title: Reversible inactivation of purified leukocyte integrin CR3 (CD11b/CD18, alpha m beta 2) by removal of divalent cations from a cryptic site.
abstract: Integrins exhibit reversible changes in their ability to bind ligands and these changes enable transient cell adhesion. We recently showed that leukocyte integrin CR3 (complement receptor type three, CD11b/CD18, alpha m beta 2) may be purified in a form that is either capable or incapable of binding soluble, monomeric ligand and that "inactive" CR3 may be rendered capable of binding ligand by addition of an anti-CR3 mAb known as KIM-127 (Cai and Wright, JBC. 270: 14358, 1995). Here, we demonstrate that active CR3 may be rendered inactive by treatment of immobilized receptor with EDTA. EDTA-treated CR3 failed to bind ligand even in the presence of mM Ca2+ and Mg2+, suggesting that EDTA-treatment caused a change in the receptor that is not readily reversed. EDTA-treated receptor did, however, bind ligand upon addition of KIM-127 plus Mg2+ with an affinity (17.8 +/- 4.5 nM) similar to untreated, active receptor (12.5 +/- 4.7 nM). EDTA-treated CR3 thus exhibits the properties of inactive CR3, in which the ligand binding site is cryptic but subject to exposure by KIM-127. A candidate for the cryptic ligand binding site is the I-domain, a Mg2+-binding region in the alpha chain of CR3. We found that monomeric C3bi binds directly to recombinant I-domain in a Mg(2+)-dependent fashion with an affinity of 300 +/- 113 nM. These results thus suggest that CR3 may be inactivated by removing tightly bound divalent cation from a cryptic site in CR3.
authors.affiliation: Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York, NY 10021, USA.
authors.forename: T Q
authors.initials: TQ
authors.lastname: Cai
authors.forename: S K
authors.initials: SK
authors.lastname: Law
authors.forename: H R
authors.initials: HR
authors.lastname: Zhao
authors.forename: S D
authors.initials: SD
authors.lastname: Wright
mesh_terms: D000911:Antibodies, Monoclonal; D001483:Base Sequence; D001665:Binding Sites / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid; D006863:Hydrogen-Ion Concentration; D016177:Macrophage-1 Antigen / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D013696:Temperature
chemical_list: D000911:Antibodies, Monoclonal; D002413:Cations, Divalent; D016177:Macrophage-1 Antigen; D010446:Peptide Fragments; D004492:Edetic Acid; D008274:Magnesium; D002118:Calcium