Regulation of integrin function: evidence that bivalent-cation-induced conformational changes lead to the unmasking of ligand-binding sites within integrin alpha5 beta1.
The molecular mechanisms that regulate integrin-ligand binding are unknown; however, bivalent cations are essential for integrin activity. According to recent models of integrin tertiary structure, sites involved in ligand recognition are located on the upper face of the seven-bladed beta-propeller formed by the N-terminal repeats of the alpha subunit and on the von Willebrand factor A-domain-like region of the beta subunit. The epitopes of function-altering monoclonal antibodies (mAbs) cluster in these regions of the alpha and beta subunits; hence these mAbs can be used as probes to detect changes in the exposure or shape of the ligand-binding sites. Bivalent cations were found to alter the apparent affinity of binding of the inhibitory anti-alpha5 mAbs JBS5 and 16, the inhibitory anti-beta1 mAb 13, and the stimulatory anti-beta1 mAb 12G10 to alpha5 beta1. Analysis of the binding of these mAbs to alpha5beta1 over a range of Mn2+, Mg2+ or Ca2+ concentrations demonstrated that there was a concordance between the ability of cations to elicit conformational changes and the ligand-binding potential of alpha5 beta1. Competitive ELISA experiments provided evidence that the domains of the alpha5 and beta1 subunits recognized by mAbs JBS5/16 and 13/12G10 are spatially close, and that the distance between these two domains is increased when alpha5 beta1 is occupied by bivalent cations. Taken together, our findings suggest that bivalent cations induce a conformational relaxation in the integrin that results in exposure of ligand-binding sites, and that these sites lie near an interface between the alpha subunit beta-propeller and the beta subunit putative A-domain.
Mould
A P
AP
Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Manchester, M13 9PT, UK. pmould@fs1.scg.man.ac.uk
Garratt
A N
AN
Puzon-McLaughlin
W
W
Takada
Y
Y
Humphries
M J
MJ
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001665:Binding Sites / Q000502:physiology; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent / Q000494:pharmacology*; D018604:Epitope Mapping; D016023:Integrins / Q000737:chemistry*; D008024:Ligands; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D017354:Point Mutation / Q000235:genetics; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000187:drug effects
D000911:Antibodies, Monoclonal; D002413:Cations, Divalent; D016023:Integrins; D008024:Ligands; D008345:Manganese; D008274:Magnesium; D002118:Calcium