title: A human skeletal overgrowth mutation increases maximal velocity and blocks desensitization of guanylyl cyclase-B.
abstract: C-type natriuretic peptide (CNP) increases long bone growth by stimulating guanylyl cyclase (GC)-B/NPR-B/NPR2. Recently, a Val to Met missense mutation at position 883 in the catalytic domain of GC-B was identified in humans with increased blood cGMP levels that cause abnormally long bones. Here, we determined how this mutation activates GC-B. In the absence of CNP, cGMP levels in cells expressing V883M-GC-B were increased more than 20 fold compared to cells expressing wild-type (WT)-GC-B, and the addition of CNP only further increased cGMP levels 2-fold. In the absence of CNP, maximal enzymatic activity (Vmax) of V883M-GC-B was increased 15-fold compared to WT-GC-B but the affinity of the enzymes for substrate as revealed by the Michaelis constant (Km) was unaffected. Surprisingly, CNP decreased the Km of V883M-GC-B 10-fold in a concentration-dependent manner without increasing Vmax. Unlike the WT enzyme the Km reduction of V883M-GC-B did not require ATP. Unexpectedly, V883M-GC-B, but not WT-GC-B, failed to inactivate with time. Phosphorylation elevated but was not required for the activity increase associated with the mutation because the Val to Met substitution also activated a GC-B mutant lacking all known phosphorylation sites. We conclude that the V883M mutation increases maximal velocity in the absence of CNP, eliminates the requirement for ATP in the CNP-dependent Km reduction, and disrupts the normal inactivation process.
authors.affiliation: Department of Pharmacology, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Jerid W
authors.initials: JW
authors.lastname: Robinson
authors.forename: Deborah M
authors.initials: DM
authors.lastname: Dickey
authors.forename: Kohji
authors.initials: K
authors.lastname: Miura
authors.forename: Toshimi
authors.initials: T
authors.lastname: Michigami
authors.forename: Keiichi
authors.initials: K
authors.lastname: Ozono
authors.forename: Lincoln R
authors.initials: LR
authors.lastname: Potter
mesh_terms: D015153:Blotting, Western; D001846:Bone Development / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D006152:Cyclic GMP / Q000378:metabolism; D006801:Humans; D009154:Mutation; D020098:Natriuretic Peptide, C-Type / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000235:genetics / Q000502:physiology; D017461:Receptors, Atrial Natriuretic Factor / Q000235:genetics / Q000378:metabolism*; D000099242:Atrial Natriuretic Factor Receptor B
chemical_list: D006152:Cyclic GMP; D020098:Natriuretic Peptide, C-Type; D017461:Receptors, Atrial Natriuretic Factor; D000099242:Atrial Natriuretic Factor Receptor B
keywords: Achondroplasia; Bone growth; C-type natriuretic peptide; CNP; Dwarfism; GC; Guanylate cyclase; NP; Natriuretic peptides; WT; cGMP; guanylyl cyclase; natriuretic peptide; wild type