Skeletal overgrowth-causing mutations mimic an allosterically activated conformation of guanylyl cyclase-B that is inhibited by 2,4,6,-trinitrophenyl ATP

Activating mutations in the receptor for C-type natriuretic peptide (CNP), guanylyl cyclase B (GC-B, also known as Npr2 or NPR-B), increase cellular cGMP and cause skeletal overgrowth, but how these mutations affect GTP catalysis is poorly understood. The A488P and R655C mutations were compared with the known mutation V883M. Neither mutation affected GC-B concentrations. The A488P mutation decreased the EC₅₀ 5-fold, increased V_max 2.6-fold, and decreased the K_m 13-fold, whereas the R655C mutation decreased the EC₅₀ 5-fold, increased the V_max 2.1-fold, and decreased the K_m 4.7-fold. Neither mutation affected maximum activity at saturating CNP concentrations. Activation by R655C did not require disulfide bond formation. Surprisingly, the A488P mutant only activated the receptor when it was phosphorylated. In contrast, the R655C mutation converted GC-B-7A from CNP-unresponsive to CNP-responsive. Interestingly, neither mutant was activated by ATP, and the K_m and Hill coefficient of each mutant assayed in the absence of ATPwere similar to those of wild-typeGC-Bassayed in the presence of ATP. Finally, 1 mM 2,4,6,-trinitrophenyl ATP inhibited all three mutants by as much as 80% but failed to inhibit WTGC-B. We conclude that 1) the A488P and R655C missense mutations result in a GC-B conformation that mimics the allosterically activated conformation, 2) GC-B phosphorylation is required for CNP-dependent activation by the A488P mutation, 3) the R655C mutation abrogates the need for phosphorylation in receptor activation, and 4) an ATP analog selectively inhibits the GC-B mutants, indicating that a pharmacologic approach could reduce GC-B dependent human skeletal overgrowth.