From the Departments of Dermatology and Cell Biology
and the Emory Skin Diseases Research Center, Emory University
School of Medicine, Atlanta, Georgia 30322 and the
§ Department of Biochemistry and Pathobiology, Medical
Faculty of University of Halle, 06097 Halle/Saale, Germany
Received for publication, June 8, 2002, and in revised form, October 31, 2002
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ABSTRACT |
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p0071, a member of the armadillo protein
family, localizes to both adherens junctions and desmosomes in
epithelial cells and exhibits homology to the adherens junction protein
p120 and the desmosomal protein plakophilin-1. p0071 is also present at
dermal microvascular endothelial intercellular junctions and
colocalizes with VE-cadherin, an endothelium-specific cadherin that
associates with both actin and intermediate filament networks. To
define the role of p0071 in junction assembly, p0071 was tested for
interactions with other components of the endothelial junctional
complex. In transient expression assays, p0071 colocalized with and
formed complexes with both VE-cadherin and desmoplakin. Deletion
analysis using the yeast two-hybrid system revealed that the armadillo repeat domain of p0071 bound directly to VE-cadherin. Site-directed mutagenesis experiments demonstrated that p0071 and p120 bound to the
same region on the cytoplasmic tail of VE-cadherin and that
overexpression of p0071 could displace p120 from intercellular junctions. In contrast to VE-cadherin, desmoplakin was found to associate with the non-armadillo head domain of p0071. Cotransfections and triple-label immunofluorescence analysis revealed that
VE-cadherin colocalization with desmoplakin in transfected COS cells
required p0071, suggesting that p0071 may couple VE-cadherin to
desmoplakin. Based on previous findings that both VE-cadherin and
desmoplakin play central roles in vasculogenesis, these new results
suggest that p0071 may play an important role in endothelial junction assembly and in the morphogenic events associated with vascular remodeling.
Vascular endothelial cells form a continuous cell layer along the
wall of blood vessels and participate in a wide range of biological
processes that regulate vascular function. One important function of
the endothelial lining is to control the movement of solutes and fluid
from the vascular space to the tissues (1, 2). The loss of cell
adhesion between endothelial cells results in tissue edema,
inflammation, and poor wound healing. In addition, these cell-cell
contacts also function as plasma membrane attachment sites for
cytoskeletal networks, such as actin and intermediate filaments,
thereby influencing cell shape and tissue integrity (3-5). It is now
apparent that intercellular junctions are macromolecular complexes that
integrate informational cues derived from cell adhesion events with
intracellular signaling pathways that regulate cell proliferation,
apoptosis, and gene expression (6, 7).
The mechanisms by which adhesive interactions are established between
vascular endothelial cells have been studied extensively (8).
Endothelial cells express a unique cadherin, VE-cadherin (where VE is
vascular endothelial; cadherin-5), which plays a central role in the
establishment and maintenance of endothelial monolayer integrity and
angiogenesis (9, 10). Significantly, endothelial cells assemble unique
junctional complexes that couple VE-cadherin to both actin and
intermediate filament networks (11), although the precise nature of
these protein interactions remains only partially characterized. Like
other classical cadherins such as E-cadherin, VE-cadherin associates
with the cytoplasmic proteins Desmoplakin, a member of the plakin family of cytoskeletal
cross-linking proteins, is an abundant constituent of the desmosomal plaque and functions to couple intermediate filaments to
membrane-associated adhesive junctions (13, 14). Previous studies
demonstrated that the carboxyl terminus of desmoplakin interacts
directly with intermediate filament networks (15-18), whereas the
amino terminus of desmoplakin associates with desmosomes through
interactions with plakoglobin (19, 20) and the plakophilins (21-23).
In addition to playing fundamental roles in desmosome assembly and epidermal integrity (24), desmoplakin also plays important roles in
adherens junction formation (24, 25). Interestingly, vascular and
lymphatic endothelial cells, which do not assemble true desmosomes, also express desmoplakin (26, 27). In cultured human umbilical vascular
endothelial cells and primary dermal microvascular endothelial cells,
desmoplakin was shown to localize to intercellular junctions (11, 28).
Recent studies also indicate that desmoplakin plays an important role
in vasculogenesis (29), underscoring the need to determine how
desmoplakin interacts with other components of endothelial
intercellular junctions.
An exciting development over the last several years is the realization
that intercellular junctions contain a newly identified subset of
armadillo family proteins termed the p120/plakophilin subfamily (30,
31). In general, p120 assembles into actin-associated adherens
junctions, whereas the plakophilins assemble into intermediate filament-based desmosomes. Interestingly, one member of the subfamily (termed p0071) assembles into both adherens junctions and desmosomes (32). This dual targeting of p0071 to both types of junctions raises
the possibility that p0071 plays a role in the molecular cross-talk
that occurs between different intercellular junctions, thus influencing
a range of cellular events that are regulated by either the actin or
intermediate filament cytoskeleton. To understand how p0071 might
perform specific roles in junction assembly, we initiated an
investigation into the subcellular localization of p0071 in dermal
microvascular endothelial cells. In addition, we tested p0071 for
interactions with other intercellular junction proteins using transient
transfection of COS-7 and 293 cells, co-immunoprecipitation, and yeast
two-hybrid analysis. The data indicate that p0071 associates with both
adherens junction and desmosomal proteins and that p0071 is a cadherin-
and desmoplakin-binding protein.
cDNA Constructs
p0071--
A cDNA construct encoding full-length human p0071
was generated as described previously (32) and subcloned into the
NotI site of the pKS vector (Stratagene, La Jolla, CA).
Expression in eukaryotic cells was performed with the FLAG-tagged
expression system pCMV-Tag4 or pCMV-Tag2 vector, containing a carboxyl-
or an amino-terminal FLAG epitope, respectively (Stratagene).
p0071-(1-1016)-FLAG was constructed using the 3.0-kb
NotI/SalI fragment from the pKS vector containing
the p0071 insert and subcloned into pCMV-Tag4. Similarly,
p0071-(1-553)-FLAG was generated from a 1.8-kb
NotI/PstI fragment and subcloned into pCMV-Tag4.
p0071-(554-1016)-FLAG was generated from a 1.8-kb
PstI/SalI fragment and subcloned into the
pCMV-Tag2 vector. To generate full-length FLAG-tagged p0071 (p0071-(1-1192)-FLAG), a point mutation was introduced to eliminate the stop codon using the primer pair 5'-GAC TCA TGG GTG GCG GAT CAA GCT
TCC CAA CAG AGG and 5'-CCT CTG TTG GGA AGC TTG ATC CGC CAC CCA TGA GTC
and the QuikChangeTM site-directed mutagenesis kit
(Stratagene). The resulting 3.5-kb BamHI/HindIII
fragment was subcloned into the pCMV-Tag4 vector. The
p0071-(1-508)-FLAG construct was generated by PCR using a p0071
5'-primer (5'-CGC GGA TCC AGA GGA ATG CCA GCT
CCT GAG CAG GCC), which generates a BamHI site (underlined)
at the 5'-end of p0071-(1-508)-FLAG, and the p0071-(1-508)-FLAG
3'-primer (5'-TTT AAC GTC GAC CTA CTT ATC
GTC GTC ATC CTT GTA ATC GGT GCC ATC ATC AGC CGG CAC), which
generates the FLAG tag (italicized) and a 3'-SalI site
(underlined). p0071-(509-992)-FLAG was also generated using a p0071
5'-primer (5'-CGC GGA TCC AGA GGA ATG ACA AGA
TCC CCA TCA ATA GAC), which generates a BamHI site
(underlined) at the 5'-end of the construct, and the
p0071-(509-992)-FLAG 3'-primer (5'-CCG CTC GAG
CTA CTT ATC GTC GTC ATC CTT GTA ATC CCA TAA TGT ATT CAA GAC
CTG GGC), which generates the FLAG tag (italicized) and a
3'-XhoI site (underlined). p0071-(992-1192)-FLAG was
generated by PCR using a p0071 5'-primer (5'-CGC GGA
TCC AGA GGA ATG CAA TAT CGG GAC CTC CGG AGC), which
generates a BamHI site (underlined) at the 5'-end of
p0071-(992-1192)-FLAG, and the p0071-(992-1192)-FLAG 3' primer
(5'-CCG CTC GAG CTA CTT ATC GTC GTC ATC
CTT GTA ATC CAC CCA TGA GTC TGG GGA CCC), which generates a FLAG
tag (italicized) and a 3'-XhoI site (underlined). The PCR
products were subcloned into donor vectors of the Creator system
(Clontech, Palo Alto, CA), and PCR-generated
regions were sequenced to verify that errors were not introduced into
the coding sequence. Expression of all constructs was further verified
by Western blotting and immunofluorescence. A diagram illustrating the
different domains of the recombinant proteins used in this work is
shown in Fig. 1.
VE-cadherin--
Full-length cDNA encoding human
VE-cadherin, subcloned into the pECE vector using the SV40 promoter,
was generated as described previously (9, 33). The cDNA clone
encoding the cytoplasmic domain of human VE-cadherin was generated by
PCR as described previously (34). The cytoplasmic domain was recovered
from the TA vector (Invitrogen) by restriction digestion with
EcoRI/XhoI and subcloned into the Creator system
donor vector. To generate the triple-alanine mutation in the
VE-cadherin juxtamembrane domain (E652A/M653A/D654A), we used
the QuikChange site-directed mutagenesis kit and the primer pair 5'-GAG
GGC GGC GGC GCG GCG GCC ACC ACC AGC TAC G and 5'-CGT AGC TGG TGG TGG
CCG CCG CGC CGC CGC CCT C.
Desmoplakin--
cDNAs encoding full-length desmoplakin with
a carboxyl-terminal Myc epitope tag, the first 584 amino acids of
desmoplakin (DPNTP, for desmoplakin
N-terminal polypeptide) (35), and a truncated desmoplakin polypeptide lacking the amino-terminal domain (DP Yeast Two-Hybrid Assays
Yeast two-hybrid vectors encoding the Gal4 DNA-binding domain
(pLP-GBK) or transcription activation domain (pLP-GAD) were purchased
from Clontech. The p0071 constructs described above were subcloned from the Creator system donor vector into the Creator system acceptor vector pLP-GBK. The VE-cadherin cytoplasmic domain was
subcloned into the Creator system donor vector, followed by recombination subcloning into the acceptor vector pLP-GAD. All constructs were then verified by sequencing. To assay for interactions between proteins, 5-10 µg of plasmid DNA was transformed into the
yeast strain AH109 (Clontech) using LiAc, and
double transformants were selected by growth in the absence of leucine
and tryptophan. Expression of the HIS and
ADE reporter genes was analyzed by monitoring colony growth
on plates lacking histidine, adenine, leucine, and tryptophan.
Cell Lines and Transfections
The monkey kidney cell line COS-7 and the human embryonic kidney
cell line 293 were routinely cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT) and penicillin/streptomycin/amphotericin B
(Invitrogen). Primary cultures of human dermal microvascular endothelial cells were purchased from the Emory Skin Diseases Research
Center (Core B) and cultured in MDCB131 (Invitrogen) supplemented with
10% fetal bovine serum, 100 µg/ml cAMP (Sigma), 1 µg/ml
hydrocortisone (Sigma), 10 ng/ml epidermal growth factor (Intergen Co.,
Purchase, NY), and penicillin/streptomycin/amphotericin B (36). For
transient transfection experiments, a subclone of COS-7 cells
(COS-7-20) was transfected by calcium phosphate precipitation. The
cells were fixed and processed for immunofluorescence analysis after
24-48 h.
Antibodies and Immunofluorescence Microscopy
The distribution of p0071 in microvascular endothelial cells and
transiently transfected COS-7 cells was analyzed by immunofluorescence. Cells grown on coverslips were rinsed in phosphate-buffered saline and
fixed in methanol at Immunoprecipitation and Western Blot Analysis
Immunoprecipitation was carried out as described previously (19,
21). Briefly, cells were scraped into Tris-buffered saline containing
0.5% Triton X-100, vortexed, and subjected to centrifugation at
14,000 × g. Antibody M2 (directed against the FLAG
tag) conjugated to agarose beads (Sigma) was incubated with the cell
lysate for 1 h at 4 °C. Immune complexes were captured by
centrifugation, and the beads were washed four times in Tris-buffered
saline containing 0.5% Triton X-100 for 10 min with gentle rotation at
4 °C. Immune complexes were released by incubation in reducing
SDS-PAGE sample buffer at 95 °C and then separated on 7.5%
acrylamide gels and transferred to nitrocellulose according to standard
protocols. Membranes were washed and incubated with horseradish
peroxidase-coupled secondary antibodies, and bound antibodies were
visualized by chemiluminescence reagent (Amersham Biosciences).
p0071 Is Localized at Intercellular Junctions in Primary Cultures
of Dermal Microvascular Endothelial Cells--
The precise
localization of p0071 in different cell types and the mechanisms by
which p0071 incorporates into junctions have been only partially
characterized. p0071 has been shown previously to localize at cell-cell
contacts in epithelial compartments of various tissues (32, 38) and was
found to be expressed and assembled into intercellular junctions of a
variety of cultured epithelial cell lines, including HeLa, A431, and
HaCaT (32). Using previously characterized antibodies specific for
p0071 (32), the distribution of p0071 was examined and compared with
that of VE-cadherin in cultured primary human microvascular endothelial cells. In confluent monolayers of primary microvascular endothelial cells, p0071 localized at intercellular junctions (Fig.
2A) and colocalized with
VE-cadherin (Fig. 2, C and D). These results are
consistent with previous studies indicating that p0071 is broadly
expressed and assembled into intercellular junctions in a variety of
cell types and tissues (32).
p0071 Binds VE-cadherin--
The localization of p0071 at
cell-cell borders suggested that this protein may interact with other
components within the intercellular junctions of microvascular
endothelial cells, such as VE-cadherin. To begin the analysis of p0071
binding partners, a FLAG-tagged p0071 construct (p0071-(1-1016)-FLAG)
was generated. When expressed in COS-7 cells, the p0071-(1-1016)-FLAG
protein localized to both the cytoplasm and intercellular junctions
(Fig. 3B). To determine whether p0071 interacts with VE-cadherin, p0071-(1-1016)-FLAG was
cotransfected with VE-cadherin (Fig. 3, C and D).
p0071-(1-1016)-FLAG (Fig. 3C) and VE-cadherin (Fig.
3D) exhibited extensive colocalization, primarily at
cell-cell borders. Moreover, in cells coexpressing p0071-(1-1016)-FLAG
and VE-cadherin, p0071 recruitment to intercellular junctions was
dramatically increased. The central armadillo domain of p0071, but not
the non-armadillo head domain, colocalized with VE-cadherin when
cotransfected into COS-7 cells (data not shown), suggesting that the
armadillo domain of p0071 binds to VE-cadherin. To test for direct
interactions between p0071 and VE-cadherin, full-length p0071 and the
cytoplasmic domain of VE-cadherin were examined for direct binding
using the yeast two-hybrid system. Direct interactions between proteins
were assayed by growth in the absence of histidine and adenine (Fig.
3E). In agreement with the COS-7 cell transient transfection
assays, growth in the absence of histidine and adenine was consistently
observed when yeast were cotransformed with plasmids encoding p0071 in
the DNA-binding domain vector and the VE-cadherin cytoplasmic domain in
the transcription activation domain vector (Fig. 3E).
Similar results were obtained using p0071 Binds to the Juxtamembrane Domain of VE-cadherin--
Recent
studies have identified a core region within the juxtamembrane domain
of E-cadherin responsible for binding to the armadillo protein p120
(39). The juxtamembrane domain sequence is highly conserved among type
I and II classical cadherins (31). To test whether p0071 and p120
compete for binding sites at cell-cell junctions, full-length
FLAG-tagged p0071 was transiently expressed in COS-7 cells. The cells
were then processed for immunofluorescence microscopy using antibodies
directed against the FLAG epitope tag to detect exogenously expressed
p0071 along with antibodies directed against endogenous p120. In
untransfected cells, p120 was detected in a continuous pattern along
cell-cell contacts (Fig. 4, A
and B). In contrast, p120 staining was dramatically reduced
at the borders of adjacent cells expressing the FLAG-tagged p0071
protein (Fig. 4, C and D), suggesting that p0071
occupies membrane-binding sites in a mutually exclusive manner with
p120. To determine whether p0071 and p120 bind to the same region of VE-cadherin, a triple-alanine substitution at residues 652-654 of the
VE-cadherin juxtamembrane domain was generated and tested for the
ability to bind to plakoglobin, p0071, or p120 in the yeast two-hybrid
system. Protein-protein interactions were monitored by growth in the
absence of histidine and adenine. Although plakoglobin bound to the
mutated VE-cadherin cytoplasmic domain, neither p0071 (Fig.
4E) nor p120 (data not shown) interacted with the mutated cytoplasmic tail of VE-cadherin. Similar results were obtained when
full-length VE-cadherin with the E652A/M653A/D654A mutation was
expressed in COS-7 cells; the VE-cadherin mutant failed to colocalize
with p0071 as observed by dual-label immunofluorescence (data not
shown). These data indicate that p0071 binds to the juxtamembrane
domain of VE-cadherin and that it may compete with p120 for binding to
this highly conserved region of cadherins.
p0071 Forms Complexes with the Intermediate Filament-binding
Protein Desmoplakin--
Vascular endothelial cells assemble unique
intercellular junctions that are thought to couple VE-cadherin not only
to actin microfilaments, but also to the vimentin intermediate filament cytoskeleton. Several studies have demonstrated that the intermediate filament-binding protein desmoplakin is expressed in endothelial cells
and assembled into endothelial intercellular junctions (26, 27).
Furthermore, desmoplakin and VE-cadherin have been shown to co-assemble
into intercellular junctions of cultured endothelial cells (11, 28),
and recent studies indicate that desmoplakin plays a central role in
blood vessel formation during development (29). Based on these previous
reports and on the observation that p0071 localizes to both adherens
junctions and desmosomes (32), p0071 was tested for interactions with
desmoplakin. p0071-(1-1016)-FLAG and full-length Myc-tagged
desmoplakin were coexpressed in COS-7 cells (Fig.
5). In transfected cells, dual-label
immunofluorescence demonstrated that p0071 colocalized with full-length
desmoplakin (Fig. 5, A and B). The two proteins
were often detected in a filamentous staining pattern and in
perinuclear aggregates, resulting from the alignment of desmoplakin
along intermediate filament networks when expressed in cultured cells
(15, 16). Furthermore, exogenous p0071 targeted to both desmosomes and
adherens junctions in transiently transfected A431 cells (data not
shown). In contrast to plakophilin-1 (40), p0071 did not appear
to enhance the recruitment of full-length desmoplakin to intercellular
junctions in transfected COS-7 cells (Fig. 5, A and
B).
To determine the domain of desmoplakin required for colocalization with
p0071, the Myc-tagged desmoplakin mutant lacking the amino-terminal
domain (DP
To determine whether p0071 binds directly to the amino-terminal domain
of desmoplakin, interactions between p0071 and the desmoplakin
amino-terminal polypeptide DPNTP (35) were tested using the yeast
two-hybrid system. Growth in the absence of histidine and adenine was
used as a reporter for protein interactions (Fig. 7A). As reported previously
(19), DPNTP interacted directly with plakoglobin, but failed to
interact with the VE-cadherin cytoplasmic domain. We were also unable
to detect interactions between DPNTP and p120 (data not shown).
However, direct interactions between multiple domains of p0071 and
DPNTP were consistently observed by yeast two-hybrid analysis (Fig.
7A). To further investigate p0071 domains that interact with
desmoplakin, the p0071 head or arm domain was coexpressed with
full-length desmoplakin in COS-7 cells (Fig. 7,
B-E). As expected, full-length desmoplakin aligned along intermediate filament networks (Fig. 7, B and
D). The amino-terminal head domain of p0071 consistently
colocalized with desmoplakin in a filamentous pattern (Fig. 7,
B and C). In contrast, the armadillo domain of
p0071 was unable to associate with desmoplakin when transiently
expressed in COS-7 cells (Fig. 7, D and E). Fig.
7F summarizes colocalization results using COS-7 cells
transfected with various domains of p0071 and full-length desmoplakin.
The results of extensive cotransfection analysis in COS-7 cells suggest that the non-armadillo head domain of p0071 associates with the amino-terminal domain of desmoplakin. However, as indicated by the
yeast two-hybrid analysis, it is likely that other domains of p0071
outside of the head domain also contribute to the interaction with the
amino-terminal domain of desmoplakin.
p0071 Couples VE-cadherin to Desmoplakin--
Desmoplakin and
VE-cadherin have been shown to colocalize in human dermal microvascular
endothelial cells. However, the association is not through a direct
linkage, but rather through a complex with plakoglobin (11). To examine
if p0071 could perform a role similar to that of plakoglobin and link
VE-cadherin to desmoplakin, triple-label immunofluorescence studies of
transiently transfected COS-7 cells were undertaken. Full-length
Myc-tagged desmoplakin and VE-cadherin were coexpressed in COS-7
cells in the absence (Fig. 8,
A-D) or presence (Fig. 8, E-H) of
p0071-(1-1192)-FLAG. In the absence of p0071, VE-cadherin failed to
colocalize with desmoplakin (Fig. 8, A-D). In contrast,
VE-cadherin exhibited striking colocalization with desmoplakin in the
presence of p0071 (Fig. 8, E-H). These data suggest that
p0071 plays a role in linking the endothelial cadherin to the vimentin
intermediate filament network, although other proteins are likely to
participate in this linkage.
Previous studies demonstrated that the amino-terminal domain of
desmoplakin interacts with the armadillo family protein plakoglobin, which in turn links desmoplakin to the endothelial cadherin,
VE-cadherin (11). The results of the present study indicate that the
armadillo protein p0071 is also assembled into intercellular junctions
of dermal microvascular endothelial cells and that VE-cadherin recruits p0071 to cell-cell borders. In addition, expression of p0071 resulted in the colocalization of VE-cadherin and desmoplakin in transient expression assays. Using the yeast two-hybrid system, p0071 was shown
to be a direct binding partner for both VE-cadherin and the
amino-terminal domain of desmoplakin. These findings suggest that, in
endothelial intercellular junctions, p0071 functions as a protein that
links VE-cadherin to the intermediate filament cytoskeleton.
p0071 is most closely related to members of the p120 subfamily of
armadillo proteins. This group of proteins also includes the
plakophilins, a family of desmosomal and nuclear proteins that are
directly involved in the recruitment of desmoplakin to desmosomes in
epithelial cells (21-23). Although amino acid sequence analysis has
revealed similarities between p0071 and plakophilin-1-3 (32, 41),
there are distinct differences in the tissue distributions of these
proteins. The plakophilins are primarily expressed in epithelial
tissues that assemble desmosomes, although cells lacking desmosomes
have also been described to contain plakophilin-1-3 (41-43). In
contrast, p0071 exhibits a broad tissue distribution and is unique
among the p120/plakophilin family members in that p0071 is the only
member of this subfamily described to date that assembles into both
adherens junctions and desmosomes. However, the distribution of the
various plakophilins in endothelial cells is not well characterized,
and the manner in which vascular endothelial cells utilize
p120/plakophilin family members to assemble adhesive junctions suited
to the regulation of vascular permeability is poorly understood.
Although p0071 interacts with desmoplakin in a manner similar to the
plakophilins, amino acid sequence analysis indicated that p0071 is more
closely related to the p120 family members An important finding in this study is that p0071 appears to physically
link VE-cadherin to desmoplakin. As shown in Fig. 8 and as described
previously (11), VE-cadherin did not colocalize with desmoplakin when
coexpressed transiently in COS-7 cells. However, in the presence of
full-length p0071, VE-cadherin and desmoplakin were found to colocalize
extensively. Both yeast two-hybrid and COS-7 cell transient
transfection experiments demonstrated that the central armadillo domain
of p0071 bound directly to VE-cadherin. Multiple domains of p0071 were
found to bind to desmoplakin in yeast two-hybrid experiments, but only
the head domain of p0071 colocalized with full-length desmoplakin in
COS-7 cells (Fig. 7). These results suggest that desmoplakin may bind
to multiple sites on p0071, but that the head domain is the primary
binding domain in the context of a cadherin complex. Consistent with
this interpretation, neither the head nor arm domain of p0071 alone was
sufficient to drive colocalization between VE-cadherin and desmoplakin
in triple-transfection experiments in COS-7 cells (data not shown).
Based on the ability of these two domains of p0071 to interact
differentially with desmoplakin and VE-cadherin, these observations
suggest that p0071 may actually link VE-cadherin to desmoplakin, with
the p0071 head domain binding to desmoplakin and the p0071 arm domain
interacting with VE-cadherin. In this manner, p0071 appears unique
among the other members of the p120/plakophilin family. For example,
p120 does not appear to play any direct role in linking classical
cadherins to the actin cytoskeleton. Likewise, plakophilin-1 binds
strongly to desmoplakin, but a direct role for plakophilin-1 in linking
the desmosomal cadherins to desmoplakin has not been demonstrated. It
is likely that plakophilin-1 plays a more important role in lateral
associations among desmosomal components (21, 22). Recently,
plakophilin-2 was found to bind to a number of intercellular junction
proteins, including desmoplakin and several members of the desmosomal
cadherin family (23). The detailed manner in which the plakophilins
contribute to junction assembly remains unclear, and understanding how
these proteins contribute to junction assembly is an important area of investigation.
The suggestion that p0071 functions as a linker between VE-cadherin and
desmoplakin predicts that this armadillo family protein may have some
overlapping function with plakoglobin. Like p0071, plakoglobin is
widely expressed and assembles into both adherens junctions and
desmosomes (48, 49). Unlike In addition to the junctional binding partners for p0071 described here
and by Hatzfeld et al.,1 p0071 has also been
shown to interact with presenilin (56) and several PDZ
(PSD-90/Dlg/ZO-1) motif-containing
proteins (57, 58). The carboxyl-terminal domain of p0071 (along with
ARVCF and
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin and plakoglobin, members of
the armadillo protein family (12). Both plakoglobin and
-catenin
bind directly to the cytoplasmic domain of VE-cadherin, thus providing
a link to the actin cytoskeleton through their association with
-catenin, a vinculin homolog that plays a key role in linking the
cadherin complex to the actin cytoskeleton. In addition, plakoglobin
(but not
-catenin) provides a link from VE-cadherin to the
intermediate filament network by recruiting desmoplakin, an
intermediate filament-binding protein, to intercellular junctions
(11).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic diagram of the cDNA constructs
used in this study. A, p0071 constructs encoding
full-length (FL) p0071 or various domains of the protein
were constructed and tested in mammalian expression assays and in the
yeast two-hybrid system. PDZ-BD, PDZ domain-binding domain.
B, shown are full-length VE-cadherin and its
cytoplasmic domain. Site-directed mutagenesis was used to alter the
region encoding amino acids 652-654 to E652A/M653A/D654A
(652-654EMD-AAA). JMD, juxtamembrane
domain; CBD, catenin-binding domain. C,
constructs encoding full-length human desmoplakin with a
carboxyl-terminal Myc epitope tag (DP.myc) or the mutant
lacking the amino-terminal domain (DP N) were used for mammalian
expression assays. DPNTP (DP-NTP) is the 584-amino acid
region of the desmoplakin amino-terminal polypeptide and was used in
yeast two-hybrid experiments. aa, amino acids. Refer to
"Experimental Procedures" for additional information.
N) (15, 16) were generously provided by Dr. K. J. Green (Fig. 1).
20 °C for 4 min. Alternatively, cells were
fixed in 3.7% paraformaldehyde in phosphate-buffered saline, followed
by permeabilization in 0.5% Triton X-100 in phosphate-buffered saline.
VE-cadherin was monitored using mouse anti-cadherin-5 monoclonal
antibody (Transduction Laboratories, Lexington, KY). Desmoplakin
was detected using rabbit polyclonal antibody NW6, directed against the
desmoplakin carboxyl-terminal domain (37), or the Myc epitope tag
(Bethyl Laboratories, Inc., Montgomery, TX). Endogenous p0071 was
analyzed with a rabbit polyclonal antibody (32), whereas FLAG-tagged
p0071 was detected by an anti-FLAG tag monoclonal antibody
(Stratagene). Appropriate species cross-absorbed secondary antibodies
conjugated to rhodamine, fluorescein, or various Alexa Fluors
(Molecular Probes, Inc., Eugene, OR) were used for dual-label
immunofluorescence. Control experiments were carried out routinely to
verify that fluorescence was not due to secondary antibody
cross-reactivity. A Leica DMR-E fluorescence microscope equipped with
narrow band-pass filters and a Hamamatsu Orca camera was used. Images
were captured and processed using Open Lab software (Improvision, Inc.,
Lexington, MA).
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
p0071 localization in dermal microvascular
endothelial cells. Primary cultures of dermal microvascular
endothelial cells were processed for immunofluorescence using a rabbit
polyclonal antibody (Ab) directed against p0071 (32) and a
monoclonal antibody directed against VE-cadherin (Cad-5).
p0071 was detected predominantly at intercellular junctions
(A). No staining was observed in the absence of the
anti-p0071 primary antibody (B). Dual-label
immunofluorescence demonstrated colocalization of p0071 (C)
with VE-cadherin (D).
-galactosidase as a reporter for
direct interactions (data not shown), indicating that p0071 and
VE-cadherin are direct binding partners. To further define the domain
of p0071 that binds to VE-cadherin, deletion mutants of p0071
(p0071-(1-508), p0071-(509-992), and p0071-(324-1192)) were
generated and analyzed for the ability to bind to the VE-cadherin
cytoplasmic domain (Fig. 3E). The results indicate that the
central armadillo domain of p0071 (amino acids 509-992) is necessary
and sufficient for binding to the cytoplasmic domain of VE-cadherin.
These observations are consistent with the hypothesis that, like the
related proteins ARVCF (armadillo repeat gene deleted in velo cardio
facial syndrome),
-catenin, and p120, p0071 is a
cadherin-binding protein.
View larger version (63K):
[in a new window]
Fig. 3.
p0071 colocalizes with VE-cadherin in COS-7
cell transient transfections and binds directly to VE-cadherin in the
yeast two-hybrid system. A cDNA construct encoding
p0071-(1-1016)-FLAG was expressed in COS-7 cells and detected using an
antibody (Ab) directed against the FLAG epitope tag. FLAG
staining was not observed in untransfected COS-7 cells (A).
In transfected (T) cells, the p0071-(1-1016)-FLAG
polypeptide was observed at intercellular junctions and in other
regions of the cytoplasm and plasma membrane (B). In cells
cotransfected with VE-cadherin (VE-cad), both cadherin
(C) and the p0071-(1-1016)-FLAG polypeptide (D)
accumulated at intercellular junctions and exhibited extensive
colocalization. Yeast two-hybrid experiments were carried out to test
for direct interactions using growth in the absence of histidine and
adenine as a reporter for direct interactions between VE-cadherin and
multiple p0071 polypeptides (E). p0071 polypeptides were
expressed as fusions with the Gal4 DNA-binding domain and tested for
interactions either with the empty activation domain (AD)
vector or with VE-cadherin in the Gal4 transactivation domain vector
(VE-cadcyto). Constructs encoding the p0071 central
armadillo domain consistently interacted with the VE-cadherin
cytoplasmic tail. FL, full-length.
View larger version (63K):
[in a new window]
Fig. 4.
p0071 displaces p120 from intercellular
junctions and binds directly to the p120-binding site on the
VE-cadherin cytoplasmic domain. Full-length FLAG-tagged p0071 was
expressed in COS-7 cells, and the distribution of endogenous p120 was
monitored. After 48 h, cells were fixed and processed for
immunofluorescence to detect FLAG-tagged p0071 (A and
C) and endogenous p120 (B and D). Note
that in adjacent cells that were both transfected with p0071-FLAG
(C), p120 staining was absent (D,
arrows). The putative p120-binding site within the
VE-cadherin cytoplasmic domain was mutated (E652A/M653A/D654A
(VE-cad652-654EMD-AAA)) and tested for interactions
with plakoglobin or p0071 in the yeast two-hybrid system
(E). The mutated VE-cadherin tail interacted with
plakoglobin, which binds to the distal catenin-binding domain of
VE-cadherin, but failed to interact with p0071. Ab,
antibody.
View larger version (140K):
[in a new window]
Fig. 5.
p0071 colocalizes with full-length
desmoplakin, but not with the desmoplakin mutant lacking the
amino-terminal domain. p0071-(1-1016)-FLAG was cotransfected in
COS-7 cells with full-length Myc-tagged desmoplakin (DP.myc;
A and B) or the desmoplakin mutant lacking the
amino-terminal domain (DP N) (C and D).
p0071-(1-1016)-FLAG (B) exhibited extensive colocalization
with full-length desmoplakin (A), but failed to colocalize
with DP
N (C and D). Note that the
filamentous pattern of both desmoplakin polypeptides results from the
binding of the desmoplakin carboxyl-terminal domain to endogenous
intermediate filament networks (15). Ab, antibody.
N-Myc) was coexpressed with p0071-(1-1016) in COS-7 cells
(Fig. 5, C and D). As with full-length
desmoplakin, DP
N aligned along intermediate filament networks in a
filamentous pattern (Fig. 5D). However, p0071 (Fig.
5C) failed to colocalize with DP
N (Fig. 5D),
suggesting that the desmoplakin amino-terminal domain is required for
interactions with p0071. To further investigate the ability of these
proteins to interact, co-immunoprecipitation studies were performed
(Fig. 6). Protein complexes were captured from transiently transfected 293 cells using anti-FLAG tag antibody M2-agarose beads. Full-length desmoplakin and DP
N were detected by
Western blot analysis using the Myc epitope tag. Full-length desmoplakin consistently co-immunoprecipitated with p0071, whereas the
amino-terminally truncated desmoplakin failed to co-precipitate with
p0071 (Fig. 6B). Parallel Western blots verified that p0071 (Fig. 6C) was captured by the immunoprecipitation procedure
and that both full-length desmoplakin and DP
N were expressed in each transfection (Fig. 6A). Together with the colocalization
data shown in Fig. 5, these experiments suggest that p0071 interacts with the amino-terminal region of desmoplakin.
View larger version (36K):
[in a new window]
Fig. 6.
p0071 co-immunoprecipitates with
desmoplakin. p0071-(1-1016)-FLAG was cotransfected in 293 cells
with either full-length Myc-tagged desmoplakin
(DP.myc) or the desmoplakin mutant lacking the
amino-terminal domain (DP N). Both full-length desmoplakin
(FL-DP) and DP
N were detected in whole cell lysates
of transfected 293 cells using an antibody directed against the c-Myc
epitope tag (A). When p0071-(1-1016)-FLAG was captured from
293 cell lysates using antibody M2-agarose beads, full-length
desmoplakin (but not DP
N) was detected in the complex with
p0071-(1-1016)-FLAG (B). Expression of the p0071
protein was demonstrated by Western blotting using a rabbit
polyclonal antibody directed against p0071
(C). IP, immunoprecipitate.
View larger version (73K):
[in a new window]
Fig. 7.
The p0071 head domain interacts with
desmoplakin. cDNA constructs encoding various domains
of p0071 were tested for interactions with the desmoplakin
amino-terminal domain using the yeast two-hybrid system. Growth in the
absence of histidine and adenine was used as a reporter for protein
interactions (A). Each construct was tested for
self-activation, and none of the p0071 constructs activated
transcription as determined by growth in the absence of histidine and
adenine (A and data not shown). In addition to the
two-hybrid analysis, the head (B and C) and the
central (D and E) armadillo domains of p0071 were
tested for colocalization with desmoplakin in COS-7 cell transient
transfections. Desmoplakin was often detected along intermediate
filaments and colocalized extensively with the head domain of
desmoplakin (B and C), but failed to colocalize
with the arm domain of p0071 (D and E). These
results, along with the results of additional colocalization
experiments, are summarized in F. FL,
full-length; VE-cadcyto, VE-cadherin cytoplasmic
domain; Ab, antibody; ND, not determined;
PDZ-BD, PDZ domain-binding domain.
View larger version (48K):
[in a new window]
Fig. 8.
VE-cadherin colocalizes with desmoplakin in
the presence of p0071 in transfected COS-7 cells.
Full-length Myc-tagged desmoplakin and full-length human
VE-cadherin were transiently coexpressed in the absence
(A-D) or presence (E-H) of p0071 in COS-7
cells. In the absence of p0071, desmoplakin (A) was detected
in a filamentous pattern, and VE-cadherin (C) was diffuse at
the cell surface, with some VE-cadherin accumulating at cell-cell
contacts. In contrast, in the presence of p0071, desmoplakin
(E), p0071-FLAG (F), and VE-cadherin
(G) exhibited extensive colocalization (H).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin and ARVCF than
to plakophilin-1-3 (41). Interestingly, p0071 exhibited strong
interactions with the classical cadherins VE-cadherin (Fig. 3) and
E-cadherin (data not shown).1
Furthermore, p0071 interacted with the VE-cadherin juxtamembrane domain
in a region that is highly conserved in the classical cadherins. Mutational analysis of VE-cadherin indicated that a triple-amino acid
substitution of the juxtamembrane domain abrogated both p0071 (Fig. 4)
and p120 (data not shown) binding. When expressed transiently in COS-7
cells, p0071 also displaced p120 from intercellular junctions. The fact
that a triple-alanine substitution abrogated binding to both p120 and
p0071, combined with the observation that p0071 displaced p120 from
cell-cell junctions, strongly suggests that p0071 may bind to the
p120-binding site on classical cadherins and compete for p120 binding
to cadherins. This prediction is similar to models proposed for other
members of the p120 family (45, 46). A major challenge will be to
discern how cells use this complex repertoire of armadillo proteins to
modulate the functional state of adhesive contacts. In the case of
endothelial cells, one possibility is that, by increasing p0071
expression, the cadherin-associated pool of p120 would be decreased,
thereby shifting VE-cadherin association from actin-based to
intermediate filament-based. Recent studies by Shasby et al.
(47) indicate that, under quiescent conditions, VE-cadherin is tightly
associated with vimentin and that this interaction is dramatically
reduced upon exposure of endothelial cells to histamine. It will be
interesting to determine what signaling pathways regulate p0071
expression and how this correlates with endothelial barrier function
and, more broadly, with the balance between endothelial cell adhesion and cell migration.
-catenin, which associates predominantly
with adherens junctions, plakoglobin is thought to facilitate
cross-talk between adherens junctions and desmosomes (50). The
hypothesis that p0071 may be able to substitute for some of these
functions of plakoglobin is supported by knockout experiments in which
various components of the endothelial adhesion complex were ablated.
VE-cadherin-null embryos were found to undergo normal vasculogenesis,
but then failed to properly remodel the vascular system and ultimately
died at embryonic day 9.5 (51, 52). Conventional approaches to knock
out the desmoplakin gene result in early embryonic lethality (53), well
before initiation of vasculogenesis, thus precluding the ability to
assess the role of desmoplakin in vascular development. However, an
important role for desmoplakin in blood vessel morphogenesis was
revealed more recently using a tetraploid aggregation approach to
rescue the early embryonic lethality of the desmoplakin-null mutation (29). Like VE-cadherin, desmoplakin was found to be required for blood
vessel remodeling, and embryos lacking desmoplakin exhibited markedly
fewer capillaries. In contrast, plakoglobin-null animals exhibit heart
and skin defects, but a vascular phenotype has not been described for
the plakoglobin-knockout mice (54, 55). Because plakoglobin plays a
role in linking desmoplakin to VE-cadherin, it is likely that, in the
plakoglobin-null animals, other armadillo family proteins compensate
for plakoglobin in linking VE-cadherin and desmoplakin. We have been
unable to demonstrate a role for
-catenin in this linkage. However,
based on the ability of p0071 to bind to both VE-cadherin and
desmoplakin and to mediate VE-cadherin and desmoplakin colocalization,
it is possible that p0071 may compensate for some plakoglobin functions
in blood vessels in the plakoglobin-null animals.
-catenin) terminates with a PDZ domain-binding motif (30, 31) and interacts with the PDZ motif-containing protein PAPIN (59) and
with the LAP protein Erbin (57, 58). LAP family proteins such as Erbin
and Scribble play important roles in the development of cell polarity
during morphogenic events in Caenorhabditis elegans and
Drosophila (44). The ability of p0071 to interact with
cadherins and PDZ motif-containing proteins strongly suggests that
p0071 and the other PDZ domain-binding proteins ARVCF and
-catenin
function not only as structural components of intercellular junctions,
but also as scaffolds for the recruitment of signaling molecules
involved in cell polarity and cell migration. It will be of significant
interest to determine how p0071 may integrate VE-cadherin-mediated
adhesion with the function of signaling proteins involved in cell
polarity, particularly in the context of vascular morphogenesis
and the loss of endothelial barrier function associated with inflammation.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. K. J. Green, A. B. Reynolds, and E. Dejana for antibodies and cDNA reagents that made this work possible.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants R01 AR48266-01, R03 AR47147, K01 AR002039, HP30 AR042687, and T32 AR007587; the Dermatology Foundation; and Deutsche Forschungsgemeinschaft Grant Hal 791/3-3.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.
¶ To whom correspondence should be addressed: Dept. of Dermatology, Emory University School of Medicine, Atlanta, GA 30322. Tel.: 404-727-8517; Fax: 404-727-5878; E-mail: akowalc@emory.edu.
Published, JBC Papers in Press, November 7, 2002, DOI 10.1074/jbc.M205693200
1 M. Hatzfeld, K. J. Green, and H. Sauter, J. Cell Science, in press.
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