Physiological Consequences of Increased Phosphorylation-Dependent Activation of Guanylyl Cyclase-A and -B

Abstract

Guanylyl Cyclase (GC)-A and GC-B are homologous, cell surface, enzyme receptors that catalyze the formation of the second messenger cyclic guanosine monophosphate (cGMP) from guanosine triphosphate (GTP). Atrial natriuretic peptide (ANP) and B-type NP (BNP) bind and activate GC-A, whereas C-type NP (CNP) binds and activates GC-B. GC-A primarily regulates cardiovascular function, which includes decreasing blood pressure, blood volume, and cardiac hypertrophy, but also regulates metabolism, immune function, and hearing. In contrast, GC-B is best known for regulating the female reproductive system and long bone growth, strength, and density. Both GC-A and GC-B contain an extracellular ligand-binding domain, a single membrane-spanning region, and an intracellular region that consists of a kinase homology regulatory domain (KHD), a helix-loop-helix dimerization domain and C-terminal GC catalytic domain. The KHD is phosphorylated on multiple, conserved serine and threonine residues. Phosphorylation is absolutely required to transduce the ligand-binding activation signal to the catalytic domains of GC-A and GC-B. Dephosphorylation of the KHD inactivates the enzymes and renders them unresponsive to ligand. When I began my studies, little was known about how phosphorylation of these GC receptors regulate physiological processes in animals. In close collaboration with Dr. Laurinda Jaffe’s laboratory, our group generated novel, knock-in mouse models that express mutated forms of either GC-A or GC-B that are activated like the phosphorylated wild-type (WT) enzymes but cannot be inactivated by dephosphorylation. These enzymes contain glutamate substitutions for the known phosphorylation sites, which mimic the negative charges of the phosphorylated residues on the WT receptors. However, unlike the WT receptors, the negative charges cannot be enzymatically removed. Hence, the glutamate-substituted enzymes exhibit increased and prolonged natriuretic peptide-dependent GC activity compared to the WT enzymes. Because the GC-A mutant has 8 glutamate substitutions, and the GC-B mutant has 7 glutamate substitutions, the mutated enzymes are called GC-A-8E and GC-B-7E, respectively. Standard double allele screening technology was used to knock in the GC-B-7E construct into each WT locus for the two GC-B alleles to produce the GC-B7E/7E mice. In contrast, CRISPR-CAS9 technology was used to knock in the GC-A-8E construct into the two endogenous alleles for GC-A to make the GC-A8E/8E mice. My research on these mice revealed demonstrable, physiological consequences of increased phosphorylation-dependent activation of GC-A and GC-B. GC-A8E/8E mice exhibit increased and prolonged cGMP synthesis in the presence ANP and/or BNP, which led to reduced ERK1/2 phosphorylation, reduced cardiomyocyte cross-sectional area, reduced cardiac hypertrophy, and increased systolic function in male, but not female, mice. Regarding GC-B, previous work in the Potter and Jaffe laboratories showed in cell culture that dephosphorylation is required for FGFR3-dependent inhibition of GC-B. My research on the GC-B7E/7E mice indicates that dephosphorylation of GC-B is required for FGFR3-dependent achondroplasia. Furthermore, my work suggests that GC-B-dependent increases in bone formation and bone strength in mice require decreases in osteoclasts as well as increases in osteoblasts and that this only occurs during an early developmental window, which may have significant clinical implications for the treatment of achondroplasia in children and osteoporosis in adults.