目的构建与海肾荧光素酶（Rluc）融合的人Neurotensin-R1（HumanNeurotensinreceptorl，NTS1 or NTSR1）真核表达载体，用于生物发光共振能量转移法检测人NTS与其它受体间的相互作用以及研究Neurotensin—R介导的细胞内信号转导机制。方法以质粒peDNA3．1-hNTS1为模板，PCR方法扩增人NTS。扩增的人NTS以及质粒pRluc—pcDNA3．1用NotI和XbaI双酶切，然后将这2种酶切产物按常规方法连接、转化至大肠杆菌Top10中，该菌在培养箱中孵育12～16h后，挑取菌落培养，提取质粒，进行酶切鉴定，最后进行测序。将测序正确的重组载体用脂质体法转染人胚胎肾（humanembryonickidney293，HEK293）细胞。最后，通过共聚焦显微镜观察经过免疫荧光染色的细胞以及Westernblot鉴定人Neu—rotensinl—R的表达。结果通过PCR扩增出一条1257bp的基因片段，序列与GenBank（NM-002531）相同。Westernblot中大约90kDa处有一蛋白条带，与预期大小相同。人Neurotensin—R表达在细胞膜上。结论成功构建了pRluc—hNeurotensinl—R重组表达载体，建立了该质粒转染HEK293的细胞模型。可被用于检测与其它受体间的相互作用以及研究Neurotensinl—R介导的细胞内信号转导机制，这将有助于探究疾病的发病机制及开发新的药物靶点。
G-protein-coupled receptors（GPCRs） are G-protein-coupled heptaspanning-membrane receptors.This group has thousands of members and is one of the important drug targets,accounting for 40%-50%of the drugs currently on the market.In the last decade,there has been a substantial re-evaluation of the assumption that GPCRs exist primarily as monomeric polypeptides, with support increasing for a model in which GPCRs can exist as homo- or hetero- dimers or even high-order oligomers.GPCRs dimers are hot research topics.Recent reports suggest that homo- or hetero- dimers exhibit ＂specific＂ functional properties which are distinct from monomeric receptors,involving agonist recognition,signaling,trafficking,and so on.Meanwhile,the occurrence of dimers with different pharmacological and signaling properties opens a completely new field in the search for novel drug targets useful to combat a variety of diseases and with potentially fewer side effects.In this paper,we will mainly review their specific structures and signal transduction,which help us reach for the high-hanging fruits in GPCRs drug discovery.
缓激肽受体是G蛋白偶联受体家族的重要成员之一,它所介导的信号转导对于维持机体内心血管系统动态平衡和炎症方面发挥了重要的作用。近年来,随着荧光共振能量转移(fluorescence resonance energy transfer,FRET)和生物发光共振能量转移(bioluminescence resonance energy trans-fer,BRET)等技术的相继出现,发现缓激肽受体不仅以单体形式存在,还可能以二聚体甚至是高阶寡聚体的形式参与细胞内的病理生理过程。与单体相比,二聚体(或高阶寡聚体)的信号转导和病理功能都产生了相应变化。本文就缓激肽受体及其二聚体所介导的信号途径、生理病理过程及新的研究技术做一简要综述。
