Mechanism of Proteasomal Degradation of Inositol Trisphosphate Receptors in CHO-K1 Cells

Abstract

myo-Inositol 1,4,5-trisphosphate receptor (IP3R) degradation occurs in response to carbachol (Cch) stimulation of CHO-K1 cells. The response was mediated by endogenous muscarinic receptors and was blocked by atropine or proteasomal inhibitors. We have used these cells to identify the sites of ubiquitination on IP3Rs and study the role of Ca2+ and substrate recognition properties of the degradation system using exogenously expressed IP3R constructs. Employing caspase-3 for IP3R cleavage, we show that Cch promotes polyubiquitination in the N-terminal domain and monoubiquitination in the C-terminal domain. The addition of extracellular Ca2+ to Ca2+-depleted Chinese hamster ovary (CHO) cells initiates IP3R degradation provided Cch is present. This effect is inhibited by thapsigargin. The data suggest that both a sustained elevation of IP3 and a minimal content of Ca2+ in the endoplasmic reticulum lumen is required to initiate IP3R degradation. Transient transfection of IP3R constructs into CHO cells indicated the selective degradation of only the SI(+) splice variant of the type I IP3R. This was also the splice form present endogenously in these cells. A pore-defective, nonfunctional SI(+) IP3R mutant (D2550A) was also degraded in Cch-stimulated cells. The Cch-mediated response in CHO cells provides a convenient model system to further analyze the Ca2+ dependence and structural requirements of the IP3R proteasomal degradation pathway. myo-Inositol 1,4,5-trisphosphate receptor (IP3R) degradation occurs in response to carbachol (Cch) stimulation of CHO-K1 cells. The response was mediated by endogenous muscarinic receptors and was blocked by atropine or proteasomal inhibitors. We have used these cells to identify the sites of ubiquitination on IP3Rs and study the role of Ca2+ and substrate recognition properties of the degradation system using exogenously expressed IP3R constructs. Employing caspase-3 for IP3R cleavage, we show that Cch promotes polyubiquitination in the N-terminal domain and monoubiquitination in the C-terminal domain. The addition of extracellular Ca2+ to Ca2+-depleted Chinese hamster ovary (CHO) cells initiates IP3R degradation provided Cch is present. This effect is inhibited by thapsigargin. The data suggest that both a sustained elevation of IP3 and a minimal content of Ca2+ in the endoplasmic reticulum lumen is required to initiate IP3R degradation. Transient transfection of IP3R constructs into CHO cells indicated the selective degradation of only the SI(+) splice variant of the type I IP3R. This was also the splice form present endogenously in these cells. A pore-defective, nonfunctional SI(+) IP3R mutant (D2550A) was also degraded in Cch-stimulated cells. The Cch-mediated response in CHO cells provides a convenient model system to further analyze the Ca2+ dependence and structural requirements of the IP3R proteasomal degradation pathway. The activation of inositol 1,4,5-trisphosphate receptors (IP3Rs) 2The abbreviations used are: IP3, myo-inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; Cch, carbachol; Ab, antibody; NEM, N-ethylmaleimide; ER, endoplasmic reticulum; GnRH, gonadotrophic hormone; ALLM, N-acetyl-l-leucinyl-l-leucinyl-l-methioninal; ALLN, N-acetyl-l-leucinyl-l-leucinyl-l-norleucinal; ZL3VS, peptide vinyl sulfone carboxybenzyl-leucyl-leucylleucine vinyl sulfone; PLC, phospholipase C; CHO, Chinese hamster ovary; Ub, ubiquitin; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl)ester. 2The abbreviations used are: IP3, myo-inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; Cch, carbachol; Ab, antibody; NEM, N-ethylmaleimide; ER, endoplasmic reticulum; GnRH, gonadotrophic hormone; ALLM, N-acetyl-l-leucinyl-l-leucinyl-l-methioninal; ALLN, N-acetyl-l-leucinyl-l-leucinyl-l-norleucinal; ZL3VS, peptide vinyl sulfone carboxybenzyl-leucyl-leucylleucine vinyl sulfone; PLC, phospholipase C; CHO, Chinese hamster ovary; Ub, ubiquitin; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl)ester. by IP3 initiates Ca2+ mobilization from the ER and triggers the Ca2+ signal that underlies alterations in cell function elicited by a diverse array of cell surface stimuli (1Berridge M.J. Bootman M.D. Roderick H.L. Nat. Rev. Mol. Cell. Biol. 2003; 4: 517-529Crossref PubMed Scopus (4151) Google Scholar, 2Patel S. Joseph S.K. Thomas A.P. Cell Calcium. 1999; 25: 247-264Crossref PubMed Scopus (370) Google Scholar). A commonly observed characteristic of cells is that they adapt their responses when chronically stimulated. In the case of cell surface receptors, this is usually the result of phosphorylation and/or internalization. Wojcikiewicz et al. (3Wojcikiewicz R.J.H. Nakade S. Mikoshiba K. Nahorski S.R. J. Neurochem. 1992; 59: 383-386Crossref PubMed Scopus (23) Google Scholar, 4Wojcikiewicz R.J.H. Furuichi T. Nakade S. Mikoshiba K. Nahorski S.R. J. Biol. Chem. 1994; 269: 7963-7969Abstract Full Text PDF PubMed Google Scholar) were the first to note that chronic stimulation of cultured SHSY-5Y neuroblastoma cells with carbachol for 6 h causes 90% loss of type I IP3R protein by a mechanism involving the marked acceleration of IP3R degradation. Subsequently, similar effects on IP3R degradation have been described in many different experimental systems with many different agonists (5Oberdorf J. Webster J. Zhu C.Z. Luo S.G. Wojcikiewicz R.J.H. Biochem. J. 1999; 339: 453-461Crossref PubMed Scopus (71) Google Scholar, 6Sharma K. Wang L. Zhu Y. Bokkala S. Jospeh S.K. J. Biol. Chem. 1997; 272: 14617-14623Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 7Sipma H. Deelman L. DeSmedt H. Missiaen L. Parys J. Vanlingen S. Henning R.H. Casteels R. Cell Calcium. 1998; 23: 11-21Crossref PubMed Scopus (45) Google Scholar, 8Bokkala S. Joseph S.K. J. Biol. Chem. 1997; 272: 12454-12461Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 9Parrington J. Brind S. De Smedt H. Gangeswaran R. Lai F.A. Wojcikiewicz R. Carroll J. Dev. Biol. 1998; 203: 451-461Crossref PubMed Scopus (99) Google Scholar, 10Brind S. Swann K. Carroll J. Dev. Biol. 2000; 223: 251-265Crossref PubMed Scopus (108) Google Scholar, 11Jellerette T. He C.H. Wu H. Parys J.B. Fissore R.A. Dev. Biol. 2000; 223: 238-250Crossref PubMed Scopus (147) Google Scholar, 12Lee B. Gai W. Laychock S.G. Endocrinology. 2001; 142: 1744-1751Crossref PubMed Scopus (23) Google Scholar, 13Kume S. Saneyoshi T. Mikoshiba K. Dev. Growth Differ. 2000; 42: 327-335Crossref PubMed Google Scholar, 14Tovey S.C. De Smet P. Lipp P. Thomas D. Young K.W. Missiaen L. De Smedt H. Parys J.B. Berridge M.J. Thuring J. Holmes A. Bootman M.D. J. Cell Sci. 2002; 114: 3979-3989Crossref Google Scholar, 15Willars G.B. Royall J.E. Nahorski S.R. El Gehani F. Everest H. McArdle C.A. J. Biol. Chem. 2001; 276: 3123-3129Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 16Wojcikiewicz R.J. Ernst S.A. Yule D.I. Gastroenterology. 1999; 116: 1194-1201Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Down-regulation of IP3R protein is associated with a decrease in the ability of IP3 to mobilize Ca2+ (4Wojcikiewicz R.J.H. Furuichi T. Nakade S. Mikoshiba K. Nahorski S.R. J. Biol. Chem. 1994; 269: 7963-7969Abstract Full Text PDF PubMed Google Scholar, 8Bokkala S. Joseph S.K. J. Biol. Chem. 1997; 272: 12454-12461Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). More has shown been recently it that the frequency of elementary Ca2+ puffs that trigger propagating Ca2+ waves in cells is decreased by chronic agonist stimulation (14Tovey S.C. De Smet P. Lipp P. Thomas D. Young K.W. Missiaen L. De Smedt H. Parys J.B. Berridge M.J. Thuring J. Holmes A. Bootman M.D. J. Cell Sci. 2002; 114: 3979-3989Crossref Google Scholar). The physiological significance of IP3R down-regulation is unknown. However, down-regulation of receptors would be expected to inhibit the global Ca2+ signal elicited by all Ca2+ mobilizing agonists this is the only of the signal system that is in would and have for A is the response of cells to receptor agonists C.A. J. L. J. 2002; PubMed Scopus Google Scholar). receptors a C-terminal and these receptors and only However, sustained activation of receptors to of the of to the cell surface with receptor agonists a and degradation of and this is associated with a marked in the Ca2+ signal G.B. Royall J.E. Nahorski S.R. El Gehani F. Everest H. McArdle C.A. J. Biol. Chem. 2001; 276: 3123-3129Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, R.J. Webster K. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The mechanism of IP3R degradation has been have shown that chronic elevation of IP3 and IP3 to the receptor required to IP3R degradation Furuichi T. Mikoshiba K. Wojcikiewicz R.J.H. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Wojcikiewicz R.J.H. Biochem. J. 2000; PubMed Scopus (32) Google Scholar). has been that the sustained elevation of IP3 causes the IP3R to a that sites that The the IP3 The of the IP3R in ubiquitination have been of cells with the has been to inhibit IP3R that Ca2+ also a role in IP3R degradation (4Wojcikiewicz R.J.H. Furuichi T. Nakade S. Mikoshiba K. Nahorski S.R. J. Biol. Chem. 1994; 269: 7963-7969Abstract Full Text PDF PubMed Google Scholar). In the present study we have IP3R degradation in CHO-K1 cells with This system has been used to the of IP3Rs that and to further the role of Ca2+ in IP3R degradation. The ability to CHO-K1 cells with IP3R constructs has of the substrate recognition properties of the IP3R degradation ALLN, and were from was from J. was a of for the of was from caspase-3 was from the a protein using described R. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The caspase-3 was by S. type I was to present the of the type I IP3R and has been K. De P. PubMed Scopus Google Scholar, S. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). N-terminal was to of the type I IP3R This was used using the peptide a The type IP3R was from to was from was from the Cell of the of type I IP3R splice variant in was the of Thomas of J. Biol. Chem. Full Text PDF PubMed Google Scholar). The the type I IP3R SI(+) variant was the of J. S. J. 1998; Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The mutant in D. Joseph S.K. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) was to the SI(+) IP3R using The constructs were using described D. Joseph S.K. J. 2000; PubMed Scopus Google Scholar). Cell and cells were from and from The cells were in minimal and The cells were of and with carbachol for the indicated the of the the was and the were in The cells were into a and was by for of protein was on and the were to of the was for in a and of Ca2+ in CHO cells on were by described Thomas A.P. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the cells were with by with in in the of for The were with and in with cells were into a with of with or Ca2+ and the of with a were of and with of for cell was from the using the described Thomas A.P. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of IP3R ubiquitination CHO-K1 cells were for h with or proteasomal and for h with The cells were for with and in with The was to The was with The were on to and with a of IP3R that we a by that was with the type I IP3R into of using a transfection h the were for h with a of proteasomal of ZL3VS, and The were with described in and with in of the was with caspase-3 in of a and of this was with and was used the The were from the by of with and The was in and with to the The and the were on and with IP3R to the of IP3R Down-regulation in CHO-K1 on IP3R degradation were in cells with S. Joseph S.K. J. Biol. Chem. 1997; 272: 12454-12461Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, S. Joseph S.K. 1999; 23: PubMed Scopus Google Scholar). cells have to with IP3R and cell their IP3R degradation response to We cells that were to transfection for IP3R degradation that carbachol (Cch) addition to CHO-K1 cells IP3R Cch effects were when the were for the type IP3R The effect was to Cch also Ca2+ in these cells IP3R degradation. further these effects we and and of that IP3R down-regulation was h and and 6 h of Cch stimulation This of response is with has been observed for IP3R down-regulation in systems S. Joseph S.K. J. Biol. Chem. 1997; 272: 12454-12461Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). the response for IP3R down-regulation using and The response for the carbachol effect was and with effects and was carbachol was the agonist with effects and only a down-regulation with the that the carbachol effects on down-regulation blocked by atropine suggest that IP3R down-regulation is mediated by endogenous muscarinic receptors in CHO-K1 in CHO-K1 cells in response to Ca2+ were in cells in extracellular Ca2+ and with Cch or Ca2+ in cells with of to Cch 6 both the of 2The effect of on IP3R degradation. CHO cells were with carbachol for 6 h in the or of and were for type I IP3R by The of used were atropine and of the IP3R when with the of and by and from carbachol The data the for to The role of different in IP3R degradation was using The of Cch-mediated IP3R degradation was observed with the proteasomal of the peptide ALLN, is proteasomal degradation of IP3Rs in cells S. Joseph S.K. J. Biol. Chem. 1997; 272: 12454-12461Abstract Full Text Full Text PDF PubMed Scopus (78) Google only a effect on Cch-mediated IP3R degradation. A effect of was also This is effect and to the of the was the of effects of ALLM, and would the of and the data would suggest that the system in Cch-mediated IP3R degradation is the proteasomal pathway. in first to that the proteasomal degradation of IP3Rs was by CHO cell were with Cch in the or of and the IP3R was for with The data show that IP3R only when the cells were with a of Cch and in the of the type I IP3R that sites for in the present study was to identify in the IP3R that of The we to was to the IP3R with a that would the IP3R A that this is is to IP3Rs J. Furuichi T. Mikoshiba K. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Neurochem. 2000; PubMed Scopus Google Scholar). to endogenous to IP3R were by the of the this we to the CHO cells with However, transfection with endogenous IP3Rs from CHO cells with Cch and of the it was to a the expected of IP3R, that endogenous receptors these A of endogenous receptors by has also been observed in cells R.J. Webster K. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). In this be to the transfection of was by the signal we CHO cells with both and IP3R these a in IP3R was observed when IP3R was from CHO cells with Cch and the of caspase-3 sites in the type I IP3R and the expected of and C-terminal for of these have a to the J. Furuichi T. Mikoshiba K. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Neurochem. 2000; PubMed Scopus Google Scholar). This would be expected to a C-terminal and a N-terminal IP3R were from CHO cells with and type I IP3R that were with Cch in the of proteasomal inhibitors. IP3R was from these with and with The were with to the of the by with a and a signal that from to The was with the and to the expected C-terminal caspase-3 it a we that the C-terminal of IP3Rs be The signal in the was with the N-terminal the C-terminal We that polyubiquitination is to the N-terminal of the IP3R. IP3R was with N-terminal Ab, a of was observed the expected and This that in sites in addition to The of the N-terminal is with would the polyubiquitination sites to this The of the signal would suggest in the of the and/or the of sites in the N-terminal domain. The of Ca2+ in IP3R the Ca2+ in CHO-K1 cells in response to a of Cch that triggers IP3R degradation. Cch addition elicited a Ca2+ signal that for a a IP3R a and Ca2+ The responses observed in cells that were with Cch for 6 h was would be IP3Rs were degraded by Cch further the role of we the cells for with or extracellular show that both blocked Cch-mediated in with on cell (4Wojcikiewicz R.J.H. Furuichi T. Nakade S. Mikoshiba K. Nahorski S.R. J. Biol. Chem. 1994; 269: 7963-7969Abstract Full Text PDF PubMed Google Scholar). The effect of the Ca2+ by the cell is shown in blocked Cch-mediated IP3R degradation when to Cch when the was different into the cells Cch addition was to the degradation of IP3R observed 6 This that a Ca2+ is required for IP3R degradation and that the that the IP3R to degradation a of Cch A similar was from in the Cch was by the addition of atropine data show that carbachol for h has the effect the to carbachol for The of the that and inhibit IP3R degradation is by the Ca2+ of phospholipase in CHO cells K.W. R.A. Nahorski S.R. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). it has been that chronic elevation of IP3 and IP3 to the IP3R for IP3R degradation Furuichi T. Mikoshiba K. Wojcikiewicz R.J.H. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Wojcikiewicz R.J.H. Biochem. J. 2000; PubMed Scopus (32) Google the effect of Ca2+ and mobilizing of and we the shown in Ca2+ was with and the cells were with This in a Ca2+ elevation that was and to This elevation of Ca2+ is to IP3R degradation of is to Ca2+ in the This be a marked elevation of Ca2+ when extracellular Ca2+ is to cells this we that the addition of Ca2+ also marked IP3R degradation and when Ca2+ was h of by the addition of The effect Ca2+ was or was for receptor stimulation Ca2+ addition in the of Cch IP3R degradation or Ca2+ The effect of Ca2+ addition was blocked by with proteasomal The addition of to Ca2+ Ca2+ into the ER lumen the elevation of Ca2+ in the or the activation of addition was to inhibit the IP3R degradation by Ca2+ that Ca2+ into the lumen of the ER is for IP3R degradation The of ER Ca2+ is further by the shown in CHO cells were for with Ca2+ to the of extracellular Ca2+ with these to IP3R degradation. of IP3R ability of Cch to the degradation of IP3Rs was in CHO cells have shown that type I IP3Rs were degraded IP3Rs in cells Furuichi T. Mikoshiba K. Wojcikiewicz R.J.H. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) or were degraded all in cells R.J. Webster K. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). In the study it was shown that the was a substrate for We that the and N-terminal constructs were degraded in CHO cells However, IP3R was also degraded This was observed a of by with Ab, would both endogenous and The to the degradation expected for endogenous receptors suggest that the receptor a effect in these The type I IP3R sites that of the IP3R constructs used in these from the and to the splice variant J. Biol. Chem. Full Text PDF PubMed Google Scholar). and that the variant be degraded when into CHO cells. be to the using we of the that only cells the splice the form is present in all T. H. Furuichi T. J. Mikoshiba K. Sci. S. A. PubMed Scopus Google Scholar). We to a the splice domain F. H. T. Furuichi T. Mikoshiba K. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) and that it the IP3R from the endogenous receptor in CHO cells and The was used to the splice in CHO cells and that only the SI(+) splice variant was degraded for this for the SI(+) form be that it the endogenously form of the IP3R in CHO cells. this we the IP3R in from CHO cells with and the of the N-terminal with the from and cell from SI(+) and cells a of and to the of both SI(+) and in this F. H. T. Furuichi T. Mikoshiba K. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). CHO cells only a to the SI(+) has been that Ca2+ a IP3R and the of Ca2+ in IP3R degradation (5Oberdorf J. Webster J. Zhu C.Z. Luo S.G. Wojcikiewicz R.J.H. Biochem. J. 1999; 339: 453-461Crossref PubMed Scopus (71) Google Scholar, R.J. Webster K. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). We have shown that the in the domain of the IP3R is a Ca2+ D. Joseph S.K. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, D. Joseph S.K. J. 2000; PubMed Scopus Google Scholar). The data in show that this mutant expressed in the SI(+) was degraded in response to Cch We that is for a in the Ca2+ for degradation is by Ca2+ and by endogenous In the present study we have of the mechanism of IP3R degradation sites of receptor Ca2+ dependence of IP3R and splice variant of the degradation of these were using CHO-K1 cells and Cch have shown that Cch for h type I IP3Rs in CHO cells or muscarinic that to phospholipase in CHO cells muscarinic receptors, to (4Wojcikiewicz R.J.H. Furuichi T. Nakade S. Mikoshiba K. Nahorski S.R. J. Biol. Chem. 1994; 269: 7963-7969Abstract Full Text PDF PubMed Google Scholar). In the present study we used the CHO-K1 to endogenous muscarinic receptors by Zhu J. PubMed Scopus Google Scholar). However, in these cells a mobilization of Ca2+ in response to Cch stimulation and IP3R degradation have that Cch addition to CHO-K1 in the of muscarinic receptors be by Zhu J. PubMed Scopus Google Scholar). This that of endogenous muscarinic receptors in the CHO-K1 cells of responses when by the of Cch in the present Cch-mediated Ca2+ and IP3R degradation were blocked by we have to identify the muscarinic CHO-K1 cells the receptor also Cch-mediated IP3R the response for Cch was different from the CHO-K1 cells A of IP3R degradation is that it is by ubiquitination S. Joseph S.K. J. Biol. Chem. 1997; 272: 12454-12461Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 16Wojcikiewicz R.J. Ernst S.A. Yule D.I. Gastroenterology. 1999; 116: 1194-1201Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, R.J. Webster K. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, Furuichi T. Mikoshiba K. Wojcikiewicz R.J.H. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). have shown that IP3R ubiquitination S. Wojcikiewicz R.J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google that also a role in the ER degradation of S. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, Mol. 2003; PubMed Scopus Google Scholar). In the present study we the IP3R that and that both and different of the The monoubiquitination is in the C-terminal of the This when in cells with polyubiquitination and IP3R degradation in Furuichi T. Mikoshiba K. Wojcikiewicz R.J.H. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google this that monoubiquitination is to IP3R degradation. However, the of a role in the of a of cell surface receptors K. Biochem. Sci. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) and a similar role in the of IP3Rs Joseph S.K. Biochem. J. 2003; PubMed Scopus Google Scholar). to be to N-terminal of the receptor the domain. is that the of to the the of the or with of IP3, the receptor a to degradation. We have the role of Ca2+ in IP3R degradation. with domain suggest that of IP3 is in CHO cells K.W. R.A. Nahorski S.R. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The experimental that sustained elevation of IP3 is a for IP3R degradation Furuichi T. Mikoshiba K. Wojcikiewicz R.J.H. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Wojcikiewicz R.J.H. Biochem. J. 2000; PubMed Scopus (32) Google Scholar). the of IP3R degradation when Ca2+ is be the of a of IP3 effect of Ca2+ on the degradation We show in the present study that the of Ca2+ CHO to in a initiates IP3R degradation provided the cells have been to this Ca2+ is expected to IP3 this be the for IP3R degradation the effect of Ca2+ addition was blocked by thapsigargin. these with the activation of (4Wojcikiewicz R.J.H. Furuichi T. Nakade S. Mikoshiba K. Nahorski S.R. J. Biol. Chem. 1994; 269: 7963-7969Abstract Full Text PDF PubMed Google Scholar, K.W. R.A. Nahorski S.R. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) or the elevation of Ca2+ would of Ca2+ into the lumen of the The of IP3R degradation when Ca2+ is be by first the with Ca2+ This that be a minimal of ER Ca2+ that is to degradation. data we that both a sustained IP3 elevation and Ca2+ for IP3R degradation. with has been shown to IP3R ubiquitination R.J. Webster K. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). This the that Ca2+ is required for the of IP3R this a for or Ca2+ of IP3Rs with or a IP3Rs to a IP3 Ca2+ promotes in the receptor K. A. Mikoshiba K. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google ubiquitination and Ca2+ different of IP3R degradation. A recently study has the role of Ca2+ in the ubiquitination and degradation of IP3Rs in cells Wojcikiewicz R.J. Biochem. J. PubMed Scopus Google Scholar). The that the of and ubiquitination of the Ca2+ signal mediated by GnRH, and it was that in Ca2+ with and to In a of CHO cells with was to the Ca2+ signal and the IP3R degradation The mutant expressed in cells was and in response to GnRH, with a the study in Wojcikiewicz R.J. Biochem. J. PubMed Scopus Google and the present in that both Ca2+ and IP3 to the IP3R required for IP3R degradation. Transient transfection in CHO cells with IP3R constructs have provided into the of the degradation We that only the SI(+) splice variant of the type I IP3R was degraded and that this was also the only form endogenously in CHO cells. The of the splice in the domain of the the and SI(+) of the type have marked in or function J. S. J. 1998; Full Text Full Text PDF PubMed Scopus (48) Google Scholar, J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google a study in the Ca2+ of the 59: PubMed Scopus Google Scholar). We have also observed that the of surface in the domain different for the SI(+) and S.K. S.K. S. Biochem. J. Scopus Google Scholar). the splice to be both and endogenous were the only the degradation of receptors used the into neuroblastoma cells Furuichi T. Mikoshiba K. Wojcikiewicz R.J.H. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) or cells R.J. Webster K. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). However, the splice of the endogenous receptor in these cells has been The for the SI(+) splice variant is to be to ubiquitination the variant was only a of IP3R ubiquitination was in the present and in ubiquitination sites splice constructs be by have that of a C-terminal has a effect on IP3R ubiquitination of the receptor was R.J. Webster K. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, Furuichi T. Mikoshiba K. Wojcikiewicz R.J.H. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the IP3R in the ER is to and also have structural requirements that by the of or splice The of a IP3R degradation system in CHO cells and the ability to these cells with mutant IP3Rs to further the structural of the IP3R proteasomal degradation pathway. We Mikoshiba for the of and for with Ca2+