Differential localization of VE- and N-cadherins in human endothelial cells: VE-cadherin competes with N-cadherin for junctional localization - PubMed (original) (raw)

Differential localization of VE- and N-cadherins in human endothelial cells: VE-cadherin competes with N-cadherin for junctional localization

P Navarro et al. J Cell Biol. 1998.

Abstract

The two major cadherins of endothelial cells are neural (N)-cadherin and vascular endothelial (VE)- cadherin. Despite similar level of protein expression only VE-cadherin is located at cell-cell contacts, whereas N-cadherin is distributed over the whole cell membrane. Cotransfection of VE-cadherin and N-cadherin in CHO cells resulted in the same distribution as that observed in endothelial cells indicating that the behavior of the two cadherins was not cell specific but related to their structural characteristics. Similar amounts of alpha- and beta-catenins and plakoglobin were associated to VE- and N-cadherins, whereas p120 was higher in the VE-cadherin complex. The presence of VE-cadherin did not affect N-cadherin homotypic adhesive properties or its capacity to localize at junctions when cotransfectants were cocultured with cells transfected with N-cadherin only. To define the molecular domain responsible for the VE-cadherin-dominant activity we prepared a chimeric construct formed by VE-cadherin extracellular region linked to N-cadherin intracellular domain. The chimera lost the capacity to exclude N-cadherin from junctions indicating that the extracellular domain of VE-cadherin alone is not sufficient for the preferential localization of the molecule at the junctions. A truncated mutant of VE-cadherin retaining the full extracellular domain and a short cytoplasmic tail (Arg621-Pro702) lacking the catenin-binding region was able to exclude N-cadherin from junctions. This indicates that the Arg621-Pro702 sequence in the VE-cadherin cytoplasmic tail is required for N-cadherin exclusion from junctions. Competition between cadherins for their clustering at intercellular junctions in the same cell has never been described before. We speculate that, in the endothelium, VE- and N-cadherin play different roles; whereas VE-cadherin mostly promotes the homotypic interaction between endothelial cells, N-cadherin may be responsible for the anchorage of the endothelium to other surrounding cell types expressing N-cadherin such as vascular smooth muscle cells or pericytes.

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Figures

Figure 6

Figure 6

N- and VE-cadherin distribution in mixed cultures of CHO N and CHO N/VE. CHO N and double-transfected CHO N/VE cells were seeded separately in small aggregates and cocultured on glass coverslips. Cells were grown to confluence, fixed, and then double immunostained with antibodies against VE- and N-cadherins. N-cadherin expressed by CHO N/VE cells can localize at cell junctions when there is homophilic interaction with CHO N cells (arrows). However, in the same cells, only VE-cadherin clusters at junctions when CHO N/VE are in contact with other CHON/VE cells (arrowheads). Bar, 30 μm.

Figure 1

Figure 1

(A) Immunofluorescence staining of VE-cadherin (a) and N-cadherin (b) in cultured endothelium. VE-cadherin is located at cell–cell contacts whereas N-cadherin shows a diffuse localization. (B) Expression of VE- and N-cadherins by Western immunoblot. Cell lysates from the same number of cells were loaded in each lane, separated by SDS-PAGE, blotted to nitrocellulose membranes, and then immunodetected with antibodies specific for VE- or N-cadherins. Molecular weight markers are indicated on the right. Bar, 30 μm.

Figure 1

Figure 1

(A) Immunofluorescence staining of VE-cadherin (a) and N-cadherin (b) in cultured endothelium. VE-cadherin is located at cell–cell contacts whereas N-cadherin shows a diffuse localization. (B) Expression of VE- and N-cadherins by Western immunoblot. Cell lysates from the same number of cells were loaded in each lane, separated by SDS-PAGE, blotted to nitrocellulose membranes, and then immunodetected with antibodies specific for VE- or N-cadherins. Molecular weight markers are indicated on the right. Bar, 30 μm.

Figure 2

Figure 2

Immunohistological localization of VE- (a and c) and N-cadherins (b and d) in an artery and a vein from human lymph node tissue sections. VE-cadherin is localized at cell–cell contacts (arrowheads) whereas N-cadherin staining was always diffuse in endothelial cells. Note the positive staining of arterial smooth muscle cells (arrow). Bar, 50 μm.

Figure 3

Figure 3

Characterization of CHO cells cotransfected with N- and VE-cadherin. (A) Western blot analysis of cadherin expression in CHO cells transfected either with N-cadherin alone (CHO N) or with both N- and VE-cadherins (CHO N/VE). Molecular weight markers are indicated on the left. Clones expressing comparable levels of the two proteins were selected for immunofluorescence analysis. (B) Immunofluorescence staining of CHO cells transfected either with N-cadherin alone (a, CHO N) or with both N- and VE-cadherin (CHO N/VE). In b and c, the same field of CHO N/VE cells has been stained with VE- (b) and N-cadherin–specific (c) antibodies. N-cadherin goes to cell–cell contact in N-cadherin–transfected cells but coexpression with VE-cadherin (CHO N/VE) excludes N-cadherin from cell junctions. Bar, 30 μm.

Figure 3

Figure 3

Characterization of CHO cells cotransfected with N- and VE-cadherin. (A) Western blot analysis of cadherin expression in CHO cells transfected either with N-cadherin alone (CHO N) or with both N- and VE-cadherins (CHO N/VE). Molecular weight markers are indicated on the left. Clones expressing comparable levels of the two proteins were selected for immunofluorescence analysis. (B) Immunofluorescence staining of CHO cells transfected either with N-cadherin alone (a, CHO N) or with both N- and VE-cadherin (CHO N/VE). In b and c, the same field of CHO N/VE cells has been stained with VE- (b) and N-cadherin–specific (c) antibodies. N-cadherin goes to cell–cell contact in N-cadherin–transfected cells but coexpression with VE-cadherin (CHO N/VE) excludes N-cadherin from cell junctions. Bar, 30 μm.

Figure 4

Figure 4

Adhesion of CHO N-cadherin transfectants to different cell monolayers. CHO cells transfected with N-cadherin (CHO N) were labeled with [125I]iododeoxyuridine and the same number of cells were seeded over different cell monolayers: endothelial cells (EC), CHO control cells (CHO), CHO cells transfected with VE-cadherin (VE), and CHO cells transfected with N-cadherin (N). The experiments were done in the presence (Ca2 +) or absence (EGTA) of calcium. CHO N cells adhered only to endothelial cells and CHO N cell monolayers in a significant way. The absence of calcium abolished this effect. Data are mean ± SD of five replicates from a typical experiments out of three performed.

Figure 5

Figure 5

Heterotypic aggregation between CHO N single transfectants and CHO N/VE double transfectants. CHO N/VE cells were labeled with a fluorescent probe and allowed to aggregate with unlabeled CHO N cells in suspension in the presence of calcium. The figure shows a typical cell aggregate (a) formed by ∼50% by CHO N/VE (fluorescent, b) and by CHO N cells (unlabeled, a). Bar, 60 μm.

Figure 7

Figure 7

Catenin and p120 association to cadherins in ECs and CHO N/VE cells. (A) Equivalent samples of VE- or N-cadherin immunocomplexes from EC were immunoblotted with antibodies against VE- or N-cadherins, α-catenin, β-catenin, and plakoglobin. (B) Equivalent samples of VE- or N-cadherin immunocomplexes from CHO N/VE cotransfected cells were immunoblotted with antibodies against VE- or N-cadherins, α-catenin, and β-catenin. (C) Equivalent samples of VE- or N-cadherin immunocomplexes from ECs or CHO N/VE cells were immunoblotted with an antibody against p120. Molecular weight markers are indicated on the right. Comparable amounts of α- and β-catenins and plakoglobin were associated to VE- and N-cadherins. Higher quantities of p120 were found in VE-cadherin in comparison to N-cadherin complex.

Figure 8

Figure 8

Tyrosine phosphorylation analysis of cadherin–catenin complex in ECs and CHO N/VE cotransfected cells. Similar number of cells were immunoprecipitated with VE- or N-cadherin antibodies and blotted with an antibody against phosphotyrosine. The bands corresponding to the molecular weight of cadherin, β-catenin, and plakoglobin are indicated on the left. VE-cadherin phosphorylation level is much higher than that of N-cadherin. Molecular weight markers are indicated on the right.

Figure 9

Figure 9

Effect of VE-cadherin mutants and chimeric constructs on N-cadherin clustering at junctions. (A) Schematic representation of the different molecules used for cotransfection: VEecNcyt, a chimeric protein formed by the extracellular region of VE-cadherin and the cytoplasmic domain of N-cadherin; tVE, a truncated form of VE-cadherin lacking the last 82 aas of the COOH terminal end; and VEectNcyt, a chimeric protein formed by the extracellular region of VE-cadherin and a truncated N-cadherin cytoplasmic domain lacking the last 77 aas. (B) Immunofluorescence analysis of typical double-transfected clones. Cells were stained with antibodies directed to the extracellular domain of VE cadherin (BV9 mAb; a, c, and e) or N-cadherin (8C11mAb; b, d, and f). The clones were selected for immunofluorescence studies on the basis of their capacity to express comparable amount of N-cadherin and mutant VE-cadherin constructs by Western blot analysis. In cells cotransfected with VEecNcyt (a), N-cadherin could cluster at intercellular contacts (b). In contrast, when the cells were cotransfected with the

t

VE mutant (c), N-cadherin was excluded from junctions (d) and this effect was specific because the cells cotransfected with VEec

t

Ncyt (e) mutant shows N-cadherin localization at cell–cell contacts (f). Bar, 30 μm.

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