The Specificity and Function of the Metal-binding Sites in the Integrin beta sub(3) A-domain

• Published in: The Journal of biological chemistry 2006-08, Vol.281 (32), p.23034-23041
• Main Authors: Pesho, Michelle M, Bledzka, Kamila, Michalec, Lidia, Cierniewski, Czeslaw S, Plow, Edward F
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X

The A-domains within integrin beta subunits contain three metal sites termed the metal ion-dependent adhesion site (MIDAS), site adjacent to the metal ion-dependent adhesion site (ADMIDAS), and ligand-induced metal-binding site (LIMBS), and these sites are involved in ligand engagement. The selectivity of these metal sites and their role in ligand binding have been investigated by expressing a fragment corresponding to the beta sub(3) A-domain, beta sub(3)-(109-352), and single point mutants in which each of the cation-binding sites has been disabled. Equilibrium dialysis experiments identified three Mn super(2+)- and two Ca super(2+)-binding sites with the LIMBS being the site that did not bind Ca super(2+). Although the ADMIDAS could bind Ca super(2+), it did not bind Mg super(2+). These results indicate that the Ca super(2+)-specific site that inhibits ligand binding is the ADMIDAS. Two different assay systems, surface plasmon resonance and a microtiter plate assay, demonstrated that the beta sub(3) A-domain fragment bound fibrinogen in the presence of 0.1 mM Ca super(2+) but not in 3 mM Ca super(2+). This behavior recapitulated the effects of Ca super(2+) on fibrinogen binding to alpha sub(v) beta sub(3) but not alpha sub(IIb) beta sub(3). Disabling any of the three cation-binding sites abrogated fibrinogen binding. These results indicate that the specificities of the three metal-binding sites for divalent cations are distinct and that each site can regulate the ligand binding potential of the beta sub(3) A-domain.