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Nuclear Run-On Assays for Measurement of Adrenergic Receptor Transcription Rate

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Adrenergic Receptor Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 126))

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Abstract

The functionally diverse group of G-protein-coupled receptors (GPCRs) is a superfamily of membrane receptors. They include receptors for many different signaling molecules, such as peptide and nonpeptide hormones, neurotransmitters, chemokines, prostanoids, and proteinases. The principal function of GPCRs is to transmit information about the extracellular environment to the interior of the cell by interacting with the heterotrimeric G-proteins and, thereby, participate in regulation of many cellular functions. In view of their major importance, it is not surprising that GPCR-mediated responses are subject to dynamic regulation by a number of mechanisms. These regulatory mechanisms have important roles in fine-tuning signals from multiple receptor signaling pathways. Multiple mechanisms contribute to the regulation of GPCRs and their transmembrane signaling. Posttranslational modifications of the receptors, such as phosphorylation, may modulate receptor function; in addition, changes in receptor gene expression can lead to alterations in sensitivity and responsiveness of cells to various signaling molecules (13).

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References

  1. Bohm, S. K., Grady, E. F., and Bunnett, N. W. (1997) Regulatory mechanisms that modulate signalling by G-protein-coupled receptors. Biochem. J. 322, 1–18.

    PubMed  CAS  Google Scholar 

  2. Ferguson, S. S. and Caron, M. G. (1998) G protein-coupled receptor adaptation mechanisms. Semin. Cell. Dev. Biol. 9, 119–127.

    Article  PubMed  CAS  Google Scholar 

  3. Collins, S., Caron, M. G., and Lefkowitz, R. J. (1992) From ligand binding to gene expression: new insights into the regulation of G-protein-coupled receptors. Trends Biochem. Sci. 17, 37–39.

    Article  PubMed  CAS  Google Scholar 

  4. Scanga, D. R. and Schwinn, D. A. (1998) Transcriptional regulation of alpha-1 adrenergic receptors. Front Biosci. 3, d348–353.

    PubMed  CAS  Google Scholar 

  5. Heck, D. A. and Bylund, D. B. (1998) Differential down-regulation of alpha-2 adrenergic receptor subtypes. Life Sci. 62, 1467–1472.

    Article  PubMed  CAS  Google Scholar 

  6. Lohse, M. J., Engelhardt, S., Danner, S., and Bohm, M. (1996) Mechanisms of beta-adrenergic receptor desensitization: from molecular biology to heart failure. Basic Res. Cardiol. 91(Suppl. 2), 29–34.

    Article  PubMed  CAS  Google Scholar 

  7. Collins, S., Altschmied, J., Herbsman, O., Caron, M. G., Mellon, P. L., and Lefkowitz, R. J. (1990) A cAMP response element in the beta 2-adrenergic receptor gene confers transcriptional autoregulation by cAMP. J. Biol. Chem. 265, 19,330–19,335.

    PubMed  CAS  Google Scholar 

  8. Sakaue, M. and Hoffman, B. B. (1991) cAMP regulates transcription of the alpha 2A adrenergic receptor gene inHT-29 cells. J. Biol. Chem. 266, 5743–5749.

    PubMed  CAS  Google Scholar 

  9. McGraw, D. W., Chai, S. E., Hiller, F. C., and Cornett, L. E. (1995) Regulation of the beta 2-adrenergic receptor and its mRNA in the rat lung by dexamethasone. Exp. Lung Res. 21, 535–546.

    Article  PubMed  CAS  Google Scholar 

  10. Thomas, R. F., Holt, B. D., Schwinn, D. A., and Liggett, S. B. (1992) Long-term agonist exposure induces upregulation of beta 3-adrenergic receptor expression via multiple cAMP response elements. Proc. Natl. Acad. Sci. USA 89, 4490–4494.

    Article  PubMed  CAS  Google Scholar 

  11. Sakaue, M. and Hoffman, B. B. (1991) Glucocorticoids induce transcription and expression of the alpha 1B adrenergic receptor gene in DTT1 MF-2 smooth muscle cells. J. Clin. Invest. 88, 385–389.

    Article  PubMed  CAS  Google Scholar 

  12. Kiely, J., Hadcock, J. R., Bahouth, S. W., and Malbon, C. C. (1994) Glucocorticoids down-regulate beta 1-adrenergic-receptor expression by suppressing transcription of the receptor gene. Biochem. J. 302, 397–403.

    PubMed  CAS  Google Scholar 

  13. Hu, Z. W., Shi, X. Y., and Hoffman, B. B. (1996) Insulin and insulin-like growth factor I differentially induce alpha1-adrenergic receptor subtype expression in rat vascular smooth muscle cells. J. Clin. Invest. 98, 1826–1834.

    Article  PubMed  CAS  Google Scholar 

  14. Gong, G., Johnson, M. L., and Pettinger, W. A. (1995) Testosterone regulation of renal alpha 2B-adrenergic receptor mRNA levels. Hypertension 25, 350–355.

    PubMed  CAS  Google Scholar 

  15. Devedjian, J. C., Fargues, M., Denis-Pouxviel, C., Daviaud, D., Prats, H., and Paris, H. (1991) Regulation of the alpha 2A-adrenergic receptor in the HT29 cell line. Effects of insulin and growth factors. J. Biol. Chem. 266, 14,359–14,366.

    PubMed  CAS  Google Scholar 

  16. Hu, Z.-W., Shi, X.-Y., Okazaki, M., and Hoffman, B. B. (1995) Angiotensin II induces transcription and expression of alpha1 adrenergic receptors in cultured rat vascular smooth muscle cells. Am. J. Physiol. 268, H1006–1014.

    PubMed  CAS  Google Scholar 

  17. Greenberg, M. E. and Bender, T. P. (1997) Identification of newly transcribed RNA, in Current Protocols in Molecular Biology, (Ausubel, F. M., et al., eds.), John Wiley, New York, pp. 4.10.1–4.10.11.

    Google Scholar 

  18. Hattori, M., Tugores, A., Veloz, L., Karin, M., and Brenner, D. A. (1990) A simplified method for the preparation of transcriptionally active liver nuclear extracts. DNA Cell. Biol. 9, 777–781.

    Article  PubMed  CAS  Google Scholar 

  19. Merscher, S., Hanselmann, R., Welter, C., and Dooley, S. (1994) Nuclear runoff transcription analysis using chemiluminescent detection. Biotechniques 16, 1024–1026.

    PubMed  CAS  Google Scholar 

  20. Haley, J. D. and Waterfield, M. D. (1991) Contributory effects of de novo transcription and premature transcript termination in the regulation of human epidermal growth factor receptor proto-oncogene RNA synthesis. J. Biol. Chem. 266, 1746–1753.

    PubMed  CAS  Google Scholar 

  21. Kindy, M. S., McCormack, J. E., Buckler, A. J., Levine, R. A., and Sonenshein, G. E. (1987) Independent regulation of transcription of the two strands of the c-myc gene. Mol. Cell. Biol. 7, 2857–2862.

    PubMed  CAS  Google Scholar 

  22. Sell, C., Chen, H. M., and Baserga, R. (1992) A simple method to generate single-stranded probes for run-on transcription assays. Biotechniques 12, 692–694.

    PubMed  CAS  Google Scholar 

  23. Fei, H. and Drake, T. A. (1993) A rapid nuclear runoff transcription assay. Biotechniques 15, 838.

    PubMed  CAS  Google Scholar 

  24. Celano, P., Berchtold, C., and Casero, R. A. Jr. A simplification of the nuclear run-off transcription assay. Biotechniques 7, 942–944.

    Google Scholar 

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© 2000 Humana Press Inc., Totowa, NJ

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Hu, ZW., Hoffman, B.B. (2000). Nuclear Run-On Assays for Measurement of Adrenergic Receptor Transcription Rate. In: Machida, C.A. (eds) Adrenergic Receptor Protocols. Methods in Molecular Biology™, vol 126. Humana Press. https://doi.org/10.1385/1-59259-684-3:169

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  • DOI: https://doi.org/10.1385/1-59259-684-3:169

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-602-4

  • Online ISBN: 978-1-59259-684-3

  • eBook Packages: Springer Protocols

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