Evidence for involvement of 3'-untranslated region in determining angiotensin II receptor coupling specificity to G-protein
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 (AT1A) with and without 3′-UTR differed in AT1A mRNA content and its coupling with intracellular signalling pathways. Moreover, RNA mobility-shift assay and UV cross-linking studies using the AT1A 3′-UTR probe identified a major mRNA-binding protein complex of 55kDa in Chinese-hamster ovary-K1 cells. In the present study, we have determined the functional significance of the native AT1A receptor 3′-UTR in rat liver epithelial (WB) cell lines by co-expressing the AT1A 3′-UTR sequence ‘decoy’ to compete with the native receptor 3′-UTR for its mRNA-binding proteins. PCR analysis using specific primers for the AT1A receptor and [125I]angiotensin II (AngII)-binding studies demonstrated the expression of the native AT1A receptors in WB (Bmax = 2.7pmol/mg of protein, Kd = 0.56nM). 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 [3H]thymidine and [3H]leucine incorporation respectively. Activation of [3H]thymidine and [3H]leucine correlated with a significant increase in cell number (cellular hyperplasia). In these cells, AngII stimulated GTPase activity by AT1 receptor coupling with G-protein αi. We also delineated that functional coupling of AT1A receptor with G-protein αi is an essential mechanism for AngII-mediated cellular hyperplasia in WB by specifically blocking G-protein αi 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 [3H]leucine incorporation and no increase in [3H]thymidine incorporation and cell number. Furthermore, [125I]AngII cross-linking and immunoprecipitation studies using specific G-protein α antibodies showed that in wild-type cells, the AT1A receptor coupled with G-protein αi, whereas in cells expressing the 3′-UTR ‘decoy’, the AT1A receptor coupled with G-protein αq. These findings indicate that the 3′-UTR-mediated changes in receptor function may be mediated in part by a switch from G-protein αi to G-protein αq coupling of the receptor. Our results suggest that the 3′-UTR-mediated post-transcriptional modification of the AT1A receptor is critical for regulating tissue-specific receptor functions.