Reversible inactivation of purified leukocyte integrin CR3 (CD11b/CD18, alpha m beta 2) by removal of divalent cations from a cryptic site.
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.
Cai
T Q
TQ
Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York, NY 10021, USA.
Law
S K
SK
Zhao
H R
HR
Wright
S D
SD
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
D000911:Antibodies, Monoclonal; D002413:Cations, Divalent; D016177:Macrophage-1 Antigen; D010446:Peptide Fragments; D004492:Edetic Acid; D008274:Magnesium; D002118:Calcium