Nearly all epithelial cells utilize the integrin alpha6beta4 to provide a critical connection between extracellular laminin and intracellular intermediate filaments, thereby helping to maintain typical polarized epithelial architecture. Upon disassembly of alpha6beta4 complexes in normal epithelia, the integrin engages in a new series of molecular interactions that promote epithelial cell invasion and tumorigenesis. Consistent with this, alpha6beta4 is upregulated during epithelial mesenchymal transition (EMT). Our preliminary data indicates that certain laminin-binding integrins, including alpha6beta4 are atypical among integrins insofar as being palmitoylated, and associating laterally with other transmembrane proteins, such as tetraspanin proteins. Furthermore, available data strongly supports our hypothesis that for the lamininbinding integrins, lateral associations with other proteins controls integrin-dependent adhesion strength, signaling, and cell morphology. To test this hypothesis, we will 1) make three different types of mutations in alpha6beta4 (palmitoylation sites, tetraspanin-association sites) to disrupt lateral associations.Then we will examine the effects of such mutations on 2) tyrosine and serine phosphorylation of alpha6beta4, and 3) signaling through phosphoinositide 3-kinase, Akt, Src-family kinases, and growth factor receptors. 4) Also we will examine mutation effects on cell adhesion, adhesion strength, motility, hemidesmosome formation, and acini formation in vitro, and 5) on spontaneous carcinoma formation in mice in vivo. Only recently have we begun to appreciate that integrins (particulary the laminin-binding integrins) can be regulated in the lateral dimension by specific interactions with other transmenbrane proteins, in the context of a novel type of membrane microdomain. The work proposed in this grant application will help to fill in important gaps in our knowledge regarding the contribution of integrin microdomains. Furthermore, we should gain new insights into alpha6beta4 as it plays such a pivotal role in the transition of normal epithelial cells from a stable, polarized state to an invasive carcinoma. Finally, our uncovering of specific alpha6beta4 domains and associated pathways that are critical during carcinogenesis may suggest novel targets for therapeutic intervention.
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