Functional role for the angiotensin II receptor (AT1A) 3'-untranslated region in determining cellular responses to agonist: evidence for recognition by RNA binding proteins.
Functional role for the angiotensin II receptor (AT1A) 3'-untranslated region in determining cellular responses to agonist: evidence for recognition by RNA binding proteins.
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血管紧张素 II 受体 (AT1A) 3-非翻译区在确定细胞对激动剂反应中的功能作用：RNA 结合蛋白识别的证据。
DOI：
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发表时间：
1998
影响因子：
4.1
通讯作者：
Kenneth M. Baker
中科院分区：
文献类型：
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作者：
T. Thekkumkara;Walter G. Thomas;Thomas J. Motel;Kenneth M. Baker
我们通过稳定转染带有或不带有 845 bp 3'-UTR 的受体基因的编码区，证明了血管紧张素 II (Ang II) 受体亚型 AT1A mRNA 的 3'-非翻译区 (3'-UTR) 在中国仓鼠卵巢 (CHO-K1) 细胞中的功能作用。本研究中使用了表达相似水平的细胞表面受体（具有 3'-UTR，Bmax=571 fmol/mg 蛋白质；没有 3'-UTR，Bmax=663 fmol/mg 蛋白质）的两种细胞系。两种细胞系均表达高亲和力受体（有 3'-UTR，Kd=0.83 nM；无 3'-UTR，Kd=0.82 nM），并且在 GTP[S] 存在的情况下用 125I 标记的 Ang II 进行的结合研究表明，两者均与异源三聚体 G 蛋白偶联。尽管有这些相似之处，但受体介导的细胞信号传导途径仍观察到显着差异。在没有 3'-UTR 的细胞中，Ang II 刺激 cAMP 积累增加（比对照高 11 倍），而在有 3'-UTR 的细胞中没有观察到刺激，这与之前在大多数内源性 Ang II 受体 (AT1) 表达细胞中观察到的结果一致。在没有 3'-UTR 的细胞中，Ang II 对 cAMP 的激活与 DNA 合成的抑制相关，通过[3H]胸苷掺入来确定。 Ang II 介导的反应被 EXP3174（一种选择性非肽受体拮抗剂）阻断。我们还观察到有和没有 3'-UTR 的细胞响应 Ang II 的细胞内钙瞬时曲线的差异。在具有 3'-UTR 的细胞中，在 Ang II 刺激后观察到细胞内钙的持续水平，而没有 3'-UTR 的细胞在 Ang II 治疗的 50 秒内显示出完全恢复到基础水平。尽管表达的外源基因受到组成型表达启动子（巨细胞病毒启动子）的控制，但 Northern 印迹分析显示，与具有 3'-UTR 的细胞相比，没有 3'-UTR 的细胞中 AT1A mRNA 的积累量要高得多。对具有和不具有 3'-UTR 的细胞中 AT1A mRNA 的衰减率的分析表明，通常不稳定的 AT1A 受体 mRNA 通过去除其 3'-UTR 变得高度稳定，从而确定了 3'-UTR 在 mRNA 不稳定中的作用。有趣的是，两种细胞在细胞表面表达相似水平的受体，表明3'-UTR也参与受体蛋白向质膜的有效翻译和/或易位。我们假设受体的这些 3'-UTR 介导的功能受到 RNA 结合蛋白的调节。为了鉴定 AT1A 3'-UTR 可能的 RNA 结合蛋白，从亲代 CHO-K1 细胞中制备细胞提取物，并进行 3'-UTR 结合测定、电泳迁移率变化测定和 UV 交联研究。鉴定出 55 kDa 的主要细胞蛋白，该蛋白与 3'-UTR 特异性相互作用。我们的数据表明，AT1A 的 3'-UTR 可以控制特定的受体功能，可能是通过 RNA 结合蛋白选择性识别 3'-UTR。
We demonstrate a functional role for the 3'-untranslated region (3'-UTR) of the angiotensin II (Ang II) receptor subtype AT1A mRNA in Chinese hamster ovary (CHO-K1) cells by stably transfecting the coding region of the receptor gene with or without the 845 bp 3'-UTR. Two cell lines expressing similar levels of cell-surface receptors (with 3'-UTR, Bmax=571 fmol/mg protein; without 3'-UTR, Bmax=663 fmol/mg protein) were used in the present study. Both cell lines expressed high-affinity receptors (with 3'-UTR, Kd=0.83 nM; without 3'-UTR, Kd=0.82 nM), and binding studies with 125I-labelled Ang II in the presence of GTP[S] demonstrated that both coupled to heterotrimeric G-proteins. Despite these similarities, significant differences were observed for receptor-mediated cell signalling pathways. In cells without the 3'-UTR, Ang II stimulated an increase in cAMP accumulation (11-fold above control) and in cells with the 3'-UTR no stimulation was observed, which was consistent with previous observations in most endogenous Ang II receptor (AT1)-expressing cells. Activation of cAMP by Ang II in cells without the 3'-UTR correlated with an inhibition of DNA synthesis, determined by [3H]thymidine incorporation. Ang II-mediated responses were blocked by EXP3174, a selective non-peptide receptor antagonist. We also observed differences in the transient profiles of intracellular calcium between cells with and without the 3'-UTR in response to Ang II. In cells with the 3'-UTR, a sustained level of intracellular calcium was observed after Ang II stimulation, whereas cells without the 3'-UTR displayed a full return to basal level within 50 s of Ang II treatment. Even though the expressed exogenous gene is under the control of a constitutively expressing promoter (cytomegalovirus promoter), Northern-blot analysis revealed a considerably greater accumulation of AT1A mRNA in cells without the 3'-UTR compared with cells with the 3'-UTR. Analysis of the decay rate of the AT1A mRNA in cells with and without the 3'-UTR revealed that the normally unstable AT1A receptor mRNA became highly stable by removing its 3'-UTR, identifying a role for the 3'-UTR in mRNA destabilization. Interestingly, both cells express similar levels of receptors at the cell surface, suggesting that the 3'-UTR is also involved in the efficient translation and/or translocation of the receptor protein to the plasma membrane. We hypothesized that these 3'-UTR-mediated functions of the receptor are regulated by RNA-binding proteins. To identify possible RNA-binding proteins for the AT1A 3'-UTR, cellular extracts were prepared from parental CHO-K1 cells and 3'-UTR-binding assays, electrophoretic mobility-shift assays and UV crosslinking studies were performed. A major cellular protein of 55 kDa was identified, which specifically interacted with the 3'-UTR. Our data suggest that the 3'-UTR of the AT1A can control specific receptor functions, perhaps via selective recognition of the 3'-UTR by RNA-binding proteins.