Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1995 May 1;307(Pt 3):831–841. doi: 10.1042/bj3070831

Thyroid hormone regulation of transmembrane signalling in neonatal rat ventricular myocytes by selective alteration of the expression and coupling of G-protein alpha-subunits.

S W Bahouth 1
PMCID: PMC1136724  PMID: 7741715

Abstract

Thyroid hormone exerts profound effects on the activity of the hormone-sensitive adenylate cyclase system in the heart. Distinct guanine nucleotide-binding regulatory proteins (G-proteins) mediate stimulatory and inhibitory influences on adenylate cyclase activity. To examine whether the effects of thyroid hormone on adenylate cyclase involve specific changes in G-protein subunit expression, the influence of tri-iodothyronine (T3) on the biosynthesis and activity of G-proteins in neonatal rat ventricular myocytes was determined. In myocytes challenged with T3 for 5 days, Gs alpha levels increased by 4 +/- 0.5-fold, whereas Gi2 alpha levels declined by more than 80%. T3 down-regulated Gi2 alpha mRNA by 60% within 3 days, but had no effect on Gs alpha mRNA. The basis for the decline in Gi2 alpha mRNA was the T3-mediated suppression of Gi2 alpha gene transcription by 80 +/- 9% within 4 h. The decline in Gi2 alpha mRNA in response to T3 produced a 2-fold decrease in relative rate of synthesis of Gi2 alpha but not in its half-life (46 +/- 7 h). Gs alpha synthesis was not altered by T3, but the half-life of Gs alpha increased from 50 +/- 6 h in control cells to 72 +/- 8 h in T3-treated cells. In addition, T3 provoked the translocation of Gs alpha from the cytoplasmic to the membranous compartment. Membranous Gs alpha increased from 30 +/- 6% to 61 +/- 7% of total cellular Gs alpha, whereas cytoplasmic Gs alpha declined from 68 +/- 6% to 33 +/- 8% within 1 day of exposure to T3. T3-mediated up-regulation of Gs alpha enhanced the activation of myocardial adenylate cyclase by the stimulatory pathway whereas the down-regulation of Gi2 alpha attenuated the deactivation of myocardial adenylate cyclase by the inhibitory pathway.

Full text

PDF
833

Images in this article

834Figure 1
on p.834
834Figure 2
on p.834
835Figure 3
on p.835
836Figure 4
on p.836
836Figure 5
on p.836
837Figure 6
on p.837

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bahouth S. W. Effects of chemical and surgical sympathectomy on expression of beta-adrenergic receptors and guanine nucleotide-binding proteins in rat submandibular glands. Mol Pharmacol. 1992 Dec;42(6):971–981. [PubMed] [Google Scholar]
  2. Bahouth S. W. Thyroid hormones transcriptionally regulate the beta 1-adrenergic receptor gene in cultured ventricular myocytes. J Biol Chem. 1991 Aug 25;266(24):15863–15869. [PubMed] [Google Scholar]
  3. Bilezikian J. P., Loeb J. N. The influence of hyperthyroidism and hypothyroidism on alpha- and beta-adrenergic receptor systems and adrenergic responsiveness. Endocr Rev. 1983 Fall;4(4):378–388. doi: 10.1210/edrv-4-4-378. [DOI] [PubMed] [Google Scholar]
  4. Birnbaumer L., Abramowitz J., Brown A. M. Receptor-effector coupling by G proteins. Biochim Biophys Acta. 1990 May 7;1031(2):163–224. doi: 10.1016/0304-4157(90)90007-y. [DOI] [PubMed] [Google Scholar]
  5. Blondel B., Roijen I., Cheneval J. P. Heart cells in culture: a simple method for increasing the proportion of myoblasts. Experientia. 1971 Mar 15;27(3):356–358. doi: 10.1007/BF02138197. [DOI] [PubMed] [Google Scholar]
  6. Bowman L. H. rDNA transcription and pre-rRNA processing during the differentiation of a mouse myoblast cell line. Dev Biol. 1987 Jan;119(1):152–163. doi: 10.1016/0012-1606(87)90217-x. [DOI] [PubMed] [Google Scholar]
  7. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  8. Brent G. A., Moore D. D., Larsen P. R. Thyroid hormone regulation of gene expression. Annu Rev Physiol. 1991;53:17–35. doi: 10.1146/annurev.ph.53.030191.000313. [DOI] [PubMed] [Google Scholar]
  9. Chen J., Iyengar R. Inhibition of cloned adenylyl cyclases by mutant-activated Gi-alpha and specific suppression of type 2 adenylyl cyclase inhibition by phorbol ester treatment. J Biol Chem. 1993 Jun 15;268(17):12253–12256. [PubMed] [Google Scholar]
  10. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  11. Cleveland D. W., Lopata M. A., MacDonald R. J., Cowan N. J., Rutter W. J., Kirschner M. W. Number and evolutionary conservation of alpha- and beta-tubulin and cytoplasmic beta- and gamma-actin genes using specific cloned cDNA probes. Cell. 1980 May;20(1):95–105. doi: 10.1016/0092-8674(80)90238-x. [DOI] [PubMed] [Google Scholar]
  12. Codina J., Yatani A., Grenet D., Brown A. M., Birnbaumer L. The alpha subunit of the GTP binding protein Gk opens atrial potassium channels. Science. 1987 Apr 24;236(4800):442–445. doi: 10.1126/science.2436299. [DOI] [PubMed] [Google Scholar]
  13. Degtyarev M. Y., Spiegel A. M., Jones T. L. Increased palmitoylation of the Gs protein alpha subunit after activation by the beta-adrenergic receptor or cholera toxin. J Biol Chem. 1993 Nov 15;268(32):23769–23772. [PubMed] [Google Scholar]
  14. Dillmann W. H., Barrieux A., Neeley W. E., Contreras P. Influence of thyroid hormone on the in vitro translational activity of specific mRNAs in the rat heart. J Biol Chem. 1983 Jun 25;258(12):7738–7745. [PubMed] [Google Scholar]
  15. Federman A. D., Conklin B. R., Schrader K. A., Reed R. R., Bourne H. R. Hormonal stimulation of adenylyl cyclase through Gi-protein beta gamma subunits. Nature. 1992 Mar 12;356(6365):159–161. doi: 10.1038/356159a0. [DOI] [PubMed] [Google Scholar]
  16. Gilman A. G. G proteins: transducers of receptor-generated signals. Annu Rev Biochem. 1987;56:615–649. doi: 10.1146/annurev.bi.56.070187.003151. [DOI] [PubMed] [Google Scholar]
  17. Glass C. K., Franco R., Weinberger C., Albert V. R., Evans R. M., Rosenfeld M. G. A c-erb-A binding site in rat growth hormone gene mediates trans-activation by thyroid hormone. Nature. 1987 Oct 22;329(6141):738–741. doi: 10.1038/329738a0. [DOI] [PubMed] [Google Scholar]
  18. Grassie M. A., McCallum J. F., Parenti M., Magee A. I., Milligan G. Lack of N terminal palmitoylation of G protein alpha subunits reduces membrane association. Biochem Soc Trans. 1993 Nov;21(4):499S–499S. doi: 10.1042/bst021499s. [DOI] [PubMed] [Google Scholar]
  19. Graziano M. P., Freissmuth M., Gilman A. G. Purification of recombinant Gs alpha. Methods Enzymol. 1991;195:192–202. doi: 10.1016/0076-6879(91)95166-h. [DOI] [PubMed] [Google Scholar]
  20. Hadcock J. R., Port J. D., Malbon C. C. Cross-regulation between G-protein-mediated pathways. Activation of the inhibitory pathway of adenylylcylclase increases the expression of beta 2-adrenergic receptors. J Biol Chem. 1991 Jun 25;266(18):11915–11922. [PubMed] [Google Scholar]
  21. Hadcock J. R., Ros M., Watkins D. C., Malbon C. C. Cross-regulation between G-protein-mediated pathways. Stimulation of adenylyl cyclase increases expression of the inhibitory G-protein, Gi alpha 2. J Biol Chem. 1990 Sep 5;265(25):14784–14790. [PubMed] [Google Scholar]
  22. Hargrove J. L., Schmidt F. H. The role of mRNA and protein stability in gene expression. FASEB J. 1989 Oct;3(12):2360–2370. doi: 10.1096/fasebj.3.12.2676679. [DOI] [PubMed] [Google Scholar]
  23. Hazeki O., Ui M. Modification by islet-activating protein of receptor-mediated regulation of cyclic AMP accumulation in isolated rat heart cells. J Biol Chem. 1981 Mar 25;256(6):2856–2862. [PubMed] [Google Scholar]
  24. Hepler J. R., Gilman A. G. G proteins. Trends Biochem Sci. 1992 Oct;17(10):383–387. doi: 10.1016/0968-0004(92)90005-t. [DOI] [PubMed] [Google Scholar]
  25. Higashijima T., Ferguson K. M., Sternweis P. C., Smigel M. D., Gilman A. G. Effects of Mg2+ and the beta gamma-subunit complex on the interactions of guanine nucleotides with G proteins. J Biol Chem. 1987 Jan 15;262(2):762–766. [PubMed] [Google Scholar]
  26. Hilal-Dandan R., Urasawa K., Brunton L. L. Endothelin inhibits adenylate cyclase and stimulates phosphoinositide hydrolysis in adult cardiac myocytes. J Biol Chem. 1992 May 25;267(15):10620–10624. [PubMed] [Google Scholar]
  27. Ishikawa Y., Katsushika S., Chen L., Halnon N. J., Kawabe J., Homcy C. J. Isolation and characterization of a novel cardiac adenylylcyclase cDNA. J Biol Chem. 1992 Jul 5;267(19):13553–13557. [PubMed] [Google Scholar]
  28. Iyengar R. Hysteretic activation of adenylyl cyclases. II. Mg ion regulation of the activation of the regulatory component as analyzed by reconstitution. J Biol Chem. 1981 Nov 10;256(21):11042–11050. [PubMed] [Google Scholar]
  29. James A. F., Xie L. H., Fujitani Y., Hayashi S., Horie M. Inhibition of the cardiac protein kinase A-dependent chloride conductance by endothelin-1. Nature. 1994 Jul 28;370(6487):297–300. doi: 10.1038/370297a0. [DOI] [PubMed] [Google Scholar]
  30. Jones D. T., Reed R. R. Molecular cloning of five GTP-binding protein cDNA species from rat olfactory neuroepithelium. J Biol Chem. 1987 Oct 15;262(29):14241–14249. [PubMed] [Google Scholar]
  31. Kenney F., Lee K. L., Reel J. R., Hoel D. G. Regulation of tyrosine alpha-ketoglutarate transaminase in rat liver. IX. Studies of the mechanisms of hormonal inductions in cultured hepatoma cells. J Biol Chem. 1970 Nov 10;245(21):5806–5812. [PubMed] [Google Scholar]
  32. Klein I., Levey G. S. New perspectives on thyroid hormone, catecholamines, and the heart. Am J Med. 1984 Feb;76(2):167–172. doi: 10.1016/0002-9343(84)90768-x. [DOI] [PubMed] [Google Scholar]
  33. Kozasa T., Itoh H., Tsukamoto T., Kaziro Y. Isolation and characterization of the human Gs alpha gene. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2081–2085. doi: 10.1073/pnas.85.7.2081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Krupinski J., Coussen F., Bakalyar H. A., Tang W. J., Feinstein P. G., Orth K., Slaughter C., Reed R. R., Gilman A. G. Adenylyl cyclase amino acid sequence: possible channel- or transporter-like structure. Science. 1989 Jun 30;244(4912):1558–1564. doi: 10.1126/science.2472670. [DOI] [PubMed] [Google Scholar]
  35. Krupinski J., Lehman T. C., Frankenfield C. D., Zwaagstra J. C., Watson P. A. Molecular diversity in the adenylylcyclase family. Evidence for eight forms of the enzyme and cloning of type VI. J Biol Chem. 1992 Dec 5;267(34):24858–24862. [PubMed] [Google Scholar]
  36. Kupfer L. E., Bilezikian J. P., Robinson R. B. Regulation of alpha and beta adrenergic receptors by triiodothyronine in cultured rat myocardial cells. Naunyn Schmiedebergs Arch Pharmacol. 1986 Nov;334(3):275–281. doi: 10.1007/BF00508782. [DOI] [PubMed] [Google Scholar]
  37. Levine M. A., Feldman A. M., Robishaw J. D., Ladenson P. W., Ahn T. G., Moroney J. F., Smallwood P. M. Influence of thyroid hormone status on expression of genes encoding G protein subunits in the rat heart. J Biol Chem. 1990 Feb 25;265(6):3553–3560. [PubMed] [Google Scholar]
  38. Levis M. J., Bourne H. R. Activation of the alpha subunit of Gs in intact cells alters its abundance, rate of degradation, and membrane avidity. J Cell Biol. 1992 Dec;119(5):1297–1307. doi: 10.1083/jcb.119.5.1297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Libby P. Long-term culture of contractile mammalian heart cells in a defined serum-free medium that limits non-muscle cell proliferation. J Mol Cell Cardiol. 1984 Sep;16(9):803–811. doi: 10.1016/s0022-2828(84)80004-8. [DOI] [PubMed] [Google Scholar]
  40. Limbird L. E., Hickey A. R., Lefkowitz R. J. Unique uncoupling of the frog erythrocyte adenylate cyclase system by manganese. Loss of hormone and guanine nucleotide-sensitive enzyme activities without loss of nucleotide-sensitive, high affinity agonist binding. J Biol Chem. 1979 Apr 25;254(8):2677–2683. [PubMed] [Google Scholar]
  41. Loganzo F., Jr, Fletcher P. W. Follicle-stimulating hormone increases the turnover of G-protein alpha i-1- and alpha i-2-subunit messenger RNA in Sertoli cells by a mechanism that is independent of protein synthesis. Mol Endocrinol. 1993 Mar;7(3):434–440. doi: 10.1210/mend.7.3.8483480. [DOI] [PubMed] [Google Scholar]
  42. Logothetis D. E., Kurachi Y., Galper J., Neer E. J., Clapham D. E. The beta gamma subunits of GTP-binding proteins activate the muscarinic K+ channel in heart. Nature. 1987 Jan 22;325(6102):321–326. doi: 10.1038/325321a0. [DOI] [PubMed] [Google Scholar]
  43. Malbon C. C., Moreno F. J., Cabelli R. J., Fain J. N. Fat cell adenylate cyclase and beta-adrenergic receptors in altered thyroid states. J Biol Chem. 1978 Feb 10;253(3):671–678. [PubMed] [Google Scholar]
  44. Malbon C. C., Rapiejko P. J., Mangano T. J. Fat cell adenylate cyclase system. Enhanced inhibition by adenosine and GTP in the hypothyroid rat. J Biol Chem. 1985 Feb 25;260(4):2558–2564. [PubMed] [Google Scholar]
  45. Malbon C. C., Rapiejko P. J., Watkins D. C. Permissive hormone regulation of hormone-sensitive effector systems. Trends Pharmacol Sci. 1988 Jan;9(1):33–36. doi: 10.1016/0165-6147(88)90240-4. [DOI] [PubMed] [Google Scholar]
  46. Masters S. B., Martin M. W., Harden T. K., Brown J. H. Pertussis toxin does not inhibit muscarinic-receptor-mediated phosphoinositide hydrolysis or calcium mobilization. Biochem J. 1985 May 1;227(3):933–937. doi: 10.1042/bj2270933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Michel-Reher M. B., Gross G., Jasper J. R., Bernstein D., Olbricht T., Brodde O. E., Michel M. C. Tissue- and subunit-specific regulation of G-protein expression by hypo- and hyperthyroidism. Biochem Pharmacol. 1993 Apr 6;45(7):1417–1423. doi: 10.1016/0006-2952(93)90040-4. [DOI] [PubMed] [Google Scholar]
  48. Moxham C. P., George S. T., Graziano M. P., Brandwein H. J., Malbon C. C. Mammalian beta 1- and beta 2-adrenergic receptors. Immunological and structural comparisons. J Biol Chem. 1986 Nov 5;261(31):14562–14570. [PubMed] [Google Scholar]
  49. Nevins J. R. The pathway of eukaryotic mRNA formation. Annu Rev Biochem. 1983;52:441–466. doi: 10.1146/annurev.bi.52.070183.002301. [DOI] [PubMed] [Google Scholar]
  50. Ono K., Tsujimoto G., Sakamoto A., Eto K., Masaki T., Ozaki Y., Satake M. Endothelin-A receptor mediates cardiac inhibition by regulating calcium and potassium currents. Nature. 1994 Jul 28;370(6487):301–304. doi: 10.1038/370301a0. [DOI] [PubMed] [Google Scholar]
  51. Rodgers J. R., Johnson M. L., Rosen J. M. Measurement of mRNA concentration and mRNA half-life as a function of hormonal treatment. Methods Enzymol. 1985;109:572–592. doi: 10.1016/0076-6879(85)09116-9. [DOI] [PubMed] [Google Scholar]
  52. Roth D. A., Urasawa K., Leiber D., Insel P. A., Hammond H. K. A substantial proportion of cardiac Gs is not associated with the plasma membrane. FEBS Lett. 1992 Jan 13;296(1):46–50. doi: 10.1016/0014-5793(92)80400-b. [DOI] [PubMed] [Google Scholar]
  53. Rudolph U., Brabet P., Kaplan J., Hasty P., Bradley A., Birnbaumer L. Targeting of the Gi2 alpha gene in ES cells with replacement and insertion vectors. J Recept Res. 1993;13(1-4):619–637. doi: 10.3109/10799899309073683. [DOI] [PubMed] [Google Scholar]
  54. Salomon Y., Londos C., Rodbell M. A highly sensitive adenylate cyclase assay. Anal Biochem. 1974 Apr;58(2):541–548. doi: 10.1016/0003-2697(74)90222-x. [DOI] [PubMed] [Google Scholar]
  55. Simpson P., Savion S. Differentiation of rat myocytes in single cell cultures with and without proliferating nonmyocardial cells. Cross-striations, ultrastructure, and chronotropic response to isoproterenol. Circ Res. 1982 Jan;50(1):101–116. doi: 10.1161/01.res.50.1.101. [DOI] [PubMed] [Google Scholar]
  56. Stiles G. L., Caron M. G., Lefkowitz R. J. Beta-adrenergic receptors: biochemical mechanisms of physiological regulation. Physiol Rev. 1984 Apr;64(2):661–743. doi: 10.1152/physrev.1984.64.2.661. [DOI] [PubMed] [Google Scholar]
  57. Tang W. J., Gilman A. G. Adenylyl cyclases. Cell. 1992 Sep 18;70(6):869–872. doi: 10.1016/0092-8674(92)90236-6. [DOI] [PubMed] [Google Scholar]
  58. Tang W. J., Gilman A. G. Type-specific regulation of adenylyl cyclase by G protein beta gamma subunits. Science. 1991 Dec 6;254(5037):1500–1503. doi: 10.1126/science.1962211. [DOI] [PubMed] [Google Scholar]
  59. Taussig R., Iñiguez-Lluhi J. A., Gilman A. G. Inhibition of adenylyl cyclase by Gi alpha. Science. 1993 Jul 9;261(5118):218–221. doi: 10.1126/science.8327893. [DOI] [PubMed] [Google Scholar]
  60. Tsai J. S., Chen A. Effect of L-triiodothyronine on (--)3H-dihydroalprenolol binding and cyclic AMP response to (--)adrenaline in cultured heart cells. Nature. 1978 Sep 14;275(5676):138–140. doi: 10.1038/275138a0. [DOI] [PubMed] [Google Scholar]
  61. Watkins D. C., Rapiejko P. J., Ros M., Wang H. Y., Malbon C. C. G-protein mRNA levels during adipocyte differentiation. Biochem Biophys Res Commun. 1989 Dec 29;165(3):929–934. doi: 10.1016/0006-291x(89)92692-2. [DOI] [PubMed] [Google Scholar]
  62. Williams L. T., Lefkowitz R. J., Watanabe A. M., Hathaway D. R., Besch H. R., Jr Thyroid hormone regulation of beta-adrenergic receptor number. J Biol Chem. 1977 Apr 25;252(8):2787–2789. [PubMed] [Google Scholar]
  63. Yanagisawa M., Kurihara H., Kimura S., Tomobe Y., Kobayashi M., Mitsui Y., Yazaki Y., Goto K., Masaki T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature. 1988 Mar 31;332(6163):411–415. doi: 10.1038/332411a0. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES