A tyrosine residue on the cytoplasmic tail of endothelial PECAM plays a critical role in leukocyte transmigratio

PECAM function is required for leukocyte transendothelial migration (TEM). While phosphorylation of both cytoplasmic tyrosines 663 and 686 is involved in PECAM signaling in other biologic processes, its role in TEM is unknown. ECV304 cells lack endogenous VE-Cadherin and PECAM and do not support TEM. When these cells are transfected with VE-Cadherin, they make tight monolayers that support leukocyte adhesion but not transmigration. Transfection of VE-Cadherin expressing cells with wild-type PECAM (ECV-WT) yields monolayers with PECAM expressed at the borders that do promote transmigration. This TEM can be blocked by the appropriate anti-PECAM mAb. We generated PECAM constructs bearing Y to F mutations in the cytoplasmic tyrosines and transfected these plasmids into VE-Cadherin bearing ECV304 cells. In cell monolayers expressing Y663F, TEM was reduced by more than 80% compared to cells expressing the wild type PECAM. Mutation of tyrosine 686 had no effect. Immunoprecipitation studies showed that the nonmutated tyrosines on both constructs were phosphorylated. Furthermore, the failure of Y663F to promote TEM was not due to inability to recruit certain downstream signaling molecules.;The reduced ability of the Y663F mutant to support TEM may be due to a defect in signaling or in PECAM trafficking and localization. We were unable to demonstrate that native PECAM in human endothelial cells became phosphorylated during TEM. Therefore we focused our attention on a potential role for the tyrosine in PECAM trafficking. Recycling of PECAM between the junction and a novel surface-connected intracellular membrane compartment is critical for TEM. There was less PECAM in the compartment in Y663F expressing cells and the mutated PECAM had lower kinetics of constitutive recycling compared to ECV WT or Y868F. During TEM across HUVEC, endothelial PECAM is targeted around the transmigrating leukocyte. ECV-WT and Y686F were able to target recycled PECAM around transmigrating monocytes. In contrast, Y663F showed an absence of targeted recycling of PECAM during monocyte TEM.;In summary, we have shown that tyrosine residue 663 on the cytoplasmic tail of endothelial PECAM is necessary for leukocyte TEM to occur. Our data underscore the importance of targeted recycling of PECAM during monocyte transmigration and show that mutation of tyrosine 663 alters PECAM's recycling properties. (Abstract shortened by UMI.).