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Abstract
Integrin α6β4–mediated adhesion interactions play key roles in keratinocyte and epithelial tumor cell biology. In order to evaluate how α6β4 adhesion interactions contribute to these important cellular processes, the authors generated soluble versions of the integrin by recombinant expression of the subunit ectodomains fused to a human immunoglobulin G (IgG) Fc constant domain. Coexpression of the appropriate subunits enabled dimerization, secretion and purification of stable Fc-containing α6β4 heterodimers. The soluble proteins exhibited the same metal ion and ligand dependency in their binding characteristics as intact α6β4. Using these reagents in combination with anti-β4 antibodies, the authors identified two distinct functional epitopes on the β4 subunit. They demonstrated the involvement of one epitope in adhesion interactions and the other in regulating adhesion-independent growth in α6β4-expressing tumor cell lines. The availability of these soluble integrin reagents and the data provided herein help to further delineate the structure-function relationships regulating α6β4 signaling biology.
Notes
1Abbreviations: BSA, bovine serum albumin; CHO, chinese hamster ovary; CMV, cytomegalovirus; DTT, dithiothreitol; ELISA, enzyme-linked immunosorbent assay; Erk, extracellular signal-related kinase; FACS, fluorescence-activated cell sorting; FAK, focal adhesion kinase; FBS, fetal bovine serum; HPLC, high-performance liquid chromatography; HRP, horseradish peroxidase; Ig, immunoglobulin; IE, intermediate-early; ILK, integrin-linked kinase; IRS2, insulin receptor substrate 2; LTβR, lymphotoxin beta receptor; MEM, minimum essential medium; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PE, phycoerythrin; PI3K, phosphoinositide 3-kinase; PMSF, phenyl methane sulfonyl fluoride; PNGase F, peptide-N-glycosidase F; PTH, phenylthionhydantoin; RU, resonance units; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SEC, size exclusion chromatography; Shc, Src homology domain containing; TBS, Tris-buffered saline; VCAM, vascular cell adhesion molecule.