Wu Zhiping, Bowen Wayne D
Department of Molecular Pharmacology, Physiology and Biotechnology, Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912, USA.
J Biol Chem. 2008 Oct 17;283(42):28198-215. doi: 10.1074/jbc.M802099200. Epub 2008 Jun 6.
Sigma-1 receptor (sigma-1R) agonists enhance inositol 1,4,5-trisphosphate (IP3)-dependent calcium release from endoplasmic reticulum by inducing dissociation of ankyrin B 220 (ANK 220) from the IP3 receptor (IP3R-3), releasing it from inhibition. MCF-7 breast tumor cells express little or no sigma-1R and were used here to investigate the effect of receptor overexpression and the role of its N- and C-terminal segments in function. We stably expressed intact sigma-1R (amino acids (aa) 1-223; lines 11 and 41), N-fragment (aa 1-100; line K3), or C-fragment (aa 102-223; line sg101). C-fragment expressed as a peripheral membrane-bound protein that was removable from the endoplasmic reticulum membrane by chaotropic salt wash, consistent with lack of a putative transmembrane domain. The expressed sigma-1R, N-fragment, and C-fragment exhibited normal, low affinity, and no 3H-pentazocine binding activity, respectively. All transfected lines showed constitutive enhancement of bradykinin (BDK)-induced calcium release, because of a decrease in BDK ED50 values. Interestingly, sigma-1R and C-fragment had high activities, whereas the N-fragment was much less active. The antagonist BD1063 behaved as an inverse agonist in sigma-1R cells, whereas C-fragment was insensitive to ligand regulation. Like BDK, vasopressin- and ATP-induced calcium release was enhanced with the same pattern in cell lines. Anti-IP3R-3 immunoprecipitates from cells expressing sigma-1R or C-fragment contained significantly less ANK 220 compared with untransfected or N-fragment cells, indicating a higher amount of ankyrin-free IP3R-3. Anti-ankyrin B immunoprecipitates contained sigma-1R or C-fragment, with markedly lower levels of N-fragment present. These results suggest that sigma-1R overexpression drives sigma agonist-independent dissociation of ANK 220 from IP3R-3, resulting in activation. The C-terminal segment plays a key role in the interaction.
σ-1受体（sigma-1R）激动剂通过诱导锚蛋白B 220（ANK 220）从肌醇1,4,5-三磷酸（IP3）受体（IP3R-3）上解离，使其解除抑制，从而增强内质网中依赖IP3的钙释放。MCF-7乳腺肿瘤细胞几乎不表达或不表达sigma-1R，在此用于研究受体过表达的影响及其N端和C端片段在功能中的作用。我们稳定表达完整的sigma-1R（氨基酸（aa）1-223；11和41号线）、N片段（aa 1-100；K3号线）或C片段（aa 102-223；sg101号线）。C片段表达为外周膜结合蛋白，可通过离液盐洗涤从内质网膜上移除，这与缺乏假定的跨膜结构域一致。表达的sigma-1R、N片段和C片段分别表现出正常、低亲和力和无3H-喷他佐辛结合活性。所有转染细胞系均显示缓激肽（BDK）诱导的钙释放组成性增强，这是由于BDK半数有效剂量（ED50）值降低。有趣的是，sigma-1R和C片段具有高活性，而N片段活性则低得多。拮抗剂BD1063在sigma-1R细胞中表现为反向激动剂，而C片段对配体调节不敏感。与BDK一样，血管加压素和ATP诱导的钙释放在细胞系中以相同模式增强。与未转染或N片段细胞相比，表达sigma-1R或C片段的细胞的抗IP3R-3免疫沉淀物中ANK 220含量显著减少，表明无锚蛋白的IP3R-3含量更高。抗锚蛋白B免疫沉淀物中含有sigma-1R或C片段，而N片段含量明显较低。这些结果表明，sigma-1R过表达驱动ANK 220与IP3R-3发生不依赖于sigma激动剂的解离，从而导致激活。C端片段在这种相互作用中起关键作用。
