Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1999 Jun 1;340(Pt 2):529–538.

Visualization of distinct patterns of subcellular redistribution of the thyrotropin-releasing hormone receptor-1 and gqalpha /G11alpha induced by agonist stimulation.

T Drmota 1, J Novotny 1, G W Gould 1, P Svoboda 1, G Milligan 1
PMCID: PMC1220281  PMID: 10333499

Abstract

The rat thyrotropin-releasing hormone receptor-1 (TRHR-1) was modified by the addition of green fluorescent protein (GFP) and expressed stably in HEK293 cells. Extensive overlap of plasma membrane distribution of autofluorescent TRHR-1-GFP with that of the phosphoinositidase C-linked G-proteins Gqalpha/G11alpha, identified by indirect immunofluorescence, was monitored concurrently. Addition of thyrotropin-releasing hormone resulted in rapid separation of TRHR-1-GFP and Gqalpha/G11alpha signals as the receptor was internalized. This situation persisted for more than an hour. At longer time periods a fraction of the cellular Gqalpha/G11alpha was also internalized, although much of the Gqalpha/G11alpha immunoreactivity remained associated with the plasma membrane. Parallel experiments, in which the cellular distribution of TRHR-1-GFP and Gqalpha/G11alpha immunoreactivity were monitored in sucrose-gradient fractions following cell disruption, also demonstrated a rapid, agonist-induced movement of TRHR-1-GFP away from the plasma membrane to low-density vesicular fractions. At later time points, a fraction of the cellular Gqalpha/G11alpha immunoreactivity was also redistributed to overlapping, but non-identical, low-density-vesicle-containing fractions. Pretreatment of the cells with cytochalasin D or nocodazole prevented agonist-induced redistribution of G-protein but not TRHR-1-GFP, further indicating resolution of the mechanics of these two processes. The combination of a GFP-modified receptor and immunostaining of the G-proteins activated by that receptor allows, for the first time, concurrent analysis of the varying dynamics and bases of internalization and redistribution of two elements of the same signal-transduction cascade.

Full Text

The Full Text of this article is available as a PDF (550.2 KB).

Selected References

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

  1. Aragay A. M., Katz A., Simon M. I. The G alpha q and G alpha 11 proteins couple the thyrotropin-releasing hormone receptor to phospholipase C in GH3 rat pituitary cells. J Biol Chem. 1992 Dec 15;267(35):24983–24988. [PubMed] [Google Scholar]
  2. Ashworth R., Yu R., Nelson E. J., Dermer S., Gershengorn M. C., Hinkle P. M. Visualization of the thyrotropin-releasing hormone receptor and its ligand during endocytosis and recycling. Proc Natl Acad Sci U S A. 1995 Jan 17;92(2):512–516. doi: 10.1073/pnas.92.2.512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barak L. S., Ferguson S. S., Zhang J., Martenson C., Meyer T., Caron M. G. Internal trafficking and surface mobility of a functionally intact beta2-adrenergic receptor-green fluorescent protein conjugate. Mol Pharmacol. 1997 Feb;51(2):177–184. doi: 10.1124/mol.51.2.177. [DOI] [PubMed] [Google Scholar]
  4. Bomsel M., Parton R., Kuznetsov S. A., Schroer T. A., Gruenberg J. Microtubule- and motor-dependent fusion in vitro between apical and basolateral endocytic vesicles from MDCK cells. Cell. 1990 Aug 24;62(4):719–731. doi: 10.1016/0092-8674(90)90117-w. [DOI] [PubMed] [Google Scholar]
  5. Cao J., O'Donnell D., Vu H., Payza K., Pou C., Godbout C., Jakob A., Pelletier M., Lembo P., Ahmad S. Cloning and characterization of a cDNA encoding a novel subtype of rat thyrotropin-releasing hormone receptor. J Biol Chem. 1998 Nov 27;273(48):32281–32287. doi: 10.1074/jbc.273.48.32281. [DOI] [PubMed] [Google Scholar]
  6. Drmota T., Gould G. W., Milligan G. Real time visualization of agonist-mediated redistribution and internalization of a green fluorescent protein-tagged form of the thyrotropin-releasing hormone receptor. J Biol Chem. 1998 Sep 11;273(37):24000–24008. doi: 10.1074/jbc.273.37.24000. [DOI] [PubMed] [Google Scholar]
  7. Drmota T., Novotny J., Kim G. D., Eidne K. A., Milligan G., Svoboda P. Agonist-induced internalization of the G protein G11alpha and thyrotropin-releasing hormone receptors proceed on different time scales. J Biol Chem. 1998 Aug 21;273(34):21699–21707. doi: 10.1074/jbc.273.34.21699. [DOI] [PubMed] [Google Scholar]
  8. Ferguson S. S., Zhang J., Barak L. S., Caron M. G. Role of beta-arrestins in the intracellular trafficking of G-protein-coupled receptors. Adv Pharmacol. 1998;42:420–424. doi: 10.1016/s1054-3589(08)60778-4. [DOI] [PubMed] [Google Scholar]
  9. Gershengorn M. C., Osman R. Molecular and cellular biology of thyrotropin-releasing hormone receptors. Physiol Rev. 1996 Jan;76(1):175–191. doi: 10.1152/physrev.1996.76.1.175. [DOI] [PubMed] [Google Scholar]
  10. Girotti M., Banting G. TGN38-green fluorescent protein hybrid proteins expressed in stably transfected eukaryotic cells provide a tool for the real-time, in vivo study of membrane traffic pathways and suggest a possible role for ratTGN38. J Cell Sci. 1996 Dec;109(Pt 12):2915–2926. doi: 10.1242/jcs.109.12.2915. [DOI] [PubMed] [Google Scholar]
  11. Hsieh K. P., Martin T. F. Thyrotropin-releasing hormone and gonadotropin-releasing hormone receptors activate phospholipase C by coupling to the guanosine triphosphate-binding proteins Gq and G11. Mol Endocrinol. 1992 Oct;6(10):1673–1681. doi: 10.1210/mend.6.10.1333052. [DOI] [PubMed] [Google Scholar]
  12. Htun H., Barsony J., Renyi I., Gould D. L., Hager G. L. Visualization of glucocorticoid receptor translocation and intranuclear organization in living cells with a green fluorescent protein chimera. Proc Natl Acad Sci U S A. 1996 May 14;93(10):4845–4850. doi: 10.1073/pnas.93.10.4845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ibarrondo J., Joubert D., Dufour M. N., Cohen-Solal A., Homburger V., Jard S., Guillon G. Close association of the alpha subunits of Gq and G11 G proteins with actin filaments in WRK1 cells: relation to G protein-mediated phospholipase C activation. Proc Natl Acad Sci U S A. 1995 Aug 29;92(18):8413–8417. doi: 10.1073/pnas.92.18.8413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Itadani H., Nakamura T., Itoh J., Iwaasa H., Kanatani A., Borkowski J., Ihara M., Ohta M. Cloning and characterization of a new subtype of thyrotropin-releasing hormone receptors. Biochem Biophys Res Commun. 1998 Sep 8;250(1):68–71. doi: 10.1006/bbrc.1998.9268. [DOI] [PubMed] [Google Scholar]
  15. Kallal L., Gagnon A. W., Penn R. B., Benovic J. L. Visualization of agonist-induced sequestration and down-regulation of a green fluorescent protein-tagged beta2-adrenergic receptor. J Biol Chem. 1998 Jan 2;273(1):322–328. doi: 10.1074/jbc.273.1.322. [DOI] [PubMed] [Google Scholar]
  16. Kim G. D., Carr I. C., Anderson L. A., Zabavnik J., Eidne K. A., Milligan G. The long isoform of the rat thyrotropin-releasing hormone receptor down-regulates Gq proteins. J Biol Chem. 1994 Aug 5;269(31):19933–19940. [PubMed] [Google Scholar]
  17. Koenig J. A., Edwardson J. M. Endocytosis and recycling of G protein-coupled receptors. Trends Pharmacol Sci. 1997 Aug;18(8):276–287. doi: 10.1016/s0165-6147(97)01091-2. [DOI] [PubMed] [Google Scholar]
  18. Lefkowitz R. J. G protein-coupled receptors. III. New roles for receptor kinases and beta-arrestins in receptor signaling and desensitization. J Biol Chem. 1998 Jul 24;273(30):18677–18680. doi: 10.1074/jbc.273.30.18677. [DOI] [PubMed] [Google Scholar]
  19. McKenzie F. R., Milligan G. Prostaglandin E1-mediated, cyclic AMP-independent, down-regulation of Gs alpha in neuroblastoma x glioma hybrid cells. J Biol Chem. 1990 Oct 5;265(28):17084–17093. [PubMed] [Google Scholar]
  20. Milligan G. Agonist regulation of cellular G protein levels and distribution: mechanisms and functional implications. Trends Pharmacol Sci. 1993 Nov;14(11):413–418. doi: 10.1016/0165-6147(93)90064-Q. [DOI] [PubMed] [Google Scholar]
  21. Mitchell F. M., Buckley N. J., Milligan G. Enhanced degradation of the phosphoinositidase C-linked guanine-nucleotide-binding protein Gq alpha/G11 alpha following activation of the human M1 muscarinic acetylcholine receptor expressed in CHO cells. Biochem J. 1993 Jul 15;293(Pt 2):495–499. doi: 10.1042/bj2930495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pippig S., Andexinger S., Lohse M. J. Sequestration and recycling of beta 2-adrenergic receptors permit receptor resensitization. Mol Pharmacol. 1995 Apr;47(4):666–676. [PubMed] [Google Scholar]
  23. Poduslo J. F. Glycoprotein molecular-weight estimation using sodium dodecyl sulfate-pore gradient electrophoresis: comparison of tris-glycine and tris-borate-EDTA buffer systems. Anal Biochem. 1981 Jun;114(1):131–139. doi: 10.1016/0003-2697(81)90463-2. [DOI] [PubMed] [Google Scholar]
  24. Ransnäs L. A., Svoboda P., Jasper J. R., Insel P. A. Stimulation of beta-adrenergic receptors of S49 lymphoma cells redistributes the alpha subunit of the stimulatory G protein between cytosol and membranes. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7900–7903. doi: 10.1073/pnas.86.20.7900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rizzuto R., Brini M., Pizzo P., Murgia M., Pozzan T. Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells. Curr Biol. 1995 Jun 1;5(6):635–642. doi: 10.1016/s0960-9822(95)00128-x. [DOI] [PubMed] [Google Scholar]
  26. Sellar R. E., Taylor P. L., Lamb R. F., Zabavnik J., Anderson L., Eidne K. A. Functional expression and molecular characterization of the thyrotrophin-releasing hormone receptor from the rat anterior pituitary gland. J Mol Endocrinol. 1993 Apr;10(2):199–206. doi: 10.1677/jme.0.0100199. [DOI] [PubMed] [Google Scholar]
  27. Shah B. H., MacEwan D. J., Milligan G. Gonadotrophin-releasing hormone receptor agonist-mediated down-regulation of Gq alpha/G11 alpha (pertussis toxin-insensitive) G proteins in alpha T3-1 gonadotroph cells reflects increased G protein turnover but not alterations in mRNA levels. Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):1886–1890. doi: 10.1073/pnas.92.6.1886. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Svoboda P., Kim G. D., Grassie M. A., Eidne K. A., Milligan G. Thyrotropin-releasing hormone-induced subcellular redistribution and down-regulation of G11alpha: analysis of agonist regulation of coexpressed G11alpha species variants. Mol Pharmacol. 1996 Apr;49(4):646–655. [PubMed] [Google Scholar]
  29. Svoboda P., Kvapil P., Insel P. A., Ransnäs L. A. Plasma-membrane-independent pool of the alpha subunit of the stimulatory guanine-nucleotide-binding regulatory protein in a low-density-membrane fraction of S49 lymphoma cells. Eur J Biochem. 1992 Sep 15;208(3):693–698. doi: 10.1111/j.1432-1033.1992.tb17236.x. [DOI] [PubMed] [Google Scholar]
  30. Tarasova N. I., Stauber R. H., Choi J. K., Hudson E. A., Czerwinski G., Miller J. L., Pavlakis G. N., Michejda C. J., Wank S. A. Visualization of G protein-coupled receptor trafficking with the aid of the green fluorescent protein. Endocytosis and recycling of cholecystokinin receptor type A. J Biol Chem. 1997 Jun 6;272(23):14817–14824. doi: 10.1074/jbc.272.23.14817. [DOI] [PubMed] [Google Scholar]
  31. Wedegaertner P. B., Bourne H. R., von Zastrow M. Activation-induced subcellular redistribution of Gs alpha. Mol Biol Cell. 1996 Aug;7(8):1225–1233. doi: 10.1091/mbc.7.8.1225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wise A., Lee T. W., MacEwan D. J., Milligan G. Degradation of G11 alpha/Gq alpha is accelerated by agonist occupancy of alpha 1A/D, alpha 1B, and alpha 1C adrenergic receptors. J Biol Chem. 1995 Jul 21;270(29):17196–17203. doi: 10.1074/jbc.270.29.17196. [DOI] [PubMed] [Google Scholar]
  33. Yu R., Hinkle P. M. Signal transduction, desensitization, and recovery of responses to thyrotropin-releasing hormone after inhibition of receptor internalization. Mol Endocrinol. 1998 May;12(5):737–749. doi: 10.1210/mend.12.5.0110. [DOI] [PubMed] [Google Scholar]
  34. von Zastrow M., Kobilka B. K. Ligand-regulated internalization and recycling of human beta 2-adrenergic receptors between the plasma membrane and endosomes containing transferrin receptors. J Biol Chem. 1992 Feb 15;267(5):3530–3538. [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Multimedia adjunct
bj3400529add.gif (1.4MB, gif)

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

RESOURCES