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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 375-382 |
| Number of pages | 8 |
| Journal | Bone |
| Volume | 56 |
| Issue number | 2 |
| DOIs | |
| State | Published - Oct 1 2013 |
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Keywords
- Achondroplasia
- Bone growth
- CGMP
- Dwarfism
- Guanylate cyclase
- Natriuretic peptides
Cite this
A human skeletal overgrowth mutation increases maximal velocity and blocks desensitization of guanylyl cyclase-B. / Robinson, Jerid W.; Dickey, Deborah M; Miura, Kohji; Michigami, Toshimi; Ozono, Keiichi; Potter, Lincoln R.
In: Bone, Vol. 56, No. 2, 01.10.2013, p. 375-382.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - A human skeletal overgrowth mutation increases maximal velocity and blocks desensitization of guanylyl cyclase-B
AU - Robinson, Jerid W.
AU - Dickey, Deborah M
AU - Miura, Kohji
AU - Michigami, Toshimi
AU - Ozono, Keiichi
AU - Potter, Lincoln R
PY - 2013/10/1
Y1 - 2013/10/1
N2 - 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.
AB - 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.
KW - Achondroplasia
KW - Bone growth
KW - CGMP
KW - Dwarfism
KW - Guanylate cyclase
KW - Natriuretic peptides
UR - http://www.scopus.com/inward/record.url?scp=84881112147&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84881112147&partnerID=8YFLogxK
U2 - 10.1016/j.bone.2013.06.024
DO - 10.1016/j.bone.2013.06.024
M3 - Article
C2 - 23827346
AN - SCOPUS:84881112147
VL - 56
SP - 375
EP - 382
JO - Bone
JF - Bone
SN - 8756-3282
IS - 2
ER -