Cytoplasmic domain of E-selectin contains a non-tyrosine endocytosis signal

E-selectin is an activation-dependent, endothelial cell-restricted adhesion molecule that is internalized and degraded rapidly once expressed on the cell surface. Tyrosine-containing structural motifs play an important role in the internalization of a number of integral proteins, and the membrane-proximal E-selectin cytoplasmic tyrosine residue (Tyr582) conforms to the endocytosis motif proposed previously. To determine the endocytosis motif in E-selectin, we selectively introduced truncation, substitution, and deletion mutations to the cytoplasmic tail of E-selectin. We analyzed the internalization kinetics of surface-expressed wild-type and mutant E-selectin constructs in transiently transfected Chinese hamster ovary cells using 125I-labeled E-selectin monoclonal antibody (125I-P6E2) in an acid elution assay. Interestingly, truncation immediately membrane proximal to Tyr582 (DeltaDGS construct) did not alter internalization kinetics significantly (DeltaDGS versus wild-type, mean surface half-life = 42 versus 45 min, respectively). Thus, it appears that the tyrosine residues are not required for internalization of E-selectin. Additional analyses indicated that Ser581 was necessary but alone was insufficient for surface E-selectin endocytosis. Thus, we conclude that there exists a novel non-tyrosine-containing endocytosis signal in the cytoplasmic tail which involves Ser581 and residues membrane-proximal to it.