Key result
Npr2-7A mutation abolishes CNP-induced cGMP generation and perturbs sensory axon bifurcation in mice.
Phosphorylation of seven serine and threonine residues in the kinase homology domain of Npr2 is essential for Npr2-mediated cGMP signaling, sensory axon bifurcation, and bone growth.
Npr2 juxtamembrane phosphorylation is required for sensory axon bifurcation; leaves open whether this mechanism extends to other cGMP-dependent processes.
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 theNpr27A/7Amutant. 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 theNpr27A/7Amutation causes dwarfism as described for global Npr2 mutants. In conclusion, ourin vivostudies 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 STATEMENTThe 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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Schmidt et al. (2018) studied Sensory axon bifurcation and bone growth (animal model). Npr2-7A mutation (alanine substitution of 7 serine/threonine residues) vs. Wild-type Npr2 was evaluated on CNP-induced cGMP generation and sensory axon bifurcation. Substitution of seven regulatory serine and threonine residues in the kinase homology domain of Npr2 with alanine abolished CNP-induced cGMP generation and perturbed sensory axon bifurcation in mice.
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