¹Department of Medical Biochemistry, Graduate School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan

²Department of Gastroenterology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan

³Department of Nephrology and Endocrinology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan

Correspondence to: Y Hata, E-mail: yuhammch@med.tmd.ac.jp

PAPIN has six PDZ domains and interacts with p0071, a catenin-related protein. Recent studies have revealed that catenins determine the subcellular localization of some PDZ proteins. We have examined whether the localization of PAPIN is determined by p0071 in epithelial cells. PAPIN was localized not only on the lateral membrane but also on the apical membrane, where p0071 was absent. The targeting to both membranes was mediated by the middle region of PAPIN and did not require the p0071-interacting PDZ domain. In cells that came into contact, PAPIN was diffusely distributed on the plasma membrane, while p0071 was concentrated at immature cell-cell contacts. When epithelial cells were exposed to the low concentration of calcium, p0071 was internalized, whereas PAPIN remained on the plasma membrane. We also confirmed that the interaction with p0071 was not essential for the membrane targeting of ERBIN, a recently identified p0071- and ErbB2-binding protein. PAPIN, p0071, and ERBIN formed a complex in 293T cells. Furthermore, ERBIN and ErbB2 were colocalized with PAPIN on the lateral membrane. These findings suggest that PAPIN, p0071, and ERBIN come to the cell-cell contacts independently and interact with each other on the lateral membrane.

Oncogene (2002) 21, 7042-7049. doi:10.1038/sj.onc.1205852

p0071; catenin; ERBIN; ErbB2; PAPIN; PDZ

Introduction

p0071 is a protein with the 10 armadillo-repeats (Hatzfeld and Nachtsheim, 1996). p0071 is very homologous to neural plakophilin-related armadillo-repeat protein (NPRAP)/-catenin (Paffenholz and Franke, 1997; Zhou et al., 1997). Like other catenins, p0071 is localized at the cell junctions and interacts with various proteins. We have discovered a novel p0071-binding molecule with multiple PSD-95/Dlg-A/ZO-1 (PDZ) domains and named it plakophilin-related armadillo-repeat protein-interacting protein (PAPIN) (Deguchi et al., 2000). PAPIN consists of 2766 amino acids. It has four PDZ domains in the N-terminal region and two PDZ domains in the carboxyl-terminal region. The middle region between the fourth and the fifth PDZ domains is composed of about 1780 amino acids and does not show any significant homology to known proteins. p0071 binds to the second PDZ domain by its carboxyl-terminal PDZ-binding motif. Recently, a human homolog of PAPIN named AIPC has been identified (Chaib et al., 2001). AIPC transcript and protein are up-regulated in prostate tumor cells, suggesting that PAPIN may be involved in the regulation of cell proliferation and polarity. Furthermore, a very recent report has revealed that p0071 interacts with ERBIN, a member of the LAP family (Borg et al., 2000; Bryant and Huwe, 2000; Jaulin-Bastard et al., 2002; Izawa et al., 2002b). ERBIN has the leucine-rich repeat in the N-terminal region and one PDZ domain in the carboxyl-terminal region, which binds to p0071. ERBIN determines the localization of ErbB2, which is implicated in tumorigenesis (Borg et al., 2000; Yarden and Sliwkowski, 2001). Thereby, both p0071-interacting proteins, PAPIN and ERBIN, are related with oncogenesis.

-Catenin, the most representative catenin, has a PDZ-binding motif and binds to PDZ proteins, including Lin-7, membrane-associated guanylate kinase with inverted organization (MAGI-1) (also called brain angiogenesis inhibitor-associated protein 1), and synaptic scaffolding molecule (S-SCAM) (Dobrosotskaya et al., 1997; Shiratsuchi et al., 1998; Hirao et al., 1998; Perego et al., 2000; Ide et al., 1999; Dobrosotskaya and James, 2000; Nishimura et al., 2000, 2002). These interactions are implicated in the determination of the localization of PDZ proteins. Lin-7 is fixed on the plasma membrane by -catenin (Perego et al., 2000). -Catenin recruits MAGI-1 and S-SCAM to the lateral membrane in epithelial cells and to synapses in neurons, respectively (Dobrosotskaya and James, 2000; Nishimura et al., 2000, 2002). By analogy, we have raised the question whether p0071 determines the subcellular localization of its interacting proteins.

In this study, we first describe that PAPIN is localized on the apical membrane as well as on the lateral membrane in Madin Darby canine kidney (MDCK) cells. We determined using green fluorescent protein (GFP)-tagged constructs which region of PAPIN is required for the membrane localization and whether the interaction with p0071 is involved in this process. Furthermore, we examined whether p0071 plays roles to determine the localization of ERBIN.

Results

PAPIN is localized on the apical membrane in Madin Darby canine kidney cells

In the previous study, we reported that PAPIN was detected not only at the cell-cell contacts but also on the apical membranes in the bronchial epithelial cells (Deguchi et al., 2000). We first confirmed the apical localization of PAPIN. We generated stable transformants of MDCK cells expressing GFP-tagged PAPIN. GFP-PAPIN-1 was localized on the lateral and apical membranes (Figure 1A). The apical localization was more clearly confirmed in the three dimensional culture (Figure 1B).

PAPIN localizes to the plasma membrane where p0071 is not localized

GFP-PAPIN-1 and the endogenous p0071 were colocalized on the lateral membrane in confluent MDCK cells (Figure 2A). p0071 is not localized on the apical membranes. Therefore, the apical membrane targeting of PAPIN is presumably independent of the interaction with p0071. To make a comparison between the membrane targeting of PAPIN and p0071, we examined how PAPIN and p0071 were localized in cells before the formation of mature cell-cell contacts. GFP-p0071 was accumulated at the borders where MDCK cells came into contact (Figure 2B). In contrast, GFP-PAPIN-1 was distributed on the membranes and was not concentrated at immature cell-cell contacts (Figure 2C). During the cytokinesis, GFP-PAPIN-1 was diffusely distributed on the plasma membrane, while p0071 was concentrated at the contractile ring (Figure 2D). Furthermore, we exposed MDCK cells to the low concentration of calcium in DMEM containing 10 mM EGTA for 30 min. p0071 underwent the endocytosis, whereas PAPIN remained on the plasma membrane (Figure 2E). All these findings indicate that the localization of PAPIN on the plasma membrane is independent of p0071.

The lateral membrane localization of PAPIN is also independent of the interaction with p0071

It is still possible that the lateral membrane targeting of PAPIN depends on the binding to p0071 and that without p0071, PAPIN is recruited to only the apical membrane but not to the lateral membrane. To exclude this possibility, we tested which regions of PAPIN were involved in the lateral membrane localization. We prepared GFP-tagged constructs containing various regions of PAPIN and generated stable transformants of MDCK cells expressing these proteins (Figure 3A). GFP-PAPIN-15 lacking the last two PDZ domains was localized on the membranes like the full length of PAPIN, suggesting that the carboxyl-terminal PDZ domains are not necessary for the lateral membrane localization (Figure 3B panel a). Consistently, GFP-PAPIN-5 containing the last two PDZ domains was mostly distributed in the cytosol, although it was partially localized on the membranes (Figure 3B panel b). GFP-PAPIN-6 containing the first four PDZ domains was distributed in the cytosol (Figure 3B panel c). These findings suggest that none of the six PDZ domains is involved in the lateral membrane localization of PAPIN. In contrast, GFP-PAPIN-18 containing the middle region between the fourth and the fifth PDZ domains was localized on the lateral membrane as well as the apical membrane (Figure 3B panel d). The second PDZ domain of PAPIN is involved in the interaction with p0071 but is not required for the lateral localization of PAPIN. We also overexpressed the carboxyl-terminal region of p0071 (Myc-pp0071-6) in stable transformants of MDCK cells expressing GFP-PAPIN-1. The carboxyl-terminal region of p0071 was diffusely distributed in the cytosol and in the nucleus but the localization of GFP-PAPIN-1 at cell-cell contacts was not affected (data not shown). All these results mean that p0071 does not play an essential role to recruit PAPIN to the lateral membrane.

ERBIN is colocalized with p0071 but the interaction with p0071 is not essential for the membrane targeting of ERBIN

All the data above indicate that PAPIN is colocalized with p0071 at the mature cell-cell contacts but its subcellular localization is determined independently of p0071. We next tested whether the localization of ERBIN correlated to that of p0071. GFP-p0071 was localized only on the lateral membrane and colocalized with ERBIN (Figure 4A). Furthermore, GFP-p0071 and ERBIN were also colocalized at immature cell-cell contacts (Figure 4B). In MDCK cells exposed to the low concentration of calcium in DMEM containing 10 mM EGTA for 30 min, GFP-p0071 and ERBIN were internalized together (Figure 4C). To test whether the subcellular localization of ERBIN required the domain interacting with p0071, we prepared various GFP-constructs of ERBIN and expressed them in MDCK cells (Figure 5A). GFP-ERBIN-1 was localized at the lateral membrane like the endogenous ERBIN (data not shown). GFP-ERBIN-2 with the leucine-rich repeat and GFP-ERBIN-3 with the middle and the carboxyl-terminal regions were localized at the cell-cell contacts like the full-length of ERBIN (Figure 5B, panels a and b). GFP-ERBIN-5 containing the p0071-binding carboxyl-terminal PDZ domain was distributed mainly in the nucleus and the cytosol, and partially accumulated on the lateral membrane (Figure 5B panel c). The membrane targeting of ERBIN depends on more than one region, and ERBIN can be recruited to cell-cell contacts without the interaction with p0071, as the subcellular localization of GFP-ERBIN-2 indicates.

PAPIN and ERBIN in the glycerol density gradient

As shown in Figure 3, GFP-PAPIN-18 containing the middle region is targeted to the membrane. There remains, however, a possibility that GFP-PAPIN-18 interacts with the endogenous PAPIN and is targeted to the membrane indirectly. Similarly, GFP-ERBIN-2 and GFP-ERBIN-3 may be localized at cell-cell contacts through the interaction with the endogenous ERBIN. We examined whether PAPIN and ERBIN formed homomultimers using the glycerol density gradient centrifugations. By comparison with known protein standards, the sedimentation coefficient of GFP-PAPIN-1 was evaluated to be 17.6 S, which corresponded to 512 kDa (Figure 6A). Furthermore, the bottom fraction contained the aggregation of GFP-PAPIN-1. The evaluated sedimentation coefficient of ERBIN was 9.4 S, which corresponded to 209 kDa (Figure 6B). The molecular weights of GFP-PAPIN-1 and ERBIN are estimated from SDS-PAGE to be 260 and 180 kDa, respectively. Thereby, GFP-PAPIN-1 migrates as a dimer, while ERBIN migrates as a monomer in the glycerol density gradient.

PAPIN, p0071, and ERBIN forms a complex

The deletion mutant of p0071 lacking the carboxyl-terminal PDZ binding motif (GFP-p0071-3) was also localized at cell-cell contacts (data not shown). Thereby, PAPIN, p0071, and ERBIN are likely to be localized at cell-cell contacts independently. We next tested whether these molecules formed a complex. Because the endogenous p0071 was relatively resistant to the detergent extraction, we overexpressed GFP-PAPIN-1 and p0071 in 293T cells. We immunoprecipitated GFP-PAPIN-1 with the anti-PAPIN antibody. p0071 and the endogenous ERBIN was coimmunoprecipitated with GFP-PAPIN-1 (Figure 7). The immunofluorescence study showed that ERBIN was colocalized with GFP-PAPIN-1 on the lateral membrane in MDCK cells (Figure 8A). These findings support the possibility that PAPIN forms a complex with p0071 and ERBIN on the lateral membrane. ErbB2 was also colocalized with PAPIN on the lateral membrane, suggesting that PAPIN may interact with ErbB2 through p0071 and ERBIN, although we did not directly confirm that the complex contained ErbB2 (Figure 8B).

Discussion

The epithelial junctions are important in the regulation of cell polarity (Tsukita et al., 1996; Stevenson and Keon, 1998; Nagafuchi, 2001). Mutations of the junctional components disrupt cell polarity and lead to oncogenesis. The structures of cell junctions are organized through various protein-protein interactions. Junctional membrane-associated proteins interact with various molecules and play a central role in the formation of junctions. Recently, proteins with PDZ domains have attracted attention, because they provide scaffolds for junctional components. Lin-7 has one PDZ domain and binds GABA transporter to fix it on the membrane (Perego et al., 1999). MAGI-1 has a modular structure with six PDZ domains, two WW domains, and one guanylate kinase domain (Dobrosotskaya et al., 1997; Shiratsuchi et al., 1998). MAGI-1 binds RapGEP, mNET1, and megalin (Mino et al., 2000; Dobrosotskaya, 2001; Patrie et al., 2001). -Catenin, which binds to E-cadherin at epithelial cell junctions, interacts with both Lin-7 and MAGI-1 and recruits them to cell junctions (Perego et al., 2000; Dobrosotskaya and James, 2000). We have also found that -catenin is involved in the synaptic targeting of S-SCAM, the neuronal isoform of MAGI-1 (Nishimura et al., 2002). These examples suggest that -catenin binds to cadherins and recruits PDZ proteins, which bind other molecules to cell junctions.

PAPIN has six PDZ domains and interacts with p0071. p0071 belongs to a catenin family. By analogy with the -catenin-dependent membrane targeting of MAGI-1 and S-SCAM, we conjectured that p0071 might recruit PAPIN to cell junctions. In the previous study, we detected the signal recognized by the anti-PAPIN antibody on the apical membrane in bronchial epithelial cells. We confirmed the apical localization of PAPIN in MDCK cells using GFP-tagged proteins. The apical localization is not mediated by the interaction with p0071, because p0071 is localized only on the lateral membrane. Furthermore, PAPIN localizes to the plasma membrane, where p0071 is absent, during the formation of cell-cell contacts, in the middle of cytokinesis, and under the condition that p0071 is internalized. We also confirmed that the deletion construct of PAPIN without the p0071-binding PDZ domain in MDCK cells was localized at the cell-cell contact. Because GFP-PAPIN-1 formed a dimer in the glycerol density gradient, we cannot exclude the possibility that the deletion construct is localized at cell-cell contacts through binding to the endogenous PAPIN, which interacts with p0071. However, the overexpressed carboxyl-terminus of p0071 did not affect the localization of GFP-PAPIN-1, suggesting that the interaction of PAPIN with p0071 is not important for the localization of PAPIN at cell-cell contacts. All these data implicate that PAPIN interacts with some component of the plasma membrane other than p0071.

Jaulin-Bastard et al. (2002) explored ERBIN interactors and obtained p0071 and later Izawa et al. (2002b) reported the same interaction. It was also reported that the overexpressed carboxyl-terminus of p0071 disrupts the integrity of cell junctions in HeLa cells. Conversely, we have searched for p0071-interacting molecules and also obtained ERBIN (Ohno's unpublished observation). ERBIN has been originally identified as an ErbB2-interacting protein and has a leucine-rich repeat and a PDZ domain (Borg et al., 2000; Bryant and Huwe, 2000). The molecular organization of ERBIN is similar to those of Densin-180 and Scribble and belongs to a LAP family (Apperson et al., 1996; Bilder and Perrimon, 2000). Izawa et al. (2002a) have reported the complex formation of Densin-180 with NPRAP/-catenin and N-cadherin at synapses. NPRAP/-catenin is very homologous to p0071 and is a neural isoform of p0071. These reports about ERBIN and Densin-180 indicate that the interaction of catenins with a LAP family protein is common for epithelial junctions and synapses and may be important in the organization of cell junctions. Because the localization of ERBIN is identical with that of p0071 under all the tested conditions, we speculated that ERBIN unlike PAPIN might be recruited by p0071 to cell-cell contacts. GFP-ERBIN-2 containing only the N-terminal region, however, is localized like the full-length of ERBIN, suggesting that the interaction with p0071 is not essential for the targeting of ERBIN to cell-cell contacts. GFP-ERBIN-3 that contains the middle and carboxyl-terminal regions is also accumulated at the cell-cell contacts. GFP-ERBIN-5 that contains only the carboxyl-terminal region is partially localized at the cell-cell contacts. Because ERBIN behaves as a monomer in the glycerol density gradient, GFP-ERBIN-2 and GFP-ERBIN-3 are unlikely to interact with the endogenous ERBIN. ERBIN may attach itself to unidentified component(s) of the lateral membrane by more than one region. The deletion construct of p0071 lacking the PDZ-binding motif is also localized at cell junctions (data not shown). Thereby, PAPIN, p0071, and ERBIN come to cell junctions independently, although the molecular mechanism of the targeting of these proteins remains elucidated. The result of the immunoprecipitation from 293T cells suggests that PAPIN, p0071, and ERBIN interact with each other at cell junctions. A recent study has revealed that the message of PAPIN is increased in prostate cancer cells (Chaib et al., 2001). EGF receptor signaling is known to play roles in the tumorigenesis of the prostate gland (Kim et al., 1999). We could not directly detect the complex formation of PAPIN with ErbB2, but the colocalization of GFP-PAPIN-1 with ErbB2 on the lateral membrane implies that PAPIN may interact with ErbB2 through p0071 and ERBIN. The overexpressed PAPIN may modulate ErbB2-dependent signaling and cause prostate cancer.

Materials and methods

Construction of expression vectors

pLGFPC-2 and pLGFPN-2 were generated from pLGFPC (Clontech) and pLGFPN (Clontech), respectively, and described previously (Nishimura et al., 2000). Various expression vectors used in this study were constructed by conventional molecular biology techniques and PCR method using pLGFPC-2, pLGFPN-2, pClneo (Promega), and pGex4T-1 (Amersham Pharmacia Biotech). The cDNA of human ERBIN was obtained by PCR from human brain cDNA. The following constructs contain the following amino acids of PAPIN or ERBIN: pLGFPN PAPIN-1; 1-2766; pLGFPC PAPIN-5, 2298-2766; pLGFPC PAPIN-6, 1-749; pLGFPN PAPIN-15, 1-2489; pLGFPC PAPIN-18, 1511-2538; pGex4T-1 PAPIN-15, 1-340; pLGFPC ERBIN-1, 1-1371; pLGFPC ERBIN-2, 1-413; pLGFPC ERBIN-3, 412-1371; pLGFPC ERBIN-5, 1262-1371; and pGex 4T-1 ERBIN-PDZ, 1262-1371. pLGFPC p0071 and pClneo p0071 contain the full-length of human p0071. We obtained p0071 by PCR using human brain cDNA as a template. The original sequence deposited in GenBank (accession number X81889) encodes the protein with 1211 amino acids. The carboxyl-terminal amino acid sequence is DSWVYDQDAQQRNSFFLTLFRLR (single letter codes indicate amino acids). However, our PCR product terminates after DSWV. The nucleotide sequence of our PCR product is gac | tca | tgg | gtg | tag# , while the original sequence is gac | tca | tgg | gtg | tac# . The difference may be due to the artifact caused by PCR. However, we have searched the database and found that the sequence from human genome (XM043502) also has tag instead of tac. Furthermore, the mouse p0071 (BC004592) terminates with DSWV. Therefore, we consider that the human p0071 is composed of 1192 amino acids and has a canonical PDZ-binding motif. pLGFPC p0071-3 contains the amino acid residues 1-1189 of p0071. pClneo Myc p0071-6 contains the carboxyl-terminal 211 amino acids of p0071.

Antibodies

The rabbit anti-p0071 antibodies were described previously (Deguchi et al., 2000). The mouse monoclonal anti-PAPIN antibody was raised against the product of pGex4T-1 PAPIN-15. The rabbit anti-ERBIN antibody was raised against the product of pGex4T-1 ERBIN-PDZ. The mouse anti-ErbB2 antibody and rhodamine-conjugated secondary antibody for dual labeling were purchased from Chemicon. Mouse anti-Myc antibody 9E10 was obtained from American Type Culture Collection.

Cell culture

MDCK, phoenix ampho, and 293T cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (Life Technologies), 100 units/ml penicillin, and 100 units/ml streptomycin under 5% CO₂ at 37^°C. To generate stable transformants of MDCK cells, phoenix ampho cells were transfected with pLGFP constructs using the mammalian transfection kit (Stratagene). After 48 h culture, the medium was collected and used as a stock of virus. MDCK cells were infected with the virus, and selected with the medium containing 1 g/l of G418 (Sigma-Aldrich). At least two independent stable transformants were analysed for each construct.

Three dimensional culture

Stable transformants of MDCK cells expressing GFP-PAPIN-1 were isolated with trypsin and triturated into a single-cell suspension. Cells were diluted to 1´10⁴ cells/ml in a type I collagen neutral solution (CELLEGEN, Koken, Tokyo, Japan). The culture was incubated at 37°C to allow the collagen solution to gel and then overlayered with medium. Cultures were grown for 6 days, until cysts with a closed monolayer of cells were formed. The images were obtained by a confocal microscopy (Olympus FV300-BX).

Immunocytostaining

MDCK cells were fixed with 4% (w/v) formaldehyde in phosphate buffered saline (PBS) at room temperature for 15 min and blocked with 50 mM glycine in PBS for 30 min. The samples were incubated with 0.2% (w/v) Triton X-100 in PBS for 15 min and subsequently with 1% (w/v) bovine serum albumin in PBS at room temperature for 30 min. The samples were incubated with various first antibodies and visualized with the rhodamine-conjugated secondary antibody. The images were obtained by confocal microscopy (Zeiss LSM 510).

Immunoprecipitation

293T cells were transfected with pClneo p0071 alone or the combination of pLGFPN PAPIN-1 and pClneo p0071 using the mammalian transfection kit (Stratagene). The cells from one 10 cm plate were homogenized in 200 l of the lysis buffer A (25 mM Tris/HCl pH 8.0, 50 mM NaCl, 1% (w/v) Triton X-100, 1% (w/v) sodium deoxycholate, 1 mM EGTA, 2 mM Na₃VO₄, and 50 mM NaF) and centrifuged at 100 000 g for 15 min at 4°C. One hundred and twenty five l of the supernatant was incubated with 2.5 l of the monoclonal anti-PAPIN ascites fixed on 5 l of protein G Sepharose 4 fast flow beads. After the beads were washed, the precipitates were analysed in SDS-PAGE and immunoblotted with the appropriate antibodies.

Glycerol density gradient

Phoenix ampho cells expressing GFP-PAPIN-1 of five 10-cm plates or MDCK cells of two 10-cm plates were homogenized in 0.4 ml of the lysis buffer A containing 2% (v/v) glycerol, and centrifuged at 100 000 g for 30 min. The supernatant was charged onto 4.4 ml of 5 to 15% (v/v) glycerol density gradient in buffer A, overlaid with 0.1 ml of buffer A, and centrifuged at 4°C at 100 000 g for 19 h. Eighteen fractions were collected from the gradient, and analysed by the immunoblotting with the anti-PAPIN antibody and anti-ERBIN antibody.

Acknowledgements

This study was supported by grants-in-aids for Scientific Research and Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science, and Technology, a grant from NOVARTIS Foundation (Japan) for the Promotion of Science (1999), and a grant from Yamanouchi Foundation for Research on Metabolic Disorders (2000). We thank Dr Gary Nolan (Stanford University) for phoenix ampho cells. We also thank Ms C Rokukawa and Ms M Miyahara-Tenkatsu for skilful technical assistance.

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Figure 1 The apical membrane localization of PAPIN in MDCK cells. (A) The fluorescence microscopic image of stable transformants of MDCK cells expressing GFP-PAPIN-1. Bar, 5 m. (B) GFP-PAPIN-1 in MDCK cells in the three-dimensional culture. Stable transformants of MDCK cells expressing GFP-PAPIN-1 were cultured for 6 days in the three-dimensional culture. Bar, 20 m

Figure 2 The localization of PAPIN on the plasma membrane where p0071 is absent. (A) The colocalization of GFP-PAPIN-1 with p0071 at the cell-cell contacts in confluent MDCK cells. Stable transformants of MDCK cells expressing GFP-PAPIN-1 were immunostained with the affinity-purified anti-p0071 antibody. Panel a, GFP-PAPIN-1; panel b, p0071; panel c, the superimposed image of panels a and b. (B) The fluorescence microscopic image of stable transformants of MDCK cells expressing GFP-p0071 that came into contact. GFP-p0071 was accumulated at the border between the cells (arrows). (C) The fluorescence microscopic image of MDCK cells expressing GFP-PAPIN-1 that came into contact. GFP-PAPIN-1 was distributed on the plasma membrane and not concentrated at the cell border. (D) GFP-PAPIN-1 and p0071 during cytokinesis. GFP-PAPIN-1 was diffusely distributed on the plasma membrane, whereas p0071 was accumulated on the contractile ring as revealed by the anti-p0071 antibody. Panel a, GFP-PAPIN-1; panel b, p0071; panel c, the superimposed image of panels a and b. (E) GFP-PAPIN-1 and p0071 in MDCK cells exposed to the low concentration of calcium in DMEM containing 10 mM EGTA for 30 min. GFP-PAPIN-1 remained on the plasma membrane, while the anti-p0071 antibody showed that p0071 was internalized. Panel a, GFP-PAPIN-1; panel b, p0071; panel c, the superimposed image of panels a and b. All bars, 5 m

Figure 3 The subcellular localization of various GFP-PAPIN in MDCK cells. (A) The schematic drawing of various GFP-PAPIN. GFP is indicated as an oval. The PDZ domains are shown as rectangles. The numbers indicate the numbers of the initial and last amino acid residues of each construct. (B) The fluorescence microscopic images of MDCK cells expressing various GFP-constructs. Panel a, GFP-PAPIN-15; panel b, GFP-PAPIN-5; panel c, GFP-PAPIN-6; panel d, GFP-PAPIN-18. All bars, 5 m

Figure 4 The colocalization of ERBIN with p0071 in MDCK cells. (A) The colocalization of GFP-p0071 with ERBIN. Stable transformants of MDCK cells expressing GFP-p0071 were immunostained with the affinity-purified anti-ERBIN antibody. Panel a, GFP-p0071; panel b, ERBIN; panel c, the superimposed image of panels a and b. (B) GFP-p0071 and ERBIN were co localized at the cell-cell borders in MDCK cells that came into contact (arrows). ERBIN was detected by the affinity-purified anti-ERBIN antibody. Panel a, GFP-p0071; panel b, ERBIN; and panel c, the superimposed image of panels a and b. (C) GFP-p0071 and ERBIN were internalized together, when MDCK cells were exposed to the low concentration of calcium in DMEM containing 10 mM EGTA for 30 min. ERBIN was detected by the affinity-purified anti-ERBIN antibody. Panel a, GFP-p0071; panel b, ERBIN; panel c, the superimposed image of panels a and b. All bars, 5 m

Figure 5 The subcellular localization of various GFP-ERBIN in MDCK cells. (A) The schematic drawing of various GFP-ERBIN. GFP is indicated as an oval. The leucine-rich repeat (LRP) and the PDZ domain are shown as hatched and white rectangles, respectively. The numbers indicate the numbers of the initial and last amino acid residues of each construct. (B) The fluorescence microscopic images of MDCK cells expressing various GFP-constructs. Panel a, GFP-ERBIN-2; panel b, GFP-ERBIN-3; panel c, GFP-ERBIN-5. All bars, 5 m

Figure 6 GFP-PAPIN-1 and ERBIN in the glycerol density gradient. The extracts of phoenix ampho cells expressing GFP-PAPIN-1 or MDCK cells were layered onto the linear glycerol density gradient. After centrifugation, 18 fractions were collected from the gradients and analysed. GFP-PAPIN-1 and ERBIN were detected with the anti-PAPIN and the anti-ERBIN antibodies, respectively. Numbers on the top indicate the numbers of fractions. The positions of protein standards (2.0 S equine myoglobin (17.5 kDa); 3.7 S chicken ovalbumin (44 kDa); 7.8 S bovine -globulin (158 kDa); 21.7 S bovine thyroglobulin (670 kDa)) are indicated by arrows. Asterisks show the peak positions of GFP-PAPIN-1 and ERBIN. (A) GFP-PAPIN-1. (B) ERBIN

Figure 7 The interaction of PAPIN with p0071 and ERBIN in 293T cells. 293T cells were transfected with pClneo p0071 alone or pClneo p0071 and pLGFPN PAPIN-1. The extracts of cells were immunoprecipitated with the mouse monoclonal anti-PAPIN ascites and the precipitates were immunoblotted with the anti-PAPIN, anti-p0071, or anti-ERBIN antibody. Lanes 1 and 3, the original extracts; lanes 2 and 4, the immunoprecipitates; lanes 1 and 2, transfected with pClneo p0071 and pLGFPN PAPIN-1; lanes 3 and 4, transfected with pClneo p0071 alone. Top panel, the immunoblot with the anti-PAPIN antibody. Middle panel, the immunoblot with the anti-p0071 antibody. Bottom panel, the immunoblot with the anti-ERBIN antibody

Figure 8 Colocalization of GFP-PAPIN-1 with ERBIN and ErbB2. (A) The colocalization of GFP-PAPIN-1 with ERBIN at the cell-cell contacts in MDCK cells. Stable transformants of MDCK cells expressing GFP-PAPIN-1 were immunostained with the affinity-purified anti-ERBIN antibody. Panel a, GFP-PAPIN-1; panel b, ERBIN; and panel c, the superimposed image of panels a and b. All bars, 5 m. (B) The colocalization of GFP-PAPIN-1 with ErbB2 at the cell-cell contacts in MDCK cells. Stable transformants of MDCK cells expressing GFP-PAPIN-1 were immunostained with the anti-ErbB2 antibody. Panel a, GFP-PAPIN-1; panel b, ErbB2; panel c, the superimposed image of panels a and b. All bars, 5 m