 |
INTRODUCTION |
Erythrocyte protein 4.1R is an 80-kDa cytoskeletal protein
critical in circulating red cells for the dynamic organization and
maintenance of the spectrin/actin cytoskeleton and for the attachment
of the cytoskeleton to the cell membrane through interactions with
integral membrane proteins such as glycophorin C and band 3 (1).
In nonerythroid cells, an additional and prevalent 135-kDa 4.1R isoform
class has been detected. It contains a 209-amino acid extension at its
N terminus end (2).
Multiple isoforms of 4.1R exist. They arise through complex alternative
pre-mRNA splicing pathways (2, 3), post-translational modifications
(4), and use of at least two translation initiation sites (5, 6). In
nucleated erythroid and nonerythroid cells, a spectrum of proteins
ranging from 30 to 210 kDa has been detected that contains epitopes for
4.1R (7, 8). Unlike the strict peripheral distribution of 4.1R in
mature erythrocytes, 4.1R isoforms in nonerythroid cells are localized
in the nucleus and nuclear matrix (9-13), at points of cell-cell or
cell-matrix contact (14, 15), and in centrosomal and Golgi structures
(16, 17). In addition, 4.1R may also interact with microtubules and
stress fibers (14, 18). However, the precise identity of 4.1R isoforms in these subcellular structures, their binding partners, and their biological significance in nonerythroid cells are not well understood.
The 80-kDa erythrocyte 4.1R protein is composed of four different
domains as defined by chymotryptic digestion (19). The 30-kDa
N-terminal domain (aa1
207-474; GenBankTM/EBI Data Bank accession number J03796)
interacts with glycophorin C (20), calmodulin (21), p55 (20), and band
3 (22). The 30-kDa domain has also been shown to bind to CD44 (23) and
pICln, a protein involved in cellular volume control (24). The 10-kDa domain (aa 579-624) forms a ternary complex with spectrin and actin
(25-27). The function of the 16-kDa (aa 472-578) and 22-24-kDa (aa
625-774) domains in erythrocytes is not known. In nonerythroid cells,
4.1R interacts with calmodulin in a
Ca2+-dependent manner through its N-terminal
extension (28), with human CASK through its 30-kDa domain (29),
with an 
-subunit of the human nuclear shuttle complex importin
(Rch1) through its 30- and 10-kDa domains (12), and with the
immunophilin FKBP13 (30) and NuMA (13) through its 22-24-kDa
C-terminal domain.
Like 4.1R, many members of the protein 4.1 superfamily, including
ezrin, radixin, moesin, merlin, and myosin, are known to associate with
the plasma membrane (reviewed in Ref. 31). A Drosophila
homolog of protein 4.1, Coracle, is localized at septate junctions in
ectodermally derived epithelial cells (32). Drosophila Neurexin and DLG (Discs Large),
homologs of proteins that are known to bind to 4.1, glycophorin C, and
human DLG, respectively, are also localized at the septate junctions
(33, 34). Collectively, these studies suggest the localization of 4.1 to the septate junction, an invertebrate specific junction with
molecular components analogous to the vertebrate tight junction (TJ)
(35). In addition to membrane binding activity, members of this family
participate in important cell signaling events. For example, merlin is
involved in growth regulation (36), and ezrin, radixin, and moesin are
involved in Rho-dependent signaling pathways (37, 38).
To explore the function of 4.1R isoform(s) in nonerythroid cells, we
searched for their binding partners. In this study, using a combination
of molecular/genetic, biochemical, and immunofluorescence studies, we
establish that two nonerythroid 4.1R isoforms bind to the C-terminal
proline-rich domain of X-104, a human homolog of the canine protein
ZO-2 (henceforth referred to as hZO-2), through its C-terminal domain.
ZO-2 belongs to the membrane-associated guanylate kinase (MAGUK) family
of proteins and is known to organize the TJ in association with other
TJ-associated proteins (39-41). We report here that these isoforms of
4.1R colocalize with ZO-2 and occludin and also form an intracellular
complex with ZO-2, ZO-1, occludin, actin, and -spectrin at MDCK cell
TJs. Our results suggest that in nonerythroid cells, 4.1R may have a
role in the organization and function of the TJ by establishing a
direct link between the TJ and the actin cytoskeleton.
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EXPERIMENTAL PROCEDURES |
Construction of Expression Vectors--
Construction of
full-length 4.1R (135-kDa/pAS2-1); 80-kDa 4.1R (80-kDa/pAS2-1); its
30-kDa (30-kDa/pAS2-1), 16-kDa (16-kDa/pAS2-1), 10-kDa (10-kDa/pAS2-1),
and 22-24-kDa (22-24-kDa/pAS2-1) domains; or its N-terminal extension
(HP; HP/pAS2-1) (see Fig. 1A and GenBankTM
accession number J03796) in frame with the Gal4 DNA-binding domain in
the yeast two-hybrid vectors pAS2-1 and pGBT9 have been described (13).
Full-length hZO-2, ZO-2, ZO-1, occludin, or their segments were
subcloned in frame with the Gal4 transactivation domain in the yeast
two-hybrid vector pACT2 by PCR amplification of the desired nucleotide
sequences using the ExpandTM Long Template PCR system
(Roche Molecular Biochemicals), a GeneAmpTM PCR System 9700 (Applied Biosystems, Foster City, CA), and custom oligonucleotide
primers (Life Technologies, Inc.) by standard molecular biology methods
(42).
The entire coding sequence of hZO-2 (aa 1-1168, FL-hZO-2/pACT2;
GenBankTM accession number L27476 as corrected in Ref. 43)
was amplified from a plasmid containing full-length hZO-2 in
pBluescript II SK(
) obtained from M. Koenig (44). The nucleotide
sequences corresponding to amino acids 1-189 (N-ZO-2/pACT2) and
189-1174 (ZO-2
N/pACT2) of ZO-2 (GenBankTM accession
number L27152) were amplified from a plasmid containing full-length
ZO-2 in pBluescript SK(+) obtained from B. R. Stevenson (39).
Full-length human occludin (aa 1-504, FL-hOc/pACT2;
GenBankTM accession number U49185) and its N-terminal
cytoplasmic domain (aa 1-57, N-hOc/pACT2) and C-terminal cytoplasmic
domain (aa 250-504, C-hOc/pACT2) were amplified from phOc6 (45). The
nucleotides corresponding to amino acids 1-1044 (N-hZO-1/pACT2) and
1045-1736 (C-hZO-1/pACT2) of human ZO-1 (GenBankTM
accession number L14837) were amplified from a plasmid containing full-length human ZO-1 in pBluescript SK(+) obtained from A. S. Fanning and J. M. Anderson (35).
Construction of deletion constructs representing different exons of the
22-24-kDa domain of 4.1R, such as exons 17-21/pGBT9, exons
18-21/pGBT9, exons 18-20/pGBT9, exon 19/pGBT9, exon 20/pGBT9, exon
21/pGBT9, exons 20-21/pGBT9, exon 21-(762-775)/pGBT9, exon 21-(745-761)/pAS2-1, and exons 19/21/pGBT9, has been described (13).
The construct exons 19-21/pGBT9 was derived from 22-24-kDa/pAS2-1 using custom oligonucleotide primers 2093-2112 and 2354-2374
(GenBankTM accession number J03796) as described above; it
was cloned into the SalI and PstI sites of pGBT9.
Deletion constructs of hZO-2 representing amino acids 980-1118
(hZO-2980-1118/pACT2), 980-1073 (hZO-2980-1073/pACT2),
980-1028 (hZO-2980-1028/pACT2), 1113-1168
(hZO-21113-1168/pACT2), 1072-1118 (hZO-21072-1118/pACT2), and
1054-1118 (hZO-21054-1118/pACT2) were derived from
FL-hZO-2/pACT2.
Constructs used for expression of 4.1R/glutathione S-transferase (GST)
fusion proteins (135-kDa/GST, 80-kDa/GST, HP/GST, 30-kDa/GST, 16-kDa/GST, 10-kDa/GST, and 24-kDa/GST) have been described (13). For
in vitro transcription and translation, cDNA sequences
corresponding to aa 2863-3325 (ZO-2-1/TOPO) and 3089-3653
(ZO-2-2/TOPO) of hZO-2 (GenBankTM accession number L27476),
aa 4354-5588 (ZO-1-1/TOPO) and 5587-6467 (ZO-1-2/TOPO) of hZO-1
(GenBankTM accession number L14837), and aa 461-919
(Ocl-1/TOPO) and 923-1687 (Ocl-2/TOPO) of human occludin
(GenBankTM accession number U49185) were amplified with the
addition of the Kozak consensus sequence and the translation initiation site (GCCACCATG) incorporated at the 5'-end and a stop codon at the
3'-end. The amplified products were cloned into the pCR-Blunt II-TOPO
vector (Invitrogen, Carlsbad, CA). To express 4.1R/green fluorescence
protein (GFP) fusion proteins, the desired 4.1R cDNA sequences were
amplified by PCR and cloned in frame into the SalI and
SmaI sites of the pEGFP-C1 or pEGFP-C2 vector
(CLONTECH, Palo Alto, CA). HP
10-kDa/GFP,
10-kDa/GFP, and 22-24-kDa/GFP were amplified from 135-kDa/pAS2-1,
10-kDa/pAS2-1, and 24-kDa/pAS2-1, respectively. 10+24-kDa/GFP was
generated by two-step PCR as described (13) using primer sets
2026-2040 and 2294-2393 for amplification of 10-kDa/pAS2-1 and primer
sets 2307-2336 and 2795-2816 (46) for amplification of
22-24-kDa/pAS2-1. 10-kDa/GFP and 10+24-kDa/GFP contained the
alternatively spliced exons 14-16. Accuracy of the reading frame and
the authenticity of all deletions were confirmed by manual dideoxy sequencing.
Yeast Two-hybrid Screening and 
-Galactosidase Assay--
The
Gal4-based MATCHMAKER Two-hybrid System II
(CLONTECH) was followed for the yeast two-hybrid
assays as described earlier (13). Plasmid vectors pAS2-1 or pGBT9 and
pACT2, encoding the Gal4 DNA-binding domain (Gal4-BD) and
Gal4-activating domain (Gal4-AD), respectively, were used to express
hybrid proteins. Saccharomyces cerevisiae strain Y190 was
transformed with full-length 4.1R (135-kDa/pAS2-1) or its 22-24- and
10-kDa domains (22-24-kDa/pAS2-1 and 10-kDa/pAS2-1, respectively). Transformants were analyzed for expression of the bait
protein by Western blotting. To screen for proteins that interact with
4.1R in yeast two-hybrid assays, a human brain cDNA library in
Gal4-AD vector pACT2 was screened by sequential transformation of Y190
expressing full-length 4.1R (135 kDa) or its 10- or 22-24-kDa domain
as a Gal4-BD fusion protein (see "Construction of Expression Vectors" for details). Transformants were selected in
synthetic medium lacking Trp, His, and Leu containing 25 mM 3-aminotriazole. DNA was isolated from positive clones,
and the Gal4-AD plasmids were recovered in Escherichia coli
strain HB101 and further tested for specificity by cotransformation
into Y190 with 135-kDa/pAS2-1, 10-kDa/pAS2-1, or 22-24 kDa/pAS2-1 or
with pAS2-1 alone. The Gal4-AD plasmids recovered from positive clones
were sequenced as described above.
For domain mapping, plasmids carrying inserts fused to Gal4-BD or
Gal4-AD were cotransformed into Y190 and assayed for -galactosidase activity on nitrocellulose filters. In some cases, liquid
-galactosidase assays using o-nitrophenyl
-D-galactopyranoside or chlorophenol red--D-galactopyranoside were used as described in the
CLONTECH manual.
Cell Culture and Transient Transfection--
MDCK cells
(American Type Culture Collection CRL 1772) were used in this study
because they are known to express TJ proteins and to form TJs at
confluent density (47). We have described the culture conditions for
MDCK cells and have documented the expression of 4.1R in this cell line
at both the protein and RNA levels (13). Transfection was performed
using LipofectAMINE reagent (Life Technologies, Inc.) according to the
manufacturer's protocol. Cells were plated on
poly-D-lysine-coated coverslips in a six-well tissue
culture plate 1 day prior to transfection. Cells at 50-70% confluency
were transfected with 2 µg of plasmid DNA. After 36-48 h, cells on
coverslips were removed, fixed, and processed for immunofluorescence
staining with anti-ZO-1 Ab as described below.
Antibodies--
Antibodies to synthetic peptides of segments of
the 4.1R 22-24-kDa domain (GVLLTAQTITSETPSSTTTTKITKC, exon 19;
anti-24-kDa Ab), 16-kDa domain (TQAQTRQASALIDRPAPHFERC, exon 12;
anti-16-kDa Ab), and 10-kDa domain (MESVPEPRPSEWDK, exon 17;
anti-10-kDa Ab) and to the recombinant (GST-fused) N-terminal extension
of 4.1R (anti-HP Ab) have been characterized (2, 13). Anti-ZO-2 Ab, anti-ZO-1 Ab, and anti-occludin Ab were purchased from Zymed
Laboratories Inc. (South San Francisco, CA). Anti-actin
monoclonal antibody was purchased from Sigma. Anti-p53 Ab was purchased
from Oncogene Research Products (Cambridge, MA). Rabbit IgG was
purchased from Pierce. Anti-spectrin ( and ) Ab was a kind
gift from Dr. Robert J. Bloch (University of Maryland, Baltimore).
Immunoprecipitation and Immunoblotting--
Immunoprecipitation
of 4.1R, ZO-1, ZO-2, and occludin was performed using MDCK cell
extracts. About 4 × 106 cells were collected by
scraping with a rubber policeman, washed three times with ice-cold PBS,
resuspended in immunoprecipitation buffer (20 mM Tris-HCl,
150 mM NaCl, 2% CHAPS, 0.1% (w/v) SDS, 1 mg/ml bovine
serum albumin, 0.2 mM EDTA, 5 mM iodoacetamide, 4 µg/ml aprotinin, 4 µg/ml leupeptin, 4 µg/ml antipain, 12.5 µg/ml chymostatin, 12 µg/ml pepstatin, 130 µg/ml 
-aminocaproic
acid, 200 µg/ml p-aminobenzamidine, and 1 mM
phenylmethylsulfonyl fluoride, pH 7.5), and given 20 strokes in a
tight-fitting glass homogenizer. The homogenate was centrifuged at
14,000 rpm for 15 min at 4 °C; the supernatant was collected; and
the protein content was determined using a protein determination kit
(Bio-Rad).
Rabbit preimmune serum equivalent to 50 µg of IgG was added to 2 mg of proteins and incubated for 2 h at 4 °C, followed by the
addition of 200 µl of a 50% suspension of protein A-Sepharose CL-4B
(Amersham Pharmacia Biotech) in immunoprecipitation buffer for 2 h
at 4 °C. The supernatant was collected by centrifugation at
3000 × g for 5 min at 4 °C and split equally into
seven tubes. Preimmune serum, affinity-purified anti-HP Ab, anti-ZO-1
Ab, anti-ZO-2 Ab, anti-occludin Ab, rabbit IgG, or anti-p53 Ab (an
irrelevant antibody as a control) containing 6 µg of IgG was added to
different tubes and incubated for 2 h at 4 °C. Protein
A-Sepharose CL-4B (100 µl of a 50% suspension) was then added and
incubated overnight at 4 °C on a rocking platform. Following
incubation with protein A-Sepharose, the samples were centrifuged at
3000 × g for 10 s at 4 °C and washed 10 times
with 1 ml of immunoprecipitation buffer. The samples were resuspended
in 80 µl of SDS sample buffer (62.5 mM Tris-HCl, 10%
glycerol, 2% (w/v) SDS, 5% 2-mercaptoethanol, and 10 µg/ml
bromphenol blue, pH 6.8) and boiled in a water bath for 5 min. The
samples were centrifuged at 10,000 × g for 15 min at
4 °C, and the supernatants were fractionated on 6-12%
SDS-polyacrylamide gels.
Transfer of proteins to nitrocellulose or polyvinylidene difluoride
membranes and detection of 4.1R, ZO-1, ZO-2, occludin, actin, and
spectrin were carried out by immunoblotting using an ECL detection kit
(Amersham Pharmacia Biotech) as described earlier (13). Quantitation of
proteins from chemiluminograms was done with NIH Image software for the
Apple Macintosh computer. To visualize proteins, the gels were stained
with GelCode® SilverSNAPTM from Pierce.
In Vitro Binding Assay--
Recombinant 4.1R/GST proteins were
expressed and affinity-purified by coupling to glutathione-Sepharose
beads as described (13). The recombinant proteins were quantitated by
Coomassie Brilliant Blue staining using bovine serum albumin as a
standard. Desired [35S]methionine-labeled segments of
hZO-2, hZO-1, and human occludin were in vitro translated
using a TNT® SP6 Quick Coupled transcription/translation
system (reticulocyte lysate system; Promega) according to the
manufacturer's recommendations. The translation products were
quantitated based on percent of methionine incorporation and
35S-specific activity. For in vitro binding,
equal amounts of labeled proteins were incubated with 20 µg of
4.1R/GST fusion proteins coupled to glutathione-Sepharose beads for
2 h at 4 °C in 300 µl of binding buffer (50 mM
Tris, pH 7.4, 50 mM NaCl, 10 mM sodium pyrophosphate, 1.5 mM sodium orthovanadate, 4 µg/ml
aprotinin, 4 µg/ml leupeptin, 4 µg/ml antipain, 12.5 µg/ml
chymostatin, 12 µg/ml pepstatin, 130 µg/ml -aminocaproic
acid, 200 µg/ml p-aminobenzamidine, and 1 mM
phenylmethylsulfonyl fluoride). After incubation, the beads were washed
10 times with 1 ml of binding buffer containing 0.5% Triton X-100 each
time. The beads were then resuspended in 30 µl of SDS sample buffer,
boiled for 5 min, and analyzed by SDS-polyacrylamide gel
electrophoresis (12% gel; National Diagnostics, Inc., Atlanta). The
gels were treated with EnlightningTM (NEN Life Sciences
Products), and binding of 35S-labeled proteins was detected
by fluorography.
To determine the dissociation constants between 4.1R and ZO-2, ZO-1, or
occludin, 5 µg of 24-kDa/GST or GST (as a control) coupled to
glutathione-Sepharose beads was incubated with 0.0025-0.015 µg of
35S-labeled translated products as described above. After
extensive washing, the bound proteins were eluted with 50 mM Tris-HCl containing 20 mM glutathione, pH
8.0. The amount of bound and free labeled protein was determined from
35S-specific activity and adjusted for binding to the
GST-conjugated Sepharose beads. A Scatchard plot of the data was
generated. Each experiment was repeated three times for estimation of
Kd.
Fluorescence and Confocal Microscopy--
MDCK cells were
grown on poly-D-lysine-coated coverslips to confluence. The
coverslips were washed three times with PBS and fixed in freshly
prepared 2% paraformaldehyde in PBS for 25 min at room temperature.
Cells were washed with PBS and permeabilized with a solution of 0.2%
Triton X-100 and 10% normal goat serum in PBS for 15 min. The cells
were blocked with 10% normal goat serum and 50 mM
NH4Cl for 1.5 h at room temperature and washed three
times with PBS containing 0.3% bovine serum albumin. Cells were
incubated with primary antibodies (1:100 dilution of anti-occludin Ab,
1:100 dilution of anti-ZO-2 Ab, 1:200 dilution of anti-ZO-1 Ab, 1:50
dilution of anti-10-kDa Ab, 1:50 dilution of anti-16-kDa Ab, and 1:25
dilution of anti-HP Ab) for 1 h at room temperature and washed
three times as described above. They were then incubated with a 1:100
dilution of Texas Red-conjugated goat anti-rabbit IgG (Jackson
ImmunoResearch Laboratories, Inc.) or a 1:50 dilution of fluorescein
isothiocyanate-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch
Laboratories, Inc.) at room temperature for 1 h and washed three
times again as described above. When double staining was desired, after
the first set of staining, cells were blocked with unconjugated goat
anti-rabbit Fab fragment (10 µg/ml in PBS; Jackson ImmunoResearch
Laboratories, Inc.) for 1 h at room temperature and washed
three times as described above. Incubation with the second set of
antibodies was the same as with the first set of antibodies. Mounting
of the coverslips and processing of images using a Noran confocal laser
scanning image system and Intervision software (Noran Instruments Inc.,
Middleton, WI) were the same as described previously (13).
Immunoelectron Microscopy--
Confluent MDCK cells were washed
with PBS and fixed in 4% paraformaldehyde and 0.1% glutaraldehyde in
PBS overnight at 4 °C. The cells were then infiltrated with 15%
polyvinylpyrrolidone and 1.95 M sucrose in PBS (48), and
ultrathin cryosections were cut at 100 °C with a Reichert FCS
cryoultramicrotome (Leica Inc.). Cryosections were stretched using 2.3 M sucrose and mounted on Formvar/carbon-coated grids.
Sections were blocked with 10% normal goat serum and 20 mM
glycine in PBS for 30 min. Sections were sequentially incubated for 60 min with anti-ZO-2 (1:20 dilution) or anti-HP (1:5 dilution) antibody
and AuroProbe EM goat anti-rabbit IgG (1:50 dilution; Amersham
Pharmacia Biotech). The sections were post-fixed in 2% glutaraldehyde
in PBS. The immunogold-labeled sections were examined in an electron
microscope (EM 10, Carl Zeiss, Inc.).
| |
RESULTS |
Identification of hZO-2 as a 4.1R-binding Protein--
We first
attempted to identify 4.1R-binding partners by use of the yeast
two-hybrid method. We screened ~107 transformants of a
human brain cDNA library (constructed in Gal4-AD vector pACT2)
using full-length 4.1R (135-kDa/pAS2-1) and its 22-24-kDa
(22-24-kDa/pAS2-1) and 10-kDa (10-kDa/pAS2-1) domains (Fig.
1A) as bait constructs. 124 positive clones were obtained; five clones encoded a segment of the
C-terminal domain of X-104 (referred to as hZO-2; aa 980-1168,
hZO-2N/pACT2; GenBankTM accession number L27476 as
corrected in Ref. 43) (Fig. 1B). These clones of hZO-2 were
obtained using 135-kDa/pAS2-1 and 22-24-kDa/pAS2-1 as bait constructs,
but not 10-kDa/pAS2-1. Secondary screening and cotransformation of
fresh yeast cells with the hZO-2N/pACT2 and 135-kDa/pAS2-1,
22-24-kDa/pAS2-1, or pAS2-1 vector alone confirmed that
hZO-2N/pACT2 bound specifically to Gal4-BD fusion proteins of
135-kDa 4.1R or its 22-24-kDa domain.
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Fig. 1.
Schematic representation of 4.1R and X-104
(hZO-2) peptides found to interact in the yeast two-hybrid screening of
a human brain cDNA library. A, the organization of
protein 4.1R (P 4.1R) and its 22-24-kDa domain was used as
the bait in the yeast two-hybrid assays. Different exons of the 4.1R
gene and corresponding chymotryptic domains are shown. The
asterisks represent the alternatively spliced exons.
B, shown is a schematic representation of the structural
organization of the hZO-2 protein and its segments encompassed by the
positive clones obtained from the yeast two-hybrid screen.
GUK, guanylate kinase; +, basic domain; , acidic domain;
, alternatively spliced.
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The X-104 (hZO-2) gene was identified in relation to the Friedreich
ataxia locus protein (44) and by cDNA direct selection (49). It was
subsequently shown to be identical to human ZO-2 (43). hZO-2 belongs to
the MAGUK protein family along with the other TJ-associated proteins
ZO-1 and ZO-3 (40). Although isoforms of hZO-2 have not been described,
hZO-2 contains a region (amino acids 1000-1036) (50) homologous to the
-motif of canine ZO-2 (36 amino acids) and amino acids 991-1028 of
chicken ZO-2, which are alternatively spliced (39, 50, 51). All five
hZO-2 clones that interacted with 4.1R in yeast two-hybrid assays
encoded amino acids 980-1168, which correspond to the C-terminal
proline-rich domain and the putative -motif of hZO-2.
Colocalization of 4.1R and ZO-2 in MDCK Cell Tight
Junctions--
To verify the interaction of 4.1R with ZO-2 and to
locate the site(s) of their intracellular interaction, we examined the subcellular distribution of 4.1R and ZO-2 in confluent MDCK cells by
double-label immunofluorescence microscopy. As shown in Fig. 2 (A and B), ZO-2
(red) and 4.1R (green) localized at the cell-cell contacts and displayed honeycomb-like staining patterns. Both ZO-2 and
4.1R also showed some diffuse cytoplasmic staining. Cytoplasmic staining of ZO-2 has also been observed by others (51). By
superimposing Fig. 2 (A and B), the
yellow color (Fig. 2C) produced due to the combination of the green and red colors suggested
the colocalization of 4.1R and ZO-2 at cell-cell junctions of confluent
MDCK cells.
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Fig. 2.
Immunofluorescence and ultrastructural
localization of 4.1R and tight junction proteins in confluent MDCK
cells. A-F, subcellular colocalization of 4.1R, ZO-2,
and occludin in confluent MDCK cells. Confluent MDCK cells were fixed
and processed for immunofluorescence using antibodies for 4.1R, ZO-2,
and occludin as described under "Experimental Procedures." A
scanning laser confocal microscope was used to collect sequential
0.35-µm-thick face-on (A, B, D, and
E) or transverse (D' and E') sections.
ZO-2 (A) and 4.1R (B) or occludin (D)
and 4.1R (E) localize at cell-cell contacts in a continuous
fashion. The yellow color in C and F
indicates the colocalization of ZO-2 and 4.1R or occludin and 4.1R,
respectively. A-C are two-dimensional confocal images, and
D-F are three-dimensional confocal images.
Bar = 25 µm. Note that the transverse sections
of occludin (D') and 4.1R (E') indicate that both
4.1R and occludin localize at the tight junction. G and
H, immunoelectron micrographs of the tight junctional
complex region in MDCK cells stained with anti-ZO-2 Ab or anti-HP Ab,
respectively. The gold particles for ZO-2 (10 nm) and 4.1R (5 nm) are
concentrated in the tight junctional complex of MDCK cells.
Bar = 200 nm.
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Occludin has been shown to exclusively localize at TJs of epithelial
and endothelial cells (52, 53) in association with ZO-2 and ZO-1. To
verify the localization of 4.1R at tight junctions, we performed double
immunofluorescent staining of confluent MDCK cells with 4.1R and
occludin. As shown in Fig. 2, occludin (red; panel
D) and 4.1R (green; panel E) localized at
apical cell borders and, to some extent, were diffuse in the cytoplasm.
A transverse view of the cells taken at the position of the
dotted line (in Fig. 2, D and E)
showed that both occludin (Fig. 2D') and 4.1R (Fig.
2E') localized at the TJ along the apical side of the cells. As shown in Fig. 2F, 4.1R precisely colocalized with
occludin at the MDCK cell TJs. Colocalization of 4.1R with ZO-2 and
occludin at MDCK cell TJs was also observed when cells were stained
with anti-10-kDa Ab or anti-16-kDa Ab (data not shown). The
colocalization of 4.1R with occludin and ZO-2 indicated that 4.1R
localizes specifically to TJs of confluent MDCK cells.
To study the subcellular localization of ZO-2 and 4.1R in more detail,
we performed high resolution immunogold electron microscopy using fixed
confluent MDCK cells. Anti-ZO-2 Ab was used as a control. As shown in
Fig. 2 (G and H), labeling for ZO-2 (10-nm gold
particles) and 4.1R (5-nm gold particles) was found concentrated at the
tight junctions as clusters (arrows). This further supports
our contention that 4.1R localizes at tight junctions.
In Vivo Association of 4.1R and ZO-2--
To examine the
interaction of 4.1R with ZO-2 in vivo, we performed
immunoprecipitation using confluent MDCK cell extracts. The extracts
were subjected to immunoprecipitation with anti-HP Ab or anti-ZO-2 Ab.
Preimmune serum (rabbit), purified rabbit IgG, and anti-p53 Ab (an
irrelevant Ab) were used as controls. Analysis of immunoprecipitates by
immunoblotting using anti-HP Ab showed that anti-ZO-2 Ab coprecipitated
two polypeptides of 
135 and 150 kDa; these comigrated with two
polypeptides of similar molecular mass that were immunoprecipitated by
anti-HP Ab (Fig. 3A).
Similarly, analysis of the immunoprecipitates by immunoblotting using
anti-ZO-2 Ab showed that ZO-2 coprecipitated with 4.1R (Fig. 3B). Neither 4.1R nor ZO-2 was coprecipitated with preimmune
serum, rabbit IgG, or anti-p53 Ab (Fig. 3, A and
B). These results suggest that 4.1R and ZO-2 are associated
together in vivo. Analysis of the same immunoprecipitates by
immunoblotting using anti-24-kDa Ab also revealed the presence of
135- and 150-kDa polypeptides (Fig. 3C), confirming
that these polypeptides are 4.1R isoforms. However, these isoforms of
4.1R were not detected in anti-HP Ab or anti-ZO-2 Ab supernatants by
anti-24-kDa Ab, but were detected by anti-HP Ab. Because
anti-24-kDa Ab is raised against exon 19, these data suggest that most
of the 135- and 150-kDa 4.1R isoforms that contain alternative exon 19 coprecipitate with ZO-2 and therefore are not detected in the
supernatant, whereas those isoforms not containing exon 19 do not
coprecipitate with ZO-2 and thus are detected by anti-HP Ab. This is
consistent with the results of yeast two-hybrid mapping data that exons
19-21 of 4.1R are required for interaction with ZO-2 (Fig.
4B).
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Fig. 3.
4.1R and ZO-2 coprecipitate in confluent MDCK
cell extracts. MDCK extracts were prepared as described under
"Experimental Procedures" and subjected to immunoprecipitation
using different antibodies. The immunoprecipitates were analyzed by
immunoblotting using anti-HP Ab (A), anti-ZO-2 Ab
(B), and anti-24-kDa Ab (C). (In C,
anti-ZO-2 Ab appears to coprecipitate more 4.1R than anti-HP Ab because
of unequal loading.) One-fourth of the immunoprecipitates of anti-HP
Ab, preimmune serum, anti-ZO-2 Ab, rabbit IgG, and anti-p53 Ab and
one-eighth of the supernatant fractions of anti-HP Ab (anti-HP Ab
sup.) and anti-ZO-2 Ab (anti-ZO-2 Ab sup.)
immunoprecipitates were loaded in order.
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Fig. 4.
The amino acids through which 4.1R and hZO-2
interact reside within the C-terminal 96 amino acids of 4.1R and amino
acids 1054-1168 of the proline-rich domain of hZO-2.
A, 4.1R and hZO-2 interact through their C-terminal domains.
4.1R and hZO-2 peptides and their segments were expressed as Gal4-BD or
Gal4-AD fusion proteins, respectively, in yeast strain Y190 by
cotransformation and were assayed for the expression of the reporter
genes (see "Experimental Procedures" for details). Plus
signs indicate the expression of the reporter genes
lacZ and HIS3 (and thus the interaction between
the peptides), and minus signs indicate non-expression of
the reporter genes (and thus no interaction between the peptides).
B, the C-terminal 96 amino acids of 4.1R are sufficient for
its interaction with hZO-2. Schematic diagrams of the various exons
within the 22-24-kDa domain of 4.1R (white rectangles)
fused to the DNA-binding domain of Gal4 (shaded rectangles)
used in the two-hybrid assay are shown. The names of the plasmids that
encoded each construct are given to the right of each schematic
diagram. Plasmid pAS2-1 expresses the Gal4 DNA-binding domain alone and
was used as a negative control. These plasmids were cotransformed with
hZO-2/pACT2 expressing amino acids 980-1168 of hZO-2 fused to the
activation domain of Gal4. C, amino acids 1054-1118 of
hZO-2 are sufficient for its interaction with 4.1R. Schematic diagrams
of the various hZO-2 polypeptides fused to the activation domain of
Gal4 (hatched) used in the two-hybrid assay are shown.
Plasmid pACT2, which expresses the Gal4 activation domain alone, was
used as a negative control (data not shown). These plasmids were
cotransformed with pGBT9 or pAS2-1 expressing the C-terminal domain of
4.1R or its exons 19-21 fused to Gal4-BD.
-gal, -galactosidase; GUK,
guanylate kinase; Alt. Spl., alternatively
spliced.
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We determined the efficiencies of immunoprecipitation and
coprecipitation to discern the fractions of 4.1R and ZO-2 that
associate together. Quantitation from three different experiments
(representative gels are shown in Fig. 3, A and
B) showed that 80-90% of 4.1R was precipitated by anti-HP
Ab, which in turn coprecipitated 35-48% of ZO-2. In addition, >95%
of ZO-2 was precipitated by anti-ZO-2 Ab, which coprecipitated 45-55%
of 4.1R. Analysis of the post-extraction pellet (insoluble fraction)
revealed that only 5-10% of both 4.1R and ZO-2 were not extracted
(data not shown). This is consistent with the notion that both 4.1R and
ZO-2 move out of the nucleus at confluent density in MDCK cells (75,
76). Thus, ~35-45% of total cellular 4.1R and ZO-2 associate
together in confluent MDCK cell TJs. Taken together, all of these
results strongly suggest that about half of the cytoplasmic 135-kDa
nonerythroid isoforms of 4.1R in MDCK cells interact with ZO-2.
The complex splicing pattern of 4.1R generates distinct isoforms with
insignificant differences in molecular mass, but with a significant
effect on their subcellular targeting. Although the isoform(s) of 4.1R
that interact with hZO-2 are also of the same higher molecular class
(~135 kDa) that interacts with NuMA in interphase nuclei and during
mitosis (13), the same isoform(s) may not interact with NuMA because of
their distinct subcellular localization. However, it has been shown
that a given 4.1R isoform can adopt several localizations within the
cell (11).
The Amino Acid Residues Required for 4.1R and hZO-2 Interaction
Reside within the C-terminal 96 Amino Acids of 4.1R and Amino Acids
1054-1118 of hZO-2--
To map the minimal segments of 4.1R and hZO-2
or ZO-2 that interact, we examined the interactions between different
domains or their segments of 4.1R and hZO-2 or ZO-2 in the yeast
two-hybrid assays. We expressed different domains or segments of 4.1R
as fusion products of the Gal4 DNA-binding domain in pAS2-1 and
cotransformed them into Y190 with different segments of hZO-2 or ZO-2.
The latter were expressed as Gal4-AD fusion products in pACT2, as shown
in Fig. 4A. Full-length 4.1R (135 kDa), its 80-kDa isoform,
and its 22-24-kDa domain interacted with 1) full-length hZO-2, 2) a
segment of the C-terminal proline-rich domain of hZO-2 (aa 980-1168), and 3) amino acids 187-1174 of ZO-2. They did not interact with the N-terminal part of ZO-2 (aa 1-189) (Fig. 4A). The
N-terminal extension (HP) and the 30-, 16-, and 10-kDa domains of 4.1R
failed to interact with hZO-2 or ZO-2. None of the domains of 4.1R,
when expressed as Gal4-BD fusion proteins in Y190, expressed the
reporter gene(s) by themselves or in combination with Gal4-AD in pACT2 alone (data not shown). These data suggest that the C-terminal domains
of 4.1R and hZO-2 or ZO-2 are necessary and sufficient for their interaction.
As illustrated in Fig. 4B, exons 17 and 18 were not required
for interaction of hZO-2 with 4.1R. Fusion proteins consisting of exons
19-21 of 4.1R (exons 19-21/pGBT9) bound to hZO-2, but none of these
exons alone (exon 19/pGBT9, exon 20/pGBT9, or exon 21/pGBT9) was
sufficient for binding. Amino acids 980-1168 were encoded by the hZO-2
clones obtained from the two-hybrid screening. As shown in Fig.
4C, the C-terminal truncation of the fusion protein from
amino acids 1119 to 1168 did not impede binding, but further truncation
from the C-terminal end (aa 1074-1168) abolished the binding of hZO-2
to 4.1R. From the N-terminal end, truncation of the hZO-2 fusion
protein from its N terminus up to amino acid 1053 did not affect its
binding to 4.1R. The fusion protein encoding amino acids 1054-1118 of
hZO-2 bound to 4.1R. The binding strength was comparable to the fusion
protein encoding amino acids 980-1168 of hZO-2, as assessed by
expression of the reporter gene lacZ (data not shown).
Therefore, it appears that amino acids 1054-1118 of hZO-2 are
sufficient for its interaction with 4.1R. These results suggest that
the amino acids through which 4.1R and hZO-2 interact reside within
amino acids encoded by exons 19-21 of 4.1R and amino acids 1054-1118
of hZO-2.
Protein 4.1R Associates with Tight Junction Proteins ZO-1, ZO-2,
and Occludin in Confluent MDCK Cells--
To examine the association
of 4.1R with the TJ protein complex, we asked if it occurred in
association with other TJ components such as ZO-1 and occludin
(54-56). Therefore, we performed immunoprecipitation using confluent
MDCK cell extracts and looked for 4.1R in the immunoprecipitates of
ZO-1 and occludin and vice versa. Rabbit preimmune serum,
purified rabbit IgG, and anti-p53 Ab (an irrelevant Ab) were used as
negative controls to rule out nonspecific aggregation. ZO-2, which is
known to coprecipitate with and bind directly to ZO-1 (57, 58) and
occludin (41, 58), was used as a positive control.
As stated earlier and shown in Fig.
5A, two 4.1R isoforms (135
and 150 kDa) were immunoprecipitated by anti-HP Ab. These isoforms
of 4.1R were also found to coprecipitate with ZO-2, ZO-1, and occludin
when immunoprecipitation was carried out with anti-ZO-2 Ab, anti-ZO-1
Ab, and anti-occludin Ab, respectively, but not with preimmune serum,
rabbit IgG, or anti-p53. Analysis of the same immunoprecipitates by
immunoblotting using antibodies specific for the 16-kDa domain of 4.1R
(anti-16-kDa Ab) also revealed the ~135- and ~150-kDa 4.1R isoforms
in the immunoprecipitates of anti-HP Ab, anti-ZO-2 Ab, anti-ZO-1 Ab,
and anti-occludin Ab. No other 4.1R isoforms were seen in the
immunoprecipitates of ZO-2, ZO-1, and occludin, suggesting that the
isoforms of 4.1R that associate with these TJ proteins are of the
135-kDa molecular mass class. This study also confirmed the 150-kDa
protein as a 4.1R isoform.
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Fig. 5.
4.1R coprecipitates with ZO-1, ZO-2, and
occludin in confluent MDCK cell extracts. Confluent MDCK cell
lysates were subjected to immunoprecipitation using different
antibodies as described under "Experimental Procedures." The
immunoprecipitates were analyzed by immunoblotting using anti-HP Ab
(A), anti-16-kDa Ab (B), anti-ZO-2 Ab
(C), anti-occludin Ab (D), anti-ZO-1 Ab
(E), anti-actin monoclonal antibody (F), or
anti-spectrin Ab (G). H is a silver-stained gel
of Bio-Rad prestained molecular mass markers (first lane)
and immunoprecipitates of anti-HP Ab (second lane) and
preimmune (Preimm.) serum (third lane). The
positions of 4.1R (135- and 150-kDa isoforms), ZO-1, ZO-2, occludin,
actin, and spectrin identified by immunoblot analysis of a duplicate
gel are indicated.
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Analysis of duplicates of the blot shown in Fig. 5A by
immunoblotting using anti-ZO-2 Ab (Fig. 5C),
anti-occludin Ab (Fig. 5D), and anti-ZO-1 Ab (Fig.
5E) showed that ZO-1, ZO-2, occludin, and two 4.1R isoforms
(135 and 150 kDa) coprecipitated together. None of these proteins
were detected in immunoprecipitates of any of the control antibodies
(Fig. 5, A-E). These results suggest that two 4.1R
isoforms, ZO-2, ZO-1, and occludin associate together in
vivo.
To examine the involvement of other 4.1R-binding cytoskeletal proteins
such as actin and spectrin in this complex, we analyzed the anti-HP Ab,
anti-ZO-2 Ab, anti-ZO-1 Ab, and anti-occludin Ab immunoprecipitates by
immunoblotting using the relevant antibodies. As shown in Fig.
5F, actin was found to coprecipitate with 4.1R, ZO-2, ZO-1,
and occludin, but not with p53, preimmune serum, or rabbit IgG
immunoprecipitates. Anti-spectrin Ab detected both the - and
-isoforms of spectrin in MDCK cell lysates (Fig. 5G). Interestingly, only -spectrin was detected in the anti-HP Ab, anti-ZO-2 Ab, anti-ZO-1 Ab, and anti-occludin Ab immunoprecipitates. The absence of actin and spectrin (despite their abundance in the cell)
in immunoprecipitates of preimmune serum, rabbit IgG, and anti-p53 Ab
indicated that their presence in the immunoprecipitates of anti-HP Ab,
anti-ZO-2 Ab, anti-ZO-1 Ab, and anti-occludin Ab was due to a specific
intracellular association with these proteins. The coprecipitation of
actin and -spectrin with the TJ proteins was not surprising because
the C-terminal half of ZO-1 has been shown to cosediment with actin
filaments (58, 59), and -spectrin has been shown to coprecipitate
with ZO-1 (60, 61).
To examine the presence of other 4.1R-binding and/or TJ-associated
proteins in this protein complex, anti-HP Ab immunoprecipitates were
examined by SDS-polyacrylamide gel electrophoresis and silver staining
(Fig. 5H). About 17 protein bands were visualized in the
anti-HP Ab immunoprecipitates, but not in the preimmune serum immunoprecipitates. The molecular mass of some of these proteins was
identical to that of some of the TJ-associated proteins. A duplicate of
the gel in Fig. 5H was transferred to polyvinylidene difluoride membrane and analyzed by immunoblotting for the
identification of 4.1R, ZO-1, ZO-2, occludin, actin, and spectrin using
the relevant antibodies (data not shown). The protein bands on the
silver-stained gel that were identified by immunoblotting are labeled
(Fig. 5H). These results suggest that 4.1R isoform(s) and TJ
proteins ZO-1, ZO-2, and occludin (and possibly others) along with
actin and -spectrin associate together in vivo.
Protein 4.1R Does Not Associate with Tight Junction Proteins ZO-1,
ZO-2, and Occludin in Nonconfluent MDCK Cells--
To examine whether
or not the association of 4.1R isoforms is TJ-specific, we repeated the
immunoprecipitation experiments in nonconfluent (<50% confluent) MDCK
cell lysates (TJ not organized). As shown in Fig.
6A, in contrast to confluent
cell lysates, only one isoform of 4.1R (135 kDa) was precipitated by
anti-HP Ab. Because 135-kDa 4.1R was expressed in confluent as well as
nonconfluent cells, but ~150-kDa 4.1R was detected in confluent cells
only, it appears that ~150-kDa 4.1 might play a critical role in
tight junction organization. However, our initial efforts to delineate the proper exon combination of the ~150-kDa isoform have not
yielded precise identity of this isoform. The characterization of the ~150-kDa isoform is in progress. Anti-ZO-1 Ab, anti-ZO-2 Ab, and anti-occludin Ab failed to co-immunoprecipitate 4.1R in the
nonconfluent state (Fig. 6A), even though ZO-1 was
coprecipitated with ZO-2 and vice versa (Fig. 6, B and
C). The fractions of ZO-2 and ZO-1 that coprecipitated were
also less compared with those of the confluent cell lysates. Occludin
also did not coprecipitate with 4.1R, ZO-1, ZO-2, or any of the
controls (Fig. 6D). Analysis of the immunoprecipitates by
immunoblotting showed that neither actin (Fig. 6E) nor
spectrin (Fig. 6F) was coprecipitated with 4.1R, ZO-1, ZO-2,
or occludin. The difference in results observed in contrast to
confluent cells was not due to non-expression of the above proteins
because all these proteins were detected in whole cell lysate (Fig. 6,
A-F). The results from Figs. 5 and 6 suggest that the
association of 4.1R, ZO-1, ZO-2, occludin, actin, and -spectrin
together in MDCK cells is dependent on TJ formation, which is organized
only when these cells become confluent.
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Fig. 6.
4.1R does not coprecipitate with ZO-1, ZO-2,
and occludin in nonconfluent MDCK cell extracts. Nonconfluent MDCK
cell lysates were subjected to immunoprecipitation using different
antibodies as described under "Experimental Procedures." The
immunoprecipitates were analyzed by immunoblotting using anti-HP Ab
(A), anti-anti-ZO-2 Ab (B), anti-ZO-1 Ab
(C), anti-occludin Ab (D), anti-actin monoclonal
antibody (mAb; E), or anti-spectrin Ab
(F). The positions of actin in E and of - and
-spectrins in F are indicated. Note that only the 135-kDa
isoform of 4.1R was immunoprecipitated by anti-HP Ab from nonconfluent
MDCK cell extracts. The 150-kDa 4.1R isoform was also not detected in
nonconfluent MDCK cell lysate. Preimm., preimmune.
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Protein 4.1R Binds to ZO-2, ZO-1, and Occludin in
Vitro--
Because the results from immunoprecipitation experiments
suggested that 4.1R associates with TJ proteins ZO-2, ZO-1, and
occludin in vivo, we asked if there were additional binary
interactions between 4.1R and ZO-1 or occludin. We constructed yeast
expression vectors expressing full-length ZO-1, occludin, or their
different segments in frame with Gal4-AD and performed yeast two-hybrid assays with 4.1R and its subdomains. The results of filter lift -galactosidase activity assays were inconclusive. Therefore, chlorophenol red--D-galactopyranoside-based liquid
-galactosidase activity assays were performed. As shown in Fig.
7A, the N-terminal cytoplasmic
domain of occludin (aa 1-57) did not show substantial -galactosidase activity over the control. However, full-length occludin (data not shown) as well as its C-terminal cytoplasmic domain
(aa 250-504) showed weak interactions with the 135- and 80-kDa
isoforms and C-terminal domain of 4.1R. A weak interaction was
also observed between the N-terminal cytoplasmic domain of occludin (aa
1-57) and HP of 4.1R. In addition, weak interactions were detected
between the 135- and 80-kDa isoforms and the 24-kDa domain of 4.1R and
the C-terminal proline-rich domain (aa 1045-1737) (Fig.
7B), but not the N-terminal part (aa 1-1044) of ZO-1 (data not shown).
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Fig. 7.
Interaction of 4.1R with occludin and ZO-1 in
yeast two-hybrid assays. A, binary interaction between
4.1R and occludin in yeast two-hybrid assays. 4.1R and occludin
peptides and their segments were expressed as Gal4-BD or Gal4-AD fusion
proteins, respectively, in S. cerevisiae strain Y190 by
cotransformation and were assayed for the expression of the reporter
gene lacZ by the chlorophenol
red--D-galactopyranoside
(CPRG)-based liquid assay (see "Experimental
Procedures" for details). The results are presented as percent of
control ± S.D. (n = 3). B, binary
interaction between 4.1R and ZO-1 in yeast two-hybrid assays. 4.1R and
ZO-1 peptides and their segments were expressed as Gal4-BD or Gal4-AD
fusion proteins, respectively, in S. cerevisiae strain Y190
by cotransformation and were assayed for the expression of the reporter
gene lacZ by the chlorophenol
red--D-galactopyranoside-based liquid assay (see
"Experimental Procedures" for details). The results are presented
as percent of control ± S.D. (n = 3).
-gal, -galactosidase; GUK,
guanylate kinase.
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To confirm direct binding of 4.1R to ZO-2, ZO-1, or occludin, we
performed in vitro binding assays. Different isoforms and domains of 4.1R were expressed as GST fusion proteins and purified by
coupling to GST-Sepharose beads as described (13). In vitro binding was performed between 4.1R/GST and in vitro
translated [35S]methionine-labeled hZO-2 (aa 905-1054,
hZO-2-1/TOPO; or aa 980-1168, hZO-2-2/TOPO), hZO-1 (aa 1042-1454,
ZO-1-1/TOPO; or aa 1453-1747, ZO-1-2/TOPO), or occludin (aa 130-283,
Ocl-1/TOPO; or aa 284-522, Ocl-2/TOPO). As shown in Fig.
8 (A-F), aa 980-1168 of
hZO-2, aa 1042-1454 of ZO-1, and aa 284-522 of occludin bound to
135-kDa/GST, 80-kDa/GST, and 24-kDa/GST, but not to 30-kDa/GST,
16-kDa/GST, 10-kDa/GST, HP/GST, or GST alone. The binding constants of
ZO-2, ZO-1, and occludin with the 24-kDa domain of 4.1R were
determined. Each binding was saturable; and from Scatchard analyses,
Kd values of ~4.5 × 10
8, 7.5 × 107, and 7.1 × 107 M for hZO-2, ZO-1, and
occludin (Fig. 8G) were revealed, respectively. These
results suggest direct binding of ZO-2, ZO-1, and occludin to 4.1R and
are consistent with results from the yeast two-hybrid assays.
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Fig. 8.
Protein 4.1R binds to hZO-2, hZO-1, and
occludin in vitro. GST alone or 4.1R/GST fusion
proteins coupled to glutathione-Sepharose beads were incubated with
in vitro translated [35S]methionine-labeled
segments of hZO-2, ZO-1, or occludin. After washing, the proteins
complexed to beads were analyzed by SDS-polyacrylamide gel
electrophoresis and detected by fluorography. A, in
vitro binding between 4.1R/GST and amino acids 905-1059 of hZO-2.
This segment of hZO-2 did not bind to 4.1R isoforms or to any of the
4.1R domains. B, in vitro binding between
4.1R/GST and amino acids 980-1168 of hZO-2. This segment of hZO-2
bound to the 135- and 80-kDa 4.1R isoforms and to the 4.1R 24-kDa
domain. C, in vitro binding between 4.1R/GST and
amino acids 1453-1747 of hZO-1. This segment of hZO-1 did not bind to
4.1R isoforms or to any of the 4.1R domains. D, in
vitro binding between 4.1R/GST and amino acids 1042-1454 of
hZO-1. This segment of hZO-1 bound to the 135- and 80-kDa 4.1R isoforms
and to the 4.1R 24-kDa domain. E, in vitro
binding between 4.1R/GST and amino acids 130-283 of human occludin.
This segment of human occludin did not bind to 4.1R isoforms or to any
of the 4.1R domains. F, in vitro binding between
4.1R/GST and amino acids 284-522 of human occludin. This segment of
occludin bound to the 135- and 80-kDa 4.1R isoforms and to the 4.1R
24-kDa domain. G, quantitative analysis of binding between
the 24-kDa domain of 4.1R and aa 980-1168 of hZO-2, aa 1042-1454 of
ZO-1, and aa 284-522 of human occludin. A glutathione-Sepharose bead
slurry containing 24-kDa/GST was incubated with 0.0025-0.015 µg of
in vitro translated [35S]methionine-labeled
hZO-2, hZO-1, or occludin (from the top). The amount of translated
peptide that bound to the bead product in each case was
quantitated as described under "Experimental Procedures." Each
value represents the mean value of triplicate experiments. The binding
was saturable, and Scatchard analysis (inset) indicated
Kd values of ~4.5 × 108, 7.5 × 107, and 7.1 × 107 M for hZO-2, ZO-1, and
occludin, respectively.
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Recruitment of 4.1R/GFP Fusion Proteins to Tight Junctions in
Confluent MDCK Cells--
To verify that 4.1R interacts with ZO-2
and/or other tight junction-associated proteins in intact cells, we
constructed expressions vectors with segments of 4.1R fused to GFP and
transfected them into cultured MDCK cells. Because results from yeast
two-hybrid and in vitro binding assays suggest that the
22-24-kDa domain of 4.1R is sufficient for interaction with ZO-2,
ZO-1, and occludin, cells were transfected with 24-kDa/GFP. GFP alone
and 10-kDa/GFP were used as controls. However, as shown in Fig.
9, GFP alone, 10-kDa/GFP, or 24-kDa/GFP
was not recruited to cell-cell junctions (Fig. 9, A-C), but
10+24-kDa/GFP was efficiently recruited to cell-cell junctions (Fig. 9,
D and E). The state of confluency and
localization of ZO-1 at cell-cell junctions in the same cells were
revealed by staining the cells with anti-ZO-1 Ab (Fig. 9, A'-E'). Confocal microscopy also revealed that
the 10+24-kDa/GFP fusion proteins colocalized with ZO-2 and ZO-1 in
cultured MDCK cell tight junctions (data not shown). All these fusion
proteins were also concentrated in the nucleus.
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Fig. 9.
Localization of transiently expressed
4.1R/GFP fusion proteins in confluent MDCK cells. GFP or the
10-kDa/GFP, 24-kDa/GFP, or 10+24-kDa/GFP fusion proteins were
exogenously and transiently expressed in MDCK cells. The cells were
grown to confluence, fixed, and stained with anti-ZO-1 Ab (in
red; A'-E') for localization of ZO-1.
Expression of GFP or GFP fusion proteins was visualized by
green fluorescence (A-E). 10+24-kDa/GFP fusion
proteins were recruited to cell-cell junctions, but not GFP alone,
10-kDa/GFP, or 24-kDa/GFP. Anti-ZO-1 antibody-stained cells that
correspond to cells transfected with GFP or 4.1R/GFP fusion proteins
are indicated with arrows in corresponding pictures.
Magnification × 40.
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DISCUSSION |
Several studies suggest that the cortical cytoskeleton is involved
in the structural and functional organization of TJs (reviewed in Ref.
56; Ref. 62), but little is known about the molecule(s) that link these
two distinct structures. In this study, we demonstrate that two
nonerythroid isoforms of 4.1R (~135 and ~150 kDa) interact with
hZO-2. In addition to ZO-2, these isoforms of 4.1R also associate with
other TJ proteins such as ZO-1 and occludin and the cytoskeletal proteins -spectrin and actin in one protein complex. Endogenous components of TJs also efficiently recruit tagged 4.1R segments containing the spectrin/actin- and ZO-2-binding domains to TJs. We thus
hypothesize that 4.1R isoforms in epithelial cells may participate in
regulation of TJ by mediating a direct link between the TJ proteins
with the underlying cytoskeleton.
ZO-2 belongs to the MAGUK protein family. MAGUK proteins such as p55,
human DLG, and the human LIN2 homolog are known to associate with the
cortical actin cytoskeleton. In erythrocytes, the MAGUK protein p55
links the transmembrane protein glycophorin C to the spectrin/actin
cytoskeleton through the 30-kDa domain of protein 4.1R (20). Human DLG
and the human LIN2 homolog are also known to bind to 4.1R in epithelial
cells (15, 29). Similarly, ZO-1, a member of the MAGUK family of
proteins, binds to ZAK, a serine-threonine kinase (63), and links the
transmembrane protein occludin to cytoskeleton by binding to F-actin
(59). However, the interaction between hZO-2 and 4.1R was unexpected
because hZO-2 lacks the 4.1R-binding motif used by the MAGUK proteins
p55, human DLG, and human LIN2, a conserved lysine-rich sequence motif
located between the SH3 and guanylate kinase domains (15, 20, 29).
Although the C-terminal domain encoded by exons 19-21 of 4.1R
interacts with multiple PDZ domains containing hZO-2, the expected PDZ
domain-tail interaction (64) was not observed. Our data suggest that
the amino acids required for the interaction of 4.1R and hZO-2 reside
within the amino acids encoded by exons 19-21 of the C-terminal domain
of 4.1R and amino acids 1054-1118 of the proline-rich domain of hZO-2.
Because deletion of exon 19, 20, or 21 abolishes this interaction, it
appears that the amino acids of 4.1R that interact with hZO-2 may be
distributed in these three exons and that there may be multiple contact
sites between these proteins. It is also possible that the presence of
these three exons of 4.1R together gives rise to a particular folding of 4.1R required for its interaction with hZO-2 or ZO-2. A recent study
shows that these amino acids of 4.1R are highly conserved among other
4.1R-like genes (30), raising the possibility that these gene products
may also interact with hZO-2 and ZO-2.
ZO-2 is known to associate with the cytoplasmic surface of the TJ (51,
57). It shares a strong homology with ZO-1, especially within the
conserved MAGUK domains (39). It is expressed in almost every tissue
(44) and associates with ZO-1 through its second PDZ domain at both the
adherens junctions and TJs (41, 51, 58, 65). ZO-2 binds directly to the
C-terminal 147 amino acids of occludin (66) and coprecipitates with
occludin and -catenin (41). It is a component of the TJ along with
ZO-1, ZO-3, cingulin, 7H6 antigen, symplekin, several unidentified
proteins, and transmembrane proteins such as occludin (reviewed in Ref. 56) and members of the claudin family (67).
ZO-1, ZO-2, and occludin have been shown to localize at TJs of
confluent MDCK cells (51-53, 61). Using anti-HP Ab, an antibody specific for nonerythroid isoforms of 4.1R, we found that 4.1R epitopes
are also localized at TJs of MDCK cells by confocal microscopy and high
resolution electron microscopy (Fig. 2, B and H,
respectively). Confocal microscopic analysis of double-labeled
confluent MDCK cells showed that these 4.1R epitopes colocalize at TJs
with ZO-2 and occludin (Fig. 2, C and F). In
immunoprecipitation studies using the same antibodies, ZO-2 and two
nonerythroid 4.1R isoforms were found to coprecipitate together. About
40-50% of 4.1R and ZO-2 appear to remain associated together under
our experimental conditions. Unlike ZO-2 and ZO-1, which also localize
at adherens junction in addition to TJs, occludin is exclusively
localized at the TJs (52, 53). We found that 4.1R, ZO-1, ZO-2, and
occludin coprecipitated together. This association of 4.1R with ZO-2,
ZO-1, or occludin was not seen in cells that were not confluent (and thus in which the tight junction was not organized) (Fig. 6), strongly
suggesting that these proteins associate together at TJs.
Coprecipitation does not reveal whether there is a direct interaction
between 4.1R and ZO-1 or occludin. We thus tested this hypothesis in
yeast two-hybrid and in vitro binding assays. As shown in
Fig. 7, only weak interactions between the C-terminal domain of 4.1R
and the proline-rich domain of ZO-1 (Fig. 7B) or the
C-terminal cytoplasmic domain of occludin (Fig. 7A) were
observed in yeast two-hybrid assays. These interactions were only
25-30% of the interaction between 4.1R and hZO-2 or ZO-2 as detected in yeast two-hybrid assays. ZO-2, ZO-1, and occludin bound to 4.1R in
in vitro binding assays (Fig. 8). Interestingly, a recent study suggested that a third protein might mediate the interaction between ZO-1 and ZO-2 (58). Similarly, a mutant occludin that bound to
ZO-1 in vitro failed to localize at TJs (68). Both ZO-1 and
ZO-2 also localize to TJs in occludin-deficient TJs (41, 69). These
findings imply that association of other factors, in addition to ZO-1
and ZO-2, may be required for localization of occludin at TJs.
Both the 135- and 80-kDa isoforms interact with ZO-2 in yeast
two-hybrid assays, whereas HP alone does not. Thus, our results do not
define the precise isoform(s) of 4.1R that interact with ZO-2. Because
of the complex splicing pathway that 4.1R undergoes, it is difficult to
know exactly which exons are included in the isoform(s) that interact
with ZO-2. However, epitopes for anti-HP Ab are observed at TJs;
moreover, the two isoforms of 4.1R that coprecipitate with ZO-2 and
other TJ proteins are exclusively of the higher molecular mass.
Finally, no 80-kDa isoform containing exon 19 (which is required for
its interaction with ZO-2) is expressed in MDCK cells (13). We thus
believe that isoforms of 4.1R that interact with ZO-2 are of the
135-kDa class or of similar molecular mass. The 80-kDa isoform binds to
ZO-2 in yeast two-hybrid and in vitro binding assays, but
does not coprecipitate with ZO-2. These data are compatible because the
increased concentration and proximity of molecules containing the
binding sites in yeast two-hybrid and in vitro binding
assays could allow the 80-kDa isoform to interact with ZO-2. In the
intact cells, factors such as post-translational modifications and
compartmentalization or presence of modifying cofactors will affect
their interaction. In the same context, the amino acids encoded by
exons 19-21 of 4.1R are required and sufficient for interaction of
4.1R with ZO-2 in vitro, but not in vivo. Indeed,
the 22-24-kDa domain is not sufficient to target GFP to tight
junctions, although per se it binds well to ZO-2. These
amino acids are highly conserved among the 4.1-like gene family (30).
It can thus be argued that other 4.1R-like gene products interact with
ZO-2. However, as discussed above, our results strongly implicate 4.1R
isoforms as opposed to 4.1-like gene products because the anti-HP Ab
used in our assays does not react with other 4.1-like proteins.
A noteworthy finding is the association of 4.1R with ZO-1, ZO-2,
occludin, -spectrin, and actin apparently in one protein complex,
implying a connection of TJs to the cortical cytoskeleton, as
previously suggested (Refs. 62 and 70; reviewed in Ref. 56). Several
studies also suggest that actin plays a role in regulation of TJ
permeability (71, 72). ZO-1 has been shown to cosediment with F-actin
(58, 59) and to colocalize with actin (60, 72-74). ZO-1, ZO-2, and
occludin cosediment with -spectrin (68). ZO-1 has been suggested to
link the actin cytoskeleton to TJ via an "actin/ZO-1/occludin"
linkage (58, 59). The C-terminal parts of ZO-2 and ZO-3 show poor
homology to the C-terminal half of ZO-1, which cosediments with
actin. ZO-2 and ZO-3 also lack the lysine-rich "protein 4.1-binding
motif" found in other MAGUK proteins. These observations suggest that
ZO-1 may be the only MAGUK protein at the TJ that can bind to actin and
thereby serve as the structural and signaling component of TJs. In
contrast, a recent in vitro study suggests that ZO-1, ZO-2,
and occludin can directly interact with F-actin (66).
The following evidence from our study suggests that 4.1R may provide or
supplement the linkage between the TJ and the cortical cytoskeleton.
First, ZO-2, ZO-1, and occludin were found to interact with 4.1R, which
in turn is known to bind to the cytoskeletal proteins actin and
spectrin. Second, the association of actin and -spectrin with ZO-1,
ZO-2, or occludin was not observed in nonconfluent cells when 4.1R also
failed to interact with these proteins. Third, ZO-2, ZO-1, and occludin
interacted with 4.1R in in vitro binding and yeast
two-hybrid assays. Finally, GFP-tagged segments of 4.1R that contained
both the spectrin/actin- and ZO-2-binding domains were recruited to
TJs, but the segments that contained only the spectrin/actin-binding
domain or the ZO-2 binding domain were not. Thus, as shown in Fig.
10, ZO-2, like other MAGUKs, may be
establishing a link between the TJ and the actin-based cytoskeleton via
a different binding interaction with 4.1R. This function of 4.1R in
nonerythroid cells is not established, but unpublished data from our
laboratory suggest that the 135-kDa nonerythroid isoform of 4.1R forms
a ternary complex with F-actin and fodrin (nonerythroid spectrin)
in vitro.2
Interaction of 4.1R with members of the MAGUK family, ZO-1, and ZO-2
could assist in recruitment of other proteins to coordinate cell
signaling. Other members of the protein 4.1 superfamily such as merlin,
ezrin, radixin, and moesin are known to be involved in different
signaling pathways (36, 37). It is thus tempting to speculate that 4.1R
may have a role in signal transduction between the TJ and the
cytoskeleton. A similar role of 4.1R has been suggested for the human
CASK and 4.1R interaction (29).
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Fig. 10.
Schematic representation of a possible role
of 4.1R in linking the tight junction proteins with the cortical actin
cytoskeleton.
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ABSTRACT
INTRODUCTION
