The interacting binding domains of the β4 integrin and calcium-activated chloride channels (CLCAs) in metastasis

CLCA (chloride channel, calcium-activated) proteins are novel pulmonary vascular addresses for bloodborne, lung-metastatic cancer cells. They facilitate vascular arrest of cancer cells via adhesion to beta(4) integrin and promote early, intravascular, metastatic growth. Here we identify the interacting binding domains of endothelial CLCA proteins (e.g. hCLCA2, mCLCA5, mCLCA1, and bCLCA2) and beta(4) integrin. Endothelial CLCAs share a common beta(4)-binding motif (beta(4)BM) in their 90- and 35-kDa subunits of the sequence F(S/N) R(I/L/ V)(S/T) S, which is located in the second extracellular domain of the 90-kDa CLCA and near the N terminus of the 35-kDa CLCA, respectively. Using enzyme-linked immunosorbent, pull-down, and adhesion assays, we showed that glutathione S-transferase fusion proteins of beta(4)BMs from the 90- and 35-kDa CLCA subunits bind to the beta(4) integrin in a metal ion-dependent manner. Fusion proteins from fibronectin and the integrins beta(1) and beta(3) served as negative controls. beta(4)BM fusion proteins competitively blocked the beta(4)/CLCA adhesion and prevented lung colonization of MDA-MB-231 breast cancer cells. A disrupted beta(4)BM in hCLCA1, which is not expressed in endothelia, failed to interact with beta(4) integrin. The corresponding CLCA-binding domain of the beta(4) integrin is localized to the specific determining loop (SDL). Again enzyme-linked immunosorbent, pull-down, and adhesion assays were used to confirm the interaction with CLCA proteins using a glutathione S-transferase fusion protein representing the C-terminal two-thirds of beta(4) SDL (amino acids 184 - 203). A chimeric beta(4) integrin in which the indicated SDL sequence had been replaced with the corresponding sequence from the beta(1) integrin failed to bind hCLCA2. The dominance of the CLCA ligand in beta(4) activation and outside-in signaling is discussed in reference to our previous report that beta(4)/CLCA ligation elicits selective signaling via focal adhesion kinase to promote metastatic growth.