Our overall goal is to understand at the molecular level how integrins work. This includes detailing how cells regulate the function of integrins at the cell surface and understanding how integrin heterodimers change conformation upon ligand binding or activation. Integrins are involved in many developmental processes and also function in numerous fully differentiated cells. With respect to known pathological conditions, integrins are essential for blood clotting (and pathological thrombosis), for proper function of the immune system, for the control of proliferation and, when that control fails, in the metastasis of tumors. The PS integrins of Drosophila are very similar to vertebrate integrins, and provide unique methodologies to examine integrin function. We will pursue three different lines of investigation that have grown out of recent experiments; all of these promise to uncover new molecular properties of integrin function. Specifically, we propose to: 1) Determine requirements for the ?PS PSI domain and serine linker motif. Based on preliminary evidence, we propose that the association of N-terminal PSI domain with the ? subunit stalk is dynamic, and that rearrangements of neighboring disulfide bonds are involved in structural changes in this region. We will examine this hypothesis directly. 2) Characterize a collection of integrin point mutants, specifically looking for changes that alter integrin affinity. The original selection for these alleles ruled out mutants that would eliminate integrin function, but we suspect that a number of these mutations will affect equilibria between integrin conformational states, and their study will provide a closer look at integrin dynamics. 3) The cytoplasmic protein talin has been proposed to be a common component of integrin activation, but regulation of ligand affinity for Drosophila ?PS2?PS is talin independent. Re-examination of the evidence from vertebrate cells suggests that talin may not modulate ligand affinity, but rather strengthen ligand binding, making it essentially irreversible. We will pursue experiments that will address this hypothesis, potentially redefining the role of talin in integrin regulation. ? ? ?
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