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. 2003 Mar 1;370(Pt 2):631–639. doi: 10.1042/BJ20020960

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

Thomas J Thekkumkara 1, Stuart L Linas 1
PMCID: PMC1223184  PMID: 12431186

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.

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Selected References

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  1. Balmer L. A., Beveridge D. J., Jazayeri J. A., Thomson A. M., Walker C. E., Leedman P. J. Identification of a novel AU-Rich element in the 3' untranslated region of epidermal growth factor receptor mRNA that is the target for regulated RNA-binding proteins. Mol Cell Biol. 2001 Mar;21(6):2070–2084. doi: 10.1128/MCB.21.6.2070-2084.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Burns C. M., Chu H., Rueter S. M., Hutchinson L. K., Canton H., Sanders-Bush E., Emeson R. B. Regulation of serotonin-2C receptor G-protein coupling by RNA editing. Nature. 1997 May 15;387(6630):303–308. doi: 10.1038/387303a0. [DOI] [PubMed] [Google Scholar]
  3. Chiu A. T., Herblin W. F., McCall D. E., Ardecky R. J., Carini D. J., Duncia J. V., Pease L. J., Wong P. C., Wexler R. R., Johnson A. L. Identification of angiotensin II receptor subtypes. Biochem Biophys Res Commun. 1989 Nov 30;165(1):196–203. doi: 10.1016/0006-291x(89)91054-1. [DOI] [PubMed] [Google Scholar]
  4. Daaka Y., Luttrell L. M., Lefkowitz R. J. Switching of the coupling of the beta2-adrenergic receptor to different G proteins by protein kinase A. Nature. 1997 Nov 6;390(6655):88–91. doi: 10.1038/36362. [DOI] [PubMed] [Google Scholar]
  5. Earp H. S., Huckle W. R., Dawson T. L., Li X., Graves L. M., Dy R. Angiotensin II activates at least two tyrosine kinases in rat liver epithelial cells. Separation of the major calcium-regulated tyrosine kinase from p125FAK. J Biol Chem. 1995 Nov 24;270(47):28440–28447. doi: 10.1074/jbc.270.47.28440. [DOI] [PubMed] [Google Scholar]
  6. Gavis E. R., Lehmann R. Translational regulation of nanos by RNA localization. Nature. 1994 May 26;369(6478):315–318. doi: 10.1038/369315a0. [DOI] [PubMed] [Google Scholar]
  7. Georgoussi Z., Milligan G., Zioudrou C. Immunoprecipitation of opioid receptor-Go-protein complexes using specific GTP-binding-protein antisera. Biochem J. 1995 Feb 15;306(Pt 1):71–75. doi: 10.1042/bj3060071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gibbons G. H., Pratt R. E., Dzau V. J. Vascular smooth muscle cell hypertrophy vs. hyperplasia. Autocrine transforming growth factor-beta 1 expression determines growth response to angiotensin II. J Clin Invest. 1992 Aug;90(2):456–461. doi: 10.1172/JCI115881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gu Wei, Pan Feng, Zhang Honglai, Bassell Gary J., Singer Robert H. A predominantly nuclear protein affecting cytoplasmic localization of beta-actin mRNA in fibroblasts and neurons. J Cell Biol. 2002 Jan 7;156(1):41–51. doi: 10.1083/jcb.200105133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Higashita R., Li L., Van Putten V., Yamamura Y., Zarinetchi F., Heasley L., Nemenoff R. A. Galpha16 mimics vasoconstrictor action to induce smooth muscle alpha-actin in vascular smooth muscle cells through a Jun-NH2-terminal kinase-dependent pathway. J Biol Chem. 1997 Oct 10;272(41):25845–25850. doi: 10.1074/jbc.272.41.25845. [DOI] [PubMed] [Google Scholar]
  11. Jackson R. J. Cytoplasmic regulation of mRNA function: the importance of the 3' untranslated region. Cell. 1993 Jul 16;74(1):9–14. doi: 10.1016/0092-8674(93)90290-7. [DOI] [PubMed] [Google Scholar]
  12. Jard S., Cantau B., Jakobs K. H. Angiotensin II and alpha-adrenergic agonists inhibit rat liver adenylate cyclase. J Biol Chem. 1981 Mar 25;256(6):2603–2606. [PubMed] [Google Scholar]
  13. Kveberg Lise, Bryceson Yenan, Inngjerdingen Marit, Rolstad Bent, Maghazachi Azzam A. Sphingosine 1 phosphate induces the chemotaxis of human natural killer cells. Role for heterotrimeric G proteins and phosphoinositide 3 kinases. Eur J Immunol. 2002 Jul;32(7):1856–1864. doi: 10.1002/1521-4141(200207)32:7<1856::AID-IMMU1856>3.0.CO;2-B. [DOI] [PubMed] [Google Scholar]
  14. Le Cam A., Legraverend C. Transcriptional repression, a novel function for 3' untranslated regions. Eur J Biochem. 1995 Aug 1;231(3):620–627. [PubMed] [Google Scholar]
  15. Lövkvist Wallström E., Takao K., Wendt A., Vargiu C., Yin H., Persson L. Importance of the 3' untranslated region of ornithine decarboxylase mRNA in the translational regulation of the enzyme. Biochem J. 2001 Jun 1;356(Pt 2):627–634. doi: 10.1042/0264-6021:3560627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mazumder B., Fox P. L. Delayed translational silencing of ceruloplasmin transcript in gamma interferon-activated U937 monocytic cells: role of the 3' untranslated region. Mol Cell Biol. 1999 Oct;19(10):6898–6905. doi: 10.1128/mcb.19.10.6898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. McLatchie L. M., Fraser N. J., Main M. J., Wise A., Brown J., Thompson N., Solari R., Lee M. G., Foord S. M. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature. 1998 May 28;393(6683):333–339. doi: 10.1038/30666. [DOI] [PubMed] [Google Scholar]
  18. Natarajan R., Gonzales N., Hornsby P. J., Nadler J. Mechanism of angiotensin II-induced proliferation in bovine adrenocortical cells. Endocrinology. 1992 Sep;131(3):1174–1180. doi: 10.1210/endo.131.3.1505459. [DOI] [PubMed] [Google Scholar]
  19. Nickenig G., Murphy T. J. Enhanced angiotensin receptor type 1 mRNA degradation and induction of polyribosomal mRNA binding proteins by angiotensin II in vascular smooth muscle cells. Mol Pharmacol. 1996 Oct;50(4):743–751. [PubMed] [Google Scholar]
  20. Oppermann M., Freedman N. J., Alexander R. W., Lefkowitz R. J. Phosphorylation of the type 1A angiotensin II receptor by G protein-coupled receptor kinases and protein kinase C. J Biol Chem. 1996 May 31;271(22):13266–13272. doi: 10.1074/jbc.271.22.13266. [DOI] [PubMed] [Google Scholar]
  21. Ostareck-Lederer A., Ostareck D. H., Standart N., Thiele B. J. Translation of 15-lipoxygenase mRNA is inhibited by a protein that binds to a repeated sequence in the 3' untranslated region. EMBO J. 1994 Mar 15;13(6):1476–1481. doi: 10.1002/j.1460-2075.1994.tb06402.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Paillasson S., Van De Corput M., Dirks R. W., Tanke H. J., Robert-Nicoud M., Ronot X. In situ hybridization in living cells: detection of RNA molecules. Exp Cell Res. 1997 Feb 25;231(1):226–233. doi: 10.1006/excr.1996.3464. [DOI] [PubMed] [Google Scholar]
  23. Ranganathan G., Li C., Kern P. A. The translational regulation of lipoprotein lipase in diabetic rats involves the 3'-untranslated region of the lipoprotein lipase mRNA. J Biol Chem. 2000 Dec 29;275(52):40986–40991. doi: 10.1074/jbc.M008775200. [DOI] [PubMed] [Google Scholar]
  24. Rastinejad F., Blau H. M. Genetic complementation reveals a novel regulatory role for 3' untranslated regions in growth and differentiation. Cell. 1993 Mar 26;72(6):903–917. doi: 10.1016/0092-8674(93)90579-f. [DOI] [PubMed] [Google Scholar]
  25. Raymond J. R. Multiple mechanisms of receptor-G protein signaling specificity. Am J Physiol. 1995 Aug;269(2 Pt 2):F141–F158. doi: 10.1152/ajprenal.1995.269.2.F141. [DOI] [PubMed] [Google Scholar]
  26. Ross A. F., Oleynikov Y., Kislauskis E. H., Taneja K. L., Singer R. H. Characterization of a beta-actin mRNA zipcode-binding protein. Mol Cell Biol. 1997 Apr;17(4):2158–2165. doi: 10.1128/mcb.17.4.2158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sachs A. B. Messenger RNA degradation in eukaryotes. Cell. 1993 Aug 13;74(3):413–421. doi: 10.1016/0092-8674(93)80043-e. [DOI] [PubMed] [Google Scholar]
  28. Sadoshima J., Izumo S. Signal transduction pathways of angiotensin II--induced c-fos gene expression in cardiac myocytes in vitro. Roles of phospholipid-derived second messengers. Circ Res. 1993 Sep;73(3):424–438. doi: 10.1161/01.res.73.3.424. [DOI] [PubMed] [Google Scholar]
  29. Schorb W., Booz G. W., Dostal D. E., Conrad K. M., Chang K. C., Baker K. M. Angiotensin II is mitogenic in neonatal rat cardiac fibroblasts. Circ Res. 1993 Jun;72(6):1245–1254. doi: 10.1161/01.res.72.6.1245. [DOI] [PubMed] [Google Scholar]
  30. Simonds W. F., Goldsmith P. K., Codina J., Unson C. G., Spiegel A. M. Gi2 mediates alpha 2-adrenergic inhibition of adenylyl cyclase in platelet membranes: in situ identification with G alpha C-terminal antibodies. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7809–7813. doi: 10.1073/pnas.86.20.7809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tay A., Maxwell P., Li Z. G., Goldberg H., Skorecki K. Cytosolic phospholipase A2 gene expression in rat mesangial cells is regulated post-transcriptionally. Biochem J. 1994 Dec 1;304(Pt 2):417–422. doi: 10.1042/bj3040417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Thekkumkara T. J., Du J., Dostal D. E., Motel T. J., Thomas W. G., Baker K. M. Stable expression of a functional rat angiotensin II (AT1A) receptor in CHO-K1 cells: rapid desensitization by angiotensin II. Mol Cell Biochem. 1995 May 10;146(1):79–89. doi: 10.1007/BF00926885. [DOI] [PubMed] [Google Scholar]
  33. Thekkumkara T. J., Du J., Zwaagstra C., Conrad K. M., Krupinski J., Baker K. M. A role for cAMP in angiotensin II mediated inhibition of cell growth in AT1A receptor-transfected CHO-K1 cells. Mol Cell Biochem. 1995 Nov 8;152(1):77–86. doi: 10.1007/BF01076466. [DOI] [PubMed] [Google Scholar]
  34. Thekkumkara T. J., Thomas W. G., Motel T. J., Baker K. M. Functional role for the angiotensin II receptor (AT1A) 3'-untranslated region in determining cellular responses to agonist: evidence for recognition by RNA binding proteins. Biochem J. 1998 Jan 15;329(Pt 2):255–264. doi: 10.1042/bj3290255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Thomas W. G., Thekkumkara T. J., Motel T. J., Baker K. M. Stable expression of a truncated AT1A receptor in CHO-K1 cells. The carboxyl-terminal region directs agonist-induced internalization but not receptor signaling or desensitization. J Biol Chem. 1995 Jan 6;270(1):207–213. doi: 10.1074/jbc.270.1.207. [DOI] [PubMed] [Google Scholar]
  36. Wolf G., Killen P. D., Neilson E. G. Intracellular signaling of transcription and secretion of type IV collagen after angiotensin II-induced cellular hypertrophy in cultured proximal tubular cells. Cell Regul. 1991 Mar;2(3):219–227. doi: 10.1091/mbc.2.3.219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Yang Q., McDermott P. J., Duzic E., Pleij C. W., Sherlock J. D., Lanier S. M. The 3'-untranslated region of the alpha2C-adrenergic receptor mRNA impedes translation of the receptor message. J Biol Chem. 1997 Jun 13;272(24):15466–15473. doi: 10.1074/jbc.272.24.15466. [DOI] [PubMed] [Google Scholar]

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