RESUMO
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-B7E/7E 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. Importantly, how GC-B increases bone mass is not known. Here, we injected 12-week-old, wild type mice once daily for 28 days with or without BMN-111 (Vosoritide), a proteolytically resistant CNP analog. We found that BMN-111 treated mice had elevated levels of osteocalcin and collagen 1 C-terminal telopeptide (CTX) as well as increased osteoblasts and osteoclasts. In BMN-111 injected mice, tibial mRNAs for Rank ligand and osteoprotegrin were increased and decreased, respectively, whereas sclerostin mRNA was elevated 400-fold, consistent with increased osteoclast activity and decreased osteoblast activity. Mineral apposition rates and trabecular bone mass were not elevated in response to BMN-111. Because 9-week-old male GC-B7E/7E mice have increased bone mass but do not exhibit increased mineral apposition rates, we examined 4-week-old male GC-B7E/7E mice and found that these animals had increased serum osteocalcin, but not CTX. Importantly, tibias from these mice had 37% more osteoblasts, 26% fewer osteoclasts as well as 36% and 40% higher mineral apposition and bone formation rates, respectively. We conclude that GC-B-dependent bone formation is coupled to an early juvenile process that requires both increased osteoblasts and decreased osteoclasts.
Assuntos
Peptídeo Natriurético Tipo C , Osteoclastos , Animais , Colágeno , GMP Cíclico , Masculino , Camundongos , Peptídeo Natriurético Tipo C/genética , Peptídeo Natriurético Tipo C/metabolismo , Osteoblastos/metabolismo , Osteocalcina , Osteoclastos/metabolismo , Osteogênese , Ligante RANK , RNA MensageiroRESUMO
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-A8E/8E mice that express an enzyme that cannot be inactivated by dephosphorylation. GC-A activity, but not protein, was increased in heart and kidney membranes from GC-A8E/8E mice. Activities were threefold higher in female compared to male cardiac ventricles. Plasma cGMP and testosterone were elevated in male and female GC-A8E/8E mice, but aldosterone was only increased in mutant male mice. Plasma and urinary creatinine concentrations were decreased and increased, respectively, but blood pressure and heart rate were unchanged in male GC-A8E/8E mice. Heart weight to body weight ratios for GC-A8E/8E male, but not female, mice were 12% lower with a 14% reduction in cardiomyocyte cross-sectional area. Subcutaneous injection of fsANP, a long-lived ANP analog, increased plasma cGMP and decreased aldosterone in male GC-AWT/WT and GC-A8E/8E mice at 15 min, but only GC-A8E/8E mice had elevated levels of plasma cGMP and aldosterone at 60 min. fsANP reduced ventricular ERK1/2 phosphorylation to a greater extent and for a longer time in the male mutant compared to WT mice. Finally, ejection fractions were increased in male but not female hearts from GC-A8E/8E mice. We conclude that increased phosphorylation-dependent GC-A activity decreases cardiac ERK activity, which results in smaller male hearts with improved systolic function.
Assuntos
Cardiomegalia , Sistema de Sinalização das MAP Quinases , Fosforilação , Receptores do Fator Natriurético Atrial , Caracteres Sexuais , Animais , Cardiomegalia/enzimologia , Cardiomegalia/genética , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Receptores do Fator Natriurético Atrial/genética , Receptores do Fator Natriurético Atrial/metabolismoRESUMO
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. Crossing GC-B7E/7E mice with FGFR3G380R/G380R mice increased naso-anal and long (tibia and femur), but not cranial, bone length twice as much as crossing GC-B7E/7E mice with FGFR3WT/WT mice from 4 to 16 weeks of age. Consistent with increased GC-B activity rescuing ACH, long bones from the GC-B7E/7E/FGFR3G380R/G380R mice were not shorter than those from GC-BWT/WT/FGFR3WT/WT mice. At 2 weeks of age, male but not female FGFR3G380R/G380R mice had shorter long bones and smaller growth plate hypertrophic zones, whereas female but not male GC-B7E/7E mice had longer bones and larger hypertrophic zones. In 2-week-old males, crossing FGFR3G380R/G380R mice with GC-B7E/7E mice increased long bone length and hypertrophic zone area to levels observed in mice expressing WT versions of both receptors. We conclude that preventing GC-B dephosphorylation rescues reduced axial and appendicular skeleton growth in a mouse model of achondroplasia.
Assuntos
Acondroplasia/genética , Desenvolvimento Ósseo/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Receptores do Fator Natriurético Atrial/genética , Animais , Tamanho Corporal/genética , Fêmur/crescimento & desenvolvimento , Lâmina de Crescimento/crescimento & desenvolvimento , Lâmina de Crescimento/patologia , Camundongos , Camundongos Transgênicos , Tamanho do Órgão , Fosforilação , Receptores do Fator Natriurético Atrial/metabolismo , Crânio/crescimento & desenvolvimento , Tíbia/crescimento & desenvolvimentoRESUMO
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-B7E/7E knockin mice, expressing a glutamate-substituted, "pseudophosphorylated," form of GC-B, exhibit increased CNP-dependent GC activity. Since mutations that constitutively activate GC-B in the absence of CNP result in low bone mineral density in humans, we determined the skeletal phenotype of 9-week old male GC-B7E/7E mice. Unexpectedly, GC-B7E/7E mice have significantly greater tibial and L5 vertebral trabecular bone volume fraction, tibial trabecular number, and tibial bone mineral density. Cortical cross-sectional area, cortical thickness, periosteal diameter and cortical cross-sectional moment of inertia were also significantly increased in GC-B7E/7E tibiae. Three-point bending measurements demonstrated that the mutant tibias and femurs had greater ultimate load, stiffness, energy to ultimate load, and energy to failure. No differences in microhardness indicated similar bone quality at the tissue level between the mutant and wildtype bones. Procollagen 1 N-terminal propeptide and osteocalcin were elevated in serum, and osteoblast number per bone perimeter and osteoid width per bone perimeter were elevated in tibias from the mutant mice. In contrast to mutations that constitutively activate GC-B, we report that mutations that enhance GC-B activity only in the presence of its natural ligand, increase bone mass, bone strength, and the number of active osteoblasts at the bone surface.
Assuntos
Guanilato Ciclase , Peptídeo Natriurético Tipo C , Animais , Densidade Óssea , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Masculino , Camundongos , Osteoblastos/metabolismo , Fosforilação , Receptores do Fator Natriurético Atrial/genética , Receptores do Fator Natriurético Atrial/metabolismoRESUMO
cGMP signaling elicited by activation of the transmembrane receptor guanylyl cyclase Npr2 (also known as guanylyl cyclase B) by the ligand CNP controls sensory axon bifurcation of DRG and cranial sensory ganglion (CSG) neurons entering the spinal cord or hindbrain, respectively. Previous studies have shown that Npr2 is phosphorylated on serine and threonine residues in its kinase homology domain (KHD). However, it is unknown whether phosphorylation of Npr2 is essential for axon bifurcation. Here, we generated a knock-in mouse line in which the seven regulatory serine and threonine residues in the KHD of Npr2 were substituted by alanine (Npr2-7A), resulting in a nonphosphorylatable enzyme. Real-time imaging of cGMP in DRG neurons with a genetically encoded fluorescent cGMP sensor or biochemical analysis of guanylyl cyclase activity in brain or lung tissue revealed the absence of CNP-induced cGMP generation in the Npr27A/7A mutant. Consequently, bifurcation of axons, but not collateral formation, from DRG or CSG in this mouse mutant was perturbed at embryonic and mature stages. In contrast, axon branching was normal in a mouse mutant in which constitutive phosphorylation of Npr2 is mimicked by a replacement of all of the seven serine and threonine sites by glutamic acid (Npr2-7E). Furthermore, we demonstrate that the Npr27A/7A mutation causes dwarfism as described for global Npr2 mutants. In conclusion, our in vivo studies provide strong evidence that phosphorylation of the seven serine and threonine residues in the KHD of Npr2 is an important regulatory element of Npr2-mediated cGMP signaling which affects physiological processes, such as axon bifurcation and bone growth.SIGNIFICANCE STATEMENT The branching of axons is a morphological hallmark of virtually all neurons. It allows an individual neuron to innervate different targets and to communicate with neurons located in different regions of the nervous system. The natriuretic peptide receptor 2 (Npr2), a transmembrane guanylyl cyclase, is essential for the initiation of bifurcation of sensory axons when entering the spinal cord or the hindbrain. By using two genetically engineered mouse lines, we show that phosphorylation of specific serine and threonine residues in juxtamembrane regions of Npr2 are required for its enzymatic activity and for axon bifurcation. These investigations might help to understand the regulation of Npr2 and its integration in intracellular signaling systems.
Assuntos
Axônios/fisiologia , Gânglios Sensitivos/fisiologia , Receptores do Fator Natriurético Atrial/fisiologia , Serina/metabolismo , Treonina/metabolismo , Animais , Feminino , Gânglios Espinais/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação/fisiologia , Gravidez , Células Receptoras Sensoriais/fisiologia , Serina/genética , Treonina/genéticaRESUMO
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.
Assuntos
Desenvolvimento Ósseo , Fatores de Crescimento de Fibroblastos/metabolismo , Processamento de Proteína Pós-Traducional , Receptores do Fator Natriurético Atrial/metabolismo , Animais , GMP Cíclico/metabolismo , Camundongos , Fosforilação , Transdução de SinaisRESUMO
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. Cells expressing a phosphomimetic mutant of GC-B that cannot be inactivated by dephosphorylation because it contains glutamate substitutions for all known phosphorylation sites showed no decrease in GC-B activity in response to FGF. We conclude that FGF rapidly inactivates GC-B by a reversible dephosphorylation mechanism, which may contribute to the signaling network by which activated FGFR3 causes dwarfism.
Assuntos
Nanismo/genética , Peptídeo Natriurético Tipo C/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Receptores do Fator Natriurético Atrial/genética , Animais , Condrócitos/metabolismo , GMP Cíclico/genética , Modelos Animais de Doenças , Nanismo/metabolismo , Nanismo/patologia , Ácido Glutâmico/metabolismo , Humanos , Fosforilação , Ratos , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Receptores do Fator Natriurético Atrial/metabolismo , Transdução de SinaisRESUMO
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. 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.
Assuntos
Guanilato Ciclase/química , Receptores do Fator Natriurético Atrial/genética , Animais , Nanismo/metabolismo , Retículo Endoplasmático/metabolismo , Glicosilação , Humanos , MutaçãoRESUMO
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.
Assuntos
Nanismo/diagnóstico , Nanismo/genética , Heterozigoto , Mutação , Fenótipo , Receptores do Fator Natriurético Atrial/genética , Alelos , Criança , Pré-Escolar , Feminino , Frequência do Gene , Estudos de Associação Genética , Variação Genética , Humanos , Masculino , Linhagem , Receptores do Fator Natriurético Atrial/metabolismo , Análise de Sequência de DNARESUMO
T cells are known to potentiate the bone anabolic activity of intermittent parathyroid hormone (iPTH) treatment. One of the involved mechanisms is increased T cell secretion of Wnt10b, a potent osteogenic Wnt ligand that activates Wnt signaling in stromal cells (SCs). However, additional mechanisms might play a role, including direct interactions between surface receptors expressed by T cells and SCs. Here we show that iPTH failed to promote SC proliferation and differentiation into osteoblasts (OBs) and activate Wnt signaling in SCs of mice with a global or T cell-specific deletion of the T cell costimulatory molecule CD40 ligand (CD40L). Attesting to the relevance of T cell-expressed CD40L, iPTH induced a blunted increase in bone formation and failed to increase trabecular bone volume in CD40L(-/-) mice and mice with a T cell-specific deletion of CD40L. CD40L null mice exhibited a blunted increase in T cell production of Wnt10b and abrogated CD40 signaling in SCs in response to iPTH treatment. Therefore, expression of the T cell surface receptor CD40L enables iPTH to exert its bone anabolic activity by activating CD40 signaling in SCs and maximally stimulating T cell production of Wnt10b.
Assuntos
Anabolizantes/farmacologia , Osso e Ossos/efeitos dos fármacos , Ligante de CD40/imunologia , Hormônio Paratireóideo/farmacologia , Linfócitos T/imunologia , Anabolizantes/administração & dosagem , Animais , Ligante de CD40/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hormônio Paratireóideo/administração & dosagemRESUMO
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 10â 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.
Assuntos
GMP Cíclico/metabolismo , Células da Granulosa/metabolismo , Hormônio Luteinizante/metabolismo , Meiose/fisiologia , Oócitos/fisiologia , Receptores do Fator Natriurético Atrial/metabolismo , Análise de Variância , Animais , Western Blotting , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Ativação Enzimática , Ensaio de Imunoadsorção Enzimática , Feminino , Imunoprecipitação , Peptídeo Natriurético Tipo C/metabolismo , Folículo Ovariano/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Ratos , Receptores do Fator Natriurético Atrial/agonistasRESUMO
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.
Assuntos
Desenvolvimento Ósseo/fisiologia , Receptores do Fator Natriurético Atrial/metabolismo , Western Blotting , Desenvolvimento Ósseo/genética , Linhagem Celular , GMP Cíclico/metabolismo , Humanos , Mutação , Peptídeo Natriurético Tipo C/genética , Peptídeo Natriurético Tipo C/metabolismo , Fosforilação/genética , Fosforilação/fisiologia , Receptores do Fator Natriurético Atrial/genéticaRESUMO
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.
Assuntos
Trifosfato de Adenosina/metabolismo , Regulação Alostérica/fisiologia , Modelos Biológicos , Peptídeos Natriuréticos/metabolismo , Receptores do Fator Natriurético Atrial/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Sítio Alostérico/genética , Animais , Dimerização , Ativação Enzimática/fisiologia , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , Camundongos , Mutagênese , Mutação Puntual/genética , Receptores do Fator Natriurético Atrial/química , Análise de RegressãoRESUMO
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.
Assuntos
Hormônio Luteinizante/metabolismo , Oócitos/metabolismo , Folículo Ovariano/enzimologia , Receptores do Fator Natriurético Atrial/metabolismo , Animais , GMP Cíclico/metabolismo , Feminino , Células da Granulosa/metabolismo , Hormônio Luteinizante/farmacologia , Meiose/efeitos dos fármacos , Camundongos , Peptídeo Natriurético Tipo C/metabolismo , Peptídeo Natriurético Tipo C/farmacologia , Oócitos/citologia , Folículo Ovariano/citologia , Receptores do Fator Natriurético Atrial/antagonistas & inibidoresRESUMO
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 (32)PO(4) 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.
Assuntos
Receptores do Fator Natriurético Atrial/metabolismo , Substituição de Aminoácidos , Animais , Linhagem Celular , Humanos , Mutação de Sentido Incorreto , Mapeamento de Peptídeos/métodos , Peptídeos/genética , Peptídeos/metabolismo , Fosforilação/fisiologia , Estrutura Terciária de Proteína/fisiologia , Ratos , Receptores do Fator Natriurético Atrial/genéticaRESUMO
Molecular stress responses to pesticide exposures represent an understudied area of cnidarian transcriptome investigations. The organochlorine pesticide lindane is known to disrupt normal neuron function. Cnidarians with simple nervous systems are recognized as sensitive indicators of water quality, yet nothing is known about cnidarian responses to lindane. Sea anemones (Aiptasia pallida) were exposed for 4h to lindane (20 µg/l). Because anemones have neurons and lindane is known to target neurons, it is anticipated that cnidarian stress responses will include changes in transcription of genes associated with neurons. Representational Difference Analysis (RDA) was utilized to isolate differentially transcribed genes in the anemones exposed to the pesticide. After two rounds of RDA hybridizations, 148 amplified fragments ranging in size from 150 to 800 bp were cloned. Sequencing and bioinformatic analyses of 106 clones revealed 56 different gene fragments. Virtual Northern dot blots were used as a preliminary screening tool to identify the most responsive RDA products. To further characterize the specificity of response, additional anemones were exposed to a series of lindane concentrations (0, 0.2, 2.0, 10, and 20 µg/l). Northern dot blots were subsequently used to develop expression profiles for selected RDA products over the range of pesticide concentrations. The seven most responsive RDA products represent genes with products associated with neuron development, immune responses, and Ca(2+) binding/transport. The resulting expression profiles illustrate that these RDA products exhibit various degrees of concentration specificity with some RDA products being significantly up-regulated at 20 µg/l while other RDA products are most responsive at concentrations <20 µg/l. Results also demonstrate how RDA can be used to identify potentially important biomarkers of organochlorine exposure while generating new hypotheses about important phenomena such as endocrine disruption in cnidarians.
Assuntos
Expressão Gênica/efeitos dos fármacos , Hexaclorocicloexano/toxicidade , Anêmonas-do-Mar/genética , Poluentes Químicos da Água/toxicidade , Animais , DNA Complementar/metabolismo , Disruptores Endócrinos/toxicidade , Inseticidas/toxicidade , Anêmonas-do-Mar/efeitos dos fármacos , Anêmonas-do-Mar/metabolismoRESUMO
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 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.
Assuntos
Trifosfato de Adenosina/metabolismo , Carbazóis/farmacologia , Inibidores Enzimáticos/farmacologia , Guanosina Trifosfato/metabolismo , Receptores do Fator Natriurético Atrial/metabolismo , Sítios de Ligação , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Humanos , Ligação Proteica/efeitos dos fármacos , Receptores do Fator Natriurético Atrial/antagonistas & inibidores , Estaurosporina/análogos & derivadosRESUMO
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. EXPERIMENTAL APPROACH: Whole cell cGMP concentrations or ³²P-cGMP accumulation in membrane preparations measured the effects of indolocarbazoles on the enzymatic activity GC-A and GC-B from transfected 293T or endogenously expressing 3T3-L1 cells. KEY RESULTS: Gö6976 blocked cellular CNP-dependent cGMP elevations in 293T-GC-B cells. The t(½) for Gö6976 inhibition was 7 s and IC50 was 380 nM. Gö6976 increased the EC50 for CNP 4.5-fold, but increasing the CNP concentration did not overcome the inhibition. Half of the inhibition was lost 1 h after removal of Gö6976 from the medium. Cellular exposure to Gö6976 reduced basal and natriuretic peptide-dependent, but not detergent-dependent, GC-A and GC-B activity. Inhibition was also observed when Gö6976 was added directly to the cyclase assay. A constitutively phosphorylated form of GC-B was similarly inhibited. CONCLUSIONS AND IMPLICATIONS: These data demonstrate that Gö6976 potently, rapidly and reversibly inhibited GC-A and GC-B via a process that did not require intact cells, known phosphorylation sites or inactivation of all catalytic sites. This is the first report of an intracellular inhibitor of a transmembrane guanylyl cyclase and the first report of a non-kinase target for Gö6976.