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Regulation of the Natriuretic Peptide Receptor 2 (Npr2) by Phosphorylation of Juxtamembrane Serine and Threonine Residues Is

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Regulation of the Natriuretic Peptide Receptor 2 (Npr2) by Phosphorylation of Juxtamembrane Serine and Threonine Residues Is This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. Research Articles: Development/Plasticity/Repair Regulation of the natriuretic peptide receptor 2 (Npr2) by phosphorylation of juxtamembrane serine and threonine residues is essential for bifurcation of sensory axons Hannes Schmidt1,2, Deborah M. Dickey3, Alexandre Dumoulin1,4, Marie Octave2, Jerid W. Robinson3, Ralf Kühn1, Robert Feil2, Lincoln R. Potter3 and Fritz G. Rathjen1 1Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 10, 13092 Berlin, Germany 2Interfaculty Institute of Biochemistry, University of Tübingen, Hoppe-Seyler-Str. 4, 72076 Tübingen, Germany 3Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Medical School, 6-155 Jackson Hall, 321 Church St., Minneapolis, MN 55455, USA 4Berlin Institute of Health, Anna-Louisa-Karsch-Str. 2, 10178 Berlin, Germany DOI: 10.1523/JNEUROSCI.0495-18.2018 Received: 8 February 2018 Revised: 28 August 2018 Accepted: 18 September 2018 Published: 24 September 2018 Author contributions: H.S., D.M.D., R.K., R.F., L.P., and F.G.R. designed research; H.S., D.M.D., A.D., M.O., J.R., R.K., and F.G.R. performed research; H.S., D.M.D., A.D., M.O., J.R., R.K., R.F., L.P., and F.G.R. analyzed data; H.S., R.F., L.P., and F.G.R. edited the paper; H.S. and F.G.R. wrote the paper; L.P. and F.G.R. wrote the first draft of the paper. Conflict of Interest: The authors declare no competing financial interests. The technical help of Madlen Driesner, Mechthild Henning, Anne Banerjee and Karola Bach is greatly acknowledged. We thank Stefanie Peters for help with FRET/cGMP measurements of embryonic DRG neurons and Siu-Pok Yee, Leia Shuhaibar and Laurinda Jaffe (University of Connecticut) for providing Npr27E/7E embryos and mice. This work was supported by DFG grants SFB 665/B2 to FGR and FOR 2060 (FE 438/5-2 and FE 438/6-2 to RF, and SCHM 2371/1 to HS), National Institutes of Health grant R01GM098309 and Hormone Receptor Fund to LRP and the grants from the Fund for Science to LRP and RF. Correspondence: Fritz G. Rathjen, email: [email protected], or, Hannes Schmidt, email: [email protected] Cite as: J. Neurosci ; 10.1523/JNEUROSCI.0495-18.2018 Alerts: Sign up at www.jneurosci.org/cgi/alerts to receive customized email alerts when the fully formatted version of this article is published. Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process. Copyright © 2018 the authors 1 Regulation of the natriuretic peptide receptor 2 (Npr2) by phosphorylation of juxtamembrane 2 serine and threonine residues is essential for bifurcation of sensory axons 3 4 5 Hannes Schmidt1,2, Deborah M. Dickey3), Alexandre Dumoulin1,4), Marie Octave2,5), Jerid W. 6 Robinson3), Ralf Kühn1,6), Robert Feil2), Lincoln R. Potter3) and Fritz G. Rathjen1) 7 8 9 10 1) Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 10, 13092 Berlin, 11 Germany 12 2) Interfaculty Institute of Biochemistry, University of Tübingen, Hoppe-Seyler-Str. 4, 72076 13 Tübingen, Germany 14 3) Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 15 Medical School, 6-155 Jackson Hall, 321 Church St., Minneapolis, MN 55455, USA 16 4) Present address: Institute of Molecular Life Sciences, University of Zürich, 17 Winterthurerstrasse 190, 8057 Zürich, Switzerland 18 5) Present address: Institute of Experimental and Clinical Research (IREC), Université 19 catholique de Louvain, Avenue Hippocrate, 55, 1200 Brussels, Belgium 20 6) Berlin Institute of Health, Anna-Louisa-Karsch-Str. 2, 10178 Berlin, Germany 21 22 23 24 Correspondence: 25 Fritz G. Rathjen, email: [email protected] 26 or 27 Hannes Schmidt, email: [email protected] 28 29 30 Running title: Regulation of Npr2 by phosphorylation 31 32 Key words: axon bifurcation, sensory axons, cGMP signaling, Npr2, phosphorylation 33 34 35 1 36 Abstract 37 38 cGMP signaling elicited by activation of the transmembrane receptor guanylyl cyclase 39 Npr2 (also known as GC-B) by the ligand CNP controls sensory axon bifurcation of dorsal 40 root ganglion (DRG) and cranial sensory ganglion (CSG) neurons entering the spinal cord or 41 hindbrain, respectively. Previous studies have shown that Npr2 is phosphorylated on serine 42 and threonine residues in its kinase homology domain (KHD). However it is unknown 43 whether phosphorylation of Npr2 is essential for axon bifurcation. Here, we generated a 44 knock-in mouse line in which the seven regulatory serine and threonine residues in the KHD 45 of Npr2 were substituted by alanine (Npr2-7A), resulting in a non-phosphorylatable enzyme. 46 Real-time imaging of cGMP in DRG neurons with a genetically encoded fluorescent cGMP 47 sensor or biochemical analysis of guanylyl cyclase activity in brain or lung tissue revealed the 48 absence of CNP-induced cGMP generation in the Npr2 7A/7A mutant. Consequently, 49 bifurcation of axons, but not collateral formation, from DRG or CSG in this mouse mutant 50 was perturbed at embryonic and mature stages. In contrast, axon branching was normal in a 51 mouse mutant in which constitutive phosphorylation of Npr2 is mimicked by a replacement of 52 all of the seven serine and threonine sites by glutamic acid (Npr2-7E). Furthermore, we 53 demonstrate that the Npr27A/7A mutation causes dwarfism as described for global Npr2 54 mutants. In conclusion our in vivo studies provide strong evidence that phosphorylation of the 55 seven serine and threonine residues in the KHD of Npr2 is an important regulatory element of 56 Npr2-mediated cGMP signaling which affects physiological processes such as axon 57 bifurcation and bone growth. 58 59 60 Significant statement 61 62 The branching of axons is a morphological hallmark of virtually all neurons. It allows an 63 individual neuron to innervate different targets and to communicate with neurons located in 64 different regions of the nervous system. The natriuretic peptide receptor 2 (Npr2) – a 65 transmembrane guanylyl cyclase – is essential for the initiation of bifurcation of sensory 66 axons when entering the spinal cord or the hindbrain. By using two genetically engineered 67 mouse lines we show that phosphorylation of specific serine and threonine residues in 68 juxtamembrane regions of Npr2 are required for its enzymatic activity and for axon 69 bifurcation. These investigations might help to understand the regulation of Npr2 and its 70 integration in intracellular signaling systems. 2 71 Introduction 72 73 Axonal branching is one of the key processes that determine the formation of complex 74 neuronal circuits during embryonic and postnatal stages of nervous system development. 75 Branching enables an individual axon to establish multiple contacts and it allows an 76 individual neuron to communicate with other neurons which might be located in different 77 regions. Therefore, branching provides the physical basis for the distribution and integration 78 of information. However, the molecular mechanisms that control branch formation in vivo are 79 not fully understood (Armijo-Weingart and Gallo, 2017). The projections of sensory axons 80 within the spinal cord or the hindbrain represent an attractive system to characterize 81 components implicated in branching of axons (Schmidt and Rathjen, 2010;Gibson and Ma, 82 2011;Dumoulin et al., 2018). Upon entering the spinal cord or hindbrain the incoming axons 83 from neurons of dorsal root ganglia (DRG) or cranial sensory ganglia (CSG), respectively, 84 split up into a T-like bifurcation giving rise to two daughter axons that extend in opposite 85 direction. The natriuretic peptide receptor 2 (Npr2) - also termed guanylyl cyclase B (GC-B) - 86 plays a central role in the process of sensory axon bifurcation. In loss-of-function mutations of 87 the receptor Npr2, sensory axons no longer form these T-like branches and instead turn only 88 in either ascending or descending direction. Identical results were obtained when the cGMP- 89 dependent kinase I (cGKI) (Zhao et al., 2009;Schmidt et al., 2002) or the Npr2 ligand C-type 90 natriuretic peptide (CNP) were inactivated in mice (Schmidt et al., 2007;Schmidt et al., 91 2009;Ter-Avetisyan et al., 2014;Zhao and Ma, 2009). More recently, this signalling pathway 92 was shown to be of equivalent importance for the bifurcation of afferents of mesencephalic 93 trigeminal neurons (MTN) within the developing hindbrain (Ter-Avetisyan et al., 2018). A 94 critical missing link of this signaling pathway are the downstream targets of cGKIα in 95 somatosensory growth cones. However, by analyzing collateral branching or growth cone 96 behavior in cell culture experiments indicated that cGMP signaling might regulate 97 microtubule dynamics (Xia et al., 2013;Akiyama et al., 2016). Furthermore, although the 98 guidance receptors robo1 and 2 and their ligands slit1 and 2 did not perturb bifurcation itself, 99 their absence caused sensory axons to enter the spinal cord prematurely (Ma and Tessier- 100 Lavigne, 2007). The nitric oxide-sensitive guanylyl cyclases (NO-GCs) are not found in DRG 101 neurons at early embryonic stages and thus are not implicated in sensory axon branching in 102 vivo (Schmidt et al., 2009). The lack of axon bifurcation in DRG neurons results in altered 103 termination fields of primary afferents from the skin in the spinal cord. Furthermore, 104 behavioral testing indicated that noxious heat perception and nociception induced by chemical 105 irritants and the maximal biting force are impaired in mouse mutants with a conditional 106 inactivation of Npr2 in DRG or MTN neurons, whereas responses to mechanical stimulation 107 and motor coordination are not affected (Troster et al., 2018;Ter-Avetisyan et al., 2018).
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