Induction of Fibroblast Spreading by Mn2+: a Possible Role for Unusual Binding Sites for Divalent Cations in Receptors for Proteins Containing Arg-Gly-Asp

Mn2+ at low (microM) concentrations fulfils the divalent cation requirement for spreading of BHK21 cells on fibronectin. At much higher concentrations, Mn2+ (and to a small extent also Mg2+) induces spreading on haemoglobin, not normally an adhesive protein. Since high Mn2+ also induces spreading of BHK variants unresponsive to exogenous fibronectin, it is unlikely to be acting as a cofactor for secreted cellular fibronectin or by stimulating its secretion. High Ca2+, but not Mg2+, inhibits the induction of spreading by Mn2+ on haemoglobin. Pre-treatment of cells with high concentrations of trypsin decreases the rate of spreading induced by Mg2+ on fibronectin, and by Mn2+ on haemoglobin, to similar extents. High and low Mn2+ could induce spreading, either by different mechanisms or through a common pathway. In the second case, at both concentrations, Mn2+ could act by binding to Ca2+/Mg2+ sites in one or more receptors for adhesion proteins. This would require binding of Mn2+ or Mg2+ to these sites to activate the receptors in the absence of adhesion proteins, and the effect of such proteins to be to increase the affinity of the sites for metal ions. The sites in question may be formed by sequences homologous to those found in the extracellular domains of the vitronectin receptor and platelet membrane glycoprotein IIb. Although very similar to the Ca2+-binding loop in the EF hand of calmodulin, these sequences more closely resemble bacterial galactose-binding protein in lacking one of the conserved co-ordinating side-chains.