3R Isoforms Research Articles

FKBP12 associates tightly with the skeletal muscle type 1 ryanodine receptor, but not with other intracellular calcium release channels

This study compared the relative levels of ryanodine receptor (RyR) isoforms, inositol 1,4,5-trisphosphate receptor (IP 3R) isoforms, and calcineurin, plus their association with FKBP12 in brain, skeletal and cardiac tissue. FKBP12 demonstrated a very tight, high affinity association with skeletal muscle microsomes, which was displaced by FK506. In contrast, FKBP12 was not tightly associated with brain or cardiac microsomes and did not require FK506 for removal from these organelles. Furthermore, of the proteins solubilised from skeletal muscle, cardiac muscle and brain microsomes, only skeletal muscle RyR1 bound to an FKBP12–glutathione- S-transferase fusion protein, in a high affinity FK506 displaceable manner. These results suggest that RyR1 has distinctive FKBP12 binding properties when compared to RyR2, RyR3, all IP 3R isoforms and calcineurin.

Modulation of inositol 1,4,5-trisphosphate binding to the various inositol 1,4,5-trisphosphate receptor isoforms by thimerosal and cyclic ADP-ribose

Three different genes encode the inositol 1,4,5-trisphosphate (IP 3) receptor (IP 3R), an intracellular Ca 2+ channel involved in cellular Ca 2+ signaling. The IP 3-binding characteristics of the various IP 3R isoforms differ, but until now no specific activators or inhibitors of IP 3 binding have been described. We compared the effects of oxidizing reagents, in particular thimerosal, and of cyclic ADP-ribose (cADPR) on IP 3 binding to the various IP 3R isoforms. We therefore expressed the N-terminal 581 amino acids of the three IP 3R isoforms as recombinant proteins in the soluble fraction of Escherichia coli (ligand-binding sites [lbs] 1, 2, and 3) as well as the full-length IP 3R1 and IP 3R3 in Spodoptera frugiperda (Sf9) insect cells. Thimerosal (100 μM) stimulated IP 3 binding to lbs-1 (1.4-fold) and lbs-3 (2.5-fold), but had no effect on lbs-2. Thimerosal acted on lbs-1 and lbs-3 by decreasing the K d for IP 3 binding (from 46 ± 4 nM to 20 ± 2 nM and from 54 ± 21 nM to 19 ± 7 nM for lbs-1 and -3, respectively) without modifying the B max. Similarly, IP 3 binding to microsomes of Sf9 insect cells overexpressing the full-length IP 3R1 was 1.2-fold stimulated by thimerosal. Thimerosal, however, did not affect IP 3 binding to Sf9–IP 3R3 microsomes, suggesting that in situ thimerosal will only directly affect ligand binding to the type 1 isoform. cADPR (50 μM) stimulated IP 3 binding to Sf9–IP 3R1 microsomes (1.5-fold), but not to Sf9–IP 3R3 microsomes. In addition, cADPR inhibited IP 3 binding to lbs-1 and lbs-2 by decreasing the affinity for IP 3 1.8- and 2.8-fold, respectively, while IP 3 binding to lbs-3 was not affected. These results suggest that a regulatory site for cADPR is present in the ligand-binding domain of IP 3R1 and 2, but not of IP 3R3.

Isoform-Specific Function of Single Inositol 1,4,5-Trisphosphate Receptor Channels

The inositol 1,4,5-trisphosphate receptor (InsP 3R) family of Ca 2+ release channels is central to intracellular Ca 2+ signaling in mammalian cells. The InsP 3R channels release Ca 2+ from intracellular compartments to generate localized Ca 2+ transients that govern a myriad of cellular signaling phenomena (Berridge, 1993. Nature. 361:315–325; Joseph, 1996. Cell Signal. 8:1–7; Kume et al., 1997. Science. 278:1940–1943; Berridge, 1997. Nature. 368:759–760). Most cells express multiple InsP 3R isoforms, but only the function of the single type 1 InsP 3R channel is known. Here the single-channel function of single type 2 InsP 3R channel is defined for the first time. The type 2 InsP 3R forms channels with permeation properties similar to that of the type 1 receptor. The InsP 3 regulation and Ca 2+ regulation of type 1 and type 2 InsP 3R channels are strikingly different. Both InsP 3 and Ca 2+ are more effective at activating single type 2 InsP 3R, indicating that single type 2 channels mobilize substantially more Ca 2+ than single type 1 channels in cells. Furthermore, high cytoplasmic Ca 2+ concentrations inactivate type 1, but not type 2, InsP 3R channels. This indicates that type 2 InsP 3R channel is different from the type 1 channel in that its activity will not be inherently self-limiting, because Ca 2+ passing through an active type 2 channel cannot feed back and turn the channel off. Thus the InsP 3R identity will help define the spatial and temporal nature of local Ca 2+ signaling events and may contribute to the segregation of parallel InsP 3 signaling cascades in mammalian cells.

Agonist-induced down-regulation of type 1 and type 3 inositol 1,4,5-tris-phosphate receptors in A7r5 and DDT 1 MF-2 smooth muscle cells

Prolonged stimulation of rat A7r5 aortic smooth muscle cells with 3 μM vasopressin, or of hamster DDT 1 MF-2 smooth muscle cells with 10 μM bradykinin or 100 μM histamine led within 4 h to a 40–50% down-regulation of the type 1 InsP 3 receptor (InsP 3R-1) and of the type 3 InsP 3 receptor (InsP 3R-3). InsP 3R down-regulation was a cell- and agonist-specific process, since several other agonists acting on PLC-coupled receptors did not change the expression level of the InsP 3R isoforms in these cell types and since no agonist-induced down-regulation of InsP 3Rs was observed in HeLa cells. Down-regulation of InsP 3Rs was prevented by an inhibitor of proteasomal protease activity, N-acetyl-Leu-Leu-norleucinal (ALLN). The Ca 2+ channel blocker verapamil (2 μM) also induced InsP 3R-1 down-regulation (43%) in A7r5 cells, which was inhibited by ALLN. In A7r5 cells transiently transfected with a cDNA construct, bearing a luciferase coding sequence under control of the rat InsP3R-1 promoter, reduced luciferase activity could be demonstrated upon stimulation of cells with vasopressin or verapamil. Thus, besides enhanced protein degradation, a reduction of InsP 3R promoter activity might contribute to the down-regulation of InsP 3Rs in A7r5 cells. We next investigated the effect of InsP 3R down-regulation on Ca 2+ responses in A7r5 cells. A rightward shift in the dose-response curve for InsP 3 induced Ca 2 release was observed in permeabilized monolayers of vasopressinpretreated A7r5 cells (EC 50 630 nM and 400 nM for pretreated and non-pretreated cells, respectively). The Ca 2+ responses to threshold doses of vasopressin were markedly reduced in intact vasopressin-pretreated cells. We conclude that prolonged agonist-exposure leads to down-regulation of InsP 3Rs in A7r5 and DDT 1 MF-2 smooth muscle cells. The mechanism of down-regulation likely involves proteasomal degradation and reduction of InsP 3R promoter activity. Moreover, down-regulation of InsP 3Rs resulted in desensitization of Ca 2+ release from InsP 3 sensitive stores.

The InsP 3 receptor and intracellular Ca 2+ signaling

The inositol 1,4,5-trisphosphate receptor (InsP 3R) is a ligand-gated Ca 2+-release channel on intracellular Ca 2+ store sites (such as the endoplasmic reticulum), and plays an important role in intracellular Ca 2+ signaling in a wide variety of cell types. Recent studies have shown that binding of inositol 1,4,5-trisphosphate (InsP 3) to InsP 3R isoforms is differentially regulated by Ca 2+, and that InsP 3R functions are finely regulated by phosphorylation via tyrosine kinases and protein kinase C, by dephosphorylation via calcineurin, and by binding to FKBP (FK506-binding protein). In addition, transient receptor potential (TRP) and TRP-like proteins appear to couple conformationally with the InsP 3R for capacitative Ca 2+ entry. The importance of InsP 3R signaling in neuronal function has been demonstrated by gene targeting in mice and by studies of T-cell receptor signaling, apoptosis, meiotic maturation, and cytokinesis.

Identification and characterization of inositol 1,4,5-trisphosphate receptors in rat testis

PCR analysis and immunoblotting with isoform specific antibodies was used to identify the presence of type I, II and III inositol 1,4,5-trisphosphate receptors (InsP 3Rs) in rat testis. PCR analysis also revealed that rat testis express both forms of the S1 splice variant (S1 + and S1 −), but only the S2 − form of the S2 splice variant of the type I InsP 3 receptor. PCR analysis was also used to identify InsP 3R isoform expression at a cellular level using myoid, Sertoli and germ cells derived from the testis of Wistar rats. The extent of [ 3H]-Ins P 3 binding was found to be 9 times lower for testicular microsomes than for cerebellar microsomes, with a B max of 1.4 pmoles/mg protein compared to 12.5 pmoles/mg protein for cerebellar microsomes. The K d for InsP 3 binding to its receptor in testicular microsomes was 60 ± 10 nM which was similar to that found for cerebellar microsomes (80 ± 20 nM). InsP 3-induced Ca 2+ release (IICR) in testicular microsomes was found to have an EC 50 (concentration which causes a half-maximal response) of 0.5 ± 0.03 μM, also similar to that seen for cerebellar microsomes (0.3 μM). Maximal IICR occurred at about 20 μM InsP 3, with up to 4% of total intracellular Ca 2+ stores being mobilized as compared to between 10–30% for cerebellar microsomes. Time resolved IICR using stopped-flow spectrofluorimetry, showed the kinetics of IICR for this testis preparation to be monophasic with a maximum rate constant of 0.15 s −1 at 30 μM InsP 3. The rate constants are 7 times slower than values for cerebellar microsomes under similar conditions (∼1 s −1) and taken together with the binding data support the proposal that the receptor density/Ca 2+ store is ∼8 times lower than seen in cerebellar microsomal vesicles. The pharmacological properties as assessed using heparin and InsP 3 analogues also confirmed similar behaviour for testicular InsP 3Rs and cerebellar InsP 3Rs.

Introducing operational diagnostic systems into general health care — Results of the ICD-10 primary health care (PHC) study in German-speaking countries

Tau is a microtubule-binding protein expressed in neurons, and the equal ratios between 4-repeat (4R) and 3-repeat (3R) isoforms are maintained in normal adult brain function. Dysregulation of 3R:4R ratio causes tauopathy, and human neurons that recapitulate tau isoforms in health and disease will provide a platform for elucidating pathogenic processes involving tau pathology. We carried out extensive characterizations of tau isoforms expressed in human neurons derived by microRNA-induced neuronal reprogramming of adult fibroblasts. Transcript and protein analyses showed that miR neurons expressed all six isoforms with the 3R:4R isoform ratio equivalent to that detected in human adult brains. Also, miR neurons derived from familial tauopathy patients with a 3R:4R ratio altering mutation showed increased 4R tau and the formation of insoluble tau with seeding activity. Our results collectively demonstrate the utility of miRNA-induced neuronal reprogramming to recapitulate endogenous tau regulation comparable with the adult brain in health and disease.

Rat basophiliv leukemia cells as model system for inositol 1,4,5-trisphosphate receptor IV, a receptor of the type II family: functional comparison and immunological detection

This study concerns the detection and analysis of the highly homologous type II-like inositol 1,4,5-trisphosphate (InsP 3) receptors (InsP 3R-II, -IV and -V). We have particularly investigated RBL-2H3 cells, which at the mRNA level predominantly expressed InsP 3RIV [De Smedt H. Missiaen L. Parys JB. et al. (1994) Determination of relative amounts of inositol trisphosphate receptor mRNA isoforms by ratio polymerase chain reaction. J. Biol. Chem., 269, 21691–21698]. When measured in identical experimental conditions, microsomes from RBL-2H3 cells were characterized by a much higher InsP 3 binding affinity (Kd 3.8 ± 0.8 nM, B max 0.40 ± 0.08 pmol/mg protein) than microsomes from A7r5 cells (K d 65 ± 7 nM, B max 0.65 ± 0.08 pmol/mg protein) or from cerebellum (K d 135 ± 14 nM, B max 7.35 ± 1.13 pmol/mg protein). An affinity-purified antibody against the C-terminus of type II-like InsP 3Rs detected, after SDS-PAGE and immunoblotting, a 250 kD protein in RBL-2H3 and C 3H10T1/2 cells, but not in other cell types. An isoform-specific antibody against the C-terminus of InsP 3R-I was used to determine the presence of the various InsP 3R-I splice isoforms at the protein level. The 273 kD (brain), 261 kD (peripheral tissues) and 256 kD ( Xenopus oocytes) isoforms were recognized. Expression of InsP 3R-I in RBL-2H3 cells was very low. Taken together, our results support the hypothesis that InsP 3R isoforms may differ to a large extent in their affinity for InsP3 and suggest that RBL-2H3 cells are a useful model for the study of InsP 3R-IV.

A new splice variant of the inositol-1,4,5-triphosphate (IP 3) receptor

In this study we have identified a new splice variant of the IP 3 receptor (IP 3R) transcript in a number of mouse cell lines (e.g. moust T.-lynphoma cells, mouse splenic lymphocytes and mouse NIH 3T3 fibroblast cell lines) using the reverse transcriptase-polymerase chain reacton. This varinet IP 3 receptor (designated as IP 3RV-S2, ≈ 453bp) is larger than the non-neuronal form (402 bp) but smaller than the neuronal form (522 bp) of the IP 3 receptors. Nucleotide sequencing data indicate that this new isoform (IP 3RV-S2) containes a 51 nucleotide insertion within the non-neuronal form of IP 3R at the S2 splice site. During mitogenic stimulation by Con A, the ratio between IP 3R (non-neuronal form) and IP3RV-SE (variant isoform) in mosue splenic T-lymphocytes increased approximately 1.5-fold. The change in relative amounts of these IP 3 receptors isolforms during mitogenic-stimulation suggests that T-lymphocytes may have different requirements for the IP 3 isoforms in order to control intracellular calcium mobilization. The selective expression of these two IP 3R isoforms (IP3RV-S2 and non-neuronal IP 3R) may be critically important for the onset of signal transduction and cell activation.

Presence of Inositol 1,4,5-Trisphosphate Receptor, Calreticulin, and Calsequestrin in Eggs of Sea Urchins and Xenopus laevis

The presence of inositol 1,4,5-trisphosphate receptor (InsP 3R), calreticulin, and calsequestrin was demonstrated in eggs of sea urchins ( Lytechinus pictus, Lytechinus variegatus, and Strongylocentroutus purpuratus) and Xenopus laevis. Binding of inositol 1,4,5-trisphosphate (InsP 3) to microsomes of L. pictus eggs was inhibited by heparin and NaCl. An affinity-purified antibody against the C-terminal of the type I InsP 3R, which recognizes InsP 3R isoforms of rabbit brain (273 kDa) and Xenopus oocytes and eggs (256 kDa), reacted with a 373-kDa protein in sea urchin eggs. The 373-kDa protein was tentatively identified as the sea urchin egg InsP 3R. Observations with fluorescence microscopy indicated that the InsP 3R is present throughout the cytoplasm of sea urchin eggs in a pattern consistent with the distribution of endoplasmic reticulum. Small differences in the relative amount of reaction deposits in cortex vs subcortex were noted among the species of sea urchins examined. Reaction product was also localized to the periphery of female pronuclei in eggs of all three sea urchins. InsP 3R reactivity was present in the perinuclear region, along the periphery of the germinal vesicle, and throughout the animal and vegetal hemispheres of Xenopus oocytes. A similar cytoplasmic staining pattern was also observed in eggs, although islands of reactivity, much larger than those in oocytes, were present in the animal hemisphere of eggs. Calreticulin and calsequestrin in sea urchin eggs had the same molecular mass as in rabbit brain (56 and 60 kDa, respectively), but differed from those present in Xenopus oocytes/eggs (61 and 57 kDa, respectively). The distribution of calreticulin and calsequestrin in both sea urchin and Xenopus oocytes and eggs was similar to that observed for the InsP 3R. These results are discussed in relation to previous studies of Ca 2+ regulation during egg development and fertilization and suggest that in the oocytes and eggs of the species examined, InsP 3-sensitive Ca 2+ stores play an important role in the regulation of cellular Ca 2+.