101. Reversible Inactivation of Purified Leukocyte Integrin CR3 (CD11b/CD18, βmβ2) by Removal of Divalent Cations from a Cryptic Site
- Author
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Tian-Quan Cai, Hui-Ren Zhao, S. K. Alex Law, and Samuel D. Wright
- Subjects
musculoskeletal diseases ,Cations, Divalent ,Neutrophils ,Molecular Sequence Data ,Integrin ,Macrophage-1 Antigen ,CD18 ,Complement receptor ,Divalent ,Magnesium ,Cell adhesion ,Receptor ,Edetic Acid ,chemistry.chemical_classification ,Binding Sites ,Base Sequence ,Dose-Response Relationship, Drug ,biology ,Temperature ,Antibodies, Monoclonal ,hemic and immune systems ,General Medicine ,Hydrogen-Ion Concentration ,Ligand (biochemistry) ,Peptide Fragments ,Protein Structure, Tertiary ,Biochemistry ,chemistry ,Integrin alpha M ,biology.protein ,Calcium - 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.
- Published
- 1995