Skip to main content
NCBI home page
Molecular Biology of the Cell logoLink to Molecular Biology of the Cell
. 1996 Jun;7(6):949–960. doi: 10.1091/mbc.7.6.949

Inositol 1,4,5-trisphosphate receptors in endocrine cells: localization and association in hetero- and homotetramers.

F C Nucifora Jr 1, A H Sharp 1, S L Milgram 1, C A Ross 1
PMCID: PMC275945  PMID: 8817000

Abstract

The inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular calcium channel involved in coupling cell membrane receptors to calcium signal transduction pathways within cells including endocrine cells. Several isoforms (I, II, and III) of IP3Rs have been identified, which are encoded by separate genes, and are expressed in many tissues with differing patterns of cellular expression. We have generated specific affinity-purified polyclonal anti-peptide antibodies to each of the three isoforms. Western blot analysis of RINm5F and ATt20 cells shows high levels of endogenously expressed type I and type III IP3R, but undetectable levels of type II. Immunofluorescence studies revealed an endoplasmic reticulum-like pattern similar to BiP, an ER marker. In contrast with previous claims, both type I and type III IP3Rs were absent from the secretory granules of ATt20 cells. Western blots of sucrose gradients and gel filtration probed with antibodies to either type I or type III showed a molecular weight of greater than 1,000 kDa consistent with a tetrameric structure. Co-immunoprecipitation experiments indicated that most of the receptors were present as heterotetramers. Homotetramers were identified for the type III IP3R; however, type I homotetramers were undetectable. These data suggest that molecular association of IP3Rs into heterotetrameric forms can contribute to the complexity of the regulation of Ca2+ release from ER by IP3Rs within cells.

Full text

PDF
949

Images in this article

952Image
on p.952
953Image
on p.953
954Image
on p.954
955Image
on p.955
956Image
on p.956
957Image
on p.957
958Image
on p.958
958Image
on p.958

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. Bezprozvanny I., Watras J., Ehrlich B. E. Bell-shaped calcium-response curves of Ins(1,4,5)P3- and calcium-gated channels from endoplasmic reticulum of cerebellum. Nature. 1991 Jun 27;351(6329):751–754. doi: 10.1038/351751a0. [DOI] [PubMed] [Google Scholar]
  4. Blondel O., Bell G. I., Seino S. Inositol 1,4,5-trisphosphate receptors, secretory granules and secretion in endocrine and neuroendocrine cells. Trends Neurosci. 1995 Apr;18(4):157–161. doi: 10.1016/0166-2236(95)93894-4. [DOI] [PubMed] [Google Scholar]
  5. Blondel O., Moody M. M., Depaoli A. M., Sharp A. H., Ross C. A., Swift H., Bell G. I. Localization of inositol trisphosphate receptor subtype 3 to insulin and somatostatin secretory granules and regulation of expression in islets and insulinoma cells. Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7777–7781. doi: 10.1073/pnas.91.16.7777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Danoff S. K., Ferris C. D., Donath C., Fischer G. A., Munemitsu S., Ullrich A., Snyder S. H., Ross C. A. Inositol 1,4,5-trisphosphate receptors: distinct neuronal and nonneuronal forms derived by alternative splicing differ in phosphorylation. Proc Natl Acad Sci U S A. 1991 Apr 1;88(7):2951–2955. doi: 10.1073/pnas.88.7.2951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ehrlich B. E., Watras J. Inositol 1,4,5-trisphosphate activates a channel from smooth muscle sarcoplasmic reticulum. Nature. 1988 Dec 8;336(6199):583–586. doi: 10.1038/336583a0. [DOI] [PubMed] [Google Scholar]
  9. Ferris C. D., Huganir R. L., Bredt D. S., Cameron A. M., Snyder S. H. Inositol trisphosphate receptor: phosphorylation by protein kinase C and calcium calmodulin-dependent protein kinases in reconstituted lipid vesicles. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2232–2235. doi: 10.1073/pnas.88.6.2232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ferris C. D., Huganir R. L., Snyder S. H. Calcium flux mediated by purified inositol 1,4,5-trisphosphate receptor in reconstituted lipid vesicles is allosterically regulated by adenine nucleotides. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2147–2151. doi: 10.1073/pnas.87.6.2147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ferris C. D., Snyder S. H. Inositol phosphate receptors and calcium disposition in the brain. J Neurosci. 1992 May;12(5):1567–1574. doi: 10.1523/JNEUROSCI.12-05-01567.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Finch E. A., Turner T. J., Goldin S. M. Calcium as a coagonist of inositol 1,4,5-trisphosphate-induced calcium release. Science. 1991 Apr 19;252(5004):443–446. doi: 10.1126/science.2017683. [DOI] [PubMed] [Google Scholar]
  13. Fujimoto T., Nakade S., Miyawaki A., Mikoshiba K., Ogawa K. Localization of inositol 1,4,5-trisphosphate receptor-like protein in plasmalemmal caveolae. J Cell Biol. 1992 Dec;119(6):1507–1513. doi: 10.1083/jcb.119.6.1507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Furuichi T., Mikoshiba K. Inositol 1, 4, 5-trisphosphate receptor-mediated Ca2+ signaling in the brain. J Neurochem. 1995 Mar;64(3):953–960. doi: 10.1046/j.1471-4159.1995.64030953.x. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Hajnóczky G., Thomas A. P. The inositol trisphosphate calcium channel is inactivated by inositol trisphosphate. Nature. 1994 Aug 11;370(6489):474–477. doi: 10.1038/370474a0. [DOI] [PubMed] [Google Scholar]
  17. Halvorsen S. W., Berg D. K. Subunit composition of nicotinic acetylcholine receptors from chick ciliary ganglia. J Neurosci. 1990 Jun;10(6):1711–1718. doi: 10.1523/JNEUROSCI.10-06-01711.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Huovila A. P., Eder A. M., Fuller S. D. Hepatitis B surface antigen assembles in a post-ER, pre-Golgi compartment. J Cell Biol. 1992 Sep;118(6):1305–1320. doi: 10.1083/jcb.118.6.1305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Husten E. J., Eipper B. A. Purification and characterization of PAM-1, an integral membrane protein involved in peptide processing. Arch Biochem Biophys. 1994 Aug 1;312(2):487–492. doi: 10.1006/abbi.1994.1336. [DOI] [PubMed] [Google Scholar]
  20. Iino M., Endo M. Calcium-dependent immediate feedback control of inositol 1,4,5-triphosphate-induced Ca2+ release. Nature. 1992 Nov 5;360(6399):76–78. doi: 10.1038/360076a0. [DOI] [PubMed] [Google Scholar]
  21. Jayaraman T., Ondriasová E., Ondrias K., Harnick D. J., Marks A. R. The inositol 1,4,5-trisphosphate receptor is essential for T-cell receptor signaling. Proc Natl Acad Sci U S A. 1995 Jun 20;92(13):6007–6011. doi: 10.1073/pnas.92.13.6007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kasai H., Li Y. X., Miyashita Y. Subcellular distribution of Ca2+ release channels underlying Ca2+ waves and oscillations in exocrine pancreas. Cell. 1993 Aug 27;74(4):669–677. doi: 10.1016/0092-8674(93)90514-q. [DOI] [PubMed] [Google Scholar]
  23. Khan A. A., Steiner J. P., Klein M. G., Schneider M. F., Snyder S. H. IP3 receptor: localization to plasma membrane of T cells and cocapping with the T cell receptor. Science. 1992 Aug 7;257(5071):815–818. doi: 10.1126/science.1323146. [DOI] [PubMed] [Google Scholar]
  24. Khan A. A., Steiner J. P., Snyder S. H. Plasma membrane inositol 1,4,5-trisphosphate receptor of lymphocytes: selective enrichment in sialic acid and unique binding specificity. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2849–2853. doi: 10.1073/pnas.89.7.2849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Khodakhah K., Ogden D. Functional heterogeneity of calcium release by inositol trisphosphate in single Purkinje neurones, cultured cerebellar astrocytes, and peripheral tissues. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):4976–4980. doi: 10.1073/pnas.90.11.4976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Maranto A. R. Primary structure, ligand binding, and localization of the human type 3 inositol 1,4,5-trisphosphate receptor expressed in intestinal epithelium. J Biol Chem. 1994 Jan 14;269(2):1222–1230. [PubMed] [Google Scholar]
  29. Meldolesi J. Multifarious IP3 receptors. Curr Biol. 1992 Jul;2(7):393–394. doi: 10.1016/0960-9822(92)90092-o. [DOI] [PubMed] [Google Scholar]
  30. Meldolesi J., Pozzan T. IP3 receptors and secretory granules. Trends Neurosci. 1995 Aug;18(8):340–341. doi: 10.1016/0166-2236(95)93924-m. [DOI] [PubMed] [Google Scholar]
  31. Meldolesi J., Pozzan T. IP3 receptors and secretory granules. Trends Neurosci. 1995 Aug;18(8):340–341. doi: 10.1016/0166-2236(95)93924-m. [DOI] [PubMed] [Google Scholar]
  32. Mignery G. A., Newton C. L., Archer B. T., 3rd, Südhof T. C. Structure and expression of the rat inositol 1,4,5-trisphosphate receptor. J Biol Chem. 1990 Jul 25;265(21):12679–12685. [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. Milgram S. L., Eipper B. A., Mains R. E. Differential trafficking of soluble and integral membrane secretory granule-associated proteins. J Cell Biol. 1994 Jan;124(1-2):33–41. doi: 10.1083/jcb.124.1.33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Milgram S. L., Mains R. E., Eipper B. A. COOH-terminal signals mediate the trafficking of a peptide processing enzyme in endocrine cells. J Cell Biol. 1993 Apr;121(1):23–36. doi: 10.1083/jcb.121.1.23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Miyawaki A., Furuichi T., Maeda N., Mikoshiba K. Expressed cerebellar-type inositol 1,4,5-trisphosphate receptor, P400, has calcium release activity in a fibroblast L cell line. Neuron. 1990 Jul;5(1):11–18. doi: 10.1016/0896-6273(90)90029-f. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Nakade S., Maeda N., Mikoshiba K. Involvement of the C-terminus of the inositol 1,4,5-trisphosphate receptor in Ca2+ release analysed using region-specific monoclonal antibodies. Biochem J. 1991 Jul 1;277(Pt 1):125–131. doi: 10.1042/bj2770125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Nakagawa T., Okano H., Furuichi T., Aruga J., Mikoshiba K. The subtypes of the mouse inositol 1,4,5-trisphosphate receptor are expressed in a tissue-specific and developmentally specific manner. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6244–6248. doi: 10.1073/pnas.88.14.6244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Nakagawa T., Shiota C., Okano H., Mikoshiba K. Differential localization of alternative spliced transcripts encoding inositol 1,4,5-trisphosphate receptors in mouse cerebellum and hippocampus: in situ hybridization study. J Neurochem. 1991 Nov;57(5):1807–1810. doi: 10.1111/j.1471-4159.1991.tb06385.x. [DOI] [PubMed] [Google Scholar]
  42. Newton C. L., Mignery G. A., Südhof T. C. Co-expression in vertebrate tissues and cell lines of multiple inositol 1,4,5-trisphosphate (InsP3) receptors with distinct affinities for InsP3. J Biol Chem. 1994 Nov 18;269(46):28613–28619. [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. Ross C. A., Meldolesi J., Milner T. A., Satoh T., Supattapone S., Snyder S. H. Inositol 1,4,5-trisphosphate receptor localized to endoplasmic reticulum in cerebellar Purkinje neurons. Nature. 1989 Jun 8;339(6224):468–470. doi: 10.1038/339468a0. [DOI] [PubMed] [Google Scholar]
  46. Schell M. J., Danoff S. K., Ross C. A. Inositol (1,4,5)-trisphosphate receptor: characterization of neuron-specific alternative splicing in rat brain and peripheral tissues. Brain Res Mol Brain Res. 1993 Mar;17(3-4):212–216. doi: 10.1016/0169-328x(93)90004-9. [DOI] [PubMed] [Google Scholar]
  47. Sharp A. H., Dawson T. M., Ross C. A., Fotuhi M., Mourey R. J., Snyder S. H. Inositol 1,4,5-trisphosphate receptors: immunohistochemical localization to discrete areas of rat central nervous system. Neuroscience. 1993 Apr;53(4):927–942. doi: 10.1016/0306-4522(93)90478-x. [DOI] [PubMed] [Google Scholar]
  48. Sharp A. H., Loev S. J., Schilling G., Li S. H., Li X. J., Bao J., Wagster M. V., Kotzuk J. A., Steiner J. P., Lo A. Widespread expression of Huntington's disease gene (IT15) protein product. Neuron. 1995 May;14(5):1065–1074. doi: 10.1016/0896-6273(95)90345-3. [DOI] [PubMed] [Google Scholar]
  49. Simpson P. B., Challiss R. A., Nahorski S. R. Neuronal Ca2+ stores: activation and function. Trends Neurosci. 1995 Jul;18(7):299–306. doi: 10.1016/0166-2236(95)93919-o. [DOI] [PubMed] [Google Scholar]
  50. Sugiyama T., Furuya A., Monkawa T., Yamamoto-Hino M., Satoh S., Ohmori K., Miyawaki A., Hanai N., Mikoshiba K., Hasegawa M. Monoclonal antibodies distinctively recognizing the subtypes of inositol 1,4,5-trisphosphate receptor: application to the studies on inflammatory cells. FEBS Lett. 1994 Nov 7;354(2):149–154. doi: 10.1016/0014-5793(94)01099-4. [DOI] [PubMed] [Google Scholar]
  51. Supattapone S., Danoff S. K., Theibert A., Joseph S. K., Steiner J., Snyder S. H. Cyclic AMP-dependent phosphorylation of a brain inositol trisphosphate receptor decreases its release of calcium. Proc Natl Acad Sci U S A. 1988 Nov;85(22):8747–8750. doi: 10.1073/pnas.85.22.8747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Supattapone S., Worley P. F., Baraban J. M., Snyder S. H. Solubilization, purification, and characterization of an inositol trisphosphate receptor. J Biol Chem. 1988 Jan 25;263(3):1530–1534. [PubMed] [Google Scholar]
  53. Südhof T. C., Newton C. L., Archer B. T., 3rd, Ushkaryov Y. A., Mignery G. A. Structure of a novel InsP3 receptor. EMBO J. 1991 Nov;10(11):3199–3206. doi: 10.1002/j.1460-2075.1991.tb04882.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Takahashi T., Momiyama A., Hirai K., Hishinuma F., Akagi H. Functional correlation of fetal and adult forms of glycine receptors with developmental changes in inhibitory synaptic receptor channels. Neuron. 1992 Dec;9(6):1155–1161. doi: 10.1016/0896-6273(92)90073-m. [DOI] [PubMed] [Google Scholar]
  55. 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]
  56. Vernallis A. B., Conroy W. G., Berg D. K. Neurons assemble acetylcholine receptors with as many as three kinds of subunits while maintaining subunit segregation among receptor subtypes. Neuron. 1993 Mar;10(3):451–464. doi: 10.1016/0896-6273(93)90333-m. [DOI] [PubMed] [Google Scholar]
  57. Zhou A., Mains R. E. Endoproteolytic processing of proopiomelanocortin and prohormone convertases 1 and 2 in neuroendocrine cells overexpressing prohormone convertases 1 or 2. J Biol Chem. 1994 Jul 1;269(26):17440–17447. [PubMed] [Google Scholar]

Articles from Molecular Biology of the Cell are provided here courtesy of American Society for Cell Biology

RESOURCES