Molecular characteristics determining entry into or exclusion from the endothelial cell (EC) lateral border recycling compartment (LBRC)

The LBRC is a recently discovered membrane compartment in EC. It contains molecules critical for leukocyte transmigration, PECAM, CD99, and JAM‐A, but excludes other junction components like VE‐cadherin. Since a mutation in the cytoplasmic tail (C‐tail) of PECAM prevented localization to the LBRC, we first determined whether the C‐tail of PECAM could bring a molecule not expressed on EC into the LBRC. A chimera with the extracellular domain (ECD) of Tac and C‐tail of PECAM localized to junctions, entered the LBRC, and recycled like PECAM. However, the control protein, Tac, entered the LBRC too, even though it was expressed diffusely. This suggested that entrance to the LBRC might be default and VE‐cadherin is selectively excluded. We constructed a VE‐cadherin/PECAM (CP) chimera with the ECD of VE‐cadherin and C‐tail of PECAM. We found CP went to junctions but was excluded from the LBRC, like VE‐cadherin. These findings suggested ECD excluded VE‐cadherin and CP from the LBRC. We then generated truncated VE‐cadherin and CP with deletion of the homophilic interaction domain EC1 or its specific motif RVDAE. Both VE‐cadherin and CP chimera lacking RVDAE motif or EC1 domain DID enter the LBRC. This suggests that homophilic interactions of VE‐cadherin with other VE‐cadherin molecules stabilize it at cell borders and prevent entry into the LBRC. Supported by AHA Postdoctoral Fellowship to GF and R01 HL046849 to WAM