Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Agonist- or light-dependent phosphorylation of muscarinic acetylcholine receptor m2 subtypes (m2 receptors) or rhodopsin by G protein-coupled receptor kinase 2 (GRK2) was found to be inhibited by calmodulin in a Ca2+-dependent manner. The phosphorylation was fully inhibited in the absence of G protein betagamma subunits and partially inhibited in the presence of betagamma subunits. The dose-response curve for stimulation by betagamma subunits of the m2 and rhodopsin phosphorylation was shifted to the higher concentration of betagamma subunits by addition of Ca2+-calmodulin. The phosphorylation by GRK2 of a glutathione S-transferase fusion protein containing a peptide corresponding to the central part of the third intracellular loop of m2 receptors (I3-GST) was not affected by Ca2+-calmodulin in the presence or absence of betagamma subunits, but the agonist-dependent stimulation of I3-GST phosphorylation by an I3-deleted m2 receptor mutant in the presence of betagamma subunits was suppressed by Ca2+-calmodulin. These results indicate that Ca2+-calmodulin does not directly interact with the catalytic site of GRK2 but inhibits the kinase activity of GRK2 by interfering with the activation of GRK2 by agonist-bound m2 receptors and G protein betagamma subunits. In agreement with the assumption that GRK2 activity is suppressed by the increase in intracellular Ca2+, the sequestration of m2 receptors expressed in Chinese hamster ovary cells was found to be attenuated by the treatment with a Ca2+ ionophore, A23187.
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PMID:Ca2+-dependent inhibition of G protein-coupled receptor kinase 2 by calmodulin. 906 79

G protein-coupled receptor kinases (GRKs) specifically phosphorylate and regulate the activated form of multiple G protein-coupled receptors. Recent studies have revealed that GRKs are also subject to regulation. In this regard, GRK2 and GRK5 can be phosphorylated and either activated or inhibited, respectively, by protein kinase C. Here we demonstrate that calmodulin, another mediator of calcium signaling, is a potent inhibitor of GRK activity with a selectivity for GRK5 (IC50 approximately 50 nM) > GRK6 >> GRK2 (IC50 approximately 2 microM) >> GRK1. Calmodulin inhibition of GRK5 is mediated via a reduced ability of the kinase to bind to both receptor and phospholipid. Interestingly, calmodulin also activates autophosphorylation of GRK5 at sites distinct from the two major autophosphorylation sites on GRK5. Moreover, calmodulin-stimulated autophosphorylation directly inhibits GRK5 interaction with receptor even in the absence of calmodulin. Using glutathione S-transferase-GRK5 fusion proteins either to inhibit calmodulin-stimulated autophosphorylation or to bind directly to calmodulin, we determined that an amino-terminal domain of GRK5 (amino acids 20-39) is sufficient for calmodulin binding. This domain is abundant in basic and hydrophobic residues, characteristics typical of calmodulin binding sites, and is highly conserved in GRK4, GRK5, and GRK6. These studies suggest that calmodulin may serve a general role in mediating calcium-dependent regulation of GRK activity.
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PMID:Regulation of G protein-coupled receptor kinases by calmodulin and localization of the calmodulin binding domain. 921 66

Endothelial nitric-oxide synthase (eNOS) and caveolin-1 are associated within endothelial plasmalemmal caveolae. It is not known, however, whether eNOS and caveolin-1 interact directly or indirectly or whether the interaction affects eNOS activity. To answer these questions, we have cloned the bovine caveolin-1 cDNA and have investigated the eNOS-caveolin-1 interaction in an in vitro binding assay system using glutathione S-transferase (GST)-caveolin-1 fusion proteins and baculovirus-expressed bovine eNOS. We have also mapped the domains involved in the interaction using an in vivo yeast two-hybrid system. Results obtained using both in vitro and in vivo protein interaction assays show that both N- and C-terminal cytosolic domains of caveolin-1 interact directly with the eNOS oxygenase domain. Interaction of eNOS with GST-caveolin-1 fusion proteins significantly inhibits enzyme catalytic activity. A synthetic peptide corresponding to caveolin-1 residues 82-101 also potently and reversibly inhibits eNOS activity by interfering with the interaction of the enzyme with Ca2+/calmodulin (CaM). Regulation of eNOS in endothelial cells, therefore, may involve not only positive allosteric regulation by Ca2+/CaM, but also negative allosteric regulation by caveolin-1.
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PMID:Direct interaction of endothelial nitric-oxide synthase and caveolin-1 inhibits synthase activity. 922 13

The fusion protein technique was used to prepare an artificial polyfunctional protein from calmodulin (CaM) and glutathione S-transferase (GST). The fusion protein was designed, expressed, and then assembled to the glutathione self-assembled gold surface. The protein assembly was confirmed through enzyme binding assay and enzyme immunoassay. Specific binding of the fusion protein to glutathione self-assembled on the gold surface was assessed via a quartz crystal microbalance (QCM). The fusion protein was reversibly adsorbed and desorbed by the competitive binding of glutathione present in a solution, thus showing that the binding of the fusion protein was specific and had a highly oriented molecular configuration.
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PMID:Self-assembling of glutathione S-transferase/calmodulin fusion protein on chemically modified gold surface. 923 34

Structural and functional analyses were used to investigate the regulation of the inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) by Ca2+. To define the structural determinants for Ca2+ binding, cDNAs encoding GST fusion proteins that covered the complete linear cytosolic sequence of the InsP3R-1 were expressed in bacteria. The fusion proteins were screened for Ca2+ and ruthenium red binding through the use of 45Ca2+ and ruthenium red overlay procedures. Six new cytosolic Ca2+-binding regions were detected on the InsP3R in addition to the one described earlier (Sienaert, I., De Smedt, H., Parys, J. B., Missiaen, L., Vanlingen, S., Sipma, H., and Casteels, R. (1996) J. Biol. Chem. 271, 27005-27012). Strong 45Ca2+ and ruthenium red binding domains were localized in the N-terminal region of the InsP3R as follows: two Ca2+-binding domains were located within the InsP3-binding domain, and three Ca2+ binding stretches were localized in a 500-amino acid region just downstream of the InsP3-binding domain. A sixth Ca2+-binding stretch was detected in the proximity of the calmodulin-binding domain. Evidence for the involvement of multiple Ca2+-binding sites in the regulation of the InsP3R was obtained from functional studies on permeabilized A7r5 cells, in which we characterized the effects of Ca2+ and Sr2+ on the EC50 and cooperativity of the InsP3-induced Ca2+ release. The activation by cytosolic Ca2+ was due to a shift in EC50 toward lower InsP3 concentrations, and this effect was mimicked by Sr2+. The inhibition by cytosolic Ca2+ was caused by a decrease in cooperativity and by a shift in EC50 toward higher InsP3 concentrations. The effect on the cooperativity occurred at lower Ca2+ concentrations than the inhibitory effect on the EC50. In addition, Sr2+ mimicked the effect of Ca2+ on the cooperativity but not the inhibitory effect on the EC50. The different [Ca2+] and [Sr2+] dependencies suggest that three different cytosolic interaction sites were involved. Luminal Ca2+ stimulated the release without affecting the Hill coefficient or the EC50, excluding the involvement of one of the cytosolic Ca2+-binding sites. We conclude that multiple Ca2+-binding sites are localized on the InsP3R-1 and that at least four different Ca2+-interaction sites may be involved in the complex feedback regulation of the release by Ca2+.
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PMID:Molecular and functional evidence for multiple Ca2+-binding domains in the type 1 inositol 1,4,5-trisphosphate receptor. 932 22

A ligand-insensitive form of the human epidermal growth factor receptor (EGFR) was enriched by Ca2+-dependent calmodulin-affinity chromatography purification. The basic amphiphilic segment Arg645-Arg-Arg-His-Ile-Val-Arg-Lys-Arg-Thr654-Leu-Arg-Arg-Le u-Leu-Gln 660, located within the cytoplasmic juxtamembrane domain of this receptor, was purified as a fusion protein with glutathione S-transferase and shown to bind calmodulin in a Ca2+-dependent manner. An apparent dissociation constant of 0.4 microM calmodulin (Kd'(CaM)) and an apparent affinity constant of 0.5 microM free Ca2+ (Ka'(Ca)) were measured for this binding process. Binding of calmodulin at the juxtamembrane site prevented the phosphorylation of residue Thr-654 by protein kinase C, and an apparent inhibition constant of 0.5-1 microM calmodulin (Ki'(CaM)) was determined. Conversely, phosphorylation of this site by protein kinase C prevented its subsequent interaction with calmodulin. We therefore propose that cross talk between signaling pathways mediated by calmodulin and protein kinase C occurs at the juxtamembrane domain of the EGFR. This calmodulin-binding sequence is highly conserved among protein tyrosine kinases of the vertebrate EGFR family.
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PMID:The human epidermal growth factor receptor contains a juxtamembrane calmodulin-binding site. 942 43

Eukaryotic P-type ATPases use energy to drive the transport of cations across membranes. A complete P-ATPase gene (CpATPase1) has been isolated from Cryptosporidium parvum, one of the opportunistic pathogens in AIDS patients. The complete gene encodes 1528 amino acids, predicting a protein of 169 kDa. A hydropathy profile of the protein suggested there are eight transmembrane domains (TM). Expression of the gene was confirmed both by Northern blot analysis and RT-PCR. A fragment of the gene has been expressed as a 49 kDa GST-fusion protein. This protein was used to produce rabbit antiserum and fluorescent labeling has localized the protein to the sporozoite apical and perinuclear regions. SDS-PAGE and Western blot analysis show a 160 kDa major protein, close to the predicted size. The protein shares greatest overall identity and similarity to a putative organellar Ca2+ P-ATPase described for Plasmodium falciparum. Unlike P. falciparum, but consistent with all genes so far isolated from C. parvum, the gene contains no introns. The Ca2+ P-ATPases from these two Apicomplexa are large and do not have motifs predicting calmodulin-binding.
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PMID:Molecular analysis of a P-type ATPase from Cryptosporidium parvum. 949 52

The nucleotide sequences of cDNAs encoding two isoforms of Arabidopsis glutamate decarboxylase, designated GAD1 (57.1 kDa) and GAD2 (56.1 kDa) and sharing 82% identical amino acid sequences, were determined. The recombinant proteins bound [35S] calmodulin (CaM) in the presence of calcium, and a region of 30-32 amino acids from the C-terminal of each isoform was sufficient for CaM binding when fused to glutathione S-transferase. Full-length GAD1 and GAD2 were expressed in Sf9 insect cells infected with recombinant baculovirus vectors. Recombinant proteins were partially purified by CaM affinity chromatography and were found to exhibit glutamate decarboxylase activity, which was dependent on the presence of Ca2+/CaM at pH 7.3. Southern hybridizations with GAD gene-specific probes suggest that Arabidopsis possesses one gene related to GAD1 and one to GAD2. Northern hybridization and western blot analysis revealed that GAD1 was expressed only in roots and GAD2 in roots, leaves, inflorescence stems and flowers. Our study provides the first evidence for the occurrence of multiple functional Ca2+/CaM-regulated GAD gene products in a single plant, suggesting that regulation of Arabidopsis GAD activity involves modulation of isoform-specific gene expression and stimulation of the catalytic activity of GAD by calcium signalling via CaM.
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PMID:Two isoforms of glutamate decarboxylase in Arabidopsis are regulated by calcium/calmodulin and differ in organ distribution. 970 69

Endothelial nitric-oxide synthase (eNOS) is targeted to caveoli through interaction with caveolin-1 (cav-1). cav-1 binding to a consensus site in the eNOS oxygenase domain is proposed to antagonize calmodulin (CaM) binding and thereby inhibit eNOS nitric oxide (NO) synthesis. To study the mechanism, we examined how cav-1 scaffolding domain peptide (amino acids 82-101; cav-1P) would affect NO synthesis, NADPH oxidation, cytochrome c reduction, and ferricyanide reduction by full-length eNOS or its isolated oxygenase and reductase domains. Cav-1P equivalently inhibited NO synthesis and NADPH oxidation by full-length eNOS in a manner reversible by CaM but did not affect NADPH-independent NO synthesis by full-length eNOS or its oxygenase domain, indicating inhibition required the reductase domain. Similar concentrations of cav-1P inhibited cytochrome c reduction by full-length eNOS or the reductase domain (amino acids 492-1205) in a CaM-reversible manner, indicating cav-1P interaction with reductase or full-length eNOS are equivalent. Ferricyanide reduction was unaffected by cav-1P in all cases. Immunoblotting showed that full-length eNOS, eNOS oxygenase, and eNOS reductase all bound to an immobilized glutathione S-transferase-cav-1 fusion protein. Thus, cav-1 interacts independently with both oxygenase and reductase domains of eNOS. The reductase interaction occurs independent of a cav-1 binding motif, is CaM-reversible, and is of sufficient affinity to match cav-1P inhibition of NO synthesis by full-length eNOS. We propose that cav-1 binding to eNOS reductase compromises its ability to bind CaM and to donate electrons to the eNOS heme, thereby inhibiting NO synthesis.
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PMID:Interaction between caveolin-1 and the reductase domain of endothelial nitric-oxide synthase. Consequences for catalysis. 971 42

Ca2+/calmodulin-dependent protein kinase II (CaM-KII) regulates numerous physiological functions, including neuronal synaptic plasticity through the phosphorylation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors. To identify proteins that may interact with and modulate CaM-KII function, a yeast two-hybrid screen was performed by using a rat brain cDNA library. This screen identified a unique clone of 1.4 kb, which encoded a 79-aa brain-specific protein that bound the catalytic domain of CaM-KII alpha and beta and potently inhibited kinase activity with an IC50 of 50 nM. The inhibitory protein (CaM-KIIN), and a 28-residue peptide derived from it (CaM-KIINtide), was highly selective for inhibition of CaM-KII with little effect on CaM-KI, CaM-KIV, CaM-KK, protein kinase A, or protein kinase C. CaM-KIIN interacted only with activated CaM-KII (i. e., in the presence of Ca2+/CaM or after autophosphorylation) by using glutathione S-transferase/CaM-KIIN precipitations as well as coimmunoprecipitations from rat brain extracts or from HEK293 cells cotransfected with both constructs. Colocalization of CaM-KIIN with activated CaM-KII was demonstrated in COS-7 cells transfected with green fluorescent protein fused to CaM-KIIN. In COS-7 cells phosphorylation of transfected alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors by CaM-KII, but not by protein kinase C, was blocked upon cotransfection with CaM-KIIN. These results characterize a potent and specific cellular inhibitor of CaM-KII that may have an important role in the physiological regulation of this key protein kinase.
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PMID:Characterization of a calmodulin kinase II inhibitor protein in brain. 972


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