Structural Transition of Cellular Integrins and Applications Thereof

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SUMMARY - Integrins are α/β heterodimeric cell surface receptors, which, via their ability to bind ligands through extracellular domains and to recruit a series of effector proteins in proximity to cytoplasmic tails, regulate diverse biological processes and play critical roles in many human diseases. Normal biological functions of integrins, such as αIIbβ3-mediated hemostasis, are regulated by a tightly-controlled balance between activated and deactivated states. Inappropriate activation of αIIbβ3 integrin in platelets causes thrombosis, making αIIbβ3 a validated anti-thrombotic target. Resting αIIbβ3 assumes a compact bent conformation. Upon activation induced by proteins binding to either the extracellular or cytoplasmic face, it undergoes a long-range conformational rearrangement characterized by an extension and opening of the headpiece and separation of the leg domains. Such conformational activation is required for high-affinity ligand binding and returning it to the resting state, i.e. deactivation, is important for maintaining appropriate αIIbβ3 function. Although structural studies revealed multiple static conformations of integrin, representing the inactive, intermediate, and active states, it remains elusive how the α and β subunits, both of which consist of multiple domains, act in concert to perform the reversible large- scale structural transitions between the active and inactive states. Little is known about the contribution of cell membrane in regulating integrin conformational changes. Our recent structural and functional studies of β3 in the absence of α subunit revealed novel aspects of integrin conformational regulation on the cell surface, suggesting previously uncharacterized roles of the cell membrane and other elements. We have identified a series of ligand- competitive inactivating inhibitors, which can displace the activating ligand from αIIbβ3 without inducing conformational changes. Furthermore, we have adapted the ex vivo production of iPS-derived human platelets to αIIbβ3 signaling studies. Based on these findings, the Aim 1 of this grant seeks to examine the molecular mechanism of integrin conformational activation and deactivation and dissect the regulatory functions of the cell membrane and other undefined factors. Using the inactivating inhibitors as tool compounds, we will define the intrinsic structure features that govern integrin conformational deactivation utilizing a combination of novel crystallographic, biochemical and biophysical approaches. The acquired structural information will be used to explore novel concepts of modulating integrin function by targeting conformational changes using small-molecule and antibody regulators. The second aim will extend our studies on integrin cytoplasmic tails to examining the composition and dynamics of the supramolecular signaling complex formed intracellularly following agonist- and ligand-induced αIIbβ3 activation, which will be performed in genetically engineered human platelets using proximity labeling and proteomics approaches. Collectively, our proposed extracellular and intracellular studies will provide new molecular insights into structural and signaling regulation of integrins, which will advance our understanding of integrin biology and may identify novel therapeutic targets for modulating integrin function.