Airway epithelial injury plays an important role in a number of common airway diseases. Interactions with extracellular matrix are central to tissue repair. We have thus begun to characterize airway epithelial cell surface receptors for matrix proteins. Given the unique environment in which these cells function, we hypothesized that they might possess novel adhesion receptors. The homology based polymerase chain reaction (PCR) was used to amplify DNA fragments encoding 3 novel subunits of members of the integrin family of adhesion receptors from airway epithelial cells. One of these, beta6, pairs with the known integrin alpha subunit, alpha/v, to form one of the principal fibronectin-binding proteins in airway epithelial cells. This proposal will further characterize the cellular distribution, extracellular ligand(s), regulation, and functional significance of this novel integrin heterodimer (alpha/v beta6). PCR and immunofluorescence microscopy will be used to characterize the distribution of alpha/v beta6, and its relationship to other alpha/v -containing integrins. The extracellular ligand(s) of alpha/v beta6 will be determined by affinity chromatography and the functional significance of alpha/v beta6 in cell attachment to these ligands and in cell migration into wounds will be assessed. Previous data from this laboratory has shown that airway epithelial cells respond in a highly polarized fashion to the cytokine transforming growth factor beta1 (TGFbeta1), a known stimulus to the regulation of other integrins. In this proposal, the polarity of regulation of alpha/v beta6 and other alpha/v-containing integrins will be examined by Northern blotting and by selective biotinylation of either the apical or basolateral surface. In vivo regulation of alpha/v-containing integrins will be assessed by tissue immunofluorescence in a model of airway epithelial injury and repair. The effects of heterologous expression of alpha/v beta6 will be examined in both fibroblasts (NIH 3T3), and epithelial cells (HeLa), to determine the role of this protein in expression of the epithelial phenotype and the importance of other epithelial characteristics for full functional expression. Finally, the role of the unique cytoplasmic domain of beta6 will be examined by affinity chromatography, and by expression of mutants containing cytoplasmic deletions and chimeric proteins composed of the beta6 extracytoplasmic domain and the cytoplasmic domain of the alternative alpha/v partners, beta3 and beta5. Through these studies we hope to learn more about the functional significance of one of the principal adhesion receptors on airway epithelial cells as a first step toward determining the role of adhesion receptors in the deranged repair processes that characterize the development of airway diseases.
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