Integrins are a family of alpha/beta heterodimeric receptors used by cells to interact with the extracellular matrix. Signals triggered by integrins regulate cell proliferation, differentiation and cell survival. Central to integrin function is the ability to mediate cell adhesion. Integrin beta subunit cytoplasmic domains are required for all steps in the cell adhesion process. Although several cytoskeletal and signaling proteins have been demonstrated to bind to beta cytoplasmic tails, it is not known whether any of these protein interactions regulate cell adhesion. In addition, signaling proteins activated by integrins, such as Ras, phosphoinositide-3 kinase and PKC, can regulate cell adhesion. However, how these signaling pathways are influenced by integrin beta cytoplasmic domains is not known. Therefore, this grant focuses on understanding the molecular mechanisms by which integrin beta cytoplasmic tails regulate cell adhesion of fibroblastic cells in culture. In previous studies, we have expressed beta cytoplasmic tails as isolated domains connected to the extracellular domain of the interleukin-2 (IL-2) receptor. Using this approach, we have demonstrated that these chimeric receptors can function as dominant negative mutants and inhibit cell adhesion. In addition, we have shown that clustering these chimeric receptors on the cell surface of suspended cells can activate signaling pathways by mechanisms similar to endogenous integrins. Therefore, depending upon experimental design, we can either use the chimeras as inhibitors of cell adhesion, or we can use them as probes to determine which signaling pathways can be activated by isolated beta cytoplasmic tails. In this grant, I propose to continue with both approaches to determine the molecular pathways by which beta cytoplasmic tails regulate cell adhesion. The experiments proposed focus on identifying the protein interactions and signaling pathways inhibited by the expression of the chimeras. In parallel experiments, we propose to determine whether beta cytoplasmic tails are sufficient to activate pathways which regulate adhesion. Also, since integrin function is mediated through the formation of adhesion-dependent multiprotein complexes linking integrins to the cell's signaling and cytoskeketal networks, we will compare the ability of wild-type and mutant beta tails to trigger the formation of these complexes. In addition, we will determine how the formation of these complexes are affected by the dominant negative phenotype of the chimera. These experiments will aid in identifying which components of these complexes are required to regulate cell adhesion. In summary, this proposal is directed at a mechanistic understanding of how integrin beta cytoplasmic tails influence the regulation of cell adhesion.
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