Attachment of the PSGL-1 cytoplasmic domain to the actin cytoskeleton is essential for leukocyte rolling on P-selectin

P-selectin glycoprotein ligand-1 (PSGL-1) serves as the leukocyte ligand for P-selectin, and many of the structural features of its ectodomain required for interactions with P-selectin have been uncovered. In contrast, the function of the highly conserved PSGL-1 cytoplasmic domain has not been explored, Stable transfectants expressing similar levels of either wild-type PSGL-1 or truncated PSGL-1 in which only 4 cytoplasmic residues were retained (designated PSGL-1Deltacyto), were analyzed. Transfectants expressing full-length PSGL-1 rolled well on P-selectin. In contrast, rolling was almost completely absent in cells transfected with PSGL-1Deltacyto, even at low shear. Importantly, cells expressing truncated PSGL-1 were able to bind soluble P-selectin and to bind COS cells overexpressing P-selectin, demonstrating that the P-selectin binding site on the PSGL-1Deltacyto transfectants was intact and was capable of recognizing P-selectin. Impaired rolling by PSGL-1Deltacyto transfectants was not due to alterations in subcellular localization because both wild-type and truncated PSGL-1 had similar surface distributions on K562 transfectants. Treatment of cells expressing native PSGL-1 with actin cytoskeletal toxins inhibited adhesion in a dose-dependent way. PSGL-1 was associated with the actin cytoskeleton, and this interaction was greatly impaired In PSGL-1Deltacyto-expressing cells. The PSGL-1 cytoplasmic domain Interacted selectively with the ezrin/radixin/moesin (ERM) protein moesin, but not with other ERM proteins or several other cytoskeletal linker proteins. Pharmacologic disruption of interactions between moesin and F-actin In cells expressing PSGL-1 resulted In a dose-dependent Inhibition of rolling on P-selectin. Thus, attachment of PSGL-1 to the leukocyte cortical cytoskeleton is essential for leukocyte rolling on P-selectin. (Blood. 2002;99:4494-4502) (C) 2002 by The American Society of Hematology.