Insights into subunit interactions within the insect olfactory receptor complex using FRET
Insights into subunit interactions within the insect olfactory receptor complex using FRET
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DOI：
10.1016/j.ibmb.2012.11.002
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发表时间：
2013-02-01
影响因子：
3.8
通讯作者：
Newcomb, Richard D.
中科院分区：
文献类型：
--
作者：
German, Pablo F.;van der Poel, Selene;Newcomb, Richard D.
昆虫嗅觉受体是一类新型的配体门控阳离子通道，能够对低浓度的挥发性有机化合物产生反应。它们参与检测与配偶识别，食物定位和捕食者回避相关的气味。这些受体形成复合物，目前认为该复合物包含至少两个亚基成员：非经典Orco离子通道亚基和配体结合受体亚基。整合膜蛋白SNMP 1和2也与嗅觉功能相关，其中SNMP 1是黑腹果蝇中顺式乙酸牛痘酯接收所需的。为了研究这些膜蛋白之间的蛋白质-蛋白质相互作用，我们测量了分子间的福斯特/荧光共振能量转移（FRET）在活的昆虫细胞受体光漂白。使用杆状病毒介导的表达在High Five（TM）细胞中产生含有青色荧光蛋白或黄色荧光蛋白的融合蛋白。大多数重组产物具有融合蛋白的预期大小，并且位于细胞内膜内。我们能够显示FRET效率，为配体结合OR、Or 22 a和Orco（Or 22 a-Or 22 a、Or 22 a Orco、Orco Orco）的同聚和异聚相互作用提供证据。没有证据表明SNMP 1和Orco之间或SNMP 2和Orco或Or 22 a之间存在相互作用。然而，SNMP 1和Or 22 a的融合蛋白确实显示了FRET的相互作用，这表明SNMP 1可能与至少一些昆虫嗅觉受体复合物相互作用。总之，这项研究支持以前观察到的同源和异源之间的相互作用Orco和配体结合OR，Or 22 a，并确定了一种新的相互作用Or 22 a和SNMP 1。(C)2012爱思唯尔有限公司保留所有权利。
Insect olfactory receptors (ORs) are a novel family of ligand-gated cation channels that can respond to volatile organic compounds at low concentrations. They are involved in the detection of odorants associated with mate recognition, food localisation and predator avoidance. These receptors form a complex that is currently thought to contain at least two subunit members: the non-canonical Orco ion channel subunit and a ligand-binding receptor subunit. The integral membrane proteins SNMP1 and 2 are also associated with olfactory function, with SNMP1 required for cis-vaccinyl acetate reception in Drosophila melanogaster. In order to investigate protein protein interactions among these membrane proteins we measured intermolecular Forster/Fluorescence Resonance Energy Transfer (FRET) in live insect cells by acceptor photobleaching. Fusion proteins containing Cyan Fluorescent Protein or Yellow Fluorescent Protein were produced using baculovirus-mediated expression in High Five (TM) cells. The majority of the recombinant products were of the expected size for the fusion proteins and located within intracellular membranes. We were able to show FRET efficiencies providing evidence for homomeric and heteromeric interactions of the ligand-binding OR, Or22a, and Orco (Or22a-Or22a, Or22a Orco, Orco Orco). There was no evidence for an interaction between SNMP1 and Orco or between SNMP2 and Orco or Or22a. However, fusion proteins of SNMP1 and Or22a did show an interaction by FRET, suggesting SNMP1 may interact with at least some insect olfactory receptor complexes. In summary, this study supports previously observed homomeric and heteromeric interactions between Orco and the ligand-binding OR, Or22a, and identifies a novel interaction between Or22a and SNMP1. (C) 2012 Elsevier Ltd. All rights reserved.