scholarly journals Interaction between 3′ Untranslated Region of Calcitonin Receptor Messenger Ribonucleic Acid (RNA) and Adenylate/Uridylate (AU)-Rich Element Binding Proteins (AU-Rich RNA-Binding Factor 1 and Hu Antigen R)

Endocrinology ◽  
2004 ◽  
Vol 145 (4) ◽  
pp. 1730-1738 ◽  
Author(s):  
Shigemitsu Yasuda ◽  
Seiki Wada ◽  
Yukitomo Arao ◽  
Masakazu Kogawa ◽  
Fujio Kayama ◽  
...  
Keyword(s):  
Ribonucleic Acid ◽  
Untranslated Region ◽  
Binding Proteins ◽  
Rna Binding ◽  
Calcitonin Receptor ◽  
Messenger Ribonucleic Acid ◽  
Binding Factor ◽  
Hu Antigen R

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

1998 ◽  
Vol 329 (2) ◽  
pp. 255-264 ◽  
Author(s):  
J. Thomas THEKKUMKARA ◽  
G. Walter THOMAS ◽  
J. Thomas MOTEL ◽  
M. Kenneth BAKER
Keyword(s):  
Angiotensin Ii ◽  
Cell Surface ◽  
Intracellular Calcium ◽  
Untranslated Region ◽  
Functional Role ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Ang Ii ◽  
In Cells

We demonstrate a functional role for the 3ʹ-untranslated region (3ʹ-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 3ʹ-UTR. Two cell lines expressing similar levels of cell-surface receptors (with 3ʹ-UTR, Bmax = 571 fmol/mg protein; without 3ʹ-UTR, Bmax = 663 fmol/mg protein) were used in the present study. Both cell lines expressed high-affinity receptors (with 3ʹ-UTR, Kd = 0.83 nM; without 3ʹ-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 3ʹ-UTR, Ang II stimulated an increase in cAMP accumulation (11-fold above control) and in cells with the 3ʹ-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 3ʹ-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 3ʹ-UTR in response to Ang II. In cells with the 3ʹ-UTR, a sustained level of intracellular calcium was observed after Ang II stimulation, whereas cells without the 3ʹ-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 3ʹ-UTR compared with cells with the 3ʹ-UTR. Analysis of the decay rate of the AT1A mRNA in cells with and without the 3ʹ-UTR revealed that the normally unstable AT1A receptor mRNA became highly stable by removing its 3ʹ-UTR, identifying a role for the 3ʹ-UTR in mRNA destabilization. Interestingly, both cells express similar levels of receptors at the cell surface, suggesting that the 3ʹ-UTR is also involved in the efficient translation and/or translocation of the receptor protein to the plasma membrane. We hypothesized that these 3ʹ-UTR-mediated functions of the receptor are regulated by RNA-binding proteins. To identify possible RNA-binding proteins for the AT1A 3ʹ-UTR, cellular extracts were prepared from parental CHO-K1 cells and 3ʹ-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 3ʹ-UTR. Our data suggest that the 3ʹ-UTR of the AT1A can control specific receptor functions, perhaps via selective recognition of the 3ʹ-UTR by RNA-binding proteins.

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