Gβ5 prevents the RGS7-Gαo interaction through binding to a distinct Gγ-like domain found in RGS7 and other RGS proteins

The G protein β subunit Gβ5 deviates significantly from the other four members of Gβ-subunit family in amino acid sequence and subcellular localization. To detect the protein targets of Gβ5 in vivo, we have isolated a native Gβ5 protein complex from the retinal cytosolic fraction and identified the protein tightly associated with Gβ5 as the regulator of G protein signaling (RGS) protein, RGS7. Here we show that complexes of Gβ5 with RGS proteins can be formed in vitro from the recombinant proteins. The reconstituted Gβ5-RGS dimers are similar to the native retinal complex in their behavior on gel-filtration and cation-exchange chromatographies and can be immunoprecipitated with either anti-Gβ5 or anti-RGS7 antibodies. The specific Gβ5-RGS7 interaction is determined by a distinct domain in RGS that has a striking homology to Gγ subunits. Deletion of this domain prevents the RGS7-Gβ5 binding, although the interaction with Gα is retained. Substitution of the Gγ-like domain of RGS7 with a portion of Gγ1 changes its binding specificity from Gβ5 to Gβ1. The interaction of Gβ5 with RGS7 blocked the binding of RGS7 to the Gα subunit Gαo, indicating that Gβ5 is a specific RGS inhibitor.

Atrial natriuretic peptide-stimulated Ca2+ reabsorption in rabbit kidney requires membrane-targeted, cGMP-dependent protein kinase type II

Atrial natriuretic peptide (ANP) and nitric oxide (NO) are key regulators of ion and water transport in the kidney. Here, we report that these cGMP-elevating hormones stimulate Ca2+ reabsorption via a novel mechanism specifically involving type II cGMP-dependent protein kinase (cGK II). ANP and the NO donor, sodium nitroprusside (SNP), markedly increased Ca2+ uptake in freshly immunodissected rabbit connecting tubules (CNT) and cortical collecting ducts (CCD). Although readily increasing cGMP, ANP and SNP did not affect Ca2+ and Na+ reabsorption in primary cultures of these segments. Immunoblot analysis demonstrated that cGK II, and not cGK I, was present in freshly isolated CNT and CCD but underwent a complete down-regulation during the primary cell culture. However, upon adenoviral reexpression of cGK II in primary cultures, ANP, SNP, and 8-Br-cGMP readily increased Ca2+ reabsorption. In contrast, no cGMP-dependent effect on electrogenic Na+ transport was observed. The membrane localization of cGK II proved to be crucial for its action, because a nonmyristoylated cGK II mutant that was shown to be localized in the cytosol failed to mediate ANP-stimulated Ca2+ transport. The Ca2+-regulatory function of cGK II appeared isotype-specific because no cGMP-mediated increase in Ca2+ transport was observed after expression of the cytosolic cGK Iβ or a membrane-bound cGK II/Iβ chimer. These results demonstrate that ANP- and NO-stimulated Ca2+ reabsorption requires membrane-targeted cGK II.

Mechanism of Ca2+-dependent nuclear accumulation of calmodulin

The intracellular Ca2+ receptor calmodulin (CaM) coordinates responses to extracellular stimuli by modulating the activities of its various binding proteins. Recent reports suggest that, in addition to its familiar functions in the cytoplasm, CaM may be directly involved in rapid signaling between cytoplasm and nucleus. Here we show that Ca2+-dependent nuclear accumulation of CaM can be reconstituted in permeabilized cells. Accumulation was blocked by M13, a CaM antagonist peptide, but did not require cytosolic factors or an ATP regenerating system. Ca2+-dependent influx of CaM into nuclei was not blocked by inhibitors of nuclear localization signal-mediated nuclear import in either permeabilized or intact cells. Fluorescence recovery after photobleaching studies of CaM in intact cells showed that influx is a first-order process with a rate constant similar to that of a freely diffusible control molecule (20-kDa dextran). Studies of CaM efflux from preloaded nuclei in permeablized cells revealed the existence of three classes of nuclear binding sites that are distinguished by their Ca2+-dependence and affinity. At high [Ca2+], efflux was enhanced by addition of a high affinity CaM-binding protein outside the nucleus. These data suggest that CaM diffuses freely through nuclear pores and that CaM-binding proteins in the nucleus act as a sink for Ca2+-CaM, resulting in accumulation of CaM in the nucleus on elevation of intracellular free Ca2+.

Convergent evolution of Trichomonas vaginalis lactate dehydrogenase from malate dehydrogenase

Lactate dehydrogenase (LDH) is present in the amitochondriate parasitic protist Trichomonas vaginalis and some but not all other trichomonad species. The derived amino acid sequence of T. vaginalis LDH (TvLDH) was found to be more closely related to the cytosolic malate dehydrogenase (MDH) of the same species than to any other LDH. A key difference between the two T. vaginalis sequences was that Arg91 of MDH, known to be important in coordinating the C-4 carboxyl of oxalacetate/malate, was replaced by Leu91 in LDH. The change Leu91Arg by site-directed mutagenesis converted TvLDH into an MDH. The reverse single amino acid change Arg91Leu in TvMDH, however, gave a product with no measurable LDH activity. Phylogenetic reconstructions indicate that TvLDH arose from an MDH relatively recently.

A deoxyribonucleotidase in mitochondria: Involvement in regulation of dNTP pools and possible link to genetic disease

Three cytosolic and one plasma membrane-bound 5′-nucleotidases have been cloned and characterized. Their various substrate specificities suggest widely different functions in nucleotide metabolism. We now describe a 5′-nucleotidase in mitochondria. The enzyme, named dNT-2, dephosphorylates specifically the 5′- and 2′(3′)-phosphates of uracil and thymine deoxyribonucleotides. The cDNA of human dNT-2 codes for a 25.9-kDa polypeptide with a typical mitochondrial leader peptide, providing the structural basis for two-step processing during import into the mitochondrial matrix. The deduced amino acid sequence is 52% identical to that of a recently described cytosolic deoxyribonucleotidase (dNT-1). The two enzymes share many catalytic properties, but dNT-2 shows a narrower substrate specificity. Mitochondrial localization of dNT-2 was demonstrated by the mitochondrial fluorescence of 293 cells expressing a dNT-2-green fluorescent protein (GFP) fusion protein. 293 cells expressing fusion proteins without leader peptide or with dNT-1 showed a cytosolic fluorescence. During in vitro import into mitochondria, the preprotein lost the leader peptide. We suggest that dNT-2 protects mitochondrial DNA replication from overproduction of dTTP, in particular in resting cells. Mitochondrial toxicity of dTTP can be inferred from a severe inborn error of metabolism in which the loss of thymidine phosphorylase led to dTTP accumulation and aberrant mitochondrial DNA replication. We localized the gene for dNT-2 on chromosome 17p11.2 in the Smith–Magenis syndrome-critical region, raising the possibility that dNT-2 is involved in the etiology of this genetic disease.

Calcium release from the endoplasmic reticulum of higher plants elicited by the NADP metabolite nicotinic acid adenine dinucleotide phosphate

Higher plants share with animals a responsiveness to the Ca2+ mobilizing agents inositol 1,4,5-trisphosphate (InsP3) and cyclic ADP-ribose (cADPR). In this study, by using a vesicular 45Ca2+ flux assay, we demonstrate that microsomal vesicles from red beet and cauliflower also respond to nicotinic acid adenine dinucleotide phosphate (NAADP), a Ca2+-releasing molecule recently described in marine invertebrates. NAADP potently mobilizes Ca2+ with a K1/2 = 96 nM from microsomes of nonvacuolar origin in red beet. Analysis of sucrose gradient-separated cauliflower microsomes revealed that the NAADP-sensitive Ca2+ pool was derived from the endoplasmic reticulum. This exclusively nonvacuolar location of the NAADP-sensitive Ca2+ pathway distinguishes it from the InsP3- and cADPR-gated pathways. Desensitization experiments revealed that homogenates derived from cauliflower tissue contained low levels of NAADP (125 pmol/mg) and were competent in NAADP synthesis when provided with the substrates NADP and nicotinic acid. NAADP-induced Ca2+ release is insensitive to heparin and 8-NH2-cADPR, specific inhibitors of the InsP3- and cADPR-controlled mechanisms, respectively. However, NAADP-induced Ca2+ release could be blocked by pretreatment with a subthreshold dose of NAADP, as previously observed in sea urchin eggs. Furthermore, the NAADP-gated Ca2+ release pathway is independent of cytosolic free Ca2+ and therefore incapable of operating Ca2+-induced Ca2+ release. In contrast to the sea urchin system, the NAADP-gated Ca2+ release pathway in plants is not blocked by L-type channel antagonists. The existence of multiple Ca2+ mobilization pathways and Ca2+ release sites might contribute to the generation of stimulus-specific Ca2+ signals in plant cells.

An ethylene-induced cDNA encoding a lipase expressed at the onset of senescence

A cDNA clone encoding a lipase (lipolytic acyl hydrolase) expressed at the onset of petal senescence has been isolated by screening a cDNA expression library prepared from carnation flowers (Dianthus caryophyllus). The cDNA contains the lipase consensus sequence, ITFAGHSLGA, and encodes a 447-amino acid polypeptide with a calculated molecular mass of 50.2 kDa that appears to be a cytosolic protein. Over-expression of the clone in Escherichia coli yielded a protein of the expected molecular weight that proved capable of deesterifying fatty acids from p-nitrophenylpalmitate, tri-linolein, soybean phospholipid, and Tween in both in vitro and in situ assays of enzyme activity. The abundance of the lipase mRNA increases just as carnation flowers begin to senesce, and expression of the gene is also induced by treatment with ethylene. Southern blot analyses of carnation genomic DNA have indicated that the lipase is a single copy gene. The lipase gene is also expressed in carnation leaves and is up-regulated when the leaves are treated with ethylene. Deesterification of membrane lipids and ensuing loss of membrane structural integrity are well established early events of plant senescence, and the expression pattern of this lipase gene together with the lipolytic activity of its cognate protein indicate that it plays a fundamentally central role in mediating the onset of senescence.

A calcium influx is triggered and propagates in the zygote as a wavefront during in vitro fertilization of flowering plants

In this paper, we report direct measurement of an influx of extracellular Ca2+ induced by gamete fusion in flowering plants. This result was obtained during maize in vitro fertilization with the use of an extracellular Ca2+-selective vibrating probe. Ca2+ influx recorded at the surface of isolated egg cells, with or without adhesion of a male sperm cell, was close to zero and stable over time. Gamete fusion, however, triggered a Ca2+ influx in the vicinity of the sperm entry site with a delay of 1.8 ± 0.6 sec. The Ca2+ influx spread subsequently through the whole egg cell plasma membrane as a wavefront, progressing at an estimated rate of 1.13 μm⋅sec−1. Once established, Ca2+ influx intensities were sustained, monotonic and homogeneous over the whole egg cell, with an average peak influx of 14.92 pmol⋅cm−2⋅sec−1 and an average duration of 24.4 min. The wavefront spread of channel activation correlates well with the cytological modifications induced by fertilization, such as egg cell contraction, and with the cytosolic Ca2+ (c[Ca2+]) elevation previously reported. Calcium influx was inhibited effectively by gadolinium, possibly implicating mechanosensitive channels. Furthermore, artificial influxes created by incubation with Ca2+ ionophores mimicked some aspects of egg activation. Taken together, these results suggest that, during fertilization in higher plants, gamete membrane fusion starts the first embryonic events by channel opening and Ca2+ influx. In turn, c[Ca2+] may work as a trigger and possibly a space and time coordinator of many aspects of egg activation.

Glutathione biosynthesis in Arabidopsis trichome cells

In Arabidopsis thaliana, trichome cells are specialized unicellular structures with uncertain functions. Based on earlier observations that one of the genes involved in cysteine biosynthesis (Atcys-3A) is highly expressed in trichomes, we have extended our studies in trichome cells to determine their capacity for glutathione (GSH) biosynthesis. First, we have analyzed by in situ hybridization the tissue-specific expression of the genes Atcys-3A and sat5, which encode O-acetylserine(thio)lyase (OASTL) and serine acetyltransferase (SAT), respectively, as well as gsh1 and gsh2, which encode γ-glutamylcysteine synthetase and glutathione synthetase, respectively. The four genes are highly expressed in leaf trichomes of Arabidopsis, and their mRNA accumulate to high levels. Second, we have directly measured cytoplasmic GSH concentration in intact cells by laser-scanning microscopy after labeling with monochlorobimane as a GSH-specific probe. From these measurements, cytosolic GSH concentrations of 238 ± 25, 80 ± 2, and 144 ± 19 μM were estimated for trichome, basement, and epidermal cells, respectively. Taking into account the volume of the cells measured using stereological techniques, the trichomes have a total GSH content more than 300-fold higher than the basement and epidermal cells. Third, after NaCl treatment, GSH biosynthesis is markedly decreased in trichomes. Atcys-3A, sat5, gsh1, and gsh2 mRNA levels show a decrease in transcript abundance, and [GSH]cyt is reduced to 47 ± 5 μM. These results suggest the important physiological significance of trichome cells related to GSH biosynthesis and their possible role as a sink during detoxification processes.

Calcium-dependent enzyme activation and vacuole formation in the apical granular region of pancreatic acinar cells

The pancreatic acinar cell produces powerful digestive enzymes packaged in zymogen granules in the apical pole. Ca2+ signals elicited by acetylcholine or cholecystokinin (CCK) initiate enzyme secretion by exocytosis through the apical membrane. Intracellular enzyme activation is normally kept to a minimum, but in the often-fatal human disease acute pancreatitis, autodigestion occurs. How the enzymes become inappropriately activated is unknown. We monitored the cytosolic Ca2+ concentration ([Ca2+]i), intracellular trypsin activation, and its localization in isolated living cells with specific fluorescent probes and studied intracellular vacuole formation by electron microscopy as well as quantitative image analysis (light microscopy). A physiological CCK level (10 pM) eliciting regular Ca2+ spiking did not evoke intracellular trypsin activation or vacuole formation. However, stimulation with 10 nM CCK, evoking a sustained rise in [Ca2+]i, induced pronounced trypsin activation and extensive vacuole formation, both localized in the apical pole. Both processes were abolished by preventing abnormal [Ca2+]i elevation, either by preincubation with the specific Ca2+ chelator 1,2-bis(O-aminophenoxy)ethane-N,N-N′,N′-tetraacetic acid (BAPTA) or by removal of external Ca2+. CCK hyperstimulation evokes intracellular trypsin activation and vacuole formation in the apical granular pole. Both of these processes are mediated by an abnormal sustained rise in [Ca2+]i.

Heterogeneity of subcellular localization and electrophoretic mobility of survival motor neuron (SMN) protein in mammalian neural cells and tissues

Spinal muscular atrophy is caused by defects in the survival motor neuron (SMN) gene. To better understand the patterns of expression of SMN in neuronal cells and tissues, we raised a polyclonal antibody (abSMN) against a synthetic oligopeptide from SMN exon 2. AbSMN immunostaining in neuroblastoma cells and mouse and human central nervous system (CNS) showed intense labeling of nuclear “gems,” along with prominent nucleolar immunoreactivity in mouse and human CNS tissues. Strong cytoplasmic labeling was observed in the perikarya and proximal dendrites of human spinal motor neurons but not in their axons. Immunoblot analysis revealed a 34-kDa species in the insoluble protein fractions from human SY5Y neuroblastoma cells, embryonic mouse spinal cord cultures, and human CNS tissue. By contrast, a 38-kDa species was detected in the cytosolic fraction of SY5Y cells. We conclude that SMN protein is expressed prominently in both the cytoplasm and nucleus in multiple types of neurons in brain and spinal cord, a finding consistent with a role for SMN as a determinant of neuronal viability.

Ribonuclease A variants with potent cytotoxic activity

Select members of the bovine pancreatic ribonuclease A (RNase A) superfamily are potent cytotoxins. These cytotoxic ribonucleases enter the cytosol, where they degrade cellular RNA and cause cell death. Ribonuclease inhibitor (RI), a cytosolic protein, binds to members of the RNase A superfamily with inhibition constants that span 10 orders of magnitude. Here, we show that the affinity of a ribonuclease for RI plays an integral role in defining the potency of a cytotoxic ribonuclease. RNase A is not cytotoxic and binds RI with high affinity. Onconase, a cytotoxic RNase A homolog, binds RI with low affinity. To disrupt the RI-RNase A interaction, three RNase A residues (Asp-38, Gly-88, and Ala-109) that form multiple contacts with RI were replaced with arginine. Replacing Asp-38 and Ala-109 with an arginine residue has no effect on the RI–RNase interaction. In addition, these variants are not cytotoxic. In contrast, replacing Gly-88 with an arginine residue yields a ribonuclease (G88R RNase A) that retains catalytic activity in the presence of RI and is cytotoxic to a transformed cell line. Replacing Gly-88 with aspartate also yields a ribonuclease (G88D RNase A) with a decreased affinity for RI and cytotoxic activity. The cytotoxic potency of onconase, G88R RNase A, and G88D RNase A correlate with RI evasion. We conclude that ribonucleases that retain catalytic activity in the presence of RI are cytotoxins. This finding portends the development of a class of chemotherapeutic agents based on pancreatic ribonucleases.

Activation of iron regulatory protein-1 by oxidative stress in vitro

Iron regulatory protein-1 (IRP-1), a central cytoplasmic regulator of cellular iron metabolism, is rapidly activated by oxidative stress to bind to mRNA iron-responsive elements. We have reconstituted the response of IRP-1 to extracellular H2O2 in a system derived from murine B6 fibroblasts permeabilized with streptolysin-O. This procedure allows separation of the cytosol from the remainder of the cells (cell pellet). IRP-1 in the cytosolic fraction fails to be directly activated by addition of H2O2. IRP-1 activation requires the presence of a nonsoluble, possibly membrane-associated component in the cell pellet. The streptolysin-O-based in vitro system faithfully recapitulates characteristic hallmarks of IRP-1 activation by H2O2 in intact cells. We show that the H2O2-mediated activation of IRP-1 is temperature dependent and sensitive to treatment with calf intestinal alkaline phosphatase (CIAP). Although IRP-1 activation is unaffected by addition of excess ATP or GTP to this in vitro system, it is negatively affected by the nonhydrolyzable nucleotide analogs adenylyl-imidodiphosphate and guanylyl-imidophosphate and completely blocked by ATP-γS and GTP-γS. The in vitro reconstitution of this oxidative stress-induced pathway has opened a different avenue for the biochemical dissection of the regulation of mammalian iron metabolism by oxidative stress. Our data show that H2O2 must be sensed to stimulate a pathway to activate IRP-1.

A molecular mechanism for signaling between seven-transmembrane receptors: evidence for a redistribution of G proteins

Although activation of one seven-transmembrane receptor can influence the response of a separate seven-transmembrane receptor, e.g., the phenomenon of synergism, the underlying mechanism(s) for this signaling process is unclear. The present study investigated communication between two receptors that exhibit classical synergism, e.g., human platelet thrombin and thromboxane A2 receptors. Activation of thrombin receptors caused an increase in ligand affinity of thromboxane A2 receptors. This effect (i) was shown to be specific, since a similar increase in ligand affinity was not caused by ADP or A23187; (ii) did not require cytosolic components, e.g., kinases, proteases, phosphatases, etc., because it occurred in isolated platelet membranes; (iii) was G protein-mediated because it was blocked by an Gαq C terminus antibody; and (iv) was associated with a net increase in Gαq coupling to thromboxane A2 receptors. Collectively, these data provide evidence that seven-transmembrane receptors that share a common Gα subunit can communicate with each other via a redistribution of their G proteins. Thus, activation of thrombin receptors increases Gαq association with thromboxane A2 receptors thereby shifting them to a higher affinity state. This signaling phenomenon, which modulates receptor-ligand affinity, may serve as a molecular mechanism for cellular adaptive processes such as synergism.

Novel role of phosphorylation in Fe–S cluster stability revealed by phosphomimetic mutations at Ser-138 of iron regulatory protein 1

Animals regulate iron metabolism largely through the action of the iron regulatory proteins (IRPs). IRPs modulate mRNA utilization by binding to iron-responsive elements (IRE) in the 5′ or 3′ untranslated region of mRNAs encoding proteins involved in iron homeostasis or energy production. IRP1 is also the cytosolic isoform of aconitase. The activities of IRP1 are mutually exclusive and are modulated through the assembly/disassembly of its [4Fe–4S] cluster, reversibly converting it between an IRE-binding protein and cytosolic aconitase. IRP1 is also phosphoregulated by protein kinase C, but the mechanism by which phosphorylation posttranslationally increases IRE binding activity has not been fully defined. To investigate this, Ser-138 (S138), a PKC phosphorylation site, was mutated to phosphomimetic glutamate (S138E), aspartate (S138D), or nonphosphorylatable alanine (S138A). The S138E IRP1 mutant and, to a lesser extent, the S138D IRP1 mutant were impaired in aconitase function in yeast when grown aerobically but not when grown anaerobically. Purified wild-type and mutant IRP1s could be reconstituted to active aconitases anaerobically. However, when exposed to oxygen, the [4Fe–4S] cluster of the S138D and S138E mutants decayed 5-fold and 20-fold faster, respectively, than was observed for wild-type IRP1. Our findings suggest that stability of the Fe–S cluster of IRP1 can be regulated by phosphorylation and reveal a mechanism whereby the balance between the IRE binding and [4Fe–4S] forms of IRP1 can be modulated independently of cellular iron status. Furthermore, our results show that IRP1 can function as an oxygen-modulated posttranscriptional regulator of gene expression.