Structural determinants of the integrin transmembrane domain required for bidirectional signal transmission across...

Studying the tight activity regulation of platelet-specific integrin α_IIbβ₃ is foundational and paramount to our understanding of integrin structure and activation. α_IIbβ₃ is essential for the aggregation and adhesion function of platelets in hemostasis and thrombosis. Structural and mutagenesis studies have previously revealed the critical role of α_IIbβ₃ transmembrane (TM) association in maintaining the inactive state. Gain-of-function TM mutations were identified and shown to destabilize the TM association leading to integrin activation. Studies using isolated TM peptides have suggested an altered membrane embedding of the β₃ TM α-helix coupled with α_IIbβ₃ activation. However, controversies remain as to whether and how the TM α-helices change their topologies in the context of full-length integrin in native cell membrane. In this study, we utilized proline scanning mutagenesis and cysteine scanning accessibility assays to analyze the structure and function correlation of the α_IIbβ₃ TM domain. Our identification of loss-of-function proline mutations in the TM domain suggests the requirement of a continuous TM α-helical structure in transmitting activation signals bidirectionally across the cell membrane, characterized by the inside-out activation for ligand binding and the outside-in signaling for cell spreading. Similar results were found for α_Lβ₂ and α₅β₁ TM domains, suggesting a generalizable mechanism. We also detected a topology change of β₃ TM α-helix within the cell membrane, but only under conditions of cell adhesion and the absence of α_IIb association. Our data demonstrate the importance of studying the structure and function of the integrin TM domain in the native cell membrane.