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J. Biol. Chem., Vol. 279, Issue 46, 48079-48084, November 12, 2004
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From the Division of Infectious Diseases, Children's Hospital of Philadelphia, Pennsylvania 19104
Received for publication, August 9, 2004 , and in revised form, September 8, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several PDZ domain-containing proteins have been localized to the cytoplasmic region of the TJ. These include the structurally related membrane-associated guanylate kinase proteins zonula occludens (ZO)-1, ZO-2, and ZO-3, the membrane-associated guanylate kinase inverted proteins, the partitioning proteins, and the multi-PDZ domain protein 1 (MUPP1). These proteins likely function to link TJ-associated transmembrane proteins to intracellular signaling molecules.
The CAR C terminus resembles hydrophobic C-terminal peptide motifs known to interact with PDZ protein domains, such as those contained within membrane-associated guanylate kinase proteins and other scaffolding proteins localized to the TJ region. CAR associates with ZO-1, as demonstrated by coprecipitation from polarized epithelia and by the relocalization of ZO-1 that occurs in CAR-transfected Chinese hamster ovary (CHO) cells (5). CAR may also interact with a component of the adherens junction, 
-catenin, as has been reported in A549 cells (6). In addition, yeast-two-hybrid studies have shown that CAR interacts with Ligand of Numb-X (LNX) (7), a PDZ protein believed to regulate Notch signaling in the central nervous system; however, LNX is not known to associate with TJs (8, 9).
To define the associations mediated by the cytoplasmic tail of CAR, we performed a yeast two-hybrid screen and identified MUPP1 as a CAR-interacting protein. The localization of MUPP1 to the area of the TJ was disrupted when CAR was absent, although the distribution of other TJ-associated components such as ZO-1 remained unchanged. Taken together, these data indicate that CAR interacts directly with MUPP1 and that this interaction is involved in restricting MUPP1 to the cytoplasmic region of the TJ.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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tail), CAR with deletion of its hydrophobic C-terminal motif (CHO-CARAQS), or with vector alone (CHO-pcDNA) were cultured in nucleoside-free 
-minimum essential medium containing 10% dialyzed fetal calf serum (5). Caco-2 cells were cultured in high glucose Dulbecco's modified Eagle's medium containing 20% fetal bovine serum, 1% nonessential amino acids, and penicillin/streptomycin. Cells were plated in 12-mm Transwell-Col inserts (0.4-µm pore size) (Costar) at a density of 5 x 105 cells or in collagen-coated chamber slides at a density of 2 x 104 cells/well.
Plasmids, siRNAs, and Transfections—Plasmid GW1 containing a hemagglutinin (HA)-tagged full-length MUPP1 cDNA and the pGEXMUPP1 (PDZ 12–13) plasmid were kindly provided by Ronald T. Javier (Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX) (10). pGEX-MUPP1 (PDZ10) was provided by Shoichiro Tsukita (Department of Cell Biology, Faculty of Medicine, Kyoto University, Japan) (11). CHO cells were transiently transfected with 2 µg of pGW1-HAMUPP1 using FuGENE 6 according to the manufacturer's protocol (Roche Applied Science) and assayed 48 h later.
Double-stranded siRNAs targeted against human CAR (sense, 5'-GGUGGAUCAAGUGAUUAUU-3' and antisense, 5'-AAUAAUCACUUGAUCCACC-3'), GAPDH, and a control with no similarity to any human gene sequence were designed and synthesized by Ambion (Austin, TX). Caco-2 cells grown on collagen-coated chamber slides were transfected with duplex siRNAs using Oligofectamine (Invitrogen). Cells were assayed 24–48 h following transfection.
Antibodies—CAR-specific antiserum was generated in rabbits and affinity-purified as described (12). Rabbit polyclonal antibodies against MUPP1 were kindly provided by Shoichiro Tsukita and Ronald T. Javier (10, 11). Mouse anti-ZO-1 and rabbit anti-proteasome subunit antibodies were purchased from Zymed Laboratories, Inc.. Antibodies to HA, GAPDH, early endosome antigen-1, Rab7, lysosomal integral membrane proteins-II (LIMP II), and lysosome-associated membrane protein 1 (LAMP I) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-CAR monoclonal antibody RmcB and sheep anti-MUPP1 were purchased from Upstate (Lake Placid, NY). Control MOPC21 was purchased from Sigma. Oregon Green-conjugated anti-HA antibody was purchased from Clontech. Fluorochrome-conjugated secondary antibodies for immunofluorescence were purchased from Jackson ImmunoResearch (West Grove, PA). Horseradish peroxidase-conjugated antibodies were obtained from Santa Cruz Biotechnology.
Yeast Two-hybrid Screening—DNA encoding the mCAR1 cytoplasmic domain (CCHRK 
DGSIV) was inserted into plasmid pGBKT7 (Clontech), in-frame with the yeast GAL4 DNA binding domain. The resulting plasmid (CAR-BD) was used as bait in a yeast two-hybrid screen of an 11-day mouse embryo library constructed in pGADT7 using protocols provided with the Clontech Matchmaker Two-Hybrid System 3. Saccharomyces cerevisiae strain AH109 was transformed with both library and bait plasmids, and 
2.5 x 106 dual transformants were screened for growth on medium deficient in histidine and adenine and for production of -galactosidase. Library plasmids were recovered from putative positive clones and further tested to eliminate any that induced GAL4 activation independently of any bait plasmid, in the presence of binding domain alone, or in the presence of binding domain fused to a nonspecific control protein (human lamin C). Of 11 cDNAs that were obtained, one encoded PDZ domain 13 of MUPP1; the other 10 encoded a PDZ domain derived from another protein.
Immunofluorescence Microscopy—CHO cells plated on glass chamber slides or siRNA-transfected Caco-2 cells on collagen-coated chamber slides were washed in phosphate-buffered saline and fixed in 1% paraformaldehyde in phosphate-buffered saline for 15 min at room temperature, washed, and permeabilized with 1% Triton X-100 for 10 min at room temperature. Cells were incubated with the indicated primary antibodies for 1 h at room temperature, washed, incubated with fluorochrome-conjugated secondary antibodies for 30 min at room temperature, washed, and mounted with Vectashield (Vector Laboratories, Burlingame, CA) containing 4',6-diamidino-2-phenylindole (DAPI). All cells were postfixed with 4% paraformaldehyde. Images were captured with an Olympus fluorescence microscope (Melville, NY).
Caco-2 cells grown on transwell inserts were permeabilized with methanol for 5 min at -20 °C, washed briefly in phosphate-buffered saline, and blocked with 5% bovine serum albumin for 30 min at 37 °C. Cells were incubated with monoclonal anti-CAR RmcB and polyclonal anti-MUPP1 for 1 h at room temperature. Cells were washed and incubated with anti-mouse fluorescein isothiocyanate and anti-rabbit Texas Red in 10% goat serum for 30 min at room temperature, washed, and postfixed with 4% paraformaldehyde. Images were captured with a confocal laser-scanning microscope (Leica, Exton, PA).
Reverse Transcription-PCR—Total RNA was isolated with RNA STAT 60 (Tel-Test, Friendswood, TX) and treated with DNase (Ambion). For complementary DNA synthesis, 1 µg of total RNA was used in a 20-µl reaction containing 1 mM dNTPs, 2.5 mM oligo(dT), 0.1 volume of 10x buffer (supplied by manufacturer), and 2500 units/ml murine leukemia virus reverse transcriptase (Invitrogen). The reverse transcription reaction was carried out at 1 cycle in a thermal cycler at 42 °C for 50 min, followed by a 15-min incubation at 70 °C. PCR was carried out with TaqDNA polymerase (Roche) for 25 cycles with primers to CAR (sense primer 5'-GCT TAG TCC CGA AGA CCA-3' and antisense 5'-GTG GCA CAT CTT CCC TGA-3') and those to GAPDH (sense primer 5'-ACC ACC AAC TGC TTA GCA-3' and antisense 5'-CCC TGT TGC TGT AGC CAA-3') added simultaneously to the PCR. PCR products were separated on a 1.5% agarose gel containing ethidium bromide
Immunoprecipitations and Western Blotting—Lysates of CHO cells transiently transfected with HA-MUPP1 were prepared with 0.1% Triton X-100 extraction buffer containing phenylmethanesulfonyl fluoride. Cells were collected with a rubber policeman in lysis buffer and sonicated briefly. Insoluble material was cleared by centrifugation for 5min at 4 °C. For coimmunoprecipitation of MUPP1 and CAR followed by immunoblotting for CAR, lysates were incubated with an anti-HA monoclonal antibody, anti-CAR monoclonal antibody RmcB, or control monoclonal antibody MOPC 21 for 1 h at 4 °C. For coimmunoprecipitation of MUPP1 and CAR followed by immunoblotting for MUPP1, lysates were incubated with anti-MUPP1 polyclonal antibody, RmcB, or control MOPC 21 overnight at 4 °C. Sepharose G beads were added for an additional 1 h. After centrifugation, the beads were washed in cell lysis buffer and then heated at 95 °C for 10 min in Laemmli sample buffer. Following a brief centrifugation, the supernatant was run on a 4–15% Tris-HCl gel (Bio-Rad) and transferred to a polyvinylidene difluoride membrane. The membrane was blocked overnight in Tris-buffered saline containing Twenn-20 (TBS-T) with 5% milk and probed with rabbit anti-CAR or blocked in 10% donkey serum and probed with anti-sheep MUPP1 followed by horseradish peroxidase-conjugated antibody to rabbit or sheep immunoglobulin (Jackson ImmunoResearch), developed with ECL reagents (Amersham Biosciences), and exposed to film.
Streptag-SUMO-hCAR Fusion Protein—A bacterial expression vector encoding the full-length human CAR cytoplasmic domain was constructed and expressed in a modified pET expression vector (provided by Steven Weeks, Drexel University, Philadelphia, PA) encoding a linear fusion consisting of an N-terminal streptactin-tag (IBA-GmbH) for affinity purification followed by SUMO (Invitrogen) linked to the cytoplasmic domain of CAR. The cytoplasmic domain of CAR was amplified by PCR and cloned into the modified pET vector between a BSAI site created at the 3'-end of SUMO, and an XhoI site using the following primers, sense primer, 5'-TTTTTTGGTCTCACATGTGCTGTCGTAAAAAGCGC-3' and antisense primer, 5'-TTTTTTCTCGAGCTATACTATAGACCCATCCTTGCT-3'.
The pET-CAR expression vector was introduced into competent Escherichia coli Rosetta (DE3) cells (Novagen). Overnight cultures grown in LB were diluted 1:5 into a final culture volume of 25 ml. At an A600 of 0.6 the culture was exposed to 1 mM isopropyl-1-thio--D-galactopyranoside (Fisher), and bacteria were collected after 4 h. The bacterial cell pellet was resuspended in phosphate-buffered saline containing 1 mM phenylmethanesulfonyl fluoride, 0.5 M NaCl, and 1% Triton X-100. The cells were lysed by sonication, and the cell debris was removed by centrifugation at 10,000 rpm for 10 min. The supernatant was incubated with 2 ml of a 50% slurry of Streptactin-MacroPrep beads (IBA-GmbH) for 2 h. at 4 °C. The beads were washed four times with cold phosphate-buffered saline, and the protein was eluted from the beads with 2.5 mM desthiobiotin. The desthiobiotin was removed by dialyzing the protein into 20 mM Hepes, pH 6.7.
In Vitro Binding Assays—GST fusion proteins expressed in E. coli were purified using glutathione-Sepharose 4B beads (Amersham Biosciences). SUMO or SUMO-hCAR (1 µg) was added to a pull-down buffer (1% Triton X-100, 100 mM NaCl, 0.2 mM phenylmethanesulfonyl fluoride, 5% fetal bovine serum) and incubated with GST, GSTMUPP1PDZ10, or GST-MUPP1PDZ12–13 bound to glutathione-Sepharose 4B beads for 1 h at 4 °C. Beads were sedimented by centrifugation, washed in pull-down buffer, and heated at 95 °C for 10 min in Laemmli sample buffer. Following a brief centrifugation, the supernatant was run on a 4–15% Tris-HCl gel and transferred to a polyvinylidene difluoride membrane. The membranes were blocked overnight in 5% bovine serum albumin at 4 °C and incubated with anti-Streptavidin monoclonal antibody. Alternatively, samples were run on 4–15% gels and stained with Coomassie Blue to control for loading of GST and GST fusion proteins.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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tail) or deletion of the C-terminal DGSIV motif (CARAQS), was expressed throughout the cytoplasm, with no localization to cell-cell contacts (Figs. 4A). Unlike full-length CAR, CARtail and CARAQS did not coprecipitate with MUPP1, and MUPP1 did not coprecipitate with truncated CAR (Fig. 3). Thus, the association between CAR and MUPP1, and recruitment of MUPP1 to sites of cell contact, depends on the presence of the putative PDZ-binding motif at the CAR C terminus. Given the results of the yeast two-hybrid screen, and the in vitro pull-down experiment shown above, it is likely that CAR association with MUPP1 involves an interaction between the C-terminal motif and MUPP1 PDZ domain 13. Localization of MUPP1 in the Absence of CAR—The experiments in CHO cells indicated that CAR recruits MUPP1 to sites of contact between transfected CHO cells. To determine whether endogenous CAR is involved in MUPP1 recruitment to the tight junction of polarized epithelial cells, we used RNA interference technology to silence CAR expression. siRNAs specific to CAR were transiently transfected into Caco-2 cells. RT-PCR was performed at 48-h post-transfection to determine the extent of CAR mRNA knockdown by siRNA transfection. Caco-2 cells transfected with a control siRNA with no sequence similarity to any human gene sequence showed a high level of CAR expression, indicating that transfection of cells with a nonspecific siRNA had no effect on CAR mRNA expression (Fig. 5A, right). However, transfection of cells with a siRNA specific for CAR significantly reduced endogenous CAR mRNA levels (Fig. 5A, left). To determine whether the decrease of mRNA levels resulted in a reduction in protein expression, control and CAR siRNA-transfected lysates were subjected to Western blot analysis. CAR protein expression was significantly reduced in CAR siRNA-transfected cells compared with controls (Fig. 5B). Immunoblots were stripped and reprobed for GAPDH to control for equal protein loading. The residual levels of CAR mRNA and protein most likely reflect the limitation of silencing by transient transfection in this cell type.
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-catenin (data not shown).
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To determine whether these bodies resulted from the relocalization or recycling of MUPP1 through endocytic vesicles or from its being targeted for degradation, we attempted to stain MUPP1 intracellular bodies with antibodies specific for endosomal markers and for components of the ubiquitin degradation pathway. MUPP1-positive bodies did not express the early endosome marker early endosome antigen 1 (13), the late endosome marker Rab7 (14), the lysosomal markers LIMP II (Fig. 7), or the late endosome/lysosomal marker LAMP I (data not shown) (15). In addition, MUPP1-positive bodies did not colocalize with internalized fluorochrome-labeled dextran (data not shown), further indicating that MUPP1 was not relocalized to endocytic compartments. MUPP1-positive intracellular bodies did not stain with antibodies specific for ubiquitin or the 26 S proteasome (data not shown). These data indicated that CAR is involved in the localization of MUPP1 to the TJ and that in the absence of CAR, MUPP1 may be retargeted to specialized vesicles within the cytoplasm.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MUPP1 localizes to the TJ and interacts with junctional adhesion molecule and with members of the claudin family of transmembrane proteins (11, 16). The loss of CAR within the TJ by siRNA transfection led to pronounced relocalization of MUPP1 from the apical TJ complex to punctate bodies within the apical domain. We were unable to identify or define the nature of the MUPP1 bodies, and it remains unclear whether they result from recycling of MUPP1 from the TJ or the whether targeting of MUPP1 to the TJ is disrupted in the absence of CAR. MUPP1 is known to interact with the junctional adhesion molecule and claudin-1, and it is not known whether loss of these components would have a similar effect. The evidence in CHO cells indicates that CAR is capable of recruiting MUPP1 to sites of cell contact and therefore may indicate that loss of CAR leads to improper targeting of MUPP1 to the TJ. Other junctional proteins including junctional adhesion molecule, claudin-1, and ZO-1 have been shown to reside in unique storage organelles following a calcium switch (17). Therefore, it is possible that the absence of CAR leads to the destabilization of the junction and relocalization of MUPP1 to similar organelles. The loss of membrane-associated MUPP1 has been postulated to correlate with the transforming ability of two viral oncoproteins, adenovirus E4-ORF1 and high risk Papillomavirus type 18 E6 (10). Interestingly, in the presence of E4-ORF1, MUPP1 has been noted to relocate to intracellular bodies within the cytoplasm similar in appearance to those induced by silencing of endogenous CAR.
In addition to TJ proteins, MUPP1 interacts with several important mediators of cellular signaling, which may indicate its role as an adaptor for intracellular signaling molecules. By yeast two-hybrid screening, MUPP1 interacts with serotonin 5-hydroxytryptamine type 2C receptors (18, 19), kinase negative c-Kit (20), the tandem plextrin homology domain protein (21), and the membrane-spanning NG2 proteoglycan (22).
In several human tumors, CAR expression is down-regulated during progression to malignancy (23–25), and the expression of transfected CAR in CAR-deficient tumor cells leads to alterations in cell cycle regulation and decreased proliferation (23). It is interesting to note that MUPP1 is targeted by viral transforming proteins and that other junctional PDZ proteins regulate cell proliferation (26, 27). It remains to be determined whether the effects of CAR on cell proliferation are mediated through its interaction with MUPP1 or other junctional proteins.
It has been reported that intercellular junctions are disrupted when adenovirus fiber is applied to the basal surface of a polarized epithelial monolayer (6). The mechanism by which this occurs has not been determined, and it will be interesting to learn whether CAR interactions with TJ proteins are affected during the process of virus entry into cells.
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FOOTNOTES |
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To whom correspondence should be addressed: 1202 Abramson Research Center, Children's Hospital of Philadelphia, 3615 Civic Center Blvd., Philadelphia, PA 19104. Tel.: 215-590-3771; Fax: 215-590-2025; E-mail: bergelson{at}email.chop.edu.
1 The abbreviations used are: CAR, coxsackievirus and adenovirus receptor; TJ, tight junction; ZO, zonula occludens; MUPP1, multi-PDZ domain protein 1; CHO, Chinese hamster ovary; siRNA, small interfering RNA; HA, hemagglutinin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LAMP I, lysosome-associated membrane protein 1; LIMP II, lysosomal integral membrane proteins II; GST, glutathione S-transferase; hCAR, human CAR; mCAR, mouse CAR.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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