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. 1990 Jan;9(1):61–67. doi: 10.1002/j.1460-2075.1990.tb08080.x

A cerebellar Purkinje cell marker P400 protein is an inositol 1,4,5-trisphosphate (InsP3) receptor protein. Purification and characterization of InsP3 receptor complex.

N Maeda 1, M Niinobe 1, K Mikoshiba 1
PMCID: PMC551630  PMID: 2153079

Abstract

P400 protein is a 250 kd glycoprotein, characteristic of the cerebellum, which is accumulated at the endoplasmic reticulum, at the plasma membrane and at the post-synaptic density of Purkinje cells. In this study, we purified inositol 1,4,5-trisphosphate (InsP3) receptor from mouse cerebellum and examined the possibility that P400 protein is identical with cerebellar InsP3 receptor protein. InsP3 receptor was solubilized with Triton X-100 from a post-nuclear fraction of ddY mouse cerebellum and was purified with high yield by sequential column chromatography on DE52, heparin-agarose, lentil lectin-Sepharose and hydroxylapatite. In these chromatographies, P400 protein co-migrated completely with the InsP3 binding activity. The purified receptor is a 250 kd protein with a Bmax of 2.1 pmol/microgram and a KD of 83 nM. It reacted with three different monoclonal antibodies against P400 protein, indicating that P400 protein is the same substance as the InsP3 receptor (P400/InsP3 receptor protein). Electron microscopy of the purified receptor showed a square shape with sides approximately 25 nm long. Binding assays of the cerebella of Purkinje cell-degeneration (pcd) mice with [3H]InsP3 demonstrated that the InsP3 binding sites in the cerebellum are distributed exclusively on the Purkinje cells. Immunohistochemical analysis indicated that P400/InsP3 receptor is present at the dendrites, cell bodies, axons and synaptic boutons of the Purkinje cells.

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Selected References

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

  1. Anderson K., Lai F. A., Liu Q. Y., Rousseau E., Erickson H. P., Meissner G. Structural and functional characterization of the purified cardiac ryanodine receptor-Ca2+ release channel complex. J Biol Chem. 1989 Jan 15;264(2):1329–1335. [PubMed] [Google Scholar]
  2. Berridge M. J. Inositol trisphosphate and diacylglycerol: two interacting second messengers. Annu Rev Biochem. 1987;56:159–193. doi: 10.1146/annurev.bi.56.070187.001111. [DOI] [PubMed] [Google Scholar]
  3. Berridge M. J., Irvine R. F. Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature. 1984 Nov 22;312(5992):315–321. doi: 10.1038/312315a0. [DOI] [PubMed] [Google Scholar]
  4. Crepel F., Dupont J. L., Gardette R. Selective absence of calcium spikes in Purkinje cells of staggerer mutant mice in cerebellar slices maintained in vitro. J Physiol. 1984 Jan;346:111–125. doi: 10.1113/jphysiol.1984.sp015010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Higashida H. Acetylcholine release by bradykinin, inositol 1,4,5-trisphosphate and phorbol dibutyrate in rodent neuroblastoma cells. J Physiol. 1988 Mar;397:209–222. doi: 10.1113/jphysiol.1988.sp016996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hirano A., Dembitzer H. M. The fine structure of staggerer cerebellum. J Neuropathol Exp Neurol. 1975 Jan;34(1):1–11. doi: 10.1097/00005072-197501000-00001. [DOI] [PubMed] [Google Scholar]
  8. Inui M., Saito A., Fleischer S. Purification of the ryanodine receptor and identity with feet structures of junctional terminal cisternae of sarcoplasmic reticulum from fast skeletal muscle. J Biol Chem. 1987 Feb 5;262(4):1740–1747. [PubMed] [Google Scholar]
  9. Ito M., Kano M. Long-lasting depression of parallel fiber-Purkinje cell transmission induced by conjunctive stimulation of parallel fibers and climbing fibers in the cerebellar cortex. Neurosci Lett. 1982 Dec 13;33(3):253–258. doi: 10.1016/0304-3940(82)90380-9. [DOI] [PubMed] [Google Scholar]
  10. Ito M., Sakurai M., Tongroach P. Climbing fibre induced depression of both mossy fibre responsiveness and glutamate sensitivity of cerebellar Purkinje cells. J Physiol. 1982 Mar;324:113–134. doi: 10.1113/jphysiol.1982.sp014103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kano M., Kato M. Quisqualate receptors are specifically involved in cerebellar synaptic plasticity. Nature. 1987 Jan 15;325(6101):276–279. doi: 10.1038/325276a0. [DOI] [PubMed] [Google Scholar]
  12. Kornfeld K., Reitman M. L., Kornfeld R. The carbohydrate-binding specificity of pea and lentil lectins. Fucose is an important determinant. J Biol Chem. 1981 Jul 10;256(13):6633–6640. [PubMed] [Google Scholar]
  13. Kuno M., Gardner P. Ion channels activated by inositol 1,4,5-trisphosphate in plasma membrane of human T-lymphocytes. Nature. 1987 Mar 19;326(6110):301–304. doi: 10.1038/326301a0. [DOI] [PubMed] [Google Scholar]
  14. Maeda N., Niinobe M., Inoue Y., Mikoshiba K. Developmental expression and intracellular location of P400 protein characteristic of Purkinje cells in the mouse cerebellum. Dev Biol. 1989 May;133(1):67–76. doi: 10.1016/0012-1606(89)90297-2. [DOI] [PubMed] [Google Scholar]
  15. Maeda N., Niinobe M., Nakahira K., Mikoshiba K. Purification and characterization of P400 protein, a glycoprotein characteristic of Purkinje cell, from mouse cerebellum. J Neurochem. 1988 Dec;51(6):1724–1730. doi: 10.1111/j.1471-4159.1988.tb01151.x. [DOI] [PubMed] [Google Scholar]
  16. Maeda N., Wada K., Yuzaki M., Mikoshiba K. Autoradiographic visualization of a calcium channel antagonist, [125I]omega-conotoxin GVIA, binding site in the brains of normal and cerebellar mutant mice (pcd and weaver). Brain Res. 1989 Jun 5;489(1):21–30. doi: 10.1016/0006-8993(89)90004-8. [DOI] [PubMed] [Google Scholar]
  17. Mallet J., Huchet M., Pougeois R., Changeux J. P. Anatomical, physiological and biochemical studies on the cerebellum from mutant mice. III. Protein differences associated with the weaver, staggerer and nervous mutations. Brain Res. 1976 Feb 20;103(2):291–312. doi: 10.1016/0006-8993(76)90800-3. [DOI] [PubMed] [Google Scholar]
  18. Mattson M. P. Neurotransmitters in the regulation of neuronal cytoarchitecture. Brain Res. 1988 Apr-Jun;472(2):179–212. doi: 10.1016/0165-0173(88)90020-3. [DOI] [PubMed] [Google Scholar]
  19. Meyer T., Stryer L. Molecular model for receptor-stimulated calcium spiking. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5051–5055. doi: 10.1073/pnas.85.14.5051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mikoshiba K., Changeux J. P. Morphological and biochemical studies on isolated molecular and granular layers from bovine cerebellum. Brain Res. 1978 Mar 10;142(3):487–504. doi: 10.1016/0006-8993(78)90911-3. [DOI] [PubMed] [Google Scholar]
  21. Mikoshiba K., Huchet M., Changeux J. P. Biochemical and immunological studies on the P400 protein, a protein characteristic of the Purkinje cell from mouse and rat cerebellum. Dev Neurosci. 1979;2(6):254–275. doi: 10.1159/000112489. [DOI] [PubMed] [Google Scholar]
  22. Mikoshiba K., Okano H., Tsukada Y. P400 protein characteristic to Purkinje cells and related proteins in cerebella from neuropathological mutant mice: autoradiographic study by 14C-leucine and phosphorylation. Dev Neurosci. 1985;7(3):179–187. doi: 10.1159/000112286. [DOI] [PubMed] [Google Scholar]
  23. Murphy S. N., Miller R. J. A glutamate receptor regulates Ca2+ mobilization in hippocampal neurons. Proc Natl Acad Sci U S A. 1988 Nov;85(22):8737–8741. doi: 10.1073/pnas.85.22.8737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Shah J., Cohen R. S., Pant H. C. Inositol trisphosphate-induced calcium release in brain microsomes. Brain Res. 1987 Sep 1;419(1-2):1–6. doi: 10.1016/0006-8993(87)90562-2. [DOI] [PubMed] [Google Scholar]
  26. Sladeczek F., Récasens M., Bockaert J. A new mechanism for glutamate receptor action: phosphoinositide hydrolysis. Trends Neurosci. 1988 Dec;11(12):545–549. doi: 10.1016/0166-2236(88)90183-x. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Worley P. F., Baraban J. M., Colvin J. S., Snyder S. H. Inositol trisphosphate receptor localization in brain: variable stoichiometry with protein kinase C. Nature. 1987 Jan 8;325(7000):159–161. doi: 10.1038/325159a0. [DOI] [PubMed] [Google Scholar]
  30. Worley P. F., Baraban J. M., Snyder S. H. Inositol 1,4,5-trisphosphate receptor binding: autoradiographic localization in rat brain. J Neurosci. 1989 Jan;9(1):339–346. doi: 10.1523/JNEUROSCI.09-01-00339.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Yamamoto H., Maeda N., Niinobe M., Miyamoto E., Mikoshiba K. Phosphorylation of P400 protein by cyclic AMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase II. J Neurochem. 1989 Sep;53(3):917–923. doi: 10.1111/j.1471-4159.1989.tb11792.x. [DOI] [PubMed] [Google Scholar]
  33. Yano K., Higashida H., Inoue R., Nozawa Y. Bradykinin-induced rapid breakdown of phosphatidylinositol 4,5-bisphosphate in neuroblastoma X glioma hybrid NG108-15 cells. J Biol Chem. 1984 Aug 25;259(16):10201–10207. [PubMed] [Google Scholar]

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