Functional consequences of phosphomimetic mutations at key cAMP-dependent protein kinase phosphorylation sites in the type 1 inositol 1,4,5-trisphosphate receptor

Regulation of Ca(2+) release through inositol 1,4,5-trisphosphate receptors (InsP(3)R) has important consequences for defining the particular spatio-temporal properties of intracellular Ca(2+) signals. In this study, regulation of Ca(2+) release by phosphorylation of type 1 InsP(3)R (InsP(3)R-1) was investigated by constructing "phosphomimetic" charge mutations in the functionally important phosphorylation sites of both the S2+ and S2- InsP(3)R-1 splice variants. Ca(2+) release was investigated following expression in Dt-40 3ko cells devoid of endogenous InsP(3)R. In cells expressing either the S1755E S2+ or S1589E/S1755E S2- InsP(3)R-1, InsP(3)-induced Ca(2+) release was markedly enhanced compared with nonphosphorylatable S2+ S1755A and S2- S1589A/S1755A mutants. Ca(2+) release through the S2- S1589E/S1755E InsP(3)R-1 was enhanced approximately 8-fold over wild type and approximately 50-fold when compared with the nonphosphorylatable S2- S1589A/S1755A mutant. In cells expressing S2- InsP(3)R-1 with single mutations in either S1589E or S1755E, the sensitivity of Ca(2+) release was enhanced approximately 3-fold; sensitivity was midway between the wild type and the double glutamate mutation. Paradoxically, forskolin treatment of cells expressing either single Ser/Glu mutation failed to further enhance Ca(2+) release. The sensitivity of Ca(2+) release in cells expressing S2+ S1755E InsP(3)R-1 was comparable with the sensitivity of S2- S1589E/S1755E InsP(3)R-1. In contrast, mutation of S2+ S1589E InsP(3)R-1 resulted in a receptor with comparable sensitivity to wild type cells. Expression of S2- S1589E/S1755E InsP(3)R-1 resulted in robust Ca(2+) oscillations when cells were stimulated with concentrations of alpha-IgM antibody that were threshold for stimulation in S2- wild type InsP(3)R-1-expressing cells. However, at higher concentrations of alpha-IgM antibody, Ca(2+) oscillations of a similar period and magnitude were initiated in cells expressing either wild type or S2- phosphomimetic mutations. Thus, regulation by phosphorylation of the functional sensitivity of InsP(3)R-1 appears to define the threshold at which oscillations are initiated but not the frequency or amplitude of the signal when established.     

ATP binding to a unique site in the type-1 S2- inositol 1,4,5-trisphosphate receptor defines susceptibility to phosphorylation by protein kinase A

The subtype- and splice variant-specific modulation of inositol 1,4,5-trisphosphate receptors (InsP3R) by interaction with cellular factors plays a fundamental role in defining the characteristics of Ca2+ release in individual cell types. In this study, we investigate the binding properties and functional consequences of the expression of a putative nucleotide binding fold (referred to as the ATPC site) unique to the S2- splice variant of the type-1 InsP3R (InsP3R-1), the predominant splice variant in peripheral tissue. A glutathione S-transferase fusion protein encompassing amino acids 1574-1765 of the S2- InsP3R-1 and including the glycine-rich motif Gly-Tyr-Gly-Glu-Lys-Gly bound ATP specifically as measured by fluorescent trinitrophenyl-ATP binding. This binding was completely abrogated by a point mutation (G1690A) in the nucleotide binding fold. The functional sensitivity of S2- InsP3R-1 constructs was evaluated in DT40-3KO-M3 cells, a null background for InsP3R, engineered to express muscarinic M3 receptors. The S2- InsP3R-1 containing the G1690A mutation was markedly less sensitive to agonist stimulation than wild type S2- InsP3R-1 or receptors containing a similar (Gly --> Ala) mutation in the established nucleotide binding sites in InsP3R-1 (the ATPA and ATPB sites). The ATP sensitivity of InsP3-induced Ca2+ release, however, was not altered by the G1690A mutation when measured in permeabilized DT40-3KO cells, suggesting a unique role for the ATPC site. Ca2+ release was dramatically potentiated following activation of cAMP-dependent protein kinase in DT40-3KO cells transiently expressing wild type S2- InsP3R or Gly --> Ala mutations in the ATPA and ATPB sites, but phosphorylation of the receptor and the potentiation of Ca2+ release were absent in cells expressing the G1690A mutation in S2- InsP3R. These data indicate that ATP binding specifically to the ATPC site in S2- InsP3R-1 controls the susceptibility of the receptor to protein kinase A-mediated phosphorylation, contributes to the functional sensitivity of the S2- InsP3R-1 and ultimately the sensitivity of cells to agonist stimulation.     

Selective phosphorylation of the IP3R-I in vivo by cGMP-dependent protein kinase in smooth muscle

This study examined the expression of inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) types and PKG isoforms in isolated gastric smooth muscle cells and determined the ability of PKG and PKA to phosphorylate IP(3)Rs and inhibit IP(3)-dependent Ca(2+) release, which mediates the initial phase of agonist-induced contraction. PKG-Ialpha and PKG-Ibeta were expressed in gastric smooth muscle cells, together with IP(3)-R-associated cG-kinase substrate, a protein that couples PKG-Ibeta to IP(3)R-I. IP(3)R-I and IP(3)R-III were also expressed, but only IP(3)R-I was phosphorylated by PKA and PKG in vitro and exclusively by PKG in vivo. Sequential phosphorylation by PKA and by PKG-Ialpha in vitro showed that PKA phosphorylated the same site as PKG (presumably S(1755)) and an additional PKA-specific site (S(1589)). In intact muscle cells, agents that activated PKG or both PKG and PKA induced IP(3)R-I phosphorylation that was reversed by the PKG inhibitor (8R,9S,11s)-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,1H,-2,7b,11a-trizadizo-benzo9(a,g)cycloocta(c,d,e)-trinden-1-one. Agents that activated PKA induced IP(3)R-I phosphorylation in permeabilized but not intact muscle cells, implying that PKA does not gain access to IP(3)R-I in intact muscle cells. The pattern of IP(3)R-I phosphorylation in vivo and in vitro was more consistent with phosphorylation by PKG-Ialpha. Phosphorylation of IP(3)R-I in microsomes by PKG, PKA, or a combination of PKG and PKA inhibited IP(3)-induced Ca(2+) release to the same extent, implying that inhibition was mediated by phosphorylation of the PKG-specific site. We conclude that IP(3)R-I is selectively phosphorylated by PKG-I in intact smooth muscle resulting in inhibition of IP(3)-dependent Ca(2+) release.     

Targeted phosphorylation of inositol 1,4,5-trisphosphate receptors selectively inhibits localized Ca2+ release and shapes oscillatory Ca2+ signals

The current study provides biochemical and functional evidence that the targeting of protein kinase A (PKA) to sites of localized Ca(2+) release confers rapid, specific phosphoregulation of Ca(2+) signaling in pancreatic acinar cells. Regulatory control of Ca(2+) release by PKA-dependent phosphorylation of inositol 1,4, 5-trisphosphate (InsP(3)) receptors was investigated by monitoring Ca(2+) dynamics in pancreatic acinar cells evoked by the flash photolysis of caged InsP(3) prior to and following PKA activation. Ca(2+) dynamics were imaged with high temporal resolution by digital imaging and electrophysiological methods. The whole cell patch clamp technique was used to introduce caged compounds and to record the activity of a Ca(2+)-activated Cl(-) current. Photolysis of low concentrations of caged InsP(3) evoked Cl(-) currents that were inhibited by treatment with dibutryl-cAMP or forskolin. In contrast, PKA activators had no significant inhibitory effect on the activation of Cl(-) current evoked by uncaging Ca(2+) or by the photolytic release of higher concentrations of InsP(3). Treatment with Rp-adenosine-3',5'-cyclic monophoshorothioate, a selective inhibitor of PKA, or with Ht31, a peptide known to disrupt the targeting of PKA, largely abolished forskolin-induced inhibition of Ca(2+) release. Further evidence for the targeting of PKA to the sites of Ca(2+) mobilization was revealed using immunocytochemical methods demonstrating that the R(IIbeta) subunit of PKA was localized to the apical regions of acinar cells and co-immunoprecipitated with the type III but not the type I or type II InsP(3) receptors. Finally, we demonstrate that the pattern of signaling evoked by acetylcholine can be converted to one that is more "CCK-like" by raising cAMP levels. Our data provide a simple mechanism by which distinct oscillatory Ca(2+) patterns can be shaped.     

Modulation of cytosolic calcium signaling by protein kinase A-mediated phosphorylation of inositol 1,4,5-trisphosphate receptors

Calcium release via intracellular Ca2+ release channels is a central event underpinning the generation of numerous, often divergent physiological processes. In electrically non-excitable cells, this Ca2+ release is brought about primarily through activation of inositol 1,4,5-trisphosphate receptors and typically takes the form of calcium oscillations. It is widely believed that information is carried in the temporal and spatial characteristics of these signals. Furthermore, stimulation of individual cells with different agonists can generate Ca2+ oscillations with dramatically different spatial and temporal characteristics. Thus, mechanisms must exist for the acute regulation of Ca2+ release such that agonist-specific Ca2+ signals can be generated. One such mechanism by which Ca2+ signals can be modulated is through simultaneous activation of multiple second messenger pathways. For example, activation of both the InsP3 and cAMP pathways leads to the modulation of Ca2+ release through protein kinase A mediated phosphoregulation of the InsP3R. Indeed, each InsP3R subtype is a potential substrate for PKA, although the functional consequences of this phosphorylation are not clear. This review will focus on recent advances in our understanding of phosphoregulation of InsP3R, as well as the functional consequences of this modulation in terms of eliciting specific cellular events.     

Regulation by Ca2+ and inositol 1,4,5-trisphosphate (InsP3) of single recombinant type 3 InsP3 receptor channels. Ca2+ activation uniquely distinguishes types 1 and 3 insp3 receptors

The inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP3R) is an endoplasmic reticulum-localized Ca2+ -release channel that controls complex cytoplasmic Ca(2+) signaling in many cell types. At least three InsP3Rs encoded by different genes have been identified in mammalian cells, with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. To examine regulation of channel gating of the type 3 isoform, recombinant rat type 3 InsP3R (r-InsP3R-3) was expressed in Xenopus oocytes, and single-channel recordings were obtained by patch-clamp electrophysiology of the outer nuclear membrane. Gating of the r-InsP3R-3 exhibited a biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). In the presence of 0.5 mM cytoplasmic free ATP, r-InsP3R-3 gating was inhibited by high [Ca2+]i with features similar to those of the endogenous Xenopus type 1 Ins3R (X-InsP3R-1). Ca2+ inhibition of channel gating had an inhibitory Hill coefficient of approximately 3 and half-maximal inhibiting [Ca2+]i (Kinh) = 39 microM under saturating (10 microM) cytoplasmic InsP3 concentrations ([InsP3]). At [InsP3] < 100 nM, the r-InsP3R-3 became more sensitive to Ca2+ inhibition, with the InsP(3) concentration dependence of Kinh described by a half-maximal [InsP3] of 55 nM and a Hill coefficient of approximately 4. InsP(3) activated the type 3 channel by tuning the efficacy of Ca2+ to inhibit it, by a mechanism similar to that observed for the type 1 isoform. In contrast, the r-InsP3R-3 channel was uniquely distinguished from the X-InsP3R-1 channel by its enhanced Ca2+ sensitivity of activation (half-maximal activating [Ca2+]i of 77 nM instead of 190 nM) and lack of cooperativity between Ca2+ activation sites (activating Hill coefficient of 1 instead of 2). These differences endow the InsP3R-3 with high gain InsP3-induced Ca2+ release and low gain Ca2+ -induced Ca2+ release properties complementary to those of InsP3R-1. Thus, distinct Ca2+ signals may be conferred by complementary Ca2+ activation properties of different InsP3R isoforms.     

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.     

Phosphorylation of the inositol 1,4,5-trisphosphate receptor by cyclic nucleotide-dependent kinases in vitro and in rat cerebellar slices in situ

We have examined cyclic nucleotide-regulated phosphorylation of the neuronal type I inositol 1,4,5-trisphosphate (IP3) receptor immunopurified from rat cerebellar membranes in vitro and in rat cerebellar slices in situ. The isolated IP3 receptor protein was phosphorylated by both cAMP- and cGMP-dependent protein kinases on two distinct sites as determined by thermolytic phosphopeptide mapping, phosphopeptide 1, representing Ser-1589, and phosphopeptide 2, representing Ser-1756 in the rat protein (Ferris, C. D., Cameron, A. M., Bredt, D. S., Huganir, R. L., and Snyder, S. H. (1991) Biochem. Biophys. Res. Commun. 175, 192-198). Phosphopeptide maps show that cAMP-dependent protein kinase (PKA) labeled both sites with the same time course and same stoichiometry, whereas cGMP-dependent protein kinase (PKG) phosphorylated Ser-1756 with a higher velocity and a higher stoichiometry than Ser-1589. Synthetic decapeptides corresponding to the two phosphorylation sites (peptide 1, AARRDSVLAA (Ser-1589), and peptide 2, SGRRESLTSF (Ser-1756)) were used to determine kinetic constants for the phosphorylation by PKG and PKA, and the catalytic efficiencies were in agreement with the results obtained by in vitro phosphorylation of the intact protein. In cerebellar slices prelabeled with [32P]orthophosphate, activation of endogenous kinases by incubation in the presence of cAMP/cGMP analogues and specific inhibitors of PKG and PKA induced in both cases a 3-fold increase in phosphorylation of the IP3 receptor. Thermolytic phosphopeptide mapping of in situ labeled IP3 receptor by PKA showed labeling on the same sites (Ser-1589 and Ser-1756) as in vitro. In contrast to the findings in vitro, PKG preferentially phosphorylated Ser-1589 in situ. Because both PKG and the IP3 receptor are specifically enriched in cerebellar Purkinje cells, PKG may be an important IP3 receptor regulator in vivo.     

Calcium-binding protein 1 is an inhibitor of agonist-evoked, inositol 1,4,5-trisphosphate-mediated calcium signaling

Intracellular calcium signals are responsible for initiating a spectrum of physiological responses. The caldendrins/calcium-binding proteins (CaBPs) represent mammal-specific members of the CaM superfamily. CaBPs display a restricted pattern of expression in neuronal/retinal tissues, suggesting a specialized role in Ca2+ signaling in these cell types. Recently, it was reported that a splice variant of CaBP1 functionally interacts with inositol 1,4,5-trisphosphate (InsP3) receptors to elicit channel activation in the absence of InsP3 (Yang, J., McBride, S., Mak, D.-O. D., Vardi, N., Palczewski, K., Haeseleer, F., and Foskett, J. K. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7711-7716). These data indicate a new mode of InsP3 receptor modulation and hence control of intracellular Ca2+ concentration ([Ca2+]i) in neuronal tissues. We have analyzed the biochemistry of the long form splice variant of CaBP1 (L-CaBP1) and show that, in vitro, a recombinant form of the protein is able to bind Ca2+ with high affinity and undergo a conformational change. We also describe the localization of endogenous and overexpressed L-CaBP1 in the model neuroendocrine PC12 cell system, where it was associated with the plasma membrane and Golgi complex in a myristoylation-dependent manner. Furthermore, we show that overexpressed L-CaBP1 is able to substantially suppress rises in [Ca2+]i in response to physiological agonists acting on purinergic receptors and that this inhibition is due in large part to blockade of release from intracellular Ca2+ stores. The related protein neuronal calcium sensor-1 was without effect on the [Ca2+]i responses to agonist stimulation. Measurement of [Ca2+] within the ER of permeabilized PC12 cells demonstrated that LCaBP1 directly inhibited InsP3-mediated Ca2+ release. Expression of L-CaBP1 also inhibited histamine-induced [Ca2+]i oscillations in HeLa cells. Together, these data suggest that L-CaBP1 is able to specifically regulate InsP3 receptor-mediated alterations in [Ca2+]i during agonist stimulation.     

The contribution of serine residues 1588 and 1755 to phosphorylation of the type I inositol 1,4,5-trisphosphate receptor by PKA and PKG

Type I inositol 1,4,5-trisphosphate receptors can be phosphorylated by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG). To define the site-specificity of these events we analyzed the phosphorylation of mutant receptors expressed in intact cells. These studies showed that S(1588) and S(1755), the serine residues within kinase consensus sequences, are equally sensitive to PKA, that phosphorylation events at these sites are independent of each other, and that PKG predominantly phosphorylates S(1588). These findings provide the basis for understanding the functional consequences of type I inositol 1,4,5-trisphosphate receptor phosphorylation.     

ATP regulation of recombinant type 3 inositol 1,4,5-trisphosphate receptor gating

A family of inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) Ca2+ release channels plays a central role in Ca2+ signaling in most cells, but functional correlates of isoform diversity are unclear. Patch-clamp electrophysiology of endogenous type 1 (X-InsP3R-1) and recombinant rat type 3 InsP3R (r-InsP3R-3) channels in the outer membrane of isolated Xenopus oocyte nuclei indicated that enhanced affinity and reduced cooperativity of Ca2+ activation sites of the InsP3-liganded type 3 channel distinguished the two isoforms. Because Ca2+ activation of type 1 channel was the target of regulation by cytoplasmic ATP free acid concentration ([ATP](i)), here we studied the effects of [ATP]i on the dependence of r-InsP(3)R-3 gating on cytoplasmic free Ca2+ concentration ([Ca2+]i. As [ATP]i was increased from 0 to 0.5 mM, maximum r-InsP3R-3 channel open probability (Po) remained unchanged, whereas the half-maximal activating [Ca2+]i and activation Hill coefficient both decreased continuously, from 800 to 77 nM and from 1.6 to 1, respectively, and the half-maximal inhibitory [Ca2+]i was reduced from 115 to 39 microM. These effects were largely due to effects of ATP on the mean closed channel duration. Whereas the r-InsP3R-3 had a substantially higher Po than X-InsP3R-1 in activating [Ca2+]i (< 1 microM) and 0.5 mM ATP, the Ca2+ dependencies of channel gating of the two isoforms became remarkably similar in the absence of ATP. Our results suggest that ATP binding is responsible for conferring distinct gating properties on the two InsP3R channel isoforms. Possible molecular models to account for the distinct regulation by ATP of the Ca2+ activation properties of the two channel isoforms and the physiological implications of these results are discussed. Complex regulation by ATP of the types 1 and 3 InsP3R channel activities may enable cells to generate sophisticated patterns of Ca2+ signals with cytoplasmic ATP as one of the second messengers.     

cAMP inhibits IP(3)-dependent Ca(2+) release by preferential activation of cGMP-primed PKG

The singular effects and interplay of cAMP- and cGMP-dependent protein kinase (PKA and PKG) on Ca(2+) mobilization were examined in dispersed smooth muscle cells. In permeabilized muscle cells, exogenous cAMP and cGMP inhibited inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release and muscle contraction via PKA and PKG, respectively. A combination of cAMP and cGMP caused synergistic inhibition that was exclusively mediated by PKG and attenuated by PKA. In intact muscle cells, low concentrations (10 nM) of isoproterenol and sodium nitroprusside (SNP) inhibited agonist-induced, IP(3)-dependent Ca(2+) release and muscle contraction via PKA and PKG, respectively. A combination of isoproterenol and SNP increased PKA and PKG activities: the increase in PKA activity reflected inhibition of phosphodiesterase 3 activity by cGMP, whereas the increase in PKG activity reflected activation of cGMP-primed PKG by cAMP. Inhibition of Ca(2+) release and muscle contraction by the combination of isoproterenol and SNP was preferentially mediated by PKG. In light of studies showing that PKG phosphorylates the IP(3) receptor in intact and permeabilized muscle cells, whereas PKA phosphorylates the receptor in permeabilized cells only, the results imply that inhibition of IP(3)-induced Ca(2+) release is mediated exclusively by PKG. The effect of PKA on agonist-induced Ca(2+) release probably reflects inhibition of IP(3) formation.     

Functional characterization of thapsigargin and agonist-insensitive acidic Ca2+ stores in Drosophila melanogaster S2 cell lines

The role of acidic intracellular calcium stores in calcium homeostasis was investigated in the Drosophila Schneider cell line 2 (S2) by means of free cytosolic calcium ([Ca2+]i) and intracellular pH (pHi) imaging together with measurements of total calcium concentrations within intracellular compartments. Both a weak base (NH4Cl, 15 mM) and a Na+/H+ ionophore (monensin, 10 microM) evoked cytosolic alkalinization followed by Ca2+ release from acidic intracellular Ca2+ stores. Pretreatment of S2 cells with either thapsigargin (1 microM), an inhibitor of endoplasmic reticulum Ca(2+)-ATPases, or with the Ca2+ ionophore ionomycin (10 microM) was without effect on the amplitude of Ca2+ release evoked by alkalinization. Application of the cholinergic agonist carbamylcholine (100 microM) to transfected S2-DM1 cells expressing a Drosophila muscarinic acetylcholine receptor (DM1) emptied the InsP3-sensitive Ca2+ store but failed to affect the amplitude of alkalinization-evoked Ca2+ release. Glycyl-L-phenylalanine-beta-naphthylamide (200 microM), a weak hydrophobic base known to permeabilize lysosomes by osmotic swelling, triggered Ca2+ release from internal stores, while application of brefeldin A (10 microM), an antibiotic which disperses the Golgi complex, resulted in a smaller increase in [Ca2+]i. These results suggest that the alkali-evoked calcium release is largely attributable to lysosomes, a conclusion that was confirmed by direct measurements of total calcium content of S2 organelles. Lysosomes and endoplasmic reticulum were the only organelles found to have concentrations of total calcium significantly higher than the cytosol. However, NH4Cl (15 mM) reduced the level of total calcium only in lysosomes. Depletion of acidic Ca2+ stores did not elicit depletion-operated Ca2+ entry. They were refilled upon re-exposure of cells to normal saline ([Ca2+]o = 2 mM), but not by thapsigargin-induced [Ca2+]i elevation in Ca(2+)-free saline.     

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.     

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.     

Kinetics of Ca2+ release and contraction induced by photolysis of caged D-myo-inositol 1,4,5-trisphosphate in smooth muscle. The effects of heparin, procaine, and adenine nucleotides.

The kinetics of Ca2+ release and contraction induced by photolytic release of inositol 1,4,5-trisphosphate (InsP3) were determined in permeabilized smooth muscle. The rate of Ca2+ release was half-maximal at 1 microM InsP3. The concentration-dependent delay of Ca2+ release at saturating InsP3 concentration was approximately 10 ms and within the uncertainty of the measurements. The relationship between the delay and InsP3 concentration showed no evidence of a high level (n = 4 or higher) of cooperativity but could not distinguish between no cooperativity (n = 1) or a low level (n = 2) of cooperativity. Submaximal [InsP3] caused only partial Ca2+ release from the InsP3-sensitive stores. InsP3-induced Ca2+ release was markedly potentiated by ATP or by adenosine 5'-(beta,gamma-methylene-triphosphate), but neither the rate nor the amplitude of release was significantly affected by procaine (2-5 mM). Heparin increased the delay between photolysis and Ca2+ release, indicating that the off rate of inert ligand(s) bound to InsP3 receptors may contribute to the physiological delay in Ca2+ release. There was a much longer (370 ms +/- 45 S.E.) delay between the rise of Ca2+ and force development, presumably reflecting events preceding and associated with myosin light chain phosphorylation.     

Increase in Ca2+ permeability of intracellular Ca2+ store membrane of saponin-treated guinea pig peritoneal macrophages by inositol 1,4,5-trisphosphate

Inositol 1,4,5-trisphosphate (InsP3) releases Ca2+ from the non-mitochondrial Ca2+ store site of various types of cells. To study the mechanisms of the Ca2+ release from the store site, the effect of InsP3 on the passive Ca2+ release and influx, and the active Ca2+ uptake in the presence of oxalate, was examined using saponin-treated guinea pig peritoneal macrophages. InsP3 stimulated the passive Ca2+ release and influx. Although InsP3 slightly inhibited the active Ca2+ uptake in the presence of oxalate, it seems unlikely that the Ca2+ release by this agent is caused by the inhibition of the Ca2+ uptake, because the addition of apyrase or hexokinase (which removes ATP within 30 s, so that no more Ca2+ can be accumulated) or vanadate (which inhibits the Ca2+ uptake) resulted in very slow release of Ca2+. These results suggest that the Ca2+ permeability of the Ca2+ store membrane is increased by InsP3. InsP3 did not cause an increase in the Ca2+ permeability of phospholipid vesicles (liposomes), indicating that this agent may bring about Ca2+ release by a specific effect on the physiologically relevant Ca2+ channels or carriers in the non-mitochondrial Ca2+ store site. The passive Ca2+ release by InsP3 was enhanced by ATP and an unhydrolyzable ATP analogue, 5'-adenylyimidodiphosphate, but not by ADP or AMP. The passive Ca2+ release by InsP3 was observed even at 0 degree C.     

Distribution of inositol 1,4,5-trisphosphate receptor isoforms, SERCA isoforms and Ca binding proteins in RBLm2H3 rat basophilic leukemia cells

RBL-2H3 rat basophilic leukemia cells were homogenized and fractionated. A fraction F3 obtained by differential centrifugation was 6-fold enriched in [3H]-inositol 1,4,5-trisphosphate (InsP3) binding activity, while the NADH-cytochrome c oxidoreductase and sulphatase-C activities were only 3.8- and 2.9-fold enriched, respectively. Furthermore, the three InsP3 receptor (InsP3R) isoforms, two sarco/endoplasmic reticulum Ca ²⁺ ATPase (SERCA) isoforms (2b and 3) as well as four Ca ²⁺ binding proteins (calreticulin, calnexin, protein disulfide isomerase (PDI) and BiP), were present in this fraction. Fraction F3 was, therefore, further purified on a discontinuous sucrose density gradient, and the 3 resulting fractions were analyzed. The InsP₃ binding sites were distributed over the gradient and did not co-migrate with the RNA. We examined the relative content of the three InsP3R isoforms, of both SERCA2b and 3, as well as that of the four Ca ²⁺ binding proteins in fraction F3 and the sucrose density gradient fractions. Ins P₃R-1 and InsP3R-2 showed a similar distribution, with the highest level in the light and intermediate density fractions. InsP₃R-3 distributed differently, with the highest level in the intermediate density fraction. Both SERCA isoforms distributed similarly to InsP₃R-1 and InsP₃R-2. SERCA3 was present at a very low level in the high density fraction. Calreticulin and BiP showed a pattern similar to that of InsP₃R-1 and InsP₃R-2 and the SERCs. PDI was clearly enriched in the light density fraction while calnexin was broadly distributed. These results indicate a heterogeneous distribution of the three InsP₃R isoforms, the two SERCA isoforms and the four Ca²⁺ binding proteins investigated. This heterogeneity may underlie specialization of the Ca²⁺ stores and the subsequent initiation of intracellular Ca²⁺ signals.     

Regulation of the cardiac ryanodine receptor by protein kinase-dependent phosphorylation.

The exogenous addition of the catalytic subunit of cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), or calmodulin (CaM) induced rapid phosphorylation of the ryanodine receptor (Ca2+ release channel) in canine cardiac microsomes treated with 1 mM [gamma-32P]ATP. Added protein kinase C (PKC) also phosphorylated the cardiac ryanodine receptor but at a relatively slow rate. The observed level of PKA-, PKG-, or PKC-dependent phosphorylation of the ryanodine receptor was comparable to the maximum level of [3H]ryanodine binding in cardiac microsomes, whereas the level of CaM-dependent phosphorylation was about 4 times greater. Phosphorylation by PKA, PKG, and PKC increased [3H]ryanodine binding in cardiac microsomes by 22 +/- 5, 17 +/- 4, and 15 +/- 9% (average +/- SD, n = 4-5), respectively. In contrast, incubation of microsomes with 5 microM CaM alone and 5 microM CaM plus 1 mM ATP decreased [3H]ryanodine binding by 38 +/- 14 and 53 +/- 15% (average +/- SD, n = 6), respectively. Phosphopeptide mapping and phosphoamino acid analysis provided evidence suggesting that PKA, PKG, and PKC predominantly phosphorylate serine residue(s) in the same phosphopeptide (peptide 1), whereas the endogenous CaM-kinase phosphorylates serine residue(s) in a different phosphopeptide (peptide 4). Photoaffinity labeling of microsomes with photoreactive 125I-labeled CaM revealed that CaM bound to a high molecular weight protein, which was immunoprecipitated by a monoclonal antibody against the cardiac ryanodine receptor. These results suggest that protein kinase-dependent phosphorylation and CaM play important regulatory roles in the function of the cardiac sarcoplasmic reticulum Ca2+ release channel.