Basement membranes contribute to the organization of tissue and play dynamic roles in embryonic development and tumorigenesis. The biological effects of basement membranes can in large part be attributed to the ability of integrins to promote cell adhesion, regulate cell migration, and profoundly influence cell proliferation and differentiation. To elucidate the effects of laminins on epithelial cells, we have focused on the alpha6beta4 integrin, a major laminin receptor which is structurally and functionally distinct from all other known integrins. The expression of alpha6beta4 is restricted to the proliferative compartment in normal tissues and the levels of alpha6beta4 are often elevated in squamous carcinomas, suggesting that alpha6beta4 may participate to the control of cell proliferation. Our previous studies have indicated that upon binding to extracellular ligand alpha6beta4 becomes phosphorylated on tyrosine by the action of an integrin-associated kinase, combines sequentially with the adaptors Shc and Grb2, and thereby activates Ras signaling. Our data have also indicated that the phosphorylation of a Tyrosine Activation Motif (TAM) located in the Connecting Segment of beta4 controls the association of alpha6beta4 with hemidesmosomal cytoskeleton, presumable via the recruitment of a signaling molecule containing two tandem Src Homology 2 (SH2) domains. Finally, we have provided evidence that Epidermal Growth Factor (EGF) causes tyrosine phosphorylation of the beta4 tail, but this event is not followed by recruitment of Shc and association with the hemidesmosomal cytoskeleton; rather, EGF induces disassembly of hemidesmosomes and augments alpha6beta4-dependent cell migration. In this proposal, we plan to: 1) elucidate the mechanism of recruitment and tyrosine phosphorylation of Shc in response to alpha6beta4 ligation by mutagenesis methods; 2) examine the effects of alpha6beta4 signaling on the regulation of cell cycle by a combination of biochemical and cell biological methods; 3) identify and characterize protein(s) interacting with the beta4 TAM by affinity chromatography and a modified yeast two- hybrid system; 4) examine the mechanism and functional consequences of EGF-mediated tyrosine phosphorylation of beta4 by a combination of mutagenesis and cell biological methods; and 5) explore the intracellular functions of alpha6beta4 in vivo by gene targeting methods. We hope that these studies will help to clarify the function of alpha6beta4 in normal and neoplastic cells.
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