Identification of a putative nuclear localization sequence within ANG II AT1A receptor associated with nuclear activation
Identification of a putative nuclear localization sequence within ANG II AT1A receptor associated with nuclear activation
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DOI：
10.1152/ajpcell.00337.2006
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发表时间：
2007-04-01
影响因子：
5.5
通讯作者：
Ullian, Michael E.
中科院分区：
文献类型：
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作者：
Morinelli, Thomas A.;Raymond, John R.;Ullian, Michael E.
血管紧张素II（ANG II）1型（AT（1））受体与其他G蛋白偶联受体类似，在激动剂相互作用后经历脱敏和内化，以及潜在的核定位.有证据表明羧基末端尾可能参与受体的核定位.在本研究中，我们研究了负责核转位现象和由此产生的核活化的特定区域的受体的羧基末端尾。检测稳定表达野生型AT（1A）受体-绿色荧光蛋白（AT（1A）R/ GFP）构建体或推定的核定位序列（NLS）[K307 Q] AT（1A）R/ GFP（KQ/ AT（1A）R/ GFP）的定点突变的人胚肾细胞在受体核运输和核活化方面的差异.野生型/ GFP构建体和KQ/ AT（1A）R/ GFP突变体的受体表达、细胞内信号传导和ANG II诱导的内化是相似的.激光扫描共聚焦显微镜显示，在表达AT（1A）R/ GFP的细胞中，在ANG II（100 nM）刺激30分钟内，受体被转运到核区域并与核纤层蛋白B共定位，而KQ/ AT（1A）R/ GFP突变体未能显示核定位。用抗GFP抗体对核裂解物进行免疫印迹证实了这些观察结果。野生型受体的核定位与EGR- 1和PTGS- 2基因的转录增加相关，而核缺陷型KQ/ AT（1A）R/ GFP突变体仅显示EGR- 1基因的转录增加.这些结果表明，NLS（KKFKKY; aa 307 - 312）位于AT（1A）受体的胞质尾区，该受体的核定位与考克斯- 2基因PTGS- 2的特异性转录激活相对应。
Angiotensin II ( ANG II) type 1 ( AT(1)) receptors, similar to other G protein- coupled receptors, undergo desensitization and internalization, and potentially nuclear localization, subsequent to agonist interaction. Evidence suggests that the carboxy- terminal tail may be involved in receptor nuclear localization. In the present study, we examined the carboxyterminal tail of the receptor for specific regions responsible for the nuclear translocation phenomenon and resultant nuclear activation. Human embryonic kidney cells stably expressing either a wild- type AT(1A) receptor- green fluorescent protein ( AT(1A)R/ GFP) construct or a site- directed mutation of a putative nuclear localization sequence ( NLS) [ K307Q] AT(1A)R/ GFP ( KQ/ AT(1A)R/ GFP), were examined for differences in receptor nuclear trafficking and nuclear activation. Receptor expression, intracellular signaling, and ANG II- induced internalization of the wild- type/ GFP construct and of the KQ/ AT(1A)R/ GFP mutant was similar. Laser scanning confocal microscopy showed that in cells expressing the AT(1A)R/ GFP, trafficking of the receptor to the nuclear area and colocalization with lamin B occurred within 30 min of ANG II ( 100 nM) stimulation, whereas the KQ/ AT(1A)R/ GFP mutant failed to demonstrate nuclear localization. Immunoblotting of nuclear lysates with an anti- GFP antibody confirmed these observations. Nuclear localization of the wild- type receptor correlated with increase transcription for both EGR- 1 and PTGS- 2 genes while the nucleardeficient KQ/ AT(1A)R/ GFP mutant demonstrated increases for only the EGR- 1 gene. These results suggest that a NLS ( KKFKKY; aa307 312) is located within the cytoplasmic tail of the AT(1A) receptor and that nuclear localization of the receptor corresponds with specific activation of transcription for the COX- 2 gene PTGS- 2.