PT  - JOURNAL ARTICLE
AU  - Neil M. Otto
AU  - William G McDowell
AU  - Deborah M Dickey
AU  - Lincoln R Potter
TI  - A Glutamate-Substituted Mutant Mimics the Phosphorylated and Active Form of Guanylyl Cyclase-A
AID  - 10.1124/mol.116.107995
DP  - 2017 Jan 01
TA  - Molecular Pharmacology
PG  - mol.116.107995
4099  - http://molpharm.aspetjournals.org/content/early/2017/04/17/mol.116.107995.short
4100  - http://molpharm.aspetjournals.org/content/early/2017/04/17/mol.116.107995.full
AB  - 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, 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 (Vmax), while glutamate substitutions had no effect or increased Vmax. Ala but not Glu substitution for Ser-497 increased the Michaelis constant (Km) ~400%. A GC-A mutant containing Glu substitutions for all 7 chemically identified sites (GC-A-7E) had a Km ~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 Vmax, Km, and EC50 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 Km. Double Ala substitutions for Ser-497 and either Thr-500, Ser-510 or Thr-513 in WT-GC-A increase the Km 23 to 70-fold but the same mutations in GC-A-8E only increased the Km 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.