Protein 4.1N Is Required for Translocation of Inositol 1,4,5-Trisphosphate Receptor Type 1 to the Basolateral Membrane Domain in Polarized Madin-Darby Canine Kidney Cells*,

Songbai ZhangDagger §, Akihiro MizutaniDagger , Chihiro HisatsuneDagger , Takayasu HigoDagger , Hiroko BannaiDagger , Tomohiro NakayamaDagger ||, Mitsuharu HattoriDagger , and Katsuhiko MikoshibaDagger **DaggerDagger

From the Dagger  Division of Molecular Neurobiology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, the  Laboratory for Developmental Neurobiology, Brain Science Institute, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama 351-0198, the || Department of Pediatrics, Tokyo Women's Medical University, Tokyo 162-8666, and the ** Calcium Osciallation Project, ICORP, Japan Science and Technology Corporation, Tokyo 108-0071, Japan

Received for publication, September 29, 2002, and in revised form, November 18, 2002

    ABSTRACT
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MATERIALS AND METHODS
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Protein 4.1N was identified as a binding molecule for the C-terminal cytoplasmic tail of inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) using a yeast two-hybrid system. 4.1N and IP3R1 associate in both subconfluent and confluent Madin-Darby canine kidney (MDCK) cells, a well studied tight polarized epithelial cell line. In subconfluent MDCK cells, 4.1N is distributed in the cytoplasm and the nucleus; IP3R1 is localized in the cytoplasm. In confluent MDCK cells, both 4.1N and IP3R1 are predominantly translocated to the basolateral membrane domain, whereas 4.1R, the prototypical homologue of 4.1N, is localized at the tight junctions (Mattagajasingh, S. N., Huang, S. C., Hartenstein, J. S., and Benz, E. J., Jr. (2000) J. Biol. Chem. 275, 30573-30585), and other endoplasmic reticulum marker proteins are still present in the cytoplasm. Moreover, the 4.1N-binding region of IP3R1 is necessary and sufficient for the localization of IP3R1 at the basolateral membrane domain. A fragment of the IP3R1-binding region of 4.1N blocks the localization of co-expressed IP3R1 at the basolateral membrane domain. These data indicate that 4.1N is required for IP3R1 translocation to the basolateral membrane domain in polarized MDCK cells.

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The inositol 1,4,5-trisphosphate receptor (IP3R)1 is an intracellular IP3-gated calcium (Ca2+) release channel that plays pivotal roles in fundamental processes, such as fertilization, cellular proliferation and differentiation, and vesicle secretion (1, 2). Three distinct types of IP3R (types 1-3) have been cloned (3). Three functional domains of the most characterized type 1 IP3R (IP3R1), a 2749-amino acid polypeptide, have been studied intensively. The N-terminal portion (residues 1-578) is a ligand-binding domain (4, 5). The middle portion (residues 579-2275) is the modulatory domain for various intracellular modulators (Ca2+, calmodulin, and ATP) and for phosphorylation by several protein kinases (cAMP-dependent protein kinase, protein kinase C, cGMP-dependent protein kinase, Ca2+/calmodulin-dependent protein kinase II, and tyrosine kinase) (6-10). This portion also transmits inositol 1,4,5-trisphosphate binding information necessary for channel opening (11). Near the C terminus is a clustered six membrane-spanning channel domain (residues 2276-2589) (12). However, the function of the C-terminal cytoplasmic tail (residues 2590-2749) has not been fully clarified.

To study the function of the C-terminal cytoplasmic tail of IP3R1, we searched for molecules binding the C-terminal cytoplasmic tail using a yeast two-hybrid system. Protein 4.1N, a homologue of the erythrocyte membrane cytoskeleton protein 4.1, was identified as a binding molecule for the C-terminal cytoplasmic tail of IP3R1. Protein 4.1, originally identified in red blood cells and called red blood cell protein 4.1 (4.1R), plays a critical role in the morphology and mechanical stability of the red blood cell plasma membrane (13). Three structural/functional domains have been identified in 4.1R. The N-terminal membrane-binding domain (also called the 30-kDa or FERM domain) (14) possesses binding sites for the cytoplasmic tails of integral membrane proteins such as band 3 (15, 16), glycophorin C (17, 18), and CD44 (19). An internal domain contains the spectrin-actin binding (also called the 10-kDa domain) activity required for membrane stability (20-22). The C-terminal domain (CTD, also called the 22-24-kDa domain) has recently been reported to bind to tight junction proteins, ZO-1 and ZO-2 (23), to an immunophilin, FKBP13 (24), and to nuclear mitotic apparatus protein, which appears to mediate the spindle formation in nucleated cells (25). Three homologues of 4.1R have been cloned: the widely expressed homologue 4.1G, the neuronal homologue 4.1N, and the brain homologue 4.1B. They share a high degree of homology with prototypical homologue 4.1R in the three structural/functional domains. Each 4.1 protein is characterized by three unconserved unique domains between the conserved membrane-binding domain, the spectrin-actin binding domain, and the CTD (26).

In this study, we found that 4.1N and IP3R1 associated in both subconfluent and confluent Madin-Darby canine kidney (MDCK) cells, a well studied tight polarized epithelial cell line. Both were predominantly translocated to the basolateral membrane domain when MDCK cells grew from subconfluence to confluence, whereas 4.1R, the prototypical homologue of 4.1N, was localized at the tight junction (23), and other endoplasmic reticulum (ER) marker proteins were still present in the cytoplasm in confluent MDCK cells. The localization of IP3R1 at the basolateral membrane domain was determined by its 4.1N-binding region and could be blocked by a fragment of the IP3R1-binding region of 4.1N. These data suggest that 4.1N serves to regulate IP3R1 subcellular localization.

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Plasmid Construction-- All of the plasmids were propagated in the Escherichia coli strain HB101. All PCR products of the cDNA fragments were generated in frame using Platinum® Pfx DNA polymerase (Invitrogen) and were verified by nucleotide sequencing using an ABI PRISM 377 automated sequencer (Applied Biosystems). cDNA encoding the C-terminal cytoplasmic tail of IP3R1 (IP3R1/CTT, aa 2590-2749) were generated by PCR from mouse IP3R1 cDNA and subcloned into the site of EcoRI and BamHI of pGBT9 (Clontech) to generate pGBT9-IP3R1/CTT, into the site of EcoRI and BamHI of pEGFP-C3 (Clontech) to generate GFP-IP3R1/CTT, and into the site of BamHI and EcoRI of pGEX-KG (27) to generate GST-IP3R1/CTT. Truncated constructs corresponding to different lengths of IP3R1/CTT (see Fig. 3A) were subcloned into the site of EcoRI and BamHI of pGBT9. GFP-IP3R1-N was generated by subcloning full-length mouse IP3R1 fusing with an EGFP cDNA in its N terminus into pcDNA3.1/Zeo+ (Invitrogen).2 GFP-IP3R1/Delta 18A10 was generated by replacing the fragment consisting of EcoRI to XhoI site (aa 2216-2749) in GFP-IP3R1-N with a PCR product consisting of aa 2216-2737 in mouse IP3R1. GFP-18A10 was generated by subcloning a synthesized cDNA fragment consisting of aa 2738-2749 in mouse IP3R1 into the site of EcoRI and BamHI in pEGFP-C3 (the terminological base for GFP-IP3R1/Delta 18A10 and GFP-18A10 is that the last 14 residues of IP3R1 contain the specific recognition site by 18A10 antibody) (28). pBact-STneoB-C1 (used to express IP3R1) and EGFP-SERCA2a were as described previously (29, 30). To construct the ER marker, DsRed2-KDEL, we fused the N-terminal 17 amino acids of calreticulin to the N terminus of DsRed2 (Clontech) and the ER target and retention signal, KDEL, to its C terminus by a two-step PCR as was described previously (31, 32). The resulting PCR products were subcloned into pcDNA3.1/Zeo+ (Invitrogen).

The mouse 4.1N cDNA was obtained by PCR using the primers of 5'-ATCGGAATTCATGACAACAGAGACAGGT-3' and 5'-ATCGTCTAGATCAGGATTCCTGTGGCTT-3' (the underlined letters indicate the EcoRI and XbaI sites for cloning, respectively) corresponding to full-length of mouse 4.1N sequence (accession number AF061283) using mouse cerebellum cDNA library as template and subcloned into the site of EcoRI and XbaI in pcDNA3.1/Zeo+ (Invitrogen) (pcDNA3-4.1N). The pcDNA3-HA4.1N/FL was generated by replacing the cDNA fragment of EcoRI to EcoRV consisting of aa 1-298 of 4.1N in pcDNA3-4.1N with a PCR product that contains a corresponding sequence and is amplified using a 5'-primer containing the HA tag sequence and EcoRI site and a 3'-primer containing an EcoRV site. The pcDNA3-Venus-4.1N/FL was generated by inserting a PCR product amplified using the Venus cDNA (33) as template into the site of BamHI and EcoRI in pCDNA3-4.1N. The pcDNA3-HA4.1N/Delta CTD and the pcDNA3-Venus-4.1N/Delta CTD (aa 1-766) were generated by replacing a 4.1N fragment consisting of aa 663-879 in pcDNA3-HA4.1N/FL and in pcDNA3-Venus-4.1N/FL (ApaI-ApaI), respectively, with a PCR product consisting of aa 663-766 in 4.1N. The pcDNA3-HA4.1N/CTD (aa 767-879) was generated by subcloning a PCR product of 4.1N/CTD amplified using a 5'-primer containing the EcoRI site and the HA tag sequence and a 3'-primer containing XbaI site and stop codon into the site of EcoRI and XbaI in pcDNA3.1/Zeo+. pcDNA3-Venus-4.1N/CTD (aa 767-879) was generated by subcloning two PCR products of Venus (BamHI-EcoRI fragment) and 4.1N/CTD (EcoRI-XbaI fragment) into the sites of BamHI and XbaI in pcDNA3.1/Zeo+. GST-4.1N/CTD was generated by subcloning a PCR product of 4.1N/CTD into the site of BamHI and EcoRI in pGEX-KG (27). Truncated constructs corresponding to different lengths of 4.1N (see Fig. 4A) were subcloned into the sites of EcoRI and BamHI in pGAD424 (Clontech).

Yeast Two-hybrid Assay-- The GAL4-based MATCHMAKER two-hybrid system II (Clontech) was used for the yeast two-hybrid assays. Plasmid vectors, pGBT9, and pGAD424, encoding the GAL4 DNA-binding domain and the GAL4 activating domain, respectively, were used to express hybrid proteins. To screen for proteins that interact with the C-terminal cytoplasmic tail of IP3R1 (IP3R1/CTT), a mixture of embryonic and adult human brain cDNA libraries (both from Clontech) in GAL4 activating domain vector pACT2 was screened using the C-terminal cytoplasmic tail of mouse IP3R type 1 cloned into GAL4 DNA-binding domain vector pGBT9 (see "Plasmid Construction" for details) as bait in PJ69-4A yeast. Positive clones were tested further for specificity by co-transformation into yeast either with pGBT9-IP3R1/CTT or with pGBT9 alone. DNA from positive clones were isolated, and the GAL4 activating domain plasmids were recovered in E. coli strain HB101 and sequenced. For binding region mapping, yeast was co-transformed with plasmids carrying respective inserts fused to GAL4 DNA-binding domain or GAL4 activating domain and were assayed for nutritional selection of drop-out leucine, tryptophan, adenine, and histidine and for beta -galactosidase activity on nitrocellulose filters as described in the Clontech manual.

Antibodies-- For production of anti-4.1N antibodies, a nucleotide sequence corresponding to amino acid residues 588-790 of mouse 4.1N, which has no homology to the other members of the 4.1 family, was subcloned into pRSET-C (Invitrogen). E. coli strain BL21 (DE3) was transformed with this plasmid, and His6-tagged protein was expressed and then purified over nickel columns. Japanese white rabbits and Wistar rats were immunized with the fusion protein. The rabbit antiserum was affinity-purified against GST-4.1N fragment (aa 588-790) covalently coupled to CNBr-activated Sepharose 4B (Amersham Biosciences) according to standard protocols. The anti-IP3R1 rat monoclonal antibodies 18A10, 4C11, and 10A6 and the anti-IP3R1 mouse monoclonal antibody KM1112 were described previously (34-36). H1L3 is a rabbit polyclonal anti-IP3R1 antibody prepared using a purified fusion protein corresponding to the residues 2463-2536 of mouse IP3R1 expressed in E. coli.3 Anti-ZO1 antiserum (T8754) was a generous gift from Dr. S. Tsukita of Kyoto University. Anti-GFP monoclonal antibody and anti-HA polyclonal antibody were purchased from Medical & Biological Laboratory, Ltd., and anti-Na,K-ATPase alpha 1 monoclonal antibody was from Upstate Biotechnology.

Cell Culture and Transfection-- COS-7 and MDCK cells were maintained in Dulbecco's modified Eagle's medium (Nacalai Tesque) supplemented with 10% heat-inactivated fetal bovine serum. For subconfluent and confluent MDCK cells, MDCK cells were plated at 0.6-1.0 × 105 and 2.25 × 105 cells, respectively, on 18 × 18 mm poly-L-lysine-coated coverslips in a 35-mm culture dish and cultured for 1 and 5 days, respectively. The transfections were performed using LipofectAMINE 2000 reagent (Invitrogen) according to the manufacturer's protocol. Transfected COS-7 cells were harvested 1 day after transfection. Transfected MDCK cells on coverslips were fixed 3 days after transfection and then were processed for immunofluorescence staining with antibodies as described below.

Co-immunoprecipitation, Pull-down Binding Assay, and Immunoblotting-- Lysates of subconfluent and confluent MDCK cells and of transformed E. coli, transfected COS-7 cells and whole brain of 8-week-old male ICR mice (Japan SLC, Inc. (Shizuoka Ken, Japan)) were prepared as previously described (37) in regard to the preparation the lysate of HEK293 cells and about preparation of P2 fraction from brain tissue, respectively. Co-immunoprecipitation from lysate of MDCK cells and lysate of whole mouse brain was performed as previously described (37). Pull-down binding assay was performed as a modification from the co-immunoprecipitation protocol. Briefly, lysates of E. coli and transfected COS-7 cells were first solubilized in 1% sodium deoxycholate at 36 °C for 30 min, followed by adding 0.1 volume of 1% Triton X-100 in 50 mM Tris-Cl, pH 9.0, and the preparations were centrifuged for 30 min at 100,000 × g. The supernatants were then used for in vitro binding assay. For each reaction, 1,000 µg of protein of solubilized lysate of E. coli was incubated with 30 µl of 1:1 slurry of glutathione-Sepharose 4B (Amersham Biosciences) at 4 °C for 2 h and then washed with washing buffer (4 mM Hepes, 150 mM NaCl, 0.5% Triton X-100) three times, and then the spun down complex of glutathione-Sepharose 4B with fusion protein was used. At the same time, 500 µg of protein of solubilized lysate of transfected COS-7 cells was incubated with 25 µl of 1:1 slurry of glutathione-Sepharose 4B at 4 °C for 1 h to clear any nonspecific binding to beads from the lysates. The cleared supernatant of the lysate was then added to the glutathione-Sepharose 4B-protein complex, and the mixture was incubated for 2 h or overnight at 4 °C. The complex was then spun down and washed with washing buffer three times. The proteins were eluted by boiling in 1× SDS-PAGE sample buffer for 3 min and were separated by SDS-PAGE. The proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA), and the membranes were probed with anti-GFP Ab, anti-4.1N Ab, 18A10 Ab, 4C11 Ab, or anti-HA Ab.

Fluorescence and Confocal Microscopy-- The cells on coverslips were fixed in freshly prepared 1.75% paraformaldehyde in cell culture medium for 15 min at room temperature. Then the cells were washed three times with PBS, permeabilized with 0.2% Triton X-100 in PBS for 5 min at room temperature, blocked with 2% normal goat serum in PBS for 1 h at room temperature, and washed three times with PBS. The cells were then incubated with primary antibodies (rabbit anti-4.1N Ab, H1L3 Ab (for endogenous IP3R1 in MDCK cells), KM1112 Ab (for exogenously expressed IP3R1), and anti-Na,K-ATPase alpha 1 Ab; final concentration, 2 µg/ml; anti-ZO-1 antiserum, 1:1; rat anti-4.1N antiserum (only used together with H1L3 Ab for dual immunostaining of endogenous 4.1N and IP3R1 in MDCK cells); 1:50) for 1 h at room temperature and washed three times as described above. They were then incubated with suitable Alexa Fluor 488, Alexa Fluor 594 secondary antibodies, or Alexa 594-phalloidin (Molecular Probes) at room temperature for 1 h and washed three times again as described above. Coverslips were mounted using Vectashield mounting medium (Vector Laboratories). Fluorescence images were taken using a confocal scanning microscope (FV-300, Olympus, Tokyo, Japan) attached to an inverted microscope (I × 70; Olympus) with a 60× objective.

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Identification of 4.1N as a Binding Protein for the C-terminal Cytoplasmic Tail of IP3R1-- To understand the function of the C-terminal cytoplasmic tail of IP3R1, a yeast two-hybrid screening was performed to search for binding molecules of the C-terminal cytoplasmic tail of IP3R1. Using a fusion protein of the GAL4 DNA-binding domain with the C-terminal cytoplasmic tail of IP3R1 (IP3R1/CTT; Fig. 1A) as bait, approximately ~4.0 × 106 yeast transformants were screened with a mixture of embryonic and adult human brain cDNA libraries fused to the GAL4 activation domain. By nutritional selection assay, 19 positive clones were obtained, and their cDNA inserts were sequenced. Among them, seven clones encoded sequences corresponding to various lengths of the C-terminal portion of 4.1N. The sequence of the shortest fragment corresponded to the C-terminal domain (amino acid residues 767-879) of mouse 4.1N (Fig. 1B; hereafter, this fragment is referred to as 4.1N/CTD). The interaction between 4.1N and IP3R1 was confirmed by a colony lift filter assay for beta -galactosidase activity.


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Fig. 1.   Schematic representation of mouse IP3R1 and corresponding 4.1N polypeptides found to interact in the yeast two-hybrid screening. A, the organization of mouse IP3R1 and its C-terminal cytoplasmic tail (aa 2590-2749) employed as bait in the yeast two-hybrid assay. B, a schematic representation of the structural organization of mouse 4.1N and the shortest corresponding segment encompassed by positive clones obtained as prey in the yeast two-hybrid screening. MBD, membrane-binding domain; SAB, spectrin-actin binding domain; U1, U2, and U3, unique domains 1, 2, and 3, respectively.

4.1N and IP3R1 Interact in Vitro and in Mouse Brain-- To further verify the interaction between 4.1N and IP3R1, a pull-down assay was performed using a fusion protein of GST and 4.1N/CTD (GST-4.1N/CTD). GST-4.1N/CTD attached to glutathione-Sepharose beads was incubated with the lysates of COS-7 cells transiently expressing the GFP-tagged C-terminal cytoplasmic tail of IP3R1 (GFP-IP3R1/CTT) or GFP alone. After extensive washing, proteins bound to GST-4.1N/CTD were separated by SDS-PAGE and probed with anti-GFP antibody. As shown in Fig. 2A, GST-4.1N/CTD bound to GFP-IP3R1/CTT but not to GFP. GST alone did not bind to GFP-IP3R1/CTT. These results indicate that 4.1N/CTD also binds to the C-terminal cytoplasmic tail of IP3R1 in vitro.


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Fig. 2.   4.1N binds to IP3R1 in vitro and in vivo. A, GST-4.1N/CTD or GST attached to glutathione-Sepharose beads were incubated with the lysates of COS-7 cells transiently expressing GFP-IP3R1/CTT or GFP. The applied and pull-down proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue (CBB) or probed with anti-GFP antibody. The bottom and middle panels show applied proteins. The top panel shows pull-down protein. GFP-IP3R1/CTT bound to GST-4.1N/CTD, but GFP did not; GFP-IP3R1/CTT did not bind to GST. B and C, 4.1N binds to IP3R1 in vivo. The lysate of whole mouse brain was solubilized and immunoprecipitated with anti-IP3R1 and anti-4.1N antibodies, respectively. The input and immunoprecipitated proteins were separated by SDS-PAGE and probed with anti-4.1N and anti-IP3R1 antibodies. B, 4.1N was co-immunoprecipitated by three rat anti-IP3R1 antibodies, 4C11, 18A10, and 10A6 antibodies but not by normal rat IgG. C, IP3R1 was co-immunoprecipitated by a rabbit anti-4.1N antibody but not by normal rabbit IgG. WB, Western blot.

To determine whether 4.1N binds to IP3R1 in vivo, lysates of whole mouse brain were subjected to co-immunoprecipitation with three distinct rat anti-IP3R1 monoclonal antibodies or a rabbit anti-4.1N polyclonal antibody. Co-precipitated proteins were separated by SDS-PAGE and probed with anti-4.1N and anti-IP3R1 antibodies. As shown in Fig. 2B, two bands that migrate at 135 and 100 kDa were detected with anti-4.1N antibody in the input sample and the pellets immunoprecipitated with anti-IP3R1 antibodies. These signals disappeared when the primary antibody was preincubated with the antigen protein (data not shown). The 135-kDa protein is the prototypical product of gene 4.1N. The 100-kDa protein is an isoform abundant in peripheral tissue, as reported by Walensky et al. (38). Conversely, when the reciprocal immunoprecipitation was performed using a rabbit anti-4.1N antibody, IP3R1 was co-immunoprecipitated (Fig. 2C). In the negative control experiments, neither normal rat IgG nor normal rabbit IgG immunoprecipitated 4.1N and IP3R1. Taken together, these observations indicate that 4.1N interacts with IP3R1 in vivo.

The Last 14 Amino Acids of IP3R1 Are Necessary and Sufficient for Binding to 4.1N-- To determine the minimal sequence responsible for IP3R1 binding to 4.1N, serial deletions of the C-terminal cytoplasmic tail of IP3R1 (Delta 1-Delta 5) in pGBT9 were constructed, and their associations with 4.1N/CTD were examined using a yeast two-hybrid system. As shown in Fig. 3A, whereas the C-terminal cytoplasmic tail of IP3R1 interacted with 4.1N/CTD, none of the deletion mutants interacted with 4.1N/CTD. These results indicate that the last 14 amino acids of IP3R1 are necessary for binding to 4.1N.


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Fig. 3.   The last 14 residues of IP3R1 are necessary and sufficient for binding to 4.1N. A, the C-terminal cytoplasmic tail of IP3R1 and its various lengths of truncations were subcloned into pGBT9 and co-transformed with pACT2-4.1N/CTD into yeast. Both beta -galactosidase activity and nutritional selection assay showed that although the full-length IP3R1/CTT interacted with 4.1N/CTD, none of the deletion mutants (Delta 1-Delta 5) did. B, GST-4.1N/CTD or GST attached to glutathione-Sepharose beads were incubated with the lysates of COS-7 cells transiently expressing GFP-IP3R1-N, or GFP-IP3R1/Delta 18A10, or GFP-18A10. The applied and pull-down proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue (CBB) or probed with 18A10 antibody and/or anti-GFP antibody. The bottom panel shows applied GST-4.1N/CTD or GST. The middle four panels show applied GFP-IP3R1-N, GFP-IP3R1/Delta 18A10, and GFP-18A10. The top two panels show pull-down proteins. Both GFP-IP3R1-N and GFP-18A10 were pulled down by GST-4.1N/CTD, but GFP-IP3R1/Delta 18A10 was not. Neither GFP-IP3R1-N nor GFP-18A10 was pull-down by GST. WB, Western blot.

A pull-down assay was then performed to confirm the yeast two-hybrid results. GST-4.1N/CTD fusion protein was incubated with the lysates of COS-7 cells transiently expressing GFP-IP3R1-N (GFP-tagged full-length IP3R1), GFP-IP3R1/Delta 18A10 (GFP-tagged IP3R1 lacking the last 14 amino acids), or GFP-18A10 (the GFP-tagged last 14 amino acids of IP3R1). Expression of GFP-IP3R1-N, GFP-IP3R1/Delta 18A10, and GFP-18A10 was confirmed by Western blotting assay using both anti-GFP antibody and 18A10 antibody. As shown in Fig. 3B, both GFP-IP3R1-N and GFP-18A10 were pulled down by GST-4.1N/CTD, but GFP-IP3R1/Delta 18A10 was not. Neither GFP-IP3R1-N nor GFP-18A10 was pulled down by GST. Taken together, these results clearly indicate that the last 14 amino acids of IP3R1 are necessary and sufficient for binding to 4.1N. Additionally, because the last 14 amino acids of IP3R1, which contain the specific recognition site by 18A10 antibody of IP3R1 (28), share no homology with IP3R2 and IP3R3, these data support the result of a yeast two-hybrid assay that 4.1N/CTD does not bind to the C-terminal cytoplasmic tails of either IP3R2 or IP3R3 (data not shown) and suggest that 4.1N specifically binds to IP3R1.

The CTD Domain of 4.1N Is Necessary and Sufficient for Binding to IP3R1-- The results of the yeast two-hybrid screening showed the shortest fragment of 4.1N required for binding to IP3R1 to correspond to amino acid sequence 767-879 of mouse 4.1N (Fig. 1B). To test whether other parts of 4.1N also bind to IP3R1 and to more precisely determine the IP3R1-binding site of 4.1N, serial deletions of 4.1N in pGAD424 were constructed, and their associations with IP3R1/CTT were examined using a yeast two-hybrid system. As shown in Fig. 4A, the N-terminal fragment of 4.1N corresponding to amino acids 1-766 (4.1N/Delta CTD) did not interact with the C-terminal cytoplasmic tail of IP3R1. The C-terminal domain of 4.1N (amino acids 767-879) interacted with IP3R1/CTT. However, the C-terminal domain fragment consisting of amino acids 784-879 did not.


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Fig. 4.   The CTD domain of 4.1N is necessary and sufficient for binding to IP3R1. A, the various truncations of 4.1N were subcloned into pGAD424 and co-transformed with pGBT9-IP3R1/CTT into yeast. Both beta -galactosidase activity and nutritional selection assay showed that the deletions of the C-terminal domain of 4.1N consisting of aa from 767, from 777, or from 783 to 879 interacted with IP3R1/CTT, and the fragment of 4.1N/Delta CTD (aa 1-766) and those deletions of the C-terminal domain of 4.1N consisting of aa from 784, or from 789 to 879 did not. B, GST-IP3R1/CTT or GST attached to glutathione-Sepharose beads were incubated with the lysates of COS-7 cells transiently expressing HA4.1N/FL, HA4.1N/Delta CTD (1-766), or HA4.1N/CTD (aa 767-879). The applied and pull-down proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue (CBB) or probed with anti-HA antibody. The bottom panel shows applied GST-IP3R1/CTT or GST. The middle two panels show applied HA4.1N/FL, HA4.1N/Delta CTD, and HA4.1N/CTD. The top two panels show pull-down proteins. Both HA4.1N/FL and HA4.1N/CTD were pulled down by GST-IP3R1/CTT, but HA4.1N/Delta CTD was not. Neither HA4.1N/FL nor HA4.1N/CTD was pulled down by GST. WB, Western blot.

A pull-down assay was then performed to confirm the yeast two-hybrid results. The GST-IP3R1/CTT fusion protein was incubated with lysates of COS-7 cells transiently transfected with plasmids encoding HA-tagged 4.1N fusion proteins: pcDNA3-HA4.1N/FL (HA-tagged full-length 4.1N), pcDNA3- HA4.1N/Delta CTD (HA-tagged 4.1N lacking the CTD domain, aa 1-766), or pcDNA3-HA4.1N/CTD (HA-tagged 4.1N/CTD, aa 767-879). The pull-down proteins were separated by SDS-PAGE and probed with anti-HA antibody. As shown in Fig. 4B, both HA4.1N/FL and HA4.1N/CTD were pulled down by GST-IP3R1/CTT, but HA4.1N/Delta CTD was not. Neither HA4.1N/FL nor HA4.1N/CTD was pulled down by GST alone. These results indicate that the CTD domain of 4.1N (aa 767-879) is necessary and sufficient for binding to IP3R1.

4.1N Associates with IP3R1 in Both Subconfluent and Confluent MDCK Cells-- Bush et al. (39) reported IP3R to be localized at the basolateral membrane domain as well as in the cytoplasm in confluent MDCK cells, but the molecular mechanisms underlying this localization have not been clarified. Considering that 4.1R, the prototypical homologue of 4.1N, is an erythroid membrane skeletal protein, we investigated the subcellular localization of 4.1N and its relationship with the localization of IP3R1 in MDCK cells. Subconfluent and confluent MDCK cells were fixed, permeabilized, and immunostained with anti-IP3R1 and anti-4.1N antibodies or phalloidin. In subconfluent MDCK cells, IP3R1 existed in the cytoplasm (Fig. 5A). 4.1N was distributed in both the cytoplasm and the nucleus and partially co-localized with IP3R1 in the cytoplasm (Fig. 5B). Neither IP3R1 nor 4.1N existed at the region of the plasma membrane stained by phalloidin (Fig. 5A and data not shown). In confluent MDCK cells, on the other hand, apart from slight immunofluorescence of both IP3R1 and 4.1N scattering in the cytoplasm, 4.1N and IP3R1 co-localized predominantly at the cell-cell junctional region in a punctate pattern (Fig. 5, C and D). These results indicate that both 4.1N and IP3R1 are translocated from the cytoplasm (and the nucleus) to the cell-cell junctional region when MDCK cells grow from subconfluence to confluence.


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Fig. 5.   4.1N associates with IP3R1 in both subconfluent and confluent MDCK cells. A-D, subconfluent and confluent MDCK cells were fixed, permeabilized, stained with Alexa 594-phalloidin and anti-4.1N or anti-IP3R1 antibodies, and analyzed by a confocal microscope. In subconfluent MDCK cells, IP3R1 exists in the cytoplasm and is not localized at the membrane region labeled by phalloidin (A). 4.1N is distributed in the cytoplasm and the nucleus and partially co-localizes with IP3R1 in the cytoplasm (B). In confluent MDCK cells, IP3R1 is localized at the cell-cell junction region labeled by phalloidin (C) and co-localizes with 4.1N (D). E and F, the lysates of subconfluent and confluent MDCK cells were subjected to immunoprecipitation with anti-IP3R1 antibodies, respectively. The input and immunoprecipitated proteins were separated by SDS-PAGE and probed with anti-IP3R1 and anti-4.1N antibodies. 4.1N was immunoprecipitated by two rat anti-IP3R1 antibodies, 4C11 and 18A10, but not by normal rat IgG in both subconfluent (E) and confluent (F) MDCK cells. Scale bar, 20 µm. WB, Western blot.

To determine whether 4.1N could also bind to IP3R1 in MDCK cells, the lysates of subconfluent and confluent MDCK cells were subjected to immunoprecipitation, respectively, with two rat polyclonal anti-IP3R1 antibodies. Co-precipitated proteins were separated by SDS-PAGE and probed with anti-4.1N and anti-IP3R1 antibodies. As shown in Fig. 5 (E and F), in both subconfluent and confluent MDCK cells, 4.1N was co-precipitated with IP3R1 by the two rat anti-IP3R1 antibodies but not by normal rat IgG. Taken together, these results indicate that 4.1N associates with IP3R1 in both subconfluent and confluent MDCK cells.

Both 4.1N and IP3R1 Are Localized at the Basolateral Membrane Domain in Confluent MDCK Cells-- 4.1N and IP3R1 co-localized at the cell-cell junctional region in confluent MDCK cells (Fig. 5D). However, in contrast, IP3R is reportedly localized at the basolateral membrane domain (39), 4.1R, the prototypical homologue of 4.1N, is reportedly localized at the tight junctions in confluent MDCK cells (23). To accurately determine the localization of 4.1N and IP3R1, the localization of 4.1N and IP3R1 was compared with that of ZO-1, a marker protein at the tight junction in MDCK cells, and Na,K-ATPase, a marker protein at the basolateral membrane domain in MDCK cells. As shown in the three-dimensional illustrations (Fig. 6), neither 4.1N nor IP3R1 was localized at the tight junctions immunolabeled by anti-ZO-1 antibody, but both were localized at the basolateral membrane domain immunolabeled by anti-Na,K-ATPase alpha 1 antibody. These results indicate that although 4.1R is localized at the tight junction in confluent MDCK cells (23), 4.1N co-localizes with IP3R1 at the basolateral membrane domain in confluent MDCK cells.


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Fig. 6.   Both 4.1N and IP3R1 are localized at the basolateral membrane domain but not at the tight junction in confluent MDCK cells. Confluent MDCK cells were fixed, permeabilized, immunostained with anti-4.1N or anti-IP3R1 antibodies and anti-ZO-1 or anti-Na,K ATPase alpha 1 antibodies, and analyzed by a confocal microscope. Sequential 0.2-µm-thick face-on (A1, A2, B1, and B2) and transverse sections (A'1, A'2, B'1, and B'2) were collected. The transverse sections represent the areas indicated by the line in the upper panels of facing sections, respectively. Both 4.1N (green) (A1, A'1, A2, and A'2) and IP3R1 (green) (B1, B'1, B2, and B'2) are not localized at the tight junction immunolabeled by anti-ZO-1 antibody (red) but are localized at the basolateral membrane domain immunolabeled by anti-Na,K ATPase alpha 1 antibody (red). Scale bar, 20 µm.

The 4.1N-binding Region of IP3R1 Is Responsible for Localization at the Basolateral Membrane Domain in Confluent MDCK Cells-- In both subconfluent and confluent MDCK cells, 4.1N associated with IP3R1 (Fig. 5). To examine the role of the 4.1N-IP3R1 interaction in the translocation of IP3R1 to the basolateral membrane domain, MDCK cells transiently expressing GFP-IP3R1-N, GFP-IP3R1/Delta 18A10, GFP-18A10, or GFP alone were grown to confluence and observed using a confocal microscope. As shown in Fig. 7, when endogenous 4.1N was recruited to the basolateral membrane domain, GFP-IP3R1-N (Fig. 7A) and GFP-18A10 (Fig. 7C) were also recruited to the basolateral membrane domain. However, neither GFP-IP3R1/Delta 18A10 (Fig. 7B) nor GFP alone (data not shown) was recruited to the basolateral membrane domain. Therefore, these results indicate that the 4.1N-binding region of IP3R1 is necessary and sufficient for the localization of IP3R1 at the basolateral membrane domain in confluent MDCK cells.


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Fig. 7.   The 4.1N-binding region of IP3R1 is necessary and sufficient for IP3R1 localization at the basolateral membrane domain in confluent MDCK cells. GFP-IP3R1-N, GFP-IP3R1/Delta 18A10, and GFP-18A10 were transiently expressed in MDCK cells. The cells were grown to confluence, immunostained, and analyzed by a confocal microscope. Although GFP-IP3R1-N (A) and GFP-18A10 (C) were found to be co-localized with endogenous 4.1N at the basolateral membrane domain, GFP-IP3R1/Delta 18A10 (B) was still distributed in the cytoplasm and the nucleus. Scale bar, 20 µm.

4.1N/CTD Fragment Blocks IP3R1 Localization at the Basolateral Membrane Domain in Confluent MDCK Cells-- To determine which portion of 4.1N is responsible for the localization of 4.1N at the basolateral membrane domain in confluent MDCK cells, MDCK cells were transfected with plasmids encoding Venus-tagged (a variant of yellow fluorescent protein) (33) 4.1N fusion proteins or Venus alone. In subconfluent MDCK cells, Venus-4.1N/FL and Venus-4.1N/CTD were distributed in the cytoplasm and the nucleus, and Venus-4.1N/Delta CTD was distributed in the cytoplasm (data not shown). In confluent MDCK cells, Venus-4.1N/FL (Fig. 8A) and Venus-4.1N/Delta CTD (Fig. 8B) were completely recruited to the basolateral membrane domain, and Venus-4.1N/CTD (Fig. 8C) and Venus alone (data not shown) were distributed in the cytoplasm and the nucleus. These results indicate the N-terminal portion, but not the C-terminal domain, to be responsible for the localization of 4.1N at the basolateral membrane domain.


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Fig. 8.   The 4.1N/CTD fragment blocks co-expressed IP3R1 localization at the basolateral membrane domain in confluent MDCK cells. A-C, Venus-4.1N/FL, Venus-4.1N/Delta CTD, and Venus-4.1N/CTD were transiently expressed in MDCK cells. The cells were grown to confluence, immunostained, and analyzed by a confocal microscope. Venus-4.1N/FL (A) and Venus-4.1N/Delta CTD (B) were completely recruited to the basolateral membrane domain immunolabeled by anti-Na,K-ATPase alpha 1 antibody. Venus-4.1N/CTD (C) was still distributed in the cytoplasm and the nucleus. D--F, MDCK cells were transiently co-expressed with Venus-4.1N/FL, Venus-4.1N/CTD, or Venus alone and IP3R1. The cells were grown to confluence, immunostained with anti-IP3R1 antibody, and analyzed by a confocal microscope. When IP3R1 was co-expressed with Venus-4.1N/FL, both were recruited to the vicinity of the cell-cell junction (D). When IP3R1 was co-expressed with Venus-4.1N/CTD, both were distributed in the cytoplasm and the nucleus (E). When IP3R1 was co-expressed with Venus, IP3R1 was recruited to the cell-cell junction; Venus was still distributed in the cytoplasm and the nucleus (F). Scale bar, 20 µm.

The CTD domain of 4.1N was necessary and sufficient for binding to IP3R1 (Fig. 4), and the 4.1N/CTD fragment was not localized at the basolateral domain in confluent MDCK cells (Fig. 8C). To determine whether the 4.1N/CTD fragment can block IP3R1 translocation to the basolateral domain in confluent MDCK cells, MDCK cells were co-transfected with plasmids encoding Venus-4.1N/FL, Venus-4.1N/CTD, or Venus with IP3R1. As shown in Fig. 8 (D-F), when IP3R1 was co-expressed with Venus-4.1N/FL, both were recruited to the vicinity of the cell-cell junction in confluent MDCK cells. However, when IP3R1 was co-expressed with Venus-4.1N/CTD, both remained in the cytoplasm and the nucleus in confluent MDCK cells. This co-expression with Venus did not affect the distribution of IP3R1. Taken together, these results indicate that the interaction between IP3R1 and 4.1N is responsible for IP3R1 translocation and that both the N-terminal portion and the CTD domain of 4.1N are necessary for IP3R1 translocation to the basolateral membrane domain in confluent MDCK cells.

Other ER Marker Proteins Are Still Present in the Cytoplasm-- IP3R1 is known to be predominantly localized on the ER (35, 40). Consistently, in subconfluent MDCK cells, IP3R1 was found to be localized in the cytoplasm (Fig. 5B), whereas in confluent MDCK cells, IP3R1 was found to be localized at the basolateral membrane domain (Figs. 5, C and D, and 6, B2, and B'2). To detect the ER localization in confluent MDCK cells, immunostaining for other endogenous ER marker proteins was performed. As shown in Fig. 9A, calnexin was localized in the cytoplasm and was not localized at the basolateral membrane domain. In particular, there is no immunofluorescence of calnexin in the cell-cell junctional region immunolabeled by anti-Na,K-ATPase alpha 1 antibody. Additionally, three other ER proteins, calreticulin, calsequenstrin, and sarcoplasmic/endoplasmic reticulum calcium-ATPase (SERCA2a), were also found to be localized in the cytoplasm (data not shown). These results indicate that when IP3R1 translocates to the basolateral membrane domain in confluent MDCK cells, other ER marker proteins are still localized in the cytoplasm. To further confirm these observations, MDCK cells were transiently transfected with EGFP-SERCA2a (a fusion protein of SERCA2a with EGFP) and DsRed-KDEL (a fusion protein of the ER target and retention signal peptide of calreticulin with DsRed2) and grown to confluence. Although the endogenous IP3R1 was predominantly concentrated at the basolateral membrane domain, the exogenously expressed EGFP-SERCA2a and DsRed2-KDEL were still distributed in the cytoplasm (Fig. 9, B and C). Taken together, these results suggest that although IP3R1 is localized at the basolateral membrane domain in confluent MDCK cells, the ER is not.


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Fig. 9.   Other ER marker proteins are not localized at the basolateral domain in confluent MDCK cells. A, confluent MDCK cells were fixed, permeabilized, stained with anti-Na,K ATPase alpha 1 antibody and anti-calnexin antibody, and analyzed by a confocal microscope. Calnexin is localized in the cytoplasm and is clearly absent at the cell-cell junction of the basolateral membrane domain immunolabeled by anti-Na,K ATPase alpha 1 antibody. B and C, EGFP-SERCA2a and DsRed-KDEL were transiently expressed in MDCK cells, the cells were grown to confluence and immunostained with anti-IP3R1 antibody. Although endogenous IP3R1 was localized at the basolateral membrane domain, the transiently expressed EGFP-SERCA2a (B) and DsRed-KDEL (C) were still localized in the cytoplasm. Scale bar, 20 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Bush et al. (39) have found IP3R to be localized at the basolateral membrane domain as well as in the cytoplasm in confluent MDCK cells by using antibodies against IP3R purified from the rat cerebellum. However, the molecular mechanism accounting for this localization has not been elucidated. In this study, we demonstrated an interaction between the C-terminal cytoplasmic tail of IP3R1 and the CTD domain of 4.1N using a yeast two-hybrid system, in vitro and in vivo binding assays. By employing specific antibodies against IP3R1 and against 4.1N, we found that IP3R1 and 4.1N associated in both subconfluent and confluent MDCK cells and were translocated from the cytoplasm (and the nucleus) to the basolateral membrane domain when MDCK cells grew from subconfluence to confluence. The localization of IP3R1 at the basolateral membrane domain was determined by its 4.1N-binding region and could be blocked by a fragment of 4.1N/CTD, which was necessary and sufficient for 4.1N binding to IP3R1 and could not be recruited to the basolateral membrane domain. Furthermore, we also found that although IP3R1 was localized at the basolateral membrane domain, several other endogenous or exogenously expressed ER marker proteins were still present in the cytoplasm. Our data indicate IP3R1 localization at the basolateral membrane domain in confluent MDCK cells to be regulated by an interaction between the C-terminal cytoplasmic tail of IP3R1 and the CTD domain of 4.1N.

Organization of proteins into structurally and functionally distinct membrane domains is an essential characteristic of polarized epithelial cells. The details of the mechanism by which 4.1N and IP3R1 are restricted to the basolateral membrane domain in confluent MDCK cells have not been clarified. Herein, we identified several features of the translocation process of 4.1N and IP3R1 to the basolateral membrane domain. First, although 4.1R and 4.1N share a high degree homology (26), they have different subcellular localizations. Second, the N-terminal portion (aa 1-766) of 4.1N is responsible for the translocation of 4.1N to the basolateral membrane domain. Third, the interaction between 4.1N and IP3R1 is necessary for IP3R1 translocation to the basolateral membrane domain. Two 4.1R isoforms of 135 and 150 kDa reportedly co-localize with ZO-1, ZO-2, and occludin at tight junction in confluent MDCK cells (23). In contrast, 4.1N is not localized at tight junction (Fig. 6, A1 and A'1) but is localized at the basolateral membrane domain in confluent MDCK cells (Fig. 6, A2 and A'2). Although the 4.1R/CTD fragment bound to ZO-1 and ZO-2 in vitro, it could not be recruited to the tight junction in confluent MDCK cells (23). The 4.1N/CTD also could not be recruited to the basolateral membrane in confluent MDCK cells (Fig. 8C). The N-terminal portion of 4.1N, which could be completely recruited to the basolateral membrane (Fig. 8B), contains three unique domains and a highly conserved membrane-binding domain (Fig. 1B). The different subcellular localizations of 4.1R and 4.1N might be determined by their unique domains. The membrane-binding domain of 4.1R is known to interact with integral plasma membrane proteins such as band 3 (15, 16), glycophorin C (17, 18), and CD44 (19). Although the interactions between 4.1N and these proteins have not been verified, the overall high sequence homology between 4.1N and 4.1R leads us to speculate that the N-terminal portion of 4.1N (aa 1-766) may also have the ability to interact with one or more integral plasma membrane proteins. Both of these interactions, which might be induced by signals from MDCK cells at confluence, and the unique domains of 4.1N may allow recruitment of the 4.1N-IP3R1 complex to the basolateral membrane domain.

IP3R1 is known to be predominantly localized on the ER (35, 40). In subconfluent MDCK cells, IP3R1 was indeed also found in the cytoplasm (Fig. 5B). However, in confluent MDCK cells, IP3R1 showed a subcellular localization different from that of other ER marker proteins; although IP3R1 was predominantly concentrated at the basolateral membrane domain (Figs. 5, C and D, and 6, B2 and B'2), other ER marker proteins remained in the cytoplasm and were clearly absent at the cell-cell junction of the basolateral membrane domain (Fig. 9A and data not shown). Again, we found that exogenously expressed EGFP-SERCA2a and DsRed2-KDEL were not translocated to the basolateral membrane domain in confluent MDCK cells (Fig. 9, B and C). Considering that about 10-20% of total IP3R was accessible to externally added biotin, primarily from the basolateral side in nonpermeabilized confluent MDCK cells (39), it is possible that a portion of IP3R1 is localized in the vicinity of the basolateral plasma membrane, a basolateral plasma membrane subdomain, or an associated membrane compartment and that a portion of IP3R1 exists as integral plasma membrane proteins. It is necessary to investigate the existence status of IP3R1 at the basolateral membrane domain to understand the physiological function of IP3R1 in confluent MDCK cells.

There is growing evidence suggesting that IP3R is localized to or on the plasma membrane. Immunolabeling studies in several cell lines have found a portion of the subcellular IP3R pool to be localized to the plasma membrane (41-44). A number of subcellular fractionation studies have found that IP3R often appears in the plasma membrane fraction (45-48). Tanimura et al. (49) clearly demonstrated all three isoforms of IP3R to be externally biotinylated in several cell lines. On the other hand, the CTD domain of 4.1N reportedly binds to the AMPA receptor GluR1 subunit (37) and to the D2 and D3 dopamine receptors (50), and these interactions appear to regulate the cell surface expression of these receptors. 4.1N, a membrane cytoskeletal protein, is expressed not only in neural tissue but also in non-neural tissues (38). The mechanism detected in MDCK cells by which 4.1N serves to regulate IP3R1 subcellular localization may have a general significance in other cell lines.

The possible role of IP3R1 at the basolateral membrane domain is at present uncertain. Localization of IP3R1 near the basolateral plasma membrane may allow IP3R1 to efficiently receive the inositol 1,4,5-trisphosphate signal and thereby rapidly induce a local Ca2+ increase, which may modulate nearby actin cytoskeleton through Ca2+-sensitive actin-binding proteins and play critical roles in cell adhesive and cell polarity maintenance (39). If IP3R1 exists as an integral plasma membrane protein at the basolateral membrane domain in confluent MDCK cells, IP3R1 could conceivably function as a plasma membrane Ca2+ channel with the IP3-binding domain and the 4.1N-binding C-terminal cytoplasmic tail facing the cytoplasm. Additionally, there are data supporting the hypothesis that IP3R residing on the plasma membrane can also function as a capacitative Ca2+ entry channel (51-53).

Wu et al. (54) reported that disruption of the spectrin-protein 4.1 interaction resulted in a decreased thapsigargin-induced global cytosolic Ca2+ response and in selective loss of the endothelial cell ISOC. Other reports have shown the dynamic activity of cytoskeletal actin to mediate the coupling process between Ca2+ store depletion and Ca2+ entry across the plasma membrane (55-58). In view of the fact that 4.1N is a membrane-binding protein, a spectrin-actin-binding protein, and a component of the cytoskeleton, it would be worthwhile to investigate the functional contribution of the interaction between 4.1N and IP3R1 and this interaction-based IP3R1 subcellular translocation in the process of Ca2+ entry across the plasma membrane triggered by Ca2+ store depletion.

    ACKNOWLEDGEMENTS

We thank Dr. S. Tsukita of Kyoto University for the generous gift of anti-ZO1 antiserum (T8754). We also thank Drs. T. Inoue, T. Michikawa, K. Uchida, M. Kawasaki, and T. Morimura for critically reading the manuscript and/or for fruitful discussion and M. Iwai for technical assistance. S. Z. thanks all the members of our laboratory and Dr. J. C. Luo for valuable help.

    FOOTNOTES

* This work was supported by grants from the Ministry of Education and Science of Japan (to M. H. and K. M.) and by a Uehara Memorial Foundation Research Fellowship (to S. Z.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a supplemental figure.

§ To whom correspondence may be addressed. Tel.: 81-3-5449-5316; Fax: 81-3-5449-5420; E-mail: sbzhang@ims.u-tokyo.ac.jp.

Dagger Dagger To whom correspondence may be addressed. Tel.: 81-3-5449-5316; Fax: 81-3-5449-5420; E-mail: mikosiba@ims.u-tokyo.ac.jp.

Published, JBC Papers in Press, November 19, 2002, DOI 10.1074/jbc.M209960200

2 T. Nakayama, M. Hattori, K. Uchida, T. Nakamura, Y. Tateishi, H. Bannai, M. Iwai, T. Michikawa, T. Inoue, and K. Mikoshiba, unpublished data.

3 T. Higo, M. Hattori, K. Nakamura, T. Michikawa, and K. Mikoshiba, unpublished data.

    ABBREVIATIONS

The abbreviations used are: IP3R, inositol 1,4,5-trisphosphate receptor; IP3, inositol 1,4,5-trisphosphate; IP3R1, IP3R type 1; CTD, C-terminal domain; CTT, C-terminal cytoplasmic tail; MDCK, Madin-Darby canine kidney; ER, endoplasmic reticulum; SERCA2a, sarcoplasmic/endoplasmic reticulum calcium-ATPase; aa, amino acid(s); GFP, green fluorescent protein; EGFP, enhanced GFP; HA hemagglutinin, PBS, phosphate-buffered saline; GST, glutathione S-transferase; Ab, antibody.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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