ATP Potentiates Competitive Inhibition of Guanylyl Cyclase A and B by the Staurosporine Analog, Gö6976

Abstract

Natriuretic peptides and ATP activate and Gö6976 inhibits guanylyl cyclase (GC)-A and GC-B. Here, the mechanism of inhibition was determined. Gö6976 progressively increased the Michaelis-Menten constant and decreased the Hill coefficient without reducing the maximal velocity of GC-A and GC-B. In the presence of 1 mM ATP, the K i was 1 M for both enzymes. Inhibition of GC-B was minimal in the absence of ATP, and 1 mM ATP increased the inhibition 4-fold. In a reciprocal manner, 10 M Gö6976 increased the potency of ATP for GC-B 4-fold. In contrast to a recent study (Duda, T., Yadav, P., and Sharma, R. K. (2010) FEBS J. 277, 2550 -2553), neither staurosporine nor Gö6976 activated GC-A or GC-B. This is the first study to show that Gö6976 reduces GTP binding and the first demonstration of a competitive inhibitor of a receptor guanylyl cyclase. We conclude that Gö6976 reduces GTP binding to the catalytic site of GC-A and GC-B and that ATP increases the magnitude of the inhibition.

Dephosphorylation and inactivation of NPR2 guanylyl cyclase in granulosa cells contributes to the LH-induced decrease in cGMP that causes resumption of meiosis in rat oocytes

“…3A), validated the specificity of the antibody. The relative amount of NPR2 staining in a sample of follicle membranes was 2-7% of that in a sample from overexpressing HEK cells (normalized to the amount of membrane protein loaded per lane), and this corresponded closely to the relative amount of NPR2 activity (2-4% of that reported for this cell line, Robinson and Potter, 2011;, further confirming the antibody specificity. The use of Phos-tag gel migration as an indicator of the phosphorylation state of NPR2 was validated by showing that incubation of follicle membranes under conditions that promoted or inhibited phosphatase activity resulted in faster or slower migrating forms of NPR2 ( Fig.…”

“…Preparation of crude membranes from rat follicles and measurement of guanylyl cyclase activity Crude membranes were prepared from rat follicles, and guanylyl cyclase assays were conducted as previously described for mouse follicles (Robinson and Potter, 2011;. cGMP production was measured after a 9-min assay period and was approximately linear over this period .…”

Guanylyl Cyclases A and B Are Asymmetric Dimers That Are Allosterically Activated by ATP Binding to the Catalytic Domain

“…HEK 293T, HEK 293T–GC-A, and HEK 293T–GC-B cells were maintained as previously described (58). HEK 293T cells do not contain detectable NP receptors (figs.…”

“…If not indicated, NP concentrations were 1 μM. Concentrations of cGMP were determined by radioimmuno-assay as previously described (58). Because enzymatic activity was not completely linear with time, we qualified the kinetic parameters obtained under these conditions as “apparent.”…”

Catalytically Active Guanylyl Cyclase B Requires Endoplasmic Reticulum-mediated Glycosylation, and Mutations That Inhibit This Process Cause Dwarfism

“…Guanylyl Cyclase Assays-Crude membranes were prepared in phosphatase inhibitor buffer from 293T-GC-B cells as described previously (33). All assays were performed at 37°C in a mixture containing 25 mM Hepes (pH 7.4), 50 mM NaCl, 0.1% BSA, 0.5 mM isobutylmethyl xanthine, 1 mM EDTA, 0.5 M microcystin, 10 mg/ml creatine kinase, 5 mM creatine phosphate, and 5 mM MgCl 2 .…”