Investigation of the alternatively spliced insert region of the D2L dopamine receptor by epitope substitution.
Investigation of the alternatively spliced insert region of the D2L dopamine receptor by epitope substitution.
复制标题
通过表位取代研究 D2L 多巴胺受体的选择性剪接插入区域。
DOI：
10.1016/j.neulet.2005.09.057
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Senogles,SusanE
中科院分区：
文献类型：
--
作者：
Kendall,RyanT;Senogles,SusanE
D2 多巴胺受体的选择性剪接变体具有不同的神经元功能和定位。这种七螺旋跨膜受体的长亚型 (D2L) 与短亚型的不同之处仅在于第三个细胞内环中存在 29 个氨基酸插入片段，该区域已知对 G 蛋白偶联很重要。短亚型和长亚型已被证明具有不同的 Gαi/o 蛋白偶联特异性。然而，选择性剪接插入区域在 D2 多巴胺受体功能中的确切作用需要更全面的检查。解决这个问题的一种方法是用同等长度但非同源的蛋白质序列替换整个插入区域。该报告证明了用血凝素双表位标签替换 29 个氨基酸插入片段的可行性，且不会产生可识别的功能后果。 D2L 突变体在其配体结合特征以及两种效应反应方面与野生型 D2L 受体没有区别：激动剂介导的毛喉素刺激的 cAMP 产生的抑制和激动剂刺激的 MAPK 磷酸化。这些数据证明表位取代产生功能性受体，并且选择性剪接的插入区域本身似乎在信号转导中不发挥直接作用。由于可变剪接插入区的存在，表位取代允许将序列介导的效应与结构效应分开。因此，这种新结构可能成为研究 D2 受体功能的有价值的工具。
Alternatively spliced variants of the D2dopamine receptor have distinct neuronal function and localization. The long isoform (D2L) of this heptahelical transmembrane receptor differs from the short form only by the presence of a 29-amino acid insert in the third intracellular loop—a region known to be important for G protein coupling. Short and long isoforms have been shown to have distinct Gαi/oprotein coupling specificities. However, the exact role of the alternatively spliced insert region in D2dopamine receptor function needs a more comprehensive examination. One way to address this is to substitute the entire insert region with an equivalent length, yet nonhomologous protein sequence. This report demonstrates the feasibility of replacing the 29-amino acid insert with a hemagglutinin double epitope tag with no recognizable functional consequences. The D2Lmutant is indistinguishable from the wild type D2Lreceptor in terms of its ligand binding characteristics, as well as two effector responses: the agonist-mediated inhibition of forskolin-stimulated cAMP production, and agonist-stimulated MAPK phosphorylation. These data demonstrate that the epitope substitution generates a functional receptor, and that the alternatively spliced insert region, itself, does not appear to play a direct role in signal transduction. The epitope substitution permits dissection of sequence-mediated effects from structural effects due to the presence of the alternatively spliced insert region. Thus, this new construct could be a valuable tool for the study of D2receptor function.