The physiological responses of angiotensin II (Ang II) are mediated across the cell membrane through the angiotensin II type 1 receptor (AT1R), a heptahelical membrane protein coupled to trimeric G-proteins on the cytosolic side. AT1R on binding its ligand, Ang II, leads to downregulation of cell-surface receptor and also its mRNA. We have investigated whether the 3'- and 5'-untranslated regions of the human AT1R mRNA mediate the degradation of hAT1R mRNA by post-transcriptional mechanisms in human adrenocortical carcinoma cell line (H295R cells). Protein kinase C (PKC) activator, phorbol-12-myristate-13-acetate (PMA), showed that the downregulation of hAT1R mRNA is mediated by the PKC pathway. Experiments performed in the presence of cycloheximide and/or Ang II demonstrated that protein translation is essential for hAT1R mRNA downregulation. In vitro cell-free RNA degradation assays did not show any increase in the rate of degradation of in vitro transcribed RNA in the presence of cytoplasmic extract from cells treated with Ang II, which suggested that hAT1R steady state mRNA levels may not be mediated by changes in mRNA degradation rates. Luciferase assay after transient transfection of chimeric plasmids of luciferase and hAT1R-3'-UTR showed that Ang II stabilizes the mRNA rather than increase the rate of degradation. Similar results were observed in Northern blot experiments utilizing beta-globin fusion with 3'-UTR that led to stabilization of the chimeric mRNA. Luciferase fusion constructs with both 5'- and 3'-UTRs demonstrated that UTRs are not involved in the Ang II-mediated degradation of hAT1R mRNA. Experiments using transcriptional inhibitor actinomycin D demonstrated that the hAT1R mRNA is not destabilized in response to Ang II activation in H295R cells. Nuclear run-on assay performed in the adrenocortical carcinoma cells demonstrated that the Ang II-stimulated downregulation of hAT1R is mediated by transcriptional inhibition. The transcription of hAT1R mRNA was reduced by 44 and 70% after Ang II treatment for 1 and 2 hours, respectively. Taken together, these findings suggest that the Ang II-induced downregulation of hAT1R steady state mRNA levels is transcriptionally controlled and is not mediated by post-transcriptional mechanisms.
Identifer | oai:union.ndltd.org:BGMYU2/oai:scholarsarchive.byu.edu:etd-1624 |
Date | 14 July 2005 |
Creators | Victor, Xylophone Vijai Aasee |
Publisher | BYU ScholarsArchive |
Source Sets | Brigham Young University |
Detected Language | English |
Type | text |
Format | application/pdf |
Source | Theses and Dissertations |
Rights | http://lib.byu.edu/about/copyright/ |
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