Effect of divalent cations on the affinity and selectivity of alpha4 integrins towards the integrin ligands vascular cell adhesion molecule-1 and mucosal addressin cell adhesion molecule-1: Ca2+ activation of integrin alpha4beta1 confers a distinct ligand specificity.
A microtiter plate assay measuring the binding of cells expressing integrins alpha4beta1 or alpha4beta7 to VCAM-1 and MAdCAM-1, expressed as Ig fusion proteins, was used to explore the interplay between the variables of integrin beta-chain, identity and density of ligand, and identity and concentration of activating cations. Both Mn2+ and Mg2+ supported binding of either integrin to either ligand. Ca2+ supported only the binding of alpha4beta1 to VCAM-Ig. Cation concentrations required for half-maximal binding (EC50) ranged from 0.8-280 microM for Mn2+ and 0.8-30 mM for Mg2+, being thus 2-3 logs lower for Mn2+ compared to Mg2+ independent of ligand. EC50 values for binding of alpha4beta1 to VCAM-Ig were 30-45-fold lower compared to MAdCAM-Ig, while alpha4beta7 showed an opposite 3-15-fold selectivity for MAdCAM-Ig over VCAM-Ig. The density of ligand required for adhesion via alpha4beta1 was markedly lower with Mn2+ versus Mg2+, and with VCAM-Ig versus MAdCAM-Ig. These results were interpreted in terms of a coupled equilibrium model, in which binding of activating metal ions and of integrin ligands each stabilizes activated integrin. We conclude that Mn2+ and Mg2+ bind to common regulatory sites with different affinities, producing similar activated states of the integrin. The resulting activated alpha4beta1 binds more strongly to VCAM-Ig versus MAdCAM-Ig by 30-45-fold, while similarly activated alpha4beta7 binds more strongly to MAdCAM-Ig versus VCAM-Ig by 3-15-fold. Inhibition studies showed that Ca2+ also binds to regulatory sites on both integrins. However, the Ca2+-activated state of alpha4beta1 is distinct from that achieved by Mn2+ and Mg2+, possessing increased selectivity for binding to VCAM-1 versus MAdCAM-1.
Day
Eric S
ES
BIOGEN Inc., Cambridge, MA 02142, USA.
Osborn
Laurelee
L
Whitty
Adrian
A
D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology / Q000502:physiology; D002448:Cell Adhesion / Q000502:physiology; D015815:Cell Adhesion Molecules; D006801:Humans; D007136:Immunoglobulins / Q000276:immunology / Q000378:metabolism*; D066298:In Vitro Techniques; D039041:Integrin alpha4beta1 / Q000819:agonists / Q000378:metabolism* / Q000502:physiology; D016023:Integrins / Q000819:agonists / Q000378:metabolism* / Q000502:physiology; D019169:Jurkat Cells; D016393:Lymphoma, B-Cell; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology; D008345:Manganese / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology; D009088:Mucoproteins / Q000276:immunology / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D019010:Vascular Cell Adhesion Molecule-1 / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D015815:Cell Adhesion Molecules; D007136:Immunoglobulins; D039041:Integrin alpha4beta1; D016023:Integrins; C504265:MADCAM1 protein, human; D009088:Mucoproteins; D019010:Vascular Cell Adhesion Molecule-1; C071777:integrin alpha4beta7; D008345:Manganese; D008274:Magnesium; D002118:Calcium