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. 2000 Jun 15;348(Pt 3):551–556.

Ligand binding directly stimulates ubiquitination of the inositol 1, 4,5-trisphosphate receptor.

C C Zhu 1, R J Wojcikiewicz 1
PMCID: PMC1221096  PMID: 10839985

Abstract

Down-regulation of the Ins(1,4,5)P(3) receptor is an adaptive response to the activation of certain phosphoinositidase C-linked cell-surface receptors. It is manifested as a profound decline in cellular Ins(1,4,5)P(3) receptor content, occurs with a half-time of 0.5-2 h and is due to accelerated proteolysis. It has been shown that this process is mediated by the ubiquitin/proteasome pathway and is therefore initiated by Ins(1,4,5)P(3) receptor ubiquitination. To investigate the role of ligand binding in Ins(1,4,5)P(3) receptor ubiquitination, we expressed 'exogenous' wild-type and ligand-binding-defective mutant type I Ins(1,4,5)P(3) receptors in human neuroblastoma SH-SY5Y cells, in which muscarinic receptor activation elicits Ins(1,4,5)P(3) receptor down-regulation. We found (1) that exogenous wild-type Ins(1,4,5)P(3) receptors are efficiently ubiquitinated in response to muscarinic receptor stimulation, (2) that exogenous ligand binding-defective mutant Ins(1,4,5)P(3) receptors are resistant to ubiquitination, (3) that this resistance is not caused by the removal of potential ubiquitin-conjugating sites in the mutated region, and (4) that in heterotetramers of exogenous mutant receptors and 'endogenous' receptors, only the latter are targeted for ubiquitination. These results indicate that the binding of Ins(1,4,5)P(3) directly stimulates ubiquitination of the Ins(1,4,5)P(3) receptor and that the targeting of Ins(1,4,5)P(3) receptors for ubiquitination is a highly specific process. We therefore propose that an Ins(1,4, 5)P(3)-binding-induced conformational change in the receptor exposes a degradation signal that leads to ubiquitination.

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Selected References

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  1. Baumeister W., Walz J., Zühl F., Seemüller E. The proteasome: paradigm of a self-compartmentalizing protease. Cell. 1998 Feb 6;92(3):367–380. doi: 10.1016/s0092-8674(00)80929-0. [DOI] [PubMed] [Google Scholar]
  2. Berridge M. J., Bootman M. D., Lipp P. Calcium--a life and death signal. Nature. 1998 Oct 15;395(6703):645–648. doi: 10.1038/27094. [DOI] [PubMed] [Google Scholar]
  3. Berridge M. J. Inositol trisphosphate and calcium signalling. Nature. 1993 Jan 28;361(6410):315–325. doi: 10.1038/361315a0. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Bokkala S., Joseph S. K. Angiotensin II-induced down-regulation of inositol trisphosphate receptors in WB rat liver epithelial cells. Evidence for involvement of the proteasome pathway. J Biol Chem. 1997 May 9;272(19):12454–12461. doi: 10.1074/jbc.272.19.12454. [DOI] [PubMed] [Google Scholar]
  6. Bonifacino J. S., Weissman A. M. Ubiquitin and the control of protein fate in the secretory and endocytic pathways. Annu Rev Cell Dev Biol. 1998;14:19–57. doi: 10.1146/annurev.cellbio.14.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brown K., Gerstberger S., Carlson L., Franzoso G., Siebenlist U. Control of I kappa B-alpha proteolysis by site-specific, signal-induced phosphorylation. Science. 1995 Mar 10;267(5203):1485–1488. doi: 10.1126/science.7878466. [DOI] [PubMed] [Google Scholar]
  8. Chen P., Johnson P., Sommer T., Jentsch S., Hochstrasser M. Multiple ubiquitin-conjugating enzymes participate in the in vivo degradation of the yeast MAT alpha 2 repressor. Cell. 1993 Jul 30;74(2):357–369. doi: 10.1016/0092-8674(93)90426-q. [DOI] [PubMed] [Google Scholar]
  9. Ciechanover A. The ubiquitin-proteasome pathway: on protein death and cell life. EMBO J. 1998 Dec 15;17(24):7151–7160. doi: 10.1093/emboj/17.24.7151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Clapham D. E. Calcium signaling. Cell. 1995 Jan 27;80(2):259–268. doi: 10.1016/0092-8674(95)90408-5. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Gilon T., Chomsky O., Kulka R. G. Degradation signals for ubiquitin system proteolysis in Saccharomyces cerevisiae. EMBO J. 1998 May 15;17(10):2759–2766. doi: 10.1093/emboj/17.10.2759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hershko A., Ciechanover A. The ubiquitin system. Annu Rev Biochem. 1998;67:425–479. doi: 10.1146/annurev.biochem.67.1.425. [DOI] [PubMed] [Google Scholar]
  14. Hicke L. Gettin' down with ubiquitin: turning off cell-surface receptors, transporters and channels. Trends Cell Biol. 1999 Mar;9(3):107–112. doi: 10.1016/s0962-8924(98)01491-3. [DOI] [PubMed] [Google Scholar]
  15. Hochstrasser M. Ubiquitin, proteasomes, and the regulation of intracellular protein degradation. Curr Opin Cell Biol. 1995 Apr;7(2):215–223. doi: 10.1016/0955-0674(95)80031-x. [DOI] [PubMed] [Google Scholar]
  16. Hochstrasser M. Ubiquitin-dependent protein degradation. Annu Rev Genet. 1996;30:405–439. doi: 10.1146/annurev.genet.30.1.405. [DOI] [PubMed] [Google Scholar]
  17. Johnson P. R., Swanson R., Rakhilina L., Hochstrasser M. Degradation signal masking by heterodimerization of MATalpha2 and MATa1 blocks their mutual destruction by the ubiquitin-proteasome pathway. Cell. 1998 Jul 24;94(2):217–227. doi: 10.1016/s0092-8674(00)81421-x. [DOI] [PubMed] [Google Scholar]
  18. Joseph S. K., Lin C., Pierson S., Thomas A. P., Maranto A. R. Heteroligomers of type-I and type-III inositol trisphosphate receptors in WB rat liver epithelial cells. J Biol Chem. 1995 Oct 6;270(40):23310–23316. doi: 10.1074/jbc.270.40.23310. [DOI] [PubMed] [Google Scholar]
  19. Kopito R. R. ER quality control: the cytoplasmic connection. Cell. 1997 Feb 21;88(4):427–430. doi: 10.1016/s0092-8674(00)81881-4. [DOI] [PubMed] [Google Scholar]
  20. Laney J. D., Hochstrasser M. Substrate targeting in the ubiquitin system. Cell. 1999 May 14;97(4):427–430. doi: 10.1016/s0092-8674(00)80752-7. [DOI] [PubMed] [Google Scholar]
  21. Mellgren R. L. Specificities of cell permeant peptidyl inhibitors for the proteinase activities of mu-calpain and the 20 S proteasome. J Biol Chem. 1997 Nov 21;272(47):29899–29903. doi: 10.1074/jbc.272.47.29899. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. 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]
  25. Oberdorf J., Webster J. M., Zhu C. C., Luo S. G., Wojcikiewicz R. J. Down-regulation of types I, II and III inositol 1,4,5-trisphosphate receptors is mediated by the ubiquitin/proteasome pathway. Biochem J. 1999 Apr 15;339(Pt 2):453–461. [PMC free article] [PubMed] [Google Scholar]
  26. Patel S., Joseph S. K., Thomas A. P. Molecular properties of inositol 1,4,5-trisphosphate receptors. Cell Calcium. 1999 Mar;25(3):247–264. doi: 10.1054/ceca.1999.0021. [DOI] [PubMed] [Google Scholar]
  27. Sipma H., Deelman L., Smedt H. D., Missiaen L., Parys J. B., Vanlingen S., Henning R. H., Casteels R. Agonist-induced down-regulation of type 1 and type 3 inositol 1,4,5-trisphosphate receptors in A7r5 and DDT1 MF-2 smooth muscle cells. Cell Calcium. 1998 Jan;23(1):11–21. doi: 10.1016/s0143-4160(98)90070-7. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Varshavsky A. The ubiquitin system. Trends Biochem Sci. 1997 Oct;22(10):383–387. doi: 10.1016/s0968-0004(97)01122-5. [DOI] [PubMed] [Google Scholar]
  30. Wojcikiewicz R. J., Ernst S. A., Yule D. I. Secretagogues cause ubiquitination and down-regulation of inositol 1, 4,5-trisphosphate receptors in rat pancreatic acinar cells. Gastroenterology. 1999 May;116(5):1194–1201. doi: 10.1016/s0016-5085(99)70023-5. [DOI] [PubMed] [Google Scholar]
  31. Wojcikiewicz R. J., Furuichi T., Nakade S., Mikoshiba K., Nahorski S. R. Muscarinic receptor activation down-regulates the type I inositol 1,4,5-trisphosphate receptor by accelerating its degradation. J Biol Chem. 1994 Mar 18;269(11):7963–7969. [PubMed] [Google Scholar]
  32. Wojcikiewicz R. J., He Y. Type I, II and III inositol 1,4,5-trisphosphate receptor co-immunoprecipitation as evidence for the existence of heterotetrameric receptor complexes. Biochem Biophys Res Commun. 1995 Aug 4;213(1):334–341. doi: 10.1006/bbrc.1995.2134. [DOI] [PubMed] [Google Scholar]
  33. Wojcikiewicz R. J., Nahorski S. R. Chronic muscarinic stimulation of SH-SY5Y neuroblastoma cells suppresses inositol 1,4,5-trisphosphate action. Parallel inhibition of inositol 1,4,5-trisphosphate-induced Ca2+ mobilization and inositol 1,4,5-trisphosphate binding. J Biol Chem. 1991 Nov 25;266(33):22234–22241. [PubMed] [Google Scholar]
  34. Wojcikiewicz R. J., Nakade S., Mikoshiba K., Nahorski S. R. Inositol 1,4,5-trisphosphate receptor immunoreactivity in SH-SY5Y human neuroblastoma cells is reduced by chronic muscarinic receptor activation. J Neurochem. 1992 Jul;59(1):383–386. doi: 10.1111/j.1471-4159.1992.tb08916.x. [DOI] [PubMed] [Google Scholar]
  35. Wojcikiewicz R. J., Oberdorf J. A. Degradation of inositol 1,4,5-trisphosphate receptors during cell stimulation is a specific process mediated by cysteine protease activity. J Biol Chem. 1996 Jul 12;271(28):16652–16655. doi: 10.1074/jbc.271.28.16652. [DOI] [PubMed] [Google Scholar]
  36. Wojcikiewicz R. J. Type I, II, and III inositol 1,4,5-trisphosphate receptors are unequally susceptible to down-regulation and are expressed in markedly different proportions in different cell types. J Biol Chem. 1995 May 12;270(19):11678–11683. doi: 10.1074/jbc.270.19.11678. [DOI] [PubMed] [Google Scholar]
  37. Zhu C. C., Furuichi T., Mikoshiba K., Wojcikiewicz R. J. Inositol 1,4,5-trisphosphate receptor down-regulation is activated directly by inositol 1,4,5-trisphosphate binding. Studies with binding-defective mutant receptors. J Biol Chem. 1999 Feb 5;274(6):3476–3484. doi: 10.1074/jbc.274.6.3476. [DOI] [PubMed] [Google Scholar]

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