Regulation of Angiotensin Receptor Trafficking by an Upstream Short Open Reading Frame in the mRNA 5′ Leader Sequence

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BackgroundA seven amino acid peptide (PEP7) encoded within a short open reading frame (sORF) in exon 2 of the 5′ leader sequence (5′LS) of the angiotensin type 1a receptor (AT1aR) mRNA is highly conserved in rats, mice and humans.AimOur goal is to investigate the mechanism by which PEP7 modulates AT1aR signaling cascade.MethodThe start codon of the sORF was mutated at adenine ‐108 (A−108 to T−108) to create E1,2(−108T),3‐AT1aR and cloned into the pEGFP‐N2 plasmid (construct). Human embryonic kidney (HEK) 293 cells were transfected with intact or the disrupted sORF construct. Live cell imaging in the presence of a LysoTracker dye (staining lysosomes), immunostaining and western blot analysis were performed after treatment with Ang II (100 nM).ResultsWhile the rate of vesicle formation after Ang II treatment was decreased by disrupting the PEP7 sORF [t1/2(s): E1,2,3‐AT1aR, 203s vs E1,2(−108T),3‐AT1aR, 328s; p<0.05; n=15], disruption of the PEP7 sORF did not alter the transport of AT1aR‐GFP to lysosomes within the first 25 minutes of Ang II stimulation. Immunostaining suggested that disruption of PEP7 sORF alters Ang II‐induced colocalization of AT1aR‐GFP with β‐arrestin and pERK1/2 with β‐arrestin. Western blot analysis showed that disruption of PEP7 sORF decreased the level of ERK activation in transfected HEK 293 cells. [pERK1/2 /total ERK1/2: E1,2,3‐AT1aR, 1.2 ± 0.1 vs E1,2(−108T),3‐AT1aR, 0.79 ± 0.0002, p<0.05; n=2]ConclusionPEP7 sORF regulates Ang‐II induced AT1aR internalization and signal transduction through the β‐arrestin‐pERK1/2 pathway by increasing ERK1/2 activation.Support or Funding InformationNIH R01‐HL121456 to KS.