Yu Jessica, Lee Chia-Ying, Changou Chun Austin, Cedano-Prieto Dora M, Takada Yoko K, Takada Yoshikazu
Department of Dermatology, Biochemistry and Molecular Medicine, University of California Davis School of Medicine, Sacramento, CA 95817, U.S.A.
Ph.D. Program for Translational Medicine, College of Medical Sciences and Technology, Taipei Medical University, Taipei, Taiwan.
Biochem J. 2017 Feb 15;474(4):589-596. doi: 10.1042/BCJ20160998. Epub 2016 Dec 19.
Tetraspanins play important roles in normal (e.g. cell adhesion, motility, activation, and proliferation) and pathological conditions (e.g. metastasis and viral infection). Tetraspanins interact with integrins and regulate integrin functions, but the specifics of tetraspanin-integrin interactions are unclear. Using co-immunoprecipitation with integrins as a sole method to detect interaction between integrins and full-length tetraspanins, it has been proposed that the variable region (helices D and E) of the extracellular-2 (EC2) domain of tetraspanins laterally associates with a non-ligand-binding site of integrins. We describe that, using adhesion assays, the EC2 domain of CD81, CD9, and CD151 bound to integrin αvβ3, and this binding was suppressed by cRGDfV, a specific inhibitor of αvβ3, and antibody 7E3, which is mapped to the ligand-binding site of β3. We also present evidence that the specificity loop of β3 directly bound to the EC2 domains. This suggests that the EC2 domains specifically bind to the classical ligand-binding site of αvβ3. αvβ3 was a more effective receptor for the EC2 domains than the previously known tetraspanin receptors α3β1, α4β1, and α6β1. Docking simulation predicted that the helices A and B of CD81 EC2 bind to the RGD-binding site of αvβ3. Substituting Lys residues at positions 116 and 144/148 of CD81 EC2 in the predicted integrin-binding interface reduced the binding of CD81 EC2 to αvβ3, consistent with the docking model. These findings suggest that, in contrast with previous models, the ligand-binding site of integrin αvβ3, a new tetraspanin receptor, binds to the constant region (helices A and B) of the EC2 domain.
四跨膜蛋白在正常生理状态（如细胞黏附、迁移、活化和增殖）及病理状态（如转移和病毒感染）中发挥重要作用。四跨膜蛋白与整合素相互作用并调节整合素功能，但四跨膜蛋白 - 整合素相互作用的具体细节尚不清楚。使用以整合素进行共免疫沉淀作为检测整合素与全长四跨膜蛋白之间相互作用的唯一方法，有人提出四跨膜蛋白胞外区 -2（EC2）结构域的可变区（D和E螺旋）与整合素的非配体结合位点侧向结合。我们描述了，通过黏附实验，CD81、CD9和CD151的EC2结构域与整合素αvβ3结合，并且这种结合被αvβ3的特异性抑制剂cRGDfV以及定位到β3配体结合位点的抗体7E3所抑制。我们还提供证据表明β3的特异性环直接与EC2结构域结合。这表明EC2结构域特异性地结合到αvβ3的经典配体结合位点。与先前已知的四跨膜蛋白受体α3β1、α4β1和α6β1相比，αvβ3是EC2结构域更有效的受体。对接模拟预测CD81 EC2的A和B螺旋与αvβ3的RGD结合位点结合。在预测的整合素结合界面处将CD81 EC2的第116位和144/148位的赖氨酸残基进行替换，降低了CD81 EC2与αvβ3的结合，这与对接模型一致。这些发现表明，与先前的模型相反，整合素αvβ3（一种新的四跨膜蛋白受体）的配体结合位点与EC2结构域的恒定区（A和B螺旋）结合。
