From the Department of Molecular Biology and
Biochemistry, Osaka University Graduate School of Medicine/ Faculty
of Medicine, Suita 565-0871, Japan and § KAN Research
Institute Inc., 93 Chudoji-Awatamachi, Shimogyo-ku, Kyoto
600-8815, Japan
Received for publication, September 25, 2002, and in revised form, November 8, 2002
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ABSTRACT |
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Afadin is an actin filament (F-actin)-binding
protein that is associated with the cytoplasmic tail of nectin, a
Ca2+-independent immunoglobulin-like cell-cell
adhesion molecule. Nectin and afadin strictly localize at cell-cell
adherens junctions (AJs) undercoated with F-actin bundles and are
involved in the formation of AJs in cooperation with E-cadherin in
epithelial cells. In epithelial cells of afadin ( Cells in multicellular organisms recognize their neighboring
cells, adhere to them, and form intercellular junctions. Such junctions
have essential roles in various cellular functions, including
morphogenesis, differentiation, proliferation, and migration (for
reviews, see Refs. 1-6). In polarized epithelial cells, intercellular
adhesion is mediated through a junctional complex composed of tight
junctions (TJs),1 adherens
junctions (AJs), and desmosomes. These junctional structures are
typically aligned from the apical to basal sides, although desmosomes
are independently distributed in other areas.
AJs were originally defined using ultrastructural analysis as closely
apposed plasma membrane domains reinforced by a dense cytoplasmic
plaque, where actin filament (F-actin) bundles are undercoated (7).
Molecular analysis shows that AJs are cell-cell adhesion sites where
classic cadherins function as cell adhesion molecules and where the
actin-based cytoskeleton and several cytoplasmic components are
assembled (8-10). E-cadherin, like other classical cadherins, is a
single-pass transmembrane protein whose extracellular domain mediates
homophilic recognition and adhesive binding in a
Ca2+-dependent manner (10). E-cadherin
associates with the actin cytoskeleton through peripheral membrane
proteins, including We have found that another cell-cell adhesion molecule, nectin, and its
associated F-actin-binding protein, afadin, strictly localize at AJs
undercoated with F-actin bundles (18-20). In contrast, E-cadherin is
concentrated at AJs but is more widely distributed from the apical to
basal sides of the lateral plasma membranes (2, 21). Nectin is a
Ca2+-independent immunoglobulin-like cell-cell adhesion
molecule (19, 22-26). Nectin comprises a family, which, at present,
consists of four members, nectin-1, -2, -3, and -4. All nectins have
two or three splice variants (19, 25-31). Nectin-1 was originally identified as one of the poliovirus receptor-related proteins (PRR-1)
(30). Nectin-2 was originally identified as the murine homolog of human
poliovirus receptor protein (27) but turned out to be another
poliovirus receptor-related protein (PRR-2) (29, 30). Neither PRR-1 nor
PRR-2 has thus far been shown to serve as a poliovirus receptor. PRR-1
and PRR-2 were later shown to serve as the receptors for Afadin has at least two splice variants, l- and s-afadin (18).
l-Afadin, the larger splice variant, binds nectin and, through its
F-actin binding domain, F-actin. l-afadin binds to the side of F-actin
but does not cross-link it to form bundles. Afadin also has two
Ras-associated domains, a forkhead-associated domain, a dilute
(DIL) domain, a PDZ domain, and three proline-rich domains (see Fig.
1A). F-actin binds to the region containing the third proline-rich domain (18). s-afadin, the smaller splice variant, has two
Ras-associated domains, a forkhead-associated domain, a DIL, a PDZ, and
two proline-rich domains, but lacks the F-actin-binding domain. Human
s-afadin is identical to the gene product of AF-6, a gene
that has been identified as an ALL-1 fusion partner that is
involved in acute myeloid leukemias (37). Unless otherwise specified,
afadin refers to l-afadin in this paper.
Nectin has a potency to recruit the E-cadherin- To gain the insight into these issues, we attempted here to identify an
afadin-binding protein using yeast two-hybrid screening. For this
purpose, we used the DIL domain of afadin as a bait. The DIL domain has
been found in afadin and type V myosins including dilute, Myo2, and
Myo4, but its function remains unknown (64). The recent finding that
the region of Myo4 containing its DIL domain binds to an adapter
protein, She3 (65, 66), implies that the DIL domain is involved
in the protein-protein interaction. We have identified and
characterized here a novel afadin-binding protein, named ADIP
(afadin DIL domain-interacting
protein), that binds to the DIL domain of afadin. ADIP
furthermore binds Yeast Two-hybrid Screening--
One bait vector, pGBDU-afadin
(aa 511-981), was constructed by subcloning the insert encoding the aa
residue of afadin into pGBDU-C1 (67). Another bait vector,
pGBD-mouse ADIP (mADIP)-M (aa 152-436), was constructed by subcloning
the insert encoding the aa residue of mADIP into pGBD-C1 (67).
The plasmids, pGBDU-MYO4-DIL and pGAD-SHE3, were constructed by
subcloning the fragments of MYO4 (aa 920-1471) and SHE3 (aa 1-425)
into pGBDU-C2 and pGAD-C2, respectively. Yeast two-hybrid libraries
constructed from mouse 11-day embryo, rat brain, rat lung, and human
testis cDNAs were purchased from Clontech.
Two-hybrid screening using the yeast strain PJ69-4A (MATa
trp1-901 leu2-3, 112 ura3-52 his3-200 gal4 Construction of Expression Vectors--
For full-length
cDNAs of mADIP and rat ADIP (rADIP), BLAST searches were conducted
against GenBankTM and EMBL databases. A selection of hits
obtained by BLAST searches against the human subset of
GenBankTM and EMBL sequences was used to assemble the
cDNA sequence of the human homologue of KIAA0923 (AB023140;
GenBankTM/EMBL/DDBJ). A cDNA of KIAA0923 was kindly
supplied by Dr. T. Nagase (Kazusa DNA Research Institute). The
full-length cDNAs of mADIP (accession number AF532969) and rADIP
(accession number AF532970) were generated from mouse and rat brain
cDNAs (Clontech), respectively, by reverse
transcription-coupled PCR using the following primer sets: for mADIP
cDNA, 5'-CGTAGGAGAGTGACAGGAGCTG-3' and 5'-GGTTATCGAGTTTTTCTACATGAC-3'; for rADIP cDNA,
5'-CGTAGGAGAGTGACAGGAGCTG-3' and
5'-TTCCTGTTTTTGCACTGTAGCTG-3'.
The PCR products were subcloned into pCR4B (Invitrogen), and nucleotide
sequence analysis was performed by the dideoxynucleotide termination
method using a DNA sequencer (model 3100; Applied Biosystems, Inc.).
The mammalian expression vectors, pCMV-FLAG, pCMV-T7, and pCMV-HA, were
designed to express N-terminal FLAG-, T7-, and hemagglutinin
(HA)-tagged proteins, respectively (69). The mammalian expression
vector expressing the DIL domain of afadin (aa 606-983) was
constructed with pCMV-T7. The mammalian expression vectors expressing
mADIP and rADIP, pCMV-HA-mADIP (aa 1-615), pCMV-HA-mADIP-C (aa
339-615), pCMV-FLAG-mADIP-C (aa 339-615), and pCMV-FLAG-mADIP-M (aa
152-436), were constructed with pCMV-FLAG and pCMV-HA. The mammalian
expression vector expressing Antibodies--
The GST fusion proteins with fragments of
rADIP-C (aa 159-613), mADIP-N (aa 1-226), and mADIP-C (aa 339-615)
were produced in Escherichia coli, purified, and used as
each antigen to raise polyclonal antibodies (pAbs) in rabbits. Two pAbs
against rADIP-C (aa 159-613) and mADIP-C (aa 339-615), M05 and M01,
were used after affinity purification with MBP-rADIP-C (aa 159-613)
and MBP-mADIP-C (aa 339-615), respectively. Another pAb against
mADIP-N (aa 1-226), M57, was used after affinity purification with
GST-mADIP-N (aa 1-226). A mouse anti-afadin monoclonal Ab (mAb) was
prepared as described (71). A rat anti-ZO-1 mAb was purchased from
Chemicon. A mouse anti-E-cadherin mAb was purchased from Transduction
Laboratories. Mouse anti- Cell Culture and Transfection--
MDCK cells were kindly
supplied by Dr. W. Birchmeier (Max-Delbruck-Center for Molecular
Medicine, Berlin, Germany). HEK293 and MDCK were cultured in
Dulbecco's modified Eagle's medium with 10% fetal calf serum. HEK293
cells were transfected using the CalPhos mammalian transfection kit (Clontech).
Assay for Co-immunoprecipitation of ADIP with Afadin and
Co-immunoprecipitation experiments using MDCK cells were performed as
follows. MDCK cells on two 10-cm dishes were sonicated in 2 ml of
Buffer A, followed by ultracentrifugation at 100,000 × g for 15 min. The cell extract was precleared by incubation with protein A-Sepharose CL-4B beads (Amersham Biosciences) and then
incubated with 20 µl of anti-ADIP pAb (M05)-coated protein A-Sepharose CL-4B beads at 4 °C for 18 h. After the beads were washed with Buffer A, the bound proteins were eluted by boiling the
beads in the SDS sample buffer for 10 min. The samples were then
subjected to SDS-PAGE, followed by Western blotting with the anti-ADIP,
anti-afadin, and anti- Assay for the Binding of ADIP to Afadin and
Direct binding of ADIP to the DIL domain of afadin or the C-terminal
region of Other Procedures--
Northern blotting was performed as
described (72). The mADIP cDNA fragment was radiolabeled with
Identification of a Novel Afadin-binding Protein--
To identify
an afadin-binding protein, we performed the yeast two-hybrid screening
using the region of afadin containing the DIL domain (aa 606-983) as a
bait (Fig. 1A). We screened
2 × 105 clones of a mouse embryo library and 2 × 105 clones of a rat brain library and obtained 31 and 25 positive clones, respectively. Four mouse clones and one rat clone
encoded proteins similar to the carboxyl-terminal portion of human
KIAA0923 (AB023140; GenBankTM/EMBL/DDBJ). The full-length
clones of these mouse and rat cDNAs were isolated, and they encoded
proteins composed of 615 aa with a calculated Mr
of 70,954 and 613 aa with a calculated Mr of
70,684, respectively (Fig. 1B). We named this protein ADIP
((afadin DIL domain-interacting
protein)). ADIP has three coiled-coil domains (Figs.
1B and 2A). The aa
sequences of mADIP and rADIP were 92% identical to each other and 88 and 87% identical to that of human KIAA0923, respectively (Fig.
1B).
To confirm whether the isolated cDNAs encode full-length ADIP,
HEK293 cells were transfected with pCMV-HA-mADIP, which expressed HA-tagged full-length mADIP. The cell extract was subjected to SDS-PAGE, followed by Western blotting with the anti-ADIP pAbs. Three
anti-ADIP pAbs, M57 against the N-terminal portion of mADIP (aa
1-226), M01 against the C-terminal portion of mADIP (aa 339-615), and
M05 against the C-terminal portion of rADIP (aa 159-613), were
generated. A protein with a molecular mass of about 78 kDa was detected
in the extracts of HEK293 cells expressing HA-mADIP by these three pAbs
(Fig. 1C and data not shown). When the extracts of MDCK and
HEK293 cells were subjected to SDS-PAGE, followed by Western blotting
with the three anti-ADIP pAbs, a protein with a similar molecular mass
to that of HA-tagged ADIP was detected (Fig. 1C and data not
shown). Therefore, we concluded that the isolated cDNA encodes
full-length ADIP.
In Vitro and in Vivo Binding of ADIP to Afadin--
Two-hybrid
analysis revealed that ADIP specifically bound to the DIL domain of
afadin but not to the DIL domain of yeast Myo4 (Fig. 2B).
The region containing the third coiled-coil domain (aa 339-436) of
ADIP was required for the binding to the DIL domain of afadin (Fig.
2A). We further confirmed the in vitro and
in vivo binding of ADIP to afadin. First, we performed the
immunoprecipitation analysis. HEK293 cells were co-transfected with the
T7-tagged DIL domain of afadin (T7-DIL; aa 606-983) and the
FLAG-tagged C-terminal portion of ADIP containing the third coiled-coil
domain (FLAG-mADIP-C; aa 339-615), which was isolated in the
two-hybrid screening. When FLAG-mADIP-C was immunoprecipitated from the
cell extract with the anti-FLAG mAb, T7-DIL was co-immunoprecipitated, as detected by Western blotting with the T7 mAb (Fig.
3A). Second, we performed the
affinity chromatography using MDCK cells. The extract of MDCK cells
expressing endogenous afadin was incubated with an MBP-fusion protein
of full-length mADIP (aa 1-615) immobilized on amylose-resin beads.
After the beads were washed with the lysis buffer, the bound proteins
were eluted, and the eluate was subjected to SDS-PAGE, followed by
Western blotting with the anti-afadin mAb. Afadin indeed bound to
MBP-mADIP, but not to MBP alone (Fig. 3B). Third, we
examined whether ADIP directly interacted with afadin. A GST fusion
protein of the DIL domain of afadin (GST-DIL) bound to an MBP-fusion
protein of the region of ADIP containing the all three coiled-coil
domains (aa 121-436) (MBP-mADIP-F) immobilized on amylose resin beads.
GST-DIL bound to MBP-mADIP-F, but not to MBP alone (Fig.
3C). Finally, to confirm that ADIP binds to afadin in
vivo, we examined whether endogenous afadin was
co-immunoprecipitated with endogenous ADIP from the extract of MDCK
cells. When endogenous ADIP was immunoprecipitated from the extract of
MDCK cells with anti-ADIP pAb, endogenous afadin was
co-immunoprecipitated (Fig. 3D). Afadin was not
co-immunoprecipitated with control IgG. These results together with the
two-hybrid analysis indicate that ADIP directly binds to afadin both
in vitro and in vivo.
Tissue and Subcellular Distribution of ADIP--
Northern blot
analysis using the fragment of mADIP cDNA (bp 552-3194) as a probe
detected an ~4.3-kb mRNA in all the mouse tissues
examined, including heart, brain, spleen, lung, liver, skeletal muscle,
kidney, and testis (Fig. 4A).
The smaller band (~3.0 kb) was also detected in the liver and the
testis (Fig. 4A, lanes 5 and
8). Western blot analysis using the anti-ADIP pAb, M57,
detected an ~78-kDa protein, which was the same size of ADIP detected
in HEK293 and MDCK cells and in mouse spleen, lung, and kidney (Fig.
4B, lanes 3, 4,
7, and 9). The smaller bands (~60 kDa in heart,
~76 and ~40 kDa in testis) were also detected, suggesting that
smaller splice variants of ADIP may be expressed (Fig. 4B,
lanes 1 and 8). Subcellular
fractionation analysis of ADIP in rat liver indicated that it was
enriched in the fraction rich in AJs and TJs, where afadin and
E-cadherin were also enriched (Fig. 4C, lanes
4 and 5). The reason why the Western blot
analysis of tissue distribution did not detect the ~78-kDa protein in
the liver (Fig. 4B, lanes 5) was
probably its low expression level. These results indicate that ADIP is
widely expressed, although its expression levels vary depending on
tissues.
Co-localization of ADIP with Afadin at AJs in Epithelial
Cells--
Since afadin has been shown to strictly localize at AJs
undercoated with F-actin bundles (18), we examined by
immunofluorescence microscopy whether ADIP co-localized with afadin at
AJs in MDCK cells. ADIP and afadin co-localized at the sites of
cell-cell contacts (Fig. 5A).
The cross-sectional analysis using a confocal microscopy revealed that
ADIP co-localized with afadin at the junctional complex region (data
not shown). Thus, ADIP and afadin co-localized at the cell-cell
junctions. Essentially the same results were obtained with three
anti-ADIP pAbs, M57, M01, and M05 (data not shown). ADIP and afadin
were also co-stained at the perinuclear regions, most presumably the
Golgi complex, as estimated by the co-staining with Golgi 58-kDa
protein, a marker for the Golgi complex (Fig. 5A and
data not shown), but the physiological significance of this staining is
currently unknown.
To examine the precise localization of ADIP at the junctional complex
region, the frozen sections of small intestine were triple-stained with
the anti-ADIP pAb, the anti-afadin mAb, and the anti-ZO-1 mAb, since
TJs and AJs are well separated in this cell type (49). ZO-1 is known to
be a marker for TJs (48, 49). Immunofluorescence microscopy revealed
that ADIP co-localized with afadin but localized at the slightly more
basal side than ZO-1 in the absorptive epithelia (Fig. 5, B
and C, arrows). A signal for ADIP was also
observed in the microvilli, but its significance is not clear. It may
be nonspecific staining. Finally, immunoelectron microscopy revealed
that ADIP was exclusively localized at AJs undercoated with F-actin
bundles (Fig. 6). The signal for ADIP was
rarely observed at TJs and desmosomes. This localization pattern of
ADIP was the same as those of afadin and nectin but was different from
that of E-cadherin, which is concentrated at AJs but is more widely
distributed from the apical to basal sides of the lateral plasma
membranes (18, 19). These results indicate that ADIP strictly
co-localizes with afadin and nectin at cell-cell AJs undercoated with
F-actin bundles.
The ADIP signal was not detected at focal adhesions where the vinculin
signal was detected in MDCK cells (Fig.
7A). In the heart, focal
adhesions, named costameres, are well developed and periodically
located along the lateral borders of cardiac muscle cells (76).
Vinculin localizes at costameres, whereas nectin and afadin do not (18,
19). The ADIP signal was not detected at costameres where the vinculin
signal was detected (Fig. 7B, arrows). Both the
ADIP and vinculin signals were detected at the intercalated discs,
corresponding to cell-cell AJs (Fig. 7B,
arrowheads). In addition to the signals for ADIP and
vinculin at intercalated discs, these signals were also observed in
stripes within the cardiac muscle cells, but their significance is not
clear. They may be nonspecific staining. These results indicate that
ADIP does not localize at cell-matrix junctions.
Binding of ADIP to
To confirm the binding of ADIP to We have isolated here a novel afadin-binding protein and named it
ADIP. Several lines of evidence suggest that ADIP binds to afadin
in vivo at cell-cell AJs: 1) ADIP binds to afadin, as estimated by the yeast two-hybrid analysis and
co-immunoprecipitation from the extracts of cells exogenously
expressing the fragments of ADIP; 2) recombinant ADIP directly binds to
the DIL domain of recombinant afadin in a cell-free system; 3)
endogenous ADIP and afadin are co-immunoprecipitated from the
extracts of MDCK cells; and 4) ADIP co-localizes with afadin and nectin
at cell-cell AJs. Several proteins, including E-cadherin, We have furthermore shown that ADIP binds What is the function of ADIP? Our study indicates that ADIP
binds two F-actin-binding proteins, afadin and We originally isolated afadin as an F-actin-binding protein (18). We
subsequently found that afadin binds nectin and ponsin (18, 19) and
furthermore isolated here another afadin-binding protein, ADIP, which
bound /
) mice and
(
/
) embryoid bodies, the proper organization of AJs is markedly
impaired. However, the molecular mechanism of how the nectin-afadin
system is associated with the E-cadherin-catenin system or functions in
the formation of AJs has not yet been fully understood. Here we
identified a novel afadin-binding protein, named ADIP
(afadin DIL domain-interacting protein). ADIP consists of 615 amino acids with a
calculated Mr of 70,954 and has three
coiled-coil domains. Northern and Western blot analyses in mouse
tissues indicated that ADIP was widely distributed. Immunofluorescence
and immunoelectron microscopy revealed that ADIP strictly localized at
cell-cell AJs undercoated with F-actin bundles in small intestine
absorptive epithelial cells. This localization pattern was the same as
those of afadin and nectin. ADIP was undetectable at cell-matrix AJs.
ADIP furthermore bound
-actinin, an F-actin-bundling protein known
to be indirectly associated with E-cadherin through its direct binding
to
-catenin. These results indicate that ADIP is an afadin- and
-actinin-binding protein that localizes at cell-cell AJs and may
have two functions. ADIP may connect the nectin-afadin and
E-cadherin-catenin systems through
-actinin, and ADIP may be
involved in organization of the actin cytoskeleton at AJs through
afadin and
-actinin.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-,
-, and
-catenins,
-actinin, and
vinculin (9, 11-14).
-Catenin directly interacts with the
cytoplasmic tail of E-cadherin and connects E-cadherin to
-catenin
that directly binds to F-actin (15).
-Actinin and vinculin are also
F-actin-binding proteins that directly bind to
-catenin (13, 14,
16). The association of E-cadherin with the actin cytoskeleton through
these peripheral membrane proteins strengthens the cell-cell adhesion
activity of E-cadherin (1, 17).
-herpes
virus, facilitating their entry and intercellular spread and so were
renamed HveC and HveB, respectively (31-36). It remains unknown
whether nectin-3 and nectin-4 serve as receptors for viruses. All of
the members have an extracellular domain with three immunoglobulin-like
loops, a single transmembrane region, and a cytoplasmic region.
Furthermore, all of them, except nectin-1
, -3
, and -4, have a
conserved motif of 4 amino acid (aa) residues
(Glu/Ala-X-Tyr-Val) at their carboxyl termini, and this
motif binds the PDZ domain of afadin (18, 19, 25, 26).
-catenin complex to
the nectin-based cell-cell adhesion sites through afadin and
-catenin in fibroblasts and epithelial cells (38-40). Nectin has
furthermore a potency to recruit the components of TJs including ZO-1,
claudin, occludin, and junctional adhesion molecule (JAM) to the
nectin-based cell-cell adhesion sites through afadin in fibroblasts and
epithelial cells (39-42). Claudin is a key cell-cell adhesion molecule
that forms TJ strands (43-46), and occludin and JAM are other
transmembrane proteins at TJs (45-47). Claudin, occludin, and JAM
interact with an F-actin-binding scaffold molecule, ZO-1 (48-59). In
epithelial cells of afadin (
/
) mice and (
/
) embryoid bodies,
the proper organization of AJs and TJs is impaired (60). Nectin-1 has
recently been determined by positional cloning to be responsible for
cleft lip/palate-ectodermal dysplasia, which is characterized by cleft
lip/palate, syndactyly, mental retardation, and ectodermal dysplasia
(61). In addition, we have recently found that the nectin-afadin system
is involved in the formation of synapses in cooperation with N-cadherin
in neurons (62) and that the nectin-afadin system constitutes an
important adhesion system in the organization of Sertoli cell-spermatid
junctions in the testis (63). Nectin and afadin are therefore important for the organization of a wide variety of intercellular junctions either with or independently of known cell adhesion molecules. However,
the molecular mechanism of how the nectin-afadin system is associated
with the E-cadherin-catenin system or functions in the formation of
these intercellular junctions has not yet been fully understood.
-actinin, an F-actin-bundling protein known to
be indirectly associated with E-cadherin through its direct binding to
-catenin (9, 16). These results indicate that ADIP is an afadin- and
-actinin-binding protein that localizes at cell-cell AJs and may
have two functions. ADIP may connect the nectin-afadin and
E-cadherin-catenin systems through
-actinin, and ADIP may be
involved in organization of the actin cytoskeleton at AJs through
afadin and
-actinin.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
gal80
GAL2-ADE2
LYS2::GAL1-HIS3
met2:: GAL7-lacZ) was performed as
described (67). Standard procedures for yeast manipulations were
performed as described (68).
-actinin-1 (human, BC015766;
GenBankTM), pCMV-HA-
-actinin-1-C (aa 406-892), was
constructed with pCMV-HA. The glutathione S-transferase
(GST) fusion or maltose-binding protein (MBP) fusion vectors of mADIP
and rADIP, MBP-mADIP (aa 1-615), MBP-rADIP-C (aa 159-613),
MBP-mADIP-C (aa 339-615), MBP-mADIP-F (aa 121-436), GST-rADIP-C (aa
159-613), GST-mADIP-N (aa 1-226), and GST-mADIP-C (aa 339-615), were
constructed with pGEX-KG (70) and pMal-C2 (New England Biolabs). The
GST fusion vector of the DIL domain of afadin (aa 606-983), GST-DIL,
was constructed with pGEX4T-1 (Amersham Biosciences). The GST fusion
vector of the two EF-hand domains of
-actinin-1 (aa 406-892),
GST-
-actinin-1-C, was constructed with pGEX4T-1. The GST and MBP
fusion proteins were purified using glutathione-Sepharose beads
(Amersham Biosciences) and amylose resin beads (New England Biolabs), respectively.
-actinin, anti-vinculin, and anti-FLAG-M2
mAbs were purchased from Sigma. A rat anti-HA mAb was purchased from
Roche Molecular Biochemicals. A rabbit anti-
-actinin pAb and a mouse anti-GST mAb were purchased from Santa Cruz. A mouse anti-T7 mAb was purchased from Novagen.
-Actinin--
Co-immunoprecipitation experiments using HEK293 cells
were performed as follows. HEK293 cells were transfected with the
expression plasmids in various combinations. The cells were suspended
in 1 ml of Buffer A (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10 µM
-phenylmethanesulfonyl fluoride hydrochloride, and
10 µg/ml aprotinin), sonicated for 10 s three times at 10-s intervals, and incubated on ice for 30 min. The cell extract (1.2 mg of
protein) was obtained by centrifugation at 100,000 × g
for 15 min and then precleared by incubation with protein G-Sepharose 4 Fast Flow beads (Amersham Biosciences). The cell extract was incubated
with 20 µl of anti-FLAG M2 mAb-coated protein G-Sepharose 4 Fast Flow
beads at 4 °C for 18 h. After the beads were washed with Buffer
A, the bound proteins were eluted by boiling the beads in an SDS sample
buffer (60 mM Tris-HCl, pH 6.7, 3% SDS, 2%
2-mercaptoethanol, and 5% glycerol) for 10 min. The samples were then
subjected to SDS-PAGE, followed by Western blotting with the anti-T7,
anti-FLAG, anti-HA, and anti-
-actinin Abs.
-actinin Abs.
-Actinin--
Affinity chromatography using MDCK cells were done as
follows: MDCK cells on two 10-cm dishes were sonicated in 2 ml of
Buffer A, followed by ultracentrifugation at 100,000 × g for 15 min. The supernatant was incubated with MBP or
MBP-mADIP (200 pmol each) immobilized on 20 µl (wet volume) of
amylose resin beads (New England Biolabs) at 4 °C for 18 h.
After the beads were extensively washed with Buffer A, the bound
proteins were eluted by Buffer A containing 20 mM maltose.
The eluates were boiled in the SDS sample buffer. The samples were then
subjected to SDS-PAGE, followed by Western blotting with the
anti-afadin mAb.
-actinin was done as follows: GST-DIL or
GST-
-actinin-1-C was incubated with MBP or MBP-mADIP-F (200 pmol
each) immobilized on 20 µl (wet volume) of amylose resin beads (New
England Biolabs) at 4 °C for 1 h. After the beads were
extensively washed with PBS, the bound proteins were eluted with PBS
containing 20 mM maltose. The eluates were boiled in the
SDS sample buffer. The samples were then subjected to SDS-PAGE,
followed by Western blotting with the anti-GST mAb.
-32P by a standard random priming method and used to
probe a mouse multiple tissue Northern blot
(Clontech). Subcellular fractionation of rat liver
was performed as described (73). Immunofluorescence microscopy of
cultured cells and frozen sections of mouse tissues was performed as
described (18, 19). Immunoelectron microscopy of mouse intestine
absorptive epithelial cells using the ultrathin cryosection technique
was performed as described (18). Protein concentrations were determined
with bovine serum albumin as a reference protein (74). SDS-PAGE was
performed as described by Laemmli (75).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Amino acid sequences of ADIP.
A, schematic structure of afadin. RA,
Ras-associated domain; FHA, forkhead-associated
domain. PDZ, PDZ domain; PR, proline-rich domain.
B, deduced aa sequences of rat ADIP, mouse ADIP, and human
KIAA0923. Asterisks, identical sequences. Putative
coiled-coil domains are underlined. C, comparison
of molecular masses of native and recombinant ADIP proteins.
pCMV-HA-mADIP was transfected to HEK293 cells, and the cell extract was
subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western
blotting with the anti-ADIP pAb (M57). The extracts of control HEK293
and MDCK cells were similarly subjected to SDS-PAGE, followed by
Western blotting with the anti-ADIP pAb (M57). Lane 1,
control HEK293 cells (20 µg of protein); lane 2,
pCMV-HA-mADIP-transfected HEK293 cells (20 µg of protein); lane
3, MDCK cells (20 µg of protein).
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Fig. 2.
Binding of ADIP to the DIL domain of
afadin. A, schematic structure of mouse ADIP.
CC, coiled-coil domain. Yeast two-hybrid analysis of the
afadin- or -actinin-binding regions of ADIP is shown. ADIP, afadin
(DIL domain), and
-actinin were constructed into pGBDU or pGAD and
co-transformed into the reporter yeast strains. Binding of ADIP to
afadin or
-actinin, as shown by expression of the HIS3
and ADE2 reporter genes, was monitored by scoring growth on
synthetic complete medium lacking histidine and adenine, respectively.
+, interacted;
, not interacted; NT, not tested. B, yeast
two-hybrid assay showing the specific binding of ADIP to the DIL domain
of afadin. Yeast transformants with the indicated plasmids were
streaked on synthetic complete medium lacking histidine to score
HIS3 reporter activity and incubated for 3 days at 30 °C.
The results are the representative of three independent
experiments.
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Fig. 3.
In vitro and in vivo
binding of ADIP to afadin. A,
co-immunoprecipitation of the T7-tagged DIL domain of afadin with the
FLAG-tagged mADIP-C. Expression vectors were transfected into HEK293
cells as indicated. The T7-tagged DIL domain of afadin was specifically
co-immunoprecipitated with the FLAG-tagged mADIP-C, as is shown by
Western blotting with the anti-T7 and anti-FLAG mAbs. B,
in vitro binding of afadin to MBP-mADIP. The extract of MDCK
cells was incubated with either MBP or MBP-mADIP (full-length)
immobilized on amylose resin beads. The beads were then subjected to
SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with
the anti-afadin mAb. C, in vitro binding of the
DIL domain of afadin to mADIP. The purified GST-DIL protein was
incubated with either MBP or MBP-mADIP-F (aa 121-436) immobilized on
amylose resin beads. The eluates were then subjected to SDS-PAGE (10%
polyacrylamide gel), followed by Western blotting with the anti-GST
mAb. D, co-immunoprecipitation of endogenous afadin with
endogenous ADIP from MDCK cells. The extracts of MDCK cells were
immunoprecipitated (IP) with the anti-ADIP pAb (M05) and
analyzed by Western blotting (IB) with the anti-ADIP pAb
(M05) and the anti-afadin mAb. The results are the representative of
three independent experiments.
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Fig. 4.
Tissue and subcellular distribution of
ADIP. A, Northern blotting. Mouse RNA blot membranes
(Clontech) were hybridized with the
32P-labeled fragment (bp 552-3194) of the ADIP cDNA,
followed by autoradiography. Lane 1, heart; lane
2, brain; lane 3, spleen; lane 4, lung;
lane 5, liver; lane 6, skeletal muscle;
lane 7, kidney; lane 8, testis (short exposed).
B, Western blotting. The homogenates of various mouse
tissues (30 µg of protein each) were subjected to SDS-PAGE (10%
polyacrylamide gel), followed by Western blotting with the anti-ADIP
pAb (M57). Lane 1, heart; lane 2, brain;
lane 3, spleen; lane 4, lung; lane 5,
liver; lane 6, skeletal muscle; lane 7, kidney;
lane 8, testis; lane 9, MDCK cells. C,
subcellular distribution of ADIP in rat liver. Subcellular
fractionation of rat liver was performed, and each fraction (30 µg of
protein each) was subjected to SDS-PAGE (10% polyacrylamide gel),
followed by Western blotting with the anti-ADIP pAb (M57), the
anti-afadin mAb, or the E-cadherin mAb. Lane 1, the
homogenate fraction; lane 2, the soluble fraction;
lane 3, the pellet fraction; lane 4, the fraction
rich in bile canaliculi; lane 5, the fraction rich in AJs
and TJs. The results are representative of three independent
experiments.
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Fig. 5.
Localization of ADIP at AJs in MDCK cells and
mouse small intestine absorptive epithelial cells. A,
MDCK cells were doubly stained with the anti-ADIP (M05) and anti-afadin
Abs. B, the frozen sections were triple-stained with the
anti-ADIP pAb (M01) and the anti-afadin and anti-ZO-1 mAb.
C, left column, the frozen sections
were double-stained with the anti-ADIP pAb (M01) and the
anti-afadin mAb; right column, the frozen
sections were double-stained with the anti-ADIP pAb (M01) and the
anti-ZO-1 mAb. Arrowhead, the ADIP signal co-localized with
the afadin signal; arrow, the ADIP signal localized at a
slightly more basal side than the ZO-1 signal; bars,
10 µm. The results are representative of three independent
experiments.
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Fig. 6.
Ultrastructural localization of ADIP in mouse
small intestine absorptive epithelial cells. The mouse small
intestine absorptive epithelial cells were labeled with the anti-ADIP
pAb (M01) using the ultrathin cryosection technique. Upper
right inset, the image at a high magnification;
DS, desmosome; bar, 0.1 µm.
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Fig. 7.
Absence of ADIP at cell-matrix junctions in
MDCK cells and mouse cardiac cells. A, MDCK cells were
doubly stained with the anti-ADIP (M05) and anti-vinculin Abs. MDCK
cells in this figure are less confluent than those in Fig.
5A. Bars, 10 µm. B, the frozen
sections were double-stained with the anti-ADIP pAb (M01) and the
anti-vinculin mAb. Arrowheads, intercalated disc;
arrows, costamere; bar, 50 µm. The results are
the representative of three independent experiments.
-Actinin--
To gain insight into the
function of ADIP, we attempted to identify an ADIP-binding protein(s).
As described above, ADIP has three coiled-coil domains, and the region
containing the third coiled-coil domain (aa 339-436) of ADIP is
required for the binding to the DIL domain of afadin (Fig.
2A). We performed the yeast two-hybrid screening using the
region of ADIP containing all three coiled-coil domains (aa 152-436)
as a bait (Fig. 2A). We screened 7 × 105
clones of a rat lung library and obtained 18 positive clones. Nine
clones encoded the C-terminal portion of
-actinin-1 (aa 406-892;
human, BC015766; GenBankTM).
-Actinin is a well
characterized protein that shows F-actin-cross-linking activity (77).
Four isoforms of human
-actinin have been identified: nonmuscle
actinin-1 and actinin-4 and muscle actinin-2 and actinin-3 (78-81).
ADIP bound
-actinin-2 in addition to
-actinin-1 as estimated by
yeast two-hybrid analysis (data not shown), indicating that the binding
of ADIP to
-actinin is not specific for
-actinin-1. Two-hybrid
analysis revealed that the region containing only the first coiled-coil
domain (aa 1-226) of ADIP bound
-actinin-1 (Fig. 2A) and
that the two C-terminal EF-hand motifs of
-actinin-1 (aa 740-892)
were required for its binding to ADIP (data not shown). These
results indicate that the first coiled-coil domain of ADIP binds to the
EF-hand motifs of
-actinin-1.
-actinin-1 in vivo, we
performed the immunoprecipitation analysis. HEK293 cells were
co-transfected with the HA-tagged C-terminal portion of
-actinin-1
(HA-
-actinin-1-C; aa 406-892) and FLAG-tagged ADIP (FLAG-mADIP-M;
aa 152-436). When FLAG-tagged ADIP was immunoprecipitated from the
cell extract with the anti-FLAG mAb, HA-
-actinin-1-C was
co-immunoprecipitated, as detected by Western blotting with the HA mAb
(Fig. 8A). When FLAG-mADIP-M
was immunoprecipitated with the anti-FLAG Ab from the extract of HEK293
cells transiently expressing the FLAG-tagged mADIP (FLAG-mADIP-M; aa
152-436) alone, endogenous
-actinin was co-immunoprecipitated (Fig.
8B). We next examined whether
-actinin-1 directly
interacted with ADIP in vitro. A GST fusion protein of the
C-terminal region of
-actinin-1 (GST-
-actinin-1-C) bound to an
MBP fusion protein of the region of ADIP containing all three
coiled-coil domains (aa 121-436) (MBP-mADIP-F) immobilized on amylose
resin beads. GST-
-actinin-1-C bound to MBP-mADIP-F but not to MBP
alone (Fig. 8C). Finally, when endogenous ADIP was
immunoprecipitated from the extract of MDCK cells with the anti-ADIP
pAb, endogenous
-actinin was co-immunoprecipitated (Fig.
8D).
-Actinin was not co-immunoprecipitated with control IgG. These results indicate that ADIP directly binds
-actinin in vivo and in vitro.
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Fig. 8.
In vivo binding of ADIP to
-actinin. A, co-immunoprecipitation
(IP) of the HA-tagged
-actinin-1-C with the FLAG-tagged
mADIP-M. Expression vectors were transfected into HEK293 cells as
indicated. The HA-tagged
-actinin-1-C was specifically
co-immunoprecipitated with the FLAG-tagged mADIP-M, as is shown by
Western blotting with the anti-HA and anti-FLAG mAbs. B,
co-immunoprecipitation of endogenous
-actinin with the FLAG-tagged
mADIP-M. Expression vectors were transfected into HEK293 cells as
indicated. Endogenous
-actinin-1 was specifically immunoprecipitated
with FLAG-tagged mADIP-M, as is shown by Western blotting
(IB) with the anti-
-actinin pAb and the anti-FLAG mAb.
C, in vitro binding of
-actinin-1 to mADIP.
The purified protein of GST-
-actinin-1-C (aa 406-892) was incubated
with either MBP or MBP-mADIP-F (aa 121-436) immobilized on amylose
resin beads. The eluates were then subjected to SDS-PAGE (10%
polyacrylamide gel), followed by Western blotting with the anti-GST
mAb. D, co-immunoprecipitation of endogenous
-actinin and
endogenous afadin with endogenous ADIP from MDCK cells. The extracts of
MDCK cells were immunoprecipitated with the anti-ADIP pAb (M05) and
analyzed by Western blotting with the anti-ADIP (M05),
anti-
-actinin, and anti-afadin Abs. The results are representative
of three independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-catenin,
-catenin, and
-actinin, are concentrated at AJs but more widely
distributed from the apical to basal sides of the lateral plasma
membranes (2, 21). Four proteins (vinculin, afadin, nectin, and ponsin) strictly localize at AJs, which are defined using ultrastructural analysis as closely apposed plasma membrane domains reinforced by a
dense cytoplasmic plaque to which F-actin bundles attach (7, 18, 19,
82), whereas vinculin and ponsin furthermore localize at cell-matrix
junctions (82). Ponsin is the afadin- and vinculin-binding protein
containing three Src homology 3 domains (82). ADIP is the fifth protein
that strictly localizes at AJs.
-actinin. Our results
suggest that ADIP binds to
-actinin in vivo. 1) ADIP
binds to
-actinin as estimated by the yeast two-hybrid analysis
and co-immunoprecipitation from the extracts of HEK293 cells
exogenously expressing the full-length ADIP or fragments; 2)
recombinant ADIP directly binds to recombinant
-actinin in a
cell-free system; and 3) endogenous ADIP and
-actinin are
co-immunoprecipitated from the extracts of MDCK cells.
-actinin. Afadin binds along the sides of F-actin but does not have a marked
F-actin-cross-linking activity (18).
-Actinin has an
F-actin-cross-linking activity (77). Thus, ADIP links afadin to
-actinin and functions in the formation of the specialized actin
structure at cell-cell AJs. Recently, we have shown that the
heterotypic trans-interaction between nectin-2 in Sertoli
cells and nectin-3 in spermatids is formed at Sertoli cell-spermatid
junctions, heterotypic AJs in the testis, and that each nectin-based
adhesive membrane domain exhibits one-to-one co-localization with each
actin bundle underlying Sertoli cell-spermatid junctions (63). Afadin
also co-localizes with nectin at Sertoli cell-spermatid junctions. Our
present finding that ADIP binds both afadin and
-actinin suggests
that afadin, together with ADIP and
-actinin, functions in the
formation of the actin bundle at the nectin-based cell-cell adhesion
sites. Since it has been reported that E-cadherin also associates with
-actinin through
-catenin (9, 16), two cell-cell adhesion systems
at AJs, the nectin-afadin and cadherin-catenin systems, are connected
to actin cytoskeleton through
-actinin. We have previously shown
that nectin has a potency to recruit the E-cadherin-
-catenin complex
to the nectin-based cell-cell adhesion sites through afadin and
-catenin in fibroblasts and epithelial cells (38-40). Our finding
that ADIP binds both afadin and
-actinin suggests that ADIP
serves as a linker between the nectin-afadin and cadherin-catenin systems.
-actinin in this study (Fig. 9).
Afadin binds these proteins through different regions; nectin binds to the PDZ domain (19), ponsin binds to the third proline-rich domain
(82), and ADIP binds to the DIL domain (Fig. 9). It has furthermore
been reported that afadin directly binds
-catenin, although its
binding is not strong (38, 83). In addition, the splice variant of
afadin, AF-6, binds Rap1 and profilin through the Ras-associated domain
and the carboxyl-terminal region, respectively (84), suggesting that
afadin also binds them. Thus, the F-actin-binding protein, afadin, may
serve as a scaffold molecule to organize various proteins including
other actin-binding proteins,
-catenin,
-actinin, and profilin,
at the nectin-based cell-cell adhesion sites (Fig. 9). The finding that
the proper organization of AJs and TJs is impaired in epithelial cells
of afadin (
/
)-mice and (
/
)-embryoid bodies (60), suggests that
this afadin-based organization of the various proteins is important for
the formation of AJs and TJs. It is of crucial importance to clarify
the molecular mechanism of this organization in the formation of the
junctional complex in epithelial cells.
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Fig. 9.
A schematic model for the interactions
between the nectin-afadin and cadherin-catenin systems at AJs.
Double arrows indicate direct interactions.
F-actin and peripheral membrane proteins shown in the
bottom cell are omitted in the top cell. See
details under "Discussion."
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ACKNOWLEDGEMENTS |
---|
We thank Dr. T. Nagase for providing the cDNA of KIAA0923 and Dr. W. Birchmeier for providing the MDCK cells.
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FOOTNOTES |
---|
* The investigation at Osaka University Medical School was supported by grants-in-aid for Scientific Research and for Cancer Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (2001 and 2002).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 nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) AF532969 and AF532970.
¶ To whom correspondence should be addressed: Dept. of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Osaka, Japan. Tel.: 81-6-6879-3410; Fax: 81-6-6879-3419; E-mail: ytakai@molbio.med.osaka-u.ac.jp.
Published, JBC Papers in Press, November 21, 2002, DOI 10.1074/jbc.M209832200
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ABBREVIATIONS |
---|
The abbreviations used are: TJ, tight junction; aa, amino acid(s); Ab, antibody; AJs, adherens junctions; DIL, dilute domain; F-actin, actin filament; GST, glutathione S-transferase; HA, hemagglutinin; mAb, monoclonal antibody; mADIP, mouse ADIP; MBP, maltose-binding protein; pAb, polyclonal antibody; rADIP, rat ADIP; PRR, poliovirus receptor-related protein; JAM, junctional adhesion molecule; MDCK, Madin-Darby canine kidney.
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