Abstract 022: A Short Open Reading Frame in the Angiotensin Type 1a Receptor 5' Leader Sequence Increases the Rate of Receptor Internalization

Abstract

Background: We recently found that a 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 inhibits AT1aR-mediated activation of extracellular signal-regulated protein kinases 1 and 2 (Erk1/2) without having any effect on the AT1aR-inositol trisphosphate protein kinase C pathway. To investigate the mechanism by which PEP7 selectively inhibits the AT1aR-Erk1/2 signaling cascade, the start codon of the sORF was mutated at adenine -108 (A-108 to T-108) to create E1,2(-108T),3-AT1aR followed by cloning the E1,2,3-AT1aR and the mutant E1,2(-108T),3-AT1aR into the pEGFP-N2 plasmid. Methods: Human embryonic kidney 293 (HEK-293) cells were transfected with E1,2,3-AT1aR (intact sORF construct) or E1,2(-108T),3-AT1aR (disrupted sORF construct) by lipofectamine. Forty eight to seventy two hours later, live cell time lapse images were collected before and after treatment with Ang II (100 nM) using a TE300 Spinning disk laser scanning confocal microscope. Image analysis was performed using Velocity software. Results: Within 5 min of Ang II stimulation, punctae formed and moved throughout the cell membrane in cells transfected with both EGFP tagged receptors; however, even though the punctae represent the localized accumulation of identical AT1aR proteins, there were distinct differences in the intensity and time course of punctae. The rate of vesicle formation after Ang II treatment was markedly decreased by disrupting the PEP7 sORF [t1/2 (s): E1,2,3-AT1aR, 203s (N=21) vs E1,2(-108T),3-AT1aR, 328s (N=14); p<0.0001]. Conclusion: The PEP7 sORF facilitates Ang II-induced AT1R internalization. These findings suggest that we have uncovered a new mechanism governing agonist-induced AT1aR cellular trafficking that could have implications not only for regulation of AT1aR signaling cascades but also for other trafficking proteins that contain an upstream sORF within their 5'LS.