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:4.6.1.2 (guanylate cyclase)
8,497 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Retinal guanylyl cyclase-1 (RetGC-1) is a membrane guanylyl cyclase found in photoreceptor outer segments. It consists of an apparent extracellular domain (ECD) linked by a single transmembrane segment to an intracellular domain (ICD). Guanylyl cyclase activating protein-2 (GCAP-2) is a Ca2+-binding protein that activates RetGC-1 in a Ca2+-sensitive manner. To establish whether GCAP-2 stimulates RetGC-1 through the ECD or ICD, we made deletion mutants lacking either the ECD or both the ECD and transmembrane domains (TMD) of RetGC-1. Recombinant wild type RetGC-1 and both deletion mutants were expressed in HEK 293 cells, and their sensitivities to GCAP-2, Ca2+, and ATP were compared. Our data demonstrate that both deletion mutants are regulated similarly to wild type RetGC-1 with indistinguishable EC50 values for Ca2+ and similar K1/2 values for activation by GCAP-2. This shows that GCAP-2 functions through the ICD of RetGC-1 and that removal of the ECD and TMD do not significantly alter regulation by these factors. Our data also show that ATP potentiates stimulation of guanylyl cyclase activity by GCAP-2 and that neither the ECD nor the TMD of RetGC-1 participate in its regulation by ATP.
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PMID:The membrane guanylyl cyclase, retinal guanylyl cyclase-1, is activated through its intracellular domain. 866 12

Nitric oxide (NO) has been postulated as a regulator of vascular reactivity, and the current study tested the hypothesis that NO-induced decreased sensitivity to vasoconstrictors persists following removal of NO. Endothelium-denuded segments of rat aorta were incubated 2-4 h at 37 degrees C with the NO donor S-nitroso-N-acetylpenicillamine (SNAP). Incubation produced rightward shifts in concentration response curves for phenylephrine [i.e., half-maximum effective concentration (EC50; in microM): control = 0.016, NO = 0.14], aluminum fluoride (i.e., EC50 in mM: control = 1.66, NO = 2.29), and KCl (i.e., EC50 in mM: control = 5.9, NO = 23.9). Similar shifts were seen for two other NO donors. The SNAP-induced shift was not attenuated by a guanylyl cyclase inhibitor, LY-83583 (10 microM) and was not mimicked by 8-bromoguanosine 3',5'-cyclic monophosphate (100 microM). It was attenuated by 1,4-naphthoquinone (50 microM), an inhibitor of endogenous mono-ADP ribosyltransferases. NO incubation increased cGMP content (4.6 +/- 0.8 vs. 1.5 +/- 0.15 pmol/mg protein), an increase unaffected by 1,4-naphthoquinone (3.3 +/- 1.0 pmol/mg protein) but prevented by LY-83583 (1.6 +/- 0.36 pmol/mg protein). ADP ribosylation of three proteins was observed in membranes from HEK 293 cells: 88,66, and 38 kDa. ADP ribosylation of the 38-kDa protein was stimulated in a concentration-dependent manner by NO but was not decreased by 1,4-naphthoquinone. In conclusion, NO produces a long-lasting inhibition of vascular contractility by both a cGMP-dependent and -independent mechanism. Based on the observations of 1,4-naphthoquinone, we conclude that the cGMP-independent mechanism is not stimulation of endogenous ADP ribosylation but some other covalent modification in the pathway that mediates contraction.
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PMID:Decreased sensitivity to vasoconstrictors in aortic rings after acute exposure to nitric oxide. 876 Jan 82

The recently cloned new subtype of G protein-coupled neurotensin receptor (NTRL) was stably expressed in the HEK 293 cell line in order to investigate its binding and internalization properties. The expressed receptor exhibited the typical binding characteristics of the low affinity, levocabastine-sensitive binding site previously described in rat and mouse brain and was detected as a protein with an apparent MW of 45 kDa by photoaffinity labeling. Although intracellular modulation of adenylate cyclase, guanylate cyclase and phospholipase C was not detected after application of neurotensin or levocabastine on NTRL-transfected cells, this receptor was able to internalize iodinated neurotensin. The internalization process was followed by recycling of receptors to the cell membrane. By contrast, no recycling was observed with the high affinity neurotensin receptor (NTRH). The differential intracellular routing of NTRH and NTRL after internalization is most probably the consequence of their divergent carboxy-terminal sequences.
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PMID:Stable expression of the mouse levocabastine-sensitive neurotensin receptor in HEK 293 cell line: binding properties, photoaffinity labeling, and internalization mechanism. 948 Aug 52

Natriuretic peptide receptor A (NPR-A) is the biological receptor for atrial natriuretic peptide (ANP). Activation of the NPR-A guanylyl cyclase requires ANP binding to the extracellular domain and ATP binding to a putative site within its cytoplasmic region. The allosteric interaction of ATP with the intracellular kinase homology domain (KHD) is hypothesized to derepress the carboxyl-terminal guanylyl cyclase catalytic domain, resulting in the synthesis of the second messenger, cyclic GMP. Here, we show that phosphorylation of the KHD is essential for receptor activation. Using a combination of phosphopeptide mapping techniques, we have identified six residues within the ATP-binding domain (S497, T500, S502, S506, S510, and T513) which are phosphorylated when NPR-A is expressed in HEK 293 cells. Mutation of any one of these Ser or Thr residues to Ala caused reductions in the receptor phosphorylation state, the number and pattern of phosphopeptides observed in tryptic maps, and ANP-dependent guanylyl cyclase activity. The reductions were not explained by decreases in NPR-A protein levels, as indicated by immunoblot analysis and determinations of cyclase activity in the presence of detergent. Conversion of Ser-497 to Ala resulted in the most dramatic decrease in cyclase activity (approximately 20% of wild-type activity), but conversion to an acidic residue (Glu), which mimics the charge of the phosphoserine moiety, had no effect. Simultaneous mutation of five of the phosphorylation sites to Ala resulted in a dephosphorylated receptor which was unresponsive to hormone and had potent dominant negative inhibitory activity. We conclude that phosphorylation of the KHD is absolutely required for hormone-dependent activation of NPR-A.
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PMID:Phosphorylation of the kinase homology domain is essential for activation of the A-type natriuretic peptide receptor. 952 88

The neuronal and endothelial isoforms of nitric oxide (NO) synthase (nNOS and eNOS, respectively) both catalyze the production of NO but are regulated differently. Stably transfected HEK 293 cell lines containing nNOS, eNOS, and a soluble mutant of eNOS were therefore established to compare their activity in a common cellular environment. NOS activity was determined by measuring L-[3H]citrulline production in homogenates and intact cells, the conversion of oxyhemoglobin to methemoglobin, and the production of cGMP. The results indicate that nNOS is more active than eNOS, both in unstimulated as well as calcium-stimulated cells. Under basal conditions, the soluble mutant of eNOS appeared to be slightly more active than wild-type eNOS in terms of NO and cGMP formation, suggesting that membrane association may be crucial for inhibition of basal NO release but is not required for stimulation by Ca2+-mobilizing agents. The maximal activity of soluble guanylate cyclase was significantly reduced by transfection with wild-type eNOS due to downregulation of mRNA expression. These results demonstrate that nNOS and eNOS behave differently even in an identical cellular environment.
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PMID:Comparison of neuronal and endothelial isoforms of nitric oxide synthase in stably transfected HEK 293 cells. 1166 66

The cGMP-dependent protein kinases (PKG) are emerging as important components of mainstream signal transduction pathways. Nitric oxide-induced cGMP formation by stimulation of soluble guanylate cyclase is generally accepted as being the most widespread mechanism underlying PKG activation. In the present study, PKG was found to be a target for phorbol 12-myristate 13-acetate (PMA)-responsive protein kinase C (PKC). PKG1alpha became phosphorylated in HEK-293 cells stimulated with PMA and also in vitro using purified components. PKC-dependent phosphorylation was found to activate PKG as measured by phosphorylation of vasodilator-stimulated phosphoprotein, and by in vitro kinase assays. Although there are 11 potential PKC substrate recognition sites in PKG1alpha, threonine 58 was examined due to its proximity to the pseudosubstrate domain. Antibodies generated against the phosphorylated form of this region were used to demonstrate phosphorylation in response to PMA treatment of the cells with kinetics similar to vasodilator-stimulated phosphoprotein phosphorylation. A phospho-mimetic mutation at this site (T58E) generated a partially activated PKG that was more sensitive to cGMP levels. A phospho-null mutation (T58A) revealed that this residue is important but not sufficient for PKG activation by PKC. Taken together, these findings outline a novel signal transduction pathway that links PKC stimulation with cyclic nucleotide-independent activation of PKG.
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PMID:Activation of cGMP-dependent protein kinase by protein kinase C. 1260 95

We have identified a novel membrane form of guanylate cyclase (GC) from a mouse testis cDNA library and termed it mGC-G (mouse GC-G) based on its high sequence homology to rat GC-G. It encodes a potential type I transmembrane receptor, with the characteristic domain structure common to all members of the family of membrane GCs, including an extracellular, putative ligand-binding domain, a single membrane-spanning segment and cytoplasmic protein kinase-like and cyclase catalytic domains. Real-time quantitative reverse transcriptase--PCR and Northern-blot analyses showed that mGC-G is highly and selectively expressed in mouse testis. Phylogenetic analysis based on the extracellular protein sequence revealed that mGC-G is closely related to members of the subfamily of natriuretic peptide receptor GCs. When overexpressed in HEK-293T cells (human embryonic kidney 293T cells) or COS-7 cells, mGC-G manifests as a membrane-bound glycoprotein, which can form either homomeric or heteromeric complexes with the natriuretic peptide receptor GC-A. It exhibits marked cGMP-generating GC activity; however, notably, all ligands known to activate other receptor GCs failed to stimulate enzymic activity. The unique testis-enriched expression of mGC-G, which is completely different from the broader tissue distribution of rat GC-G, suggests the existence of as-yet-unidentified ligands and unappreciated species-specific physiological functions mediated through mGC-G/cGMP signalling in the testis.
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PMID:Identification of an orphan guanylate cyclase receptor selectively expressed in mouse testis. 1471 86

GC-C (guanylate cyclase C) is the receptor for heat-stable enterotoxins, guanylin and uroguanylin peptides. Ligand binding to the extracellular domain of GC-C activates the guanylate cyclase domain leading to accumulation of cGMP. GC-C is expressed as differentially glycosylated forms in HEK-293 cells (human embryonic kidney-293 cells). In the present study, we show that the 145 kDa form of GC-C contains sialic acid and galactose residues and is present on the PM (plasma membrane) of cells, whereas the 130 kDa form is a high mannose form that is resident in the endoplasmic reticulum and serves as the precursor for the PM-associated form. Ligand-binding affinities of the differentially glycosylated forms are similar, indicating that glycosylation of GC-C does not play a role in direct ligand interaction. However, ligand-stimulated guanylate cyclase activity was observed only for the fully mature form of the receptor present on the PM, suggesting that glycosylation had a role to play in imparting a conformation to the receptor that allows ligand stimulation. Treatment of cells at 20 degrees C led to intracellular accumulation of a mature glycosylated form of GC-C that now showed ligand-stimulated guanylate cyclase activity, indicating that localization of GC-C was not critical for its catalytic activity. To determine if complex glycosylation was required for ligand-stimulated activation of GC-C, the receptor was expressed in HEK-293 cells that were deficient in N -acetylglucosaminyltransferase 1. This minimally glycosylated form of the receptor was expressed on the cell surface and could bind a ligand with an affinity comparable with the 145 kDa form of the receptor. However, this form of the receptor was poorly activated by the ligand. Therefore our studies indicate a novel role for glycosidic modification of GC-C during its biosynthesis, in imparting subtle conformational changes in the receptor that allow for ligand-mediated activation and perhaps regulation of basal activity.
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PMID:Glycosylation of the receptor guanylate cyclase C: role in ligand binding and catalytic activity. 1474 40

Carbon monoxide (CO) is increasingly recognized as a physiological messenger. CO is produced in the gastrointestinal tract with diverse functions, including regulation of gastrointestinal motility, interacting with nitric oxide (NO) to mediate neurotransmission. The aim of this study was to determine the effect of CO on the human intestinal L-type Ca(2+) channel expressed in HEK cells and in native cells using the patch-clamp technique. Extracellular solution contained 10 mM Ba(2+) as the charge carrier. Maximal peak Ba(2+) current (I(Ba)) was significantly increased by bath application of 0.2% CO to transfected HEK cells (18 +/- 3%). The NO donor S-nitroso-N-acetylpenicillamine also increased I(Ba), and CO (0.2%) increased NO production in transfected HEK cells. The CO-induced increase in I(Ba) was blocked when cells were pretreated with 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (10 microM) or inhibitors of NO synthase (NOS). The PKA inhibitor KT-5720 (0.5 microM) and milrinone (3 microM), a phosphodiesterase (PDE) III inhibitor, blocked the effect of CO on I(Ba). Similar effects were seen in freshly dissociated human intestinal smooth muscle cells. The data suggest that exogenous CO can activate native and heterologously expressed intestinal L-type Ca(2+) channels through a pathway that involves activation of NOS, increased NO, and cGMP levels, but not PKG. Rather, the pathway appears to involve PKA, partly by reducing cAMP breakdown through inhibition of PDE III. CO-induced NO production may explain the apparent discrepancy between the low affinity of guanylyl cyclase for CO and the robust cGMP production evoked by CO.
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PMID:Carbon monoxide activates human intestinal smooth muscle L-type Ca2+ channels through a nitric oxide-dependent mechanism. 1531 83

We have identified a GDAY motif in the C-terminal domain of guanylyl cyclase (guanylate cyclase)/NPRA (natriuretic peptide receptor A) sequence, which serves a dual role as an internalization signal and a recycling signal. To delineate the role of the GDAY motif in receptor internalization and sequestration, we mutated Gly920, Asp921 and Tyr923 to alanine residues (GDAY/AAAA) in the NPRA cDNA sequence. The cDNAs encoding wild-type and mutant receptors were transfected in HEK-293 cells (human embryonic kidney 293 cells). The internalization studies of ligand-receptor complexes revealed that endocytosis of 125I-ANP by HEK-293 cells expressing G920A, Y923A or GDAY/AAAA mutant receptor was decreased by almost 50% (P<0.001) when compared with cells expressing the wild-type receptor. However, the effect of D921A mutation on receptor internalization was minimal. Ligand-mediated down-regulation of G920A, Y923A and GDAY/AAAA mutant receptors was decreased by 35-40% when compared with wild-type NPRA. Subsequently, the recycling of internalized D921A and GDAY/AAAA mutant receptors from the intracellular pool was decreased by more than 40+/-4% when compared with wild-type NPRA. Recycling of G920A and Y923A mutant receptors was also decreased, but to a significantly lesser extent compared with the D921A or GDAY/AAAA mutant receptors. We conclude that the Gly920 and Tyr923 residues within the GDAY consensus motif are necessary for internalization, and that residue Asp921 is important for recycling of NPRA. The current results provide new evidence for a dual role of the GDAY sequence motif in ligand-mediated internalization, recycling and down-regulation of a single-transmembrane receptor protein NPRA.
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PMID:Internalization and trafficking of guanylyl (guanylate) cyclase/natriuretic peptide receptor A is regulated by an acidic tyrosine-based cytoplasmic motif GDAY. 1557 17


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