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Evidence for involvement of 3'-untranslated region in determining angiotensin II receptor coupling specificity to G-protein.

ABSTRACT: The mRNA 3'-untranslated region (3'-UTR) of many genes has been identified as an important regulator of the mRNA transcript itself as well as the translated product. Previously, we demonstrated that Chinese-hamster ovary-K1 cells stably expressing angiotensin receptor subtypes (AT(1A)) with and without 3'-UTR differed in AT(1A) mRNA content and its coupling with intracellular signalling pathways. Moreover, RNA mobility-shift assay and UV cross-linking studies using the AT(1A) 3'-UTR probe identified a major mRNA-binding protein complex of 55 kDa in Chinese-hamster ovary-K1 cells. In the present study, we have determined the functional significance of the native AT(1A) receptor 3'-UTR in rat liver epithelial (WB) cell lines by co-expressing the AT(1A) 3'-UTR sequence 'decoy' to compete with the native receptor 3'-UTR for its mRNA-binding proteins. PCR analysis using specific primers for the AT(1A) receptor and [(125)I]angiotensin II (AngII)-binding studies demonstrated the expression of the native AT(1A) receptors in WB (B(max)=2.7 pmol/mg of protein, K(d)=0.56 nM). Northern-blot analysis showed a significant increase in native receptor mRNA expression in 3'-UTR decoy-expressing cells, confirming the role of 3'-UTR in mRNA destabilization. Compared with vehicle control, AngII induced DNA and protein synthesis in wild-type WB as measured by [(3)H]thymidine and [(3)H]leucine incorporation respectively. Activation of [(3)H]thymidine and [(3)H]leucine correlated with a significant increase in cell number (cellular hyperplasia). In these cells, AngII stimulated GTPase activity by AT(1) receptor coupling with G-protein alpha i. We also delineated that functional coupling of AT(1A) receptor with G-protein alpha i is an essential mechanism for AngII-mediated cellular hyperplasia in WB by specifically blocking G-protein alpha i activation. In contrast with wild-type cells, stable expression of the 3'-UTR 'decoy' produced AngII-stimulated protein synthesis and cellular hypertrophy as demonstrated by a significant increase in [(3)H]leucine incorporation and no increase in [(3)H]thymidine incorporation and cell number. Furthermore, [(125)I]AngII cross-linking and immunoprecipitation studies using specific G-protein alpha antibodies showed that in wild-type cells, the AT(1A) receptor coupled with G-protein alpha i, whereas in cells expressing the 3'-UTR 'decoy', the AT(1A) receptor coupled with G-protein alpha q. These findings indicate that the 3'-UTR-mediated changes in receptor function may be mediated in part by a switch from G-protein alpha i to G-protein alpha q coupling of the receptor. Our results suggest that the 3'-UTR-mediated post-transcriptional modification of the AT(1A) receptor is critical for regulating tissue-specific receptor functions.

SUBMITTER: Thekkumkara TJ

PROVIDER: S-EPMC1223184 | biostudies-other | 2003 Mar

REPOSITORIES: biostudies-other

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Project description:We demonstrate a functional role for the 3'-untranslated region (3'-UTR) of the angiotensin II (Ang II) receptor subtype AT1A mRNA in Chinese hamster ovary (CHO-K1) cells by stably transfecting the coding region of the receptor gene with or without the 845 bp 3'-UTR. Two cell lines expressing similar levels of cell-surface receptors (with 3'-UTR, Bmax=571 fmol/mg protein; without 3'-UTR, Bmax=663 fmol/mg protein) were used in the present study. Both cell lines expressed high-affinity receptors (with 3'-UTR, Kd=0.83 nM; without 3'-UTR, Kd=0.82 nM), and binding studies with 125I-labelled Ang II in the presence of GTP[S] demonstrated that both coupled to heterotrimeric G-proteins. Despite these similarities, significant differences were observed for receptor-mediated cell signalling pathways. In cells without the 3'-UTR, Ang II stimulated an increase in cAMP accumulation (11-fold above control) and in cells with the 3'-UTR no stimulation was observed, which was consistent with previous observations in most endogenous Ang II receptor (AT1)-expressing cells. Activation of cAMP by Ang II in cells without the 3'-UTR correlated with an inhibition of DNA synthesis, determined by [3H]thymidine incorporation. Ang II-mediated responses were blocked by EXP3174, a selective non-peptide receptor antagonist. We also observed differences in the transient profiles of intracellular calcium between cells with and without the 3'-UTR in response to Ang II. In cells with the 3'-UTR, a sustained level of intracellular calcium was observed after Ang II stimulation, whereas cells without the 3'-UTR displayed a full return to basal level within 50 s of Ang II treatment. Even though the expressed exogenous gene is under the control of a constitutively expressing promoter (cytomegalovirus promoter), Northern-blot analysis revealed a considerably greater accumulation of AT1A mRNA in cells without the 3'-UTR compared with cells with the 3'-UTR. Analysis of the decay rate of the AT1A mRNA in cells with and without the 3'-UTR revealed that the normally unstable AT1A receptor mRNA became highly stable by removing its 3'-UTR, identifying a role for the 3'-UTR in mRNA destabilization. Interestingly, both cells express similar levels of receptors at the cell surface, suggesting that the 3'-UTR is also involved in the efficient translation and/or translocation of the receptor protein to the plasma membrane. We hypothesized that these 3'-UTR-mediated functions of the receptor are regulated by RNA-binding proteins. To identify possible RNA-binding proteins for the AT1A 3'-UTR, cellular extracts were prepared from parental CHO-K1 cells and 3'-UTR-binding assays, electrophoretic mobility-shift assays and UV crosslinking studies were performed. A major cellular protein of 55 kDa was identified, which specifically interacted with the 3'-UTR. Our data suggest that the 3'-UTR of the AT1A can control specific receptor functions, perhaps via selective recognition of the 3'-UTR by RNA-binding proteins.

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Evidence for involvement of 3'-untranslated region in determining angiotensin II receptor coupling specificity to G-protein.

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