PhD Defense: Parnika S. Kadam, M.S.
Thesis Advisor: Kathryn Sandberg, Ph.D.
Title: REGULATION OF ANGIOTENSIN TYPE 1 RECEPTOR FUNCTION BY AN UPSTREAM SHORT OPEN READING FRAME (SORF) IN THE MRNA 5' LEADER SEQUENCE
ABSTRACT: The angiotensin type 1 receptor (AT1R) plays a critical role in blood pressure control. In this study, we investigated the role of an upstream short open reading frame (sORF) in AT1aR signaling. We investigated Angiotensin II (Ang II)- and Sar1,Ile4,Ile8-Ang II (SII)-induced extracellular signal regulated kinases 1 and 2 (ERK1/2) signaling and its alteration in the presence of an sORF in exon (E) 2 in the AT1aR mRNA 5’ leader sequence (LS). Human embryonic kidney 293 cells (HEK293) were transfected with WT [E1,2,3-AT1aR-EGFP] or MT [E1,2(-108T),3-AT1aR-EGFP]. A single nucleotide was mutated at the start codon of the intact sORF (WT) to create a disrupted sORF (MT). Dose response curves for ERK1/2 activation in WT vs MT were significantly different for Ang II (p<0.001, N=3-4) and (SII) (p<0.01, N=3). A time course of Ang II-and SII-induced ERK1/2 activation from 0–80 minutes also showed greater ERK1/2 activation in the MT compared to WT [Ang II: p<0.01; N=3 and SII: p<0.05; N=3]. The rate of internalized AT1aR in the vesicles after Ang II (100 nM) treatment was markedly increased in the WT [t1/2 in seconds(s): WT, 118 s (n=21) vs MT, 250 s (n=9); p<0.0001]. We also examined the effect of this sORF on apoptosis by flow cytometry. The presence of the sORF (WT) increased the percentage of live EGFP(+) cells (p<0.05, N=3) and reduced the percentage of early apoptotic EGFP(+) cells (p<0.05, N=4). This study shows that the E2 sORF is a selective inhibitor of AT1aR-mediated ERK1/2 activation and could be a potential therapeutic for diseases involving AT1R dysfunction by modulating AT1R activity. This study also points out the regulatory role of 5’ LS that is often not present in the expression vectors used to study proteins by transfection or overexpression systems.
Monday, September 9, 2019 at 2:00pm to 4:00pm
Basic Science Building, 239
3900 Reservoir Road, N.W., Washington