The long-term goal of this proposal is to understand the molecular mechanism of integrin activation and signaling. Integrins are heterodimeric (alpha/beta) cell surface receptors that mediate adhesion of the cells to one another and to their surroundings (extracellular matrix). The adhesion is crucial for many biological processes, such as embryogenesis, haemostasis, the immune response and the maintenance of tissue integrity, and adhesive dysfunctions lead to numerous human disorders such as thrombosis, bleeding disorders, cancer, and chronic inflammatory diseases. Therefore, understanding the mechanism of integrin adhesion is of both physiological and pathological importance. The ability of integrins to bind their extracellular ligands is tightly regulated through a process termed inside-out signaling, i.e., a cellular signal transmits from its short (alpha and/or beta) cytoplasmic tails to the extracellular domain, transforming it from a low to a high affinity ligand binding state (integrin activation). On the other hand, ligand occupancy of the extracellular domain elicits signals that are transmitted back into the cell, which activate cascades of intracellular signaling and responses (outside-in signaling). The elegant crystal structure of an integrin (alphav/beta3) extracellular segment has recently been solved, which provides a framework for understanding how the extracellular domain binds to the ligand(s). However, a structural basis for how the intracellular segment of the receptor (cytoplasmic face) regulates the extracellular ligand binding affinity through inside-out signaling remains to be established and yet is fundamental to our understanding of integrin biology. Recent structural work from our laboratory and other biochemical data now allows us to propose a structurally-based hypothesis for the inside-out integrin activation: the alpha/beta cytoplasmic tails of a latent integrin receptor form a clasp by interacting with each other to maintain the receptor in the latent state. Binding of activators such as cytoskeletal protein talin to the cytoplasmic tail complex opens this clasp, thus initiating an inside-out conformational signaling for the receptor activation. To thoroughly evaluate this hypothesis, we propose to undertake a vigorous structural/biochemical study by focusing on alphaIIb/beta3, the major platelet integrin that is best characterized for the inside-out activation.
Aim 1 and 2 will determine NMR structures of the alphaIIb/beta3 cytoplasmic face in its latent and active forms.
Aim #3 will functionally evaluate how the latent/active cytoplasmic faces regulate the extracellular ligand binding affinity via inside-out signaling.
Aim #4 will investigate how the structural change in the cytoplasmic face propagates to the transmembrane region. We wish that the results will not only impact on the integrin biology but contribute to the development of new agents, in particular, those that target at integrin function by regulating their activation states.
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