Contributions of the extracellular and cytoplasmic domains of platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) in regulating cell-cell localization

2000 ◽  
Vol 113 (8) ◽  
pp. 1459-1469 ◽  
Author(s):  
J. Sun ◽  
C. Paddock ◽  
J. Shubert ◽  
H.B. Zhang ◽  
K. Amin ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Extracellular Domain ◽  
Cytoplasmic Domain ◽  
Proximal Region ◽  
Adjacent Cell ◽  
Cell Localization ◽  
Platelet Endothelial Cell Adhesion ◽  
Direct Cell ◽  
Membrane Proximal Region ◽  
Cell Cell

PECAM-1/CD31, a vascular cell adhesion/signaling molecule that has been implicated in a number of vascular functions (including angiogenesis and the transmigration of leukocytes through endothelium) is highly enriched at the cell-cell borders of adjacent endothelial cells. To identify the mechanisms responsible for this localization, a series of PECAM-1 mutants and chimeric PECAM-1 molecules were transfected into non-PECAM-expressing cells and the ability of the constructs to move to cell-cell borders of adjacent cells was determined using immunohistochemistry and confocal microscopy. Although neither the extracellular domain, by itself, nor the cytoplasmic domain, by itself, was sufficient to direct cell-cell localization, the combination of the extracellular and transmembrane domains with a small group of highly charged amino acids in a membrane proximal region of the cytoplasmic domain was sufficient to direct efficient localization of the molecule to cell-cell borders. Importantly, only constructs that supported PECAM-1 mediated adhesion localized to cell-cell borders. Our data are consistent with a ‘diffusion trapping’ model in which movement of PECAM-1 in the cell membrane occurs relatively freely until the ‘stablized’ extracellular domain of the molecule encounters its ligand on an adjacent cell. When this occurs, the complex is ‘captured’ at the cell-cell interface leading to localization at cell-cell borders.

scholarly journals Transmembrane control of cadherin-mediated cell adhesion: a 94 kDa protein functionally associated with a specific region of the cytoplasmic domain of E-cadherin.

1989 ◽  
Vol 1 (1) ◽  
pp. 37-44 ◽  
Author(s):  
A Nagafuchi ◽  
M Takeichi
Keyword(s):  
Cell Adhesion ◽  
Extracellular Domain ◽  
Cytoplasmic Domain ◽  
Cytoplasmic Domains ◽  
Mutant Proteins ◽  
L Cells ◽  
Binding Function ◽  
Mutant Polypeptides ◽  
E Cadherin ◽  
Cell Cell

Cadherins are a family of transmembrane glycoproteins which play a key role in Ca(2+)-dependent cell-cell adhesion. Cytoplasmic domains of these molecules are anchored to the cell cytoskeleton and are required for cadherin function. To elucidate how the function of cadherins is controlled through their cytoplasmic domains, we deleted five different regions in the cytoplasmic domain of E-cadherin. After transfecting L cells with cDNA encoding the mutant polypeptides, we assayed aggregating activity of these transfectants; all these mutant proteins were shown to have an extracellular domain with normal Ca(2+)-sensitivity and molecular weight. Two mutant polypeptides with deletions in the carboxy half of the cytoplasmic domain, however, did not promote cell-cell adhesion and had also lost the ability to bind to the cytoskeleton, whereas the mutant molecules with deletions of other regions retained the ability to promote cell adhesion and to anchor to the cytoskeleton. Thus, the cytoplasmic domain contains a subdomain which was involved in the cell adhesion and cytoskeleton-binding functions. When E-cadherin in F9 cells or in L cells transfected with wild-type or functional mutant cadherin polypeptides was solubilized with nonionic detergents and immunoprecipitated, two additional 94 and 102 kDa components were coprecipitated. The 94 kDa component, however, was not detected in the immunoprecipitates from cells expressing the mutant cadherins which had lost the adhesive function. These results suggest that the interaction of the carboxy half of the cytoplasmic domain with the 94 kDa component regulates the cell binding function of the extracellular domain of E-cadherin.

scholarly journals Deletions in the cytoplasmic domain of platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31) result in changes in ligand binding properties

1994 ◽  
Vol 124 (1) ◽  
pp. 195-203 ◽  
Author(s):  
HM DeLisser ◽  
J Chilkotowsky ◽  
HC Yan ◽  
ML Daise ◽  
CA Buck ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Ligand Binding ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Cytoplasmic Domain ◽  
Wild Type ◽  
Platelet Endothelial Cell Adhesion ◽  
Cell Adhesion Molecule 1 ◽  
Endothelial Cell Adhesion ◽  
Cell Cell

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.

Herpes simplex virus glycoprotein B (gB) mutations define structural sites in domain I, the membrane proximal region, and the cytodomain that regulate entry.

2021 ◽  
Author(s):  
Qing Fan ◽  
Richard Longnecker ◽  
Sarah A. Connolly
Keyword(s):  
Cell Fusion ◽  
Virus Entry ◽  
Proximal Region ◽  
Glycoprotein B ◽  
Viral Fusion ◽  
Viral Fusion Protein ◽  
Membrane Proximal Region ◽  
Domain V ◽  
Domain I ◽  
Cell Cell

The viral fusion protein glycoprotein B (gB) is conserved in all herpesviruses and is essential for virus entry. During entry, gB fuses viral and host cell membranes by refolding from a prefusion to a postfusion form. We previously introduced three structure-based mutations (gB-I671A/H681A/F683A) into the domain V arm of the gB ectodomain that resulted in reduced cell-cell fusion. A virus carrying these three mutations (called gB3A) displayed a small plaque phenotype and remarkably delayed entry into cells. To identify mutations that could counteract this phenotype, we serially passaged the gB3A virus and selected for revertant viruses with increased plaque size. Genomic sequencing revealed that the revertant viruses had second-site mutations in gB, including E187A, M742T, and S383F/G645R/V705I/V880G. Using expression constructs encoding these mutations, only gB-V880G was shown to enhance cell-cell fusion. In contrast, all of the revertant viruses showed enhanced entry kinetics, underscoring the fact that cell-cell fusion and virus-cell fusion are different. The results indicate that mutations in three different regions of gB (domain I, the membrane proximal region, and the cytoplasmic tail domain) can counteract the slow entry phenotype of gB3A virus. Mapping these compensatory mutations to prefusion and postfusion structural models suggests sites of intramolecular functional interactions with the gB domain V arm that may contribute to the gB fusion function. Importance The nine human herpesviruses are ubiquitous and cause a range of disease in humans. Glycoprotein B (gB) is an essential viral fusion protein that is conserved in all herpesviruses. During host cell entry, gB mediates virus-cell membrane fusion by undergoing a conformational change. Structural models for the prefusion and postfusion form of gB exist, but the details of how the protein converts from one to the other are unclear. We previously introduced structure-based mutations into gB that inhibited virus entry and fusion. By passaging this entry-deficient virus over time, we selected second-site mutations that partially restore virus entry. The location of these mutations suggest regulatory sites that contribute to fusion and gB refolding during entry. gB is a target of neutralizing antibodies and defining how gB refolds during entry could provide a basis for the development of fusion inhibitors for future research or clinical use.

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

1997 ◽  
Vol 8 (7) ◽  
pp. 1329-1341 ◽  
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.

scholarly journals Fc receptor endocytosis is controlled by a cytoplasmic domain determinant that actively prevents coated pit localization.

1992 ◽  
Vol 116 (4) ◽  
pp. 875-888 ◽  
Author(s):  
H M Miettinen ◽  
K Matter ◽  
W Hunziker ◽  
J K Rose ◽  
I Mellman
Keyword(s):  
Cytoplasmic Domain ◽  
Insoluble Fraction ◽  
Fc Receptor ◽  
Proximal Region ◽  
Wild Type ◽  
Coated Pits ◽  
Tyrosine Residues ◽  
Receptor Endocytosis ◽  
Alternative Mrna Splicing ◽  
Membrane Proximal Region

Macrophages and B-lymphocytes express two major isoforms of Fc receptor (FcRII-B2 and FcRII-B1) that exhibit distinct capacities for endocytosis. This difference in function reflects the presence of an in-frame insertion of 47 amino acids in the cytoplasmic domain of the lymphocyte isoform (FcRII-B1) due to alternative mRNA splicing. By expressing wild type and mutant FcRII cDNAs in fibroblasts, we have now examined the mechanism by which the insertion acts to prevent coated pit localization and endocytosis. We first identified the region of the FcRII-B2 cytoplasmic domain that is required for rapid internalization. Using a biochemical assay for endocytosis and an immuno-EM assay to determine coated pit localization directly, we found that the distal half of the cytoplasmic domain, particularly a region including residues 18-31, as needed for coated pit-mediated endocytosis. Elimination of the tyrosine residues at position 26 and 43, separately or together, had little effect on coated pit localization and a partial effect on endocytosis of ligand. Since the FcRII-B1 insertion occurs in the membrane-proximal region of the cytoplasmic domain (residue 6) not required for internalization, it is unlikely to act by physically disrupting the coated pit localization determinant. In fact, the insertion was found to prevent endocytosis irrespective of its position in the cytoplasmic tail and appeared to selectively exclude the receptor from coated regions. Moreover, receptors bearing the insertion exhibited a temperature- and ligand-dependent association with a detergent-insoluble fraction and with actin filaments, perhaps in part explaining the inability of FcRII-B1 to enter coated pits.

scholarly journals The CEACAM1-L Glycoprotein Associates with the Actin Cytoskeleton and Localizes to Cell–Cell Contact through Activation of Rho-like GTPases

2000 ◽  
Vol 11 (1) ◽  
pp. 65-77 ◽  
Author(s):  
Svetlana Sadekova ◽  
Nathalie Lamarche-Vane ◽  
Xiaodong Li ◽  
Nicole Beauchemin
Keyword(s):  
Cell Adhesion ◽  
Actin Cytoskeleton ◽  
Adhesion Molecule ◽  
Control Cell ◽  
Growth Inhibitor ◽  
Cytoplasmic Domain ◽  
Cell Contact ◽  
A Cell ◽  
Rho Family ◽  
Cell Cell

Associations between plasma membrane-linked proteins and the actin cytoskeleton play a crucial role in defining cell shape and determination, ensuring cell motility and facilitating cell–cell or cell–substratum adhesion. Here, we present evidence that CEACAM1-L, a cell adhesion molecule of the carcinoembryonic antigen family, is associated with the actin cytoskeleton. We have delineated the regions involved in actin cytoskeleton association to the distal end of the CEACAM1-L long cytoplasmic domain. We have demonstrated that CEACAM1-S, an isoform of CEACAM1 with a truncated cytoplasmic domain, does not interact with the actin cytoskeleton. In addition, a major difference in subcellular localization of the two CEACAM1 isoforms was observed. Furthermore, we have established that the localization of CEACAM1-L at cell–cell boundaries is regulated by the Rho family of GTPases. The retention of the protein at the sites of intercellular contacts critically depends on homophilic CEACAM1–CEACAM1 interactions and association with the actin cytoskeleton. Our results provide new evidence on how the Rho family of GTPases can control cell adhesion: by directing an adhesion molecule to its proper cellular destination. In addition, these results provide an insight into the mechanisms of why CEACAM1-L, but not CEACAM1-S, functions as a tumor cell growth inhibitor.

scholarly journals Two Cell Adhesion Molecules, Nectin and Cadherin, Interact through Their Cytoplasmic Domain–Associated Proteins

2000 ◽  
Vol 150 (5) ◽  
pp. 1161-1176 ◽  
Author(s):  
Kouichi Tachibana ◽  
Hiroyuki Nakanishi ◽  
Kenji Mandai ◽  
Kumi Ozaki ◽  
Wataru Ikeda ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Cell Lines ◽  
Cell Adhesion Molecules ◽  
Cytoplasmic Domain ◽  
Adhesion Sites ◽  
Adhesion System ◽  
Associated Proteins ◽  
Mechanism Of Interaction ◽  
E Cadherin ◽  
Cell Cell

We have found a new cell–cell adhesion system at cadherin-based cell–cell adherens junctions (AJs) consisting of at least nectin and l-afadin. Nectin is a Ca2+-independent homophilic immunoglobulin-like adhesion molecule, and l-afadin is an actin filament-binding protein that connects the cytoplasmic region of nectin to the actin cytoskeleton. Both the trans-interaction of nectin and the interaction of nectin with l-afadin are necessary for their colocalization with E-cadherin and catenins at AJs. Here, we examined the mechanism of interaction between these two cell–cell adhesion systems at AJs by the use of α-catenin–deficient F9 cell lines and cadherin-deficient L cell lines stably expressing their various components. We showed here that nectin and E-cadherin were colocalized through l-afadin and the COOH-terminal half of α-catenin at AJs. Nectin trans-interacted independently of E-cadherin, and the complex of E-cadherin and α- and β-catenins was recruited to nectin-based cell–cell adhesion sites through l-afadin without the trans-interaction of E-cadherin. Our results indicate that nectin and cadherin interact through their cytoplasmic domain–associated proteins and suggest that these two cell–cell adhesion systems cooperatively organize cell–cell AJs.

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