated with an anti-Myc antibody followed by immunoblotting with anti-Pcdh␥ antibodies. cytoplasmic domains of CNR/Pcdh␣ could interact with fulllength Pcdh␥. There appeared to be slightly more Pcdh␥ precipitated with the delCP than with the delEC protein, suggesting that the EC regions were more tightly associated than the cytoplasmic regions.
Pcdh␥ Increased the Cell Surface Expression of CNR/ Pcdh␣-In previous reports, we demonstrated that CNR/ Pcdh␣-v4 overexpressed in several cell lines was detected mainly in the cytoplasm and hardly at all on the cell surface (10,14,18). Here, we coexpressed CNR/Pcdh␣ and Pcdh␥ in HEK293T cells and observed the colocalization of these proteins. Interestingly, the cellular localization of the Myc-CNR/ Pcdh␣-v4 protein was different when it was expressed alone versus when it was coexpressed with Pcdh␥-a12 (Fig. 6). Without detergent treatment, the cell surface expression levels of the cells expressing Myc-CNR/Pcdh␣-v4 versus those coex-pressing Myc-CNR/Pcdh␣-v4 with Pcdh␥-a12 were especially different (Fig. 6A, panels c and d). When we used an anti-Myc antibody to detect the Myc-CNR/Pcdh␣-v4 protein, the regions of cellular junction were more extensively stained (appearing as lines) in the coexpressing cells (Fig. 6A, panel d) than in the cells expressing Myc-CNR/Pcdh␣-v4 alone (Fig. 6A, panel c). The anti-Myc-stained cellular junctions in the coexpressing cells were also stained by the anti-Pcdh␥ antibody, suggesting that Myc-CNR/Pcdh␣-v4 and Pcdh␥-a12 proteins were colocalized at the cell junctions (double stained in Fig. 6A, panel f ). Interactions between these proteins were also detected by immunoprecipitation analysis (Fig. 3). Both CNR/Pcdh␣-v4 and Pcdh␥-a12 were concentrated at the intercellular junctions rather than generally on the cell membrane. To further confirm these protein localizations, we used confocal laser-scanning microscopy, which also detected the colocalization of these pro- teins on the cell surface (Fig. 6B). These data demonstrated that the CNR/Pcdh␣-v4 and Pcdh␥-a12 proteins were expressed on the cell surface, and that the Pcdh␥-a12 protein could induce the surface expression of the CNR/Pcdh␣-v4 proteins. Furthermore, we tested whether the other Pcdh␥ variants possessed the same activity for the cell surface expression of Myc-CNR/Pcdh␣-v4 as did Pcdh␥-a12. Coexpression analyses with Myc-CNR/Pcdh␣-v4 and Pcdh␥-a1, -a3, -b1, -b2, or -b4 in HEK293T cells are shown in Fig. 6C. Extensive cell surface expression of the Myc-CNR/Pcdh␣-v4 protein was detected with the anti-Myc antibody when any of these Pcdh␥ variants was coexpressed with it. The expression patterns were also concentrated at the intercellular junctions, appearing as lines.
To assess the cell surface expression level of CNR/Pcdh␣ in live HEK293T cells, we performed quantitative fluorescenceactivated cell sorting analyses. Coexpression of Pcdh␥-a12 or -b2 with Myc-CNR/Pcdh␣-v4 increased the amount of Myc-CNR/Pcdh␣-v4 on the cell surface (Fig. 6D). Stained cells expressing only Myc-CNR/Pcdh␣-v4 were also detected as a single peak population. The stained-peak levels were shifted to the right, indicating the levels in cells that coexpressed Pcdh␥ were more than 5-fold higher than in the cells expressing Myc-CNR/ Pcdh␣-v4 alone. These findings indicated that many Pcdh␥ variants induced and enhanced the surface expression of the Myc-CNR/Pcdh␣-v4 protein. However, these data were obtained using the artificial Myc fusion protein. To further confirm and evaluate the cell surface expression of the native CNR/Pcdh␣ proteins, we performed surface biotinylation experiments. HEK293T cells were transfected with CNR/ Pcdh␣-v4 only, both CNR/Pcdh␣-v4 and Pcdh␥-a12, CNR/ Pcdh␣-v7 only, or both CNR/Pcdh␣-v7 and Pcdh␥-a12. After a 24-h transfection, the transfected cells were surface-biotinylated with sulfo-NHS-S-S-biotin and extracted with lysis buffer. Biotinylated proteins were then affinity-purified from the lysates with streptavidin-agarose and immunoblotted with the anti-CNR/Pcdh␣ or anti-Pcdh␥ antibody. As shown as in Fig. 6E, coexpression with Pcdh␥-a12 resulted in an increase in biotinylated CNR/Pcdh␣-v4 and -v7, whereas the amount of biotinylated Pcdh␥ was little affected by its coexpression with CNR/Pcdh␣s. These data indicated that Pcdh␥ proteins upregulated the surface expression level of the CNR/Pcdh␣ proteins on the plasma membrane. Using Chinese hamster ovary cells, we also found that the extracellular domain of Pcdh␥-␣12 induced the secretion of the extracellular domain of CNR/ Pcdh␣-v4, which was barely transported to culture medium when expressed alone (data not shown).
Surface Expression Domain of the CNR/Pcdh␣ and Pcdh␥ Proteins-To determine the regions of CNR/Pcdh␣-v4 that inhibited its cell surface expression, we transfected the fulllength cDNA or constructs encoding deletion mutants of either the cytoplasmic or extracellular domain of Myc-CNR/Pcdh␣-v4 into HEK293T cells and performed immunostaining analysis with an anti-Myc antibody. Immunoreactivity for the fulllength protein and the cytoplasmic deletion mutant was barely detected on the cell surface (Fig. 7A, panels a and b). In contrast, the extracellular domain deletion mutant was expressed well on the cell surface (Fig. 7A, panel c). This result indicated that the extracellular and transmembrane domains in the CNR/Pcdh␣-v4 protein inhibited its cell surface expression.
The coexpression of Pcdh␥ enhanced the cell surface expression of the full-length and cytoplasmic deletion mutant of CNR/ Pcdh␣-v4. These data indicated that Pcdh␥ induced the surface expression of the CNR/Pcdh␣-v4 normally inhibited by the latter's extracellular and transmembrane domains. Expression of the extracellular domain of CNR/Pcdh␣ was sufficient for Pcdh␥ to enhance its surface expression (data not shown). In contrast, the extracellular domain deletion mutant of CNR/ Pcdh␣-v4 showed a down-regulation of surface expression when the Pcdh␥ protein was coexpressed (Fig. 7A, panels d and  f ). This result suggested that the cytoplasmic interaction between the CNR/Pcdh␣ and Pcdh␥ proteins (shown in Fig. 5A) inhibits the cell surface expression of CNR/Pcdh␣.
Next, we set out to identify the region of Pcdh␥ responsible for promoting the cell surface expression of CNR/Pcdh␣. When HEK293T cells were cotransfected with Myc-CNR/Pcdh␣-v4 and full-length Pcdh␥-a12, or with deletion mutants for its cytoplasmic domain (which encoded the extracellular and transmembrane regions), or its extracellular domain, intense immunoreactivity for the Myc-CNR/Pcdh␣-v4 protein was detected on the cell surface (Fig. 7B). We also detected the induction of the cell surface expression of Myc-CNR/Pcdh␣-v4 in response to coexpression of the extracellular domain of Pcdh␥-b2 (data not shown). These results demonstrated that interactions between the extracellular domains of the CNR/ Pcdh␣ and Pcdh␥ proteins are involved in regulating the cell surface expression of CNR/Pcdh␣. DISCUSSION Molecules that mediate the specification of different synapses are most likely to be differentially expressed in neuronal populations and to be coupled to intracellular functions at the synaptic plasma membranes. The clustered CNR/Pcdh␣ and Pcdh␥ families may meet these criteria (15). These proteins are expressed differentially within single cells (10,17), mediate cell-cell adhesion (13,14), and are synaptic proteins that localize in part to synaptic membranes and to the nonsynaptic plasma membrane and the inside of the endosomal vesicles of synapses (10,15). The CNR/Pcdh␣ and Pcdh␥ gene clusters both consist of variable exons and constant exons. The constant exons of both genes encode cytoplasmic tails that are the same for all CNR/Pcdh␣ and Pcdh␥ proteins, respectively (10,11). Here we show that the CNR/Pcdh␣ and Pcdh␥ proteins associate with each other through their extracellular and cytoplasmic regions. Their interaction was detected by coimmunoprecipitation using extracts of mouse brain and neuroblastoma. This finding was supported by the partial colocalization of these molecules in cells and their enrichment in the same subcellular fractions. It has been reported that the CNR/Pcdh␣ proteins are barely expressed on the cell surface. However, here we show that Pcdh␥ can induce the cell surface expression of CNR/Pcdh␣.
The association of CNR/Pcdh␣ and Pcdh␥ in vivo and in vitro was shown for various combinations of these proteins. Because there are 12 and 22 variants in the gene clusters for CNR/ Pcdh␣ and Pcdh␥, respectively, there are 264 (12 ϫ 22) possible combinations for protein complexes between them. Their family variants are differentially expressed in neuronal cell populations; therefore, individual neurons could contain different sets of combinational variations. The extracellular and cytoplasmic domains of CNR/Pcdh␣ and Pcdh␥ were responsible for their binding, which was independent of calcium ions. It has been reported that, in cotransfected cell lines, N-and R-cadherins form stable cis-heterodimers. The first extracellular cadherin domain of N-cadherin is required for heterodimer formation, and the cis-heterodimer can form in the absence of calcium ions. Furthermore, the cis-heterodimer is a functional unit for mediating cell-cell adhesion (22). Here, we found that the CNR/Pcdh␣ and Pcdh␥ family proteins form a protein complex in cotransfected cells. These proteins formed the complexes via their extracellular domains in the absence of calcium and via their cytoplasmic regions. These protein complexes are similar to the cis-heterodimer of R-and N-cadherins with respect to their calcium independence and extracellular domain interaction.
The interaction between CNR/Pcdh␣ and Pcdh␥ could be detected in brain extracts; however, the immunostained patterns of CNR/Pcdh␣ and Pcdh␥ only partially overlapped and many single-labeled dots were also seen. These data demonstrated that CNR/Pcdh␣ and Pcdh␥ did not always interact in vivo. A recent report showed that Pcdh␥ is localized to some synapses, to nonsynaptic plasma membranes, and to axonal and dendritic tubulovesicular structures and suggested that these molecules are exchanged among synapses and intracel-lular compartments (15). It is possible that the double-and single-stained puncta we observed reflect different distributions of CNR/Pcdh␣ and Pcdh␥ in synapses and intracellular compartments.
The overlapping staining patterns of the co-overexpressed CNR/Pcdh␣ and Pcdh␥ were detected in part in the cell membranes of hippocampal neurons (Fig. 1A). Furthermore, the intercellular junctions were more extensively stained than nonjunctional membranes (Fig. 6A, panel f ). Because classic cadherins are concentrated in the cell adhesion sites of intercellular junctions (2), the enrichment of intercellular junctions in these proteins suggested that their hetero-protein complex might have a function in cell adhesion. Some of the Pcdh␥ proteins have homophilic cell adhesion activity (23), and the CNR/Pcdh␣ proteins have heterophilic cell adhesion activity for ␤ 1 integrin, but not homophilic activity (14). HEK293T cells express ␤ 1 integrin; therefore, at the intercellular junctions where cell adhesion occurs, a cis-complex of CNR/Pcdh␣ and Pcdh␥ at one membrane site could interact with integrins and Pcdh␥s in the opposite membrane. Thus, the cis-protein complex of CNR/Pcdh␣ and Pcdh␥ might have cell adhesion activity.
The cell surface expression of the CNR/Pcdh␣ protein was repressed by its extracellular domain. This inhibition was removed by the binding of CNR/Pcdh␣ to the extracellular domain of Pcdh␥. In addition, the cytoplasmic region of the CNR/ Pcdh␣ protein bound the cytoplasmic region of Pcdh␥. The interactions between these molecules were previously unknown, and may function in mediating intracellular signals. Full-length Pcdh␥ induced the surface expression of CNR/ Pcdh␣, resulting in the presence of the CNR/Pcdh␣ and Pcdh␥ complex on the cell surface. This protein complex may play a role in the intercellular junctions of the neuronal cell surface.
The molecular mechanisms of the cell surface expression of membrane proteins have been extensively studied. The extracellular, transmembrane, and cytoplasmic regions of membrane proteins have all been implicated in the regulation of their endoplasmic reticulum retention/retrieval, trafficking to their target sites, and their degradation (24). In particular, the retinoid X receptor, KKXX, and di-leucine motifs in the cytoplasmic region of glycoproteins have been shown to regulate the cell surface expression of glycoproteins. Recently, we demonstrated that CNR/Pcdh␣ that was overexpressed in a variety of cells was mainly expressed in the endoplasmic reticulum and Golgi (10,14,18) and that CNR/Pcdh␣ proteins also possess a putative KKXX-like motif in the cytoplasmic region. Here, we showed that extracellular domains of CNR/Pcdh␣ proteins negatively regulated their cell surface expression. Therefore, the retention signal of CNR/Pcdh␣ is mainly located in its extracellular domain. Only the cytoplasmic region of the CNR/Pcdh␣ protein could easily translocate to the plasma membrane and was not retained in the cytoplasm (Fig. 7A); thus, the cytoplasmic domain of the CNR/Pcdh␣ proteins (containing the putative KKXX-like motif) did not work as a retention signal of CNR/Pcdh␣.
On HEK293T cells transfected with Pcdh␥, CNR/Pcdh␣ was colocalized with Pcdh␥ on the plasma membrane. The interaction between the CNR/Pcdh␣ and Pcdh␥ extracellular domains resulted in an increase in the level of CNR/Pcdh␣ cell surface expression. Consistent with these results, it is reported that the cell surface expression of several glycoproteins is promoted by the association of their extracellular domain with the extracellular domain of other glycoproteins and that these complexes form functional receptors. For example, the interaction of calcitonin receptor-like receptor (CLCR) with the receptor activity-modifying proteins (RAMPs) or of Caspr/Paranodin with F3 results in the cell surface expression of CLCR or Caspr/Paranodin and induces the formation of functional CLCR/RAMPs or Caspr/F3 receptor (25,26). The interaction between the extracellular domains of these proteins contributes to their glycosylation and/or trafficking to the cell surface (25,26). Thus, it is possible that Pcdh␥ influences the glycosylation and/or trafficking of CNR/Pcdh␣ to the cell surface via the interaction between the extracellular domains of these proteins.
In the present study, we found a functional interaction between CNR/Pcdh␣ and Pcdh␥ proteins that causes their cell surface expression. Recently, the deletion of the Pcdh␥ locus in mice was shown to cause severe apoptosis of the interneurons in the spinal cord and to decrease the number of neuronal cells in the basal forebrain, thalamus, cerebral cortex, and hippocampus (27). The interaction of CNR/Pcdh␣ and Pcdh␥ that we report here also suggests the possibility that CNR/Pcdh␣ not only contributes to the combinatorial diversity of cell recognition proteins but is also involved in neuronal cell survival and apoptosis in concert with Pcdh␥ in the brain. However, further analysis will be required to assess the roles of the CNR/Pcdh␣ and Pcdh␥ protein complex in the development and regulation of the brain.