Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Apr 1;323(Pt 1):273–280. doi: 10.1042/bj3230273

Intracellular targeting and homotetramer formation of a truncated inositol 1,4,5-trisphosphate receptor-green fluorescent protein chimera in Xenopus laevis oocytes: evidence for the involvement of the transmembrane spanning domain in endoplasmic reticulum targeting and homotetramer complex formation.

L G Sayers 1, A Miyawaki 1, A Muto 1, H Takeshita 1, A Yamamoto 1, T Michikawa 1, T Furuichi 1, K Mikoshiba 1
PMCID: PMC1218306  PMID: 9173893

Abstract

In an attempt to define structural regions of the type I inositol 1, 4,5-trisphosphate [Ins(1,4,5)P3] receptor [Ins(1,4,5)P3R] involved in its intracellular targeting to the endoplasmic reticulum (ER), we have employed the use of green fluorescent protein (GFP) to monitor the localization of a truncated Ins(1,4,5)P3R mutant containing just the putative transmembrane spanning domain and the C-terminal cytoplasmic domain [amino acids 2216-2749; termed inositol trisphosphate receptor(ES)]. We expressed a chimeric GFP-Ins(1,4, 5)P3R(ES) fusion protein in Xenopus laevis oocytes, and used fluorescence confocal microscopy to monitor its intracellular localization. Fluorescence confocal microscopy data showed an intense fluorescence in the perinuclear region and in a reticular-network under the animal pole of the oocyte, consistent with the targeting of expressed GFP-Ins(1,4,5)P3R(ES) to perinuclear ER and ER under the animal pole. These findings are consistent with the intracellular localization of the endogenous Xenopus Ins(1,4, 5)P3R shown previously. Furthermore, electron microscopy data indicate that expressed GFP-Ins(1,4,5)P3R(ES) is in fact targeted to the ER. Sodium carbonate extraction of microsomal membranes and cross-linking experiments indicate that the expressed chimeric protein is in fact membrane anchored and able to form a homotetrameric complex. Our data provides evidence that Ins(1,4, 5)P3R(ES) constitutes the membrane spanning domain of the Ins(1,4, 5)P3R and is able to mediate homotetramer formation, without the need for the large N-terminal cytoplasmic domain. Furthermore, the localization of GFP-Ins(1,4,5)P3R(ES) on the ER indicates that an ER retention/targeting signal is contained within the transmembrane spanning domain of the inositol trisphosphate receptor.

Full Text

The Full Text of this article is available as a PDF (452.9 KB).

Selected References

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

  1. Berridge M. J. Inositol trisphosphate and calcium signalling. Nature. 1993 Jan 28;361(6410):315–325. doi: 10.1038/361315a0. [DOI] [PubMed] [Google Scholar]
  2. Berridge M. J., Irvine R. F. Inositol phosphates and cell signalling. Nature. 1989 Sep 21;341(6239):197–205. doi: 10.1038/341197a0. [DOI] [PubMed] [Google Scholar]
  3. Blondel O., Takeda J., Janssen H., Seino S., Bell G. I. Sequence and functional characterization of a third inositol trisphosphate receptor subtype, IP3R-3, expressed in pancreatic islets, kidney, gastrointestinal tract, and other tissues. J Biol Chem. 1993 May 25;268(15):11356–11363. [PubMed] [Google Scholar]
  4. Cubitt A. B., Heim R., Adams S. R., Boyd A. E., Gross L. A., Tsien R. Y. Understanding, improving and using green fluorescent proteins. Trends Biochem Sci. 1995 Nov;20(11):448–455. doi: 10.1016/s0968-0004(00)89099-4. [DOI] [PubMed] [Google Scholar]
  5. Foletti D., Guerini D., Carafoli E. Subcellular targeting of the endoplasmic reticulum and plasma membrane Ca2+ pumps: a study using recombinant chimeras. FASEB J. 1995 May;9(8):670–680. doi: 10.1096/fasebj.9.8.7768360. [DOI] [PubMed] [Google Scholar]
  6. Furuichi T., Kohda K., Miyawaki A., Mikoshiba K. Intracellular channels. Curr Opin Neurobiol. 1994 Jun;4(3):294–303. doi: 10.1016/0959-4388(94)90089-2. [DOI] [PubMed] [Google Scholar]
  7. Furuichi T., Yoshikawa S., Miyawaki A., Wada K., Maeda N., Mikoshiba K. Primary structure and functional expression of the inositol 1,4,5-trisphosphate-binding protein P400. Nature. 1989 Nov 2;342(6245):32–38. doi: 10.1038/342032a0. [DOI] [PubMed] [Google Scholar]
  8. Kume S., Muto A., Aruga J., Nakagawa T., Michikawa T., Furuichi T., Nakade S., Okano H., Mikoshiba K. The Xenopus IP3 receptor: structure, function, and localization in oocytes and eggs. Cell. 1993 May 7;73(3):555–570. doi: 10.1016/0092-8674(93)90142-d. [DOI] [PubMed] [Google Scholar]
  9. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  10. Lorenzen J. A., Dadabay C. Y., Fischer E. H. COOH-terminal sequence motifs target the T cell protein tyrosine phosphatase to the ER and nucleus. J Cell Biol. 1995 Nov;131(3):631–643. doi: 10.1083/jcb.131.3.631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Maeda N., Kawasaki T., Nakade S., Yokota N., Taguchi T., Kasai M., Mikoshiba K. Structural and functional characterization of inositol 1,4,5-trisphosphate receptor channel from mouse cerebellum. J Biol Chem. 1991 Jan 15;266(2):1109–1116. [PubMed] [Google Scholar]
  12. Maeda N., Niinobe M., Mikoshiba K. A cerebellar Purkinje cell marker P400 protein is an inositol 1,4,5-trisphosphate (InsP3) receptor protein. Purification and characterization of InsP3 receptor complex. EMBO J. 1990 Jan;9(1):61–67. doi: 10.1002/j.1460-2075.1990.tb08080.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Meldolesi J., Madeddu L., Pozzan T. Intracellular Ca2+ storage organelles in non-muscle cells: heterogeneity and functional assignment. Biochim Biophys Acta. 1990 Nov 12;1055(2):130–140. doi: 10.1016/0167-4889(90)90113-r. [DOI] [PubMed] [Google Scholar]
  14. Mignery G. A., Südhof T. C., Takei K., De Camilli P. Putative receptor for inositol 1,4,5-trisphosphate similar to ryanodine receptor. Nature. 1989 Nov 9;342(6246):192–195. doi: 10.1038/342192a0. [DOI] [PubMed] [Google Scholar]
  15. Mignery G. A., Südhof T. C. The ligand binding site and transduction mechanism in the inositol-1,4,5-triphosphate receptor. EMBO J. 1990 Dec;9(12):3893–3898. doi: 10.1002/j.1460-2075.1990.tb07609.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Miyawaki A., Furuichi T., Ryou Y., Yoshikawa S., Nakagawa T., Saitoh T., Mikoshiba K. Structure-function relationships of the mouse inositol 1,4,5-trisphosphate receptor. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4911–4915. doi: 10.1073/pnas.88.11.4911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Monkawa T., Miyawaki A., Sugiyama T., Yoneshima H., Yamamoto-Hino M., Furuichi T., Saruta T., Hasegawa M., Mikoshiba K. Heterotetrameric complex formation of inositol 1,4,5-trisphosphate receptor subunits. J Biol Chem. 1995 Jun 16;270(24):14700–14704. doi: 10.1074/jbc.270.24.14700. [DOI] [PubMed] [Google Scholar]
  18. Otsu H., Yamamoto A., Maeda N., Mikoshiba K., Tashiro Y. Immunogold localization of inositol 1, 4, 5-trisphosphate (InsP3) receptor in mouse cerebellar Purkinje cells using three monoclonal antibodies. Cell Struct Funct. 1990 Jun;15(3):163–173. doi: 10.1247/csf.15.163. [DOI] [PubMed] [Google Scholar]
  19. Parys J. B., Sernett S. W., DeLisle S., Snyder P. M., Welsh M. J., Campbell K. P. Isolation, characterization, and localization of the inositol 1,4,5-trisphosphate receptor protein in Xenopus laevis oocytes. J Biol Chem. 1992 Sep 15;267(26):18776–18782. [PubMed] [Google Scholar]
  20. Pelham H. R., Munro S. Sorting of membrane proteins in the secretory pathway. Cell. 1993 Nov 19;75(4):603–605. doi: 10.1016/0092-8674(93)90479-A. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pozzan T., Rizzuto R., Volpe P., Meldolesi J. Molecular and cellular physiology of intracellular calcium stores. Physiol Rev. 1994 Jul;74(3):595–636. doi: 10.1152/physrev.1994.74.3.595. [DOI] [PubMed] [Google Scholar]
  22. Prasher D. C., Eckenrode V. K., Ward W. W., Prendergast F. G., Cormier M. J. Primary structure of the Aequorea victoria green-fluorescent protein. Gene. 1992 Feb 15;111(2):229–233. doi: 10.1016/0378-1119(92)90691-h. [DOI] [PubMed] [Google Scholar]
  23. Ross C. A., Danoff S. K., Schell M. J., Snyder S. H., Ullrich A. Three additional inositol 1,4,5-trisphosphate receptors: molecular cloning and differential localization in brain and peripheral tissues. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4265–4269. doi: 10.1073/pnas.89.10.4265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Satoh T., Ross C. A., Villa A., Supattapone S., Pozzan T., Snyder S. H., Meldolesi J. The inositol 1,4,5,-trisphosphate receptor in cerebellar Purkinje cells: quantitative immunogold labeling reveals concentration in an ER subcompartment. J Cell Biol. 1990 Aug;111(2):615–624. doi: 10.1083/jcb.111.2.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Tokuyasu K. T. Application of cryoultramicrotomy to immunocytochemistry. J Microsc. 1986 Aug;143(Pt 2):139–149. doi: 10.1111/j.1365-2818.1986.tb02772.x. [DOI] [PubMed] [Google Scholar]
  26. Tokuyasu K. T. Use of poly(vinylpyrrolidone) and poly(vinyl alcohol) for cryoultramicrotomy. Histochem J. 1989 Mar;21(3):163–171. doi: 10.1007/BF01007491. [DOI] [PubMed] [Google Scholar]
  27. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Worley P. F., Baraban J. M., Supattapone S., Wilson V. S., Snyder S. H. Characterization of inositol trisphosphate receptor binding in brain. Regulation by pH and calcium. J Biol Chem. 1987 Sep 5;262(25):12132–12136. [PubMed] [Google Scholar]

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

RESOURCES