膜脂环境调控整合素构象与功能的研究

Integrin family is a major class of cell-surface receptors that mediate cell adhesion and migration. Integrins are heterodimeric transmembrane proteins containing α and β subunits. Integrin activity is directly regulated by its transmembrane domain topography. Recent studies find that integrin activity is regulated by the ionic interaction between the phosphate group of phospholipids and the conserved basic residue in the β transmembrane domain. This unusual ionic interaction keeps the β transmembrane domain at a tilted crossing angle and thus locks integrin on an inhibitory conformation. However, the structural details of the integrin-lipid interaction remain elusive. In T lymphocytes, Ca2+ signaling can activate integrin. Our previous study found that Ca2+ can directly bind to the phosphate group of phospholipids and thus regulate membrane receptor activity (Nature 2013). We therefore hypothesize that Ca2+ might disrupt integrin-phospholipid interaction by directly competing with the β subunit for the binding of the phosphate group of phospholipids, which should change the β subunit topology and result in integrin activation. In this project, we will focus on integrin α4β7, a lymphocyte homing receptor, to study the regulation of integrin structure and function by membrane lipids. We will use solution NMR to determine α4β7 transmemrbane heterodimer structure and study the ionic protein-lipid interaction between β7 and different phospholipid species. Moreover, we will use NMR to study how Ca2+ specifically regulate α4β7 structure and function by modulating phospholipids. Cell adhesion experiments will also be performed to verify our in vitro findings. The accomplishment of this project will greatly contribute to our further understanding of the molecular mechanism of cell adhesion and migration from a new point of view.

整合素是一类重要的细胞表面受体，介导细胞粘附与迁移。整合素由α和β亚基构成跨膜异源二聚体，其活性直接受控于它的跨膜区构象变化。β亚基跨膜区中保守的碱性残基与磷脂的磷酸根存在静电作用，维持整合素的抑制型构象。申请人所在研究组发现，钙离子可以直接结合磷脂的磷酸根，从而改变膜脂环境来调控膜受体的活化（Nature 2013）。于是我们推测钙离子也可以干扰β亚基与磷脂的静电结合，改变β亚基跨膜区的构象，从而导致整合素活化。本项目以淋巴细胞归巢受体整合素α4β7 为对象，研究膜脂环境对整合素结构和功能的调控。本项目将利用液相NMR解析α4β7跨膜区异源二聚体的三维结构；研究β7跨膜区与不同种类磷脂的相互作用；研究钙离子对α4β7构象的特异性调控；并用细胞粘附实验来验证体外发现。本项目的开展将使我们从一个全新的角度来理解细胞粘附和迁移的分子机制。

膜蛋白的跨膜区通常较为疏水，而我们通过生物信息学的分析发现，约有40%的单跨膜蛋白的跨膜区存在碱性氨基酸，其中包括广泛表达的整合素家族，而这些碱性氨基酸主要分布在靠近富含酸性磷脂的质膜内侧的位置。但这些碱性氨基酸的具体功能尚不清楚。之前针对整合素αIIbβ3的研究表明，跨膜区碱性氨基酸的突变会引起整合素的活化，但具体机制尚不清楚。我们通过NMR、分子动力学模拟和细胞生物学方法研究T细胞中重要的整合素αLβ2（又称LFA-1，lymphocyte function-associated antigen 1）发现，突变跨膜区保守碱性氨基酸K702后，会引起跨膜区二聚体的不稳定。酸性磷脂可以同时与β2亚基上的K702位点以及αL上的R1094位点相互作用，从而稳定跨膜区异源二聚体。在生理状态下，这一作用可以调控整合素αLβ2胞外段的构象以及对配体ICAM-1的亲和力。TCR活化后，胞外钙离子快速内流，引起胞内钙离子浓度升高，内流的Ca2+可以通过与酸性磷脂结合，引起αLβ2跨膜区的分离以及整合素的活化，Ca2+的这一作用只依赖于其电荷属性而不依赖于传统的Ca2+信号通路或整合素inside-out信号通路。据此，我们提出了膜内静电相互作用在调控跨膜受体构象及活性中的重要作用，同时提出了Ca2+内流调控αLβ2活性的新机制。由于碱性氨基酸在跨膜区中广泛存在，这一蛋白-磷脂静电相互作用调控膜蛋白结构与功能的机制可能具有广泛的适用性。