Regulation of Integrin 
5
1-Fibronectin Interactions by Divalent Cations
EVIDENCE FOR DISTINCT CLASSES OF BINDING SITES FOR Mn
, Mg
, AND Ca
(*)
- From the Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Manchester, M13 9PT, United Kingdom Laboratory of Developmental Biology, NIDR, National Institutes of Health, Bethesda, Maryland 20892
- § To whom correspondence should be addressed: School of Biological Sciences, University of Manchester, 2.205 Stopford Bldg., Oxford Rd., Manchester, M13 9PT, United Kingdom. Tel.: 44-161-275-5071; Fax: 44-161-275-5082.
Abstract
Integrin-ligand interactions are known to be dependent on divalent cations, although the precise role of cations in ligand
binding is still unclear. Using the interaction between α5β1 and fibronectin as a model system, we have performed a comprehensive
analysis of the effects of Mn
, Mg
, and Ca
on ligand binding. Each cation had distinct effects on the ligand-binding capacity of α5β1: Mn
promoted high levels of ligand binding, Mg
promoted low levels of binding, and Ca
failed to support binding. Studies of the effects of different combinations of cations on ligand binding indicated that the
cation-binding sites within α5β1 are not all identical, or of broad specificity, but instead each site shows a distinct preference
for one or more cations. Ca
strongly inhibited Mn
-supported ligand binding, but this inhibition was noncompetitive, suggesting that Ca
recognizes different cation-binding sites to Mn
. In contrast, Ca
acted as a direct competitive inhibitor of Mg
-supported ligand binding, implying that Ca
can displace Mg
from the integrin. However, low concentrations of Ca
greatly increased the apparent affinity of Mg
for its binding site, suggesting the existence of a distinct high affinity Ca
-binding site. Taken together, our results imply that the ligand-binding capacity of α5β1 can be regulated in a complex manner
through separate classes of binding sites for Mn
, Mg
, and Ca
.
Footnotes
-
↵* This work was supported by grants from the Wellcome Trust (to M. J. H. and A. P. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 The abbreviations used are:
- mAb
-
monoclonal antibody
- CCBD
-
central cell-binding domain of fibronectin
- BSA
-
bovine serum albumin
- MES
-
4-morpholinoethane-sulfonic acid
- ELISA
-
enzyme-linked immunosorbent assay.
-
↵2 A. P. Mould, A. N. Garrat, J. A. Askari, S. K. Akiyama, and M. J. Humphries, manuscript in preparation.
-
- Received June 19, 1995.
- Revision received August 4, 1995.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
