p120-Catenin regulates clathrin-dependent endocytosis of VE-cadherin.
ABSTRACT VE-cadherin is an adhesion molecule critical to vascular barrier function and angiogenesis. VE-cadherin expression levels are regulated by p120 catenin, which prevents lysosomal degradation of cadherins by unknown mechanisms. To test whether the VE-cadherin cytoplasmic domain mediates endocytosis, and to elucidate the nature of the endocytic machinery involved, the VE-cadherin tail was fused to the interleukin (IL)-2 receptor (IL-2R) extracellular domain. Internalization assays demonstrated that the VE-cadherin tail dramatically increased endocytosis of the IL-2R in a clathrin-dependent manner. Interestingly, p120 inhibited VE-cadherin endocytosis via a mechanism that required direct interactions between p120 and the VE-cadherin cytoplasmic tail. However, p120 did not inhibit transferrin internalization, demonstrating that p120 selectively regulates cadherin internalization rather than globally inhibiting clathrin-dependent endocytosis. Finally, cell surface labeling experiments in cells expressing green fluorescent protein-tagged p120 indicated that the VE-cadherin-p120 complex dissociates upon internalization. These results support a model in which the VE-cadherin tail mediates interactions with clathrin-dependent endocytic machinery, and this endocytic processing is inhibited by p120 binding to the cadherin tail. These findings suggest a novel mechanism by which a cytoplasmic binding partner for a transmembrane receptor can serve as a selective plasma membrane retention signal, thereby modulating the availability of the protein for endo-lysosomal processing.
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ABSTRACT: Vascular hyperpermeability induced by lipopolysaccharide (LPS) is a common pathogenic process in cases of severe trauma and sepsis. Vascular endothelial cadherin (VE-cad) is a key regulatory molecule involved in this process, although the detailed mechanism through which this molecule acts remains unclear. We assessed the role of clathrin-mediated and caveolae-mediated endocytosis of VE-cad in LPS-induced vascular hyperpermeability in the human vascular endothelial cell line CRL-2922 and determined that vascular permeability and VE-cad localization at the plasma membrane were negatively correlated after LPS treatment. Additionally, the loss of VE-cad at the plasma membrane was caused by both clathrin-mediated and caveolae-mediated endocytosis. Clathrin-mediated endocytosis was dominant early after LPS treatment, and caveolae-mediated endocytosis was dominant hours after LPS treatment. The caveolae-mediated endocytosis of VE-cad was activated through the LPS-Toll-like receptor 4 (TLR4)-Src signaling pathway. Structural changes in the actin cytoskeleton, specifically from polymerization to depolymerization, were important reasons for the switching of the VE-cad endocytosis pathway from clathrin-mediated to caveolae-mediated. Our findings suggest that clathrin-mediated and caveolae-mediated endocytosis of VE-cad contribute to LPS-induced vascular hyperpermeability, although they contribute via different mechanism. The predominant means of endocytosis depends on the time since LPS treatment.PLoS ONE 09/2014; 9(9):e106328. · 3.53 Impact Factor
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ABSTRACT: p120-catenin (p120) modulates adherens junction (AJ) dynamics by controlling the stability of classical cadherins. Among all p120 isoforms, p120-3A and p120-1A are the most prevalent. Both stabilize cadherins, but p120-3A is preferred in epithelia, while p120-1A takes precedence in neurons, fibroblasts, and macrophages. During Epithelial-to-Mesenchymal Transition (EMT), E- to N-cadherin switching coincides with p120-3A to -1A alternative splicing. These isoforms differ by a 101 amino acid "head domain" comprising the p120-1A N-terminus. Although its exact role is unknown, the head domain likely mediates developmental and cancer-associated events linked to p120-1A expression (e.g., motility, invasion, metastasis). Here, we have identified (Delta-Interacting Protein A) DIPA as the first head domain-specific binding partner and candidate mediator of isoform 1A activity. DIPA colocalizes with AJs in a p120-1A- but not 3A-dependent manner. Moreover, all DIPA family members (DIPA, Ccdc85a, and Ccdc85c) interact reciprocally with p120 family members (p120, δ-catenin, p0071, and ARVCF), suggesting significant functional overlap. During zebrafish neural tube development, both knockdown and overexpression of DIPA phenocopy N-cadherin mutations, an effect bearing functional ties to a reported mouse hydrocephalus phenotype associated with Ccdc85c. These studies identify a novel, highly conserved interaction between two protein families that may participate either individually or collectively in N-cadherin-mediated development.Molecular Biology of the Cell 07/2014; · 4.55 Impact Factor
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ABSTRACT: A key event in the progression of systemic inflammation resulting from severe trauma or shock involves microvascular hyperpermeability, which leads to excessive plasma fluid and proteins accumulating in extravascular space resulting in tissue edema. The precise molecular mechanism of the hyperpermeability response is not completely understood. Protein tyrosine kinase 6 (PTK6, also known as breast tumor kinase BRK) is a non-receptor tyrosine kinase related to Src-family proteins. Although it has also been shown that PTK6 participates in regulating epithelial barrier function, the role of PTK6 in endothelial barrier function has not been reported. In this study, we hypothesized that PTK6 is 1) expressed in vascular endothelial cells, and 2) contributes to vascular endothelial hyperpermeability in response to TNFα. Results showed that PTK6 was detected in mouse endothelial cells at the level of protein and mRNA. In addition, PTK6 knockdown attenuated TNFα induced decrease in endothelial barrier function as measured by electric cell-substrate impedance sensing (ECIS) and in vitro transwell albumin-flux assays. Furthermore, we showed that TNFα treatment of endothelial cells increased active PTK6 association with p120-catenin at endothelial cell-cell junctions. Further analysis using immunocytochemistry and immunoprecipitation demonstrated that PTK6 knockdown attenuated TNFα induced VE-cadherin internalization as well as promoting its association with p120-catenin. Our study demonstrates a novel role of PTK6 in mediating endothelial barrier dysfunction.Biochemical and Biophysical Research Communications 11/2014; · 2.28 Impact Factor
Molecular Biology of the Cell
Vol. 16, 5141–5151, November 2005
p120-Catenin Regulates Clathrin-dependent Endocytosis of
Kanyan Xiao,* Jennifer Garner,* Kathleen M. Buckley,†Peter A. Vincent,‡
Christine M. Chiasson,†Elisabetta Dejana,§Victor Faundez,?and
Andrew P. Kowalczyk*?
Departments of *Dermatology and?Cell Biology, Emory University, Atlanta, GA 30322;§FIRC Institute of
Molecular Oncology, Department of Biomolecular and Biotechnological Sciences, University of Milan, Milan
20139, Italy;†Graduate Program in Biochemistry, Cell, and Developmental Biology, Emory University,
Atlanta, GA 30322; and‡The Center for Cardiovascular Sciences, Albany Medical College, Albany, NY 12208
Submitted May 19, 2005; Revised July 11, 2005; Accepted August 15, 2005
Monitoring Editor: M. Bishr Omary
VE-cadherin is an adhesion molecule critical to vascular barrier function and angiogenesis. VE-cadherin expression levels
are regulated by p120 catenin, which prevents lysosomal degradation of cadherins by unknown mechanisms. To test
whether the VE-cadherin cytoplasmic domain mediates endocytosis, and to elucidate the nature of the endocytic
machinery involved, the VE-cadherin tail was fused to the interleukin (IL)-2 receptor (IL-2R) extracellular domain.
Internalization assays demonstrated that the VE-cadherin tail dramatically increased endocytosis of the IL-2R in a
clathrin-dependent manner. Interestingly, p120 inhibited VE-cadherin endocytosis via a mechanism that required direct
interactions between p120 and the VE-cadherin cytoplasmic tail. However, p120 did not inhibit transferrin internalization,
demonstrating that p120 selectively regulates cadherin internalization rather than globally inhibiting clathrin-dependent
endocytosis. Finally, cell surface labeling experiments in cells expressing green fluorescent protein-tagged p120 indicated
that the VE-cadherin–p120 complex dissociates upon internalization. These results support a model in which the
VE-cadherin tail mediates interactions with clathrin-dependent endocytic machinery, and this endocytic processing is
inhibited by p120 binding to the cadherin tail. These findings suggest a novel mechanism by which a cytoplasmic binding
partner for a transmembrane receptor can serve as a selective plasma membrane retention signal, thereby modulating the
availability of the protein for endo-lysosomal processing.
VE-cadherin is a member of the cadherin family of cell-cell
adhesion molecules and is expressed selectively in vascular
endothelial cells (Breviario et al., 1995; Dejana, 2004). Previ-
ous studies have established a key role for VE-cadherin in
several processes central to endothelial physiology, includ-
ing vascular barrier function and angiogenesis (Carmeliet
and Collen, 2000; Stevens et al., 2000; Dejana et al., 2001;
Vincent et al., 2004). Gene ablation and antibody inhibition
studies have revealed a critical role for VE-cadherin in vas-
cular patterning during development (Vittet et al., 1997;
Carmeliet et al., 1999; Corada et al., 2001; Crosby et al., 2005).
VE-cadherin function is also coordinated with growth factor
signaling pathways that regulate endothelial proliferation
during angiogenesis (Lampugnani et al., 2003; Spagnuolo et
al., 2004). Furthermore, antibody blocking studies raise the
possibility that cell surface VE-cadherin can be targeted by
therapeutic strategies designed to limit angiogenesis associ-
ated with tumor growth (Liao et al., 2000; Corada et al., 2002;
May et al., 2005). These studies demonstrate a central role for
VE-cadherin in vascular biology and pathophysiology, and
highlight the importance of understanding how plasma
membrane levels of VE-cadherin are regulated.
Cadherins at the cell surface are associated with cytoplas-
mic binding partners termed catenins. The catenins bind to
the carboxy-terminal tail of the cadherins and regulate cad-
herin cytoskeletal interactions and cadherin adhesive activ-
ity (Gooding et al., 2004). VE-cadherin is coupled to the actin
cytoskeleton through interactions with ?-catenin, an arma-
dillo family protein that binds to both the cadherin tail and
to actin-associating proteins such as ?-catenin (Navarro
et al., 1995). VE-cadherin is also coupled to the vimentin
intermediate filament cytoskeleton (Kowalczyk et al., 1998;
Calkins et al., 2003), and the molecules mediating these
linkages seem to be critical for normal VE-cadherin adhesive
activity and for VE-cadherin function during development
(Valiron et al., 1996; Gallicano et al., 2001). In addition to
?-catenin, the armadillo family also includes several other
related molecules, including p120-catenin (Anastasiadis and
Reynolds, 2000; Hatzfeld, 2005). Although not directly in-
volved in coupling the cadherins to the cytoskeleton, p120
has emerged as a critical regulator of cadherin adhesive
activity and cytoskeletal organization (Thoreson et al., 2000;
Anastasiadis and Reynolds, 2001; Ozawa and Ohkubo, 2001;
Pettitt et al., 2003).
This article was published online ahead of print in MBC in Press
on August 24, 2005.
Address correspondence to: Andrew P. Kowalczyk (akowalc@
Abbreviations used: IL-2R, interleukin 2 receptor; MEC, microvas-
cular endothelial cell; p120: p120-catenin.
© 2005 by The American Society for Cell Biology5141
In addition to regulating adhesion and actin cytoskeletal
organization, recent studies revealed a core function for
p120 in regulating cadherin expression levels in vertebrate
cells. Separate studies demonstrated that p120 regulates
steady-state levels of E-cadherin in epithelial cells (Davis et
al., 2003) and VE-cadherin in vascular endothelial cells (Xiao
et al., 2003a). In the absence of p120, cadherins become
destabilized and targeted for degradation. The precise
mechanism by which p120 regulates cadherin levels is not
fully understood. However, several lines of evidence sug-
gest that p120 regulates the entry of cell surface cadherins
into a lysosomal degradation pathway (Davis et al., 2003;
Xiao et al., 2003a). In the absence of p120, E-cadherin is
efficiently delivered to the cell surface, but it is then rapidly
turned over (Davis et al., 2003). Similarly, cell surface label-
ing experiments indicate that p120 regulates the lysosomal
degradation of cell surface pools of VE-cadherin (Xiao et al.,
2003a). These data suggest that p120 likely functions at the
plasma membrane to regulate cadherin internalization.
However, it is also possible that p120 regulates the fate of the
cadherin after endocytosis. p120 was found to associate with
kinesin and alter the rates of N-cadherin accumulation at
cell-cell borders (Chen et al., 2003). Collectively, these find-
ings strongly implicate p120 as a modulator of cadherin
trafficking (Kowalczyk and Reynolds, 2004). However, these
previous studies did not define a role for p120 in any specific
control point in cadherin delivery to degradative compart-
ments. Thus, it is currently unclear whether p120 regulates
cadherin levels by controlling cadherin endocytosis, target-
ing of the protein to degradative compartments after inter-
nalization, and/or recycling back to the plasma membrane.
The present study is part of a systematic effort to delineate
the role of the endocytic pathway in controlling cell surface
presentation of VE-cadherin. Herein, we report that the VE-
cadherin cytoplasmic domain mediates rapid and efficient en-
docytosis and that p120 regulates this critical step in VE-cad-
herin trafficking. Using a gain of function approach in which
the VE-cadherin tail was fused to the interleukin 2 receptor
(IL-2R) extracellular domain, we found that the cytoplasmic
domain of VE-cadherin dramatically enhances IL-2R endocy-
tosis. Furthermore, VE-cadherin internalization was found to
occur through a clathrin-mediated pathway. These data indi-
cate that the tail of VE-cadherin harbors sorting information
that mediates interactions, directly or indirectly, with compo-
nents of clathrin-dependent endocytic machinery. In addition,
p120 blocked endocytosis mediated by the VE-cadherin tail via
a mechanism that required direct interactions between p120
and the VE-cadherin juxtamembrane domain. Finally, p120
had no obvious effects on transferrin internalization, indi-
cating that p120 does not globally inhibit clathrin-dependent
endocytosis. Rather, our findings indicate that p120 func-
tions to selectively inhibit cadherin internalization from the
plasma membrane by inhibiting a process dependent upon
both the cadherin cytoplasmic domain and clathrin-depen-
dent endocytic machinery. We propose a model whereby
constitutive endocytic signals intrinsic to the cadherin tail
are suppressed by cytosolic p120-catenin. These findings
provide new insights into the mechanisms by which p120
controls cadherin expression levels and represent the first
example in which binding of a cytoplasmic protein directly
inhibits endocytosis of a transmembrane receptor.
MATERIALS AND METHODS
Primary cultures of dermal microvascular endothelial cells (MECs) from
human neonatal foreskin were obtained from the Emory Skin Diseases Re-
search Center (Core B) and cultured in MCDB131 medium (Invitrogen, Carls-
bad, CA) as described previously (Xiao et al., 2003b). The culture medium was
supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis,
MO), l-glutamine (Mediatech, Herndon, VA), cAMP (Sigma-Aldrich), hydro-
cortisone (Sigma-Aldrich), epidermal growth factor (Intergen, Purchase, NY),
and antibiotic/antimycotic (Invitrogen). Cells were typically cultured over-
night on 0.1% gelatin-coated plates and grown to 80% confluence for most
experiments. For adenovirus production, human embryonic kidney cell line
QBI-293A (Qbiogene, Carlsbad, CA) were routinely cultured in DMEM sup-
plemented with 10% FBS and antibiotic/antimycotic.
The IL-2R-VE-cadherin chimeras, p120ctn 1A, and ?-catenin cDNAs were
subcloned into the pAd-Track vector which coexpresses green fluorescent
protein (GFP) (He et al., 1998). Adenoviruses carrying the Empty Vector (EV),
VE-cadherin constructs, p120ctn 1A, ?-catenin, and p0071 were produced
using the pAdeasy adenovirus-packaging system as described previously
(Xiao et al., 2003a; Setzer et al., 2004). For most experiments, a low dose of
adenovirus was used to obtain 40–50% infection rates as monitored by GFP
MECs cultured on gelatin-coated glass coverslips were fixed in methanol for
5 min at ?20°C or 3.7% paraformaldehyde for 8 min on ice followed by either
extraction in 0.2% Triton X-100 for 5 min on ice or methanol for 1 min at
?20°C. The choice for fixation method depends on the performance of dif-
ferent antibodies. Endogenous VE-cadherin was detected using mouse mono-
clonal antibody (mAb) directed against the VE-cadherin extracellular domain
(BV-6) (Corada et al., 2001). The BV-6 antibody was fluorescently conjugated
using Alexa Fluor 555 mAb labeling kit (Molecular Probes, Eugene, OR).
IL-2R and the IL-2R-VE-cadherin chimeras were followed using an affinity-
purified anti-IL-2R IgG produced from 7G7B6 mouse hybridoma (American
Type Culture Collection, Manassas, VI) or by using an Alexa Fluor 488- or
555-conjugated anti-IL-2R IgG. VE-cadherin constructs were also followed
using a chicken antibody directed against the c-myc epitope tag (Bethyl
Laboratories, Montgomery, TX). The localization of p120ctn, ?-catenin, p0071,
and plakoglobin was determined using rabbit polyclonal antibodies against
p120ctn (Santa Cruz Biotechnology, Santa Cruz, CA), ?-catenin (NeoMarkers,
Fremont, CA), plakoglobin (Santa Cruz Biotechnology) and p0071 (Bethyl
Laboratories), respectively. The localization of early endosomes was moni-
tored using a rabbit polyclonal early endosome antigen (EEA)-1 antibody
(Affinity Bioreagents, Golden, CO). Second antibodies conjugated to various
Alexa Fluors (Molecular Probes) were used for dual or triple-label immuno-
fluorescence. Microscopy was performed using a fluorescence microscope
(model DMR-E; Leica, Wetzlar, Germany) equipped with narrow band pass
filters and a digital camera (model Orca; Hamamatsu, Bridgewater, NJ).
Images were captured, pseudocolored, and processed using Simple PCI soft-
ware (Compix, Cranberry Township, PA). In each case, the amount of vesic-
ular VE-cadherin present was quantified by counting VE-cadherin vesicles/
cell using Simple PCI software. For fluorimetry experiments, the cells were
cultured on 24-well plates, and the fluorescence was measured by Synergy HT
multi-detection microplate reader (KC-4; Bio-Tek Instruments, Winooski, VT).
Internalization assays were performed as described previously (Xiao et al.,
2003a). MECs were cultured on glass coverslips for experimentation. Fluores-
cently conjugated BV-6 antibody was incubated with cells at 4°C on ice for 30
min in MCDB131 media containing 20 mM HEPES (Mediatech). Unbound
antibody was removed by rinsing cells in ice-cold MCDB 131. Cells were
incubated at 4°C or transferred to 37°C for various amounts of time (1–30 min)
to allow the internalization of molecules from cell surface. To remove cell
surface bound antibody while retaining internalized antibody, cells were acid
washed for 30 min in phosphate-buffered saline (PBS), pH 2.7, containing 25
mM glycine and 3% bovine serum albumin (BSA). The cells were rinsed,
fixed, and processed for immunofluorescence as described above. For exper-
iments to monitor internalization of IL-2R or IL-2R-VE-cadherin chimeras,
MECs were infected with adenovirus overnight to allow time for infection
and expression of the polypeptides. The cells were surface labeled using IL-2R
antibody at 4°C and transferred to 37°C for various amounts of time. In some
experiments, cells were infected with EV, p120ctn, ?-catenin, or p0071 4–6 h
before the IL-2R-VE-cadherin chimeras to allow time for expression of the
catenins. For transferrin internalization, cells were incubated with 20 ?g/ml
Alexa Fluor 555-conjugated transferrin (Molecular Probes) at 4°C and then
allowed to internalize for 5 min. Potassium depletion was performed by
hypotonic shock of the cells in hypotonic buffer (1:1 dilution of K?depletion
buffer) for 5 min at 37°C and then incubation with K?depletion buffer (20 mM
HEPES, 140 mM NaCl, 1 mM CaCl2,1 mM MgCl2, pH 7.4) for 30 min at 37°C
(Wu et al., 2001). Cytosol acidification protocol was performed by incubating
cells with acetic acid buffer for 15 min at 37°C (Salazar and Gonzalez, 2002).
K. Xiao et al.
Molecular Biology of the Cell5142
MECs were grown to 80% confluence on 60-mm dishes in MEC growth
media. 18 h before biotinylation, MECs were infected by adenoviruses carry-
ing IL-2R control or the IL-2R-VE-cadcytochimera. Cells were washed three
times in ice-cold PBS? and labeled with EZ-Link Sulfo-NHS-SS-Biotin (Pierce
Chemical, Rockford, IL) at 0.5 mg/ml in PBS?, on ice, for 30 min. Excess
biotin was quenched by washing twice with blocking solution (50 mM NH4Cl
in PBS containing 1 mM MgCl2, and 0.1 mM CaCl2) on ice, followed by three
washes in ice-cold PBS?. Cells were incubated at 4°C or transferred to 37°C
for 5–30 min to allow internalization of the biotinylated surface molecules.
Biotin remaining at the cell surface was stripped by 100 mM sodium 2-mer-
captoethanesulfonic acid (MESNA; Sigma-Aldrich) in 50 mM Tris-HCl, pH
8.6, 100 mM NaCl, 1 mM EDTA, and 0.2% BSA as described previously
(Hammond et al., 2003). The cells were then rinsed twice with ice-cold PBS?,
and residual MESNA was quenched by 120 mM iodoacetamide (Sigma-
Aldrich) in PBS? (Hammond et al., 2003). After two additional rinses with
ice-cold PBS?, samples were lysed using cytoskeleton (CSK) buffer (50 mM
NaCl, 10 mM PIPES, 3 mM MgCl2, 300 mM sucrose, 1% Triton-X 100, 10 mM
NaF, 10 mM NaP2O7, pH 6.8) containing protease inhibitor cocktail. After
centrifugation at 14,000 rpm for 30 min, the supernatant was recovered and
incubated with UltraLink immobilized streptavidin (Pierce Chemical) for 1 h
at 4°C. Cell surface biotin-labeled proteins were captured by centrifugation,
and the beads were washed three times in CSK lysis buffer. Biotin labeled
proteins were released in 2? Laemmli buffer containing ?-mercaptoethanol at
95°C and then processed for Western blot analysis according to standard
protocols. The IL-2R extracellular domain was detected using a rabbit poly-
clonal antibody (Santa Cruz Biotechnology). Horseradish peroxidase-conju-
gated secondary antibodies (Bio-Rad, Hercules, CA) were used at 1:3000
dilution and detected using enhanced chemiluminescence (GE Healthcare,
Little Chalfont, Buckinghamshire, United Kingdom). The blots were quanti-
tated using GelPlot Software in Scion Image.
The VE-Cadherin Cytoplasmic Tail Mediates Endocytosis
Previously, we demonstrated that VE-cadherin is constitu-
tively internalized from the plasma membrane and deliv-
ered to the lysosome for degradation (Xiao et al., 2003b).
However, the experiments lacked the temporal resolution
necessary to define a function for p120-catenin in any spe-
cific step in VE-cadherin processing. To test whether the
VE-cadherin cytoplasmic domain mediates endocytosis and
to elucidate the nature of the endocytic machinery involved,
a gain of function approach was used in which the VE-
cadherin tail was fused to the IL-2R extracellular domain.
The IL-2R and the IL-2R-VE-cadcytochimera were then ex-
pressed in primary cultures of human MECs using an ad-
enoviral delivery system. Antibodies directed against the
IL-2R were used to label cell surface pools of the IL-2R or the
IL-2R-VE-cadcytochimera and the amount of internalization
of the proteins was quantified. For these experiments, MECs
were incubated at 4°C with the IL-2R antibody to label cell
surface pools of the IL-2R or the IL-2R-VE-cadcytochimera.
The cells were then washed to remove unbound antibody
and then switched to 37°C to allow for endocytosis. Cell
surface-bound antibody was distinguished from internal-
ized pools by washing cell surfaces with a low pH wash,
which removes antibody bound at the plasma membrane
but not antibody that has been internalized (Faundez et al.,
As shown in Figure 1, after labeling at 4°C with a mAb
directed against the IL-2R, both the IL-2R (Figure 1A) and
the IL-2R-VE-cadcytochimera (Figure 1C) were distributed
on the endothelial cell surface. In cells that remained at 4°C,
acid washing removed virtually all IL-2R antibody bound to
the cell surface (Figure 1, B and D). However, after incubat-
ing the cells at 37°C for 5 min, both the IL-2R (Figure 1E) and
the IL-2R-VE-cadcytochimera (Figure 1F) were detected in
intracellular vesicular compartments after acid washing the
cells. Interestingly, the degree of IL-2R-VE-cadcytointernal-
ization was substantially higher (?3-fold) than that of the
IL-2R without the VE-cadherin cytoplasmic tail (Figure 1,
E–G), indicating that the VE-cadherin tail accelerates endo-
cytosis of the IL-2R. To test this possibility further, biotiny-
lation experiments were performed to evaluate the amount
of internalization of the two proteins from the cell surface.
MECs expressing IL-2R or IL-2R-VE-cadcytowere biotinyl-
ated at 4°C and then transferred to 37°C for various amounts
of time. Stripping buffer was then used to selectively remove
biotin from cell surface proteins. Cells were then lysed and
biotinylated proteins were captured using streptavidin
beads. Western blot analysis using antibodies directed
against the IL-2R was performed to detect the amount of
IL-2R or IL-2R-VE-cadcytointernalized over time. In cells
incubated at 4°C, both IL-2R-VE-cadcytoand IL-2R were
expressed on the cell surface (Figure 1H). When the cells
were moved to 37°C, small amounts of IL-2R internalization
could be detected over time. In contrast, significant amounts
of internalized IL-2R-VE-cadcytochimera were detected as
early as 15 min after incubation at 37°C. These results are
consistent with the immunofluorescence based assay shown
in Figure 1, A–G. This gain of function approach demon-
strates that the VE-cadherin cytoplasmic tail harbors se-
quence information that functions to promote endocytosis.
To determine whether the IL-2R-VE-cadcytopolypeptide
follows the same route of internalization as endogenous
VE-cadherin, colocalization experiments were carried out
using surface labeled endogenous VE-cadherin and the IL-
2R-VE-cadcytochimera. As shown in Figure 2, A–C, endog-
enous VE-cadherin colocalized extensively with the early
endosomal marker EEA-1 (Simonsen et al., 1998) after inter-
nalization. Similar to previous results with E-cadherin
(Paterson et al., 2003), VE-cadherin exhibited partial colocal-
ization with fluorescently tagged transferrin internalized
from the cell surface (Figure 2, D–F). The IL-2R-VE-cadcyto
chimera also exhibited extensive colocalization with EEA-1
upon internalization from the cell surface (Figure 2, G–I).
Furthermore, the IL-2R-VE-cadcytochimera exhibited strik-
ing colocalization with endogenous VE-cadherin internal-
ized from the cell surface (Figure 2, J–L). These data indicate
that the IL-2R-VE-cadcytofollows the same pathway for in-
ternalization as does endogenous VE-cadherin. These results
provide further evidence that the VE-cadherin tail provides
targeting information for cadherin endocytosis.
VE-Cadherin Internalization Is Clathrin Dependent
Recent studies indicate that multiple pathways may mediate
E-cadherin internalization in various cell types, including
clathrin-dependent and clathrin-independent mechanisms
(Akhtar and Hotchin, 2001; Palacios et al., 2002; Paterson et
al., 2003; Bryant and Stow, 2004). Although multiple path-
ways for internalization can converge on transferrin-positive
compartments, the colocalization between VE-cadherin and
transferrin in endocytic compartments (Figure 2, D–E) sug-
gested that VE-cadherin may be internalized via a clathrin
mediated pathway. To test this possibility directly, clathrin-
dependent endocytosis was inhibited using K?depletion
and cytosol acidification, two well-characterized methods to
inhibit clathrin-mediated internalization (Wu et al., 2001;
Salazar and Gonzalez, 2002). Endocytosis mediated by the
VE-cadherin cytoplasmic domain was monitored by ex-
pressing the IL-2R-VE-cadcytochimeric protein in MEC as
shown in Figure 1. A myc epitope at the carboxy-terminal
tail of the VE-cadherin cytoplasmic domain was used to
verify expression of the IL-2R-VE-cadcytochimera. Similar to
the results shown in Figure 1, extensive internalization of the
IL-2R-VE-cadcytochimera was observed after 5 min at 37°C
(Figure 3D). Both K?depletion (Figure 3, E and F) and
cytosol acidification (Figure 3, G and H) dramatically inhib-
p120-Catenin Regulates Endocytosis
Vol. 16, November 20055143
ited internalization of the IL-2R-VE-cadcytopolypeptide.
These results, shown quantitatively in Figure 3I, indicate
that endocytosis of the IL-2R-VE-cadcytochimera is clathrin
Additional experiments were performed to verify that
endogenous VE-cadherin is internalized via mechanisms
similar to the IL-2R-VE-cadcytopolypeptide. For these exper-
iments, cell surface VE-cadherin was followed using a fluo-
rescently tagged mAb (BV6) directed against the VE-cad-
herin extracellular domain. In cells incubated at 4°C, BV6
decorated cell-cell junctions as well as nonjunctional cell
surface VE-cadherin (Figure 4A). Cell surface BV6 labeling
MECs at 4°C using a monoclonal IL-2R antibody. The cells were rinsed, fixed, and processed for immunofluorescence microscopy. A low pH
wash (acid wash) was used to remove antibody bound to the cell surface but not antibody that had been internalized. In cells that remained
at 4°C, acid washing the cells removed virtually all IL-2R antibody bound to the cell surface (B and D). To monitor internalization, cells were
labeled at 4°C and transferred to 37°C for 5 min. After acid washing to remove cell surface antibody, cells were processed for immunoflu-
orescence microscopy (E and F). Fluorimetry was performed to quantify the percentage of cell surface IL-2R and the IL-2R-VE-cadcytochimera
internalized after 30 min at 37°C (G). As an alternative approach, biotinylation experiments were performed (H). MECs expressing IL-2R or
IL-2R-VE-cadcytowere biotinylated at 4°C and then transferred to 37°C for 5–30 min. Striping buffer was used to selectively remove biotin
from cell surface proteins. Cells were then lysed and biotinylated proteins were captured using streptavidin beads. Western blot analysis
using antibodies directed against the IL-2R was performed to determine the percentage of IL-2R or IL-2R-VE-cadcytointernalized over time
(I). Bar, 50 ?m.
The VE-cadherin cytoplasmic tail mediates internalization. Cell surface IL-2R and IL-2R-VE-cadcytochimera were labeled in living
K. Xiao et al.
Molecular Biology of the Cell5144
was efficiently removed upon acid washing cells incubated
at 4°C (Figure 4B). After 15 min at 37°C, VE-cadherin was
internalized and targeted to early endosomes as assessed by
EEA-1 localization (Figure 4, C–E). Both K?depletion (Fig-
ure 4, F–H) and cytosol acidification (our unpublished data)
dramatically blocked endocytosis of endogenous VE-cad-
herin (Figure 4I). Together with the results shown in Figure
3, these data indicate that the VE-cadherin cytoplasmic do-
main mediates internalization via clathrin-dependent mech-
p120 Selectively Inhibits VE-Cadherin Endocytosis
In previous studies, p120 was found to inhibit the delivery
of cell surface derived VE-cadherin to lysosomes (Xiao et al.,
2003a). However, these experiments lacked the temporal
resolution required to determine whether p120 specifically
inhibited endocytosis of the cadherin. The results shown
above indicate that the IL-2R-VE-cadcytochimera is internal-
ized in a clathrin-dependent manner and routed to identical
compartments as endogenous VE-cadherin. Furthermore,
because the IL-2R-VE-cadcytopolypeptide is not engaged in
adhesive interactions, significant amounts of the IL-2R-VE-
cadcytochimera can be detected in intracellular pools within
5 min after switching cells to 37°C (Izumi et al., 2004). There-
fore, we tested the ability of p120 to inhibit endocytosis of
the IL-2R-VE-cadcytochimera using short time courses (5
min) to determine whether p120 regulates early steps in
cadherin internalization. For these experiments, IL-2R-VE-
cadcytochimera was expressed with empty vector or in
combination with p120 using an adenoviral delivery system.
In the absence of exogenous p120, substantial levels of IL-
2R-VE-cadcytochimera internalization were observed within
5 min (Figure 5, A–C). In contrast, endocytosis of the IL-2R-
VE-cadcytopolypeptide was virtually eliminated in cells
MECs was labeled by an Alexa Fluor 555-conjugated mAb (BV6) directed against the extracellular domain of VE-cadherin at 4°C. Cells were
then transferred to 37°C for 15 min. After acid washing to remove the antibodies bound to the cell surface, the localization of internalized
VE-cadherin was examined by immunofluorescence microscopy and compared with EEA-1 (A–C) and transferrin (D–F) localization. The
colocalization of internalized IL-2R-VE-cadcytoand EEA-1 was also determined (G–I). The IL-2R-VE-cadcytowas monitored by using an Alexa
Fluor 555-conjugated IL-2R antibody. Furthermore, the colocalization of internalized IL-2R-VE-cadcytoand endogenous VE-cadherin was
examined in MECs expressing IL-2R-VE-cadcyto.The IL-2R-VE-cadcytowas followed by using an Alexa Fluor 488-conjugated IL-2R antibody
and the endogenous VE-cadherin was followed by Alexa Fluor 555-conjugated BV6 VE-cadherin antibody directed against the extracellular
domain of VE-cadherin (J–L). Bar, 50 ?m.
IL-2R-VE-cadcytofollows the same internalization pathway as endogenous VE-cadherin. Cell surface VE-cadherin in untreated
p120-Catenin Regulates Endocytosis
Vol. 16, November 2005 5145
coexpressing exogenous p120 (Figure 5, D–F). Interestingly,
the p120 related protein p0071 also inhibited VE-cadherin
endocytosis (Figure 5, G–I). However, ?-catenin was unable
to inhibit internalization (Figure 5, J and K). These experi-
ments demonstrate that p120 family proteins specifically
and potently inhibit VE-cadherin endocytosis.
It is possible that p120 inhibits VE-cadherin endocytosis
selectively, or alternatively, that p120 functions as a general
inhibitor of clathrin-mediated internalization from the
plasma membrane. To distinguish these possibilities, two
approaches were taken. First, the ability of p120 to block
endocytosis of a prototypical clathrin cargo protein was
tested. For these experiments, the effect of p120 on trans-
ferrin endocytosis was tested. As shown in Figure 6, trans-
ferrin internalization was quantitatively similar in control
endothelial cells and in endothelial cells overexpressing
Cell surface IL-2R-VE-cadcytowas labeled using the IL-2R antibody
at 4°C. Cells were then kept at 4°C (A and B) or transferred to 37°C
for 5 min (C–H). At the end of the incubation period, cells were
processed for immunofluorescence using antibodies directed
against the myc epitope tag at the carboxy-terminal domain of the
IL-2R-VE-cadcytoto verify expression of the protein. After 5 min at
37°C, substantial internalization of the IL-2R-VE-cadcytowas ob-
served (C and D). Cytosol acidification (E and F) and potassium
depletion (G and H), both of which inhibit clathrin mediated endo-
cytosis, completely blocked internalization of the IL-2R-VE-cadcyto
chimera (I). Bar, 50 ?m.
IL-2R-VE-cadcytointernalization is clathrin dependent.
surface VE-cadherin was labeled using BV6 VE-cadherin antibody
conjugated to Alexa Fluor 555. Localization of VE-cadherin was
examined by immunofluorescence microscopy in cells incubated at
4°C (A and B) or transferred to 37°C for 15 min (C–H). Acid washing
removed virtually all surface bound BV6 (B). Colocalization with
EEA-1 indicated that the internalized VE-cadherin was targeted to
early endosomes (C–E). K?depletion dramatically blocked endocy-
tosis of endogenous VE-cadherin (F–H), indicating that VE-cadherin
internalization was clathrin-mediated. Results from the immunoflu-
orescence experiments are shown quantitatively in I. Bar, 50 ?m.
VE-cadherin internalization is clathrin dependent. Cell
K. Xiao et al.
Molecular Biology of the Cell5146
p120. These findings suggest that p120 does not globally
alter the kinetics of clathrin-mediated endocytic pathways.
Based on these results, we tested whether p120 binding to
the VE-cadherin tail was required for p120 to inhibit VE-
cadherin endocytosis. In previous studies, a triple amino-
acid substitution in the cadherin juxtamembrane domain
was shown to abrogate p120 binding to the cadherin tail
(Thoreson et al., 2000; Calkins et al., 2003; Xiao et al., 2003a).
Therefore, this mutation was introduced into the VE-cad-
herin cytoplasmic domain in the context of the IL-2R-VE-
cadcytochimera. The resulting polypeptide, termed IL-2R-
VE-cadJMD-AAA, encodes the VE-cadherin cytoplasmic tail
with a triple alanine substitution at amino acids 562–564
(EMD-AAA), which abrogates p120 and p0071 binding
(Calkins et al., 2003). As shown in Figure 7, p120 dramati-
cally decreased endocytosis of IL-2R-VE-cadcytochimera
(Figure 7, D–F). However, the IL-2R-VE-cadJMD-AAAcon-
struct was completely refractile to overexpression of p120;
even in cells expressing high levels of p120, substantial
internalization of the IL-2R-VE-cadJMD-AAApolypeptide was
observed (Figure 7, J–L). These data indicate that binding of
p120 to the cadherin tail is required for p120 to inhibit
VE-cadherin endocytosis. Furthermore, that p120 does not
prevent transferrin internalization indicates that p120 regu-
lates VE-cadherin endocytosis selectively rather than clath-
rin-dependent endocytosis globally.
VE-cadcytochimera was expressed with empty virus (A–C) or in
combination with p120 (D–F), p0071 (G–I), or ?-catenin (J and K).
Cell surface IL-2R-VE-cadcytowas labeled using IL-2R antibody at
4°C, and then cells were transferred to 37°C for 5 min. After acid
washing, localization of internalized IL-2R-VE-cadcytowas exam-
ined by immunofluorescence microscopy. Expression of the IL-2R-
VE-cadcytopolypeptide was verified by using a chicken antibody
directed against the myc epitope at the carboxyl terminal tail of
the IL-2R-VE-cadherin chimera (B, E, and H). Results from the
immunofluorescence experiments are shown quantitatively in L.
Bar, 50 ?m.
p120 prevents endocytosis of VE-cadherin. The IL-2R-
MECs infected with empty virus or virus carrying p120 were incu-
bated with Alexa Fluor 555-conjugated transferrin at 4°C and then
transferred to 37°C for 5 min. After acid washing, cells were fixed
and processed for immunofluorescence microscopy. The localiza-
tion of internalized transferrin was examined in both control MEC
cells (A and B) and MECs expressing p120 (C and D). Results are
shown quantitatively in E. Bar, 50 ?m.
p120 does not inhibit transferrin receptor internalization.
p120-Catenin Regulates Endocytosis
Vol. 16, November 20055147
p120 Dissociates from VE-Cadherin upon Internalization
The aforementioned data suggest that p120 binding to the
VE-cadherin at the plasma membrane stabilizes the cadherin
and prevents internalization. A prediction based on these
observations is that p120 would dissociate from the cadherin
during internalization. In previous studies, p120 did not
colocalize with vesicular pools of VE-cadherin in chloro-
quine-treated MECs. However, these previous internaliza-
tion assays were carried out over long periods (3–6 h),
allowing substantial amounts of time for p120 to dissociate
after endocytosis. To investigate whether p120 is associated
with the VE-cadherin tail upon and immediately after endo-
cytosis, p120 localization was monitored relative to the IL-
2R-VE-cadcytochimera. In cells that remained at 4°C, the
IL-2R-VE-cadcytochimera colocalized extensively with en-
dogenous p120 at the cell surface (Figure 8, A–D). In con-
trast, 5 min after internalization at 37°C, endogenous p120
failed to colocalize with the internalized IL-2R-VE-cadcyto
polypeptide, suggesting that p120 dissociated from the IL-
2R-VE-cadcytopolypeptide upon internalization (Figure 8,
E–H). Similar results were obtained when ?-catenin local-
ization was examined (our unpublished data), suggesting
that both catenins may dissociate from the VE-cadherin
cytoplasmic tail upon endocytosis. A caveat to these exper-
iments is that epitope masking of p120 could occur upon
entry of the complex into endosomes. Therefore, internaliza-
tion of endogenous VE-cadherin was followed using the
fluorescently tagged BV6 mAb in MECs expressing low
levels of GFP-tagged p120. The p120.GFP assembled into
junctions and exhibited extensive colocalization with BV6 at
the plasma membrane (Figure 8, I–L). However, p120.GFP
did not colocalize in early endosomes with the BV6-VE-
cadherin complex derived from the cell surface (Figure 8,
M–Q). These data support a model in which p120 dissociates
from the VE-cadherin tail upon clathrin-mediated internal-
ization of the cadherin from the plasma membrane.
The work presented here demonstrates that the cytoplasmic
tail of VE-cadherin targets the protein for internalization via
a clathrin-dependent pathway. Furthermore, p120 selec-
tively prevents clathrin-dependent endocytic machinery
from targeting the cadherin for endocytosis. These findings
establish a novel mechanism whereby a cytoplasmic binding
partner functions as a specific plasma membrane retention
signal for a transmembrane receptor. In addition, the results
presented here clarify the mechanism by which p120 func-
tions as a set point to regulate cadherin cell surface expres-
Previous studies in several model systems demonstrated
that p120 catenin functions as a set point, or rheostat, to
control cadherin expression levels in vascular endothelial
cells and in epithelial cells (Davis et al., 2003; Xiao et al.,
2003a; Iyer et al., 2004). Furthermore, E-cadherin is metabol-
ically unstable in tumor cell lines lacking p120, suggesting
that loss of p120 leads to a corresponding loss of E-cadherin
during tumor progression (Ireton et al., 2002; Reynolds and
Carnahan, 2004). Collectively, these findings revealed a core
function for p120 in the stabilization of cell surface cad-
herins. However, the mechanism by which p120 controls the
metabolic stability of cadherins was not clear from these
previous studies. Newly synthesized E-cadherin is delivered
to the plasma membrane efficiently in the absence of p120,
suggesting that p120 is dispensable in cadherin trafficking to
the plasma membrane after translation (Davis et al., 2003).
Similarly, the plasma membrane pool of VE-cadherin be-
comes destabilized in the absence of p120, leading to VE-
cadherin degradation via a lysosomal pathway (Xiao et al.,
2003a). However, other studies found that p120 binds to
kinesin and that this interaction modulates the rate of N-
inhibition of endocytosis. p120 was coexpressed in MEC expressing
either IL-2R-VE-cadcytoor the IL-2R-VE-cadJMD-AAA,which does not
bind p120. Cell surface IL-2R-VE-cadherin polypeptides were la-
beled using the IL-2R antibody at 4°C and then transferred to 37°C
for 5 min. After acid washing, cells were fixed and processed for
triple-labeled immunofluorescence microscopy. Internalized IL-2R-
VE-cadherin polypeptides were detected using an Alexa Fluor 555
goat anti-mouse secondary antibody (B, E, H, and K). Expression of
the IL-2R-VE-cadcytochimera was verified by using a chicken anti-
body directed against the myc epitope at the carboxy-terminal tail of
the IL-2R-VE-cadherin polypeptide (A, D, G, and J). Expression of
p120 in the cells was verified using a rabbit p120 antibody (C, F, I,
and L). Results from the immunofluorescence experiments are
shown quantitatively in M. Bar, 50 ?m.
p120 binding to the VE-cadherin tail is required for
K. Xiao et al.
Molecular Biology of the Cell 5148
cadherin accumulation at cell-cell borders upon initiation of
cell contact in response to calcium (Chen et al., 2003). Al-
though these previous studies clearly established a role for
p120 in cadherin trafficking and metabolic stability, the ex-
periments lacked the temporal resolution necessary to im-
plicate p120 in any particular step of membrane trafficking.
In the present study, we used several approaches to iden-
tify the cellular machinery involved in VE-cadherin internal-
ization and to directly test whether p120 regulates this step
in cadherin trafficking. First, a gain of function approach was
used to establish that the VE-cadherin cytoplasmic tail har-
bors the information necessary to target the cadherin for
internalization. Indeed, fusion of the VE-cadherin tail to the
IL-2R dramatically enhanced the baseline rate of IL-2R in-
ternalization (Figure 1). In addition, internalization of both
the IL-2R-VE-cadcytochimera and endogenous VE-cadherin
were mediated by a clathrin-dependent pathway (Figures 3
and 4). Interestingly, cointernalization assays demonstrated
extensive colocalization between the IL-2R-VE-cadcytochi-
mera and endogenous VE-cadherin (Figure 2). These data
demonstrate that the tail of the cadherin is sufficient not only
to mediate clathrin-dependent endocytosis but also to target
the cadherin to specific subcellular compartments after en-
The IL-2R-VE-cadcytochimera is unable to engage in ad-
hesion, and the protein is rapidly internalized from the cell
surface. Substantial levels of internalized IL-2R-VE-cadcyto
were detected within 5 min, as assessed by both fluores-
cence-based antibody internalization assays as well as cell
surface biotinylation experiments. These findings are consis-
tent with the idea that engagement of adhesion prevents
cadherin endocytosis (Izumi et al., 2004). The rapid kinetics
of endocytosis of this protein allowed us to determine
whether p120 specifically regulates VE-cadherin internaliza-
tion and thus to isolate this step in the trafficking pathway
from temporally subsequent steps in sorting, such as recy-
cling and degradation. The results indicate that p120 dra-
matically inhibits cadherin endocytosis (Figure 5). Further-
more, mutation of the p120 binding site on the cadherin
relieves this inhibition (Figure 7). These data indicate that
p120 must bind to the cadherin to function as a plasma
membrane retention signal. Interestingly, p120 had no dis-
cernible impact on transferrin internalization (Figure 6), in-
dicating that the p120 effect is selective for cadherins and not
the endocytic pathway globally. These data reveal a novel
mechanism whereby a cytoplasmic binding partner can
function as a highly selective plasma membrane retention
signal for a transmembrane binding partner. It is formally
possible that in addition to regulating the endocytosis of
cadherins, p120 may also regulate subsequent steps in cad-
herin trafficking, such as recycling (Le et al., 1999; Mary et al.,
2002; Bryant and Stow, 2004). Our data do not rule out this
possibility. As discussed above, previous work demon-
strated that p120 associates with kinesin and that this inter-
action plays a role in the movement of vesicular pools of
N-cadherin out toward intercellular junctions (Chen et al.,
upon internalization. MEC cells were infected
with adenovirus carrying the IL-2R-VE-cadcyto
chimera and localization of endogenous p120
was monitored relative to the IL-2R-VE-cadcyto
chimera. At 4°C, the IL-2R-VE-cadcytopolypep-
tide colocalized extensively with endogenous
p120 at the cell surface (A–D). However, 5 min
after internalization at 37°C, endogenous p120
failed to colocalize with the internalized IL-2R-
VE-cadcyto(E–H). To further test if p120 dissoci-
ates from VE-cadherin upon internalization, in-
ternalization of endogenous VE-cadherin was
followed using the Alexa Fluor 555-tagged BV6
mAb in MEC expressing GFP-tagged p120. The
p120.GFP assembled into junctions and exhib-
ited extensive colocalization with the BV6-VE-
cadherin complex at the plasma membrane at
4°C (I–L). However, 30 min after internalization
at 37°C, p120.GFP did not colocalize with BV6–
VE-cadherin complexes that had been internal-
ized to early endosomes (M–Q). Bar, 50 ?m.
p120 dissociates from VE-cadherin
p120-Catenin Regulates Endocytosis
Vol. 16, November 20055149
2003). These data suggest that p120 may play a role in
recycling of the cadherin after endocytosis. However, the
effects of p120 on junctional accumulation of N-cadherin
were quantitatively modest compared with the effects of
p120 on VE-cadherin internalization. In addition, although
the rates of movement of N-cadherin toward the plasma
membrane are slower if the p120 binding site on the cad-
herin is ablated, mutant N-cadherin that is unable to bind
p120 is still delivered to intercellular junctions (Chen et al.,
2003). Finally, newly synthesized E-cadherin does not seem
at all dependent upon p120 for proper delivery to the
plasma membrane (Davis et al., 2003). Thus, although p120
may have some effects on cadherin recycling, the data pre-
sented here indicate that quantitatively the most important
point of cadherin regulation by p120 is an early step in
cadherin internalization from the plasma membrane.
The model that emerges from these studies is that the
VE-cadherin tail associates with cellular machinery involved
in clathrin-dependent endocytosis and that p120 somehow
prevents these interactions. Several sequence motifs in the
VE-cadherin tail exhibit homology to di-leucine- or tyrosine-
based internalization signals. These amino acid motifs me-
diate binding to adaptor complexes, such as AP-2, and
thereby couple cargo molecules to clathrin-coated pits (Boni-
facino and Traub, 2003; Robinson, 2004). An attractive pos-
sibility is that p120 competes for such interactions and
thereby functions to prevent entry of the cadherin into the
endocytic pathway. This possibility is supported by the
observation that p120 colocalizes strongly with junctional
VE-cadherin, but it is not present in vesicular pools of VE-
cadherin that have been internalized from the plasma mem-
brane. Nonetheless, it is clear that p120 also exhibits influ-
ence over small GTPases and actin cytoskeletal organization
(Anastasiadis and Reynolds, 2001), thus raising the possibil-
ity that a more indirect mechanism of action may underlie
p120 regulation of cadherin internalization. However, the
fact that p120 must bind to the cadherin leads us to favor a
model in which p120 binding to the cadherin functions to
“cap” the cadherin tail, thereby preventing adaptor protein
interactions and subsequent endocytosis (Figure 9). Studies
are underway to distinguish between these possibilities and
to identify the function of this regulatory system in the
context of endothelial cell biology in vivo. Indeed, recent
studies from the Radice laboratory indicate that VE-cadherin
expression levels are regulated by N-cadherin, possibly
through the regulation of p120 levels (Luo and Radice, 2005).
These data suggest that p120, and perhaps other classical
cadherins, exert influence over VE-cadherin expression lev-
els during mouse vascular development.
We are grateful to Dr. Kathleen Green for insightful comments and advice
and to Dr. Gloria Salazar for assistance with the clathrin-dependent endocy-
tosis assays. Additional thanks go to members of the Kowalczyk laboratory
for helpful advice and discussion. This work was supported by National
Institutes of Health Grants R01 AR050501 and R01 AR48266 (to A.P.K.). K. X.
was supported by a postdoctoral fellowship from the American Heart Asso-
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