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.
Bibliographical noteFunding Information:
This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 665/B2 to F.G.R. and Grant FOR 2060 (FE 438/5-2 and FE 438/6-2 to R.F., and SCHM2371/1 to H.S.), National Institutes of Health Grant R01GM098309 and Hormone Receptor Fund to L.R.P., and Fund for Science Grants to L.R.P. and R.F. We thank Madlen Driesner, Mechthild Henning, Anne Banerjee, and Karola Bach for technical help; 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 Deutsche Forschungsgemeinschaft Grant SFB 665/B2 to F.G.R. and Grant FOR 2060 (FE438/5-2andFE438/6-2toR.F.,andSCHM2371/1toH.S.),NationalInstitutesofHealthGrantR01GM098309and Hormone Receptor Fund to L.R.P., and Fund for Science Grants to L.R.P. and R.F. We thank Madlen Driesner,
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- Axonal branching
- CGMP signaling
- Sensory axons