title: Crucial role of the specificity-determining loop of the integrin beta4 subunit in the binding of cells to laminin-5 and outside-in signal transduction.
abstract: Within each hemidesmosome, alpha6beta4 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 beta integrin in regulating ligand binding. Here, we investigated the function of an SDL-like motif within the extracellular I-like domain of beta4 integrin. We generated point mutations within the SDL of beta4 integrin tagged with green fluorescent protein (GFP-beta4K150A and GFP-beta4Q155L). We also generated a mutation within the I-like domain of the beta4 integrin, lying outside the SDL region (GFP-beta4V284E). We transfected constructs encoding the mutated beta4 integrins and a GFP-conjugated wild type beta4 integrin (GFP-beta4WT) into 804G cells, which assemble hemidesmosomes, and human endothelial cells, which express little endogenous beta4 integrin. In transfected 804G cells, GFP-beta4WT and GFP-beta4V284E colocalize with hemidesmosome proteins, whereas hemidesmosomal components in cells expressing GFP-beta4K150A and GFP-beta4Q155L are aberrantly localized. In endothelial cells, GFP-beta4WT and mutant proteins are co-expressed at the cell surface with alpha6 integrin. When transfected endothelial cells are plated onto laminin-5 matrix, GFP-beta4WT and GFP-beta4V284E localize with laminin-5, whereas GFP-beta4K150A and GFP-beta4Q155L do not. GFP-beta4WT and GFP-beta4V284E expressed in endothelial cells associate with the adaptor protein Shc when the cells are stimulated with laminin-5. However, GFP-beta4K150A and GFP-beta4Q155L 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 beta4 integrin subunit is required for ligand binding and is involved in outside-in signaling.
authors.affiliation: Departments of Cell and Molecular Biology and Medicine, Feinberg School of Medicine at Northwestern University, Chicago, Illinois 60611, USA.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Tsuruta
authors.forename: Susan B
authors.initials: SB
authors.lastname: Hopkinson
authors.forename: Kimberly D
authors.initials: KD
authors.lastname: Lane
authors.forename: Michael E
authors.initials: ME
authors.lastname: Werner
authors.forename: Vincent L
authors.initials: VL
authors.lastname: Cryns
authors.forename: Jonathan C R
authors.initials: JC
authors.lastname: Jones
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002469:Cell Separation; D003593:Cytoplasm / Q000378:metabolism; D003896:Desmosomes / Q000378:metabolism; D004727:Endothelium / Q000378:metabolism; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D015151:Immunoblotting; D039641:Integrin beta Chains / Q000378:metabolism; D039663:Integrin beta4 / Q000737:chemistry* / Q000502:physiology*; D007797:Laminin / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D017354:Point Mutation; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014162:Transfection
chemical_list: D039641:Integrin beta Chains; D039663:Integrin beta4; D007797:Laminin; D008024:Ligands; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins