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PECAM Tyrosine 663 is Critical for Transendothelial Migration
Author(s) -
Dasgupta Bidisha,
Mamdouh Zahra,
Muller William A.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a127-c
Subject(s) - transfection , phosphorylation , microbiology and biotechnology , tyrosine phosphorylation , cadherin , intracellular , cytoplasm , immunoprecipitation , cell culture , biology , chemistry , cell , biochemistry , genetics
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 VE‐Cadherin and PECAM and do not support TEM. When these cells are transfected with VE‐Cadherin, they form confluent 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 is blocked by anti‐PECAM mAb. PECAM constructs bearing Y to F mutations in cytoplasmic tyrosines were transfected into VE‐Cadherin bearing ECV304. In monolayers expressing Y663F, TEM was reduced by more than 80% compared to cells expressing the wild type PECAM. Mutation of Y 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. Recycling of PECAM between the junction and a novel surface‐connected intracellular membrane compartment is critical for TEM (Mamdouh et. al, Nature 2003). In HUVEC phosphorylated PECAM was predominantly located in this compartment. The Y663F expressing cells had less PECAM in this compartment and the mutated PECAM had slower kinetics of constitutive recycling compared to ECV WT or Y868F. During TEM across HUVEC recycling endothelial PECAM is targeted around the transmigrating leukocyte. ECV‐WT and Y686F were able to target recycling PECAM around transmigrating monocytes, but Y663F could not. Our data underscore the importance of targeted recycling of PECAM during monocyte transmigration and show that mutation of Y 663 alters PECAM's recycling properties.