Integrin-mediated cell adhesion can lead to migration, differentiation or proliferation. Recent evidence suggests that cytoplasmic tails of both the alpha and Beta subunit not only transmit signals into cell but are also capable of responding to intracellular signals. This proposal will focus on the """"""""inside out"""""""" signaling events that impact the activation state of the homologous integrin alphavBeta3, alphavIIBeta3 and alphavBeta5. Thus, one objective of this proposal will be to determine the role of the alphav and aIIBeta cytoplasmic tails in promoting integrin activation and ligand binding. This is based on our binding to alphavBeta3. In contrast, alphaIIbBeta3 is negatively regulated by the alphaIIbeta cytoplasmic tail. Therefore, we will determine the minimal sequence within the cytoplasmic tails of alphav and alphaIIbeta that regulate ligand binding by transfection of alphav(-) Beta3 (+) cells with mutant forms of alphav or alphaIIbeta. We will then assess their function as measured by cell adhesion and ligand binding studies. These mutant receptors will also be examined for conformational changes as detected by, protease sensitivity, resonance energy transfer and expression of neo antigens. A second objective of this proposal will be investigate the role of integrin alphavBeta5 in cell migration. This involves a novel pathway of integrin activation mediated by protein kinase C (PKC). Specifically, human carcinoma cells attach to vitronectin utilizing integrin alphavBeta5 as their exclusive vitronectin receptor yet will not migrate on this ligand without PKC stimulation. PKC activation leads to de novo protein synthesis which is required for the migration response on vitronectin but not collagen. It is known that PKC actually represents a family of nine isoenzymes and it is common for a given cell to express multiple forms. We will test the hypothesis that vitronectin and collagen migration events are regulated by distinct PKC isoforms within human carcinoma cells. We will also examine whether a region within the Beta5 cytoplasmic tail is involved in the PKC-dependent migration response on vitronectin. These studies will facilitate our understanding of the structural and functional role of the cytoplasmic tails of integrin alpha and Beta subunits leading to """"""""inside out"""""""" signalling events resulting in integrin activation, ligand binding and a novel pathway of cell motility.
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