Abstract
Cadherins play a central role in the assembly and adhesive function of adherens junctions (AJ). Adhesion and signaling functions of cadherins regulate epidermal and vascular barrier function, development, and tumor metastasis. Our group previously showed that a viral ubiquitin ligase K5, which is encoded in Human Herpesvirus 8 genome and involved in Kaposi Sarcoma, causes endocytosis and degradation of VE-cadherin (VE-cad). K5 is homologous to endogenous human membrane associated RING-CH-type ubiquitin ligase (MARCH) proteins. However, the contribution of endogenous MARCH family proteins to cadherin regulation is unclear. To assess the effect of MARCH family protein expression on epithelial and endothelial cadherins, MARCH proteins were expressed in epithelial and endothelial cells and the effect on E-cadherin (E-cad), VE-cad, and N-cadherin (N-cad) was monitored by western blot and immunofluorescence microscopy. MARCH1 had no discernable impact on E-cad, VE-cad or N-cad. MARCH8 downregulated E-cad and slightly downregulated VE-cad but not N-cad. In contrast, MARCH2, MARCH3 and MARCH4 transfection markedly downregulated VE-cad. Interestingly, MARCH2 and MARCH3 did not downregulate E-cad and N-cad, but MARCH4 downregulated E-cad, VE-cad and N-cad. These results revealed that different MARCH proteins have different cadherin specificities. To determine the amino-acid sequences within VE-cad that dictate this specificity, we generated VE-cad/N-cad chimeras by swapping the cadherin transmembrane and juxta-membrane domains (VE-cad-NcadTMDJMD, N-cad-VEcadTMDJMD). Interestingly, MARCH2 downregulated N-cad-VEcadTMDJMD but not VE-cad-NcadTMDJMD. These results indicate that MARCH proteins exhibit selectivity for different cadherins and that the amino acid sequence within the transmembrane and juxta-membrane domains is essential for this selectivity. These findings suggest that MARCH proteins may differentially regulate cadherins in epithelial and endothelial cells to modulate cell migration and barrier function during development and inflammation.
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