Translational regulation of angiotensin type 1a receptor expression and signaling by upstream AUGs in the 5′ leader sequence

Abstract

Rat angiotensin type 1a receptor (AT1aR) is regulated by four upstream AUGs present in the 5′ leader sequence (5′-LS). Disruption of all four upstream AUGs (QM) results in 2-3-fold higher levels of angiotensin type 1 receptor (AT1R) densities in transiently transfected rat aortic smooth muscle cells (A10 cells) and stably transfected Chinese hamster ovary cells. Cells expressing QM have 5-fold higher levels of angiotensin II-induced inositol phosphate production than wild type (WT). Polysome analysis showed that QM mRNA is present in heavier fractions than the WT transcript, and 5.7-fold more AT1R protein is produced by in vitro translation from QM transcripts compared with WT transcripts. The AT1aR comprises 3 exons. Exon 3 (E3) encodes the entire open reading frame and 3′-untranslated region. Exons 1 and 2 (E1 and E2) and 52 nucleotides of E3 encode the 5′-LS. The AUGs in both exons contribute to the inhibitory effect on AT1R expression but not to the same degree. Disruption of the AUGs in exon 2 (DM2) relieves half of the inhibition, whereas disruption of the AUGs in exon 1 (DM1) is without effect. Disruption of the AUGs in exon 2 results in levels of receptor expression and translation that are indistinguishable from the alternative splice variant E1,3, which we previously showed was more efficiently translated than the E1,2,3 transcript. Individual mutations revealed that only the fourth AUG increased AT1R translation. In conclusion, all four AUGs present in the 5′-LS function cumulatively to suppress AT1aR expression and signaling by inhibiting translation. These data also show that both AUGs in E2 contribute to the inhibitory cis element present in this alternatively spliced exon.