Jerid W. Robinson

Luteinizing hormone reduces the activity of the NPR2 guanylyl cyclase in mouse ovarian follicles, contributing to the cyclic GMP decrease that promotes resumption of meiosis in oocytes

In preovulatory ovarian follicles of mice, meiotic prophase arrest in the oocyte is maintained by cyclic GMP from the surrounding granulosa cells that diffuses into the oocyte through gap junctions. The cGMP is synthesized in the granulosa cells by the transmembrane guanylyl cyclase natriuretic peptide receptor 2 (NPR2) in response to the agonist C-type natriuretic peptide (CNP). In response to luteinizing hormone (LH), cGMP in the granulosa cells decreases, and as a consequence, oocyte cGMP decreases and meiosis resumes. Here we report that within 20 min, LH treatment results in decreased guanylyl cyclase activity of NPR2, as determined in the presence of a maximally activating concentration of CNP. This occurs by a process that does not reduce the amount of NPR2 protein. We also show that by a slower process, first detected at 2h, LH decreases the amount of CNP available to bind to the receptor. Both of these LH actions contribute to decreasing cGMP in the follicle, thus signaling meiotic resumption in the oocyte.

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

In mammals, the meiotic cell cycle of oocytes starts during embryogenesis and then pauses. Much later, in preparation for fertilization, oocytes within preovulatory follicles resume meiosis in response to luteinizing hormone (LH). Before LH stimulation, the arrest is maintained by diffusion of cyclic (c)GMP into the oocyte from the surrounding granulosa cells, where it is produced by the guanylyl cyclase natriuretic peptide receptor 2 (NPR2). LH rapidly reduces the production of cGMP, but how this occurs is unknown. Here, using rat follicles, we show that within 10 min, LH signaling causes dephosphorylation and inactivation of NPR2 through a process that requires the activity of phosphoprotein phosphatase (PPP)-family members. The rapid dephosphorylation of NPR2 is accompanied by a rapid phosphorylation of the cGMP phosphodiesterase PDE5, an enzyme whose activity is increased upon phosphorylation. Later, levels of the NPR2 agonist C-type natriuretic peptide decrease in the follicle, and these sequential events contribute to the decrease in cGMP that causes meiosis to resume in the oocyte.

Heterozygous mutations in natriuretic peptide receptor-B (NPR2) gene as a cause of short stature

Based on the observation of reduced stature in relatives of patients with acromesomelic dysplasia, Maroteaux type (AMDM), caused by homozygous or compound heterozygous mutations in natriuretic peptide receptor‐B gene (NPR2), it has been suggested that heterozygous mutations in this gene could be responsible for the growth impairment observed in some cases of idiopathic short stature (ISS). We enrolled 192 unrelated patients with short stature and 192 controls of normal height and identified seven heterozygous NPR2 missense or splice site mutations all in the short stature patients, including one de novo splice site variant. Three of the six inherited variants segregated with short stature in the family. Nine additional rare nonsynonymous NPR2 variants were found in three additional cohorts. Functional studies identified eight loss‐of‐function mutations in short individuals and one gain‐of‐function mutation in tall individuals. With these data, we were able to rigorously verify that NPR2 functional haploinsufficiency contributes to short stature. We estimate a prevalence of NPR2 haploinsufficiency of between 0 and 1/26 in people with ISS. We suggest that NPR2 gain of function may be a more common cause of tall stature than previously recognized.

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

An ATP-binding allosteric site could be pharmacologically targeted to alter the activity of membrane guanylyl cyclases. ATP Allosterically Regulates Guanylyl Cyclases Adenylyl cyclase (AC) and guanylyl cyclase (GC) catalyze the generation of the cyclic nucleoside monophosphates cAMP and cGMP, respectively, which act as intracellular second messengers. ACs and GCs are implicated in various cardiovascular and metabolic diseases, making them pharmacological targets; however, because their catalytic sites are conserved, development of selective activators and inhibitors has been difficult (see the Perspective by Seifert and Beste). Through enzymatic analysis of wild-type and mutant GCs, Robinson and Potter showed that the binding of ATP to an allosteric site near the GTP-binding catalytic site of these enzymes enhanced their activity in response to GC ligands. Together, these data suggest that the allosteric site of GCs may provide a potential therapeutic target to modulate enzyme activity. It is not known how natriuretic peptides and adenosine triphosphate (ATP) activate guanylyl cyclase A (GC-A) and GC-B, which generate the second messenger cyclic guanosine monophosphate. We determined that natriuretic peptides increased the maximum rate of these enzymes >10-fold in a positive cooperative manner in the absence of ATP. In the absence of natriuretic peptides, ATP shifted substrate-velocity profiles from cooperative to linear but did not increase the affinity of GCs for the substrate guanosine triphosphate (GTP) since the Michaelis constant was unchanged. However, in the presence of natriuretic peptides, ATP competed with GTP for binding to an allosteric site, which enhanced the activation of GCs by decreasing the Michaelis constant. Thus, natriuretic peptide binding was required for communication of the allosteric activation signal to the catalytic site. The ability of ATP to activate GCs decreased and enzyme potency (a measure of sensitivity to stimulation) increased with increasing GTP concentrations. Point mutations in the purine-binding site of the catalytic domain abolished GC activity but not allosteric activation. Coexpression of inactive mutants produced half the activity expected for symmetric catalytic dimers. 2′-Deoxy-ATP and 2′-deoxy-GTP were poor allosteric activators, but 2′-deoxy-GTP was an effective substrate, consistent with distinct binding requirements for the allosteric and catalytic sites. We conclude that membrane GC domains are asymmetric homodimers with distinct and reciprocally regulated catalytic and allosteric sites that bind to GTP and ATP, respectively. These data define a new membrane GC activation model and provide evidence of a previously unidentified GC drug interaction site.

A Functional Screen Provides Evidence for a Conserved, Regulatory, Juxtamembrane Phosphorylation Site in Guanylyl Cyclase A and B

Kinase homology domain (KHD) phosphorylation is required for activation of guanylyl cyclase (GC)-A and -B. Phosphopeptide mapping identified multiple phosphorylation sites in GC-A and GC-B, but these approaches have difficulty identifying sites in poorly detected peptides. Here, a functional screen was conducted to identify novel sites. Conserved serines or threonines in the KHDs of phosphorylated receptor GCs were mutated to alanine and tested for reduced hormone to detergent activity ratios. Mutation of Ser-489 in GC-B to alanine but not glutamate reduced the activity ratio to 60% of wild type (WT) levels. Similar results were observed with Ser-473, the homologous site in GC-A. Receptors containing glutamates for previously identified phosphorylation sites (GC-A-6E and GC-B-6E) were activated to ∼20% of WT levels but the additional glutamate substitution for S473 or S489 increased activity to near WT levels. Substrate-velocity assays indicated that GC-B-WT-S489E and GC-B-6E-S489E had lower Km values and that WT-GC-B-S489A, GC-B-6E and GC-B-6E-S489A had higher Km values than WT-GC-B. Homologous desensitization was enhanced when GC-A contained the S473E substitution, and GC-B-6E-S489E was resistant to inhibition by a calcium elevating treatment or protein kinase C activation – processes that dephosphorylate GC-B. Mass spectrometric detection of a synthetic phospho-Ser-473 containing peptide was 200–1300-fold less sensitive than other phosphorylated peptides and neither mass spectrometric nor 32PO4 co-migration studies detected phospho-Ser-473 or phospho-Ser-489 in cells. We conclude that Ser-473 and Ser-489 are Km-regulating phosphorylation sites that are difficult to detect using current methods.

A human skeletal overgrowth mutation increases maximal velocity and blocks desensitization of guanylyl cyclase-B☆

C-type natriuretic peptide (CNP) increases long bone growth by stimulating guanylyl cyclase (GC)-B/NPR-B/NPR2. Recently, a Val to Met missense mutation at position 883 in the catalytic domain of GC-B was identified in humans with increased blood cGMP levels that cause abnormally long bones. Here, we determined how this mutation activates GC-B. In the absence of CNP, cGMP levels in cells expressing V883M-GC-B were increased more than 20 fold compared to cells expressing wild-type (WT)-GC-B, and the addition of CNP only further increased cGMP levels 2-fold. In the absence of CNP, maximal enzymatic activity (Vmax) of V883M-GC-B was increased 15-fold compared to WT-GC-B but the affinity of the enzymes for substrate as revealed by the Michaelis constant (Km) was unaffected. Surprisingly, CNP decreased the Km of V883M-GC-B 10-fold in a concentration-dependent manner without increasing Vmax. Unlike the WT enzyme the Km reduction of V883M-GC-B did not require ATP. Unexpectedly, V883M-GC-B, but not WT-GC-B, failed to inactivate with time. Phosphorylation elevated but was not required for the activity increase associated with the mutation because the Val to Met substitution also activated a GC-B mutant lacking all known phosphorylation sites. We conclude that the V883M mutation increases maximal velocity in the absence of CNP, eliminates the requirement for ATP in the CNP-dependent Km reduction, and disrupts the normal inactivation process.

ATP potentiates competitive inhibition of guanylyl cyclase A and B by the staurosporine analog, Gö6976: Reciprocal regulation of ATP and GTP binding

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 Ki was 1 μm 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 μm 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.

The indolocarbazole, Gö6976, inhibits guanylyl cyclase-A and -B

BACKGROUND AND PURPOSE Atrial natriuretic peptide (ANP) and B‐type natriuretic peptide (BNP) decrease vascular volume and pressure by activating guanylyl cyclase‐A (GC‐A). C‐type natriuretic peptide (CNP) activation of guanylyl cyclase‐B (GC‐B) stimulates long bone growth. This study investigated the effects of the indolocarbazole, Gö6976, on the guanylyl cyclase activity of GC‐A and GC‐B as a first step towards developing small molecule regulators of these enzymes.

Catalytically active guanylyl cyclase B requires endoplasmic reticulum-mediated glycosylation, and mutations that inhibit this process cause dwarfism

C-type natriuretic peptide activation of guanylyl cyclase B (GC-B), also known as natriuretic peptide receptor B or NPR2, stimulates long bone growth, and missense mutations in GC-B cause dwarfism. Four such mutants (L658F, Y708C, R776W, and G959A) bound 125I-C-type natriuretic peptide on the surface of cells but failed to synthesize cGMP in membrane GC assays. Immunofluorescence microscopy also indicated that the mutant receptors were on the cell surface. All mutant proteins were dephosphorylated and incompletely glycosylated, but dephosphorylation did not explain the inactivation because the mutations inactivated a “constitutively phosphorylated” enzyme. Tunicamycin inhibition of glycosylation in the endoplasmic reticulum or mutation of the Asn-24 glycosylation site decreased GC activity, but neither inhibition of glycosylation in the Golgi by N-acetylglucosaminyltransferase I gene inactivation nor PNGase F deglycosylation of fully processed GC-B reduced GC activity. We conclude that endoplasmic reticulum-mediated glycosylation is required for the formation of an active catalytic, but not ligand-binding domain, and that mutations that inhibit this process cause dwarfism.

Dephosphorylation of the NPR2 guanylyl cyclase contributes to inhibition of bone growth by fibroblast growth factor

Activating mutations in fibroblast growth factor (FGF) receptor 3 and inactivating mutations in the NPR2 guanylyl cyclase both cause severe short stature, but how these two signaling systems interact to regulate bone growth is poorly understood. Here, we show that bone elongation is increased when NPR2 cannot be dephosphorylated and thus produces more cyclic GMP. By developing an in vivo imaging system to measure cyclic GMP production in intact tibia, we show that FGF-induced dephosphorylation of NPR2 decreases its guanylyl cyclase activity in growth plate chondrocytes in living bone. The dephosphorylation requires a PPP-family phosphatase. Thus FGF signaling lowers cyclic GMP production in the growth plate, which counteracts bone elongation. These results define a new component of the signaling network by which activating mutations in the FGF receptor inhibit bone growth.