Crucial Role of the Specificity-determining Loop of the Integrin β4 Subunit in the Binding of Cells to Laminin-5 and Outside-in Signal Transduction

Abstract Within each hemidesmosome, α6β4 integrin plays a crucial role in hemidesmosome assembly by binding to laminin-5 in the basement membrane zone of epithelial tissue. Recent analyses have implicated “specificity-determining loops” (SDLs) in the I-like domain of β integrin in regulating ligand binding. Here, we investigated the function of an SDL-like motif within the extracellular I-like domain of β4 integrin. We generated point mutations within the SDL of β4 integrin tagged with green fluorescent protein (GFP-β4K150A and GFP-β4Q155L). We also generated a mutation within the I-like domain of the β4 integrin, lying outside the SDL region (GFP-β4V284E). We transfected constructs encoding the mutated β4 integrins and a GFP-conjugated wild type β4 integrin (GFP-β4WT) into 804G cells, which assemble hemidesmosomes, and human endothelial cells, which express little endogenous β4 integrin. In transfected 804G cells, GFP-β4WT and GFP-β4V284E colocalize with hemidesmosome proteins, whereas hemidesmosomal components in cells expressing GFP-β4K150A and GFP-β4Q155L are aberrantly localized. In endothelial cells, GFP-β4WT and mutant proteins are co-expressed at the cell surface with α6 integrin. When transfected endothelial cells are plated onto laminin-5 matrix, GFP-β4WT and GFP-β4V284E localize with laminin-5, whereas GFP-β4K150A and GFP-β4Q155L do not. GFP-β4WT and GFP-β4V284E expressed in endothelial cells associate with the adaptor protein Shc when the cells are stimulated with laminin-5. However, GFP-β4K150A and GFP-β4Q155L fail to associate with Shc even when laminin-5 is present, thus impacting downstream signaling. These results provide evidence that the SDL segment of the β4 integrin subunit is required for ligand binding and is involved in outside-in signaling.