MARCH Family E3 Ubiquitin Ligases Selectively Downregulate Cadherin Family Proteins

Abstract

VE‐cadherin (VE‐cad) plays a central role in the adherens junctions (AJ) of vascular endothelial cells by regulating endothelial barrier function. Our group previously showed that a viral ubiquitin ligase K5, which is encoded in Human Herpesvirus 8 genome, causes endocytosis and degradation of 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. We began by performing RT‐PCR analysis to assess which MARCH family proteins are expressed in endothelial cells. Vascular endothelial cells were found to express low levels of MARCH1 and readily detectable levels of MARCH2, MARCH3, MARCH4 and MARCH8. To assess the effect of MARCH family protein expression on VE‐cad and N‐cad, GFP‐tagged MARCH family proteins were expressed in human umbilical endothelial cells (HUVECs) and the effect on VE‐cad and N‐cad was assessed. MARCH1 had no discernable impact on either VE‐cad or N‐cad. MARCH8 slightly downregulated VE‐cad but not N‐cad. In contrast, MARCH2, MARCH3 and MARCH4 transfection markedly downregulated VE‐cad in HUVECs. Interestingly, MARCH2 and MARCH3 did not downregulate N‐cad, but MARCH4 downregulated both VE‐cad and N‐cad. These results suggest 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 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 the amino acid sequence within the transmembrane and juxta‐membrane domains is essential for the selectivity of MARCH2‐induced downregulation. These findings indicate that MARCH ubiquitin ligases exhibit selectivity for different members of the classical cadherin family. These findings suggest that MARCH family ligases may differentially regulate cadherins in vascular endothelial cells to modulate endothelial cell migration and barrier function during development and inflammation.