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)

Guanylyl cyclase activating protein (GCAP)-1 regulates photoreceptor membrane guanylyl cyclase, RetGC, in a Ca2+-sensitive manner. It contains four Ca2+-binding motifs, EF-hands, three of which are capable of binding Ca2+. GCAP-1 activates RetGC in low Ca2+ and inhibits it in high Ca2+. In this study we used deletion and substitution analysis to identify regions of GCAP-1 sequence that are specifically required for inhibition and activation. A COOH-terminal sequence within Met157 to Arg182 is required for activation but not for inhibition of RetGC. We localized one essential stretch to 5 residues from Arg178 to Arg182. Another sequence essential for activation is within the N-terminal residues Trp21 to Thr27. The region between EF-hands 1 and 3 of GCAP-1 also contains elements needed for activation of RetGC. Finally, we found that inhibition of RetGC requires the first 9 amino-terminal residues of GCAP-1, but none of the residues from Gln33 to the COOH-terminal Gly205 are specifically required for inhibition. The ability of GCAP-1 mutants to regulate RetGC was tested on total guanylyl cyclase activity present in rod outer segments. In addition, the key mutants were also shown to produce similar effects on recombinant bovine outer segment cyclases GC1 and GC2.
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PMID:Mapping sites in guanylyl cyclase activating protein-1 required for regulation of photoreceptor membrane guanylyl cyclases. 1019 59

Guanylyl cyclase activating protein-2 (GCAP-2) is a Ca2+-sensitive regulator of phototransduction in retinal photoreceptor cells. GCAP-2 activates retinal guanylyl cyclases at low Ca2+ concentration (<100 nM) and inhibits them at high Ca2+ (>500 nM). The light-induced lowering of the Ca2+ level from approximately 500 nM in the dark to approximately 50 nM following illumination is known to play a key role in visual recovery and adaptation. We report here the three-dimensional structure of unmyristoylated GCAP-2 with three bound Ca2+ ions as determined by nuclear magnetic resonance spectroscopy of recombinant, isotopically labeled protein. GCAP-2 contains four EF-hand motifs arranged in a compact tandem array like that seen previously in recoverin. The root mean square deviation of the main chain atoms in the EF-hand regions is 2.2 A in comparing the Ca2+-bound structures of GCAP-2 and recoverin. EF-1, as in recoverin, does not bind calcium because it contains a disabling Cys-Pro sequence. GCAP-2 differs from recoverin in that the calcium ion binds to EF-4 in addition to EF-2 and EF-3. A prominent exposed patch of hydrophobic residues formed by EF-1 and EF-2 (Leu24, Trp27, Phe31, Phe45, Phe48, Phe49, Tyr81, Val82, Leu85, and Leu89) may serve as a target-binding site for the transmission of calcium signals to guanylyl cyclase.
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PMID:Three-dimensional structure of guanylyl cyclase activating protein-2, a calcium-sensitive modulator of photoreceptor guanylyl cyclases. 1038 44

Guanylyl cyclase-activating proteins (GCAPs are 23-kDa Ca2+-binding proteins belonging to the calmodulin superfamily. Ca2+-free GCAPs are responsible for activation of photoreceptor guanylyl cyclase during light adaptation. In this study, we characterized GCAP1 mutants in which three endogenous nonessential Trp residues were replaced by Phe residues, eliminating intrinsic fluorescence. Subsequently, hydrophobic amino acids adjacent to each of the three functional Ca2+-binding loops were replaced by reporter Trp residues. Using fluorescence spectroscopy and biochemical assays, we found that binding of Ca2+ to GCAP1 causes a major conformational change especially in the region around the EF3-hand motif. This transition of GCAP1 from an activator to an inhibitor of GC requires an activation energy Ea = 9.3 kcal/mol. When Tyr99 adjacent to the EF3-hand motif was replaced by Cys, a mutation linked to autosomal dominant cone dystrophy in humans, Cys99 is unable to stabilize the inactive GCAP1-Ca2+ complex. Stopped-flow kinetic measurements indicated that GCAP1 rapidly loses its bound Ca2+ (k-1 = 72 s-1 at 37 degrees C) and was estimated to associate with Ca2+ at a rate (k1 > 2 x 10(8) M-1 s-1) close to the diffusion limit. Thus, GCAP1 displays thermodynamic and kinetic properties that are compatible with its involvement early in the phototransduction response.
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PMID:Conformational changes in guanylyl cyclase-activating protein 1 (GCAP1) and its tryptophan mutants as a function of calcium concentration. 1039 27

Guanylyl cyclase (GC) plays a central role in the responses of vertebrate rod and cone photoreceptors to light. cGMP is an internal messenger molecule of vertebrate phototransduction. Light stimulates hydrolysis of cGMP, causing the closure of cGMP-dependent cation channels in the plasma membranes of photoreceptor outer segments. Light also lowers the concentration of intracellular free Ca(2+) and by doing so it stimulates resynthesis of cGMP by guanylyl cyclase. The guanylyl cyclases that couple Ca(2+) to cGMP synthesis in photoreceptors are members of a family of transmembrane guanylyl cyclases that includes atrial natriuretic peptide receptors and the heat-stable enterotoxin receptor. The photoreceptor membrane guanylyl cyclases, RetGC-1 and RetGC-2 (also referred to as GC-E and GC-F), are regulated intracellularly by two Ca(2+)-binding proteins, GCAP-1 and GCAP-2. GCAPs bind Ca(2+) at three functional EF-hand structures. Several lines of biochemical evidence suggest that guanylyl cyclase activator proteins (GCAPs) bind constitutively to an intracellular domain of RetGCs. In the absence of Ca(2+) GCAP stimulates and in the presence of Ca(2+) it inhibits cyclase activity. Proper functioning of RetGC and GCAP is necessary not only for normal photoresponses but also for photoreceptor viability since mutations in RetGC and in GCAP cause photoreceptor degeneration.
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PMID:Regulation of photoreceptor membrane guanylyl cyclases by guanylyl cyclase activator proteins. 1058 Nov 51

Paramecium has a 280-kDa guanylyl cyclase. The N terminus resembles a P-type ATPase, and the C terminus is a guanylyl cyclase with the membrane topology of canonical mammalian adenylyl cyclases, yet with the cytosolic loops, C1 and C2, inverted compared with the mammalian order. We expressed in Escherichia coli the cytoplasmic domains of the protozoan guanylyl cyclase, independently and linked by a peptide, as soluble proteins. The His(6)-tagged proteins were enriched by affinity chromatography and analyzed by immunoblotting. Guanylyl cyclase activity was reconstituted upon mixing of the recombinant C1a- and C2-positioned domains and in a linked C1a-C2 construct. Adenylyl cyclase activity was minimal. The nucleotide substrate specificity was switched from GTP to ATP upon mutation of the substrate defining amino acids Glu(1681) and Ser(1748) in the C1-positioned domain to the adenylyl cyclase specific amino acids Lys and Asp. Using the C2 domains of mammalian adenylyl cyclases type II or IX and the C2-positioned domain from the Paramecium guanylyl cyclase we reconstituted a soluble, all C2 adenylyl cyclase. All enzymes containing protozoan domains were not affected by Galpha(s)/GTP or forskolin, and P site inhibitors were only slightly effective.
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PMID:A guanylyl cyclase from Paramecium with 22 transmembrane spans. Expression of the catalytic domains and formation of chimeras with the catalytic domains of mammalian adenylyl cyclases. 1075 32

Guanylyl cyclase C (GCC) is the receptor for the family of guanylin peptides and bacterial heat-stable enterotoxins (ST). The receptor is composed of an extracellular, ligand-binding domain and an intracellular domain with a region of homology to protein kinases and a guanylyl cyclase catalytic domain. We have expressed the entire intracellular domain of GCC in insect cells and purified the recombinant protein, GCC-IDbac, to study its catalytic activity and regulation. Kinetic properties of the purified protein were similar to that of full-length GCC, and high activity was observed when MnGTP was used as the substrate. Nonionic detergents, which stimulate the guanylyl cyclase activity of membrane-associated GCC, did not appreciably increase the activity of GCC-IDbac, indicating that activation of the receptor by Lubrol involved conformational changes that required the transmembrane and/or the extracellular domain. The guanylyl cyclase activity of GCC-IDbac was inhibited by Zn(2+), at concentrations shown to inhibit adenylyl cyclase, suggesting a structural homology between the two enzymes. Covalent cross-linking of GCC-IDbac indicated that the protein could associate as a dimer, but a large fraction was present as a trimer. Gel filtration analysis also showed that the major fraction of the protein eluted at a molecular size of a trimer, suggesting that the dimer detected by cross-linking represented subtle differences in the juxtaposition of the individual polypeptide chains. We therefore provide evidence that the trimeric state of GCC is catalytically active, and sequences required to generate the trimer are present in the intracellular domain of GCC.
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PMID:Biochemical characterization of the intracellular domain of the human guanylyl cyclase C receptor provides evidence for a catalytically active homotrimer. 1112 35

Guanylyl cyclase subtype A (GCA) is the main receptor that mediates the effects of atrial natriuretic peptide (ANP) in the regulation of plasma volume and blood pressure. The dynamics of the dissociation of ANP from GCA were investigated in cultured Chinese hamster ovary (CHO) cells stably transfected with wild-type (WT) or mutant GCA receptors. The rate of dissociation of specifically bound (125)I-ANP-(1-28) from intact CHOGCAWT cells at 37 degrees C was extremely rapid (K(off) = 0.49 +/- 0.02 min(-1)), whereas in isolated membranes prepared from these cells, the dissociation at 37 degrees C was >10-fold slower (K(off) = 0.035 +/- 0.006 min(-1)). The dissociation of ANP from CHOGCAWT cells showed remarkable temperature dependence. Between 22 and 37 degrees C, K(off) increased approximately 8 times, whereas between 4 and 22 degrees C, it increased only 1.5 times. Total deletion of the cytoplasmic domain or of the catalytic guanylyl cyclase sequence within this domain abolished ANP-induced increases in cGMP, dramatically slowed receptor-ligand dissociation by at least 10-fold, and abolished the temperature dependence of the dissociation of ANP. Deletion of the kinase-like domain led to maximal constitutive activation of guanylyl cyclase, markedly decreased K(off) to 0.064 +/- 0.006 min(-1), and also abolished the temperature dependence of dissociation. Substitution of Ser(506) by Ala and particularly the double substitution of Gly(505) and Ser(506) by Ala within the kinase-like domain markedly reduced ANP-induced increases in cGMP, whereas K(off) decreased modestly (albeit significantly) to 0.36 +/- 0.03 and 0.24 +/- 0.02 min(-1), respectively. As a whole, the results demonstrate for the first time that temperature per se or ATP alone cannot account for rapid GCA receptor-ligand dissociation under physiological conditions and suggest that ligand dissociation is modulated in part by the interaction of still unidentified cytosolic factors with the cytoplasmic domain of GCA.
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PMID:Molecular and cellular physiology of the dissociation of atrial natriuretic peptide from guanylyl cyclase a receptors. 1147 86

Guanylyl cyclase-activating proteins are EF-hand Ca(2+)-binding proteins that belong to the calmodulin superfamily. They are involved in the regulation of photoreceptor membrane-associated guanylyl cyclases that produce cGMP, a second messenger of vertebrate vision. Here, we investigated changes in GCAP1 structure using mutagenesis, chemical modifications, and spectroscopic methods. Two Cys residues of GCAP1 situated in spatially distinct regions of the N-terminal domain (positions 18 and 29) and two Cys residues located within the C-terminal lobe (positions 106 and 125) were employed to detect conformational changes upon Ca(2+) binding. GCAP1 mutants with only a single Cys residue at each of these positions, modified with N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine, an environmentally sensitive fluorophore, and with (1-oxy-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonate, a spin label reagent, were studied using fluorescence and EPR spectroscopy, respectively. Only minor structural changes around Cys(18), Cys(29), Cys(106), and Cys(125) were observed as a function of Ca(2+) concentration. No Ca(2+)-dependent oligomerization of GCAP1 was observed at physiologically relevant Ca(2+) concentrations, in contrast to the observation reported by others for GCAP2. Based on these results and previous studies, we propose a photoreceptor activation model that assumes changes within the flexible central helix upon Ca(2+) dissociation, causing relative reorientation of two structural domains containing a pair of EF-hand motifs and thus switching its partner, guanylyl cyclase, from an inactive (or low activity) to an active conformation.
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PMID:Calcium-sensitive regions of GCAP1 as observed by chemical modifications, fluorescence, and EPR spectroscopies. 1152 15

Guanylyl cyclase activator proteins (GCAPs) are calcium-binding proteins closely related to recoverin, neurocalcin, and many other neuronal Ca(2+)-sensor proteins of the EF-hand superfamily. GCAP-1 and GCAP-2 interact with the intracellular portion of photoreceptor membrane guanylyl cyclase and stimulate its activity by promoting tight dimerization of the cyclase subunits. At low free Ca(2+) concentrations, the activator form of GCAP-2 associates into a dimer, which dissociates when GCAP-2 binds Ca(2+) and becomes inhibitor of the cyclase. GCAP-2 is known to have three active EF-hands and one additional EF-hand-like structure, EF-1, that deviates form the EF-hand consensus sequence. We have found that various point mutations within the EF-1 domain can specifically affect the ability of GCAP-2 to interact with the target cyclase but do not hamper the ability of GCAP-2 to undergo reversible Ca(2+)-sensitive dimerization. Point mutations within the EF-1 region can interfere with both the activation of the cyclase by the Ca(2+)-free form of GCAP-2 and the inhibition of retGC basal activity by the Ca(2+)-loaded GCAP-2. Our results strongly indicate that evolutionary conserved and GCAP-specific amino acid residues within the EF-1 can create a contact surface for binding GCAP-2 to the cyclase. Apparently, in the course of evolution GCAP-2 exchanged the ability of its first EF-hand motif to bind Ca(2+) for the ability to interact with the target enzyme.
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PMID:Instead of binding calcium, one of the EF-hand structures in guanylyl cyclase activating protein-2 is required for targeting photoreceptor guanylyl cyclase. 1158 9

Guanylyl cyclase A (GCA) and soluble guanylyl cyclase (sGC) encode GCs in Dictyostelium and have a topology similar to 12-transmembrane and soluble adenylyl cyclase, respectively. We demonstrate that all detectable GC activity is lost in a cell line in which both genes have been inactivated. Cell lines with one gene inactivated were used to characterize the other guanylyl cyclase (i.e. GCA in sgc(minus sign) null cells and sGC in gca(minus sign) null cells). Despite the different topologies, the enzymes have many properties in common. In vivo, extracellular cAMP activates both enzymes via a G-protein-coupled receptor. In vitro, both enzymes are activated by GTPgammaS (K(a) = 11 and 8 microm for GCA and sGC, respectively). The addition of GTPgammaS leads to a 1.5-fold increase of V(max) and a 3.5-fold increase of the affinity for GTP. Ca(2+) inhibits both GCA and sGC with K(i) of about 50 and 200 nm, respectively. Other biochemical properties are very different; GCA is expressed mainly during growth and multicellular development, whereas sGC is expressed mainly during cell aggregation. Folic acid and cAMP activate GCA maximally about 2.5-fold, whereas sGC is activated about 8-fold. Osmotic stress strongly stimulates sGC but has no effect on GCA activity. Finally, GCA is exclusively membrane-bound and is active mainly with Mg(2+), whereas sGC is predominantly soluble and more active with Mn(2+).
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PMID:Characterization of two unusual guanylyl cyclases from dictyostelium. 1177 34


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