Modulation of the rat angiotensin type 1a receptor by an upstream short open reading frame

The rat angiotensin type 1a receptor (AT_1aR) 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 AT_1aR coding region located in exon 3 (E3). To determine the role of this E2 sORF in AT_1aR signaling, human embryonic kidney-293 (HEK293) cells were transfected with plasmids containing AT_1aR cDNA with either an intact or disrupted E2 sORF. The intact sORF attenuated the efficacy of angiotensin (Ang) II (p < 0.001) and sarcosine¹,Ile⁴,Ile⁸-Ang II (SII), (p < 0.01) to activate AT_1aR signaling through extracellular signal-related kinases 1/2 (ERK1/2). A time-course showed agonist-induced AT_1aR-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 AT_1aR plasmid containing the intact sORF. These findings have implications for the regulation of AT₁Rs in physiological and pathological conditions and warrant investigation of sORFs in the 5' leader sequence (5'LS) of other GPCRs.