| Popis: |
Integrins mediate adhesive interactions between cells and the extracellular matrix (ECM), and play central roles in organizing the cytoskeleton, and in signal transduction controlling growth, migration, proliferation and differentiation. This thesis examines the contribution of the transmembrane domain to integrin signalling by characterizing the transmembrane interaction between [beta]1 integrin and the 16 kDa subunit of the V-ATPase (16K). This interaction was first identified in a yeast two-hybrid screen and confirmed by immunoprecipitation and in 'in vitro' binding assays using bacterially expressed proteins. Immunofluorescent studies in L6 myoblasts demonstrated co-localization of 16K with [beta]1 integrin in focal adhesions and intracellular vesicles. Overexpression of 16K, or expression of a mutant form of 16K which lacks the fourth of four transmembrane helices and is unable to bind [beta]1 integrin (TM4), alters the morphology of myoblasts plated on plastic and on the ECM ligands fibronectin and laminin. The interplay between 16K and the ECM is further evidenced by the redistribution of 16K when cells bind to fibronectin. Expression of the TM4 mutant inhibits differentiation of myoblasts, enhances their migration through laminin and leads to an almost two-fold increase in the surface levels of [beta]1 integrin. The surplus [beta]1 integrin localizes to focal adhesions and correlates with a redistribution of kinase activities. Co-immunoprecipitation analyses in 293 cells using T7-epitope tagged integrin proteins with single amino acid substitutions within the transmembrane domain show that 16K interacts preferentially with a precursor form of the [beta]1 integrin molecule. Expression of 16K and the TM4 mutant both result in altered processing of [beta]1 integrin. Expression of integrin transmembrane mutants in myoblasts alters cell morphology, the distribution of phosphotyrosines, organization of the actin cytoskeleton, and acidification of vesicles. The binding of [beta]1 integrin to 16K may orchestrate these changes by regulating the activity of the V-ATPase. The interaction between [beta]1 integrin and 16K provides a link between integrins, PDGF-[beta]R, V-ATPase and the cytoskeleton, and thus may prove to be an important control point for processes such as cell migration. |
