Post-translational Regulation of the Type III Inositol 1,4,5-Trisphosphate Receptor by miRNA-506

The type III isoform of the inositol 1,4,5-trisphosphate receptor (InsP₃R3) is apically localized and triggers Ca²⁺ waves and secretion in a number of polarized epithelia. However, nothing is known about epigenetic regulation of this InsP3R isoform. We investigated miRNA regulation of InsP₃R3 in primary bile duct epithelia (cholangiocytes) and in the H69 cholangiocyte cell line, because the role of InsP₃R3 in cholangiocyte Ca²⁺ signaling and secretion is well established and because loss of InsP₃R3 from cholangiocytes is responsible for the impairment in bile secretion that occurs in a number of liver diseases. Analysis of the 3′-UTR of human InsP₃R3 mRNA revealed two highly conserved binding sites for miR-506. Transfection of miR-506 mimics into cell lines expressing InsP₃R3–3′UTR-luciferase led to decreased reporter activity, whereas co-transfection with miR-506 inhibitors led to enhanced activity. Reporter activity was abrogated in isolated mutant proximal or distal miR-506 constructs in miR-506-transfected HEK293 cells. InsP₃R3 protein levels were decreased by miR-506 mimics and increased by inhibitors, and InsP₃R3 expression was markedly decreased in H69 cells stably transfected with miR-506 relative to control cells. miR-506-H69 cells exhibited a fibrotic signature. In situ hybridization revealed elevated miR-506 expression in vivo in human-diseased cholangiocytes. Histamine-induced, InsP₃-mediated Ca²⁺ signals were decreased by 50% in stable miR-506 cells compared with controls. Finally, InsP₃R3-mediated fluid secretion was significantly decreased in isolated bile duct units transfected with miR-506, relative to control IBDU. Together, these data identify miR-506 as a regulator of InsP₃R3 expression and InsP₃R3-mediated Ca²⁺ signaling and secretion.     

The Bcl-2 Protein Family Member Bok Binds to the Coupling Domain of Inositol 1,4,5-Trisphosphate Receptors and Protects Them from Proteolytic Cleavage

Bok is a member of the Bcl-2 protein family that controls intrinsic apoptosis. Bok is most closely related to the pro-apoptotic proteins Bak and Bax, but in contrast to Bak and Bax, very little is known about its cellular role. Here we report that Bok binds strongly and constitutively to inositol 1,4,5-trisphosphate receptors (IP₃Rs), proteins that form tetrameric calcium channels in the endoplasmic reticulum (ER) membrane and govern the release of ER calcium stores. Bok binds most strongly to IP₃R1 and IP₃R2, and barely to IP₃R3, and essentially all cellular Bok is IP₃R bound in cells that express substantial amounts of IP₃Rs. Binding to IP₃Rs appears to be mediated by the putative BH4 domain of Bok and the docking site localizes to a small region within the coupling domain of IP₃Rs (amino acids 1895–1903 of IP₃R1) that is adjacent to numerous regulatory sites, including sites for proteolysis. With regard to the possible role of Bok-IP₃R binding, the following was observed: (i) Bok does not appear to control the ability of IP₃Rs to release ER calcium stores, (ii) Bok regulates IP₃R expression, (iii) persistent activation of inositol 1,4,5-trisphosphate-dependent cell signaling causes Bok degradation by the ubiquitin-proteasome pathway, in a manner that parallels IP₃R degradation, and (iv) Bok protects IP₃Rs from proteolysis, either by chymotrypsin in vitro or by caspase-3 in vivo during apoptosis. Overall, these data show that Bok binds strongly and constitutively to IP₃Rs and that the most significant consequence of this binding appears to be protection of IP₃Rs from proteolysis. Thus, Bok may govern IP₃R cleavage and activity during apoptosis.     

Functional Inositol 1,4,5-Trisphosphate Receptors Assembled from Concatenated Homo- and Heteromeric Subunits

Vertebrate genomes code for three subtypes of inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R1, -2, and -3). Individual IP₃R monomers are assembled to form homo- and heterotetrameric channels that mediate Ca²⁺ release from intracellular stores. IP₃R subtypes are regulated differentially by IP₃, Ca²⁺, ATP, and various other cellular factors and events. IP₃R subtypes are seldom expressed in isolation in individual cell types, and cells often express different complements of IP₃R subtypes. When multiple subtypes of IP₃R are co-expressed, the subunit composition of channels cannot be specifically defined. Thus, how the subunit composition of heterotetrameric IP₃R channels contributes to shaping the spatio-temporal properties of IP₃-mediated Ca²⁺ signals has been difficult to evaluate. To address this question, we created concatenated IP₃R linked by short flexible linkers. Dimeric constructs were expressed in DT40–3KO cells, an IP₃R null cell line. The dimeric proteins were localized to membranes, ran as intact dimeric proteins on SDS-PAGE, and migrated as an ∼1100-kDa band on blue native gels exactly as wild type IP₃R. Importantly, IP₃R channels formed from concatenated dimers were fully functional as indicated by agonist-induced Ca²⁺ release. Using single channel “on-nucleus” patch clamp, the channels assembled from homodimers were essentially indistinguishable from those formed by the wild type receptor. However, the activity of channels formed from concatenated IP₃R1 and IP₃R2 heterodimers was dominated by IP₃R2 in terms of the characteristics of regulation by ATP. These studies provide the first insight into the regulation of heterotetrameric IP₃R of defined composition. Importantly, the results indicate that the properties of these channels are not simply a blend of those of the constituent IP₃R monomers.     

Differential Regulation of Multiple Steps in Inositol 1,4,5-Trisphosphate Signaling by Protein Kinase C Shapes Hormone-stimulated Ca2+ Oscillations

How Ca²⁺ oscillations are generated and fine-tuned to yield versatile downstream responses remains to be elucidated. In hepatocytes, G protein-coupled receptor-linked Ca²⁺ oscillations report signal strength via frequency, whereas Ca²⁺ spike amplitude and wave velocity remain constant. IP₃ uncaging also triggers oscillatory Ca²⁺ release, but, in contrast to hormones, Ca²⁺ spike amplitude, width, and wave velocity were dependent on [IP₃] and were not perturbed by phospholipase C (PLC) inhibition. These data indicate that oscillations elicited by IP₃ uncaging are driven by the biphasic regulation of the IP₃ receptor by Ca²⁺, and, unlike hormone-dependent responses, do not require PLC. Removal of extracellular Ca²⁺ did not perturb Ca²⁺ oscillations elicited by IP₃ uncaging, indicating that reloading of endoplasmic reticulum stores via plasma membrane Ca²⁺ influx does not entrain the signal. Activation and inhibition of PKC attenuated hormone-induced Ca²⁺ oscillations but had no effect on Ca²⁺ increases induced by uncaging IP₃. Importantly, PKC activation and inhibition differentially affected Ca²⁺ spike frequencies and kinetics. PKC activation amplifies negative feedback loops at the level of G protein-coupled receptor PLC activity and/or IP₃ metabolism to attenuate IP₃ levels and suppress the generation of Ca²⁺ oscillations. Inhibition of PKC relieves negative feedback regulation of IP₃ accumulation and, thereby, shifts Ca²⁺ oscillations toward sustained responses or dramatically prolonged spikes. PKC down-regulation attenuates phenylephrine-induced Ca²⁺ wave velocity, whereas responses to IP₃ uncaging are enhanced. The ability to assess Ca²⁺ responses in the absence of PLC activity indicates that IP₃ receptor modulation by PKC regulates Ca²⁺ release and wave velocity.     

Calpain-cleaved type 1 inositol 1,4,5-trisphosphate receptor (InsP(3)R1) has InsP(3)-independent gating and disrupts intracellular Ca(2+) homeostasis

The type 1 inositol 1,4,5-trisphosphate receptor (InsP(3)R1) is a ubiquitous intracellular Ca(2+) release channel that is vital to intracellular Ca(2+) signaling. InsP(3)R1 is a proteolytic target of calpain, which cleaves the channel to form a 95-kDa carboxyl-terminal fragment that includes the transmembrane domains, which contain the ion pore. However, the functional consequences of calpain proteolysis on channel behavior and Ca(2+) homeostasis are unknown. In the present study we have identified a unique calpain cleavage site in InsP(3)R1 and utilized a recombinant truncated form of the channel (capn-InsP(3)R1) corresponding to the stable, carboxyl-terminal fragment to examine the functional consequences of channel proteolysis. Single-channel recordings of capn-InsP(3)R1 revealed InsP(3)-independent gating and high open probability (P(o)) under optimal cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) conditions. However, some [Ca(2+)](i) regulation of the cleaved channel remained, with a lower P(o) in suboptimal and inhibitory [Ca(2+)](i). Expression of capn-InsP(3)R1 in N2a cells reduced the Ca(2+) content of ionomycin-releasable intracellular stores and decreased endoplasmic reticulum Ca(2+) loading compared with control cells expressing full-length InsP(3)R1. Using a cleavage-specific antibody, we identified calpain-cleaved InsP(3)R1 in selectively vulnerable cerebellar Purkinje neurons after in vivo cardiac arrest. These findings indicate that calpain proteolysis of InsP(3)R1 generates a dysregulated channel that disrupts cellular Ca(2+) homeostasis. Furthermore, our results demonstrate that calpain cleaves InsP(3)R1 in a clinically relevant injury model, suggesting that Ca(2+) leak through the proteolyzed channel may act as a feed-forward mechanism to enhance cell death.     

Isoform- and Species-specific Control of Inositol 1,4,5-Trisphosphate (IP3) Receptors by Reactive Oxygen Species

Reactive oxygen species (ROS) stimulate cytoplasmic [Ca²⁺] ([Ca²⁺]_c) signaling, but the exact role of the IP₃ receptors (IP₃R) in this process remains unclear. IP₃Rs serve as a potential target of ROS produced by both ER and mitochondrial enzymes, which might locally expose IP₃Rs at the ER-mitochondrial associations. Also, IP₃Rs contain multiple reactive thiols, common molecular targets of ROS. Therefore, we have examined the effect of superoxide anion (O₂^⨪) on IP₃R-mediated Ca²⁺ signaling. In human HepG2, rat RBL-2H3, and chicken DT40 cells, we observed [Ca²⁺]_c spikes and frequency-modulated oscillations evoked by a O₂^⨪ donor, xanthine (X) + xanthine oxidase (XO), dose-dependently. The [Ca²⁺]_c signal was mediated by ER Ca²⁺ mobilization. X+XO added to permeabilized cells promoted the [Ca²⁺]_c rise evoked by submaximal doses of IP₃, indicating that O₂^⨪ directly sensitizes IP₃R-mediated Ca²⁺ release. In response to X+XO, DT40 cells lacking two of three IP₃R isoforms (DKO) expressing either type 1 (DKO1) or type 2 IP₃Rs (DKO2) showed a [Ca²⁺]_c signal, whereas DKO expressing type 3 IP₃R (DKO3) did not. By contrast, IgM that stimulates IP₃ formation, elicited a [Ca²⁺]_c signal in every DKO. X+XO also facilitated the Ca²⁺ release evoked by submaximal IP₃ in permeabilized DKO1 and DKO2 but was ineffective in DKO3 or in DT40 lacking every IP₃R (TKO). However, X+XO could also facilitate the effect of suboptimal IP₃ in TKO transfected with rat IP₃R3. Although in silico studies failed to identify a thiol missing in the chicken IP₃R3, an X+XO-induced redox change was documented only in the rat IP₃R3. Thus, ROS seem to specifically sensitize IP₃Rs through a thiol group(s) within the IP₃R, which is probably inaccessible in the chicken IP₃R3.     

Polycystin-2 Activation by Inositol 1,4,5-Trisphosphate-induced Ca2+ Release Requires Its Direct Association with the Inositol 1,4,5-Trisphosphate Receptor in a Signaling Microdomain

Autosomal dominant polycystic kidney disease is characterized by the loss-of-function of a signaling complex involving polycystin-1 and polycystin-2 (TRPP2, an ion channel of the TRP superfamily), resulting in a disturbance in intracellular Ca²⁺ signaling. Here, we identified the molecular determinants of the interaction between TRPP2 and the inositol 1,4,5-trisphosphate receptor (IP₃R), an intracellular Ca²⁺ channel in the endoplasmic reticulum. Glutathione S-transferase pulldown experiments combined with mutational analysis led to the identification of an acidic cluster in the C-terminal cytoplasmic tail of TRPP2 and a cluster of positively charged residues in the N-terminal ligand-binding domain of the IP₃R as directly responsible for the interaction. To investigate the functional relevance of TRPP2 in the endoplasmic reticulum, we re-introduced the protein in TRPP2^−/− mouse renal epithelial cells using an adenoviral expression system. The presence of TRPP2 resulted in an increased agonist-induced intracellular Ca²⁺ release in intact cells and IP₃-induced Ca²⁺ release in permeabilized cells. Using pathological mutants of TRPP2, R740X and D509V, and competing peptides, we demonstrated that TRPP2 amplified the Ca²⁺ signal by a local Ca²⁺-induced Ca²⁺-release mechanism, which only occurred in the presence of the TRPP2-IP₃R interaction, and not via altered IP₃R channel activity. Moreover, our results indicate that this interaction was instrumental in the formation of Ca²⁺ microdomains necessary for initiating Ca²⁺-induced Ca²⁺ release. The data strongly suggest that defects in this mechanism may account for the altered Ca²⁺ signaling associated with pathological TRPP2 mutations and therefore contribute to the development of autosomal dominant polycystic kidney disease.     

Calcium regulation of inositol 1,4,5-trisphosphate receptors

Ca2+ exerts both a stimulatory and inhibitory effect on type-I IP3R channel activity. However, the structural determinants of Ca2+ sensing in IP3Rs are not fully understood. Previous studies by others have identified eight domains of the type-I IP3R that bind 45Ca2+ when expressed as GST-fusion proteins. We have mutated six highly conserved acidic residues within the second of these domains (aa378-450) in the full-length IP3R and measured the Ca2+ regulation of IP3-mediated Ca2+ release in COS-7 cells. 45Ca2+ flux assays measured with a maximal [IP3] (1 microM) indicate that one of the mutants retained a Ca2+ sensitivity that was not significantly different from control (E411Q), three of the mutants show an enhanced Ca2+ inhibition (D426N, E428Q and E439Q) and two of the mutants were relatively insensitive to Ca2+ inhibition (D442N and D444N). IP3 dose-response relationships indicated that the sensitivity to Ca2+ inhibition and affinity for IP3 were correlated for three of the constructs. Other mutants with enhanced IP3 sensitivity (e.g. R441Q and a type-II/I IP3R chimera) were also less sensitive to Ca2+ inhibition. We conclude that the acidic residues within the aa378-450 segment are unlikely to represent a single functional Ca2+ binding domain and do not contribute to Ca2+ activation of the receptor. The different effects of the mutations may be related to their location within two clusters of acidic residues identified in the crystal structure of the ligand-binding domain [I. Bosanac, J.R. Alattia, T.K. Mal, et al., Structure of the inositol 1,4,5-trisphosphate receptor binding core in complex with its ligand, Nature 420 (2002) 696-700]. The data support the view that all IP3R isoforms may display a range of Ca2+ sensitivities that are determined by multiple sites within the protein and markedly influenced by the affinity of the receptor for IP3.     

Inositol 1,4,5-trisphosphate receptor type 1 phosphorylation and regulation by extracellular signal-regulated kinase

Type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) is a widely expressed intracellular calcium-release channel found in many cell types. The operation of IP(3)R1 is regulated through phosphorylation by multiple protein kinases. Extracellular signal-regulated kinase (ERK) has been found involved in calcium signaling in distinct cell types, but the underlying mechanisms remain unclear. Here, we present evidence that ERK1/2 and IP(3)R1 bind together through an ERK binding motif in mouse cerebellum in vivo as well as in vitro. ERK-phosphorylating serines (Ser 436) was identified in mouse IP(3)R1 and Ser 436 phosphorylation had a suppressive effect on IP(3) binding to the recombinant N-terminal 604-amino acid residues (N604). Moreover, phosphorylation of Ser 436 in R(224-604) evidently enhance its interaction with the N-terminal "suppressor" region (N223). At last, our data showed that Ser 436 phosphorylation in IP(3)R1 decreased Ca(2+) releasing through IP(3)R1 channels.     

InsP3R-associated cGMP Kinase Substrate Determines Inositol 1,4,5-Trisphosphate Receptor Susceptibility to Phosphoregulation by Cyclic Nucleotide-dependent Kinases

Ca²⁺ release through inositol 1,4,5-trisphosphate receptors (InsP₃R) can be modulated by numerous factors, including input from other signal transduction cascades. These events shape the spatio-temporal characteristics of the Ca²⁺ signal and provide fidelity essential for the appropriate activation of effectors. In this study, we investigate the regulation of Ca²⁺ release via InsP₃R following activation of cyclic nucleotide-dependent kinases in the presence and absence of expression of a binding partner InsP₃R-associated cGMP kinase substrate (IRAG). cGMP-dependent kinase (PKG) phosphorylation of only the S2+ InsP₃R-1 subtype resulted in enhanced Ca²⁺ release in the absence of IRAG expression. In contrast, IRAG bound to each InsP₃R subtype, and phosphorylation of IRAG by PKG attenuated Ca²⁺ release through all InsP₃R subtypes. Surprisingly, simply the expression of IRAG attenuated phosphorylation and inhibited the enhanced Ca²⁺ release through InsP₃R-1 following cAMP-dependent protein kinase (PKA) activation. In contrast, IRAG expression did not influence the PKA-enhanced activity of the InsP₃R-2. Phosphorylation of IRAG resulted in reduced Ca²⁺ release through all InsP₃R subtypes during concurrent activation of PKA and PKG, indicating that IRAG modulation is dominant under these conditions. These studies yield mechanistic insight into how cells with various complements of proteins integrate and prioritize signals from ubiquitous signaling pathways.     

Inositol 1,4,5-trisphosphate receptor subtypes are differentially distributed between smooth muscle and endothelial layers of rat arteries

In blood vessels, the ability to control vascular tone depends on extracellular calcium entry and the release of calcium from inositol 1,4,5-trisphosphate receptor (IP3R)-gated stores located in both the endothelial and smooth muscle cells of the vascular wall. Therefore, we examined mRNA expression and protein distribution of IP3R subtypes in intact aorta, basilar and mesenteric arteries of the rat. IP3R1 mRNA was predominantly expressed in all three arteries. Immunohistochemistry showed that IP3R1 was present in both the muscle and endothelial cell layers, while IP3R2 and IP3R3 were largely restricted to the endothelium. Weak expression of IP3R2 was observed in the smooth muscle of the basilar artery. Co-localisation studies of IP3R subtypes with known cellular elements showed no association of any of the three subtypes with the endothelial cell plasma membrane, but a close association between the subtypes and actin filaments was observed in all cell layers. IP3R2 was found to be present near the endothelial cell nucleus. We are the first to demonstrate differential IP3R subtype distribution between the cell layers of the intact vascular wall and hypothesise that this may underlie the diversity of IP3R-dependent responses, such as vasoconstriction, vasodilation and vasomotion, displayed by arteries.     

Isoform-specific regulation of the inositol 1,4,5-trisphosphate receptor by O-linked glycosylation

The inositol 1,4,5-trisphosphate receptor (InsP(3)R), an intracellular calcium channel, has three isoforms with >65% sequence homology, yet the isoforms differ in their function and regulation by post-translational modifications. We showed previously that InsP(3)R-1 is functionally modified by O-linked β-N-acetylglucosamine glycosylation (O-GlcNAcylation) (Rengifo, J., Gibson, C. J., Winkler, E., Collin, T., and Ehrlich, B. E. (2007) J. Neurosci. 27, 13813-13821). We now report the effect of O-GlcNAcylation on InsP(3)R-2 and InsP(3)R-3. Analysis of AR4-2J cells, a rat pancreatoma cell line expressing predominantly InsP(3)R-2, showed no detectable O-GlcNAcylation of InsP(3)R-2 and no significant functional changes despite the presence of the enzymes for addition (O-β-N-acetylglucosaminyltransferase) and removal (O-β-N-acetylglucosaminidase) of the monosaccharide. In contrast, InsP(3)R-3 in Mz-ChA-1 cells, a human cholangiocarcinoma cell line expressing predominantly InsP(3)R-3, was functionally modified by O-GlcNAcylation. Interestingly, the functional impact of O-GlcNAcylation on the InsP(3)R-3 channel was opposite the effect measured with InsP(3)R-1. Addition of O-GlcNAc by O-β-N-acetylglucosaminyltransferase increased InsP(3)R-3 single channel open probability. Incubation of Mz-ChA-1 cells in hyperglycemic medium caused an increase in the InsP(3)-dependent calcium release from the endoplasmic reticulum. The dynamic and inducible nature of O-GlcNAcylation and the InsP(3)R isoform specificity suggest that this form of modification of InsP(3)R and subsequent changes in intracellular calcium transients are important in physiological and pathophysiological processes.     

Ligand-induced conformational changes via flexible linkers in the amino-terminal region of the inositol 1,4,5-trisphosphate receptor

Cytoplasmic Ca2+ signals are highly regulated by various ion transporters, including the inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R), which functions as a Ca2+ release channel on the endoplasmic reticulum membrane. Crystal structures of the two N-terminal regulatory regions from type 1 IP(3)R have been reported; those of the IP(3)-binding core (IP(3)R(CORE)) with bound IP(3), and the suppressor domain. This study examines the structural effects of ligand binding on an IP(3)R construct, designated IP(3)R(N), that contains both the IP(3)-binding core and the suppressor domain. Our circular dichroism results reveal that the IP(3)-bound and IP(3)-free states have similar secondary structure content, consistent with preservation of the overall fold within the individual domains. Thermal denaturation data show that, while IP(3) has a large effect on the stability of IP(3)R(CORE), it has little effect on IP(3)R(N), indicating that the suppressor domain is critical to the stability of IP(3)R(N). The NMR data for IP(3)R(N) provide evidence for chemical exchange, which may be due to protein conformational dynamics in both apo and IP(3)-bound states: a conclusion supported by the small-angle X-ray scattering data. Further, the scattering data show that IP(3)R(N) undergoes a change in average conformation in response to IP(3) binding and the presence of Ca2+ in the solution. Taken together, these data lead us to propose that there are two flexible linkers in the N-terminal region of IP(3)R that join stably folded domains and give rise to an equilibrium mixture of conformational sub-states containing compact and more extended structures. IP(3) binding drives the conformational equilibrium toward more compact structures, while the presence of Ca2+ drives it to a more extended set.     

Inositol 1,4,5-trisphosphate 3-kinase A is a novel microtubule-associated protein: PKA-dependent phosphoregulation of microtubule binding affinity

Inositol 1,4,5-trisphosphate 3-kinase A (IP(3)K-A) is a brain specific and F-actin-binding protein. We recently demonstrated that IP(3)K-A modulates a structural reorganization of dendritic spines through F-actin remodeling, which is required for synaptic plasticity and memory formation in brain. However, detailed functions of IP(3)K-A and its regulatory mechanisms involved in the neuronal cytoskeletal dynamics still remain unknown. In the present study, we identified tubulin as a candidate of IP(3)K-A-binding protein through proteomic screening. By various in vitro and in vivo approaches, we demonstrated that IP(3)K-A was a novel microtubule-associated protein (MAP), and the N terminus of IP(3)K-A was a critical region for direct binding to tubulin in dendritic shaft of hippocampal neurons. Moreover, PKA phosphorylated Ser-119 within IP(3)K-A, leading to a significant reduction of microtubule binding affinity. These results suggest that PKA-dependent phosphorylation and microtubule binding of IP(3)K-A are involved in its regulatory mechanism for activity-dependent neuronal events such as local calcium signaling and its synaptic targeting.     

D-myo-inositol 1-phosphate as a surrogate of D-myo-inositol 1,4,5-tris phosphate to monitor G protein-coupled receptor activation

Phospholipase C beta (PLC-beta)-coupled G protein-coupled receptor (GPCR) activities traditionally are assessed by measuring Ca2+ triggered by D-myo-inositol 1,4,5-trisphosphate (IP3), a PLC-beta hydrolysis product, or by measuring the production of inositol phosphate using cumbersome radioactive assays. A specific detection of IP3 production was also established using IP3 binding proteins. The short lifetime of IP3 makes this detection very challenging in measuring GPCR responses. Indeed, this IP3 rapidly enters the metabolic inositol phosphate cascade. It has been known for decades that lithium chloride (LiCl) leads to D-myo-inositol 1-phosphate accumulation on GPCR activation by inhibiting inositol monophosphatase, the final enzyme of the IP3 metabolic cascade. We show here that IP1 can be used as a surrogate of IP3 to monitor GPCR activation. We developed a novel homogeneous time-resolved fluorescence (HTRF) assay that correlates perfectly with existing methods and is easily amenable to high-throughput screening. The IP-One assay was validated on various GPCR models. It has the advantage over the traditional Ca2+ assay of allowing the measurement of inverse agonist activity as well as the analysis of PLC-beta activity in any nontransfected primary cultures. Finally, the high assay specificity for D-myo-inositol 1 monophosphate (IP1(1)) opens new possibilities in developing selective assays to study the functional roles of the various isoforms of inositol phosphates.     

Activated inositol 1,4,5-trisphosphate receptors are modified by homogeneous Lys-48- and Lys-63-linked ubiquitin chains, but only Lys-48-linked chains are required for degradation

Inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) are large, ubiquitously expressed, endoplasmic reticulum membrane proteins that form tetrameric IP(3) and Ca(2+)-gated Ca(2+) channels. Endogenous IP(3)Rs provide very appealing tools for studying the ubiquitin-proteasome pathway in intact mammalian cells because, upon activation, they are rapidly ubiquitinated and degraded. Using mass spectrometry, we previously examined the ubiquitination of IP(3)R1 in αT3-1 pituitary gonadotrophs and found that IP(3)R1 ubiquitination is highly complex, with receptors being modified at multiple sites by monoubiquitin and polyubiquitin chains formed through both Lys-48 and Lys-63 linkages (Sliter, D. A., Kubota, K., Kirkpatrick, D. S., Alzayady, K. J., Gygi, S. P., and Wojcikiewicz, R. J. H. (2008) J. Biol. Chem. 283, 35319-35328). Here, we have extended these studies to determine whether IP(3)R2 and IP(3)R3 are similarly modified and if ubiquitination is cell type-dependent. Using mass spectrometry and linkage-specific ubiquitin antibodies, we found that all IP(3)R types are subject to ubiquitination at approximately the same locations and that, independent of cell type, IP(3)Rs are modified by monoubiquitin and Lys-48- and Lys-63-linked ubiquitin chains, although in differing proportions. Remarkably, the attached Lys-48- and Lys-63-linked ubiquitin chains are homogeneous and are segregated to separate IP(3)R subunits, and Lys-48-linked ubiquitin chains, but not Lys-63-linked chains, are required for IP(3)R degradation. Together, these data provide unique insight into the complexities of ubiquitination of an endogenous ubiquitin-proteasome pathway substrate in unperturbed mammalian cells. Importantly, although Lys-48-linked ubiquitin chains appear to trigger proteasomal degradation, the presence of Lys-63-linked ubiquitin chains suggests that ubiquitination of IP(3)Rs may have physiological consequences beyond signaling for degradation.     

Inositol(1,4,5)trisphosphate signal triggers a receptor-mediated ATP release

Intracellular signal transduction pathways involved in ATP release evoked by angiotensin II (Ang II) were investigated in cultured guinea pig Taenia coli smooth muscle cells. Ang II (0.3-1 microM) elicited substantial release of ATP from the cells, but not from a human fibroblast cell line. However, Ang II even at 10 microM failed to cause a leakage of lactate dehydrogenase (LDH) from the smooth muscle cells. The release of ATP by Ang II was suppressed by 10 microM SC52458, an AT1 receptor antagonist, not by 10 microM PD123319, an AT2 receptor antagonist. The evoked release of ATP was almost completely inhibited in the presence of 10 microM U73122, a phospholipase C inhibitor, and 0.5 microM thapsigargin, a Ca2+-ATPase inhibitor. Furthermore, the release was hampered by 50 microM BAPTA/AM, an intracellular Ca2+ chelator, but not by 0.1 microM nifedipine, a voltage gated Ca2+ channel inhibitor. The basal release of ATP was increased by BAPTA/AM, but was reduced by U-73122. Ang II enhanced instantaneously inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) accumulation in the cells. The enhancing effect was perfectly antagonized by SC52458. These findings suggest that intracellular Ca2+ signals activated via stimulation of Ins(1,4,5)P3 receptor are involved in the release of ATP evoked by Ang II.     

Role of secretory granules in inositol 1,4,5-trisphosphate-dependent Ca(2+) signaling: from phytoplankton to mammals

The majority of secretory cell calcium is stored in secretory granules that serve as the major IP₃-dependent intracellular Ca²⁺ store. Even in unicellular phytoplankton secretory granules are responsible for the IP₃-induced Ca²⁺ release that triggers exocytosis. The number of secretory granules in the cell is directly related not only to the magnitude of IP₃-induced Ca²⁺ release, which accounts for the majority of the IP₃-induced cytoplasmic Ca²⁺ release in neuroendocrine cells, but also to the IP₃ sensitivity of the cytoplasmic IP₃ receptor (IP₃R)/Ca²⁺ channels. Moreover, secretory granules contain the highest IP₃R concentrations and the largest amounts of IP₃Rs in any subcellular organelles in neuroendocrine cells. Secretory granules from phytoplankton to mammals contain large amounts of polyanionic molecules, chromogranins being the major molecules in mammals, in addition to acidic intragranular pH and high Ca²⁺ concentrations. The polyanionic molecules undergo pH- and Ca²⁺-dependent conformational changes that serve as a molecular basis for condensation-decondensation phase transitions of the intragranular matrix. Likewise, chromogranins undergo pH- and Ca²⁺-dependent conformational changes with increased exposure of the structure and increased interactions with Ca²⁺ and other granule components at acidic pH. The unique physico-chemical properties of polyanionic molecules appear to be at the center of biogenesis, and physiological functions of secretory granules in living organisms from primitive to advanced species.     

RIC-3 Exclusively Enhances the Surface Expression of Human Homomeric 5-Hydroxytryptamine Type 3A (5-HT3A) Receptors Despite Direct Interactions with 5-HT3A, -C, -D, and -E Subunits

Although five 5-hydroxytryptamine type 3 (5-HT3) subunits (A–E) have been cloned, knowledge on the regulation of their assembly is limited. RIC-3 has been identified as a chaperone specific for the pentameric ligand-gated nicotinic acetylcholine and 5-HT₃ receptors. Therefore, we examined the impact of RIC-3 on differently composed 5-HT₃ receptors with the focus on 5-HT3C, -D, and -E subunits. The influence of RIC-3 on these receptor subtypes is supported by the presence of RIC3 mRNA in tissues expressing at least one of the subunits 5-HT3C, -D, and -E. Furthermore, immunocytochemical studies on transfected mammalian cells revealed co-localization in the endoplasmic reticulum and direct interaction of RIC-3 with 5-HT3A, -C, -D, and -E. Functional and pharmacological characterization was performed using HEK293 cells expressing 5-HT3A or 5-HT3A + 5-HT3B (or -C, -D, or -E) in the presence or absence of RIC-3. Ca²⁺ influx analyses revealed that RIC-3 does not influence the 5-HT concentration-response relationship on 5-HT₃A receptors but leads to differential increases of 5-HT-induced maximum response (E_max) on cells expressing different subunits. Increases of E_max were due to analogously enhanced B_max values for binding of the 5-HT₃ receptor antagonist [³H]GR65630. The observed enhanced cell surface expression of the tested 5-HT3 subunit combinations correlated with the increased surface expression of 5-HT3A as determined by flow cytometry. In conclusion, we showed that RIC-3 can interact with 5-HT3A, -C, -D, and -E subunits and predominantly enhances the surface expression of homomeric 5-HT₃A receptors in HEK293 cells. These data implicate a possible role of RIC-3 in determining 5-HT₃ receptor composition in vivo.