Agonist-Stimulated Inositol Polyphosphate Formation in Cerebellum

The accumulation of inositol polyphosphates in the cerebellum in response to agonists has not been demonstrated. Guinea pig cerebellar slices prelabeled with [3H]inositol showed the following increases in response to 1 mM serotonin: At 15 s, there was a peak in 3H label in the second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], decreasing to a lower level in about 1 min. The level of 3H label in the putative second-messenger inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] increased rapidly up to 60 s and increased slowly thereafter. The accumulation of 3H label in various inositol phosphate isomers at 10 min, when steady state was obtained, showed the following increases due to serotonin: inositol 1,3,4-trisphosphate [Ins(1,3,4)P3], eight-fold; Ins(1,3,4,5)P4, 6.4-fold; Ins(1,4,5)P3, 75%; inositol 1,4-bisphosphate [Ins(1,4)P2], 0%; inositol 3,4-bisphosphate, 100%; inositol 1-phosphate/inositol 3-phosphate, 30%; and inositol 4-phosphate, 40%. [3H]Inositol 1,3-bisphosphate was not detected in controls, but it accounted for 7.2% of the total inositol bisphosphates formed in the serotonin-stimulated samples. The fact that serotonin did not increase the formation of Ins(1,4)P2 could be due to the fact that Ins(1,4)P2 is rapidly degraded or that Ins(1,4,5)P3 is metabolized primarily by Ins(1,4,5)P3-3'kinase to form Ins(1,3,4,5)P4. In the presence of pargyline (10 microM), [3H]Ins(1,3,4,5)P4 and [3H]Ins(1,3,4)P3 levels were increased, even at 1 microM serotonin. Ketanserin (7 microM) completely inhibited the serotonin effect, indicating stimulation of serotonin2 receptors. Quisqualic acid (100 microM) also increased the levels of [3H]Ins(1,4,5)P3, [3H]Ins(1,3,4,5)P4, and [3H]Ins(1,3,4)P3, but the profile of these increases was different.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ligand Binding Characteristics of a Glycosylphosphatidyl Inositol Membrane-Anchored HeLa Cell Folate Receptor Epitope-Related to Human Milk Folate Binding Protein

The folate receptor (FR) in HeLa cells was characterized as to ligandbinding mechanism, antigenic properties and membrane anchor in order toobtain information to be used for the design of biological agentstargeting FR in malignant tumors. The receptor displayed the followingbinding characteristics in equilibrium dialysis experiments(37°C, pH 7.4) with [³H] folate: a high-affinity type of bindingthat exhibited positive cooperativity with a Hill coefficient >1.0and an upward convex Scatchard plot, a slow radioligand dissociation atpH 7.4 becoming rapid at pH 3.5 and inhibition in the presence of otherfolates. The molecular size of the receptor was 100 kDa on gel filtrationwith Triton X-100, or similar to that of high molecular weight human milkfolate binding protein (FBP). The latter protein represents a 25 kDamolecule which equipped with a hydrophobic glycosylphosphatidylinositol (GPI) membrane anchor susceptible to cleavage byphosphatidylinositol specific phospholipase C (PI-PLC) formsmicelles of 100 kDa size with Triton X-100. The HeLa cell FRimmunoreacted with antibodies against purified human milk FBP inELISA, and in a fluorescence activated cell sorting system, whereHeLa cells exposed to increasing concentrations of antibody showed adose-dependent response. Exposure to PI-PLC decreased the fraction ofimmunolabeled cells indicating a linkage of FR to cell membranes by aGPI anchor. HeLa cells incubated with radiofolate showed a continuousuptake with time, however, with a complete suppression of uptake in thepresence of an excess of cold folate. Prewash of cells at acidic pH toremove endogenous folate increased the uptake. Binding and uptake of [³H]folate was increased in cells grown in a folate-deprived medium. The HeLaFR seems to be epitope related to human milk FBP.     

Characterisation and Distribution of Inositol Polyphosphate and Ryanodine Receptors in the Rat Brain

Abstract: The regional distribution of inositol 1,4,5-trisphosphate (InsP₃), inositol 1,3,4,5-tetrakisphosphate (InsP₄), and ryanodine binding sites has been characterised and compared in the rat brain using radioligand binding assays. Cortical [³H]InsP₃ binding indicated similar positional and stereospecificity as observed in other tissues, with 100-fold selectivity for lnsP₃ over InsP₄. Similarly, high-affinity [³²P]InsP₄ binding also showed a high degree of positional specificity, with a 1,000-fold selectivity for InsP₄ over InsP₃. Initial characterisation of [³H]ryanodine binding to cortical membranes demonstrated that specific binding was highly dependent on high salt and micromolar Ca²⁺ concentrations and inhibited by Ca²⁺ levels of >1 mM. [³H]-Ryanodine binding was also enhanced by β,γ-methylene-adenosine 5′-trisphosphate and caffeine and inhibited by magnesium and ruthenium red (K_i= 0.81 μM). However, dantrolene (300 μM) was ineffective on the binding. Therefore, although the results indicate a greater similarity to the binding properties of the Ca²⁺-induced Ca²⁺ release channel isoform present in skeletal, rather than cardiac, muscle, it does not appear to be identical. Detailed binding analysis of ryanodine and polyphosphate sites, with the exception of ruthenium red, indicated no interaction between binding sites. Ruthenium red markedly enhanced the binding of both [³H]InsP₃ and [³²P]InsP₄, an effect most probably due to nonspecific complex formation. Regional binding of InP₃, InsP₄, and ryanodine in the rat brain was of similar affinity for each ligand in each area, but the density profile for each ligand was clearly different. The highest density of InsP₃ sites was in the cerebellum, whereas the highest density of ryanodine sites was in the hippocampus. High-affinity InsP₄ sites showed less regional diversity, with highest densities in the cerebellum, cortex, and hippocampus. However, in each area studied the density of sites followed the order InsP₃ > InsP₄ > ryanodine.     

The Picrotoxinin Binding Site and Its Relationship to the GABA Receptor Complex

The binding characteristics of [3H] alpha-dihydropicrotoxinin to the picrotoxinin binding site were investigated in membrane preparations of adult rat forebrain and living cultures of rat cerebral cortex. The binding of [3H]alpha-dihydropicrotoxinin to rat forebrain was decreased by lysing, treating with Triton X-100, and heating. Coincubation with gamma-aminobutyric acid (GABA), benzodiazepines, or alterations in the Na+ or Cl- composition of the media had no effect on the binding to the rat brain preparation. However, in the living neurons in tissue culture both GABA and diazepam significantly decreased the binding of [3H]alpha-dihydropicrotoxinin. The dose-response relationships for GABA antagonism of [3H]alpha-dihydropicrotoxinin binding and for picrotoxinin antagonism of the GABA enhancement of [3H]flunitrazepam binding in cultured cortical neurons were also investigated. The Hill coefficients for these actions were reciprocal, suggesting that they result from complementary interactions between the binding sites for GABA and picrotoxinin. These data support the association of the picrotoxinin binding site with the postsynaptic GABA receptor complex.     

Structural Specificity of Polyamines in Modulating the Binding of Estrogen Receptor to Potential Z-DNA Forming Sequences

Abstract

Estrogen receptor (ER) is a gene-regulatory protein that mediates the action of estradiol. In order to examine the role of conformational dynamics of DNA in estrogenic regulation of gene expression, we studied the binding of ER to poly(dA-dC).poly(dG-dT) which undergoes transition to a left-handed Z-DNA form. This type of dinucleotide repeats are widely distributed in mammalian genome and are present in estrogen response elements. Binding affinity of ER for the polynucleotide was assessed by its ability to release ER bound to DNA-cellulose. ER binding by poly(dA-dC).poly(dG-dT) was enhanced in the presence of an endogenous polyamine, spermidine, H₂N(CH₂)₄NH(CH₂)₃NH₂. The concentration of spermidine required for facilitating 50% elution of ER (EC₅₀) was 75 μM. This EC₅₀ increased to 500 μM for a spermidine homolog, H₂N(CH₂)₈NH(CH₂)₃NH₂, demonstrating polyamine structural specificity. Spectroscopic measurements showed that the presence of 100 – 200 μM spermidine initiated changes in the conformation of the polynucleotide indicative of Z-DNA form, but a major alteration to Z-DNA spectrum occurred only at 300 μM concentration. These data suggest that ER favors DNA sequences poised for Z-DNA transition. The efficacy of spermidine homologs in facilitating ER-DNA interaction may be important in predicting their efficiency to replace cellular functions of spermidine.     

Crystal Structure of the Mineralocorticoid Receptor DNA Binding Domain in Complex with DNA

The steroid hormone receptors regulate important physiological functions such as reproduction, metabolism, immunity, and electrolyte balance. Mutations within steroid receptors result in endocrine disorders and can often drive cancer formation and progression. Despite the conserved three-dimensional structure shared among members of the steroid receptor family and their overlapping DNA binding preference, activation of individual steroid receptors drive unique effects on gene expression. Here, we present the first structure of the human mineralocorticoid receptor DNA binding domain, in complex with a canonical DNA response element. The overall structure is similar to the glucocorticoid receptor DNA binding domain, but small changes in the mode of DNA binding and lever arm conformation may begin to explain the differential effects on gene regulation by the mineralocorticoid and glucocorticoid receptors. In addition, we explore the structural effects of mineralocorticoid receptor DNA binding domain mutations found in type I pseudohypoaldosteronism and multiple types of cancer.     

Inositol Polyphosphate Multikinase Signaling: Multifaceted Functions in Health and Disease

Inositol phosphates are water-soluble intracellular signaling molecules found in eukaryotes from yeasts to mammals, which are synthesized by a complex network of enzymes including inositol phosphate kinases. Among these, inositol polyphosphate multikinase (IPMK) is a promiscuous enzyme with broad substrate specificity, which phosphorylates multiple inositol phosphates, as well as phosphatidylinositol 4,5-bisphosphate. In addition to its catalytic actions, IPMK is known to non-catalytically control major signaling events via direct protein-protein interactions. In this review, we describe the general characteristics of IPMK, highlight its pleiotropic roles in various physiological and pathological conditions, and discuss future challenges in the field of IPMK signaling pathways.     

T-cell Receptor (TCR)-Peptide Specificity Overrides Affinity-enhancing TCR-Major Histocompatibility Complex Interactions

αβ T-cell receptors (TCRs) engage antigens using complementarity-determining region (CDR) loops that are either germ line-encoded (CDR1 and CDR2) or somatically rearranged (CDR3). TCR ligands compose a presentation platform (major histocompatibility complex (MHC)) and a variable antigenic component consisting of a short “foreign” peptide. The sequence of events when the TCR engages its peptide-MHC (pMHC) ligand remains unclear. Some studies suggest that the germ line elements of the TCR engage the MHC prior to peptide scanning, but this order of binding is difficult to reconcile with some TCR-pMHC structures. Here, we used TCRs that exhibited enhanced pMHC binding as a result of mutations in either CDR2 and/or CDR3 loops, that bound to the MHC or peptide, respectively, to dissect the roles of these loops in stabilizing TCR-pMHC interactions. Our data show that TCR-peptide interactions play a strongly dominant energetic role providing a binding mode that is both temporally and energetically complementary with a system requiring positive selection by self-pMHC in the thymus and rapid recognition of non-self-pMHC in the periphery.     

The Inositol Trisphosphate Receptor in the Control of Autophagy

The second messenger myo-inositol-1,4,5-trisphosphate (IP₃) acts on the IP₃ receptor (IP₃R), an IP3-activated Ca²⁺ channel of the endoplasmic reticulum (ER). The IP₃R agonist IP3 inhibits starvation-induced autophagy. The IP₃R antagonist xestospongin B induces autophagy in human cells through a pathway that requires the obligate contribution of Beclin-1, Atg5, Atg10, Atg12 and hVps34, yet is inhibited by ER-targeted Bcl-2 or Bcl-X_L, two proteins that physically interact with IP₃R. Autophagy can also be induced by depletion of the IP₃R by small interfering RNAs. Autophagy induction by IP3R blockade cannot be explained by changes in steady state levels of Ca²⁺ in the endoplasmic reticulum (ER) and the cytosol. Autophagy induction by IP₃R blockade is effective in cells lacking the obligate mediator of ER stress IRE1. In contrast, IRE1 is required for autophagy induced by ER stress-inducing agents such a tunicamycin or thapsigargin. These findings suggest that there are several distinct pathways through which autophagy can be initiated at the level of the ER.

Addendum to:

Regulation of Autophagy by the Inositol Trisphosphate Receptor

A. Criollo, M.C. Maiuri, E. Tasdemir, I. Vitale, A.A. Fiebig, D. Andrews, J. Molgo, J. Diaz, S. Lavandero, F. Harper, G. Pierron, D. di Stefano, R. Rizzuto, G. Szabadkai and G. Kroemer

Cell Death Differ 2007; In press     

Differential Substrate Specificity of Two Inositol Transporters of Bacillus subtilis

Bacillus subtilis IolT is the major myo-inositol transporter for growth, while IolF is a minor one unable to support growth. We found that either IolT or IolF was sufficient for moderate growth using D-chiro-inositol. Conversely to IolT, IolF transported D-chiro-inositol more preferentially than myo-inositol. These results indicate that IolT and IolF are different in substrate specificity.     

Specificity of Muscarinic Acetylcholine Receptor Regulation by Receptor Activity

Abstract— Regulation of muscarinic acetylcholine receptor concentration by receptor activity in neuron-like NG108-15 hybrid cells is a highly specific process. Receptor levels, monitored by binding of [³H]quinuclidinyl benzilate ([³H]QNB), decreased 50-75% following 24-h incubation of cells with muscarinic agonists, but none of the following cellular processes was altered by this chronic receptor stimulation: (1) glycolytic energy metabolism, measured by [³H]deoxy- d -glucose ([³H]DG) uptake and retention; (2) rate of cell division; (3) transport, measured by [³H]valine and [³H]uridine uptake; (4) RNA biosynthesis, measured by [³H]uridine incorporation; (5) protein biosynthesis, measured by [³H]valine and [³⁵S]methionine incorporation into total protein and into protein fractions obtained by polyacrylamide gel electrophoresis. In contrast, chronic stimulation did cause a threefold decrease in the capacity of carbachol to stimulate phosphatidylinositol (PI) turnover, a receptor-mediated response. In addition to cholinomimetics, the neuroeffector adenosine (1 m m for 24 h) also caused a decrease in [³H]QNB binding levels, but chronic stimulation of α -adrenergic, opiate, prostaglandin E₁, and prostaglandin F_2α receptors found on NG108-15 cells caused no changes. The data indicate that loss of muscarinic receptors caused by receptor stimulation is not a consequence of fundamental changes evoked in overall cellular physiology but reflects a specific regulation of cholinoceptive cell responsiveness.     

Association of the Inositol (1,4,5)-Trisphosphate Receptor Ligand Binding Site with Phosphatidylinositol (4,5)-Bisphosphate and Adenophostin A

The inositol 1,4,5-trisphosphate receptor (InsP₃R) is activated by InsP₃ binding to amino-terminal ligand binding domain (InsP₃R-N). Recently we reported functional coupling of phosphatidylinositol (4,5)-bisphosphate (PIP₂) to the InsP₃R. Specific binding of PIP₂ to InsP₃R-N domain was postulated as a part of the InsP₃R-PIP₂ functional coupling model. Here we utilized bacterially expressed and purified InsP₃R-N domain to characterize its binding specificity for InsP₃, Adenophostin A (AdA) and the water-soluble PIP₂ analog dioctanoyl-(4,5)PIP₂ (ShPIP₂). Obtained data led us to conclude that specific InsP₃, AdA, and ShPIP₂ binding sites are located within the InsP₃R-N domain, that the extra receptor binding element responsible for enhanced binding of AdA is an integral part of the InsP₃R-N domain, that ShPIP₂ is able to displace InsP₃ from the InsP₃R-N, but InsP₃ or AdA is unable to completely displace ShPIP₂. These results support the InsP₃R-PIP₂ functional coupling model and provide novel insights into InsP₃R ligand specificity.     

多聚磷酸盐聚合酶VTC复合体的结构和功能

磷酸盐(P_i)稳态在所有生物体中都是一个受严格调控的过程,其功能障碍会导致人类肾范科尼综合征(Fanconi syndrome)、植物生长迟缓和微生物致死等多种功能紊乱。为了在P_i的生物合成需求和胞质P_i浓度过高的风险之间实现平衡,细胞以无机多聚磷酸盐(polyP)的形式将P_i储存在膜结合的酸钙小体样细胞器中。酿酒酵母液泡转运蛋白伴侣(vacuolar transporter chaperone,VTC)复合体作为已知的真核生物多聚磷酸盐聚合酶,利用ATP在胞质中合成polyP,并将其转运到液泡中储存起来以维持细胞内P_i稳态。本文从结构特征、polyP合成及polyP转运机制等方面介绍了VTC复合体结构和功能的最新研究进展,着重介绍了最近发表的完整VTC复合体的结构信息,并探讨了VTC的激活机制。     

Dual Effects of K+ Depoiarisation on Inositol Polyphosphate Production in Rat Cerebral Cortex

Depolarisation of [3H]inositol-prelabelled slices of rat cerebral cortex with elevated extracellular K+ induced a rapid and marked increase in inositol polyphosphate accumulation. Addition of the muscarinic antagonist atropine (10 microM) markedly inhibited the K+-induced accumulation of inositol tetrakisphosphate (InsP4), with only a slight reduction in stimulated inositol bis- and trisphosphate levels. Inhibitory effects on InsP4 were noted at the earliest time period measured (30 s) and suggested the involvement of released endogenous acetylcholine in part of the response. The atropine-insensitive component of depolarisation did not appear to be secondary to release of noradrenaline, histamine, or 5-hydroxytryptamine, because addition of prazosin, mepyramine, or ketanserin was without effect on the K+ response. Furthermore, secretion of a neuropeptide that could stimulate phosphoinositide hydrolysis was unlikely, because the peptidase inhibitor bacitracin was also without effect. The results suggest that endogenous acetylcholine can stimulate phosphoinositide metabolism by interacting with muscarinic receptors and that this is particularly evident on InsP4 accumulation. Atropine-insensitive responses may be secondary to Ca2+ entry via voltage-sensitive channels.     

Binding Specificity of the Lantibiotic-Binding Immunity Protein NukH

NukH is a lantibiotic-binding immunity protein that shows strong binding activity against type A(II) lantibiotics. In this study, the binding specificity of NukH was analyzed by using derivatives of nukacin ISK-1, which is a type A(II) lantibiotic produced by Staphylococcus warneri ISK-1. Interactions between cells of Lactococcus lactis transformants expressing nukH and nukacin ISK-1 derivatives were analyzed by using a quantitative peptide-binding assay. Differences in the cell-binding rates of each nukacin ISK-1 derivative suggested that three lysine residues at positions 1 to 3 of nukacin ISK-1 contribute to the effective binding of nukacin ISK-1 to nukH-expressing cells. The binding levels of mutants with lanthionine and dehydrobutyrine substitutions (S11A nukacin_4-27 and T24A nukacin_4-27, respectively) to nukH-expressing cells were considerably lower than those of nukacin_4-27, suggesting that unusual amino acids play a decisive role in NukH recognition. Additionally, it was suggested that T9A nukacin_4-27, a mutant with a 3-methyllanthionine substitution, binds to NukH via an intermolecular disulfide bond after it is weakly recognized by NukH. We succeeded in the detection of specific type A(II) lantibiotics from the culture supernatants of various bacteriocin producers by using the binding specificity of nukH-expressing cells.     

红花菜豆凝集素的糖结合专一性

经肼解、Ｂｉｏ－Ｇｅｌ Ｐ－２柱层析、ＮａＢ＾３Ｈ４和ＮａＢＨ４还原,制备各种来源的、氚标记在还原末端的、还原末端为Ｎ－乙酰氨基葡萄糖醇的混合寡糖,经Ｂｉｏ－Ｇｅｌ Ｐ－４凝胶柱分离,以及用糖苷酶酶解,制备了各种不同类型的氚标记的寡糖。这些寡糖在固定化的ＰＣＬ－Ｓｅｐｈａｒｏｓｅ柱上亲和层析,根据各种类型寡糖在ＰＣＬ－Ｓｅｐｈａｒｏｓｅ柱上的层析行为,确定红花菜豆（矮生红花变种）凝集素（ＰＣＬ）的     

Lithium Reduqes the Accumulation or Inositol Polyphosphate Second Messengers Following Cholinergic Stimulation of Cerebral Cortex Slices

Abstract: The ability of lithium to interfere with the metabolism of inositol phosphates in brain may underlie its therapeutic action in manic-depressive illness. In these experiments, lithium, at therapeutic concentrations, enhanced the accumulation of [³H]inpsitol monophosphate but suppressed the accumulation of the putative second messengers [³H]inositol 1,4,5-trisphosphate ([³H]Ins(1,4,5)P₃) and f³H]inositol 1,3,4,5-tetrakisphosphate following stimulation of cerebral cortex slices with carbachol. Mass measurements of Ins(1,4,5)P₃showed similar inhibitory effects, which could be prevented by preincubation with myo -inositol. These data may reveal the mechanism by which lithium can reduce polyphosphoinositide-midiated neurotransmission in brain.