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Lincoln R. Potter - One of the best experts on this subject based on the ideXlab platform.
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regulation of the natriuretic peptide receptor 2 NPR2 by phosphorylation of juxtamembrane serine and threonine residues is essential for bifurcation of sensory axons
The Journal of Neuroscience, 2018Co-Authors: Jerid W. Robinson, Lincoln R. Potter, Hannes Schmidt, Deborah M Dickey, Alexandre Dumoulin, Marie Octave, Ralf Kuhn, Robert Feil, Fritz G RathjenAbstract:cGMP signaling elicited by activation of the transmembrane receptor guanylyl cyclase NPR2 (also known as GC-B) by the ligand CNP controls sensory axon bifurcation of dorsal root ganglion (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 non-phosphorylatable 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 NPR2 7A/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 NPR2 7A/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. Significant 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.
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Regulation of the Natriuretic Peptide Receptor 2 (NPR2) by Phosphorylation of Juxtamembrane Serine and Threonine Residues Is Essential for Bifurcation of Sensory Axons.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2018Co-Authors: Hannes Schmidt, Jerid W. Robinson, Lincoln R. Potter, Deborah M Dickey, Alexandre Dumoulin, Marie Octave, Ralf Kuhn, Robert Feil, Fritz G RathjenAbstract: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.
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Dephosphorylation of juxtamembrane serines and threonines of the NPR2 guanylyl cyclase is required for rapid resumption of oocyte meiosis in response to luteinizing hormone.
Developmental biology, 2015Co-Authors: Leia C. Shuhaibar, Aaron B Edmund, Lincoln R. Potter, Deborah M Dickey, Tracy F. Uliasz, Siu-pok Yee, Jeremy R. Egbert, Laurinda A. JaffeAbstract: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. Here, we prevented NPR2 dephosphorylation by generating a mouse line in which the seven regulatory serines and threonines of NPR2 were changed to the phosphomimetic amino acid glutamate (NPR2–7E). NPR2–7E/7E follicles failed to show a decrease in enzyme activity in response to LH, and the cGMP decrease was attenuated; correspondingly, LH-induced meiotic resumption was delayed. Meiotic resumption in response to EGF receptor activation was likewise delayed, indicating that NPR2 dephosphorylation is a component of the pathway by which EGF receptor activation mediates LH signaling. We also found that most of the NPR2 protein in the follicle was present in the mural granulosa cells. These findings indicate that NPR2 dephosphorylation in the mural granulosa cells is essential for the normal progression of meiosis in response to LH and EGF receptor activation. In addition, these studies provide the first demonstration that a change in phosphorylation of a transmembrane guanylyl cyclase regulates a physiological process, a mechanism that may also control other developmental events.
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Dephosphorylation of juxtamembrane serines and threonines of the NPR2 guanylyl cyclase regulates oocyte meiotic resumption
BMC Clinical Pharmacology, 2015Co-Authors: Leia C. Shuhaibar, Aaron B Edmund, Lincoln R. Potter, Siu-pok Yee, Jeremy R. Egbert, Laurinda A. JaffeAbstract:Background The meiotic cell cycle of mammalian oocytes starts during embryogenesis and then pauses until luteinizing hormone (LH) restarts the cycle. This meiotic arrest is maintained by cGMP, which is produced in the granulosa cells by C-type natriuretic peptide (CNP) activation of NPR2 [1]. LH decreases cGMP in the granulosa cells, and via equilibration through gap junctions, cyclic GMP also decreases in the oocyte, thus releasing the meiotic arrest [2]. LH causes dephosphorylation and inactivation of NPR2 [3,4], but whether NPR2 dephosphorylation is required for meiotic resumption is not known. Seven regulatory NPR2 phosphorylation sites have been identified (Fig. 1) [5,6]. Here, we generated a knock-in mouse where each site was mutated to glutamate (NPR2-7E), resulting in a “constitutively phosphorylated” enzyme that we used to investigate the role of NPR2 dephosphorylation in the rapid resumption of meiosis in response to LH.
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heterozygous mutations in natriuretic peptide receptor b NPR2 gene as a cause of short stature
Human Mutation, 2015Co-Authors: Sophie R Wang, Jerid W. Robinson, Christina M Jacobsen, Aaron B Edmund, Robert C Olney, Jennifer E Moon, Timothy C. Miller, Verónica Mericq, Heather Carmichael, Lincoln R. PotterAbstract: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.
Hannes Schmidt - One of the best experts on this subject based on the ideXlab platform.
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NPR2 null mutants show initial overshooting followed by reduction of spiral ganglion axon projections combined with near-normal cochleotopic projection
Cell and Tissue Research, 2019Co-Authors: Hannes Schmidt, Bernd FritzschAbstract:NPR2 (natriuretic peptide receptor 2) affects bifurcation of neural crest or placode-derived afferents upon entering the brain stem/spinal cord, leading to a lack of either rostral or caudal branches. Previous work has shown that early embryonic growth of cochlear and vestibular afferents is equally affected in this mutant but later work on postnatal NPR2 point mutations suggested some additional effects on the topology of afferent projections and mild functional defects. Using multicolor lipophilic dye tracing, we show that absence of NPR2 has little to no effect on the initial patterning of inner ear afferents with respect to their dorsoventral cochleotopic-specific projections. However, in contrast to control animals, we found a variable degree of embryonic extension of auditory afferents beyond the boundaries of the anterior cochlear nucleus into the cerebellum that emanates only from apical spiral ganglion neurons. Such expansion has previously only been reported for Hox gene mutants and implies an unclear interaction of Hox codes with NPR2-mediated afferent projection patterning to define boundaries. Some vestibular ganglion neurons expand their projections to reach the cochlear apex and the cochlear nuclei, comparable to previous findings in Neurod1 mutant mice. Before birth, such expansions are reduced or lost leading to truncated projections to the anteroventral cochlear nucleus and expansion of low-frequency fibers of the apex to the posteroventral cochlear nucleus.
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regulation of the natriuretic peptide receptor 2 NPR2 by phosphorylation of juxtamembrane serine and threonine residues is essential for bifurcation of sensory axons
The Journal of Neuroscience, 2018Co-Authors: Jerid W. Robinson, Lincoln R. Potter, Hannes Schmidt, Deborah M Dickey, Alexandre Dumoulin, Marie Octave, Ralf Kuhn, Robert Feil, Fritz G RathjenAbstract:cGMP signaling elicited by activation of the transmembrane receptor guanylyl cyclase NPR2 (also known as GC-B) by the ligand CNP controls sensory axon bifurcation of dorsal root ganglion (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 non-phosphorylatable 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 NPR2 7A/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 NPR2 7A/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. Significant 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.
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Regulation of the Natriuretic Peptide Receptor 2 (NPR2) by Phosphorylation of Juxtamembrane Serine and Threonine Residues Is Essential for Bifurcation of Sensory Axons.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2018Co-Authors: Hannes Schmidt, Jerid W. Robinson, Lincoln R. Potter, Deborah M Dickey, Alexandre Dumoulin, Marie Octave, Ralf Kuhn, Robert Feil, Fritz G RathjenAbstract: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.
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Distinct submembrane localisation compartmentalises cardiac NPR1 and NPR2 signalling to cGMP.
Nature communications, 2018Co-Authors: Hariharan Subramanian, Hannes Schmidt, Alexander Froese, Peter Jönsson, Julia Gorelik, Viacheslav O. NikolaevAbstract:Natriuretic peptides (NPs) are important hormones that regulate multiple cellular functions including cardiovascular physiology. In the heart, two natriuretic peptide receptors NPR1 and NPR2 act as membrane guanylyl cyclases to produce 3′,5′-cyclic guanosine monophosphate (cGMP). Although both receptors protect from cardiac hypertrophy, their effects on contractility are markedly different, from little effect (NPR1) to pronounced negative inotropic and positive lusitropic responses (NPR2) with unclear underlying mechanisms. Here we use a scanning ion conductance microscopy (SICM) approach combined with Forster resonance energy transfer (FRET)-based cGMP biosensors to show that whereas NPR2 is uniformly localised on the cardiomyocyte membrane, functional NPR1 receptors are found exclusively in membrane invaginations called transverse (T)-tubules. This leads to far-reaching CNP/NPR2/cGMP signals, whereas ANP/NPR1/cGMP signals are highly confined to T-tubular microdomains by local pools of phosphodiesterase 2. This provides a previously unrecognised molecular basis for clearly distinct functional effects engaged by different cGMP producing membrane receptors.
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Loss of Axon Bifurcation in Mesencephalic Trigeminal Neurons Impairs the Maximal Biting Force in NPR2-Deficient Mice.
Frontiers in cellular neuroscience, 2018Co-Authors: Gohar Ter-avetisyan, Hannes Schmidt, Alexandre Dumoulin, Anthony Herrel, Johanna Strump, Shoaib Afzal, Fritz G RathjenAbstract:Bifurcation of axons from dorsal root ganglion (DRG) and cranial sensory ganglion (CSG) neurons is mediated by a cGMP-dependent signaling pathway composed of the ligand C-type natriuretic peptide (CNP), the receptor guanylyl cyclase NPR2 and the cGMP-dependent protein kinase I (cGKI). Here, we demonstrate that mesencephalic trigeminal neurons (MTN) which are the only somatosensory neurons whose cell bodies are located within the CNS co-express NPR2 and cGKI. Afferents of MTNs form Y-shaped branches in rhombomere 2 where the ligand CNP is expressed. Analyzing mouse mutants deficient for CNP or NPR2 we found that in the absence of CNP-induced cGMP signaling MTN afferents no longer bifurcate and instead extend either into the trigeminal root or caudally in the hindbrain. Since MTNs provide sensory information from jaw closing muscles and periodontal ligaments we measured the bite force of conditional mouse mutants of NPR2 (NPR2flox/flox;Engr1Cre ) that lack bifurcation of MTN whereas the bifurcation of trigeminal afferents is normal. Our study revealed that the maximal biting force of both sexes is reduced in NPR2flox/flox;Engr1Cre mice as compared to their NPR2flox/flox littermate controls. In conclusion sensory feedback mechanisms from jaw closing muscles or periodontal ligaments might be impaired in the absence of MTN axon bifurcation.
Huijuan Zhu - One of the best experts on this subject based on the ideXlab platform.
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Clinical Characteristics of short stature patients with NPR2 mutation and the therapeutic response to rhGH.
The Journal of clinical endocrinology and metabolism, 2020Co-Authors: Hanting Liang, Hui Miao, Hongbo Yang, Linjie Wang, Fengying Gong, Hui Pan, Huijuan ZhuAbstract:CONTEXT Natriuretic peptide receptor 2 gene (NPR2) is a causative gene of idiopathic short stature (ISS) with an incidence rate of 2%-6%. The clinical characteristics of patients with NPR2 heterozygous mutation are atypical, and data on the efficacy of recombinant human growth hormone (rhGH) treatment in patients with NPR2 mutation are limited. OBJECTIVES To report six cases with NPR2 mutation and explore the characteristics of patients with NPR2 mutation and their therapeutic response to rhGH. DESIGN, SETTINGS AND PATIENTS Six Chinese short stature patients in our hospital with NPR2 mutation by whole-o exome sequencing were included. We also searched all previously published NPR2 mutation cases as of August 10, 2020, and the information of their medical history, mutations and rhGH treatment were recorded and summarized. RESULTS The clinical characteristics of patients with NPR2 heterozygous mutation mainly included short stature, facial anomalies, and skeletal dysplasia. Skeletal dysplasia mainly included brachydactyly (56.2%), shortened metacarpals or metatarsals (particularly 4 th‒5 th; 26.1%) and clinodactyly (21.7%). rhGH treatment significantly improved the height SDS of patients with NPR2 heterozygous mutation (median -2.1 vs -2.9, P
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clinical characteristics of short stature patients with NPR2 mutation and the therapeutic response to rhgh
The Journal of Clinical Endocrinology and Metabolism, 2020Co-Authors: Hanting Liang, Hui Miao, Hongbo Yang, Linjie Wang, Fengying Gong, Hui Pan, Huijuan ZhuAbstract:CONTEXT Natriuretic peptide receptor 2 gene (NPR2) is a causative gene of idiopathic short stature (ISS) with an incidence rate of 2%-6%. The clinical characteristics of patients with NPR2 heterozygous mutation are atypical, and data on the efficacy of recombinant human growth hormone (rhGH) treatment in patients with NPR2 mutation are limited. OBJECTIVES To report six cases with NPR2 mutation and explore the characteristics of patients with NPR2 mutation and their therapeutic response to rhGH. DESIGN, SETTINGS AND PATIENTS Six Chinese short stature patients in our hospital with NPR2 mutation by whole-o exome sequencing were included. We also searched all previously published NPR2 mutation cases as of August 10, 2020, and the information of their medical history, mutations and rhGH treatment were recorded and summarized. RESULTS The clinical characteristics of patients with NPR2 heterozygous mutation mainly included short stature, facial anomalies, and skeletal dysplasia. Skeletal dysplasia mainly included brachydactyly (56.2%), shortened metacarpals or metatarsals (particularly 4 th‒5 th; 26.1%) and clinodactyly (21.7%). rhGH treatment significantly improved the height SDS of patients with NPR2 heterozygous mutation (median -2.1 vs -2.9, P<0.001), especially in girls. The height SDS change was negatively correlated with initial age of treatment (r=-4.77; P=0.034) and height SDS change of patients with NPR2 heterozygous mutation in GC domain was significantly higher than that of ECD domain (median 1.9 vs 0.6, P=0.019). CONCLUSIONS ISS patients with skeletal deformities should be tested for NPR2 mutation. rhGH treatment is beneficial for short stature patients with NPR2 heterozygous mutation and need further studies.
Jerid W. Robinson - One of the best experts on this subject based on the ideXlab platform.
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regulation of the natriuretic peptide receptor 2 NPR2 by phosphorylation of juxtamembrane serine and threonine residues is essential for bifurcation of sensory axons
The Journal of Neuroscience, 2018Co-Authors: Jerid W. Robinson, Lincoln R. Potter, Hannes Schmidt, Deborah M Dickey, Alexandre Dumoulin, Marie Octave, Ralf Kuhn, Robert Feil, Fritz G RathjenAbstract:cGMP signaling elicited by activation of the transmembrane receptor guanylyl cyclase NPR2 (also known as GC-B) by the ligand CNP controls sensory axon bifurcation of dorsal root ganglion (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 non-phosphorylatable 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 NPR2 7A/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 NPR2 7A/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. Significant 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.
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Regulation of the Natriuretic Peptide Receptor 2 (NPR2) by Phosphorylation of Juxtamembrane Serine and Threonine Residues Is Essential for Bifurcation of Sensory Axons.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2018Co-Authors: Hannes Schmidt, Jerid W. Robinson, Lincoln R. Potter, Deborah M Dickey, Alexandre Dumoulin, Marie Octave, Ralf Kuhn, Robert Feil, Fritz G RathjenAbstract: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.
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Dephosphorylation of the NPR2 guanylyl cyclase contributes to inhibition of bone growth by fibroblast growth factor
eLife, 2017Co-Authors: Leia C. Shuhaibar, Jerid W. Robinson, Valentina Baena, Tracy F. Uliasz, Siu-pok Yee, Jeremy R. Egbert, Giulia Vigone, Ninna P. Shuhaibar, Deborah Kaback, Robert FeilAbstract: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.
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Dephosphorylation of the NPR2 guanylyl cyclase contributes to inhibition of bone growth by fibroblast growth factor
2017Co-Authors: Leia C. Shuhaibar, Jerid W. Robinson, Robert Feil, Valentina Baena, Siu-pok Yee, Jeremy R. Egbert, Giulia Vigone, Ninna P. Shuhaibar, Deborah Kaback, Melanie C. FisherAbstract:Activating mutations in fibroblast growth factor (FGF) receptor 3 and inactivating mutations in the NPR2 guanylyl cyclase cause similar forms of dwarfism, but how these two signaling systems interact to regulate bone growth is poorly understood. Here, by use of a mouse model in which NPR2 cannot be dephosphorylated, we show that bone elongation is opposed when NPR2 is dephosphorylated and thus produces less cyclic GMP. By developing an in vivo imaging system to measure cyclic GMP levels in intact tibia, we show that FGF-induced dephosphorylation of NPR2 decreases its guanylyl cyclase activity in growth plate chondrocytes in living bone. Thus FGF signaling lowers cyclic GMP 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 cause dwarfism.
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heterozygous mutations in natriuretic peptide receptor b NPR2 gene as a cause of short stature
Human Mutation, 2015Co-Authors: Sophie R Wang, Jerid W. Robinson, Christina M Jacobsen, Aaron B Edmund, Robert C Olney, Jennifer E Moon, Timothy C. Miller, Verónica Mericq, Heather Carmichael, Lincoln R. PotterAbstract: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.
Fritz G Rathjen - One of the best experts on this subject based on the ideXlab platform.
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regulation of the natriuretic peptide receptor 2 NPR2 by phosphorylation of juxtamembrane serine and threonine residues is essential for bifurcation of sensory axons
The Journal of Neuroscience, 2018Co-Authors: Jerid W. Robinson, Lincoln R. Potter, Hannes Schmidt, Deborah M Dickey, Alexandre Dumoulin, Marie Octave, Ralf Kuhn, Robert Feil, Fritz G RathjenAbstract:cGMP signaling elicited by activation of the transmembrane receptor guanylyl cyclase NPR2 (also known as GC-B) by the ligand CNP controls sensory axon bifurcation of dorsal root ganglion (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 non-phosphorylatable 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 NPR2 7A/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 NPR2 7A/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. Significant 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.
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Regulation of the Natriuretic Peptide Receptor 2 (NPR2) by Phosphorylation of Juxtamembrane Serine and Threonine Residues Is Essential for Bifurcation of Sensory Axons.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2018Co-Authors: Hannes Schmidt, Jerid W. Robinson, Lincoln R. Potter, Deborah M Dickey, Alexandre Dumoulin, Marie Octave, Ralf Kuhn, Robert Feil, Fritz G RathjenAbstract: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.
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Loss of Axon Bifurcation in Mesencephalic Trigeminal Neurons Impairs the Maximal Biting Force in NPR2-Deficient Mice.
Frontiers in cellular neuroscience, 2018Co-Authors: Gohar Ter-avetisyan, Hannes Schmidt, Alexandre Dumoulin, Anthony Herrel, Johanna Strump, Shoaib Afzal, Fritz G RathjenAbstract:Bifurcation of axons from dorsal root ganglion (DRG) and cranial sensory ganglion (CSG) neurons is mediated by a cGMP-dependent signaling pathway composed of the ligand C-type natriuretic peptide (CNP), the receptor guanylyl cyclase NPR2 and the cGMP-dependent protein kinase I (cGKI). Here, we demonstrate that mesencephalic trigeminal neurons (MTN) which are the only somatosensory neurons whose cell bodies are located within the CNS co-express NPR2 and cGKI. Afferents of MTNs form Y-shaped branches in rhombomere 2 where the ligand CNP is expressed. Analyzing mouse mutants deficient for CNP or NPR2 we found that in the absence of CNP-induced cGMP signaling MTN afferents no longer bifurcate and instead extend either into the trigeminal root or caudally in the hindbrain. Since MTNs provide sensory information from jaw closing muscles and periodontal ligaments we measured the bite force of conditional mouse mutants of NPR2 (NPR2flox/flox;Engr1Cre ) that lack bifurcation of MTN whereas the bifurcation of trigeminal afferents is normal. Our study revealed that the maximal biting force of both sexes is reduced in NPR2flox/flox;Engr1Cre mice as compared to their NPR2flox/flox littermate controls. In conclusion sensory feedback mechanisms from jaw closing muscles or periodontal ligaments might be impaired in the absence of MTN axon bifurcation.
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Bifurcation of axons from cranial sensory neurons is disabled in the absence of NPR2-induced cGMP signaling.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2014Co-Authors: Gohar Ter-avetisyan, Fritz G Rathjen, Hannes SchmidtAbstract:Axonal branching is a prerequisite for the establishment of complex neuronal circuits and their capacity for parallel information processing. Previously, we have identified a cGMP signaling pathway composed of the ligand C-type natriuretic peptide (CNP), its receptor, the guanylyl cyclase natriuretic peptide receptor 2 (NPR2), and the cGMP-dependent kinase Iα (cGKIα) that regulates axon bifurcation of dorsal root ganglion (DRG) neurons in the spinal cord. Now we asked whether this cascade also controls axon bifurcation elsewhere in the nervous system. An NPR2-lacZ reporter mouse line was generated to clarify the pattern of the CNP receptor expression. It was found that during the period of axonal outgrowth, NPR2 and cGKIα were strongly labeled in neurons of all cranial sensory ganglia (gV, gVII, gVIII, gIX, and gX). In addition, strong complementary expression of CNP was detected in the hindbrain at the entry zones of sensory afferents. To analyze axon branching in individual NPR2-positive neurons, we generated a mouse mutant expressing a tamoxifen-inducible variant of Cre recombinase expressed under control of the NPR2-promoter (NPR2-CreER(T2)). After crossing this strain with conditional reporter mouse lines, we revealed that the complete absence of NPR2 activity indeed prohibited the bifurcation of cranial sensory axons in their entrance region. Consequently, axons only turned in either an ascending or descending direction, while collateral formation and growth of the peripheral arm was not affected. These findings indicate that in neurons of the cranial sensory ganglia, as in DRG neurons, cGMP signals are necessary for the execution of an axonal bifurcation program.
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A genetic strategy for the analysis of individual axon morphologies in cGMP signalling mutant mice.
Methods in molecular biology (Clifton N.J.), 2013Co-Authors: Hannes Schmidt, Gohar Ter-avetisyan, Fritz G RathjenAbstract:One of the many physiological functions of cyclic guanosine 3',5' monophosphate (cGMP) signalling is the regulation of a specific mode of axonal branching. The bifurcation of axons from dorsal root ganglion (DRG) neurons at the dorsal root entry zone of the embryonic spinal cord is triggered by a cGMP -signalling pathway comprising the ligand C-type natriuretic peptide (CNP), the cGMP-producing natriuretic peptide receptor 2 (NPR2), and the cGMP-dependent protein kinase Iα (cGKIα). Absence of any of these components causes a loss of bifurcation and sensory axons instead only turn in either a rostral or a caudal direction. In this chapter we describe a genetic strategy to study the impact of cGMP signalling on the arborization of individual DRG neurons in mice. Expression of an alkaline phosphatase (AP) reporter is selectively induced in NPR2-positive DRG neurons by tamoxifen-dependent activation of a Cre -recombinase under the control of the NPR2 promoter. This approach might also be employed for the analysis of axonal branching in neuronal subsets expressing NPR2 elsewhere in the nervous system.
