Systematic biochemical characterization of the SAM domains in Eph receptor family from Mus Musculus
Systematic biochemical characterization of the SAM domains in Eph receptor family from Mus Musculus
复制标题
小家鼠 Eph 受体家族 SAM 结构域的系统生化特征
DOI：
10.1016/j.bbrc.2016.04.059
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发表时间：
2016
影响因子：
3.1
通讯作者：
Liu Wei
中科院分区：
文献类型：
--
作者：
Wang Yue;Li Qingxia;Zheng Yunhua;Li Gang;Liu Wei
Eph受体家族是受体酪氨酸激酶家族中最大的一个亚家族，在轴突导向、突触形成、动脉/静脉分化和肿瘤发生等方面具有重要作用。Eph受体的激活需要受体多聚化。据报道，Eph受体的胞内C-末端SAM结构域通过homo SAM-SAM相互作用介导Eph受体的自缔合。本研究在大肠杆菌中系统表达并纯化了小家鼠14种Eph受体的SAM结构域蛋白，FPLC（fast protein liquid chromatography）结果表明重组SAM结构域具有高度的同源性。使用CD（圆二色性）光谱，我们发现，所有的SAM域的二级结构是典型的α螺旋折叠和显着相似。热稳定性试验表明，它们在溶液中相当稳定。SEC-MALS（尺寸排阻色谱结合多角度光散射）结果表明，200 μM Eph SAM结构域在尺寸排阻色谱中表现为良好的单体。更重要的是，DLS（动态光散射）结果显示，在200 μM或2000 μM蛋白浓度下，绝大多数SAM结构域在溶液中没有多聚化，这表明单独的SAM结构域不足以介导Eph受体的聚合。总之，我们的研究提供了系统的生化特性的Eph受体SAM结构域，并暗示其在Eph受体介导的信号通路中的作用。
The Eph receptor family is the largest subfamily of receptor tyrosine kinases and well-known for their pivotal roles in axon guidance, synaptogenesis, artery/venous differentiation and tumorigenesis, etc. Activation of the Eph receptor needs multimerization of the receptors. The intracellular C-terminal SAM domain of Eph receptor was reported to mediate self-association of Eph receptors via the homo SAM–SAM interaction. In this study, we systematically expressed and purified the SAM domain proteins of all fourteen Eph receptors ofMus musculusinEscherichia coli.The FPLC (fast protein liquid chromatography) results showed the recombinant SAM domains were highly homogeneous. Using CD (circular dichroism) spectrometry, we found that the secondary structure of all the SAM domains was typically alpha helical folded and remarkably similar. The thermo-stability tests showed that they were quite stable in solution. SEC-MALS (size exclusion chromatography coupled with multiple angle light scattering) results illustrated 200 μM Eph SAM domains behaved as good monomers in the size-exclusion chromatography. More importantly, DLS (dynamic light scattering) results revealed the overwhelming majority of SAM domains was not multimerized in solution either at 200 μM or 2000 μM protein concentration, which indicating the SAM domain alone was not sufficient to mediate the polymerization of Eph receptor. In summary, our studies provided the systematic biochemical characterizations of the Eph receptor SAM domains and implied their roles in Eph receptor mediated signaling pathways.