Mutation Analysis of the Short Cytoplasmic Domain of the Cell-Cell Adhesion Molecule CEACAM1 Identifies Residues That Orchestrate Actin Binding and Lumen Formation

Platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a member of the immunoglobulin superfamily present on platelets, endothelial cells, and leukocytes that may function as a vascular cell adhesion molecule. The purpose of this study was to examine the role of the cytoplasmic domain in PECAM-1 function. To accomplish this, wild-type and mutated forms of PECAM-1 cDNA were transfected into murine fibroblasts and the functional characteristics of the cells analyzed. Wild-type PECAM-1 localized to the cell-cell borders of adjacently transfected cells and mediated heterophilic, calcium-dependent L-cell aggregation that was inhibitable by a polyclonal and two monoclonal anti-PECAM-1 antibodies. A mutant protein lacking the entire cytoplasmic domain did not support aggregation or move to cell-cell borders. In contrast, both forms of PECAM-1 with partially truncated cytoplasmic domains (missing either the COOH-terminal third or two thirds of the cytoplasmic domain) localized to cell-cell borders in 3T3 cells in a manner analogous to the distribution seen in cultured endothelial cells. L-cells expressing these mutants demonstrated homophilic, calcium-independent aggregation that was blocked by the polyclonal anti-PECAM-1 antibody, but not by the two bioactive monoclonal antibodies. Although changes in the cytoplasmic domain of other receptors have been shown to alter ligand-binding affinity, to our knowledge, PECAM-1 is the first example of a cell adhesion molecule where changes in the cytoplasmic domain result in a switch in the basic mechanism of adhesion leading to different ligand-binding specificity. Variations in the cytoplasmic domain could thus be a potential mechanism for regulating PECAM-1 activity in vivo.

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Cytoplasmic Tail Regulates the Intercellular Adhesion Function of the Epithelial Cell Adhesion Molecule

Molecular and Cellular Biology ◽

10.1128/mcb.18.8.4833 ◽

1998 ◽

Vol 18 (8) ◽

pp. 4833-4843 ◽

Cited By ~ 56

Author(s):

Maarten Balzar ◽

Hellen A. M. Bakker ◽

Inge H. Briaire-de-Bruijn ◽

Gert Jan Fleuren ◽

Sven O. Warnaar ◽

...

Keyword(s):

Cell Adhesion ◽

Adhesion Molecule ◽

Cell Adhesion Molecule ◽

Solid Phase ◽

Cytoplasmic Tail ◽

Cytoplasmic Domain ◽

Specific Cell ◽

Actin Microfilaments ◽

Intercellular Adhesions ◽

Cell Cell

ABSTRACT Ep-CAM, an epithelium-specific cell-cell adhesion molecule (CAM) not structurally related to the major families of CAMs, contains a cytoplasmic domain of 26 amino acids. The chemical disruption of the actin microfilaments, but not of the microtubuli or intermediate filaments, affected the localization of Ep-CAM at the cell-cell boundaries, suggesting that the molecule interacts with the actin-based cytoskeleton. Mutated forms of Ep-CAM were generated with the cytoplasmic domain truncated at various lengths. All of the mutants were transported to the cell surface in the transfectants; however, the mutant lacking the complete cytoplasmic domain was not able to localize to the cell-cell boundaries, in contrast to mutants with partial deletions. Both the disruption of the actin microfilaments and a complete truncation of the cytoplasmic tail strongly affected the ability of Ep-CAM to mediate aggregation of L cells. The capability of direct aggregation was reduced for the partially truncated mutants but remained cytochalasin D sensitive. The tail truncation did not affect the ability of the transfectants to adhere to solid-phase-adsorbed Ep-CAM, suggesting that the ability to form stable adhesions and not the ligand specificity of the molecule was affected by the truncation. The formation of intercellular adhesions mediated by Ep-CAM induced a redistribution to the cell-cell boundaries of α-actinin, but not of vinculin, talin, filamin, spectrin, or catenins. Coprecipitation demonstrated direct association of Ep-CAM with α-actinin. Binding of α-actinin to purified mutated and wild-type Ep-CAMs and to peptides representing different domains of the cytoplasmic tail of Ep-CAM demonstrates two binding sites for α-actinin at positions 289 to 296 and 304 to 314 of the amino acid sequence. The results demonstrate that the cytoplasmic domain of Ep-CAM regulates the adhesion function of the molecule through interaction with the actin cytoskeleton via α-actinin.

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622: Von Hippel-Lindau Inactivation Downregulates the Cell-Cell Adhesion Molecule E Cadherin in Renal Cancer Cells

The Journal of Urology ◽

10.1016/s0022-5347(18)34862-6 ◽

2005 ◽

Vol 173 (4S) ◽

pp. 170-170

Author(s):

Maxine G. Tran ◽

Miguel A. Esteban ◽

Peter D. Hill ◽

Ashish Chandra ◽

Tim S. O'Brien ◽

...

Keyword(s):

Cell Adhesion ◽

Cancer Cells ◽

Renal Cancer ◽

Adhesion Molecule ◽

Cell Adhesion Molecule ◽

Von Hippel Lindau ◽

E Cadherin ◽

Cell Cell

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Purification of a 92-kDa cytoplasmic protein tightly associated with the cell-cell adhesion molecule E-cadherin (uvomorulin)

Journal of Biological Chemistry ◽

10.1016/s0021-9258(20)64353-8 ◽

1991 ◽

Vol 266 (7) ◽

pp. 4514-4520

Author(s):

P D McCrea ◽

B M Gumbiner

Keyword(s):

Cell Adhesion ◽

Adhesion Molecule ◽

Cell Adhesion Molecule ◽

Cytoplasmic Protein ◽

E Cadherin ◽

Cell Cell

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Vascular-endothelial-cadherin modulates endothelial monolayer permeability

Journal of Cell Science ◽

10.1242/jcs.112.12.1915 ◽

1999 ◽

Vol 112 (12) ◽

pp. 1915-1923 ◽

Cited By ~ 3

Author(s):

P.L. Hordijk ◽

E. Anthony ◽

F.P. Mul ◽

R. Rientsma ◽

L.C. Oomen ◽

...

Keyword(s):

Cell Adhesion ◽

Actin Cytoskeleton ◽

Adhesion Molecule ◽

Cell Adhesion Molecule ◽

Transendothelial Migration ◽

Stress Fibers ◽

Vascular Endothelial ◽

Monolayer Permeability ◽

Actin Stress Fibers ◽

Cell Cell

Vascular endothelial (VE)-cadherin is the endothelium-specific member of the cadherin family of homotypic cell adhesion molecules. VE-cadherin, but not the cell adhesion molecule platelet/endothelial cell adhesion molecule (PECAM-1), markedly colocalizes with actin stress fibers at cell-cell junctions between human umbilical vein endothelial cells. Inhibition of VE-cadherin-mediated, but not PECAM-1-mediated, adhesion induced reorganization of the actin cytoskeleton, loss of junctional VE-cadherin staining and loss of cell-cell adhesion. In functional assays, inhibition of VE-cadherin caused increased monolayer permeability and enhanced neutrophil transendothelial migration. In a complementary set of experiments, modulation of the actin cytoskeleton was found to strongly affect VE-cadherin distribution. Brief stimulation of the beta2-adrenergic receptor with isoproterenol induced a loss of actin stress fibers resulting in a linear, rather than ‘jagged’, VE-cadherin distribution. The concomitant, isoproterenol-induced, reduction in monolayer permeability was alleviated by a VE-cadherin-blocking antibody. Finally, cytoskeletal reorganization resulting from the inactivation of p21Rho caused a diffuse localization of VE-cadherin, which was accompanied by reduced cell-cell adhesion. Together, these data show that monolayer permeability and neutrophil transendothelial migration are modulated by VE-cadherin-mediated cell-cell adhesion, which is in turn controlled by the dynamics of the actin cytoskeleton.

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Role of Nectin in Formation of E-Cadherin–based Adherens Junctions in Keratinocytes: Analysis with the N-Cadherin Dominant Negative Mutant

Molecular Biology of the Cell ◽

10.1091/mbc.e02-10-0632 ◽

2003 ◽

Vol 14 (4) ◽

pp. 1597-1609 ◽

Cited By ~ 19

Author(s):

Yoshinari Tanaka ◽

Hiroyuki Nakanishi ◽

Shigeki Kakunaga ◽

Noriko Okabe ◽

Tomomi Kawakatsu ◽

...

Keyword(s):

Cell Adhesion ◽

Adhesion Molecule ◽

Cell Adhesion Molecule ◽

Adherens Junctions ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Adhesion Activity ◽

Negative Mutant ◽

E Cadherin ◽

Cell Cell

E-Cadherin is a Ca2+-dependent cell-cell adhesion molecule at adherens junctions (AJs) of epithelial cells. A fragment of N-cadherin lacking its extracellular region serves as a dominant negative mutant (DN) and inhibits cell-cell adhesion activity of E-cadherin, but its mode of action remains to be elucidated. Nectin is a Ca2+-independent immunoglobulin-like cell-cell adhesion molecule at AJs and is associated with E-cadherin through their respective peripheral membrane proteins, afadin and catenins, which connect nectin and cadherin to the actin cytoskeleton, respectively. We showed here that overexpression of nectin capable of binding afadin, but not a mutant incapable of binding afadin, reduced the inhibitory effect of N-cadherin DN on the cell-cell adhesion activity of E-cadherin in keratinocytes. Overexpressed nectin recruited N-cadherin DN to the nectin-based cell-cell adhesion sites in an afadin-dependent manner. Moreover, overexpression of nectin enhanced the E-cadherin–based cell-cell adhesion activity. These results suggest that N-cadherin DN competitively inhibits the association of the endogenous nectin-afadin system with the endogenous E-cadherin-catenin system and thereby reduces the cell-cell adhesion activity of E-cadherin. Thus, nectin plays a role in the formation of E-cadherin–based AJs in keratinocytes.

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Basal-Cell Adhesion Molecule (B-CAM) is Induced in Epithelial Skin Tumors and Inflammatory Epidermis, and is Expressed at Cell–Cell and Cell–Substrate Contact Sites

Journal of Investigative Dermatology ◽

10.1046/j.1523-1747.2000.00189.x ◽

2000 ◽

Vol 115 (6) ◽

pp. 1047-1053 ◽

Cited By ~ 33

Author(s):

Margarete Schön ◽

Viktor Hogenkamp ◽

B. Gregor Wienrich ◽

Michael P. Schön ◽

C. Eberhard Klein ◽

...

Keyword(s):

Cell Adhesion ◽

Adhesion Molecule ◽

Basal Cell ◽

Cell Adhesion Molecule ◽

Skin Tumors ◽

Contact Sites ◽

Cell Substrate ◽

Cell Cell

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α-Catenin-independent Recruitment of ZO-1 to Nectin-based Cell-Cell Adhesion Sites through Afadin

Molecular Biology of the Cell ◽

10.1091/mbc.12.6.1595 ◽

2001 ◽

Vol 12 (6) ◽

pp. 1595-1609 ◽

Cited By ~ 67

Author(s):

Shigekazu Yokoyama ◽

Kouichi Tachibana ◽

Hiroyuki Nakanishi ◽

Yasunori Yamamoto ◽

Kenji Irie ◽

...

Keyword(s):

Cell Adhesion ◽

Actin Cytoskeleton ◽

Tight Junctions ◽

Cell Lines ◽

Adhesion Molecule ◽

Binding Protein ◽

Actin Binding ◽

Actin Binding Protein ◽

Adhesion Sites ◽

Cell Cell

ZO-1 is an actin filament (F-actin)–binding protein that localizes to tight junctions and connects claudin to the actin cytoskeleton in epithelial cells. In nonepithelial cells that have no tight junctions, ZO-1 localizes to adherens junctions (AJs) and may connect cadherin to the actin cytoskeleton indirectly through β- and α-catenins as one of many F-actin–binding proteins. Nectin is an immunoglobulin-like adhesion molecule that localizes to AJs and is associated with the actin cytoskeleton through afadin, an F-actin–binding protein. Ponsin is an afadin- and vinculin-binding protein that also localizes to AJs. The nectin-afadin complex has a potency to recruit the E-cadherin–β-catenin complex through α-catenin in a manner independent of ponsin. By the use of cadherin-deficient L cell lines stably expressing various components of the cadherin-catenin and nectin-afadin systems, and α-catenin–deficient F9 cell lines, we examined here whether nectin recruits ZO-1 to nectin-based cell-cell adhesion sites. Nectin showed a potency to recruit not only α-catenin but also ZO-1 to nectin-based cell-cell adhesion sites. This recruitment of ZO-1 was dependent on afadin but independent of α-catenin and ponsin. These results indicate that ZO-1 localizes to cadherin-based AJs through interactions not only with α-catenin but also with the nectin-afadin system.

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Thrombospondin-1, a natural inhibitor of angiogenesis, regulates platelet-endothelial cell adhesion molecule-1 expression and endothelial cell morphogenesis.

Molecular Biology of the Cell ◽

10.1091/mbc.8.7.1329 ◽

1997 ◽

Vol 8 (7) ◽

pp. 1329-1341 ◽

Cited By ~ 52

Author(s):

N Sheibani ◽

P J Newman ◽

W A Frazier

Keyword(s):

Endothelial Cells ◽

Cell Adhesion ◽

Endothelial Cell ◽

Adhesion Molecule ◽

Cell Adhesion Molecule ◽

Cell Contact ◽

Platelet Endothelial Cell Adhesion ◽

Cell Adhesion Molecule 1 ◽

Endothelial Cell Adhesion ◽

Cell Cell

Expression of thrombospondin-1 (TS1) in polyoma middle-sized T (tumor)-transformed mouse brain endothelial cells (bEND.3) restores a normal phenotype and suppresses their ability to form hemangiomas in mice. We show that TS1 expression results in complete suppression of platelet-endothelial cell adhesion molecule-1 (PECAM-1) expression and altered cell-cell interactions in bEND.3 cells. To further investigate the role of PECAM-1 in regulation of endothelial cell-cell interactions and morphogenesis, we expressed human (full length) or murine (delta 15) PECAM-1 isoforms in TS1-transfected bEND.3 (bEND/TS) cells. Expression of either human or murine PECAM-1 resulted in an enhanced ability to organize and form networks of cords on Matrigel, an effect that was specifically blocked by antibodies to PECAM-1. Anti-PECAM-1 antibodies also inhibited tube formation in Matrigel by normal human umbilical vein endothelial cells. However, PECAM-1-transfected bEND/TS cells did not regain the ability to form hemangiomas in mice and the expressed PECAM-1, unlike the endogenous PECAM-1 expressed in bEND.3 cells, failed to localize to sites of cell-cell contact. This may be, in part, attributed to the different isoforms of PECAM-1 expressed in bEND.3 cells. Using reverse transcription-polymerase chain reaction, we determined that bEND.3 cells express mRNA encoding six different PECAM-1 isoforms, the isoform lacking both exons 14 and 15 (delta 14&15) being most abundant. Expression of the murine delta 14&15 PECAM-1 isoform in bEND/TS cells resulted in a similar phenotype to that described for the full-length human or murine delta 15 PECAM-1 isoform. The delta 14&15 isoform, despite the lack of exon 14, failed to localize to sites of cell-cell contact even in clones that expressed it at very high levels. Thus, contrary to recent reports, lack of exon 14 is not sufficient to result in junctional localization of PECAM-1 isoforms in bEND/TS cells.

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