CD44 interaction with ankyrin and IP3 receptor in lipid rafts promotes hyaluronan-mediated Ca2+ signaling leading to nitric oxide production and endothelial cell adhesion and proliferation

In this study, we have showed that aortic endothelial cells (GM7372A cell line) express CD44v10 [a hyaluronan (HA) receptor], which is significantly enriched in cholesterol-containing lipid rafts (characterized as caveolin-rich plasma membrane microdomains). HA binding to CD44v10 promotes recruitment of the cytoskeletal protein, ankyrin and inositol 1,4,5-triphosphate (IP3) receptor into cholesterol-containing lipid rafts. The ankyrin repeat domain (ARD) of ankyrin is responsible for binding IP3 receptor to CD44v10 at lipid rafts and subsequently triggering HA/CD44v10-mediated intracellular calcium (Ca2+) mobilization leading to a variety of endothelial cell functions such as nitric oxide (NO) production, cell adhesion and proliferation. Further analyses indicate (i) disruption of lipid rafts by depleting cholesterol from the membranes of GM7372A cells (using methyl-beta-cyclodextrin treatment) or (ii) interference of endogenous ankyrin binding to CD44 and IP3 receptor using overexpression of ARD fragments (by transfecting cells with ARDcDNA) not only abolishes ankyrin/IP3 receptor accumulation into CD44v10/cholesterol-containing lipid rafts, but also blocks HA-mediated Ca2+ signaling and endothelial cell functions. Taken together, our findings suggest that CD44v10 interaction with ankyrin and IP3 receptor in cholesterol-containing lipid rafts plays an important role in regulating HA-mediated Ca2+ signaling and endothelial cell functions such as NO production, cell adhesion and proliferation.     

Inositol 1,4,5-trisphosphate receptor (type 1) phosphorylation and modulation by Cdc2

Calcium (Ca2+) release from the endoplasmic reticulum (ER) controls numerous cellular functions including proliferation, and is regulated in part by inositol 1,4,5-trisphosphate receptors (IP3Rs). IP3Rs are ubiquitously expressed intracellular Ca2+-release channels found in many cell types. Although IP3R-mediated Ca2+ release has been implicated in cellular proliferation, the biochemical pathways that modulate intracellular Ca2+ release during cell cycle progression are not known. Sequence analysis of IP3R1 reveals the presence of two putative phosphorylation sites for cyclin-dependent kinases (cdks). In the present study, we show that cdc2/CyB, a critical regulator of eukaryotic cell cycle progression, phosphorylates IP3R1 in vitro and in vivo at both Ser(421) and Thr(799) and that this phosphorylation increases IP3 binding. Taken together, these results indicate that IP3R1 may be a specific target for cdc2/CyB during cell cycle progression.     

Inositol trisphosphate receptors in smooth muscle cells

Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are a family of tetrameric intracellular calcium (Ca(2+)) release channels that are located on the sarcoplasmic reticulum (SR) membrane of virtually all mammalian cell types, including smooth muscle cells (SMC). Here, we have reviewed literature investigating IP(3)R expression, cellular localization, tissue distribution, activity regulation, communication with ion channels and organelles, generation of Ca(2+) signals, modulation of physiological functions, and alterations in pathologies in SMCs. Three IP(3)R isoforms have been identified, with relative expression and cellular localization of each contributing to signaling differences in diverse SMC types. Several endogenous ligands, kinases, proteins, and other modulators control SMC IP(3)R channel activity. SMC IP(3)Rs communicate with nearby ryanodine-sensitive Ca(2+) channels and mitochondria to influence SR Ca(2+) release and reactive oxygen species generation. IP(3)R-mediated Ca(2+) release can stimulate plasma membrane-localized channels, including transient receptor potential (TRP) channels and store-operated Ca(2+) channels. SMC IP(3)Rs also signal to other proteins via SR Ca(2+) release-independent mechanisms through physical coupling to TRP channels and local communication with large-conductance Ca(2+)-activated potassium channels. IP(3)R-mediated Ca(2+) release generates a wide variety of intracellular Ca(2+) signals, which vary with respect to frequency, amplitude, spatial, and temporal properties. IP(3)R signaling controls multiple SMC functions, including contraction, gene expression, migration, and proliferation. IP(3)R expression and cellular signaling are altered in several SMC diseases, notably asthma, atherosclerosis, diabetes, and hypertension. In summary, IP(3)R-mediated pathways control diverse SMC physiological functions, with pathological alterations in IP(3)R signaling contributing to disease.     

IP(3)-mediated Ca(2+) signals in human neuroblastoma SH-SY5Y cells with exogenous overexpression of type 3 IP(3) receptor

Human neuroblastoma SH-SY5Y cells, predominantly expressing type 1 inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R), were stably transfected with IP(3)R type 3 (IP(3)R3) cDNA. Immunocytochemistry experiments showed a homogeneous cytoplasmic distribution of type 3 IP(3)Rs in transfected and selected high expression cloned cells. Using confocal Ca(2+) imaging, carbachol (CCh)-induced Ca(2+) release signals were studied. Low CCh concentrations (< or = 750 nM) evoked baseline Ca(2+) oscillations. Transfected cells displayed a higher CCh responsiveness than control or cloned cells. Ca(2+) responses varied between fast, large Ca(2+) spikes and slow, small Ca(2+) humps, while in the clone only Ca(2+) humps were observed. Ca(2+) humps in the transfected cells were associated with a high expression level of IP(3)R3. At high CCh concentrations (10 microM) Ca(2+) transients in transfected and cloned cells were similar to those in control cells. In the clone exogenous IP(3)R3 lacked the C-terminal channel domain but IP(3)-binding capacity was preserved. Transfected cells mainly expressed intact type 3 IP(3)Rs but some protein degradation was also observed.We conclude that in transfected cells expression of functional type 3 IP(3)Rs causes an apparent higher affinity for IP(3). In the clone, the presence of degraded receptors leads to an efficient cellular IP(3) buffer and attenuated IP(3)-evoked Ca(2+) release.     

Inositol (1,4,5)-trisphosphate receptor microarchitecture shapes Ca2+ puff kinetics

Inositol (1,4,5)-trisphosphate receptors (IP(3)Rs) release intracellular Ca(2+) as localized Ca(2+) signals (Ca(2+) puffs) that represent the activity of small numbers of clustered IP(3)Rs spaced throughout the endoplasmic reticulum. Although much emphasis has been placed on estimating the number of active Ca(2+) release channels supporting Ca(2+) puffs, less attention has been placed on understanding the role of cluster microarchitecture. This is important as recent data underscores the dynamic nature of IP(3)R transitions between heterogeneous cellular architectures and the differential behavior of IP(3)Rs socialized into clusters. Here, we applied a high-resolution model incorporating stochastically gating IP(3)Rs within a three-dimensional cytoplasmic space to demonstrate: 1), Ca(2+) puffs are supported by a broad range of clustered IP(3)R microarchitectures; 2), cluster ultrastructure shapes Ca(2+) puff characteristics; and 3), loosely corralled IP(3)R clusters (>200 nm interchannel separation) fail to coordinate Ca(2+) puffs, owing to inefficient triggering and impaired coupling due to reduced Ca(2+)-induced Ca(2+) release microwave velocity (<10 nm/s) throughout the channel array. Dynamic microarchitectural considerations may therefore influence Ca(2+) puff occurrence/properties in intact cells, contrasting with a more minimal role for channel number over the same simulated conditions in shaping local Ca(2+) dynamics.     

IP(3) receptors: the search for structure

Inositol (1,4,5)-trisphosphate receptors (IP(3)R) are intracellular Ca(2+) channels that are regulated by Ca(2+) and IP(3), and are modulated by many additional signals. They thereby allow both receptors that stimulate IP(3) formation and Ca(2+) to control release of Ca(2+) from intracellular stores. IP(3)Rs share many features with their close relatives, ryanodine receptors; each provides insight into the structure and function of the other. The structural basis of IP(3)R behaviour is beginning to emerge from intermediate-resolution structures of the complete IP(3)R, a 2.2-A structure of the IP(3)-binding core and comparisons with the pore structures of other tetrameric cation channels. The binding of IP(3) to a site towards the N-terminal of each IP(3)R subunit promotes binding of Ca(2+). This destabilizes an inhibitory interaction between N-terminal residues and a C-terminal 'gatekeeper' sequence, enabling the pore to open.     

Identification of common binding sites for calmodulin and inositol 1,4,5-trisphosphate receptors on the carboxyl termini of trp channels

Homologues of Drosophila Trp (transient receptor potential) form plasma membrane channels that mediate Ca(2+) entry following the activation of phospholipase C by cell surface receptors. Among the seven Trp homologous found in mammals, Trp3 has been shown to interact with and respond to IP(3) receptors (IP(3)Rs) for activation. Here we show that Trp4 and other Trp proteins also interact with IP(3)Rs. The IP(3)R-binding domain also interacts with calmodulin (CaM) in a Ca(2+)-dependent manner with affinities ranging from 10 nm for Trp2 to 290 nm for Trp6. In addition, other binding sites for CaM and IP(3)Rs are present in the alpha but not the beta isoform of Trp4. In the presence of Ca(2+), the Trp-IP(3)R interaction is inhibited by CaM. However, a synthetic peptide representing a Trp-binding domain of IP(3)Rs inhibited the binding of CaM to Trp3, -6, and -7 more effectively than that to Trp1, -2, -4, and -5. In inside-out membrane patches, Trp4 is activated strongly by calmidazolium, an antagonist of CaM, and a high (50 microm) but not a low (5 microm) concentration of the Trp-binding peptide of the IP(3)R. Our data support the view that both CaM and IP(3)Rs play important roles in controlling the gating of Trp-based channels. However, the sensitivity and responses to CaM and IP(3)Rs differ for each Trp.     

DANGER, a novel regulatory protein of inositol 1,4,5-trisphosphate-receptor activity

We report the cloning and characterization of DANGER, a novel protein which physiologically binds to inositol 1,4,5-trisphosphate receptors (IP(3)R). DANGER is a membrane-associated protein predicted to contain a partial MAB-21 domain. It is expressed in a wide variety of neuronal cell lineages where it localizes to membranes in the cell periphery together with IP(3)R. DANGER interacts with IP(3)R in vitro and co-immunoprecipitates with IP(3)R from cellular preparations. DANGER robustly enhances Ca(2+)-mediated inhibition of IP(3) RCa(2+) release without affecting IP(3) binding in microsomal assays and inhibits gating in single-channel recordings of IP(3)R. DANGER appears to allosterically modulate the sensitivity of IP(3) RtoCa(2+) inhibition, which likely alters IP(3)R-mediated Ca(2+) dynamics in cells where DANGER and IP(3)R are co-expressed.     

Selective regulation of IP3-receptor-mediated Ca2+ signaling and apoptosis by the BH4 domain of Bcl-2 versus Bcl-Xl

Antiapoptotic B-cell lymphoma 2 (Bcl-2) targets the inositol 1,4,5-trisphosphate receptor (IP(3)R) via its BH4 domain, thereby suppressing IP(3)R Ca(2+)-flux properties and protecting against Ca(2+)-dependent apoptosis. Here, we directly compared IP(3)R inhibition by BH4-Bcl-2 and BH4-Bcl-Xl. In contrast to BH4-Bcl-2, BH4-Bcl-Xl neither bound the modulatory domain of IP(3)R nor inhibited IP(3)-induced Ca(2+) release (IICR) in permeabilized and intact cells. We identified a critical residue in BH4-Bcl-2 (Lys17) not conserved in BH4-Bcl-Xl (Asp11). Changing Lys17 into Asp in BH4-Bcl-2 completely abolished its IP(3)R-binding and -inhibitory properties, whereas changing Asp11 into Lys in BH4-Bcl-Xl induced IP(3)R binding and inhibition. This difference in IP(3)R regulation between BH4-Bcl-2 and BH4-Bcl-Xl controls their antiapoptotic action. Although both BH4-Bcl-2 and BH4-Bcl-Xl had antiapoptotic activity, BH4-Bcl-2 was more potent than BH4-Bcl-Xl. The effect of BH4-Bcl-2, but not of BH4-Bcl-Xl, depended on its binding to IP(3)Rs. In agreement with the IP(3)R-binding properties, the antiapoptotic activity of BH4-Bcl-2 and BH4-Bcl-Xl was modulated by the Lys/Asp substitutions. Changing Lys17 into Asp in full-length Bcl-2 significantly decreased its binding to the IP(3)R, its ability to inhibit IICR and its protection against apoptotic stimuli. A single amino-acid difference between BH4-Bcl-2 and BH4-Bcl-Xl therefore underlies differential regulation of IP(3)Rs and Ca(2+)-driven apoptosis by these functional domains. Mutating this residue affects the function of Bcl-2 in Ca(2+) signaling and apoptosis.     

心肌肥厚大鼠心肌细胞核三磷酸肌醇受体的特征

为了研究细胞核三磷酸肌醇受体在心肌肥厚中的作用,制备了腹主动脉缩窄大鼠心肌肥厚模型、用差速离心和密度梯度离心提纯心肌细胞核,以［3H］IP3为配基,采用放射受体分析心肌细胞核膜IP3R与其配体的最大结合容量(Bmax)和解离常数(Kd)。大鼠心肌细胞核上存在IP3R、CaM和PKC激动剂PMA,能显著抑制该受体与IP3的结合(P<0.05);核外［Ca2+］也能剂量依赖的抑制细胞核IP3R与IP3的结合。腹主动脉缩窄术后4周,大鼠心肌显著肥大,伴有明显的血流动力学异常,其心肌细胞核IP3R的Bmax和Kd与对照组比较分别增加1.217和2.149倍(P<0.01)。心肌细胞核上存在IP3R,并受CaM和PMA及核外［Ca     

Inositol 1,4,5-trisphosphate IP(3) receptors and their role in neuronal cell function

Inositol 1,4,5-trisphosphate (IP(3)) receptor is a Ca(2+) release channel localized on the endoplasmic reticulum (ER) and plays an important role in neuronal function. IP(3) receptor was discovered as a developmentally regulated protein missing in the cerebellar mutant mice. Recent studies indicate that IP(3)Rs are involved in early development and neuronal plasticity. IP(3) works to release IRBIT from the IP(3) binding core in addition to release Ca(2+). IRBIT binds to and activates Na, Bicarbonate cotransporter. Electron microscopic study show the IP(3) receptor has allosteric property to change its form from square to windmill in the presence of Ca(2+). IP(3)R associates with ERp44, a redox sensor, Homer, other proteins and is transported as vesicular ER on microtubules. All these data suggests IP(3) receptor/CA(2+) channel works as a signaling center inside cells.     

Subtype-specific and ER lumenal environment-dependent regulation of inositol 1,4,5-trisphosphate receptor type 1 by ERp44

Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are intracellular channel proteins that mediate Ca(2+) release from the endoplasmic reticulum (ER) and are involved in many biological processes and diseases. IP(3)Rs are differentially regulated by a variety of cytosolic proteins, but their regulation by ER lumenal protein(s) remains largely unexplored. In this study, we found that ERp44, an ER lumenal protein of the thioredoxin family, directly interacts with the third lumenal loop of IP(3)R type 1 (IP(3)R1) and that the interaction is dependent on pH, Ca(2+) concentration, and redox state: the presence of free cysteine residues in the loop is required. Ca(2+)-imaging experiments and single-channel recording of IP(3)R1 activity with a planar lipid bilayer system demonstrated that IP(3)R1 is directly inhibited by ERp44. Thus, ERp44 senses the environment in the ER lumen and modulates IP(3)R1 activity accordingly, which should in turn contribute to regulating both intralumenal conditions and the complex patterns of cytosolic Ca(2+) concentrations.     

Role of inositol 1,4,5-trisphosphate receptors in apoptosis in DT40 lymphocytes

The role of inositol 1,4,5-trisphosphate receptors (IP(3)R) in caspase-3 activation and cell death was investigated in DT40 chicken B-lymphocytes stably expressing various IP(3)R constructs. Both full-length type-I IP(3)R and a truncated construct corresponding to the caspase-3 cleaved "channel-only" fragment were able to support staurosporine (STS)-induced caspase-3 activation and cell death even when the IP(3)R construct harbored a mutation that inactivates the pore of the Ca(2+) channel (D2550A). However, a full-length wild-type IP(3)R did not promote caspase-3 activation when the 159-amino acid cytosol-exposed C-terminal tail was deleted. STS caused an increase in cytosolic free Ca(2+) in DT40 cells expressing wild-type or pore-dead IP(3)R mutants. However, in the latter case all the Ca(2+) increase originated from Ca(2+) entry across the plasma membrane. Caspase-3 activation of pore-dead DT40 cells was also more sensitive to extracellular Ca(2+) chelation when compared with wild-type cells. STS-mediated release of cytochrome c into the cytosol and mitochondrial membrane potential depolarization could also be observed in DT40 cells lacking IP(3)Rs or containing the pore-dead mutant. We conclude that nonfunctional IP(3)Rs can sustain apoptosis in DT40 lymphocytes, because they facilitate Ca(2+) entry mechanisms across the plasma membrane. Although the intrinsic ion-channel function of IP(3)Rs is dispensable for apoptosis induced by STS, the C-terminal tail of IP(3)Rs appears to be essential, possibly reflecting key protein-protein interactions with this domain.     

Sub-plasmalemmal [Ca2+]i upstroke in myocytes of the guinea-pig small intestine evoked by muscarinic stimulation: IP3R-mediated Ca2+ release induced by voltage-gated Ca2+ entry

Membrane depolarization triggers Ca(2+) release from the sarcoplasmic reticulum (SR) in skeletal muscles via direct interaction between the voltage-gated L-type Ca(2+) channels (the dihydropyridine receptors; VGCCs) and ryanodine receptors (RyRs), while in cardiac muscles Ca(2+) entry through VGCCs triggers RyR-mediated Ca(2+) release via a Ca(2+)-induced Ca(2+) release (CICR) mechanism. Here we demonstrate that in phasic smooth muscle of the guinea-pig small intestine, excitation evoked by muscarinic receptor activation triggers an abrupt Ca(2+) release from sub-plasmalemmal (sub-PM) SR elements enriched with inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and poor in RyRs. This was followed by a lesser rise, or oscillations in [Ca(2+)](i). The initial abrupt sub-PM [Ca(2+)](i) upstroke was all but abolished by block of VGCCs (by 5 microM nicardipine), depletion of intracellular Ca(2+) stores (with 10 microM cyclopiazonic acid) or inhibition of IP(3)Rs (by 2 microM xestospongin C or 30 microM 2-APB), but was not affected by block of RyRs (by 50-100 microM tetracaine or 100 microM ryanodine). Inhibition of either IP(3)Rs or RyRs attenuated phasic muscarinic contraction by 73%. Thus, in contrast to cardiac muscles, excitation-contraction coupling in this phasic visceral smooth muscle occurs by Ca(2+) entry through VGCCs which evokes an initial IP(3)R-mediated Ca(2+) release activated via a CICR mechanism.     

IP(3) receptors: toward understanding their activation

Inositol 1,4,5-trisphosphate receptors (IP(3)R) and their relatives, ryanodine receptors, are the channels that most often mediate Ca(2+) release from intracellular stores. Their regulation by Ca(2+) allows them also to propagate cytosolic Ca(2+) signals regeneratively. This brief review addresses the structural basis of IP(3)R activation by IP(3) and Ca(2+). IP(3) initiates IP(3)R activation by promoting Ca(2+) binding to a stimulatory Ca(2+)-binding site, the identity of which is unresolved. We suggest that interactions of critical phosphate groups in IP(3) with opposite sides of the clam-like IP(3)-binding core cause it to close and propagate a conformational change toward the pore via the adjacent N-terminal suppressor domain. The pore, assembled from the last pair of transmembrane domains and the intervening pore loop from each of the four IP(3)R subunits, forms a structure in which a luminal selectivity filter and a gate at the cytosolic end of the pore control cation fluxes through the IP(3)R.     

IRBIT,a novel inositol 1,4,5-trisphosphate (IP3) receptor-binding protein,is released from the IP3 receptor upon IP3 binding to the receptor

The inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) are IP(3)-gated Ca(2+) channels on intracellular Ca(2+) stores. Herein, we report a novel protein, termed IRBIT (IP(3)R binding protein released with inositol 1,4,5-trisphosphate), which interacts with type 1 IP(3)R (IP(3)R1) and was released upon IP(3) binding to IP(3)R1. IRBIT was purified from a high salt extract of crude rat brain microsomes with IP(3) elution using an affinity column with the huge immobilized N-terminal cytoplasmic region of IP(3)R1 (residues 1-2217). IRBIT, consisting of 530 amino acids, has a domain homologous to S-adenosylhomocysteine hydrolase in the C-terminal and in the N-terminal, a 104 amino acid appendage containing multiple potential phosphorylation sites. In vitro binding experiments showed the N-terminal region of IRBIT to be essential for interaction, and the IRBIT binding region of IP(3)R1 was mapped to the IP(3) binding core. IP(3) dissociated IRBIT from IP(3)R1 with an EC(50) of approximately 0.5 microm, i.e. it was 50 times more potent than other inositol polyphosphates. Moreover, alkaline phosphatase treatment abolished the interaction, suggesting that the interaction was dualistically regulated by IP(3) and phosphorylation. Immunohistochemical studies and co-immunoprecipitation assays showed the relevance of the interaction in a physiological context. These results suggest that IRBIT is released from activated IP(3)R, raising the possibility that IRBIT acts as a signaling molecule downstream from IP(3)R.     

Endothelial Ca2+ wavelets and the induction of myoendothelial feedback

When arteries constrict to agonists, the endothelium inversely responds, attenuating the initial vasomotor response. The basis of this feedback mechanism remains uncertain, although past studies suggest a key role for myoendothelial communication in the signaling process. The present study examined whether second messenger flux through myoendothelial gap junctions initiates a negative-feedback response in hamster retractor muscle feed arteries. We specifically hypothesized that when agonists elicit depolarization and a rise in second messenger concentration, inositol trisphosphate (IP(3)) flux activates a discrete pool of IP(3) receptors (IP(3)Rs), elicits localized endothelial Ca(2+) transients, and activates downstream effectors to moderate constriction. With use of integrated experimental techniques, this study provided three sets of supporting observations. Beginning at the functional level, we showed that blocking intermediate-conductance Ca(2+)-activated K(+) channels (IK) and Ca(2+) mobilization from the endoplasmic reticulum (ER) enhanced the contractile/electrical responsiveness of feed arteries to phenylephrine. Next, structural analysis confirmed that endothelial projections make contact with the overlying smooth muscle. These projections retained membranous ER networks, and IP(3)Rs and IK channels localized in or near this structure. Finally, Ca(2+) imaging revealed that phenylephrine induced discrete endothelial Ca(2+) events through IP(3)R activation. These events were termed recruitable Ca(2+) wavelets on the basis of their spatiotemporal characteristics. From these findings, we conclude that IP(3) flux across myoendothelial gap junctions is sufficient to induce focal Ca(2+) release from IP(3)Rs and activate a discrete pool of IK channels within or near endothelial projections. The resulting hyperpolarization feeds back on smooth muscle to moderate agonist-induced depolarization and constriction.     

An RGD sequence in the P2Y(2) receptor interacts with alpha(V)beta(3) integrins and is required for G(o)-mediated signal transduction

The P2Y(2) nucleotide receptor (P2Y(2)R) contains the integrin-binding domain arginine-glycine-aspartic acid (RGD) in its first extracellular loop, raising the possibility that this G protein-coupled receptor interacts directly with an integrin. Binding of a peptide corresponding to the first extracellular loop of the P2Y(2)R to K562 erythroleukemia cells was inhibited by antibodies against alpha(V)beta(3)/beta(5) integrins and the integrin-associated thrombospondin receptor, CD47. Immunofluorescence of cells transfected with epitope-tagged P2Y(2)Rs indicated that alpha(V) integrins colocalized 10-fold better with the wild-type P2Y(2)R than with a mutant P2Y(2)R in which the RGD sequence was replaced with RGE. Compared with the wild-type P2Y(2)R, the RGE mutant required 1,000-fold higher agonist concentrations to phosphorylate focal adhesion kinase, activate extracellular signal-regulated kinases, and initiate the PLC-dependent mobilization of intracellular Ca(2+). Furthermore, an anti-alpha(V) integrin antibody partially inhibited these signaling events mediated by the wild-type P2Y(2)R. Pertussis toxin, an inhibitor of G(i/o) proteins, partially inhibited Ca(2+) mobilization mediated by the wild-type P2Y(2)R, but not by the RGE mutant, suggesting that the RGD sequence is required for P2Y(2)R-mediated activation of G(o), but not G(q). Since CD47 has been shown to associate directly with G(i/o) family proteins, these results suggest that interactions between P2Y(2)Rs, integrins, and CD47 may be important for coupling the P2Y(2)R to G(o).     

Positive regulation of inositol 1,4,5-trisphosphate-induced Ca2+ release by mammalian target of rapamycin (mTOR) in RINm5F cells

The inositol 1,4,5-trisphosphate receptor (IP(3)R), a ligand-gated Ca(2+) channel, is the main regulator of intracellular Ca(2+) mobilization in non-excitable cells. An emerging body of evidence suggests that specific regulatory control of the Ca(2+) signaling pathway is modulated by the activation of additional signaling pathways. In the present study, we investigated the influence of the PI3-kinase/mammalian target of rapamycin (mTOR) pathway on the activity of the IP(3)R/Ca(2+) signaling pathway in RINm5F cells. We used a co-immunoprecipitation approach to show that mTOR physically interacts with IP(3)R-3 in an mTOR activity-dependent manner. We also showed that IP(3)R is phosphorylated by mTOR in cellulo. All the conditions known to modulate mTOR activity (IGF-1, wortmannin, rapamycin, PP242, and nutrient starvation) were shown to modify carbachol-induced Ca(2+) signaling in RINm5F cells. Lastly, we used an assay that directly measures the activity of IP(3)R, to show that mTOR increases the apparent affinity of IP(3)R. Given that mTOR controls cell proliferation and cell homeostasis, and that Ca(2+) plays a key role in these two phenomena, it follows that mTOR facilitates IP(3)R-mediated Ca(2+) release when the nutritional status of cells requires it.