title: Attachment of the PSGL-1 cytoplasmic domain to the actin cytoskeleton is essential for leukocyte rolling on P-selectin.
abstract: 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-1 Delta cyto), 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-1 Delta cyto, 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-1 Delta cyto transfectants was intact and was capable of recognizing P-selectin. Impaired rolling by PSGL-1 Delta cyto 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-1 Delta cyto- 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.
authors.affiliation: Department of Microbiology/Immunology, Northwestern University Medical School, Chicago, IL 60611, USA. krs133@northwestern.edu
authors.forename: Karen R
authors.initials: KR
authors.lastname: Snapp
authors.forename: Christine E
authors.initials: CE
authors.lastname: Heitzig
authors.forename: Geoffrey S
authors.initials: GS
authors.lastname: Kansas
mesh_terms: D000199:Actins / Q000378:metabolism* / Q000502:physiology; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism / Q000502:physiology; D006801:Humans; D007962:Leukocytes / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D019007:P-Selectin / Q000378:metabolism* / Q000502:physiology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019311:Staurosporine / Q000494:pharmacology; D014162:Transfection
chemical_list: D000199:Actins; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D019007:P-Selectin; C084882:P-selectin ligand protein; C071058:moesin; D019311:Staurosporine