Dissecting the basis of nongenomic activation of endothelial nitric oxide synthase by estradiol:: Role of ERα domains with known nuclear functions
Dissecting the basis of nongenomic activation of endothelial nitric oxide synthase by estradiol:: Role of ERα domains with known nuclear functions
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DOI：
10.1210/me.2004-0008
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发表时间：
2005-02-01
影响因子：
--
通讯作者：
Shaul, PW
中科院分区：
文献类型：
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作者：
Chambliss, KL;Simon, L;Shaul, PW
雌激素通过激活质膜（PM）相关的雌激素受体（ER）α刺激内皮型一氧化氮合酶（eNOS）。该过程需要Src和erk信号传导以及由磷酸肌醇3-激酶（PI 3激酶）-Akt激酶引起的eNOS磷酸化，其中Src和PI 3激酶在配体活化后与ER α缔合。为了阐明非基因组eNOS刺激的基础，在COS-7细胞中研究了经典核功能所必需的ER α结构域的潜在作用。在交联研究中，雌二醇-17 β（E-2）导致PM相关ER α形成二聚体。然而，eNOS激活E-2是不变的二聚化缺陷的突变体ER α（ERalphaL 511 R）。相反，缺乏核定位信号（NLS）、NLS 2、3（ER α Δ 250 -274）或DNA结合结构域（ER α Δ 185 -251）的ER α突变体（其通常靶向PM和小窝/筏）不能激活eNOS。NLS 2/NLS 3的缺失阻止了Src和erk的激活，并改变了配体诱导的PI 3激酶-ER α相互作用，阻止了eNOS磷酸化。DNA结合结构域的丢失并没有改变E-2对Src或erk的激活，但配体诱导的PI 3激酶-ER α结合和eNOS磷酸化并没有发生。因此，ERalpha偶联eNOS不需要二聚化;然而，NLS 2/NLS 3在Src活化中起作用，并且DNA结合区参与ERalpha和PI 3激酶之间的动态相互作用。
Estradiol stimulates endothelial nitric oxide synthase (eNOS) via the activation of plasma membrane (PM)-associated estrogen receptor (ER) alpha. The process requires Src and erk signaling and eNOS phosphorylation by phosphoinositide 3-kinase (PI3 kinase)-Akt kinase, with Src and PI3 kinase associating with ERalpha upon ligand activation. To delineate the basis of nongenomic eNOS stimulation, the potential roles of ERalpha domains necessary for classical nuclear function were investigated in COS-7 cells. In cross-linking studies, estradiol-17beta (E-2) caused PM-associated ERalpha to form dimers. However, eNOS activation by E-2 was unaltered for a dimerization-deficient mutant ERalpha (ERalphaL511R). In contrast, ERalpha mutants lacking the nuclear localization signals (NLS), NLS2,3 (ERalphaDelta250-274) or the DNA binding domain (ERalphaDelta185-251), which targeted normally to PM and caveolae/rafts, were incapable of activating eNOS. The loss of NLS2/NLS3 prevented Src and erk activation, and it altered ligand-induced PI3 kinase-ERalpha interaction and prevented eNOS phosphorylation. Loss of the DNA binding domain did not change E-2 activation of Src or erk, but ligand-induced PI3 kinase-ERalpha binding and eNOS phosphorylation did not occur. Thus, dimerization is not required for ERalpha coupling to eNOS; however, NLS2/NLS3 plays a role in Src activation, and the DNA binding region is involved in the dynamic interaction between ERalpha and PI3 kinase.