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© The Rockefeller University Press, 0021-9525/1998//263 $5.00
The Journal of Cell Biology, Volume 142, Number 1, , 1998 263-270

Articles

Dimerization of P-Selectin Glycoprotein Ligand-1 (PSGL-1) Required for Optimal Recognition of P-Selectin



Karen R. Snapp*, Ron Craig{ddagger}, Michael Herron§, Robert D. Nelson§, Lloyd M. Stoolman{ddagger}, and Geoffrey S. Kansas*

* Department of Microbiology/Immunology, Northwestern University Medical School, Chicago, Illinois 60611; {ddagger} Department of Pathology, University of Michigan, Ann Arbor, Michigan 48109; § Department of Dermatology, University of Minnesota, Minneapolis, Minnesota 55455

Interactions between P-selectin, expressed on endothelial cells and activated platelets, and its leukocyte ligand, a homodimer termed P-selectin glycoprotein ligand-1 (PSGL-1), mediate the earliest adhesive events during an inflammatory response. To investigate whether dimerization of PSGL-1 is essential for functional interactions with P-selectin, a mutant form of PSGL-1 was generated in which the conserved membrane proximal cysteine was mutated to alanine (designated C320A). Western blotting under both denaturing and native conditions of the C320A PSGL-1 mutant isolated from stably transfected cells revealed expression of only a monomeric form of PSGL-1. In contrast to cells cotransfected with {alpha}1-3 fucosyltransferase-VII (FucT-VII) plus PSGL-1, K562 cells expressing FucT-VII plus C320A failed to bind COS cells transfected with P-selectin in a low shear adhesion assay, or to roll on CHO cells transfected with P-selectin under conditions of physiologic flow. In addition, C320A transfectants failed to bind chimeric P-selectin fusion proteins. Both PSGL-1 and C320A were uniformly distributed on the surface of transfected K562 cells. Thus, dimerization of PSGL-1 through the single, conserved, extracellular cysteine is essential for functional recognition of P-selectin.

Key Words: selectin • adhesion • endothelium • dimerization • inflammation

Abbreviations used in this paper: ECL, enhanced chemiluminescence; HUVEC, human umbilical vein endothelial cells; ICAM-1, intercellular adhesion molecule 1; LFA-1, Limas flavus agglutinin 1; PSGL-1, P-selectin glycoprotein ligand-1; RT, reverse transcription; TBS-T, TBS-Tween.

Address all correspondence to Geoffrey S. Kansas, Department of Microbiology/Immunology, Northwestern University Medical School; 303 E. Chicago Ave., Chicago, IL 60611. Tel.: (312) 908-3237. Fax: (312) 503-1339. E-mail: gsk{at}nwu.edu


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