Chen Angela Shuyi, Kim Young Mee, Gayen Shovanlal, Huang Qiwei, Raida Manfred, Kang Congbao
Experimental Therapeutics Centre, Agency for Science, Technology and Research(A*STAR), Singapore 138669, Singapore.
Biochim Biophys Acta. 2011 Sep;1808(9):2224-32. doi: 10.1016/j.bbamem.2011.05.014. Epub 2011 May 24.
The serotonin (5-HT(1A)) receptor, a G-protein-coupled receptor (GPCR), plays important roles in serotonergic signaling in the central nervous system. The third intracellular loop (ICL3) of the 5-HT(1A) receptor has been shown to be important for the regulation of this receptor through interactions with proteins such as G-proteins and calmodulin. In this study, the ICL3 of 5-HT(1A) receptor was expressed in E. coli and purified. Gel filtration and mass spectrometry were used to confirm the molecular weight of the purified ICL3. Secondary structure analysis using circular dichroism (CD) demonstrated the presence of α-helical structures. Backbone assignment of ICL3 was achieved using three-dimensional experiments. A chemical shift index and Talos+ analysis showed that residues E326 to R339 form α-helical structure. Residues G256 to S269 of ICL3 were shown to be a novel region that has a molecular interaction with calmodulin in titration assays. Peptide derived from the ICL3 containing residues from G256 to S269 also showed molecular interaction with calmodulin.
5-羟色胺（5-HT(1A)）受体是一种G蛋白偶联受体（GPCR），在中枢神经系统的5-羟色胺能信号传导中发挥重要作用。5-HT(1A)受体的第三个细胞内环（ICL3）已被证明通过与G蛋白和钙调蛋白等蛋白质相互作用，对该受体的调节具有重要意义。在本研究中，5-HT(1A)受体的ICL3在大肠杆菌中表达并纯化。使用凝胶过滤和质谱法确认纯化的ICL3的分子量。利用圆二色性（CD）进行的二级结构分析表明存在α-螺旋结构。通过三维实验实现了ICL3的主链归属。化学位移指数和Talos+分析表明，E326至R339残基形成α-螺旋结构。在滴定实验中，ICL3的G256至S269残基被证明是与钙调蛋白发生分子相互作用的一个新区域。源自ICL3且包含G256至S269残基的肽也显示出与钙调蛋白的分子相互作用。
