Dimerization of P-selectin glycoprotein ligand-1 (PSGL-1) required for optimal recognition of P-selectin.
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 alpha1-3 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.
Snapp
K R
KR
Department of Microbiology/Immunology, Northwestern University Medical School, Chicago, Illinois 60611, USA.
Craig
R
R
Herron
M
M
Nelson
R D
RD
Stoolman
L M
LM
Kansas
G S
GS
D000409:Alanine / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002448:Cell Adhesion; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D019281:Dimerization; D005647:Fucosyltransferases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D017351:N-Acetylglucosaminyltransferases / Q000096:biosynthesis; D019007:P-Selectin / Q000378:metabolism*; D012995:Solubility; D014407:Tumor Cells, Cultured
D008024:Ligands; D008562:Membrane Glycoproteins; D019007:P-Selectin; C084882:P-selectin ligand protein; D005647:Fucosyltransferases; D017351:N-Acetylglucosaminyltransferases; C037649:N-acetyllactosaminide beta-1,6-N-acetylglucosaminyltransferase; C031692:galactoside 3-fucosyltransferase; D003545:Cysteine; D000409:Alanine