From the 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
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ABSTRACT |
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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.
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.
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/
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/ 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 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 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 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
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.
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 (
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/ 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/
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/ 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.
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 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/ 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/
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 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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/
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).
CTD and the
pcDNA3-Venus-4.1N/
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).
-galactosidase activity
on nitrocellulose filters as described in the
Clontech manual.
1 monoclonal antibody was from Upstate Biotechnology.
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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-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.
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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.
1-
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 -galactosidase activity and
nutritional selection assay showed that although the full-length
IP3R1/CTT interacted with 4.1N/CTD, none of the deletion
mutants (
1-
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/
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/
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/
18A10 was not. Neither GFP-IP3R1-N nor GFP-18A10 was
pull-down by GST. WB, Western blot.
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/
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/
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.
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
-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/
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/
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/
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/
CTD was not.
Neither HA4.1N/FL nor HA4.1N/CTD was pulled down by GST. WB,
Western blot.
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/
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.
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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.
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 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
1 antibody (red).
Scale bar, 20 µm.
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/
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/ 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/
18A10
(B) was still distributed in the cytoplasm and the nucleus.
Scale bar, 20 µm.
CTD was distributed in the cytoplasm (data not shown). In
confluent MDCK cells, Venus-4.1N/FL (Fig. 8A) and Venus-4.1N/
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/ 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/
CTD (B) were completely recruited to the
basolateral membrane domain immunolabeled by anti-Na,K-ATPase
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.
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 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
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
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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.
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FOOTNOTES |
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* 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.
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.
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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.
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