Purification, molecular cloning, and expression of the mammalian sigma(1)-binding site
Purification, molecular cloning, and expression of the mammalian sigma(1)-binding site
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DOI：
10.1073/pnas.93.15.8072
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发表时间：
1996-07-23
影响因子：
11.1
通讯作者：
Glossmann, H
中科院分区：
文献类型：
--
作者：
Hanner, M;Moebius, FF;Glossmann, H
σ-配体包括几种化学上不相关的药物，如氟哌啶醇、喷他佐辛和二甲苯胍，其结合内质网中的低分子量蛋白质家族。这些所谓的σ-受体被认为通过至今未知的机制介导σ-配体的各种药理学作用。基于它们对苯并吗啉的相反的对映选择性和不同的分子量，区分了两种亚型。我们使用苯并吗喃（+）[H-3]喷他佐辛和芳基叠氮（-）[H-3]叠氮多帕米作为特异性探针，从豚鼠肝脏中纯化了sigma（1）结合位点作为单一的30 kDa蛋白。纯化的（+）[H-3]喷他佐辛结合蛋白保留了其对氟哌啶醇、喷他佐辛和二甲苯基胍的高亲和力。经胰蛋白酶消化后获得的部分氨基酸序列与已知蛋白质无同源性，喷他佐辛标记的sigma（1）结合位点经辐射灭活后得到分子量为24 ± 2 kDa的蛋白质。利用简并寡核苷酸和cDNA文库筛选技术克隆了相应的cDNA，其开放阅读框编码一个分子量为25.3 kDa的蛋白质，至少有一个跨膜片段。在用cDNA转化的酵母细胞中表达的蛋白质显示了脑和肝脏sigma（1）结合位点的药理学特征。推导的氨基酸序列与已知的哺乳动物蛋白质结构无关，但与参与甾醇合成的真菌蛋白质具有同源性。北方印迹显示在甾醇产生组织中有高密度的sigma（1）结合位点mRNA，这也与已知的sigma（1）结合位点与类固醇如孕酮相互作用的能力一致。
Sigma-ligands comprise several chemically unrelated drugs such as haloperidol, pentazocine, and ditolylguanidine, which bind to a family of low molecular mass proteins in the endoplasmic reticulum. These so-railed sigma-receptors are believed to mediate various pharmacological effects of sigma-ligands by as yet unknown mechanisms. Based on their opposite enantioselectivity for benzomorphans and different molecular masses, two subtypes are differentiated. We purified the sigma(1)-binding site as a single 30-kDa protein from guinea pig liver employing the benzomorphan (+) [H-3]pentazocine and the arylazide (-) [H-3]azidopamil as specific probes. The purified (+) [H-3]pentazocine-binding protein retained its high affinity for haloperidol, pentazocine, and ditolylguanidine. Partial amino acid sequence obtained after trypsinolysis revealed no homolog to known proteins, Radiation inactivation of the pentazocine-labeled sigma(1)-binding site yielded a molecular mass of 24 +/- 2 kDa, The corresponding cDNA was cloned using degenerate oligonucleotides and cDNA library screening, Its open reading frame encoded a 25.3-kDa protein with at least one putative transmembrane segment. The protein expressed in yeast cells transformed with the cDNA showed the pharmacological characteristics of the brain and liver sigma(1)-binding site. The deduced amino acid sequence was structurally unrelated to known mammalian proteins but it shared homology with fungal proteins involved in sterol synthesis, Northern blots showed high densities of the sigma(1)-binding site mRNA in sterol-producing tissues, This is also in agreement with the known ability of sigma(1)-binding sites to interact with steroids, such as progesterone.