Molecular cloning and pharmacological characterization of the rat sigma1 receptor
Molecular cloning and pharmacological characterization of the rat sigma1 receptor
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DOI：
10.1016/s0006-2952(01)00666-9
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发表时间：
2001-08-01
影响因子：
5.8
通讯作者：
Pasternak, GW
中科院分区：
文献类型：
--
作者：
Mei, JF;Pasternak, GW
为了进一步了解sigma受体的药理学，我们克隆了sigma受体的大鼠同源物。我们用逆转录-聚合酶链反应（RT-PCR）和5'和3'端快速扩增cDNA末端（RACE）技术从大鼠脑组织中克隆了一个cDNA克隆（rs 2 -2），它编码223个氨基酸的全长序列。该克隆的预测蛋白质序列与鼠（93.3%）、豚鼠（93.7%）和人（96%）σ 1受体的蛋白质序列具有高度同源性。北方分析显示约1.8 kb的主要mRNA条带。RT-PCR显示在所有测试的组织中存在mRNA，在脑、脊髓、肝、胸腺、肾上腺和肾中具有高水平。当在中国仓鼠卵巢（CHO）细胞中表达时，σ 1结合水平显著增加，并且结合特征与σ 1位点一致。然而，转染前细胞系中存在的可测量水平的σ 1结合使得难以解释这些结果。为了确保结合反映了转染的蛋白质，我们在氨基末端用血凝素（HA）表位标记受体，并检查免疫沉淀受体中的结合。使用抗HA表位的抗血清的Western分析显示分子量类似于28 kDa，接近预测值。免疫纯化受体的受体结合特征与传统σ 1结合位点所见的一致。因此，rs 2 -2.HA编码具有大鼠σ 1受体特征的高亲和力[H-3]（+）-喷他佐辛结合位点。(C)2001 Elsevier Science Inc. All rights reserved.
In an effort to further understand the pharmacology of sigma receptors, we have cloned the rat homolog of the sigma, receptor. We isolated a cDNA clone (rs2-2) from rat brain tissue using reverse transcriptase-polymerase chain reaction (RT-PCR) and 5' and 3' rapid amplification of cDNA ends (RACE) that encoded a full-length sequence of 223 amino acids. The predicted protein sequence of the clone has high homology with that of the murine (93.3%), guinea pig (93.7%), and human (96%) sigma, receptors. Northern analysis showed a major mRNA band of approximately 1.8 kb. RT-PCR revealed the presence of the mRNA in all the tissues tested, with high levels in the brain, spinal cord, liver, thymus, adrenal glands, and kidneys. When expressed in Chinese hamster ovary (CHO) cells, the level of sigma, binding increased markedly, and the binding profile was consistent with sigma, sites. However, measurable levels of sigma, binding present in the cell lines before transfection made the interpretation of these results difficult. To ensure that the binding reflected the transfected protein, we tagged the receptors with a hemagglutinin (HA) epitope at the amino terminus and examined binding in immunoprecipitated receptors. Western analysis using an antisera against the HA epitope revealed a molecular weight of similar to 28 kDa, close to the predicted value. The receptor binding profile of the immunopurified receptor was consistent with that seen with traditional sigma, binding sites. Thus, rs2-2.HA encodes a high-affinity [H-3](+)-pentazocine binding site with characteristics of a rat sigma, receptor. (C) 2001 Elsevier Science Inc. All rights reserved.