Blood Research Institute, Versiti, Milwaukee, Wisconsin, USA.
Blood Research Institute, Versiti, Milwaukee, Wisconsin, USA; Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
J Biol Chem. 2021 Nov;297(5):101318. doi: 10.1016/j.jbc.2021.101318. Epub 2021 Oct 20.
Studying the tight activity regulation of platelet-specific integrin αβ is foundational and paramount to our understanding of integrin structure and activation. αβ 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 αβ 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 β TM α-helix coupled with αβ 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 αβ 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 αβ and αβ TM domains, suggesting a generalizable mechanism. We also detected a topology change of β TM α-helix within the cell membrane, but only under conditions of cell adhesion and the absence of α association. Our data demonstrate the importance of studying the structure and function of the integrin TM domain in the native cell membrane.
研究血小板特异性整合素 αβ 的紧密活性调节对于我们理解整合素结构和激活至关重要。αβ 对于止血和血栓形成中的血小板聚集和黏附功能至关重要。结构和突变研究先前揭示了 αβ 跨膜 (TM) 关联对于维持非活性状态的关键作用。已经鉴定出具有功能获得的 TM 突变，并且显示出破坏 TM 关联导致整合素激活。使用分离的 TM 肽的研究表明，β TM α-螺旋的膜嵌入发生改变，同时伴随着 αβ 的激活。然而，关于 TM α-螺旋在天然细胞膜中全长整合素的情况下是否以及如何改变其拓扑结构仍存在争议。在这项研究中，我们利用脯氨酸扫描突变和半胱氨酸扫描可及性测定来分析 αβ TM 结构域的结构和功能相关性。我们在 TM 结构域中鉴定出功能丧失的脯氨酸突变，这表明在跨细胞膜双向传递激活信号时需要连续的 TM α-螺旋结构，其特征是配体结合的内向外激活和细胞扩展的外向信号。在 αβ 和 αβ TM 结构域中也发现了相似的结果，这表明存在一种普遍适用的机制。我们还在细胞膜内检测到 β TM α-螺旋的拓扑变化，但仅在细胞黏附条件下且没有 α 关联时才会发生。我们的数据表明，在天然细胞膜中研究整合素 TM 结构域的结构和功能非常重要。
