An Upstream Short Open Reading Frame in the Angiotensin Type 1 Receptor Regulates the β‐Arrestin Signaling Pathway

Abstract

The angiotensin type 1 receptor (AT1R) plays a critical role in blood pressure control and thus furthering our understanding of its regulation has physiological and clinical significance. We previously showed a short open reading frame (sORF) in exon (E)2 of the 5' leader sequence of the AT1aR mRNA inhibits AT1R mRNA translation and receptor internalization. In this study, we investigated the role of this sORF in AT1R signaling. The sORF start codon in E2 was disrupted by site‐directed mutagenesis [E1,2(−108T),3‐AT1aR] (MT), which along with the wild type unmutated sORF [E1,2,3‐AT1aR] (WT) were cloned into an expression plasmid [EGFPN2]. Human embryonic kidney‐293 cells were then transfected with WT or MT plasmids. No significant differences were found in the time course of Ang II‐induced ERK1/2 activation between 0–80 min at angiotensin doses of 10 nm (n=3) and 100 nM (n=3); however, the peak response to 10 nM Ang II at 5 min was 1.9‐fold higher in cells transfected with MT compared to WT plasmids (p<0.05; n=4). Furthermore, peak ERK1/2 activation was 1.3‐fold higher in cells transfected with MT compared to WT plasmids after stimulation with Sar1,Ile4,Ile8‐Ang II, a biased AT1R agonist which selectively activates the β‐arrestin‐dependent pathway [pERK1/2/total ERK1/2 normalized to AT1R Bmax: WT, 1.8E‐05 ± 9.5E‐07 vs. MT, 2.3E‐05 ± 3.4E‐06, p<0.05; n=3]. A time course of Sar1,Ile4,Ile8‐Ang II‐induced ERK1/2 activation from 0–80 minutes at 30 μM also showed greater ERK1/2 activation in cells transfected with the MT compared to WT plasmids [p<0.05; n=4]. Since β‐arrestins are implicated to play a role in apoptotic signaling, we are also studying the effect of this sORF on Ang II‐induced cell proliferation and both early and late apoptosis by flow cytometry. This study supports our previous findings that a seven amino acid peptide (PEP7) encoded by this sORF is a selective inhibitor of AT1R‐mediated ERK1/2 activation. These findings not only have implications for the regulation of AT1Rs in physiological and pathological conditions but also for the superfamily of G protein coupled receptors, many of which possess sORFs of unknown function in their 5’ leader sequence.Support or Funding InformationNIH/NHLBI R01‐HL121456 to KS NIH TL1 Award TL1TR001431This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.