Chapter 4 - Inositol 1,4,5-Trisphosphate Receptor Ubiquitination.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are large (∼300kDa) proteins that associate into tetrameric ion channels in the endoplasmic reticulum (ER) membrane. Activation and opening of the channel upon binding of IP3 and Ca(2+) allows the flow of Ca(2+) ions from stores within the ER lumen to the cytosol, thereby promoting a number of Ca(2+)-dependent cellular events, such as secretion, neurotransmitter release, and cell division. Intriguingly, it appears that the same conformational change that IP3Rs undergo during activation makes them a target for degradation by the ubiquitin-proteasome pathway and that this mode of processing allows the cell to tune its internal Ca(2+) response to extracellular signals. Here, we review recent studies showing that activated IP3Rs interact with an array of proteins that mediate their degradation, that IP3Rs are modified by a complex array of ubiquitin conjugates, that this ubiquitination and degradation functions to regulate IP3-mediated Ca(2+) responses in the cell, and that mutations to different proteins involved in IP3R degradation result in a set of similar diseases.

Calcium mobilization via type III inositol 1,4,5-trisphosphate receptors is not altered by PKA-mediated phosphorylation of serines 916, 934, and 1832.

Several studies have shown that PKA-mediated phosphorylation of IP3R1 at serines S1588 and S1755 enhances the receptor's ability to mobilize Ca2+. In contrast, much less is known about whether Ca2+ mobilization via IP3R2 and IP3R3 is regulated by PKA. We report here that IP3R2 is only very weakly phosphorylated in response to PKA activation and is probably not a physiological substrate for this kinase. IP3R3, however, is known to be phosphorylated by PKA at three sites (S916, S934, and S1832) and, thus, we examined how phosphorylation of these sites affects Ca2+ mobilization in DT40-3KO cells stably expressing either exogenous wild-type or mutant IP3R3s; an antibody raised against phospho-serine 934 of IP3R3 was used to demonstrate that the exogenous IP3R3s are strongly phosphorylated in response to PKA activation. Surprisingly, our data show that IP3R3-mediated Ca2+ mobilization is unaffected by phosphorylation of S916, S934, and S1832. In contrast, phosphorylation of exogenous IP3R1 (monitored with an antibody against phospho-serine 1755) enhances Ca2+ mobilization, indicating that DT40-3KO cells have the capacity to respond to phosphorylation of IP3Rs. Overall, these data suggest that modification of Ca2+ flux may not be the primary effect of IP3R3 phosphorylation by PKA.

2-Aminoethoxydiphenyl borate inhibits inositol 1,4,5-trisphosphate receptor function, ubiquitination and downregulation, but acts with variable characteristics in different cell types.

2-Aminoethoxydiphenyl borate (2-APB) is a putative, membrane-permeable inhibitor of inositol 1,4,5-trisphosphate (InsP(3)) receptors, but it is the case that little is known about its action at the InsP(3) receptor level. Thus, we examined the effects of 2-APB on InsP(3) receptor-mediated effects in a range of cell types expressing different complements of InsP(3) receptor types. In experiments with permeabilized cells we found that 2-APB could inhibit InsP(3)-induced release of stored Ca(2+), but also that it released Ca(2+), and that the prevalence of these two effects varied between different cell types and did not correlate with the expression of a particular receptor type. These effects of 2-APB reflected an interaction distal to the ligand binding site of InsP(3) receptors, since InsP(3) binding was unaffected by 2-APB. In intact cells, we found only inhibitory effects of 2-APB on Ca(2+) mobilization, and that variation between cell types in the characteristics of this inhibition appeared to be due to differential entry of 2-APB. 2-APB also inhibited InsP(3) receptor ubiquitination and proteasomal degradation, which again was cell type dependent. In total, these data reveal a remarkable degree of variation between cell types in the effects of 2-APB, showing that its usefulness as a specific and universal inhibitor of InsP(3) receptors is limited. However, the ability of 2-APB to inhibit InsP(3) receptor ubiquitination and degradation indicates that 2-APB may block InsP(3)-induced conformational changes in the receptor, resulting in perturbation of multiple regulatory events.

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

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.

Distinct localization and function of (1,4,5)IP(3) receptor subtypes and the (1,3,4,5)IP(4) receptor GAP1(IP4BP) in highly purified human platelet membranes.

Platelet activation is associated with an increase of cytosolic Ca(++) levels. The (1,4,5)IP(3) receptors [(1,4,5)IP(3)R] are known to mediate Ca(++) release from intracellular stores of many cell types. Currently there are at least 3 distinct subtypes of (1,4, 5)IP(3)R-type I, type II, and type III-with suggestions of distinct roles in Ca(++) elevation. Specific receptors for (1,3,4,5)IP(4) belonging to the GAP1 family have also been described though their involvement with Ca(++) regulation is controversial. In this study we report that platelets contain all 3 subtypes of (1,4,5)IP(3)R but in different amounts. Type I and type II receptors are predominant. In studies using highly purified platelet plasma (PM) and intracellular membranes (IM) we report a distinct localization of these receptors. The PM fractions were found to contain the type III (1,4,5)IP(3)R and GAP1(IP4BP) in contrast to IM, which contained type I (1,4,5)IP(3)R. The type II receptor exhibited a dual distribution. In studies examining the labeling of surface proteins with biotin in intact platelets only the type III (1,4,5)IP(3)R was significantly labeled. Immunogold studies of ultracryosections of human platelets showed significantly more labeling of the PM with the type III receptor antibodies than with type I receptor antibodies. Ca(++) flux studies were carried out with the PM to demonstrate in vitro function of inositol phosphate receptors. Ca(++) release activities were present with both (1,4,5)IP(3) and (1, 3,4,5)IP(4) (EC(50) = 1.3 and 0.8 micromol/L, respectively). Discrimination of the Ca(++)-releasing activities was demonstrated with cyclic adenosine monophosphate (cAMP)-dependent protein kinase (cAMP-PK) specifically inhibiting (1,4,5)IP(3) but not (1,3,4, 5)IP(4)-induced Ca(++) flux. In experiments with both PM and intact platelets, the (1,4,5)IP(3)Rs but not GAP1(IP4BP) were found to be substrates of cAMP-PK and cGMP-PK. Thus the Ca(++) flux property of (1,3,4,5)IP(4) is insensitive to cAMP-PK. These studies suggest distinct roles for the (1,4,5)IP(3)R subtypes in Ca(++) movements, with the type III receptor and GAP1(IP4BP) associated with cation entry in human platelets and the type I receptor involved with Ca(++) release from intracellular stores.

Ryanodine and inositol trisphosphate receptors are differentially distributed and expressed in rat parotid gland.

The present study examines the cellular distribution of the ryanodine receptor/channel (RyR) and inositol 1,4,5-trisphosphate receptor (InsP3R) subtypes in parotid acini. Using fluorescently labelled 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3-propionic acid glycyl-ryanodine (BODIPYtrade mark-ryanodine) and confocal microscopy, RyRs were localized primarily to the perinuclear region (basal pole) of the acinar cell. Ryanodine, Ruthenium Red, cAMP and cADP ribose (cADPR) competed with BODIPY-ryanodine, resulting in a reduction in the fluorescence signal. However, inositol 1,4, 5-trisphosphate [Ins(1,4,5)P3] did not alter the binding of BODIPY-ryanodine. Using receptor-subtype-specific antisera, InsP3Rs (types I, II and III) were located predominantly in the apical pole of the parotid cell. The presence of these three subtypes was confirmed using reverse transcriptase PCR with RNA-specific oligonucleotide probes. Binding studies using a parotid cell-membrane fraction identified and characterized RyRs and InsP3Rs in terms of binding affinity (Kd) and maximum binding capacity (Bmax) and confirmed that cADPR displaces ryanodine from its binding sites. Ruthenium Red and 8-Br-cADP-ribose blocked Ca2+ release in permeabilized acinar cells in response to cADPR and cAMP or forskolin, whereas Ins(1,4,5)P3-induced Ca2+ release was unaffected. The localization of the RyRs and InsP3Rs in discrete regions endow broad areas of the parotid cell with ligand-activated Ca2+ channels. The consequences of the dual activation of the RyRs and InsP3Rs by physiologically relevant stimuli such as noradrenaline (norepinephrine) are considered in relation to Ca2+ signalling in the parotid gland.

Secretagogues cause ubiquitination and down-regulation of inositol 1, 4,5-trisphosphate receptors in rat pancreatic acinar cells.

BACKGROUND & AIMS: The action of several exocrine pancreas secretagogues depends on the second messenger inositol 1,4, 5-trisphosphate (IP3), which, via endoplasmic reticulum-located IP3 receptors, mobilizes intracellular Ca2+ stores. Signaling pathways like this one are regulated at multiple loci. To determine whether IP3 receptors are one of these loci, we measured IP3 receptor concentration, distribution, and modification in secretagogue-stimulated rat pancreatic acinar cells. METHODS: Isolated rat pancreatic acinar cells were exposed to cholecystokinin and other secretagogues, or rats were injected intraperitoneally with cerulein. Then samples of cells or pancreata were probed for IP3 receptor content and distribution as well as for ubiquitin association with IP3 receptors. RESULTS: Secretagogues rapidly down-regulated acinar cell IP3 receptors both in vitro and in vivo. They also elicited receptor redistribution and caused receptors to become ubiquitinated, indicating that the ubiquitin/proteasome proteolytic pathway contributes to the down-regulation. Surprisingly, however, proteasome inhibitors did not block IP3 receptor down-regulation, and phospholipase Cbeta1 and protein kinase C also were down-regulated. Thus, secretagogues simultaneously activate an additional proteolytic pathway. CONCLUSIONS: Secretagogues rapidly down-regulate IP3 receptors and other proteins involved in intracellular signaling by a mechanism that involves, but is not limited to, the ubiquitin/proteasome pathway. Loss of these proteins may account for the disruption of Ca2+ mobilization that occurs in models of acute pancreatitis, and may contribute to cell adaptation under physiological conditions.

Down-regulation of types I, II and III inositol 1,4,5-trisphosphate receptors is mediated by the ubiquitin/proteasome pathway.

Activation of certain phosphoinositidase-C-linked cell-surface receptors is known to cause an acceleration of the proteolysis of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptors and, thus, lead to Ins(1,4,5)P3-receptor down-regulation. In the current study we have sought to determine whether the ubiquitin/proteasome pathway is involved in this adaptive response. The data presented show (i) that activation of phosphoinositidase-C-linked receptors causes Ins(1,4,5)P3-receptor ubiquitination in a range of cell types (AR4-2J cells, INS-1 cells and rat cerebellar granule cells), (ii) that the Ins(1,4,5)P3-receptor down-regulation induced by activation of these receptors is blocked by proteasome inhibitors, (iii) that all known Ins(1,4,5)P3 receptors (types I, II and III) are substrates for ubiquitination, (iv) that ubiquitination occurs while Ins(1,4,5)P3 receptors are membrane-bound, (v) that Ins(1,4, 5)P3-receptor ubiquitination and down-regulation are stimulated only by those agonists that elevate Ins(1,4,5)P3 concentration persistently, and (vi) that a portion of cellular Ins(1,4,5)P3 receptors (those that are not type-I-receptor-associated) can be resistant to ubiquitination and degradation. In total these data indicate that the ubiquitin/proteasome pathway mediates Ins(1,4, 5)P3-receptor down-regulation and suggest that ubiquitination is stimulated by the binding of Ins(1,4,5)P3 to its receptor.

Inositol 1,4,5-trisphosphate receptor down-regulation is activated directly by inositol 1,4,5-trisphosphate binding. Studies with binding-defective mutant receptors.

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 InsP3 receptors 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 InsP3 formation 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.

Calcium-dependent clustering of inositol 1,4,5-trisphosphate receptors.

Rat basophilic leukemia (RBL-2H3) cells predominantly express the type II receptor for inositol 1,4,5-trisphosphate (InsP3), which operates as an InsP3-gated calcium channel. In these cells, cross-linking the high-affinity immunoglobulin E receptor (FcepsilonR1) leads to activation of phospholipase C gamma isoforms via tyrosine kinase- and phosphatidylinositol 3-kinase-dependent pathways, release of InsP3-sensitive intracellular Ca2+ stores, and a sustained phase of Ca2+ influx. These events are accompanied by a redistribution of type II InsP3 receptors within the endoplasmic reticulum and nuclear envelope, from a diffuse pattern with a few small aggregates in resting cells to large isolated clusters after antigen stimulation. Redistribution of type II InsP3 receptors is also seen after treatment of RBL-2H3 cells with ionomycin or thapsigargin. InsP3 receptor clustering occurs within 5-10 min of stimulus and persists for up to 1 h in the presence of antigen. Receptor clustering is independent of endoplasmic reticulum vesiculation, which occurs only at ionomycin concentrations >1 microM, and maximal clustering responses are dependent on the presence of extracellular calcium. InsP3 receptor aggregation may be a characteristic cellular response to Ca2+-mobilizing ligands, because similar results are seen after activation of phospholipase C-linked G-protein-coupled receptors; cholecystokinin causes type II receptor redistribution in rat pancreatoma AR4-2J cells, and carbachol causes type III receptor redistribution in muscarinic receptor-expressing hamster lung fibroblast E36(M3R) cells. Stimulation of these three cell types leads to a reduction in InsP3 receptor levels only in AR4-2J cells, indicating that receptor clustering does not correlate with receptor down-regulation. The calcium-dependent aggregation of InsP3 receptors may contribute to the previously observed changes in affinity for InsP3 in the presence of elevated Ca2+ and/or may establish discrete regions within refilled stores with varying capacity to release Ca2+ when a subsequent stimulus results in production of InsP3.

Differences among type I, II, and III inositol-1,4,5-trisphosphate receptors in ligand-binding affinity influence the sensitivity of calcium stores to inositol-1,4,5-trisphosphate.

Type I, II, and III inositol-1,4,5-trisphosphate (InsP3) receptors are expressed selectively in different cell lines and tissues. We examined whether type I, II, and III InsP3 receptors differ in ligand-binding affinity and whether such differences influence the sensitivity of Ca2+ stores to InsP3. Initially, SH-SY5Y human neuroblastoma cells, AR4-2J rat pancreatoma cells, and RINm5F rat insulinoma cells were studied because these cells express predominantly (>85%) type I, II, and III receptors, respectively. Immunopurification of receptors from these cell lines and measurement of InsP3 binding revealed that the rank order of affinity for InsP3 was type I > type II > type III (binding sites were half-maximally saturated at 1.5, 2.5, and 22.4 nM InsP3, respectively). Examination of Ca2+ store mobilization in permeabilized cells showed that InsP3 was equipotent in SH-SY5Y and AR4-2J cells but was approximately 5-fold less potent in RINm5F cells. In contrast, Ca2+ uptake and InsP3-independent Ca2+ release were very similar in the three cell types. The binding affinity of InsP3 in permeabilized SH-SY5Y, AR4-2J, and RINm5F cells correlated well with its potency as a Ca2+-mobilizing agent and with binding affinity to immunopurified type I, II, and III receptors. Thus, InsP3 receptor binding affinity seems to influence the potency of InsP3 as a Ca2+-mobilizing agent. Finally, immunopurification of type I, II, and III receptors from rat tissues revealed that the affinity differences seen in receptors purified from cultured cells are paralleled in vivo. In combination, the data from cell lines and rat tissues reveal that type I, II, and III receptors bind InsP3 with Kd values of approximately 1, approximately 2, and approximately 40 nM, respectively, and that the selective expression of a particular receptor type will influence the sensitivity of cellular Ca2+ stores to InsP3.

Phosphorylation of inositol 1,4,5-trisphosphate receptors by cAMP-dependent protein kinase. Type I, II, and III receptors are differentially susceptible to phosphorylation and are phosphorylated in intact cells.

The ability of cAMP-dependent protein kinase (PKA) to phosphorylate type I, II, and III inositol 1,4,5-trisphosphate (InsP3) receptors was examined. The receptors either were immunopurified from cell lines and then phosphorylated with purified PKA or were phosphorylated in intact cells after activating intracellular cAMP formation. The former studies showed that the type I receptor was a good substrate for PKA (0.65 mol Pi incorporated/mol receptor), whereas type II and III receptors were phosphorylated relatively weakly. The latter studies showed that despite these differences, each of the receptors was phosphorylated in intact cells in response to forskolin or activation of neurohormone receptors. Detailed examination of SH-SY5Y neuroblastoma cells, which express >/=99% type I receptor, revealed that minor increases in cAMP concentration were sufficient to cause maximal phosphorylation. Thus, VIP and pituitary adenylyl cyclase activating peptide (acting through Gs-coupled pituitary adenylyl cyclase activating peptide-I receptors) were potent stimuli of type I receptor phosphorylation, and remarkably, even slight increases in cAMP concentration induced by carbachol (acting through Gq-coupled muscarinic receptors) or other Ca2+ mobilizing agents were sufficient to cause phosphorylation. Finally, PKA enhanced InsP3-induced Ca2+ mobilization in a range of permeabilized cell types, irrespective of whether the type I, II, or III receptor was predominant. In summary, these data show that all InsP3 receptors are phosphorylated by PKA, albeit with marked differences in stoichiometry. The ability of both Gs- and Gq-coupled cell surface receptors to effect InsP3 receptor phosphorylation by PKA suggests that this process is widespread in mammalian cells and provides multiple routes by which the cAMP signaling pathway can influence Ca2+ mobilization.

Polarized expression of Ca2+ channels in pancreatic and salivary gland cells. Correlation with initiation and propagation of [Ca2+]i waves.

In polarized epithelial cells [Ca2+]i waves are initiated in discrete regions and propagate through the cytosol. The structural basis for these compartmentalized and coordinated events are not well understood. In the present study we used a combination of [Ca2+]i imaging at high temporal resolution, recording of Ca2+-activated Cl- current, and immunolocalization by confocal microscopy to study the correlation between initiation and propagation of [Ca2+]i waves and localization of Ca2+ release channels in pancreatic acini and submandibular acinar and duct cells. In all cells Ca2+ waves are initiated in the luminal pole and propagate through the cell periphery to the basal pole. All three cell types express the three known inositol 1,4,5-trisphosphate receptors (IP3Rs). Expression of IP3Rs was confined to the area just underneath the luminal and lateral membranes, with no detectable receptors in the basal pole or other regions of the cells. In pancreatic acini and SMG ducts IP3R3 was also found in the nuclear envelope. Expression of ryanodine receptor was detected in submandibular salivary gland cells but not pancreatic acini. Accordingly, cyclic ADP ribose was very effective in mobilizing Ca2+ from internal stores of submandibular salivary gland but not pancreatic acinar cells. Measurement of [Ca2+]i and localization of IP3Rs in the same cells suggests that only a small part of IP3Rs participate in the initiation of the Ca2+ wave, whereas most receptors in the cell periphery probably facilitate the propagation of the Ca2+ wave. The combined results together with our previous studies on this subject lead us to conclude that the internal Ca2+ pool is highly compartmentalized and that compartmentalization is achieved in part by polarized expression of Ca2+ channels.

Evidence that zymogen granules are not a physiologically relevant calcium pool. Defining the distribution of inositol 1,4,5-trisphosphate receptors in pancreatic acinar cells.

A key event leading to exocytosis of pancreatic acinar cell zymogen granules is the inositol 1,4,5-trisphosphate (InsP3)-mediated release of Ca2+ from intracellular stores. Studies using digital imaging microscopy and laser-scanning confocal microscopy have indicated that the initial release of Ca2+ is localized to the apical region of the acinar cell, an area of the cell dominated by secretory granules. Moreover, a recent study has shown that InsP3 is capable of releasing Ca2+ from a preparation enriched in secretory granules (Gerasimenko, O., Gerasimenko, J., Belan, P., and Petersen, O. H., (1996) Cell 84, 473-480). In the present study, we have investigated the possibility that zymogen granules express InsP3 receptors and are thus Ca2+ release sites. Immunofluorescence staining, obtained with antisera specific to types I, II, or III InsP3 receptors and analyzed by confocal fluorescence microscopy revealed that all InsP3 receptor types were present in acinar cells. The type II receptor localized exclusively to an area close to or at the luminal plasma membrane. While types I and III InsP3 receptors displayed a similar luminal distribution, these receptors were also present at low levels in nuclei. The localization of InsP3 receptor was in marked contrast to the distribution of amylase, a zymogen granule content protein. In a zymogen granule fraction prepared in an identical manner to the aforementioned report demonstrating InsP3-induced Ca2+ release, immunoblotting demonstrated the presence of types I, II, and III InsP3 receptors. Ca2+ release from this preparation in response to InsP3, but not thapsigargin, could also be demonstrated. In contrast, when the zymogen granules were further purified on a Percoll gradient, InsP3 receptors were undetectable, and InsP3 failed to release Ca2+. Transmission electron microscopy performed on both preparations showed that the Percoll-purified granule preparation consisted of essentially pure zymogen granules, whereas the granules prepared without this step were enriched in granules but also contained significant contamination by mitochondria, endoplasmic reticulum, and nuclei. It is concluded that zymogen granules do not express InsP3 receptors and thus are not a site of Ca2+ release relevant to the secretory process in the pancreatic acinar cell.

Expression and regulation of types I and II inositol 1,4,5-trisphosphate receptors in rat cerebellar granule cell preparations.

Previous studies have shown that as rat cerebellar granule cell cultures differentiate in the presence of 25 mM KCl, they "up-regulate" their ability to form inositol phosphates and release Ca2+ from internal stores in response to the activation of phosphoinositidase C-linked muscarinic and metabotropic receptors. Here we show that they simultaneously up-regulate their ability to respond to inositol 1,4,5-trisphosphate (InsP3) by increasing InsP3 receptor (InsP3R) expression. In contrast, if granule cells are maintained at the more physiological KCl concentration of 5 mM, most cells undergo apoptosis, although a significant number survive. The surviving cells, however, express few InsP3Rs, suggesting that an influx of Ca2+ through voltage-dependent channels is required for InsP3R up-regulation. In addition, we have determined that these cultures express two genetically distinct InsP3R types, but that only one, the type I receptor, is expressed in granule cells. Type II receptors are also present but are found exclusively in astrocytes, which are a minor contaminant of granule cell cultures. This segregation of InsP3R types explains a previous observation, showing that the muscarinic agonist carbachol causes the reduction or "down-regulation" of type I but not type II InsP3Rs.

Degradation of inositol 1,4,5-trisphosphate receptors during cell stimulation is a specific process mediated by cysteine protease activity.

Inositol 1,4,5-trisphosphate (InsP3) receptors are down-regulated in response to chronic activation of certain cell surface receptors because their degradation is accelerated. Studies on the nature of the down-regulatory process and the protease(s) responsible for receptor degradation are described here. InsP3 receptor down-regulation was not accompanied by parallel changes in the concentrations of several other relevant proteins (endoplasmic reticulum Ca2+-ATPase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and protein kinases alpha and epsilon). Thus, the down-regulatory process selectively targets InsP3 receptors for degradation. Furthermore, down-regulation was unaffected by brefeldin A and NH4Cl, indicating that InsP3 receptor degradation occurs without removal of receptors from the endoplasmic reticulum and independently of functional lysosomes. Analysis of InsP3 receptor immunofluorescence confirmed that the receptors are not redistributed prior to or during down-regulation. Finally, of a range of protease inhibitors tested, only N-acetyl-Leu-Leu-norleucinal blocked down-regulation. Thus, cysteine protease activity accounts for InsP3 receptor degradation and analysis of proteolysis in permeabilized cells indicates that this activity is calpain. Thus, InsP3 receptor down-regulation appears to result from the highly selective calpain-mediated degradation of InsP3 receptors. Calpain activity may be stimulated by the high concentrations of Ca2+ that are thought to be found in the vicinity of activated InsP3 receptors.

Type I, II and III inositol 1,4,5-trisphosphate receptor co-immunoprecipitation as evidence for the existence of heterotetrameric receptor complexes.

The recently described co-expression of type I, II and III inositol 1,4,5-trisphosphate (InsP3) receptors in the same cell type has raised the issue of whether these proteins exist as homotetramers or heterotetramers. To address this question, InsP3 receptors were immunoprecipitated with specific antibodies and then probed for co-immunoprecipitating proteins. This revealed that type I, II and III InsP3 receptors co-immunoprecipitate and thus, exist in heteromeric complexes. This situation was maintained when the relative abundance of InsP3 receptors was altered radically during cell differentiation. Thus, heterotetrameric InsP3 receptors are likely to contribute towards signaling in cells expressing more than one receptor type.

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.

The type I inositol 1,4,5-trisphosphate (InsP3) receptor can be rapidly depleted from cells during stimulation of phosphoinositide hydrolysis because its degradation is accelerated (Wojcikiewicz, R. J. H., Furuichi, T., Nakade, S., Mikoshiba, K., and Nahorski, S. R. (1994) J. Biol. Chem. 269, 7963-7969). The present study examines the regulatory properties of type II and III InsP3 receptors. Initially, the relative abundance of InsP3 receptors was defined in a range of cell types by quantitative immunoblotting. These studies showed that the proportions in which type I, II, and III InsP3 receptors are expressed differs greatly and that some cells (for example, AR4-2J rat pancreatoma cells) express all three receptors. Analysis of the effects of cholecystokinin and bombesin on AR4-2J cells showed that each of the InsP3 receptors could be down-regulated during activation of phosphoinositide hydrolysis, but that depletion of the type II receptor was limited. Such a discrepancy was also seen in rat cerebellar granule cells and was found to result from the type II receptor being relatively resistant to degradation. In conclusion, type I, II, and III receptors can all be down-regulated, but with different characteristics. As the relative abundance of InsP3 receptors is extremely variable, the extent to which activation of the down-regulatory process alters intracellular signaling will vary depending on which InsP3 receptors are expressed.

Muscarinic receptor-mediated inositol 1,4,5-trisphosphate formation in SH-SY5Y neuroblastoma cells is regulated acutely by cytosolic Ca2+ and by rapid desensitization.

Stimulation of muscarinic receptors expressed in SH-SY5Y human neuroblastoma cells resulted in a complex profile of inositol 1,4,5-trisphosphate (InsP3) accumulation, with a dramatic increase (six- to eightfold) over the first 10 s (the "peak" phase) and subsequently from approximately 60 s onward, maintained at a lower but sustained level (the "plateau" phase). Chelation of extracellular Ca2+ with EGTA or inhibition of Ca2+ channels with Ni2+ showed that the plateau phase was dependent upon Ca2+ entry. Furthermore, use of thapsigargin and EGTA to discharge and sequester Ca2+ from intracellular stores revealed that Ca2+ from this source was capable of supporting the peak phase of the InsP3 response. Carbachol-stimulated phosphoinositidase C activity in permeabilized SH-SY5Y cells was also shown to be highly dependent on free Ca2+ concentration (20-100 nM) and suggests that under normal conditions, InsP3 formation is enhanced by increases in cytosolic free Ca2+ concentration that accompany muscarinic receptor activation. Measurement of carbachol-stimulated total inositol phosphate accumulation in the presence of Li+ indicated that the initial rate of phosphoinositide hydrolysis (from 0 to 30 s) was about fivefold greater than that from 30 to 300 s. This rapid but partial desensitization of receptor-mediated phosphoinositide hydrolysis provides strong evidence for the mechanism underlying the changes in InsP3 accumulation over this time. Because very similar data were obtained in Chinese hamster ovary cells transfected with human m3 receptor cDNA, we suggest that although increases in cytosolic free CA2+ concentration amplify InsP3 formation during stimulation of m3 muscarinic receptors, the primary factor that governs the profile of InsP3 accumulation is rapid, but partial, desensitization.(ABSTRACT TRUNCATED AT 250 WORDS)

Aluminium inhibits muscarinic agonist-induced inositol 1,4,5-trisphosphate production and calcium mobilization in permeabilized SH-SY5Y human neuroblastoma cells.

The effects of aluminium (as Al3+) on carbachol-induced inositol 1,4,5-trisphosphate (InsP3) production and Ca2+ mobilisation were assessed in electropermeabilised human SH-SY5Y neuroblastoma cells. Al3+ had no effect on InsP3-induced Ca2+ release but appreciably reduced carbachol-induced Ca2+ release (IC50 of approximately 90 microM). Al3+ also inhibited InsP3 production (IC50 of approximately 15 microM). Dimethyl hydroxypyridin-4-one, a potent Al3+ chelator (Ks = 31), at 100 microM was able to abort and reverse the effects of Al3+ on both Ca2+ release and InsP3 production. These data suggest that, in permeabilised cells, the effect of Al3+ on the phosphoinositide-mediated signalling pathway is at the level of phosphatidylinositol 4,5-bisphosphate hydrolysis. This may reflect interference with receptor-G protein-phospholipase C coupling or an interaction with phosphatidylinositol 4,5-bisphosphate.