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Multisite phosphorylation is required for activation of guanylyl cyclase (GC)-A, also known as NPR-A or NPR1, by cardiac natriuretic peptides (NPs). Seven chemically identified sites (Ser-487, Ser-497, Thr-500, Ser-502, Ser-506, Ser-510, and Thr-513) and one functionally identified putative site (Ser-473) were reported. Single alanine substitutions for Ser-497, Thr-500, Ser-502, Ser-506, and Ser-510 reduced maximal velocity ( V max ), whereas glutamate substitutions had no effect or increased V max Ala but not Glu substitution for Ser-497 increased the Michaelis constant ( K m ) approximately 400%. A GC-A mutant containing Glu substitutions for all seven chemically identified sites (GC-A-7E) had a K m approximately 10-fold higher than phosphorylated wild-type (WT) GC-A, but one additional substitution for Ser-473 to make GC-A-8E resulted in the same V max , K m , and EC 50 as the phosphorylated WT enzyme. Adding more glutamates to make GC-A-9E or GC-A-10E had little effect on activity, and sequential deletion of individual glutamates in GC-A-8E progressively increased the K m Double Ala substitutions for Ser-497 and either Thr-500, Ser-510 or Thr-513 in WT-GC-A increased the K m 23- to 70-fold but the same mutations in GC-A-8E only increased the K m 8-fold, consistent with one site affecting the phosphorylation of other sites. Phosphate measurements confirmed that single-site Ala substitutions reduced receptor phosphate levels more than expected for the loss of a single site. We conclude that a concentrated region of negative charge, not steric properties, resulting from multiple interdependent phosphorylation sites is required for a GC-A conformation capable of transmitting the hormone binding signal to the catalytic domain., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

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 EC 50 5-fold, increased V max 13-fold, whereas the R655C mutation decreased the EC K m 13-fold, whereas the R655C mutation decreased the EC 50 5-fold, increased the V 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 max and Hill coefficient of each mutant assayed in the absence of ATP were similar to those of wild-type GC-B assayed 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 WT-GC-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.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 ATP were similar to those of wild-type GC-B assayed 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 WT-GC-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., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)