Kadam Parnika S, Mueller Susette C, Ji Hong, Liu Jun, Pai Amrita V, Ma Junfeng, Speth Robert C, Sandberg Kathryn
Department of Biochemistry, Molecular and Cellular Biology, Georgetown University, Washington, DC, United States; Department of Medicine, Georgetown University, Washington, DC, United States.
Department of Oncology, Georgetown University, Washington, DC, United States.
Peptides. 2021 Jun;140:170529. doi: 10.1016/j.peptides.2021.170529. Epub 2021 Mar 17.
The rat angiotensin type 1a receptor (ATR) is a peptide hormone G protein-coupled receptor (GPCR) that plays a key role in electrolyte homeostasis and blood pressure control. There is a highly conserved short open reading frame (sORF) in exon 2 (E2) that is downstream from exon 1 (E1) and upstream of the ATR coding region located in exon 3 (E3). To determine the role of this E2 sORF in ATR signaling, human embryonic kidney-293 (HEK293) cells were transfected with plasmids containing ATR cDNA with either an intact or disrupted E2 sORF. The intact sORF attenuated the efficacy of angiotensin (Ang) II (p < 0.001) and sarcosine,Ile,Ile-Ang II (SII), (p < 0.01) to activate ATR signaling through extracellular signal-related kinases 1/2 (ERK1/2). A time-course showed agonist-induced ATR-mediated ERK1/2 activation was slower in the presence of the intact compared to the disrupted sORF [Ang II: p < 0.01 and SII: p < 0.05]. Ang II-induced ERK1/2 activation was completely inhibited by the protein kinase C (PKC) inhibitor Ro 31-8220 regardless of whether the sORF was intact or disrupted. Flow cytometric analyses suggested the intact sORF improved cell survival; the percentage of live cells increased (p < 0.05) while the percentage of early apoptotic cells decreased (p < 0.01) in cells transfected with the ATR plasmid containing the intact sORF. These findings have implications for the regulation of ATRs in physiological and pathological conditions and warrant investigation of sORFs in the 5' leader sequence (5'LS) of other GPCRs.
大鼠血管紧张素1a受体（ATR）是一种肽激素G蛋白偶联受体（GPCR），在电解质稳态和血压控制中起关键作用。外显子2（E2）中有一个高度保守的短开放阅读框（sORF），它位于外显子1（E1）下游且在位于外显子3（E3）的ATR编码区域上游。为了确定这个E2 sORF在ATR信号传导中的作用，用含有完整或破坏的E2 sORF的ATR cDNA质粒转染人胚肾-293（HEK293）细胞。完整的sORF减弱了血管紧张素（Ang）II（p<0.001）和肌氨酸、异亮氨酸、异亮氨酸-血管紧张素II（SII）（p<0.01）通过细胞外信号相关激酶1/2（ERK1/2）激活ATR信号传导的效力。时间进程显示，与破坏的sORF相比，完整sORF存在时激动剂诱导的ATR介导的ERK1/2激活较慢[Ang II：p<0.01，SII：p<0.05]。无论sORF是否完整，蛋白激酶C（PKC）抑制剂Ro 31-8220均可完全抑制Ang II诱导的ERK1/2激活。流式细胞术分析表明完整的sORF可改善细胞存活；转染含有完整sORF的ATR质粒的细胞中活细胞百分比增加（p<0.05），而早期凋亡细胞百分比降低（p<0.01）。这些发现对生理和病理条件下ATR的调节具有重要意义，值得对其他GPCR的5'前导序列（5'LS）中的sORF进行研究。
