Functional role for the angiotensin II receptor (AT1A) 3′-untranslated region in determining cellular responses to agonist:: evidence for recognition by RNA binding proteins

We demonstrate a functional role for the 3'-untranslated region (3'-UTR) of the angiotensin II (Ang II) receptor subtype AT(1A) 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, B-max = 571 fmol/mg protein; without 3'-UTR, B-max = 663 fmol/mg protein) were used in the present study. Both cell lines expressed high-affinity receptors (with 3'-UTR, K-d = 0.83 nM; without 3'-UTR, K-d = 0.82 nM, and binding studies with I-125-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 (Ii-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 (AT(1))-expressing cells. Activation of cAMP by Ang II in cells without the 3'-UTR correlated with an inhibition of DNA synthesis, determined by [H-3]thymidine incorporation. Ang II-mediated responses were blocked by EXP3174, a selective nonpeptide 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 AT(1A) mRNA in cells without the 3'-UTR compared with cells with the 3'-UTR. Analysis of the decay rate of the AT(1A) mRNA in cells with and without the 3'-UTR revealed that the normally unstable AT(1A) 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 AT(1A) 3'-UTR, cellular extracts were prepared from parental CHO-KI 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 AT(1A) can control specific receptor functions, perhaps via selective recognition of the 3'-UTR by RNA-binding proteins.