Synergistic Ca2+ and Cu2+ requirements of the FGF1-S100A13 interaction measured by quartz crystal microbalance:: An initial step in amlexanox-reversible non-classical release of FGF1

it is known that fibroblast growth factor-1 (FGF1) lacking a conventional signal peptide sequence shows non-classical release independent of the endoplasmic reticulum-Golgi system. Recent studies reveal that FGF1 is co-released with S100A13, a Ca2+-binding protein that acts as an extracellular cargo molecule. Although both FGFI and S100A13 are Cu2+-binding proteins, the role of Cu2+, as well as that of Ca2+ in nonclassical release, remains to be clarified. In the present study we examined the requirements of both metal ions for the interaction between these two proteins. The addition of Ca2+ significantly increased the k(a) value, while decreasing the K-D value, for the interaction between Strep-tagII-S100A13 and GST-FGF1; both values were obtained by use of a quartz crystal microbalance, a real-time mass-measuring device. The EC50 of Ca2+ to enhance the interaction was 10.11 mu M. Although the addition of Cu2+ alone had no effect, it caused a marked potentiation of the Ca2+-enhanced interaction. The EC50 Of Cu2+ for the potentiation was 50.45 nM. On the other hand, the EC50 of Ca2+ and the K-D values were decreased from 11.69 to 2.07 mu M and 0.75 to 0.38 x 10(-7) M, respectively, by the addition of 200 nM Cu2+. The Cu2+-induced potentiation of this interaction was abolished by amlexanox, which inhibits non-classical release of FGFI. All of these findings suggest that synergistic effects of Ca2+ and Cu2+ play a key role in the interaction between FGFI and S100A13, which is the initial step in non-classical release of FGF1. (C) 2007 Elsevier Ltd. All rights reserved.