The Interacting Binding Domains of the β4 Integrin and Calcium-activated Chloride Channels (CLCAs) in Metastasis

Abstract CLCA (chloride channel, calcium-activated) proteins are novel pulmonary vascular addresses for blood-borne, lung-metastatic cancer cells. They facilitate vascular arrest of cancer cells via adhesion to β4 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 β4 integrin. Endothelial CLCAs share a common β4-binding motif (β4BM) 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 β4BMs from the 90- and 35-kDa CLCA subunits bind to the β4 integrin in a metal ion-dependent manner. Fusion proteins from fibronectin and the integrins β1 and β3 served as negative controls. β4BM fusion proteins competitively blocked the β4/CLCA adhesion and prevented lung colonization of MDA-MB-231 breast cancer cells. A disrupted β4BM in hCLCA1, which is not expressed in endothelia, failed to interact with β4 integrin. The corresponding CLCA-binding domain of the β4 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 β4 SDL (amino acids 184–203). A chimeric β4 integrin in which the indicated SDL sequence had been replaced with the corresponding sequence from the β1 integrin failed to bind hCLCA2. The dominance of the CLCA ligand in β4 activation and outside-in signaling is discussed in reference to our previous report that β4/CLCA ligation elicits selective signaling via focal adhesion kinase to promote metastatic growth.