Phosphorylation-Dependent Regulation of Guanylyl Cyclase (GC)-A and Other Membrane GC Receptors.

Receptor guanylyl cyclases (GCs) are single membrane spanning, multidomain enzymes, that synthesize cGMP in response to natriuretic peptides or other ligands. They are evolutionarily conserved from sea urchins to humans and regulate diverse physiologies. Most family members are phosphorylated on 4 to 7 conserved serines or threonines at the beginning of their kinase homology domains.

The Concise Guide to PHARMACOLOGY 2023/24: Catalytic receptors.

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and nearly 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.

Vicinal glutamates are better phosphomimetics: Phosphorylation is required for allosteric activation of guanylyl cyclase-A.

Multisite phosphorylation of guanylyl cyclase (GC)-A, also known as NPR-A or NPR1, is required for receptor activation by natriuretic peptides (NPs) because alanine substitutions for the first four GC-A phosphorylation sites produce an enzyme that cannot be stimulated by NPs. In contrast, single Glu substitutions for the first six chemically identified GC-A phosphorylation sites to mimic the negative charge of phosphate produced an enzyme that is activated by NPs but had an elevated Michaelis constant (Km), resulting in low activity. Here, we show that vicinal (double adjacent) Glu substitutions for the same sites to mimic the two negative charges of phosphate produced a near wild type (WT) enzyme with a low Km.

Epitope-tagged and phosphomimetic mouse models for investigating natriuretic peptide-stimulated receptor guanylyl cyclases.

The natriuretic peptide receptors NPR1 and NPR2, also known as guanylyl cyclase A and guanylyl cyclase B, have critical functions in many signaling pathways, but much remains unknown about their localization and function . To facilitate studies of these proteins, we developed genetically modified mouse lines in which endogenous NPR1 and NPR2 were tagged with the HA epitope. To investigate the role of phosphorylation in regulating NPR1 and NPR2 guanylyl cyclase activity, we developed mouse lines in which regulatory serines and threonines were substituted with glutamates, to mimic the negative charge of the phosphorylated forms (NPR1-8E and NPR2-7E).

Guanylyl Cyclase-B Dependent Bone Formation in Mice is Associated with Youth, Increased Osteoblasts, and Decreased Osteoclasts.

C-type natriuretic peptide (CNP) activation of guanylyl cyclase-B (GC-B) catalyzes the synthesis of cGMP in chondrocytes and osteoblasts. Elevated cGMP stimulates long bone growth, and inactivating mutations in CNP or GC-B reduce cGMP, which causes dwarfism. GC-B mice that express a GC-B mutant that cannot be inactivated by dephosphorylation exhibit increased CNP-dependent GC-B activity, which increases bone length, as well as bone mass and strength.

Guanylyl cyclase-A phosphorylation decreases cardiac hypertrophy and improves systolic function in male, but not female, mice.

Atrial natriuretic peptide (NP) and BNP increase cGMP, which reduces blood pressure and cardiac hypertrophy by activating guanylyl cyclase (GC)-A, also known as NPR-A or Npr1. Although GC-A is highly phosphorylated, and dephosphorylation inactivates the enzyme, the significance of GC-A phosphorylation to heart structure and function remains unknown. To identify in vivo processes that are regulated by GC-A phosphorylation, we substituted glutamates for known phosphorylation sites to make GC-A mice that express an enzyme that cannot be inactivated by dephosphorylation.

THE CONCISE GUIDE TO PHARMACOLOGY 2021/22: Catalytic receptors.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.

Prevention of guanylyl cyclase-B dephosphorylation rescues achondroplastic dwarfism.

Activating mutations in the fibroblast growth factor receptor 3 (FGFR3) or inactivating mutations in guanylyl cyclase-B (GC-B), also known as NPR-B or Npr2, cause short-limbed dwarfism. FGFR3 activation causes dephosphorylation and inactivation of GC-B, but the contribution of GC-B dephosphorylation to achondroplasia (ACH) is unknown. GC-B7E/7E mice that express a glutamate-substituted version of GC-B that cannot be inactivated by dephosphorylation were bred with mice expressing FGFR3-G380R, the most common human ACH mutation, to determine if GC-B dephosphorylation is required for ACH.

Male mice with elevated C-type natriuretic peptide-dependent guanylyl cyclase-B activity have increased osteoblasts, bone mass and bone strength.

C-type natriuretic peptide (CNP) activation of guanylyl cyclase (GC)-B, also known as NPR2, stimulates cGMP synthesis and bone elongation. CNP activation requires the phosphorylation of multiple GC-B residues and dephosphorylation inactivates the receptor. GC-B knockin mice, expressing a glutamate-substituted, "pseudophosphorylated," form of GC-B, exhibit increased CNP-dependent GC activity.

The pseudokinase domains of guanylyl cyclase-A and -B allosterically increase the affinity of their catalytic domains for substrate.

Natriuretic peptides regulate multiple physiologic systems by activating transmembrane receptors containing intracellular guanylyl cyclase domains, such as GC-A and GC-B, also known as Npr1 and Npr2, respectively. Both enzymes contain an intracellular, phosphorylated pseudokinase domain (PKD) critical for activation of the C-terminal cGMP-synthesizing guanylyl cyclase domain. Because ATP allosterically activates GC-A and GC-B, we investigated how ATP binding to the PKD influenced guanylyl cyclase activity.

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.

Dephosphorylation is the mechanism of fibroblast growth factor inhibition of guanylyl cyclase-B.

Activating mutations in fibroblast growth factor receptor 3 (FGFR3) and inactivating mutations of guanylyl cyclase-B (GC-B, also called NPRB or NPR2) cause dwarfism. FGF exposure inhibits GC-B activity in a chondrocyte cell line, but the mechanism of the inactivation is not known. Here, we report that FGF exposure causes dephosphorylation of GC-B in rat chondrosarcoma cells, which correlates with a rapid, potent and reversible inhibition of C-type natriuretic peptide-dependent activation of GC-B.

Skeletal overgrowth-causing mutations mimic an allosterically activated conformation of guanylyl cyclase-B that is inhibited by 2,4,6,-trinitrophenyl ATP.

Activating mutations in the receptor for C-type natriuretic peptide (CNP), guanylyl cyclase B (GC-B, also known as Npr2 or NPR-B), increase cellular cGMP and cause skeletal overgrowth, but how these mutations affect GTP catalysis is poorly understood. The A488P and R655C mutations were compared with the known mutation V883M. Neither mutation affected GC-B concentrations.

A Glutamate-Substituted Mutant Mimics the Phosphorylated and Active Form of Guanylyl Cyclase-A.

Multisite phosphorylation is required for activation of guanylyl cyclase (GC)-A, also known as NPR-A or NPR1, by cardiac natriuretic peptides (NPs). Seven chemically identified sites (Ser-487, Ser-497, Thr-500, Ser-502, Ser-506, Ser-510, and Thr-513) and one functionally identified putative site (Ser-473) were reported. Single alanine substitutions for Ser-497, Thr-500, Ser-502, Ser-506, and Ser-510 reduced maximal velocity (), whereas glutamate substitutions had no effect or increased Ala but not Glu substitution for Ser-497 increased the Michaelis constant () approximately 400%.

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 (125)I-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.

Dephosphorylation of juxtamembrane serines and threonines of the NPR2 guanylyl cyclase is required for rapid resumption of oocyte meiosis in response to luteinizing hormone.

The meiotic cell cycle of mammalian oocytes starts during embryogenesis and then pauses until luteinizing hormone (LH) acts on the granulosa cells of the follicle surrounding the oocyte to restart the cell cycle. An essential event in this process is a decrease in cyclic GMP in the granulosa cells, and part of the cGMP decrease results from dephosphorylation and inactivation of the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase, also known as guanylyl cyclase B. However, it is unknown whether NPR2 dephosphorylation is essential for LH-induced meiotic resumption.

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.

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).

Regulation of intraocular pressure by soluble and membrane guanylate cyclases and their role in glaucoma.

Glaucoma is a progressive optic neuropathy characterized by visual field defects that ultimately lead to irreversible blindness (Alward, 2000; Anderson et al., 2006). By the year 2020, an estimated 80 million people will have glaucoma, 11 million of which will be bilaterally blind.

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.

Guanylyl cyclases A and B are asymmetric dimers that are allosterically activated by ATP binding to the catalytic domain.

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.