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 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.
Thekkumkara
T J
TJ
Department of Medicine, C281, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262, USA.
Thomas
W G
WG
Motel
T J
TJ
Baker
K M
KM
D000804:Angiotensin II / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary; D008969:Molecular Sequence Data; D011485:Protein Binding; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011945:Receptors, Angiotensin / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013936:Thymidine / Q000378:metabolism; D014162:Transfection; D014316:Tritium
D018076:DNA, Complementary; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011945:Receptors, Angiotensin; D011994:Recombinant Proteins; D014316:Tritium; D000804:Angiotensin II; D000242:Cyclic AMP; D002118:Calcium; D013936:Thymidine