Functional reconstitution of olfactory receptor complex on baculovirus.
Functional reconstitution of olfactory receptor complex on baculovirus.
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杆状病毒嗅觉受体复合物的功能重建。
DOI：
10.1093/chemse/bjs067
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Mitsui K
中科院分区：
文献类型：
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作者：
T. Kaneko;T. Akiba;Mitsui K
尽管基因组学的最新进展已经阐明了嗅觉受体（ORs）的基因组结构，但它们中的大多数仍然是孤儿受体。异源细胞中OR的低表达水平阻碍了许多建立细胞生物OR测定系统的尝试。最近，我们证明，某些G蛋白偶联受体，如白三烯B4受体或多巴胺D1受体，有效地重建从感染的Sf 9细胞的杆状病毒出芽。芽殖病毒（BV）被证明是大部分免费的外源蛋白以外的病毒感染，导致低噪音的测定条件。利用这些条件，我们试图在BV上重建OR复合物。用携带编码腺苷酸环化酶、三聚体G蛋白和受体mOR-EG或S6的cDNA的重组杆状病毒共感染Sf 9细胞。这些蛋白质的共表达通过蛋白质印迹法检测，并且激动剂或拮抗剂依赖性受体应答使用配体依赖性环AMP产生来确认。这些结果表明BV上功能性OR复合物的成功重建。此外，还证实了OR 8B 3在BV上的表达，BV是人类孤儿OR之一。该BV表达系统有望成为筛选未知OR配体的高效工具。
Despite that recent progress in genomics has elucidated the genomic structure of the olfactory receptors (ORs), most of them are still orphan receptors. The low expression level of ORs in heterologous cells has hampered many attempts to establish cell biological OR assay systems. Recently, we demonstrated that certain G protein-coupled receptors, such as the leukotriene B4 receptor or the dopamine D1 receptor, were efficiently reconstituted on baculovirus budding from infected Sf9 cells. The budded virus (BV) was shown to be mostly free of exogenous proteins other than those related to viral infection, resulting in low-noise assay conditions. Taking advantage of these conditions, we attempted to reconstitute OR complexes on BV. Sf9 cells were coinfected with recombinant baculoviruses harboring the cDNAs encoding adenylyl cyclase, trimeric G-protein, and the receptor: mOR-EG or S6. The coexpression of these proteins was detected by western blot, and the agonist- or antagonist-dependent receptor response was confirmed using ligand-dependent cyclic AMP production. These results demonstrated the successful reconstitution of functional OR complex on BV. Additionally, the expression of OR8B3 on BV, one of human orphan ORs, was also confirmed. This BV expression system is expected to be a highly effective tool for screening unknown ligands for ORs.