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.
Chuang
P I
PI
Department of Medicine, University of Washington, Seattle, Washington 98195, USA. ichuang@u.washington.edu
Young
B A
BA
Thiagarajan
R R
RR
Cornejo
C
C
Winn
R K
RK
Harlan
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D016384:Consensus Sequence; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D019040:E-Selectin / Q000737:chemistry* / Q000378:metabolism*; D004705:Endocytosis; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D015398:Signal Transduction; D013552:Swine; D014162:Transfection; D014443:Tyrosine
D019040:E-Selectin; D011994:Recombinant Proteins; D014443:Tyrosine