Publications

• Guanyl cyclase, an enzyme catalyzing the formation of guanosine 3',5'-monophosphate from guanosine trihosphate.

  Hardman J.G., Sutherland E.W.

  J Biol Chem 244:6363-6370(1969) [PubMed] [EuropePMC]

• Structural and biochemical identity of retinal rod outer segment membrane guanylate cyclase.

  Margulis A., Goraczniak R.M., Duda T., Sharma R.K., Sitaramayya A.

  Recent molecular cloning reports show that there are at least three membrane guanylate cyclases in vertebrate retina: (1) atrial natriuretic factor receptor guanylate cyclase (ANF-RGC), (2) C-type natriuretic peptide receptor guanylate cyclase (CNP-RGC), and (3) "retinal guanylate cyclase" (RetGC) ... >> More

  Recent molecular cloning reports show that there are at least three membrane guanylate cyclases in vertebrate retina: (1) atrial natriuretic factor receptor guanylate cyclase (ANF-RGC), (2) C-type natriuretic peptide receptor guanylate cyclase (CNP-RGC), and (3) "retinal guanylate cyclase" (RetGC). The specific cellular localization of the first two cyclases is unknown, but RetGC is apparently localized in photoreceptor cells, suggesting that it participates in visual transduction. With the overall objective of identifying the guanylate cyclase that is linked to phototransduction, we compared the structural and regulatory properties of the biochemically characterized 112 kDa bovine rod outer segment membrane guanylate cyclase (ROS-GC) with those of RetGC, ANF-RGC and CNP-RGC. The N-terminal and two internal peptide sequences of purified ROS-GC had about 90% similarity with the corresponding sequences of the RetGC; the sequence identity with natriuretic peptide receptor cyclases was about 30%. A 19 amino acid long sequence from a tryptic peptide of ROS-GC had no corresponding sequence in the other three cyclases. ROS-GC was inhibited by ATP but ANF-RGC and CNP-RGC were activated by ATP in the presence of the respective peptide hormones. These results suggest that ROS-GC represents a new subtype of the membrane guanylate cyclase family that is structurally and biochemically distinct from the other retinal cyclases. << Less

  Biochem. Biophys. Res. Commun. 194:855-861(1993) [PubMed] [EuropePMC]

• Two amino acid substitutions convert a guanylyl cyclase, RetGC-1, into an adenylyl cyclase.

  Tucker C.L., Hurley J.H., Miller T.R., Hurley J.B.

  Guanylyl cyclases (GCs) and adenylyl cyclases (ACs) have fundamental roles in a wide range of cellular processes. Whereas GCs use GTP as a substrate to form cGMP, ACs catalyze the analogous conversion of ATP to cAMP. Previously, a model based on the structure of adenylate cyclase was used to predi ... >> More

  Guanylyl cyclases (GCs) and adenylyl cyclases (ACs) have fundamental roles in a wide range of cellular processes. Whereas GCs use GTP as a substrate to form cGMP, ACs catalyze the analogous conversion of ATP to cAMP. Previously, a model based on the structure of adenylate cyclase was used to predict the structure of the nucleotide-binding pocket of a membrane guanylyl cyclase, RetGC-1. Based on this model, we replaced specific amino acids in the guanine-binding pocket of GC with their counterparts from AC. A change of two amino acids, E925K together with C995D, is sufficient to completely alter the nucleotide specificity from GTP to ATP. These experiments strongly validate the AC-derived RetGC-1 structural model and functionally confirm the role of these residues in nucleotide discrimination. << Less

  Proc. Natl. Acad. Sci. U.S.A. 95:5993-5997(1998) [PubMed] [EuropePMC]

• The human photoreceptor membrane guanylyl cyclase, RetGC, is present in outer segments and is regulated by calcium and a soluble activator.

  Dizhoor A.M., Lowe D.G., Olshevskaya E.V., Laura R.P., Hurley J.B.

  A human photoreceptor membrane guanylyl cyclase, RetGC, was recently cloned and expressed, but its localization and manner of regulation were not defined. We report here that RetGC is detected primarily in outer segments of human photoreceptor cells. Recombinant RetGC can be stimulated by a solubl ... >> More

  A human photoreceptor membrane guanylyl cyclase, RetGC, was recently cloned and expressed, but its localization and manner of regulation were not defined. We report here that RetGC is detected primarily in outer segments of human photoreceptor cells. Recombinant RetGC can be stimulated by a soluble retinal-specific factor. Ca2+ interferes with stimulation of RetGC by this factor with a cooperativity coefficient of 1.7 and EC50 near 200 nM. The Ca2+ sensitivities of recombinant RetGC and of guanylyl cyclase activity from rod outer segment membranes are very similar. Our findings indicate that RetGC is a photoreceptor-specific guanylyl cyclase which is stimulated by a retinal-specific activator and inhibited by physiologically relevant concentrations of free Ca2+. The Ca2+ sensitivity of RetGC may be responsible for some of the previously reported effects of Ca2+ on light adaptation and recovery of the dark state. << Less

  Neuron 12:1345-1352(1994) [PubMed] [EuropePMC]

• Characterization of two unusual guanylyl cyclases from dictyostelium.

  Roelofs J., Van Haastert P.J.M.

  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 ... >> More

  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+). << Less

  J. Biol. Chem. 277:9167-9174(2002) [PubMed] [EuropePMC]

• A novel calcium-regulated membrane guanylate cyclase transduction system in the olfactory neuroepithelium.

  Duda T., Jankowska A., Venkataraman V., Nagele R.G., Sharma R.K.

  This report defines the identity of a calcium-regulated membrane guanylate cyclase transduction system in the cilia of olfactory sensory neurons, which is the site of odorant transduction. The membrane fraction of the neuroepithelial layer of the rat exhibited Ca(2+)-dependent guanylate cyclase ac ... >> More

  This report defines the identity of a calcium-regulated membrane guanylate cyclase transduction system in the cilia of olfactory sensory neurons, which is the site of odorant transduction. The membrane fraction of the neuroepithelial layer of the rat exhibited Ca(2+)-dependent guanylate cyclase activity, which was eliminated by the addition of EGTA. This indicated that the cyclase did not represent a rod outer segment guanylate cyclase (ROS-GC), which is inhibited by free Ca(2+). This interpretation was supported by studies with the Ca(2+) binding proteins, GCAPs (guanylate cyclase activating proteins), which stimulate photoreceptor ROS-GC in the absence of Ca(2+). They did not stimulate the olfactory neuroepithelial membrane guanylate cyclase. The olfactory neuroepithelium contained a Ca(2+) binding protein, neurocalcin, which stimulated the cyclase in a Ca(2+)-dependent fashion. The cyclase was cloned from the neuroepithelium and was found to be identical in structure to that of the previously cloned cyclase termed GC-D. The cyclase was expressed in a heterologous cell system, and was reconstituted with its Ca(2+)-dependent activity in the presence of recombinant neurocalcin. The reconstituted cyclase mimicked the native enzyme. Immunocytochemical studies showed that the guanylate cyclase coexists with neurocalcin in the apical region of the cilia. Deletion analysis showed that the neurocalcin-regulated domain resides at the C-terminal region of the cyclase. The findings establish the biochemical, molecular, and functional identity of a novel Ca(2+)-dependent membrane guanylate cyclase transduction system in the cilia of the olfactory epithelium, suggesting a mechanism of the olfactory neuroepithelial guanylate cyclase regulation fundamentally distinct from the phototransduction-linked ROS-GC. << Less

  Biochemistry 40:12067-12077(2001) [PubMed] [EuropePMC]

• The phytosulfokine (PSK) receptor is capable of guanylate cyclase activity and enabling cyclic GMP-dependent signaling in plants.

  Kwezi L., Ruzvidzo O., Wheeler J.I., Govender K., Iacuone S., Thompson P.E., Gehring C., Irving H.R.

  Phytosulfokines (PSKs) are sulfated pentapeptides that stimulate plant growth and differentiation mediated by the PSK receptor (PSKR1), which is a leucine-rich repeat receptor-like kinase. We identified a putative guanylate cyclase (GC) catalytic center in PSKR1 that is embedded within the kinase ... >> More

  Phytosulfokines (PSKs) are sulfated pentapeptides that stimulate plant growth and differentiation mediated by the PSK receptor (PSKR1), which is a leucine-rich repeat receptor-like kinase. We identified a putative guanylate cyclase (GC) catalytic center in PSKR1 that is embedded within the kinase domain and hypothesized that the GC works in conjunction with the kinase in downstream PSK signaling. We expressed the recombinant complete kinase (cytoplasmic) domain of AtPSKR1 and show that it has serine/threonine kinase activity using the Ser/Thr peptide 1 as a substrate with an approximate K(m) of 7.5 μm and V(max) of 1800 nmol min(-1) mg(-1) of protein. This same recombinant protein also has GC activity in vitro that is dependent on the presence of either Mg(2+) or Mn(2+). Overexpression of the full-length AtPSKR1 receptor in Arabidopsis leaf protoplasts raised the endogenous basal cGMP levels over 20-fold, indicating that the receptor has GC activity in vivo. In addition, PSK-α itself, but not the non-sulfated backbone, induces rapid increases in cGMP levels in protoplasts. Together these results indicate that the PSKR1 contains dual GC and kinase catalytic activities that operate in vivo and that this receptor constitutes a novel class of enzymes with overlapping catalytic domains. << Less

  J. Biol. Chem. 286:22580-22588(2011) [PubMed] [EuropePMC]

• Retinal degeneration 3 (RD3) protein inhibits catalytic activity of retinal membrane guanylyl cyclase (RetGC) and its stimulation by activating proteins.

  Peshenko I.V., Olshevskaya E.V., Azadi S., Molday L.L., Molday R.S., Dizhoor A.M.

  Retinal membrane guanylyl cyclase (RetGC) in the outer segments of vertebrate photoreceptors is controlled by guanylyl cyclase activating proteins (GCAPs), responding to light-dependent changes of the intracellular Ca(2+) concentrations. We present evidence that a different RetGC binding protein, ... >> More

  Retinal membrane guanylyl cyclase (RetGC) in the outer segments of vertebrate photoreceptors is controlled by guanylyl cyclase activating proteins (GCAPs), responding to light-dependent changes of the intracellular Ca(2+) concentrations. We present evidence that a different RetGC binding protein, retinal degeneration 3 protein (RD3), is a high-affinity allosteric modulator of the cyclase which inhibits RetGC activity at submicromolar concentrations. It suppresses the basal activity of RetGC in the absence of GCAPs in a noncompetitive manner, and it inhibits the GCAP-stimulated RetGC at low intracellular Ca(2+) levels. RD3 opposes the allosteric activation of the cyclase by GCAP but does not significantly change Ca(2+) sensitivity of the GCAP-dependent regulation. We have tested a number of mutations in RD3 implicated in human retinal degenerative disorders and have found that several mutations prevent the stable expression of RD3 in HEK293 cells and decrease the affinity of RD3 for RetGC1. The RD3 mutant lacking the carboxy-terminal half of the protein and associated with Leber congenital amaurosis type 12 (LCA12) is unable to suppress the activity of the RetGC1/GCAP complex. Furthermore, the inhibitory activity of the G57V mutant implicated in cone-rod degeneration is strongly reduced. Our results suggest that inhibition of RetGC by RD3 may be utilized by photoreceptors to block RetGC activity during its maturation and/or incorporation into the photoreceptor outer segment rather than participate in dynamic regulation of the cyclase by Ca(2+) and GCAPs. << Less

  Biochemistry 50:9511-9519(2011) [PubMed] [EuropePMC]

• Enzymatic formation of inosine 3',5'-monophosphate and of 2'-deoxyguanosine 3',5'-monophosphate. Inosinate and deoxyguanylate cyclase activity.

  Garbers D.L., Suddath J.L., Hardman J.G.

  Enzymes in particulate fractions from sea urchin sperm and in soluble fractions from rat lung were shown to catalyze the formation of inosine 3',5'-monophosphate (cyclic IMP) and of 2'-deoxyguanosine 3',5'-monophosphate (cyclic dGMP) from ITP and dGTP, respectively. With sea urchin sperm particula ... >> More

  Enzymes in particulate fractions from sea urchin sperm and in soluble fractions from rat lung were shown to catalyze the formation of inosine 3',5'-monophosphate (cyclic IMP) and of 2'-deoxyguanosine 3',5'-monophosphate (cyclic dGMP) from ITP and dGTP, respectively. With sea urchin sperm particulate fractions, Mn2+ was an essential metal cofactor for inosinate, deoxyguanylate, guanylate and adenylate cyclase activities. Heat-inactivation studies differentiated inosinate and deoxyguanylate cyclase activities from adenylate cyclase, but indicated an association of these activities with guanylate cyclase. Preincubation of sea urchin sperm particulate fractions with trypsin altered in a very similar manner guanylate, inosinate, and deoxyguanylate cyclase activities, and various metals and metal-nucleotide combinations protected the three cyclase activities to comparable degrees against trypsin. The relative guanylate, deoxyguanylate and inosinate cyclase activities at 0.1 mM nucleoside triphosphate were 1.0, 0.5 and 0.08, respectively. With these three cyclase activities, plots of reciprocal velocities against reciprocal Mn2+-nucleoside triphosphate concentrations were concave upward, suggesting positive homotropic effects. With rat lung soluble preparations, relative guanylate, deoxyguanylate, inosinate and adenylate cyclase activities at 0.09 mM nucleoside triphosphate were 1.0, 1.7, 0.1 and 0, respectively. MnGTP was a competitive inhibitor of deoxyguanylate cyclase activity (Ki equals 12.2 muM) and MndGTP was a competitive inhibitor of guanylate cyclase activity (Ki equals 16.2 muM). Inhibition studies using ITP were not conducted. When soluble fractions from rat lung were applied to Bio-Gel A 1.5 m columns, elution profiles of guanylate, deoxyguanylate and inosinate cyclase activities were similar. These results suggest that deoxyguanylate, guanylate and inosinate cyclase activities reside within the same protein molecule. << Less

  Biochim Biophys Acta 377:174-185(1975) [PubMed] [EuropePMC]