* The Lymphocyte Biology Section, The cadherins are a family of homophilic adhesion molecules that play a vital role in the formation
of cellular junctions and in tissue morphogenesis. Members of the integrin family are also involved in cell to
cell adhesion, but bind heterophilically to immunoglobulin superfamily molecules such as intracellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, or mucosal addressin cell adhesion
molecule (MadCAM)-1. Recently, an interaction between epithelial (E-) cadherin and the mucosal lymphocyte integrin, The binding of THE cadherins constitute a family of cell surface adhesion molecules that are involved in calcium-
dependent homophilic cell to cell adhesion (Takeichi, 1990 The integrins are a second family of transmembrane adhesion molecules that are involved in both cell to cell and
cell to matrix interactions. At least 15 Recently, we reported that E-cadherin on human epithelial cells may be a ligand for the mucosal lymphocyte
integrin, Given that the interaction of Materials
DNA manipulating enzymes were purchased from New England Biolabs
Inc. (Beverly, MA). Oligonucleotides were obtained from Oligotech (Boston, MA). Other chemicals were purchased from Sigma Chemical Co. (St.
Louis, MO). ICAM-1-Fc (the entire extracellular region of human
ICAM-1 fused to the hinge and Fc portion of human IgG1) was a generous gift of Dr. Lloyd Klickstein (Brigham and Women's Hospital, Boston,
MA). Purified human IgG1 was obtained from Calbiochem-Novabiochem Corp. (La Jolla, CA).
mAbs
The mAb used (all mouse IgG against human antigens) were as follows:
E4.6 (anti-E-cadherin, IgG1; Cepek et al., 1994 Cells
Human iIEL were isolated as previously described (Roberts et al., 1993 A subline of the human B lymphoblastoid cell line, JY, that expresses
the Construction of E- and P-Cadherin-Fc
Expression Vectors
E-cadherin-Fc.
A double-stranded DNA adapter containing a 5 Renal Division, Department of
Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115; § Combined Program in
Pediatric Gastroenterology and Nutrition, and Department of Pathology, Massachusetts General Hospital, Boston,
Massachusetts 02115; and
Department of Molecular and Cellular Physiology, Beckman Center, Stanford University School of
Medicine, Stanford, California 94305
Abstract
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
E
7, has been proposed. Here, we
demonstrate that a human E-cadherin-Fc fusion protein binds directly to soluble recombinant
E
7, and to
E
7 solubilized from intraepithelial T lymphocytes.
Furthermore, intraepithelial lymphocytes or transfected JY
cells expressing the
E
7 integrin adhere
strongly to purified E-cadherin-Fc coated on plastic,
and the adhesion can be inhibited by antibodies to
E
7
or E-cadherin.
E
7 integrin to cadherins is selective
since cell adhesion to P-cadherin-Fc through
E
7 requires >100-fold more fusion protein than to E-cadherin-Fc. Although the structure of the
E-chain is
unique among integrins, the avidity of
E
7 for E-cadherin can be regulated by divalent cations or phorbol myristate acetate. Cross-linking of the T cell receptor
complex on intraepithelial lymphocytes increases the
avidity of
E
7 for E-cadherin, and may provide a
mechanism for the adherence and activation of lymphocytes within the epithelium in the presence of specific foreign antigen. Thus, despite its dissimilarity to known integrin ligands, the specific molecular interaction demonstrated here indicates that E-cadherin is a
direct counter receptor for the
E
7 integrin.
Introduction
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
). The best studied human cadherins, E-, P-, N-,
and VE-cadherin, have a restricted tissue distribution: E-
and P-cadherin are expressed in epithelial tissues (Nose
and Takeichi, 1986
; Shimoyama et al., 1989a), N-cadherin
is found mainly on neural cells (Hatta et al., 1987
), and
VE-cadherin is found on vascular endothelium (Lampugnani et al., 1992
). Homophilic binding between cadherins on
adjacent cells is vital for the maintenance of strong cell to
cell adhesion in these tissues. For example, E-cadherin is
required for the formation of adherens junctions between
mature epithelial cells (Boller et al., 1985
; Gumbiner et al.,
1988
) and is involved in Langerhans cell adhesion to keratinocytes (Tang et al., 1993
), and VE-cadherin is needed
for the maintenance of lateral association between endothelial cells (Lampugnani et al., 1992
). During development
cadherins are critically involved in the cell sorting required
for tissue morphogenesis (Takeichi, 1995
), and loss of
E-cadherin function contributes to the metastasis of a variety of carcinomas (Ben-Ze'ev, 1997
). The extracellular regions of mature mammalian cadherins are comprised five
"CAD" modules of ~110 amino acids. Crystallographic
and biochemical studies indicate that cadherins probably
form dimers on the cell surface (Shapiro et al., 1995
; Nagar
et al., 1996
), and that interaction with dimeric cadherins on
opposing cell surfaces can lead to the formation of "zipper-like" cell junctions (Shapiro et al., 1995
; Tomschy et
al., 1996
).
chains associate
with 8
chains to form a large number of heterodimeric
integrins that can be classified into several major subfamilies based on their shared use of a particular
chain
(Hynes, 1992
). Members of three such subfamilies, the
1,
2, and
7 integrins, are commonly found on leukocytes. The expression of
1 integrins is widespread (for example,
5
1, CD49e/CD29, is found on T cells, granulocytes,
platelets, fibroblasts, endothelium, and epithelium), whereas
the
2 and
7 integrins have a restricted pattern of expression. For example,
L
2 (CD11a/CD18) is expressed on
most lymphocytes and many myeloid cells but not on
other cell types, and
E
7 (CD103/unclustered) is found
on >95% of intestinal intraepithelial lymphocytes (iIEL)1
and on other mucosal T cells, macrophages, and mast cells,
but on only 2% of peripheral blood lymphocytes (Cerf-Bensussan et al., 1987
; Smith et al., 1994
; Tiisala et al.,
1995
). The major ligands of the integrins fall into two categories: cell surface molecules that are members of the immunoglobulin superfamily (such as vascular cell adhesion
molecule [VCAM]-1, intracellular adhesion molecule
[ICAM]-1, 2, 3, and mucosal addressin cell adhesion molecule [MadCAM]-1) and a variety of large extracellular
proteins (such as fibronectin, vitronectin, fibrinogen, and
complement component iC3b; Hynes, 1992
). A common
feature of both groups is the presence of exposed acidic
amino acids crucial for integrin binding (Bergelson and
Hemler, 1995
). Many integrins exist in states of low or
high avidity for their ligands, and these states can be regulated from within the cell (Hynes, 1992
). For example, the
avidity of
L
2 on resting T cells can be increased by stimulation through the T cell receptor (TCR) with anti-CD3
mAbs (Dustin and Springer, 1989
; van Kooyk et al., 1989
).
E
7, and a similar interaction has been suggested in the mouse (Cepek et al., 1994
; Karecla et al.,
1995
). mAbs to E-cadherin or to
E
7 block IEL adherence to epithelial cells, and transfection of cells with
E
7
confers upon them the ability to adhere to cells transfected
with E-cadherin (Cepek et al., 1994
). E-cadherin has been
defined extensively as a homophilic adhesion molecule, and its sequence is not related to known cell surface or extracellular integrin ligands. Thus, the concept that
E
7
and E-cadherin are counter receptors is at variance with
current knowledge of both integrin and cadherin interactions. Moreover, as we and others have pointed out, the indirect methods used in the studies to date are insufficient
to conclusively demonstrate a direct physical interaction
between these two adhesion receptors (Cepek et al., 1994
;
Erle, 1995
; Karecla et al., 1995
; Takeichi, 1995
). For example, it is clear that antibodies to one integrin (e.g.,
V
3)
can cause specific transdominant inhibition of the function
of another integrin (e.g.,
5
1; Blystone et al., 1995
; Díaz-González et al., 1996
) or could result in steric hindrance of
an adjacent receptor. Furthermore, expression of an exogenous gene in a transfected cell can have profound effects
on the surface expression of a variety of other proteins
(Marks et al., 1996
), and the transfection of constructs containing integrin
1 tails into fibroblasts alters the function of endogenous integrins (LaFlamme et al., 1994
). Such effects can lead to erroneous conclusions about the interactions that are directly involved in adhesion.
E
7 on intraepithelial lymphocytes with its receptor on epithelial cells is likely to be
crucial for the normal development, function, and/or retention of lymphocytes in the epithelium (Cepek et al.,
1993
; Erle, 1995
), we sought to determine if
E
7 and
E-cadherin are directly interacting counter receptors. In
addition, we investigated whether this interaction is specific among cadherins and whether it can be regulated
through alterations in
E
7 avidity.
Materials and Methods
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
), HECD-1 (anti-E-cadherin, IgG1; Shimoyama et al., 1989b), NCC-CAD299 (anti-P-cadherin, IgG1; Shimoyama et al., 1989b), HML-1 (anti-
E
7, IgG2a; Cerf-Bensussan et al., 1987
), BerACT-8 (anti-
E
7, IgG1; Kruschwitz et al., 1991
),
E7-1 (anti-
E
7, IgG2a; Russell et al., 1994
),
E7-2 (anti-
E
7, IgG1;
Russell et al., 1994
),
E7-3 (anti-
E
7, IgG1; Russell et al., 1994
), D6.21
(anti-
L
2, IgG1; Cepek et al., 1993
), TS1/22 (anti-
L
2, IgG1; Sanchez-Madrid et al., 1982
), TS1/18 (anti-
2, IgG1; Sanchez-Madrid et al., 1983
),
ACT-1 (anti-
4
7, IgG1; Lazarovits et al., 1984
), B5G10 (anti-
4, IgG1;
Hemler et al., 1987
), 4B4 (anti-
1, IgG1; Morimoto et al., 1985
), RR1/1
(anti-ICAM-1, IgG1; Rothlein et al., 1986
), SPVT3b (anti-CD3
, IgG2a;
Spits et al., 1983
), OKT4 (anti-CD4, IgG2b; obtained from American
Type Culture Collection [ATCC], Rockville, MD), OKT8 (anti-CD8
,
IgG2a; ATCC), W6/32 (anti-MHCI, IgG2a; Barnstable et al., 1978
), TCR-
1
(anti-TCR-
, IgG1; Band et al., 1987
), P3 (control IgG1; Kohler and Milstein, 1975
), RPC5.4 (control IgG2a; Mohit and Fan, 1971
).
;
Russell et al., 1996
). The iIEL were stimulated with PHA-P (Difco Laboratories Inc., Detroit, MI) and irradiated feeder cells (80% PBMC and
20% JY lymphoblastoid cells) in 2 nM IL-2, 4% (vol/vol) heat-inactivated
FBS (Hyclone Laboratories Inc., Logan, UT), 50 µM 2-mercaptoethanol,
and Yssel's medium at 10% CO2 (Russell et al., 1996
). The iIEL lines 496 and 194 were maintained by periodic restimulation as described and were
used in adhesion assays after 2-8 wk. At the time of assay, IEL496 was
100% CD3+, 90% CD8+, 10% CD4+, 95%
L
2+ (MFI ~400), 97%
E
7+ (MFI ~700), and IEL194 was 100% CD3+, 60% CD8+, 40% CD4+,
95%
L
2+ (MFI ~500), 80%
E
7+ (MFI ~600) by FACS® analysis. Both
lines maintained expression of
E
7 without addition of exogenous TGF-
1.
4
7 integrin (JY
), was kindly provided by Dr. Martin Hemler (Dana-Farber Cancer Institute, Boston, MA; Chan et al., 1992
) and was maintained in 10% (vol/vol) heat-inactivated FBS (Hyclone Laboratories Inc.),
RPMI-1640 (GIBCO BRL, Gaithersburg, MD) at 37°C, and 5% CO2. Human embryonic kidney HEK293 cells (obtained from ATCC) were maintained in 10% (vol/vol) heat-inactivated FBS (Hyclone Laboratories Inc.)
and DME Medium (GIBCO BRL) at 37°C in 10% CO2. COS-7 cells (obtained from ATCC) were grown in 10% (vol/vol) NuSerum (Collaborative Research, Inc., Waltham, MA), 10 mM Hepes, 2 mM l-glutamine,
and DMEM (GIBCO BRL) at 10% CO2. Human breast epithelial
16E6.A5 cells were maintained as described (Cepek et al., 1993
).
Ngo MI
cohesive end, the final five codons of the human E-cadherin extracellular region, and a 3
XhoI cohesive end was produced by annealing the complimentary oligonucleotides JON1 (5
-CCGGCGTCTGTAGGAAGC-3
)
and JON2 (5
-TCGAGCTTCCTACAGACG-3
). This adapter was then
ligated to the 3
-end of an EcoRV-NgoMI fragment encoding the rest of
the extracellular region of human E-cadherin derived from the plasmid
pERF-1 (kindly provided by Dr. David Rimm, Yale University, New Haven, CT; Rimm and Morrow, 1994
). After removal of excess adapters by
centrifugation through a Centricon-100 filter (Amicon Corp., Danvers,
MA), the resulting EcoRV-XhoI fragment was introduced inframe, upstream of coding for the hinge and Fc region of human IgG1 in a derivative of pCDM8 (pCDM8/Fc; Chen and Nelson, 1996
) also cleaved with
EcoRV and XhoI. The sequence of the junctional region is shown in Fig. 1 a.
To confirm the integrity of the construct, the nucleotide sequence of the
junctional region of the E-cadherin-Fc construct was determined by double-stranded sequencing using the Sequenase kit (United States Biochemical Corp., Cleveland, OH) according to the manufacturer's protocol. Finally, the E-cadherin-Fc cDNA was excised from pCDM8 using EcoRV
and NotI and ligated into the expression vector pCEP4 (Invitrogen Corp.,
Carlsbad, CA) cleaved with PvuII and NotI.
View larger version (31K):
[in a new window]
Fig. 1.
Structure of soluble recombinant E-cadherin-Fc and
truncated E
7. (a) Structure of the human E-cadherin-Fc fusion
protein. The sequence of the extracellular juxtamembrane region
of wild-type E-cadherin and the alterations resulting from fusion
with the human Fc region are shown. Regions corresponding to
the Fc portion are shown in bold. The corresponding sequences
in P-cadherin, tgc cct gga ccc tgg aaa (encoding CPGPWK), become tgc cct gga ctc gag ctc (encoding CPGLEL) in the P-cadherin-Fc fusion protein. (b) Structure of soluble truncated
E
7.
In the
E chain, the EF hand-like repeats are labeled I to VII, the
extra "X" domain is shown in black, and the A domain in grey. In
the
7 chain, the
integrin conserved region that may resemble
an A domain (Lee et al., 1995
) is shown in grey, and the cysteine-rich repeats are hatched. The transmembrane and cytoplasmic regions that were removed from each chain are shown as dotted
lines. The change made in the cDNA sequence of each chain to
introduce a stop codon immediately before the transmembrane
region is shown in bold.
P-Cadherin-Fc.
An adapter fragment encoding the final 93 codons of
the human P-cadherin extracellular region was generated by PCR from a human P-cadherin cDNA in pBR322 (kindly provided by Dr. S. Hirohashi, National Cancer Center Research Institute, Tokyo, Japan; Shimoyama et
al., 1989b) using the primers JON4 (5-GGCGTGCCACCTACCTTATCAT-3
) and JON5 (5
-TTTTTTCTCGAGTCCAGGGCAGGTTTCGAC-3
) and cloned plaque-forming unit (PFU) polymerase (Stratagene, La Jolla, CA) according to the manufacturer's recommendations, with 25 cycles of 94°C 1 min/55°C 1 min/72°C 1 min. After digestion with BsaBI
and XhoI, this adapter was ligated to the 3
-end of a HindIII-BsaBI fragment encoding the rest of the extracellular region of human P-cadherin.
After digestion of the ligation products with HindIII and XhoI and purification of the required fragment by Agarose gel electrophoresis and USBioclean (United States Biochemical Corp.), the resulting HindIII-XhoI
fragment was introduced into pCDM8/Fc cleaved with HindIII and XhoI.
The sequence of the junctional region is indicated in the legend of Fig. 1 a.
After sequencing as described above, the P-cadherin-Fc cDNA was excised with HindIII and NotI, and inserted into pCEP4 cleaved with the
same enzymes.
Construction of E and
7 Expression Vectors
Full-Length E.
A full-length open reading frame encoding human
E was
generated from cDNA clones described previously (Shaw et al., 1994
). The final construct included the HindIII-NsiI fragment of clone 38, the
NsiI-BsaBI fragment of clone 2-54, the BsaBI-AccI fragment of clone 1-39A,
the AccI-BglII fragment of clone 2-54, and the BglII-XhoI fragment of
clone 3-15. The full-length cDNA was introduced into the XhoI site of
the expression vector pSR
-neo (Takebe et al., 1988
) to produce pSR
-neo/
E.
Truncated E.
PCR was used to introduce a stop codon and HindIII site
immediately upstream of the transmembrane region of human
E cDNA (see Fig. 1 b). PCR with the primers 5
-AAGAATGGCATTCAGTGAGC-3
and 5
-GGGAAGGTTGATGATAGGCTAAGAATGGTAC-3
amplified a product that was subsequently cleaved with BglII and HindIII
to generate a 180-bp fragment from the end of the
E extracellular region.
This fragment was then introduced into the BglII site of the full-length
E
cDNA to generate an open reading frame for the entire extracellular portion of human
E. After sequencing as described above, the truncated
E
cDNA was introduced in either the sense or antisense direction into the HindIII site of the expression vector pAPRM8 (Wong and Farrell, 1991
)
to produce pAPRM8/t
Es and pAPRM8/t
Eas.
Truncated 7.
A full-length human
7 cDNA was derived by ligation of
the XhoI-EcoRI fragment from a partial
7 cDNA (kindly provided by Dr. G.W. Krissansen, University of Auckland, New Zealand; Yuan et al.,
1990
) and the NotI-XhoI fragment of another partial
7 cDNA cloned
from an IEL cDNA library (Shaw et al., 1994
). To introduce a stop codon
and EcoRI site immediately upstream of the transmembrane region of human
7 cDNA, PCR with the primers 5
-TCGCTGCCAATGTGGAGTATG-3
and 5
-GGGGAATTCACAATGGCCTACGTGTGGTCTGC-3
was carried out. The product obtained was subsequently cleaved with
BsmI and EcoRI to generate a 400-bp fragment from the end of the
7 extracellular region. This fragment was then introduced into the BsmI and
EcoRI sites of pBluescript containing the
7 cDNA to generate an open
reading frame for the entire extracellular portion of human
7 (see Fig. 1
b). After sequencing as described above, the truncated
7 cDNA was excised from pBluescript with EcoRI and introduced in either the sense or
antisense direction into the EcoRI site of the expression vector pAPRM8
to produce pAPRM8/t
7s and pAPRM8/t
7as.
Production of Cadherin-Fc Proteins
HEK293 cells (106 cells per 75-cm2 flask) were stably transfected with 25 µg plasmid DNA using the Mammalian transfection kit (Stratagene Inc.). After growth for 24 h in nonselective medium the cells were transferred to 96-well tissue culture plates and incubated in selective medium containing 300 µg/ml hygromycin B. After 15 d, supernatants from wells containing resistant colonies were assayed for fusion proteins by ELISA.
To produce the cadherin-Fc proteins, transfected cells were grown in
triple-layer 500-cm2 flasks (Nunc, Roskilde, Denmark) in 10% (vol/vol)
Ultralow Ig FBS (GIBCO BRL), 300 µg/ml hygromycin B, and DMEM.
After 5-10 d of culture, the medium was harvested and filtered through a 0.2-µm membrane. The E- and P-cadherin-Fc fusion proteins were then
purified on separate, previously unused GammaBind G-Sepharose columns (Pharmacia Biotech Sverige, Uppsala, Sweden). The columns were
washed with TBS and 1 mM CaCl2, pH 7.4, and then eluted with 0.2 M glycine and 1 mM CaCl2, pH 2.3. Fractions containing purified fusion proteins were dialyzed into TBS and 1 mM CaCl2, pH 7.4 and then stored at
20°C. The purity of fusion protein was assessed by SDS-PAGE and
Coomassie blue staining, and the concentration was determined by Bradford assay using BSA as a standard (Bio-Rad Labs., Hercules, CA).
Production of Soluble 35S-labeled Recombinant
E
7 Integrin
Soluble recombinant E
7 was produced by COS-7 cells after transient
transfection using DEAE-dextran (Coligan et al., 1994
, Unit 10-14) with
the plasmids pAPRM8/t
Es and pAPRM8/t
7s. Control transfections
were carried out with the antisense constructs pAPRM8/t
Eas and
pAPRM8/t
7as. After incubation for 48 h in complete medium, the cells
were washed once with PBS and then 1 mCi 35S-Express (Dupont-NEN,
Boston, MA) in 6 ml 10% (vol/vol), dialyzed FBS, 5% DMEM, 85% methionine- and cysteine-free DMEM (GIBCO BRL), 10 mM Hepes, and 2 mM l-glutamine was added. After incubation at 37°C for 24 h, the
medium, containing labeled secreted proteins, was filtered through a 0.2-µm membrane.
Generation of JY-
E and JY
-Vector Cell Lines
To produce cells expressing cell surface E
7, JY
cells that express endogenous
7 integrin chain were transfected by electroporation with 4 µg
pSR
-neo/
E or with the pSR
-neo vector alone as a control. Then transfected cells were selected by culture in 0.5 mg/ml G418. To generate the
JY
-
E line,
E
7-expressing transfectants were isolated by positive selection with the anti-
E
7 mAb BerACT-8 on magnetic goat anti-mouse immunoglobulin dynabeads according to the manufacturer's recommendations (Dynal A.S., Oslo, Norway) and by flow cytometric sorting. FACS®
was carried out as previously described (Parker et al., 1990
). The clone
J6.7 was finally isolated by limiting dilution. The
E
7 expressed on JY
-
E cells was indistinguishable from that on IEL when immunoprecipitated
from 125I surface-labeled cells with the anti-
E
7 mAb, HML-1 (not shown).
Adhesion Assays
Unless otherwise stated, the wells of Linbro 96-well microtiter plates (ICN Flow Laboratories, Horsham, MA) were coated with human IgG1 Fc-containing proteins in 50 µl/well TBS and 1 mM CaCl2, pH 7.4, for 18 h at 4°C. The wells were subsequently washed twice with 20 mM Hepes, 137 mM NaCl, and 3 mM KCl, pH 7.4 (HBS), with 1 mM CaCl2, and was then blocked with 1% BSA (Calbiochem-Novabiochem Corp.), HBS, and 1 mM CaCl2 for 2 h at room temperature. In assays in which the effects of divalent cations were assessed, coating and blocking was carried out in HBS and 10 mM EDTA. In assays in which adhesion to P- and E-cadherin-Fc was compared, the wells were coated with 1 µg/well goat anti-human IgG polyclonal antibody (Zymed, San Francisco, CA) in 100 µl TBS, pH 7.4, and blocked as described above before addition of Fc fusion proteins.
IEL or transfected JY cells were labeled with BCECF-AM (Molecular
Probes, Eugene, OR) as previously described (Cepek et al., 1993
). During
labeling of JY
cells, 10% (vol/vol) heat-inactivated normal human serum
was included to block Fc receptors. Adhesion assays were carried out in
0.1% BSA and HBS with combinations of MnCl2, MgCl2, CaCl2, or 1 mM
EGTA, as indicated (see text). In antibody blocking experiments, cells or
wells were preincubated with mAbs for 10 min at 4°C as described in the
text. For cell activation experiments, cells were preincubated with antibodies or 50 ng/ml PMA at 4°C for 15 min. Adhesion assays were carried out as described previously (Cepek et al., 1993
) with the following modifications. Labeled cells were brought into contact with the microtiter plate
wells by centrifugation at 60 g for 2 min (IEL) or 1 min (JY
). After incubation at 37°C for 10 min, nonadherent cells were removed by washing
with 1 mM MnCl2, 1 mM MgCl2, 1 mM CaCl2, and HBS at 37°C unless the
effect of divalent cations was being assessed, in which case HBS alone was
used. Since in these assays the fluorescence of input cells was quenched to
some degree by the presence of adhesion buffer, but the percent bound
was determined after removing the buffer, some apparent readings of >100% are obtained.
Homophilic adhesion assays were carried out as described above with
the following modifications. 16E6.A5 cells were released from culture
dishes using 0.02% (wt/vol) trypsin, 2 mM CaCl2, and HBS to minimize
proteolysis of cadherins. After adding 2 vol of 0.04% (wt/vol) soy bean
trypsin inhibitor, HBS, and washing twice with HBS, the cells were resuspended in 0.1% BSA, HBS, and 1 mM CaCl2 and allowed to settle onto
the microtiter plate wells for 10 min at 4°C. After incubation at 37°C for 30 min, and washing twice with HBS and 1 mM CaCl2, the percentage of
bound cells was determined using a fluorogenic assay of endogenous cellular phosphatase activity (Tolosa and Shaw, 1996).
Surface Labeling of iIEL with 125I
Cultured iIEL (4 × 107) were isolated by centrifugation on Ficoll-Paque
(Pharmacia Biotech Sverige) and subjected to cell surface labeling with
2 mCi Na125I (Dupont-NEN) using the lactoperoxidase method (Coligan
et al., 1994, Unit 8-11). The labeled cells were then lysed in 0.5% Triton X-100, TBS, and 4 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride for 4 h at 4°C. Insoluble material was removed by centrifugation at
12,000 g for 20 min.
Immunoadsorption
Batches of 125I-labeled IEL lysate or 35S-labeled transfected COS-7 medium were supplemented divalent cations (see text) and precleared twice with 0.4% (vol/vol) normal rabbit serum, 1.5% (vol/vol) protein A-Sepharose (Pharmacia), and 1.5% (wt/vol) Pansorbin (Calbiochem-Novabiochem Corp.) for a total of 24 h at 4°C. Subsequently, aliquots were incubated with mouse mAbs or human IgG1 Fc-containing proteins for 3 h at 4°C. Then, 10 µl protein A-Sepharose resin was added to each tube, and the incubation at 4°C was continued for a further 3 h. For immunoprecipitations using mouse IgG1 mAbs, protein A-Sepharose precoated with rabbit anti-mouse immunoglobulin polyclonal antibody (Cappel, West Chester, PA) was used. Then the immobilized complexes were washed six times with TBS containing the same concentration of divalent cations used during the adsorption steps. For immunoadsorption from IEL lysates, 0.1% Triton X-100 was also present during washing. Proteins bound to the resin were eluted by boiling in 3% (wt/vol) SDS, 10% (vol/vol) glycerol, 50% (wt/vol) urea, and 60 mM Tris, pH 7, before analysis by SDS-PAGE.
SDS-PAGE
SDS-PAGE on 7.5% (wt/vol) polyacrylamide gels (Protogel; National Diagnostics, Atlanta, GA) was carried out as described (Coligan et al., 1994,
Unit 8-4). Samples were reduced by the inclusion of 25 mM dithiothreitol.
Radiolabeled proteins were visualized by autoradiography using Biomax
MR and MS film (Kodak, Rochester, NY) and quantitated using phosphorimaging and the ImageQuant package (Molecular Dynamics Inc., Sunnyvale, CA).
Statistical Analysis
P values testing the hypothesis that two populations had equal means were calculated using a two-tailed Welch t test (which assumes the populations follow a normal distribution, but not that they have equal variance). Also, a nonparametric two-tailed Mann-Whitney test (which does not assume normal distributions) was used to test whether the population medians were equal. Calculations were carried out using the Instat package (Graphpad Software, San Diego, CA).
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Production of Human E- and P-cadherin-Fc Fusion Proteins
Constructs encoding the extracellular portion of either human E-cadherin or P-cadherin were linked in frame to a
construct encoding the Fc region of human IgG1 (including the hinge, CH2, and CH3 domains). Transfection of
HEK293 cells and selection with hygromycin B led to the
generation of stable lines expressing soluble E-cadherin-
Fc or P-cadherin-Fc fusion proteins. The cadherin-Fc fusion proteins were expected to be dimeric due to the presence of disulfide bonds in the Fc region (Fig. 1 a), possibly
similar to cadherin dimers on the cell surface (Shapiro et
al., 1995; Nagar et al., 1996
).
After purification on protein G-Sepharose, SDS-PAGE in reducing conditions revealed the presence of proteins of the expected size for both E- and P-cadherin-Fc monomers (~120 kD, Fig. 2). Minor species of 135 or 130 kD were also present in preparations of the E- and P-cadherin fusion proteins, respectively. Since cadherins are synthesized as proproteins and the sizes of these bands match those predicted for the immature forms, it is likely that a small proportion of partially processed procadherin is present in each case. In nonreducing conditions both fusion proteins migrate at ~240 kD (Fig. 2) as expected for dimeric fusion proteins linked through disulfide bonds in the hinge of the Fc region. A small proportion of monomeric cadherin fusion protein is also present in each case. In an ELISA, E-cadherin-Fc but not P-cadherin-Fc is recognized by the antihuman E-cadherin mAb E4.6, and P-cadherin-Fc but not E-cadherin-Fc is recognized by the antihuman P-cadherin mAb NCC-CAD299, further confirming the identity of the two recombinant products (not shown).
|
E-Cadherin-Fc Supports Adhesion of IEL
Cell surface E-cadherin has been proposed to be a ligand
for the IEL integrin E
7 (Cepek et al., 1994
; Karecla et
al., 1995
). To test the capacity of the cadherin-Fc fusions
to support adhesion of IEL, we immobilized the proteins
via antihuman IgG antibody on polystyrene microtiter
plate wells. Binding of IEL to wells coated with subnanogram quantities of E-cadherin-Fc could be detected in the
presence of 1 mM MnCl2, 1 mM MgCl2, and 1 mM CaCl2
(Fig. 3 a). At 1 ng/well, ~40% of the IEL adhered, and
maximal adhesion occurred at 10 ng/well of E-cadherin-
Fc. Thus, a dose-dependent adhesion of IEL to the E-cadherin fusion protein was clear. Indeed, for IEL expressing
similar levels of
E
7 and
L
2, adhesion to human E-cadherin-Fc was similar or greater than that seen to human ICAM-1-Fc (data not shown and see Fig. 3 b). In contrast,
adhesion to P-cadherin-Fc required >100-fold more fusion protein and did not reach 100% at any coating concentration. 40% adhesion was seen to P-cadherin-Fc at
500 ng/well. No adhesion could be detected to wells coated
with 500 ng/well human IgG1 (Fig. 3 a).
|
Adhesion of PHA-activated peripheral blood lymphocytes (PBL) to E-cadherin-Fc could not be detected (data
not shown). This is consistent with the fact that only 2- 5%
of PBL express E
7 (Cerf-Bensussan et al., 1987
). The E-
and P-cadherin-Fc proteins support similar levels of adhesion of 16E6.A5 epithelial cells (Cepek et al., 1993
) that
express similar levels of E- and P-cadherin, suggesting that
both fusion proteins are equally able to support cadherin-mediated homophilic adhesion (Fig. 3 c).
Antibodies to E
7 and E-Cadherin Block IEL
Adhesion to E-Cadherin-Fc
To determine if lymphocyte E
7 was responsible for adhesion of IEL to E-cadherin-Fc, we attempted to block
the interaction with mAbs to human lymphocyte surface
integrins. The binding of IEL to E-cadherin-Fc coated directly on plastic was completely blocked by anti-
E
7
mAbs HML-1, BerACT8,
E7-1, and
E7-2 and by the
anti-E-cadherin mAb E4.6, while adhesion to ICAM-1-Fc
was not affected (Fig. 3 b and data not shown). In contrast,
the anti-
2 integrin mAb, TS1/18, the anti-
L
2 mAbs,
TS1/22 and D6.21, and the anti-ICAM-1 mAb RR1/1 all
prevented IEL adhesion to ICAM-1-Fc, but not to E-cadherin-Fc. The anti-
1 integrin mAb, 4B4, blocked adhesion of IEL to human fibronectin (not shown) but not to
E-cadherin-Fc or ICAM-1-Fc. A blocking mAb to
4
7
(ACT-1) also did not inhibit IEL adhesion to E-cadherin-
Fc or ICAM-1-Fc (Fig. 3 b). Adhesion of IEL to P-cadherin-Fc was also completely blocked by the anti-
E
7 mAb,
E7-2, but was unaffected by the anti-
L
2 mAb,
D6.21 (data not shown). Thus, adhesion of IEL to E- or
P-cadherin-Fc involves
E
7, and adhesion to ICAM-1-Fc
involves
L
2.
JY Cells Transfected with E Adhere to E-Cadherin-Fc
To confirm that expression of exogenous E
7 would confer upon a cell the ability to adhere to E-cadherin-Fc, adhesion assays were performed with JY
cells transfected
with a full-length human
E-encoding cDNA construct.
JY
cells, or JY
cells transfected with vector only (JY
-vector), expressed the
4
7 integrin, very little
1 integrin,
but no
E
7 by FACS®. JY
transfected with
E (JY
-
E)
had levels of
4 and
1 integrins similar to untransfected or
JY
-vector cells, but now also expressed
E
7 (99%
E
7+,
MFI ~80). Both JY
-vector and JY
-
E transfectants also
expressed similar levels of
L
2 (Fig. 4 a).
|
In the presence of manganese, JY cells transfected with
vector alone did not adhere to E-cadherin-Fc coated directly on microtiter plate wells at any concentration tested.
However, under the washing conditions used, 40% of JY
-
E cells adhered to 600 ng/well E-cadherin-Fc (Fig. 4 b).
In contrast, both cell lines adhered to ICAM-1-Fc (not
shown) while neither adhered to the control Fc protein human IgG1 (Fig. 4 b). The higher percent binding observed
for IEL to E-cadherin-Fc when compared with transfected JY
cells probably reflects the higher surface expression of
E
7 on IEL (on IEL, mean fluorescence intensity [MFI] ~600; on JY
-
E, MFI ~80).
The adhesion of JY-
E cells to E-cadherin-Fc was
blocked from 40% cells bound to <5% (the level seen for
JY
-vector cells) by mAbs to
E
7 (
E7-2 and BerACT8)
and E-cadherin-Fc (E4.6), but not by blocking mAbs to
L
2,
4
7,
1, or
2 integrins or to ICAM-1 (Fig. 4 c). In
contrast, the adhesion of JY
-
E cells to ICAM-1-Fc was
blocked by antibodies to
L
2 (TS1/22 and D6.21),
2 integrins (TS1/18), and ICAM-1 (RR1/1), but not by antibodies to
E
7,
4
7, or
1 integrins (data not shown). These
mAb-blocking experiments further confirm that the cell
adhesion measured is mediated by
E
7 binding to E-cadherin-Fc, or by
L
2 binding to ICAM-1-Fc.
E-Cadherin-Fc Binds Directly to 125I-labeled E
7 from
a Lysate of IEL
To demonstrate that E-cadherin molecules interact directly with E
7 molecules, the proteins that bound to E-cadherin-Fc from a 125I-labeled IEL lysate in the presence of
1 mM MnCl2, 1 mM MgCl2, and 1 mM CaCl2 were analyzed. Since the fusion protein contains a human IgG1 Fc
region that binds to protein A, a standard immunoadsorption procedure was used. Proteins of the expected size for
E
7 (175, 135, and 110 kD) and
L
2 (160 and 100 kD)
were visible on SDS-PAGE in nonreducing conditions after immunoprecipitation with anti-
E
7 and anti-
L
2
mAbs, respectively (Fig. 5). Remarkably, E-cadherin-Fc bound to radiolabeled species identical in size and relative
intensity to those seen with the anti-
E
7 mAb (Fig. 5,
compare lanes 1 and 3), but distinct from those seen with
the anti-
L
2 mAb (Fig. 5, compare lanes 3 and 8). In contrast, no radiolabeled species were detected binding to the
P-cadherin-Fc or IgG1 proteins. After immunoadsorption
with the ICAM-1-Fc, proteins of the expected size for
L
2 could be visualized only on overexposed phosphorimages (not shown). While the same number of cell equivalents were used in each of the human IgG1 containing
protein-binding experiments, ~40-fold fewer cell equivalents were required to immunoadsorb an equal amount of
radiolabeled
E
7 with the anti-
E
7 mAb compared with
E-cadherin-Fc (see legend to Fig. 5). This is consistent with the expected lower affinity of a cell adhesion receptor
interaction compared with that of an antibody-antigen interaction.
|
To further confirm that the proteins bound by anti-E
7
and the E-cadherin fusion protein were the same, batches
of lysate were precleared with mAbs to
E
7 (
E7-1),
L
2
(TS1/22), or the control mAbs RPC5.4 and P3, before exposure to E-cadherin-Fc (Fig. 5). Prior immunodepletion
with the anti-
E
7 mAb prevented the subsequent binding
of material from the IEL lysate to E-cadherin-Fc. Preclearing with the other mAbs had no such effect. Thus, the predominant 125I-labeled protein from the IEL cell surface
that interacts with E-cadherin-Fc in these conditions is
E
7.
E-Cadherin-Fc Binds to Soluble Recombinant E
7
To produce a soluble form of E
7, stop codons were introduced immediately upstream of the transmembrane
coding regions in the cDNAs encoding the human
E and
7 proteins (see Fig. 1 b, and Materials and Methods). Supernatant containing 35S-labeled soluble
E
7 was produced by transient transfection of COS-7 cells followed by
metabolic labeling. Proteins in the supernatant were then
subjected to immunoprecipitation with a panel of anti-
E
7 antibodies that recognize at least three distinct
E
epitopes (Russell et al., 1994
). In the medium from cells
transfected with plasmids encoding truncated
E and
7 in
the sense orientation, all the anti-
E
7 mAbs tested (
E7-1,
E7-2,
E7-3, HML-1, and BerACT8), but not a control
mAb (TCR-
1), precipitated two major bands of 140 and
105 kD on reducing SDS-PAGE. On nonreducing SDS-PAGE, two bands of 170 and 100 kD were observed (Fig.
6 and data not shown). These sizes correspond to those expected for the truncated
E and
7 chains, respectively. No
such proteins were precipitated from the medium of COS-7
cells transfected with constructs containing truncated
E
and
7 cDNAs in the antisense orientation. These results
confirm the secretion of a soluble form of
E
7 that retains all of the epitopes of cell surface
E
7 that were tested.
|
We then sought to demonstrate binding of recombinant
soluble E
7 integrin to the human E-cadherin-Fc fusion.
Both the anti-
E
7 mAb
E7-1 and the E-cadherin-Fc
protein were able to bind proteins of the expected size for
soluble
E
7 (Fig. 6). No detectable soluble
E
7 bound to
the control mAb RPC5.4, or the fusion proteins P-cadherin-Fc and ICAM-1-Fc. Furthermore, these proteins
were not bound by E-cadherin-Fc in medium from COS-7
cells transfected with the antisense
E and
7 constructs
(Fig. 6). Thus, E-cadherin-Fc interacts with soluble
E
7
in the absence of other cellular proteins that are present in
the IEL lysate used previously.
The Avidity of Cell Surface E
7 Is Regulated
Although the avidity of many integrins is regulated from
within the cell, similar regulation of E
7 has not previously been reported. Furthermore, the structure of the
E
chain is unique among integrins due to the presence of an
extra domain in the extracellular region, membrane distal
of the A domain, that is cleaved in the mature protein (the
"X" domain, see Fig. 1 b; Shaw et al., 1994
). This raises the
possibility that regulation of the avidity of
E
7 due to
conformational changes could be different from other integrins. Therefore, studies were performed to investigate the regulation of
E
7 avidity on JY
-
E cells and IEL, and
to compare it to the well studied
L
2 integrin.
JY cells transfected with
E adhered poorly to E-cadherin-Fc in the presence of 1 mM MgCl2 and 1 mM CaCl2
in the absence of manganese. The addition of 1 mM manganese caused a 12-fold or greater rise in the adhesion of
JY
-
E cells to E-cadherin-Fc. PMA stimulation of JY
-
E
also caused a sevenfold increase in binding to E-cadherin-Fc
(Fig. 7 a). Adhesion of JY
-
E cells to ICAM-1-Fc was
also enhanced by manganese or PMA, suggesting that
both
E
7 and
L
2 can be activated by similar means.
Relative to the effect of manganese, PMA was better able
to stimulate adhesion of JY
-
E to ICAM-1-Fc than to
E-cadherin-Fc. The reason for this difference is unclear,
but valid comparisons are difficult since
L
2 is expressed
at a higher level than
E
7 on JY
-
E cells (Fig. 3 a).
|
In contrast to JY transfectants, >90% of IEL adhered
to E-cadherin-Fc in 1 mM MgCl2 and 1 mM CaCl2 and the
addition of manganese or PMA had little enhancing effect
on adhesion (not shown). Thus IEL, which are maintained
in culture in the presence of IL-2 with periodic PHA-stimulation, have constitutively active
E
7 in these conditions.
To study regulation of
E
7 on IEL, we carried out assays
in 0.05 mM MgCl2 and 1 mM CaCl2. In these conditions of limiting MgCl2, just under 40% of IEL adhere to E-cadherin-Fc (Fig. 7 b). The addition of manganese or PMA
causes an almost twofold increase in the percentage of
IEL adhering to E-cadherin-Fc. Moreover, cross-linking
of the TCR complex with a mouse anti-CD3 mAb, followed by anti-mouse immunoglobulin polyclonal antibody, also causes a significant increase in IEL adhesion to
E-cadherin-Fc (P < 0.0001, by Welch's alternate t test, see
Fig. 7 b). Similar treatment of IEL with antibodies to
CD8
or MHC class I had no such effect. Cross-linking of
the TCR on IEL also increases the avidity of
L
2 for
ICAM-1, and of
1-integrins for fibronectin (data not
shown), as has been reported for other T cells (Dustin and
Springer, 1989
; Shimizu et al., 1990
; van Kooyk et al., 1989
).
It is known that calcium is required for the rigidification
of the structure of E-cadherin (Nagar et al., 1996; Pokutta
et al., 1994
) and for E-cadherin-mediated homotypic cell
to cell adhesion (Hyafil et al., 1981
; Yoshida and Takeichi,
1982
). However, in the presence of magnesium, even when
calcium has been depleted with EGTA, both JY
-
E cells
(Fig. 7 a) and IEL (not shown) adhere to E-cadherin-Fc.
Thus, the heterophilic interaction of E-cadherin appears
to be independent of calcium, at least in this system. It is
unlikely that this is due to restriction of conformational changes within plate-bound E-cadherin-Fc, since similar
results were found whether the fusion protein was immobilized directly on plastic or through its Fc region on antiimmunoglobulin antibodies (not shown). In contrast, the
adhesion of E-cadherin-expressing 16E6.A5 epithelial
cells to E-cadherin-Fc is dependent on calcium (not
shown). Also, since mouse E-cadherin does not have appreciable affinity for magnesium (Hyafil et al., 1981
), and
magnesium cannot support homophilic adhesion to E-cadherin-Fc (not shown), it is unlikely that magnesium substitutes for calcium in the cadherin structure. The results also
suggest that calcium is not required for the function of
E
7 integrin. This is also the case for
L
2 binding to
ICAM-1 (Fig. 7 a; Shimizu and Mobley, 1993
; Stewart et
al., 1996
).
In summary, E
7, like
L
2, can exist in both high and
low avidity states on the cell surface. In common with
L
2-mediated adhesion to ICAM-1, treatment of cells expressing
E
7 in a low avidity state with manganese, with
PMA, with anti-CD3 antibodies, or by removing calcium
and elevating magnesium, leads to increased adhesion to
E-cadherin-Fc.
The Direct Binding of E-Cadherin-Fc to Solubilized
E
7 Is Modulated by Divalent Cations
We wished to determine if the effects of divalent cations
on cell adhesion to E-cadherin-Fc were due to a direct influence on the binding of E
7 molecules to E-cadherin-
Fc molecules. The binding of E-cadherin-Fc to 125I-labeled
E
7 in an IEL lysate was analyzed by immunoadsorption as described in Fig. 5, but in the presence of various concentrations of divalent cations (Fig. 8). No binding of
E-cadherin-Fc to
E
7 was detected in the presence of 5 mM
EDTA, confirming the cation dependency of the interaction (Fig. 8 b). Binding of E-cadherin-Fc to
E
7 was clear
in the presence of 1 mM MgCl2 and 1 mM CaCl2, but was
increased eightfold by the addition of 1 mM MnCl2 (Fig. 8
b). Approximately equal quantities of
E
7 were immunoadsorbed by the anti-
E
7 mAb
E7-1 in all conditions,
confirming that the differences seen in binding to the
E-cadherin-Fc fusion protein were not due to dissociation
or degradation of the
E
7 chains (Fig. 8 a). Furthermore,
since cadherins are known to be protease sensitive in the
absence of calcium, we confirmed that an equal quantity of
E-cadherin-Fc protein was present in each condition by
Coomassie Blue staining of the immunoadsorbed material
(Fig. 8 b, ii).
|
Thus, the cation dependency of E
7-mediated cell adhesion to E-cadherin-Fc is paralleled by the cation dependency of
E
7 binding directly to E-cadherin-Fc. Also, the
association of
E with
7 chains is not dependent on calcium or magnesium, as found for mouse
E
7 (Kilshaw
and Murant, 1991
), but in contrast to mouse
4
7, an integrin that requires calcium for heterodimer formation in
similar experiments (Holzmann et al., 1989
).
![]() |
Discussion |
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Here, we demonstrate that an E-cadherin fusion protein
binds directly both to E
7 from solubilized intraepithelial
lymphocytes, and to a soluble recombinant form of
E
7.
Furthermore, purified E-cadherin fusion protein can serve
as a potent substrate for cell adhesion through the
E
7 integrin. Thus, although E-cadherin is classically thought to
be a homophilic adhesion molecule and is unlike known
integrin ligands, it is a direct counter receptor for the
E
7
integrin.
We found that E
7 exhibits selectivity in binding to
cadherins. The adhesion of IEL cells to P-cadherin-Fc requires >100-fold more fusion protein than adhesion to
E-cadherin-Fc. In both cases the adhesion is dependent on
E
7, since it is abolished by antibodies to
E
7. Thus, although human epithelial cells express both E- and P-cadherin, it is likely that E-cadherin is the primary epithelial receptor for
E
7 in vivo. The idea that
E
7 is selective in binding cadherins is consistent with the proposed role of
E
7 in tissue-specific leukocyte adhesion, and with the
finding that IEL do not adhere to all cadherin-expressing
cells. For example, IEL do not adhere to endothelial cells
that possess VE-cadherin (Cepek et al., 1994
).
Regulation of E
7 avidity for its counter receptor has
not previously been reported. An increase in IEL adhesion to epithelial cells in the presence of manganese has
been observed (Cepek et al., 1993
; Karecla et al., 1995
).
However, it is not clear that this change is due to regulation of
E
7 binding to E-cadherin since other receptors,
including
L
2 and ICAM-1, are involved in this complex
cell to cell interaction (Cepek et al., 1993
; Roberts et al.,
1993
). Here, the adhesion of cells expressing both
E
7
and
L
2 to purified E-cadherin-Fc or ICAM-1-Fc allowed us to compare the regulation of
E
7 avidity with
that of
L
2 in a system in which only a single integrin
counter receptor was available in each case. The
L
2 integrin on freshly isolated PBL has low avidity for immobilized ICAM-1 in the presence of 1 mM MgCl2 and 1 mM
CaCl2. However, in the presence of manganese ions PBL
adhere strongly to purified ICAM-1 (Dransfield et al., 1992
). Changes in the concentrations and presence of divalent cations are thought to have a direct effect on the conformation of the integrin at the cell surface, leading to increased ligand binding through an increase in the affinity
of the integrin and/or changes in clustering of the integrin
in the membrane. In addition, signaling from inside the
cell can lead to increased integrin avidity. For example,
PMA stimulation of resting PBL leads to increased adhesion to ICAM-1 through
L
2. This type of regulation may
involve changes in integrin clustering on the cell surface due to alterations in integrin association with the cytoskeleton (Lub et al., 1995
, 1997
; Stewart et al., 1996
). In contrast to resting PBL,
L
2 on a proportion of IL-2/PHA-activated PBL has high avidity for ICAM-1 (Lub et al.,
1997
). Furthermore, the avidity state of integrins transfected into different cell types can vary. For example,
L
2
expressed on K562 cells is in a constitutively inactive state,
while
L
2 expressed on L cells is constitutively active
(Lub et al., 1995
).
We find that in 1 mM MgCl2 and 1 mM CaCl2, E-transfected JY
cells exhibit poor adhesion to both E-cadherin-Fc
and ICAM-1-Fc. Thus, on these cells, both
E
7 and
L
2
exist in a low avidity state. However, these cells can be induced to adhere to both integrin ligands by the addition of
1 mM MnCl2. Since parallel changes in the direct binding
of solubilized
E
7 to E-cadherin-Fc were also observed,
it is likely that this difference in cell adhesiveness is due to
a direct effect of manganese on the conformation of the
E
7 integrin. Thus, changes in extracellular cations can regulate the affinity of
E
7 for its ligand. In contrast, cultured IEL, which are maintained in IL-2 with periodic
stimulation with PHA and feeder cells, adhere avidly to
both E-cadherin-Fc and ICAM-1-Fc in 1 mM MgCl2 and
1 mM CaCl2 even in the absence of manganese. Both
E
7
and
L
2 on IEL appear to be maintained in a constitutively active state, like
L
2 on a proportion of IL-2/PHA-stimulated peripheral blood T cells.
We also show that signaling from inside the cell is able
to increase the avidity of E
7. The addition of the phorbol
ester, PMA, which acts intracellularly to upregulate protein kinase C, leads to increased adhesion of
E-transfected JY
cells to E-cadherin-Fc or ICAM-1-Fc. In the
presence of a suboptimal concentration of magnesium,
PMA also enhanced IEL adhesion to E-cadherin-Fc. The physiological triggers for such changes in lymphocyte integrin avidity in vivo remain unclear. In vitro, chemokines
such as MCP-1, MIP-1
, and RANTES can activate lymphocyte integrins (Campbell et al., 1996
; Carr et al., 1996
;
Lloyd et al., 1996
), and cross-linking of components of the
T cell receptor can boost the adhesion of resting PBL to
ICAM-1 through
L
2 (Dustin and Springer, 1989
; van
Kooyk et al., 1989
) or to fibronectin and laminin through
1-integrins (Shimizu et al., 1990
). Here, we report that
antibody cross-linking of cell surface CD3 is similarly able
to increase IEL adhesion to E-cadherin-Fc, while cross-linking of MHC class I or CD8 receptors had no such effect. Thus, recognition by an IEL of an antigen presented
by an epithelial cell or a professional antigen-presenting
cell expressing E-cadherin (such as a Langerhans cell;
Tang et al., 1993
) could trigger increased adhesion to that cell through an upregulation of
E
7 avidity. This interaction may also provide costimulation to the T cell, since
anti-
E
7 antibodies, in common with anti-
L
2 antibodies, are known to increase T cell proliferation in the presence of suboptimal anti-CD3 (Russell et al., 1994
; Sarnacki
et al., 1992
; Wacholtz et al., 1989
), and ICAM-1 on an antigen-presenting cell can costimulate through
L
2 (Dubey
et al., 1995
). Such events may be important in arresting
lymphocytes within the epithelium when a specific antigen
is recognized. In addition, since IEL contact more than one cell when resident within the epithelium, localized upregulation of
E
7 avidity within an IEL could aid in polarizing lymphocyte interactions toward the relevant antigen-presenting cell.
X-ray crystallography and NMR studies have recently
revealed that cadherin modules adopt a tertiary structure
rather like immunoglobulin domains (Overduin et al.,
1995; Shapiro et al., 1995
; Nagar et al., 1996
). Thus E-cadherin has a structure resembling that of the known cellular
integrin ligands and can now be placed within the family
of immunoglobulin-like, integrin-binding proteins. E-cadherin may share another feature of well-defined integrin ligands; the presence of a solvent-exposed acidic residue
vital for integrin binding (Bergelson and Hemler, 1995
).
Mutation of an aspartate or glutamate residue in the CD-loop region in domain 1 of the immunoglobulin superfamily integrin ligands VCAM-1 (Osborn et al., 1994
; Renz et
al., 1994
; Vonderheide et al., 1994
; Jones et al., 1995
; Wang
et al., 1995
), ICAM-1, (Staunton et al., 1990
; Holness et al.,
1995
), and MadCAM-1 (Briskin et al., 1996
; Viney et al.,
1996
) abolishes integrin binding. The tenth immunoglobulin-like FN III repeat of fibronectin also has an acidic residue within the RGD sequence on the FG-loop that is involved in integrin binding (Main et al., 1992
). In mouse
E-cadherin, the BC loop of domain 1 contains a glutamate
residue required for adhesion of
E
7-expressing lymphocytes to E-cadherin-transfected L cells (Karecla et al., 1996
). Since this residue is conserved in human E-cadherin
and we demonstrate that E-cadherin is a direct counter receptor for
E
7, it is likely that this amino acid contributes
directly to the
E
7-binding site on E-cadherin, in a manner similar to that proposed for other integrin ligands. It is
interesting to note that while three different families of
immunoglobulin-like structures with exposed acidic amino
acids serve as integrin ligands, the face of the module involved appears to be different in each case. It is also intriguing that both E-cadherin and ICAM-1 are likely to be
dimeric on the cell surface (Miller et al., 1995
; Reilly et al.,
1995
; Nagar et al., 1996
), and that both are ligands of integrins that contain an A domain in the
chain (
E
7 and
L
2, respectively). Since it has been proposed that integrin
-chains also contain a ligand-binding A domain (Lee
et al., 1995
; Puzon-McLaughlin and Takada, 1996
), it is
possible that
E
7 and
L
2 actually possess two A domains each. As suggested for
L
2 binding to ICAM-1 (Miller et al., 1995
), it is tempting to speculate that the
binding of dimeric E-cadherin by
E
7 could involve both
these domains.
Although E-cadherin is a calcium-binding molecule, we
find that E-cadherin-Fc binding to E
7 is independent of
calcium. It seems that calcium is not directly involved in
maintenance of the
E
7 binding site on E-cadherin. Studies with recombinant soluble forms of mouse E-cadherin
and Xenopus C-cadherin suggest that homophilic cadherin
interactions do require the presence of calcium (Brieher et
al., 1996
; Tomschy et al., 1996
). Interestingly, in their
NMR studies of the first module of mouse E-cadherin, Overduin et al. (1995)
find that the addition of calcium
leads to a large shift in the orientation of histidine-79
within the HAV motif implicated in homophilic E-cadherin interaction (Blaschuk et al., 1990
; Shapiro et al.,
1995
). In contrast, no such shift is found in any of the residues in or around the BC-loop implicated in
E
7 interaction (Overduin et al., 1995
). Thus it is possible that
changes in extracellular calcium levels (for review see
Maurer et al., 1996
) would differentially regulate E-cadherin binding to
E
7 versus other E-cadherin molecules.
However, since calcium is required to rigidify and extend
E-cadherin (Pokutta et al., 1994
; Nagar et al., 1996
) and to
protect it from proteolysis (Hyafil et al., 1981
; Yoshida and
Takeichi, 1982
), the lack of calcium dependence for
E
7-mediated adhesion to the E-cadherin-Fc fusion protein
may not hold true on the cell surface.
Although the role of E
7 binding to E-cadherin in vivo
has yet to be established, the direct, specific, and regulated
binding of
E
7-expressing cells and of
E
7 itself to the
E-cadherin fusion protein very strongly suggests a physiological function for this interaction. Although there is an
increase in the number of
7-integrin positive iIEL in chimeric mice with a defect in intestinal E-cadherin expression
(Hermiston and Gordon, 1995
), this is perhaps not surprising since the observed disruption of the epithelial layer
and accompanying infectious and inflammatory response
is likely to result in the attraction of many leukocytes into
the intestine. In contrast, recently developed
E knockout mice have reduced numbers of intraepithelial IEL (Parker,
C.M., unpublished results).
The role in vivo of the low but detectable E
7-mediated adhesion of IEL to human P-cadherin is more difficult to assess. The relative abundance of E- and P-cadherin on epithelial cells depends upon the state of cell
differentiation (Hirai et al., 1989a
,b), but this may not be
the sole determinant of
E
7-mediated interactions. On a
single cell type, cadherins can display differential association with the cytoskeleton and thus exist in distinct pools on the cell surface (Salomon et al., 1992
), and E- and
P-cadherin are found in separate complexes on A431 cells
(Johnson et al., 1993
). Cell adhesion through homophilic
cadherin-cadherin interactions requires their association
with the cytoskeleton through intracellular catenins (Nagafuchi and Takeichi, 1988
). However, while E-cadherin lacking its cytoplasmic tail is unable to associate with the
catenins and cannot support strong homophilic adhesion,
it is able to support adhesion of lymphocytes expressing
E
7 (Karecla et al., 1996
). So, since different pools of cadherins may differ in their availability for interaction with
different counter receptors, it is difficult to predict whether
or not a high local concentration of a distinct population of
P-cadherin on the cell surface could contribute to
E
7-mediated adhesion.
Like human, mouse, canine, and Xenopus E-cadherin,
human P-cadherin possesses an acidic residue at the tip of
the BC loop in the first cadherin module (Shimoyama et
al., 1989b). Thus human E- and P-cadherin may share a
similar mode of binding to E
7. In contrast, mouse P-cadherin does not possess an acidic residue at this position
(Nose et al., 1987
), and it has been reported that murine
lymphocytes expressing
E
7 do not adhere to L cells transfected with mouse P-cadherin (Karecla et al., 1996
).
Although this cell to cell adhesion assay is likely to be less
sensitive than the cell to fusion protein assay used here,
these findings further complicate the question of the potential importance of
E
7 binding to P-cadherin. It is possible that in some respects the function of P-cadherin may
differ in the two species, and this may be reflected in the
different expression pattern of P-cadherin in human and
mouse (Shimoyama et al., 1989a,b).
Recently, a second direct heterophilic noncadherin
ligand of a cadherin has been identified. The Listeria surface protein internalin binds to E-cadherin and invasion of
cells expressing E-cadherin by Listeria in vitro is inhibited
by anti-E-cadherin antibodies (Mengaud et al., 1996). Together with studies suggesting that different cadherins can
bind to each other (for example N- and R-cadherin)
(Takeichi, 1995
), it is now clear that the biology of cadherin function is not limited to homophilic interactions, and is more complex than previously imagined.
![]() |
Footnotes |
---|
Address correspondence to Dr. M.B. Brenner, Brigham and Women's Hospital, Smith Building, Room 552, 75 Francis Street, Boston, MA 02115. Tel: (617) 525-1000. FAX: (617) 525-1010.
Received for publication 8 August 1997 and in revised form 10 November 1997.
This work was supported by a Wellcome Trust International Prize Travelling Research Fellowship (J.M.G. Higgins), and grants from the National Institutes of Health (NIH; M.B. Brenner), NIH grant AI01212 (D.A. Mandlebrot), the Crohn's and Colitis Foundation of America and NIH grant DK43351 (G.J. Russell), an NIH R29 First Award GM49342 and a Cancer Research Institute Investigator Award (C.M. Parker), NIH grant GM35527 (W.J. Nelson), and a postdoctoral fellowship from the National Kidney Foundation (Y.-T. Chen).
![]() |
Abbreviations used in this paper |
---|
ICAM, intracellular adhesion molecule; iIEL, intestinal intraepithelial lymphocytes; MadCAM, mucosal addressin cell adhesion molecule; MFI, mean fluorescence intensity; PBL, peripheral blood lymphocytes; TCR, T cell receptor; VCAM, vascular cell adhesion molecule.
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References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Band, H.,
F. Hochstenbach,
J. McLean,
S. Hata,
M.S. Krangel, and
M.B. Brenner.
1987.
Immunochemical proof that a novel rearranging gene encodes the
T cell receptor ![]() |
2. | Barnstable, C.J., W.F. Bodmer, G. Brown, G. Galfre, C. Milstein, A.F. Williams, and A. Ziegler. 1978. Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens-new tools for genetic analysis. Cell. 14: 9-20 |
3. | Ben-Ze'ev, A.. 1997. Cytoskeletal and adhesion proteins as tumor suppressors. Curr. Opin. Cell Biol. 9: 99-108 |
4. | Bergelson, J.M., and M.E. Hemler. 1995. Integrin-ligand binding. Do integrins use a `MIDAS touch' to grasp an Asp? Curr. Biol. 5: 615-617 |
5. | Blaschuk, O.W., R. Sullivan, S. David, and Y. Pouliot. 1990. Identification of a cadherin cell adhesion recognition sequence. Dev. Biol. 139: 227-229 |
6. |
Blystone, S.D.,
F.P. Lindberg,
S.E. LaFlamme, and
E.J. Brown.
1995.
Integrin ![]() ![]() ![]() ![]() ![]() |
7. | Boller, K., D. Vestweber, and R. Kemler. 1985. Cell-adhesion molecule uvomorulin is localized in the intermediate junctions of adult intestinal epithelial cells. J. Cell Biol. 100: 327-332 [Abstract]. |
8. | Brieher, W.M., A.S. Yap, and B.M. Gumbiner. 1996. Lateral dimerization is required for the homophilic binding activity of C-cadherin. J. Cell Biol. 135: 487-496 [Abstract]. |
9. |
Briskin, M.J.,
L. Rott, and
E.C. Butcher.
1996.
Structural requirements for mucosal vascular addressin binding to its lymphocyte receptor ![]() ![]() |
10. | Campbell, J.J., S. Qin, K.B. Bacon, C.R. Mackay, and E.C. Butcher. 1996. Biology of chemokine and classical chemoattractant receptors: differential requirements for adhesion-triggering versus chemotactic responses in lymphoid cells. J. Cell Biol. 134: 255-266 [Abstract]. |
11. |
Carr, M.W.,
R. Alon, and
T.A. Springer.
1996.
The C-C chemokine MCP-1 differentially modulates the avidity of ![]() ![]() |
12. |
Cepek, K.L.,
C.M. Parker,
J.L. Madara, and
M.B. Brenner.
1993.
Integrin ![]() ![]() |
13. |
Cepek, K.L.,
S.K. Shaw,
C.M. Parker,
G.J. Russell,
J.S. Morrow,
D.L. Rimm, and
M.B. Brenner.
1994.
Adhesion between epithelial cells and T lymphocytes mediated by E-cadherin and the ![]() ![]() |
14. | Cerf-Bensussan, N., A. Jarry, N. Brousse, B. Lisowska-Grospierre, D. Guy-Grand, and C. Griscelli. 1987. A monoclonal antibody (HML-1) defining a novel membrane molecule present on human intestinal lymphocytes. Eur. J. Immunol. 17: 1279-1285 |
15. |
Chan, B.M.,
M.J. Elices,
E. Murphy, and
M.E. Hemler.
1992.
Adhesion to vascular cell adhesion molecule 1 and fibronectin. Comparison of ![]() ![]() ![]() ![]() |
16. | Chen, Y.T., and W.J. Nelson. 1996. Continuous production of soluble extracellular domain of a type-I transmembrane protein in mammalian cells using an Epstein-Barr virus ori-P based expression vector. Anal. Biochem. 242: 276-278 |
17. | Coligan, J.E., A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober. 1994. Current Protocols in Immunology. R. Coico, editor. Current Protocols. John Wiley & Sons, Inc., New York. |
18. | Díaz-González, F., J. Forsyth, B. Steiner, and M.H. Ginsberg. 1996. Trans-dominant inhibition of integrin function. Mol. Biol. Cell. 7: 1939-1951 [Abstract]. |
19. | Dransfield, I., C. Cabañas, A. Craig, and N. Hogg. 1992. Divalent cation regulation of the function of the leukocyte integrin LFA-1. J. Cell Biol. 116: 219-226 [Abstract]. |
20. | Dubey, C., M. Croft, and S.L. Swain. 1995. Costimulatory requirements of naive CD4+ T cells. ICAM-1 or B7-1 can costimulate naive CD4 T cell activation but both are required for optimum response. J. Immunol. 155: 45-57 [Abstract]. |
21. | Dustin, M.L., and T.A. Springer. 1989. T-cell receptor cross-linking transiently stimulates adhesiveness through LFA-1. Nature. 341: 619-624 |
22. | Erle, D.J.. 1995. Intraepithelial lymphocytes. Scratching the surface. Curr. Biol. 5: 252-254 |
23. | Gumbiner, B., B. Stevenson, and A. Grimaldi. 1988. The role of the cell adhesion molecule uvomorulin in the formation and maintenance of the epithelial junctional complex. J. Cell Biol. 107: 1575-1587 [Abstract]. |
24. | Hatta, K., S. Takagi, H. Fujisawa, and M. Takeichi. 1987. Spatial and temporal expression pattern of N-cadherin cell adhesion molecules correlated with morphogenetic processes of chicken embryos. Dev. Biol. 120: 215-227 |
25. |
Hemler, M.E.,
C. Huang,
Y. Takada,
L. Schwarz,
J.L. Strominger, and
M.L. Clabby.
1987.
Characterization of the cell surface heterodimer VLA-4 and
related peptides.
J. Biol. Chem.
262:
11478-11485
|
26. | Hermiston, M.L., and J.I. Gordon. 1995. Inflammatory bowel disease and adenomas in mice expressing a dominant negative N-cadherin. Science. 270: 1203-1207 [Abstract]. |
27. | Hirai, Y., A. Nose, S. Kobayashi, and M. Takeichi. 1989a. Expression and role of E- and P-cadherin adhesion molecules in embryonic histogenesis. I. Lung epithelial morphogenesis. Development. 105: 263-270 [Abstract]. |
28. | Hirai, Y., A. Nose, S. Kobayashi, and M. Takeichi. 1989b. Expression and role of E- and P-cadherin adhesion molecules in embryonic histogenesis. II. Skin morphogenesis. Development. 105: 271-277 [Abstract]. |
29. |
Holness, C.L.,
P.A. Bates,
A.J. Little,
C.D. Buckley,
A. McDowall,
D. Bossy,
N. Hogg, and
D.L. Simmons.
1995.
Analysis of the binding site on intercellular
adhesion molecule 3 for the leukocyte integrin lymphocyte function-associated antigen 1.
J. Biol. Chem.
270:
877-884
|
30. |
Holzmann, B.,
B.W. McIntyre, and
I.L. Weissman.
1989.
Identification of a murine Peyer's patch-specific lymphocyte homing receptor as an integrin molecule with an ![]() ![]() |
31. | Hyafil, F., C. Babinet, and F. Jacob. 1981. Cell-cell interactions in early embryogenesis: a molecular approach to the role of calcium. Cell. 26: 447-454 |
32. | Hynes, R.O.. 1992. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 69: 11-25 |
33. | Johnson, K.R., J.E. Lewis, D. Li, J. Wahl, A.P. Soler, K.A. Knudsen, and M.J. Wheelock. 1993. P- and E-cadherin are in separate complexes in cells expressing both cadherins. Exp. Cell Res. 207: 252-260 |
34. | Jones, E.Y., K. Harlos, M.J. Bottomley, R.C. Robinson, P.C. Driscoll, R.M. Edwards, J.M. Clements, T.J. Dudgeon, and D.I. Stuart. 1995. Crystal structure of an integrin-binding fragment of vascular cell adhesion molecule-1 at 1.8 Å resolution. Nature. 373: 539-544 |
35. |
Karecla, P.I.,
S.J. Bowden,
S.J. Green, and
P.J. Kilshaw.
1995.
Recognition of
E-cadherin on epithelial cells by the mucosal T cell integrin ![]() ![]() ![]() ![]() |
36. |
Karecla, P.I.,
S.J. Green,
S.J. Bowden,
J. Coadwell, and
P.J. Kilshaw.
1996.
Identification of a binding site for integrin ![]() ![]() |
37. |
Kilshaw, P.J., and
S.J. Murant.
1991.
Expression and regulation of ![]() ![]() |
38. | Kohler, G., and C. Milstein. 1975. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 256: 495-497 |
39. | Kruschwitz, M., G. Fritzsche, R. Schwarting, K. Micklem, D.Y. Mason, B. Falini, and H. Stein. 1991. Ber-ACT8: new monoclonal antibody to the mucosa lymphocyte antigen. J. Clin. Path. 44: 636-645 [Abstract]. |
40. | LaFlamme, S.E., L.A. Thomas, S.S. Yamada, and K.M. Yamada. 1994. Single subunit chimeric integrins as mimics and inhibitors of endogenous integrin functions in receptor localization, cell spreading and migration, and matrix assembly. J. Cell Biol. 126: 1287-1298 [Abstract]. |
41. | Lampugnani, M.G., M. Resnati, M. Raiteri, R. Pigott, A. Pisacane, G. Houen, L.P. Ruco, and E. Dejana. 1992. A novel endothelial-specific membrane protein is a marker of cell-cell contacts. J. Cell Biol. 118: 1511-1522 [Abstract]. |
42. |
Lazarovits, A.I.,
R.A. Moscicki,
J.T. Kurnick,
D. Camerini,
A.K. Bhan,
L.G. Baird,
M. Erikson, and
R.B. Colvin.
1984.
Lymphocyte activation antigens.
I. A monoclonal antibody, anti-Act I, defines a new late lymphocyte activation antigen.
J. Immunol.
133:
1857-1862
|
43. |
Lee, J.O.,
P. Rieu,
M.A. Arnaout, and
R. Liddington.
1995.
Crystal structure of
the A domain from the ![]() |
44. | Lloyd, A.R., J.J. Oppenheim, D.J. Kelvin, and D.D. Taub. 1996. Chemokines regulate T cell adherence to recombinant adhesion molecules and extracellular matrix proteins. J. Immunol. 156: 932-938 [Abstract]. |
45. | Lub, M., Y. van Kooyk, and C.G. Figdor. 1995. Ins and outs of LFA-1. Immunol. Today. 16: 479-483 |
46. | Lub, M., Y. van Kooyk, S.J. van Vliet, and C.G. Figdor. 1997. Dual role of the actin cytoskeleton in regulating cell adhesion mediated by the integrin lymphocyte function-associated molecule-1. Mol. Biol. Cell. 8: 341-351 [Abstract]. |
47. | Main, A.L., T.S. Harvey, M. Baron, J. Boyd, and I.D. Campbell. 1992. The three-dimensional structure of the tenth type III module of fibronectin: an insight into RGD-mediated interactions. Cell. 71: 671-678 |
48. | Marks, M.S., L. Woodruff, H. Ohno, and J.S. Bonifacino. 1996. Protein targeting by tyrosine- and dileucine-based signals: evidence for distinct saturable components. J. Cell Biol. 135: 341-354 [Abstract]. |
49. | Maurer, P., E. Hohenester, and J. Engel. 1996. Extracellular calcium-binding proteins. Curr. Opin. Cell Biol. 8: 609-617 |
50. | Mengaud, J., H. Ohayon, P. Gounon, R.M. Mege, and P. Cossart. 1996. E-cadherin is the receptor for internalin, a surface protein required for entry of L. monocytogenes into epithelial cells. Cell. 84: 923-932 |
51. | Miller, J., R. Knorr, M. Ferrone, R. Houdei, C.P. Carron, and M.L. Dustin. 1995. Intercellular adhesion molecule-1 dimerization and its consequences for adhesion mediated by lymphocyte function associated-1. J. Exp. Med. 182: 1231-1241 [Abstract]. |
52. | Mohit, B., and K. Fan. 1971. Hybrid cell line from a cloned immunoglobulin-producing mouse myeloma and a nonproducing mouse lymphoma. Science. 171: 75-77 |
53. |
Morimoto, C.,
N.L. Letvin,
A.W. Boyd,
M. Hagan,
H.M. Brown,
M.M. Kornacki, and
S.F. Schlossman.
1985.
The isolation and characterization of the
human helper inducer T cell subset.
J. Immunol.
134:
3762-3769
|
54. | Nagafuchi, A., and M. Takeichi. 1988. Cell binding function of E-cadherin is regulated by the cytoplasmic domain. EMBO (Eur. Mol. Biol. Organ.) J. 7: 3679-3684 [Abstract]. |
55. | Nagar, B., M. Overduin, M. Ikura, and J.M. Rini. 1996. Structural basis of calcium-induced E-cadherin rigidification and dimerization. Nature. 380: 360-364 |
56. | Nose, A., A. Nagafuchi, and M. Takeichi. 1987. Isolation of placental cadherin cDNA: identification of a novel gene family of cell-cell adhesion molecules. EMBO (Eur. Mol. Biol. Organ.) J. 6: 3655-3661 [Abstract]. |
57. | Nose, A., and M. Takeichi. 1986. A novel cadherin cell adhesion molecule: its expression patterns associated with implantation and organogenesis of mouse embryos. J. Cell Biol. 103: 2649-2658 [Abstract]. |
58. |
Osborn, L.,
C. Vassallo,
B.G. Browning,
R. Tizard,
D.O. Haskard,
C.D. Benjamin,
I. Dougas, and
T. Kirchhausen.
1994.
Arrangement of domains, and
amino acid residues required for binding of vascular cell adhesion molecule-1 to its counter-receptor VLA-4 (![]() ![]() |
59. | Overduin, M., T.S. Harvey, S. Bagby, K.I. Tong, P. Yau, M. Takeichi, and M. Ikura. 1995. Solution structure of the epithelial cadherin domain responsible for selective cell adhesion. Science. 267: 386-389 |
60. |
Parker, C.M.,
V. Groh,
H. Band,
S.A. Porcelli,
C. Morita,
M. Fabbi,
D. Glass,
J.L. Strominger, and
M.B. Brenner.
1990.
Evidence for extrathymic changes
in the T cell receptor ![]() ![]() |
61. | Pokutta, S., K. Herrenknecht, R. Kemler, and J. Engel. 1994. Conformational changes of the recombinant extracellular domain of E-cadherin upon calcium binding. Eur. J. Biochem. 223: 1019-1026 [Abstract]. |
62. |
Puzon-McLaughlin, W., and
Y. Takada.
1996.
Critical residues for ligand binding in an I domain-like structure of the integrin beta1 subunit.
J. Biol. Chem.
271:
20438-20443
|
63. | Reilly, P.L., J.R.J. Woska, D.D. Jeanfavre, E. McNally, R. Rothlein, and B.J. Bormann. 1995. The native structure of intercellular adhesion molecule-1 (ICAM-1) is a dimer. Correlation with binding to LFA-1. J. Immunol. 155: 529-532 [Abstract]. |
64. |
Renz, M.E.,
H.H. Chiu,
S. Jones,
J. Fox,
K.J. Kim,
L.G. Presta, and
S. Fong.
1994.
Structural requirements for adhesion of soluble recombinant murine
vascular cell adhesion molecule-1 to ![]() ![]() |
65. | Rimm, D.L., and J.S. Morrow. 1994. Molecular cloning of human E-cadherin suggests a novel subdivision of the cadherin superfamily. Biochem. Biophys. Res. Commun. 200: 1754-1761 |
66. | Roberts, A.I., S.M. O'Connell, and E.C. Ebert. 1993. Intestinal intraepithelial lymphocytes bind to colon cancer cells by HML-1 and CD11a. Cancer Res. 53: 1608-1611 [Abstract]. |
67. |
Rothlein, R.,
M.L. Dustin,
S.D. Marlin, and
T.A. Springer.
1986.
A human intercellular adhesion molecule (ICAM-1) distinct from LFA-1.
J. Immunol.
137:
1270-1274
|
68. |
Russell, G.J.,
C.M. Parker,
K.L. Cepek,
D.A. Mandelbrot,
A. Sood,
E. Mizoguchi,
E.C. Ebert,
M.B. Brenner, and
A.K. Bhan.
1994.
Distinct structural and
functional epitopes of the ![]() ![]() |
69. | Russell, G.J., C.M. Parker, A. Sood, E. Mizoguchi, E.C. Ebert, A.K. Bhan, and M.B. Brenner. 1996. p126 (CDw101), a costimulatory molecule preferentially expressed on mucosal T lymphocytes. J. Immunol. 157: 3366-3374 [Abstract]. |
70. | Salomon, D., O. Ayalon, R. Patel-King, R.O. Hynes, and B. Geiger. 1992. Extrajunctional distribution of N-cadherin in cultured human endothelial cells. J. Cell Sci. 102: 7-17 [Abstract]. |
71. | Sanchez-Madrid, F., A.M. Krensky, C.F. Ware, E. Robbins, J.L. Strominger, S.J. Burakoff, and T.A. Springer. 1982. Three distinct antigens associated with human T-lymphocyte-mediated cytolysis: LFA-1, LFA-2, and LFA-3. Proc. Natl Acad. Sci. USA. 79: 7489-7493 [Abstract]. |
72. | Sanchez-Madrid, F., J.A. Nagy, E. Robbins, P. Simon, and T.A. Springer. 1983. A human leukocyte differentiation antigen family with distinct alpha-subunits and a common beta-subunit: the lymphocyte function-associated antigen (LFA-1), the C3bi complement receptor (OKM1/Mac-1), and the p150,95 molecule. J. Exp. Med. 158: 1785-1803 [Abstract]. |
73. |
Sarnacki, S.,
B. Begue,
H. Buc,
F. Le Deist, and
N. Cerf-Bensussan.
1992.
Enhancement of CD3-induced activation of human intestinal intraepithelial lymphocytes by stimulation of the ![]() |
74. | Shapiro, L., A.M. Fannon, P.D. Kwong, A. Thompson, M.S. Lehmann, G. Grübel, J.F. Legrand, J. Als-Nielsen, D.R. Colman, and W.A. Hendrickson. 1995. Structural basis of cell-cell adhesion by cadherins. Nature. 374: 327-337 |
75. |
Shaw, S.K.,
K.L. Cepek,
E.A. Murphy,
G.J. Russell,
M.B. Brenner, and
C.M. Parker.
1994.
Molecular cloning of the human mucosal lymphocyte integrin
![]() |
76. |
Shimizu, Y., and
J.L. Mobley.
1993.
Distinct divalent cation requirements for
integrin-mediated CD4+ T lymphocyte adhesion to ICAM-1, fibronectin,
VCAM-1, and invasin.
J. Immunol.
151:
4106-4115
|
77. |
Shimizu, Y.,
G.A. Van Seventer,
K.J. Horgan, and
S. Shaw.
1990.
Regulated expression and binding of three VLA (![]() |
78. | Shimoyama, Y., S. Hirohashi, S. Hirano, M. Noguchi, Y. Shimosato, M. Takeichi, and O. Abe. 1989a. Cadherin cell-adhesion molecules in human epithelial tissues and carcinomas. Cancer Res. 49: 2128-2133 [Abstract]. |
79. | Shimoyama, Y., T. Yoshida, M. Terada, Y. Shimosato, O. Abe, and S. Hirohashi. 1989b. Molecular cloning of a human Ca2+-dependent cell-cell adhesion molecule homologous to mouse placental cadherin: its low expression in human placental tissues. J. Cell Biol. 109: 1787-1794 [Abstract]. |
80. | Smith, T.J., L.A. Ducharme, S.K. Shaw, C.M. Parker, M.B. Brenner, P.J. Kilshaw, and J.H. Weis. 1994. Murine M290 integrin expression modulated by mast cell activation. Immunity 1: 393-403 |
81. | Spits, H., G. Keizer, J. Borst, C. Terhorst, A. Hekman, and J.E. de Vries. 1983. Characterization of monoclonal antibodies against cell surface molecules associated with cytotoxic activity of natural and activated killer cells and cloned CTL lines. Hybridoma. 2: 423-437 |
82. | Staunton, D.E., M.L. Dustin, H.P. Erickson, and T.A. Springer. 1990. The arrangement of the immunoglobulin-like domains of ICAM-1 and the binding sites for LFA-1 and rhinovirus. Cell. 61: 243-254 |
83. | Stewart, M.P., C. Cabañas, and N. Hogg. 1996. T cell adhesion to intercellular adhesion molecule-1 (ICAM-1) is controlled by cell spreading and the activation of integrin LFA-1. J. Immunol. 156: 1810-1817 [Abstract]. |
84. |
Takebe, Y.,
M. Seiki,
J. Fujisawa,
P. Hoy,
K. Yokota,
K. Arai,
M. Yoshida, and
N. Arai.
1988.
SR![]() |
85. | Takeichi, M.. 1990. Cadherins: a molecular family important in selective cell-cell adhesion. Annu. Rev. Biochem. 59: 237-252 |
86. | Takeichi, M.. 1995. Morphogenetic roles of classic cadherins. Curr. Opin. Cell Biol. 7: 619-627 |
87. | Tang, A., M. Amagai, L.G. Granger, J.R. Stanley, and M.C. Udey. 1993. Adhesion of epidermal Langerhans cells to keratinocytes mediated by E-cadherin. Nature. 361: 82-85 |
88. |
Tiisala, S.,
T. Paavonen, and
R. Renkonen.
1995.
![]() ![]() ![]() ![]() |
89. | Tolosa, E., and S. Shaw. 1996. A fluorogenic assay of endogenous phosphatase for assessment of cell adhesion. J. Immunol. Methods. 192: 165-172 |
90. | Tomschy, A., C. Fauser, R. Landwehr, and J. Engel. 1996. Homophilic adhesion of E-cadherin occurs by a co-operative two-step interaction of N-terminal domains. EMBO (Eur. Mol. Biol. Organ.) J. 15: 3507-3514 [Abstract]. |
91. | van Kooyk, Y., P. van de Wiel-van Kemenade, P. Weder, T.W. Kuijpers, and C.G. Figdor. 1989. Enhancement of LFA-1-mediated cell adhesion by triggering through CD2 or CD3 on T lymphocytes. Nature. 342: 811-813 |
92. | Viney, J.L., S. Jones, H.H. Chiu, B. Lagrimas, M.E. Renz, L.G. Presta, D. Jackson, K.J. Hillan, S. Lew, and S. Fong. 1996. Mucosal addressin cell adhesion molecule-1: a structural and functional analysis demarcates the integrin binding motif. J. Immunol. 157: 2488-2497 [Abstract]. |
93. | Vonderheide, R.H., T.F. Tedder, T.A. Springer, and D.E. Staunton. 1994. Residues within a conserved amino acid motif of domains 1 and 4 of VCAM-1 are required for binding to VLA-4. J. Cell Biol. 125: 215-222 [Abstract]. |
94. | Wacholtz, M.C., S.S. Patel, and P.E. Lipsky. 1989. Leukocyte function-associated antigen 1 is an activation molecule for human T cells. J. Exp. Med. 170: 431-448 [Abstract]. |
95. |
Wang, J.H.,
R.B. Pepinsky,
T. Stehle,
J.H. Liu,
M. Karpusas,
B. Browning, and
L. Osborn.
1995.
The crystal structure of an N-terminal two-domain fragment of vascular cell adhesion molecule 1 (VCAM-1): a cyclic peptide based
on the domain 1 C-D loop can inhibit VCAM-1-![]() |
96. |
Wong, W.W., and
S.A. Farrell.
1991.
Proposed structure of the F![]() |
97. | Yoshida, C., and M. Takeichi. 1982. Teratocarcinoma cell adhesion: identification of a cell-surface protein involved in calcium-dependent cell aggregation. Cell. 28: 217-224 |
98. |
Yuan, Q.,
W. Jiang,
G.W. Krissansen, and
J.D. Watson.
1990.
Cloning and sequence analysis of a novel ![]() |