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• W2000199830 abstract "Activation of certain phosphoinositidase C-linked cell surface receptors is known to cause an acceleration of the proteolysis of inositol 1,4,5-trisphosphate (InsP3) receptors and, thus, lead to InsP3 receptor down-regulation. To gain insight into this process, we examined whether or not InsP3 receptor degradation is a direct consequence of InsP3 binding by analyzing the down-regulation of exogenous wild-type and binding-defective mutant InsP3receptors expressed in SH-SY5Y human neuroblastoma cells. Stimulation of these cells with carbachol showed that wild-type exogenous receptors could be down-regulated but that the binding-defective mutant exogenous receptors were not. Thus, InsP3 binding appears to mediate down-regulation. To validate this conclusion, a comprehensive analysis of the effects of the exogenous receptors was undertaken. This showed that exogenous receptors (i) are localized appropriately within the cell, (ii) enhance InsP3-induced Ca2+ release in permeabilized cells, presumably by increasing the number of InsP3-sensitive Ca2+ channels, (iii) have minimal effects on Ca2+ mobilization and InsP3formation in intact cells, (iv) form heteromers with endogenous receptors, and (v) do not alter the down-regulation of endogenous receptors. In total, these data show that the introduction of exogenous receptors into SH-SY5Y cells does not compromise intracellular signaling or the down-regulatory process. We can thus conclude that InsP3 binding directly activates InsP3 receptor degradation. Because InsP3 binding induces a conformational change in the InsP3 receptor, these data suggest that this change provides the signal for accelerated proteolysis. Activation of certain phosphoinositidase C-linked cell surface receptors is known to cause an acceleration of the proteolysis of inositol 1,4,5-trisphosphate (InsP3) receptors and, thus, lead to InsP3 receptor down-regulation. To gain insight into this process, we examined whether or not InsP3 receptor degradation is a direct consequence of InsP3 binding by analyzing the down-regulation of exogenous wild-type and binding-defective mutant InsP3receptors expressed in SH-SY5Y human neuroblastoma cells. Stimulation of these cells with carbachol showed that wild-type exogenous receptors could be down-regulated but that the binding-defective mutant exogenous receptors were not. Thus, InsP3 binding appears to mediate down-regulation. To validate this conclusion, a comprehensive analysis of the effects of the exogenous receptors was undertaken. This showed that exogenous receptors (i) are localized appropriately within the cell, (ii) enhance InsP3-induced Ca2+ release in permeabilized cells, presumably by increasing the number of InsP3-sensitive Ca2+ channels, (iii) have minimal effects on Ca2+ mobilization and InsP3formation in intact cells, (iv) form heteromers with endogenous receptors, and (v) do not alter the down-regulation of endogenous receptors. In total, these data show that the introduction of exogenous receptors into SH-SY5Y cells does not compromise intracellular signaling or the down-regulatory process. We can thus conclude that InsP3 binding directly activates InsP3 receptor degradation. Because InsP3 binding induces a conformational change in the InsP3 receptor, these data suggest that this change provides the signal for accelerated proteolysis. When certain types of G-protein-coupled cell surface receptors (for example, M3 muscarinic receptors) are occupied by their cognate agonists, phosphoinositidase C (PIC) 1The abbreviations used are: PIC, phosphoinositidase C; CCh, carbachol; HA, hemagglutinin; InsP3, inositol 1,4,5-trisphosphate; InsP3R, wild-type mouse type I InsP3 receptor; ΔInsP3R, InsP3 binding-defective mouse type I InsP3 receptor; InsP3RHA, HA epitope-tagged InsP3R; ΔInsP3RHA, HA epitope-tagged ΔInsP3R.1The abbreviations used are: PIC, phosphoinositidase C; CCh, carbachol; HA, hemagglutinin; InsP3, inositol 1,4,5-trisphosphate; InsP3R, wild-type mouse type I InsP3 receptor; ΔInsP3R, InsP3 binding-defective mouse type I InsP3 receptor; InsP3RHA, HA epitope-tagged InsP3R; ΔInsP3RHA, HA epitope-tagged ΔInsP3R. is activated, phosphatidylinositol 4,5-bisphosphate is hydrolyzed and inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol are formed (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6147) Google Scholar). InsP3 is a second messenger that elicits calcium signals within cells that mediate many physiological processes (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6147) Google Scholar, 2Clapham D.E. Cell. 1995; 80: 259-268Abstract Full Text PDF PubMed Scopus (2254) Google Scholar). The primary effect of InsP3 is to trigger calcium release from the endoplasmic reticulum, thus raising cytoplasmic free calcium concentration ([Ca2+]i) (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6147) Google Scholar, 2Clapham D.E. Cell. 1995; 80: 259-268Abstract Full Text PDF PubMed Scopus (2254) Google Scholar). This is achieved by interaction of InsP3 with InsP3 receptors, proteins that form tetrameric complexes in the endoplasmic reticulum membrane and that act as calcium channels (3Joseph S.K. Cell. Signal. 1996; 8: 1-7Crossref PubMed Scopus (120) Google Scholar, 4Yoshida Y. Imai S. Jpn. J. Pharmacol. 1997; 74: 125-137Crossref PubMed Scopus (52) Google Scholar).Three types of InsP3 receptor, namely, types I, II, and III, have been defined; they have similar sizes (2670–2749 amino acids) and the same basic structure (3Joseph S.K. Cell. Signal. 1996; 8: 1-7Crossref PubMed Scopus (120) Google Scholar, 4Yoshida Y. Imai S. Jpn. J. Pharmacol. 1997; 74: 125-137Crossref PubMed Scopus (52) Google Scholar, 5Furuichi T. Yoshikawa S. Miyawaki A. Wada K. Maeda N. Mikoshiba K. Nature. 1989; 342: 32-38Crossref PubMed Scopus (820) Google Scholar, 6Sudhof T.C. Newton C.L. Archer III, B.T. Ushkaryov Y.A. Mignery G.A. EMBO J. 1991; 10: 3199-3206Crossref PubMed Scopus (319) Google Scholar, 7Blondel O. Takeda J. Janssen H. Seino S. Bell G.I. J. Biol. Chem. 1993; 268: 11356-11363Abstract Full Text PDF PubMed Google Scholar). For the type I InsP3 receptor, which is the predominant type in neuronal cells (3Joseph S.K. Cell. Signal. 1996; 8: 1-7Crossref PubMed Scopus (120) Google Scholar, 4Yoshida Y. Imai S. Jpn. J. Pharmacol. 1997; 74: 125-137Crossref PubMed Scopus (52) Google Scholar, 5Furuichi T. Yoshikawa S. Miyawaki A. Wada K. Maeda N. Mikoshiba K. Nature. 1989; 342: 32-38Crossref PubMed Scopus (820) Google Scholar), three domains have been defined: an InsP3-binding domain within the N-terminal 650 amino acids, a transmembrane or channel-forming domain close to the C terminus, and an intervening coupling domain (3Joseph S.K. Cell. Signal. 1996; 8: 1-7Crossref PubMed Scopus (120) Google Scholar, 4Yoshida Y. Imai S. Jpn. J. Pharmacol. 1997; 74: 125-137Crossref PubMed Scopus (52) Google Scholar, 8Mignery G.A. Sudhof T.C. EMBO J. 1990; 9: 3893-3898Crossref PubMed Scopus (273) Google Scholar). Several lines of evidence indicate that a conformational change occurs upon InsP3binding and that this is responsible for channel opening (3Joseph S.K. Cell. Signal. 1996; 8: 1-7Crossref PubMed Scopus (120) Google Scholar, 4Yoshida Y. Imai S. Jpn. J. Pharmacol. 1997; 74: 125-137Crossref PubMed Scopus (52) Google Scholar, 8Mignery G.A. Sudhof T.C. EMBO J. 1990; 9: 3893-3898Crossref PubMed Scopus (273) Google Scholar).The waning of cellular responses during persistent activation of cell surface receptors is a well documented phenomenon (9Lohse M.J. Biochim. Biophys. Acta. 1993; 1179: 171-188Crossref PubMed Scopus (400) Google Scholar) and is evident for PIC-coupled receptors (10Wojcikiewicz R.J.H. Tobin A.B. Nahorski S.R. Trends Pharmacol. Sci. 1993; 14: 279-285Abstract Full Text PDF PubMed Scopus (108) Google Scholar, 11Fisher S.K. Eur. J. Pharmacol. 1995; 288: 231-250Crossref PubMed Scopus (81) Google Scholar). Such “desensitization” is mediated by several mechanisms, some of which occur acutely (within minutes), and some of which require long-term exposure to agonists (9Lohse M.J. Biochim. Biophys. Acta. 1993; 1179: 171-188Crossref PubMed Scopus (400) Google Scholar, 10Wojcikiewicz R.J.H. Tobin A.B. Nahorski S.R. Trends Pharmacol. Sci. 1993; 14: 279-285Abstract Full Text PDF PubMed Scopus (108) Google Scholar, 11Fisher S.K. Eur. J. Pharmacol. 1995; 288: 231-250Crossref PubMed Scopus (81) Google Scholar). One of the mechanisms by which cells adapt during long-term agonist exposure is by down-regulation of cell surface receptors, which is characterized by a decline in the cellular content of these proteins (9Lohse M.J. Biochim. Biophys. Acta. 1993; 1179: 171-188Crossref PubMed Scopus (400) Google Scholar, 10Wojcikiewicz R.J.H. Tobin A.B. Nahorski S.R. Trends Pharmacol. Sci. 1993; 14: 279-285Abstract Full Text PDF PubMed Scopus (108) Google Scholar, 11Fisher S.K. Eur. J. Pharmacol. 1995; 288: 231-250Crossref PubMed Scopus (81) Google Scholar). Remarkably, it has recently been found that InsP3receptors are also subject to down-regulation upon stimulation of PIC-linked cell surface receptors (12Wojcikiewicz R.J.H. Nahorski S.R. J. Biol. Chem. 1991; 266: 22234-22241Abstract Full Text PDF PubMed Google Scholar, 13Wojcikiewicz 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, 14Wojcikiewicz R.J.H. J. Biol. Chem. 1995; 270: 11678-11683Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar, 15Bokkala S. Joseph S.K. J. Biol. Chem. 1997; 272: 12454-12461Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 16Sipma H. Deelman L. Smedt H.D. Missiaen L. Parys J.B. Vanlingen S. Henning R.H. Casteels R. Cell Calcium. 1998; 23: 11-21Crossref PubMed Scopus (45) Google Scholar), providing a novel locus of adaptation. It has also been shown that InsP3 receptor down-regulation can be induced by receptor-independent activation of PIC (17Honda Z. Takano T. Hirose N. Suzuki T. Muto A. Kume S. Mikoshiba K. Itoh K. Shimizu T. J. Biol. Chem. 1995; 270: 4840-4844Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 18Lobaugh L.A. Eisfelder B. Gibson K. Johnson G.L. Putney Jr., J.W. Mol. Pharmacol. 1996; 50: 493-500PubMed Google Scholar, 19Quick M.W. Lester H.A. Davidson N. Simon M.I. Aragay A.M. J. Biol. Chem. 1996; 271: 32021-32027Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). This phenomenon is seen with types I, II, and III InsP3 receptors in a range of cell types (12Wojcikiewicz R.J.H. Nahorski S.R. J. Biol. Chem. 1991; 266: 22234-22241Abstract Full Text PDF PubMed Google Scholar, 13Wojcikiewicz 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, 14Wojcikiewicz R.J.H. J. Biol. Chem. 1995; 270: 11678-11683Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar, 15Bokkala S. Joseph S.K. J. Biol. Chem. 1997; 272: 12454-12461Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 16Sipma H. Deelman L. Smedt H.D. Missiaen L. Parys J.B. Vanlingen S. Henning R.H. Casteels R. Cell Calcium. 1998; 23: 11-21Crossref PubMed Scopus (45) Google Scholar). For example, stimulation of M3 muscarinic receptors in SH-SY5Y human neuroblastoma cells with carbachol (CCh), a metabolically stable analogue of acetylcholine, reduces type I InsP3 receptor immunoreactivity by ∼90%, with half maximal effect at 0.5–1 h (13Wojcikiewicz 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,20Wojcikiewicz R.J.H. Oberdorf J.A. J. Biol. Chem. 1996; 271: 16652-16655Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). This reduction in InsP3 receptor content is a specific process, as the other proteins are not simultaneously down-regulated (14Wojcikiewicz R.J.H. J. Biol. Chem. 1995; 270: 11678-11683Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar, 15Bokkala S. Joseph S.K. J. Biol. Chem. 1997; 272: 12454-12461Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 16Sipma H. Deelman L. Smedt H.D. Missiaen L. Parys J.B. Vanlingen S. Henning R.H. Casteels R. Cell Calcium. 1998; 23: 11-21Crossref PubMed Scopus (45) Google Scholar, 20Wojcikiewicz R.J.H. Oberdorf J.A. J. Biol. Chem. 1996; 271: 16652-16655Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Moreover, the down-regulation is not related to changes in mRNA levels, but rather, it results from a profound acceleration of InsP3 receptor degradation (13Wojcikiewicz 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 responsible proteolytic mechanism has yet to be defined but has been proposed to involve either calpain (20Wojcikiewicz R.J.H. Oberdorf J.A. J. Biol. Chem. 1996; 271: 16652-16655Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) or the ubiquitin/proteasome pathway (15Bokkala S. Joseph S.K. J. Biol. Chem. 1997; 272: 12454-12461Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar).Previous studies have shown that InsP3 receptor down-regulation correlates with persistent increases in InsP3 concentration and is not mediated by a diacylglycerol-dependent pathway (12Wojcikiewicz R.J.H. Nahorski S.R. J. Biol. Chem. 1991; 266: 22234-22241Abstract Full Text PDF PubMed Google Scholar, 14Wojcikiewicz R.J.H. J. Biol. Chem. 1995; 270: 11678-11683Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar, 21Wojcikiewicz R.J.H. Nakade S. Mikoshiba K. Nahorski S.R. J. Neurochem. 1992; 59: 383-386Crossref PubMed Scopus (23) Google Scholar). As yet, however, it is not clear whether receptor proteolysis is initiated by InsP3 binding to its receptor, by Ca2+ signals generated following activation of InsP3 receptors or by a more indirect cell surface receptor-mediated mechanism (22Gutkind J.S. J. Biol. Chem. 1998; 273: 1839-1842Abstract Full Text Full Text PDF PubMed Scopus (689) Google Scholar). Thus, to define whether or not InsP3 binding is the signal that initiates InsP3 receptor degradation, we established SH-SY5Y cell lines stably expressing wild-type and mutant type I InsP3 receptors and focused on the characteristics of a binding-defective mutant. We found that this mutant InsP3receptor was resistant to down-regulation, whereas wild-type InsP3 receptor was appropriately down-regulated. Thus, our data indicate that InsP3 binding directly activates InsP3 receptor down-regulation.DISCUSSIONThe data presented show (i) that deletion of 37 amino acids within the ligand-binding domain of the type I InsP3 receptor renders it unable to bind InsP3 and blocks its down-regulation, (ii) that introduction of exogenous InsP3receptors into SH-SY5Y cells does not adversely affect signaling via PIC-linked receptors, and (iii) that exogenous receptors are appropriately located within the cell and form heteromers and functional Ca2+ channels with endogenous receptors. Thus, we conclude that InsP3 binding directly activates InsP3 receptor degradation.Importantly, our studies on the effects of exogenous InsP3receptors on signaling in intact SH-SY5Y cells are the first in a mammalian cell type in which both the complement of endogenous InsP3 receptors is known (SH-SY5Y cells contain >99% type I receptor (14Wojcikiewicz R.J.H. J. Biol. Chem. 1995; 270: 11678-11683Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar)), and an appreciable overexpression of InsP3 receptor has been achieved. To date, the effects of exogenous InsP3 receptors on intracellular signaling have been examined in only three other mammalian cell types (33Miyawaki A. Furuichi T. Maeda N. Mikoshiba K. Neuron. 1990; 5: 11-18Abstract Full Text PDF PubMed Scopus (109) Google Scholar, 34Mackrill J.J. Wilcox R.A. Miyawaki A. Mikoshiba K. Nahorski S.R. Challiss R.A. Biochem. J. 1996; 318: 871-878Crossref PubMed Scopus (27) Google Scholar, 37Fischer G.A. Clementi E. Raichman M. Sudhof T. Ullrich A. Meldolesi J. J. Biol. Chem. 1994; 269: 19216-19224Abstract Full Text PDF PubMed Google Scholar,38Blondel O. Bell G.I. Moody M. Miller R.J. Gibbons S.J. J. Biol. Chem. 1994; 269: 27167-27170Abstract Full Text PDF PubMed Google Scholar), the most comprehensive of these analyses being performed on 3T3 fibroblasts (37Fischer G.A. Clementi E. Raichman M. Sudhof T. Ullrich A. Meldolesi J. J. Biol. Chem. 1994; 269: 19216-19224Abstract Full Text PDF PubMed Google Scholar). However, in that study, only very limited overexpression of exogenous type I receptor was achieved (15–30% above endogenous values) and possible interaction with endogenous type II and III receptors, which make up ∼90% of the total receptor complement in this cell type (39De Smedt H. Missiaen L. Parys J.B. Henning R.H. Sienaert I. Vanlingen S. Gijsens A. Himpens B. Casteels R. Biochem. J. 1997; 322: 575-583Crossref PubMed Scopus (116) Google Scholar), was not assessed (37Fischer G.A. Clementi E. Raichman M. Sudhof T. Ullrich A. Meldolesi J. J. Biol. Chem. 1994; 269: 19216-19224Abstract Full Text PDF PubMed Google Scholar). Interestingly, however, whereas wild-type receptors had no effect on Ca2+ signaling or InsP3 formation in intact cells, a deletion mutant lacking the N-terminal InsP3-binding domain, which is analogous to the ΔInsP3RHA mutant used in the present study, enhanced the sensitivity of Ca2+ stores to InsP3 in permeabilized cells and inhibited agonist-stimulated InsP3production and [Ca2+]i increases in intact cells (37Fischer G.A. Clementi E. Raichman M. Sudhof T. Ullrich A. Meldolesi J. J. Biol. Chem. 1994; 269: 19216-19224Abstract Full Text PDF PubMed Google Scholar). These results parallel our findings inSΔInsP3RHA and indicate that the effects of exogenous binding-defective mutants are complex. In the other study in which Ca2+ signaling was analyzed in intact cells (38Blondel O. Bell G.I. Moody M. Miller R.J. Gibbons S.J. J. Biol. Chem. 1994; 269: 27167-27170Abstract Full Text PDF PubMed Google Scholar), severalfold overexpression of type III receptor in βTC-3 insulin-producing cells doubled peak [Ca2+]i in intact cells, but other parameters were not monitored. Finally, inl-cell fibroblasts, a much higher (∼8-fold) overexpression of wild-type type I receptor was achieved, and this substantially enhanced Ca2+ store sensitivity to InsP3 in permeabilized cells; however, Ca2+signaling in intact cells was not examined (33Miyawaki A. Furuichi T. Maeda N. Mikoshiba K. Neuron. 1990; 5: 11-18Abstract Full Text PDF PubMed Scopus (109) Google Scholar, 34Mackrill J.J. Wilcox R.A. Miyawaki A. Mikoshiba K. Nahorski S.R. Challiss R.A. Biochem. J. 1996; 318: 871-878Crossref PubMed Scopus (27) Google Scholar). Thus, our findings are broadly in agreement with these studies, in that overexpression of either wild-type or InsP3 binding-defective mutant receptors enhances the sensitivity of Ca2+ stores to InsP3. Surprisingly, however, this enhancement was not translated into increases in [Ca2+ ]i in intact cells in response to cell surface receptor stimulation. Although in the previous study on 3T3 cells (37Fischer G.A. Clementi E. Raichman M. Sudhof T. Ullrich A. Meldolesi J. J. Biol. Chem. 1994; 269: 19216-19224Abstract Full Text PDF PubMed Google Scholar) this apparent paradox was attributed to a substantial decrease in InsP3 production in response to cell surface receptor activation, such an explanation can not be applied to the present study, in which InsP3 production in response to CCh was not consistently or substantially affected by the presence of exogenous receptors. Thus, other, perhaps more subtle, mechanisms may serve to control [Ca2+]i in the transfected SH-SY5Y cells. Indeed, given the spatial and temporal complexity of Ca2+ release in intact cells (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6147) Google Scholar, 2Clapham D.E. Cell. 1995; 80: 259-268Abstract Full Text PDF PubMed Scopus (2254) Google Scholar) and, in particular, the ability of mobilized Ca2+ to suppress further InsP3 receptor-mediated Ca2+ release (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6147) Google Scholar, 2Clapham D.E. Cell. 1995; 80: 259-268Abstract Full Text PDF PubMed Scopus (2254) Google Scholar, 40Bezprozvanny I. Watras J. Ehrlich B.E. Nature. 1991; 351: 751-754Crossref PubMed Scopus (1428) Google Scholar), it is perhaps naive to think that an enhancement of InsP3-induced Ca2+ release in permeabilized cells will be translated into changes in [Ca2+ ]iin intact cells.The surprising ability of ΔInsP3RHA to enhance the sensitivity of Ca2+ stores to InsP3 indicates that this mutant increases the number of InsP3-sensitive Ca2+ channels, presumably by forming heterotetramers with endogenous receptors. The existence of these complexes is supported directly by the observed co-immunoprecipitation of exogenous and endogenous receptors and is important to the interpretation of our down-regulation data; the presence of ΔInsP3RHA in channels that are mediating Ca2+ release from the endoplasmic reticulum and the fact that only endogenous receptors are down-regulated from these channels indicate that Ca2+ flux alone does not activate receptor degradation. This argues against the proposal that activation of Ca2+-dependent proteases in the vicinity of the InsP3 receptor complex might be the sole mediator of receptor degradation (20Wojcikiewicz R.J.H. Oberdorf J.A. J. Biol. Chem. 1996; 271: 16652-16655Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Rather, InsP3 binding appears to be the critical signal for degradation, because only this could account for the selective degradation of endogenous receptors in ΔInsP3RHA-containing channels.Given this conclusion, it is tempting to speculate upon what it is about InsP3 binding that activates receptor degradation. InsP3 binding induces a substantial but as yet undefined conformational change in the ligand-binding domain of the type I receptor (8Mignery G.A. Sudhof T.C. EMBO J. 1990; 9: 3893-3898Crossref PubMed Scopus (273) Google Scholar), which obviously will not occur in the binding-defective mutant. This appears to be the primary consequence of InsP3binding, and it can be envisaged that such a conformational change might expose regions of the receptor that either are cleavage sites for proteases (20Wojcikiewicz R.J.H. Oberdorf J.A. J. Biol. Chem. 1996; 271: 16652-16655Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) or are sites that facilitate ubiquitin conjugation (15Bokkala S. Joseph S.K. J. Biol. Chem. 1997; 272: 12454-12461Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Intriguingly, preliminary analysis of CCh-stimulated ubiquitination in the transfected cells suggest that the latter possibility may be correct, as endogenous receptors and InsP3RHA are ubiquitinated, but ΔInsP3RHA is not.2Finally, it is important to consider the effects of the HA tag on InsP3 receptor function and down-regulation. First, the HA tag did not appear to impair InsP3 receptor function, as expression of similar levels of InsP3R and InsP3RHA had similar effects on Ca2+mobilization in permeabilized and intact cells. However, given our inability to stably express ΔInsP3R in SH-SY5Y cells, 4Although ΔInsP3R could not be expressed stably in SH-SY5Y cells, stable expression of this mutant was possible in HEK293 cells (Fig. 3), and an analogous mutant can be expressed in 3T3 cells (37Fischer G.A. Clementi E. Raichman M. Sudhof T. Ullrich A. Meldolesi J. J. Biol. Chem. 1994; 269: 19216-19224Abstract Full Text PDF PubMed Google Scholar). Thus, some cell types can tolerate ΔInsP3R. Differential tolerance may relate to differential InsP3 receptor expression; SH-SY5Y cells express almost exclusively type I receptor, whereas HEK293 and 3T3 cells express 3 and 12% type I receptor, respectively (14Wojcikiewicz R.J.H. J. Biol. Chem. 1995; 270: 11678-11683Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar, 39De Smedt H. Missiaen L. Parys J.B. Henning R.H. Sienaert I. Vanlingen S. Gijsens A. Himpens B. Casteels R. Biochem. J. 1997; 322: 575-583Crossref PubMed Scopus (116) Google Scholar). it is possible that the tag has a subtle effect that is not detected in these assays. Perhaps ΔInsP3R is too disruptive to be stably expressed in SH-SY5Y cells, and addition of the HA tag to this mutant compensates for its disruptive effect. This speculation has a basis in studies showing that interference with the InsP3 receptor C-terminal modifies receptor function (41Nakade S. Maeda N. Mikoshiba K. Biochem. J. 1991; 277: 125-131Crossref PubMed Scopus (82) Google Scholar, 42Miyazaki S. Yuzaki M. Nakada K. Shirakawa H. Nakanishi S. Nakade S. Mikoshiba K. Science. 1992; 257: 251-255Crossref PubMed Scopus (443) Google Scholar). It is also intriguing that the HA tag retards receptor down-regulation, indicating that interference with the C terminus inhibits the down-regulatory process. Thus, the C terminus may play a role in the events that lead to receptor degradation.In summary, our data show that the InsP3 receptor degradation that occurs in response to stimulation of PIC-linked cell surface receptors is activated directly by the interaction of InsP3 with its receptor. It will be intriguing to define how InsP3 binding stimulates receptor proteolysis. When certain types of G-protein-coupled cell surface receptors (for example, M3 muscarinic receptors) are occupied by their cognate agonists, phosphoinositidase C (PIC) 1The abbreviations used are: PIC, phosphoinositidase C; CCh, carbachol; HA, hemagglutinin; InsP3, inositol 1,4,5-trisphosphate; InsP3R, wild-type mouse type I InsP3 receptor; ΔInsP3R, InsP3 binding-defective mouse type I InsP3 receptor; InsP3RHA, HA epitope-tagged InsP3R; ΔInsP3RHA, HA epitope-tagged ΔInsP3R.1The abbreviations used are: PIC, phosphoinositidase C; CCh, carbachol; HA, hemagglutinin; InsP3, inositol 1,4,5-trisphosphate; InsP3R, wild-type mouse type I InsP3 receptor; ΔInsP3R, InsP3 binding-defective mouse type I InsP3 receptor; InsP3RHA, HA epitope-tagged InsP3R; ΔInsP3RHA, HA epitope-tagged ΔInsP3R. is activated, phosphatidylinositol 4,5-bisphosphate is hydrolyzed and inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol are formed (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6147) Google Scholar). InsP3 is a second messenger that elicits calcium signals within cells that mediate many physiological processes (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6147) Google Scholar, 2Clapham D.E. Cell. 1995; 80: 259-268Abstract Full Text PDF PubMed Scopus (2254) Google Scholar). The primary effect of InsP3 is to trigger calcium release from the endoplasmic reticulum, thus raising cytoplasmic free calcium concentration ([Ca2+]i) (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6147) Google Scholar, 2Clapham D.E. Cell. 1995; 80: 259-268Abstract Full Text PDF PubMed Scopus (2254) Google Scholar). This is achieved by interaction of InsP3 with InsP3 receptors, proteins that form tetrameric complexes in the endoplasmic reticulum membrane and that act as calcium channels (3Joseph S.K. Cell. Signal. 1996; 8: 1-7Crossref PubMed Scopus (120) Google Scholar, 4Yoshida Y. Imai S. Jpn. J. Pharmacol. 1997; 74: 125-137Crossref PubMed Scopus (52) Google Scholar). Three types of InsP3 receptor, namely, types I, II, and III, have been defined; they have similar sizes (2670–2749 amino acids) and the same basic structure (3Joseph S.K. Cell. Signal. 1996; 8: 1-7Crossref PubMed Scopus (120) Google Scholar, 4Yoshida Y. Imai S. Jpn. J. Pharmacol. 1997; 74: 125-137Crossref PubMed Scopus (52) Google Scholar, 5Furuichi T. Yoshikawa S. Miyawaki A. Wada K. Maeda N. Mikoshiba K. Nature. 1989; 342: 32-38Crossref PubMed Scopus (820) Google Scholar, 6Sudhof T.C. Newton C.L. Archer III, B.T. Ushkaryov Y.A. Mignery G.A. EMBO J. 1991; 10: 3199-3206Crossref PubMed Scopus (319) Google Scholar, 7Blondel O. Takeda J. Janssen H. Seino S. Bell G.I. J. Biol. Chem. 1993; 268: 11356-11363Abstract Full Text PDF PubMed Google Scholar). For the type I InsP3 receptor, which is the predominant type in neuronal cells (3Joseph S.K. Cell. 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