Plasma membrane localization and function of the estrogen receptor α variant (ER46) in human endothelial cells
Plasma membrane localization and function of the estrogen receptor α variant (ER46) in human endothelial cells
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DOI：
10.1073/pnas.0831079100
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发表时间：
2003-04-15
影响因子：
11.1
通讯作者：
Bender, JR
中科院分区：
文献类型：
--
作者：
Li, L;Haynes, MP;Bender, JR
已在一系列非内皮细胞中鉴定出雌激素受体（ER）a变体。我们以前证明，雌激素通过磷脂酰肌醇3-激酶/Akt/内皮一氧化氮合酶（eNOS）途径在EA.hy926细胞（永生化的人内皮细胞），表达一个46 kDa的ER迅速诱导一氧化氮释放。我们现在确认，由于选择性剪接，46 kDa的内皮细胞蛋白（ER 46）是全长ER α（ER 66）的氨基末端截短产物。ER 46在静息的、雌激素剥夺的细胞的质膜、胞质溶胶和细胞核中表达。流式细胞术和免疫荧光显微镜分析表明，ER 46的C端，而不是N端是抗体可访问的质膜。棕榈酰化与衣霉素和[H-3]棕榈酸标记的抑制表现出雌激素诱导的，棕榈酰化依赖质膜ER 46招聘，重组成小窝。在重建的，雌激素刺激的COS-7（ER-空）细胞，膜ER 46更有效地触发膜eNOS磷酸化比ER 66。相反，ER 66比ER 46更有效地介导雌激素反应元件转录因子基因的反式激活。这些结果表明，ER 46是本地化的，并进一步动态地以棕榈酰化依赖性的方式靶向质膜。ER 46比全长ER 66更有效地调节膜启动的雌激素作用，包括eNOS活化。这些发现可能对雌激素受体激动剂的血管特异性靶向具有重要意义。
Estrogen receptor (ER) a variants have been identified in an array of nonendothelial cells. We previously demonstrated that estrogen rapidly induces nitric oxide release via a phosphatidylinositol 3-kinase/Akt/endothelial nitric-oxide synthase (eNOS) pathway in EA.hy926 cells (immortalized human endothelial cells), which express a 46-kDa ER. We now confirm that, due to alternative splicing, the 46-kDa endothelial cell protein (ER46) is an amino-terminal truncated product of full-length ERalpha (ER66). ER46 is expressed in the plasma membrane, cytosol, and nucleus of resting, estrogen-deprived cells. Flow cytometric and immunofluorescence microscopic analyses demonstrated that the ER46 C but not N terminus is Ab-accessible in the plasma membrane. Inhibition of palmitoylation with tunicamycin and [H-3]palmitic acid labeling demonstrated an estrogen-induced, palmitoylation-clependent plasma membrane ER46 recruitment, with reorganization into caveolae. In reconstituted, estrogen-stimulated COS-7 (ER-null) cells, membrane ER46 more efficiently triggered membrane eNOS phosphorylation than ER66. Conversely, ER66 more efficiently mediated estrogen response element reporter-gene transactivation than ER46. These results demonstrate that ER46 is localized and further dynamically targeted to the plasma membrane in a palmitoylation-dependent manner. ER46 more efficiently modulates membrane-initiated estrogen actions, including eNOS activation, than full-length ER66. These findings may have important implications in vascular-specific targeting of estrogen receptor agonists.