title: Neutrophil recognition requires a Ca(2+)-induced conformational change in the lectin domain of GMP-140.
abstract: GMP-140, a receptor for myeloid cells that is expressed on surfaces of thrombin-activated platelets and endothelial cells, is a member of the selectin family of adhesion molecules that regulate leukocyte interactions with the blood vessel wall. Each selectin contains an N-terminal domain homologous to Ca(2+)-dependent lectins and mediates cell-cell contact by binding to oligosaccharide ligands in a Ca(2+)-dependent manner. The mechanisms by which Ca2+ promotes selectin-dependent cellular interactions have not been defined. We demonstrate that purified GMP-140 contains two high affinity binding sites for Ca2+ as measured by equilibrium dialysis (Kd = 22 +/- 2 microM). Occupancy of these sites by Ca2+ alters the conformation of the protein as detected by a reduction in intrinsic fluorescence emission intensity (Kd = 4.8 +/- 0.2 microM). This Ca(2+)-dependent conformational change exposes an epitope spanning residues 19-34 of the lectin domain that is recognized by a monoclonal antibody capable of blocking neutrophil adhesion to GMP-140 (half-maximal antibody binding at approximately 20 microM Ca2+). Furthermore, a synthetic peptide encoding this epitope, CQNRYTDLVAIQNKNE, inhibits neutrophil binding to GMP-140. Mg2+ also alters the conformation of the protein, but not in a manner that will support leukocyte recognition in the absence of Ca2+. There is a strong correlation between the Ca2+ levels required for neutrophil adhesion to GMP-140, for occupancy of the two Ca(2+)-binding sites, for the fluorescence-detected conformational change, and for exposure of the antibody epitope in the lectin domain. We conclude that binding of Ca2+ to high affinity sites on GMP-140 modulates the conformation of the lectin domain in a manner that is essential for leukocyte recognition.
authors.affiliation: Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City 73104.
authors.forename: J G
authors.initials: JG
authors.lastname: Geng
authors.forename: K L
authors.initials: KL
authors.lastname: Moore
authors.forename: A E
authors.initials: AE
authors.lastname: Johnson
authors.forename: R P
authors.initials: RP
authors.lastname: McEver
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000378:metabolism; D001792:Blood Platelets / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology*; D002413:Cations, Divalent; D002460:Cell Line; D004492:Edetic Acid / Q000494:pharmacology; D000939:Epitopes / Q000032:analysis; D006801:Humans; D007700:Kinetics; D037102:Lectins; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000502:physiology*; D019007:P-Selectin; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D010980:Platelet Membrane Glycoproteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D002413:Cations, Divalent; D000939:Epitopes; D037102:Lectins; D019007:P-Selectin; D010455:Peptides; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; D004492:Edetic Acid; D002118:Calcium