Although there have been several clear demonstrations of signalling through integrins, there are few examples of specialized signalling associated with a particular integrin. The underlying theme of this proposal is that unique integrin beta chain cytoplasmic domains contribute to specific signalling functions. We already have demonstrated that cytoplasmic domains of the integrin beta1 and beta5 subunits contribute similarly to the constitutive regulation of cell adhesion (inside-out signalling), but differ markedly in their mediation of several outside-in functions and signalling events. Here we propose a detailed study of the comparative biochemistry of specific beta1 and beta5 signalling pathways. A panel of human beta1 and beta5 chimeric constructs will be generated in which five subregions of cytoplasmic domain dissimilarity are individually exchanged or mutated, and then expressed in the context of either the beta1 or beta5 ectodomain. We anticipate that these wild type and mutant beta1 and beta5 constructs will give similar expression, alpha chain pairing, ligand binding, and cell adhesion in CHO cells and other cells. Then, by studying subcellular localization, cell spreading, proliferation, migration, and the transmission of mechanical forces, we will be able to pinpoint the precise amino acids responsible for the previously established functional differences between the beta1 and beta5 tails. In parallel, we will analyze biochemical pathways specifically associated with either the beta1 or beta5 tails, involving the appearance and disappearance of phosphorylated proteins. Examples include the beta1-dependent appearance of p90 (phosphorylated on serine?), and beta5-dependent effects on p60 and p65 proteins phosphorylated on tyrosine. Finally, using minimally mutated beta1 and beta5 tails as controls, we will utilize wild type cytoplasmic domain fusion proteins to identify proteins specifically associated with either the beta1 or beta5 tail. Together these experiments will allow a coordinated mapping of specific residues simultaneously contributing to distinct functions, to different signalling pathways, and to direct biochemical associations. By integrating these three approaches, we should not only gain definitive insights into specialized integrin signalling, but also we may potentially open up a new arena of sites for specific therapeutic intervention on the cytoplasmic side of integrins.
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