Latrophilin-2 mediates fluid shear stress mechanotransduction at endothelial junctions

Endothelial cell responses to fluid shear stress from blood flow are crucial for vascular development, function and disease. A complex of PECAM-1, VE-cadherin, VEGF receptors (VEGFRs) and PlexinD1 located at cell-cell junctions mediates many of these events. But available evidence suggests that another mechanosensor upstream of PECAM-1 initiates signaling. Hypothesizing that GPCR and Gα proteins may serve this role, we performed siRNA screening of Gα subunits and found that Gαi2 and Gαq/11 are required for activation of the junctional complex. We then developed a new activation assay, which showed that these G proteins are activated by flow. We next mapped the Gα residues required for activation and developed an affinity purification method that used this information to identify latrophilin-2 (Lphn-2/ADGRL2) as the upstream GPCR. Latrophilin-2 is required for all PECAM-1 downstream events tested. In both mice and zebrafish, latrophilin-2 is required for flow-dependent angiogenesis and artery remodeling. Furthermore, endothelial specific knockout demonstrates that latrophilin plays a role in flow-dependent artery remodeling. Human genetic data reveal a correlation between the latrophilin-2-encoding Adgrl2 gene and cardiovascular disease. Together, these results define a pathway that connects latrophilin-dependent G protein activation to subsequent endothelial signaling, vascular physiology and disease.

血液流动产生的流体切应力对内皮细胞的刺激效应，对于血管发育、生理功能维持及疾病发生发展均具有关键作用。定位于细胞间连接的血小板内皮细胞黏附分子-1（PECAM-1）、血管内皮钙粘蛋白（VE-cadherin）、血管内皮生长因子受体（VEGF receptors, VEGFRs）与PlexinD1所组成的复合物，介导了诸多此类信号事件。但现有研究证据显示，另有一类位于PECAM-1上游的机械感受器启动了相关信号通路。
我们提出G蛋白偶联受体（GPCR）与Gα蛋白可能承担该机械传感功能的假说，并针对Gα亚基开展了小干扰RNA（siRNA）筛选实验，结果发现Gαi2与Gαq/11是激活该连接复合物所必需的蛋白组分。随后我们构建了全新的激活检测体系，证实这类G蛋白可被流体切应力激活。
我们进一步定位了介导激活所需的Gα氨基酸残基，并基于该信息开发了亲和纯化方法，最终鉴定出Latrophilin-2（Lphn-2/ADGRL2）作为上游GPCR。实验证实，Latrophilin-2是所有已检测的PECAM-1下游信号事件的必需调控因子。在小鼠与斑马鱼模型中，Latrophilin-2均为流体依赖性血管生成与动脉重塑所必需。此外，内皮特异性敲除实验表明，Latrophilin家族蛋白参与了流体依赖性动脉重塑过程。人类遗传学数据显示，编码Latrophilin-2的Adgrl2基因与心血管疾病存在显著相关性。
综上，本研究确立了一条完整信号通路，该通路将Latrophilin依赖的G蛋白激活，与后续的内皮细胞信号转导、血管生理功能及疾病进程紧密关联起来。