An Activating Mutation in the Kinase Homology Domain of the Natriuretic Peptide Receptor-2 Causes Extremely Tall Stature Without Skeletal Deformities

Abstract Context: Natriuretic peptide receptor-B (NPR-B, GC-B in rodents; gene name NPR2) is a guanylyl cyclase-coupled receptor that mediates the effect of C-type natriuretic peptide. Homozygous mutations in human NPR-B cause acromesomelic dysplasia, type Maroteaux (OMIM 602875), an autosomal recessive skeletal dysplasia. NPR-B has an intracellular kinase homology domain (KHD), which has no kinase activity, and its functional significance in vivo is currently unknown. Objective: We examined the functional significance of a novel NPR-B KHD mutation in humans. Patients and Methods: A 28-yr-old Japanese male presented with marked short stature (118.5 cm, −9.3 sd). His limbs showed marked shortening in the middle and distal segments. His parents had relatively short stature with height z-scores of −2.75 and −0.98 (his father and mother, respectively). Direct sequencing of coding region of the NPR2 gene of the family was performed. The mutant receptor activity was investigated by saturation binding assay and cGMP measurement. Additionally, interaction between the mutant and wild type allele was investigated by the titration experiments. Results: We identified a novel missense mutation L658F in KHD of NPR-B in homozygous and heterozygous states in the patient and his parents, respectively. The mutation conferred normal binding affinity for C-type natriuretic peptide but no discernible ligand-induced cGMP production. Furthermore, L658F mutant impaired wild-type NPR-B-mediated cGMP production in a dose-dependent manner, suggesting that short stature found in L658F heterozygote can be caused by its dominant-negative effect. Conclusions: This study provides the first evidence that intact KHD of NPR-B is essential for skeletal development.

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Phosphorylation of the Kinase Homology Domain Is Essential for Activation of the A-Type Natriuretic Peptide Receptor

Molecular and Cellular Biology ◽

10.1128/mcb.18.4.2164 ◽

1998 ◽

Vol 18 (4) ◽

pp. 2164-2172 ◽

Cited By ~ 103

Author(s):

Lincoln R. Potter ◽

Tony Hunter

Keyword(s):

Natriuretic Peptide ◽

Guanylyl Cyclase ◽

Receptor Activation ◽

Dominant Negative ◽

Cyclase Activity ◽

Atp Binding ◽

Natriuretic Peptide Receptor ◽

Peptide Receptor ◽

Kinase Homology Domain ◽

Kinase Homology

ABSTRACT Natriuretic peptide receptor A (NPR-A) is the biological receptor for atrial natriuretic peptide (ANP). Activation of the NPR-A guanylyl cyclase requires ANP binding to the extracellular domain and ATP binding to a putative site within its cytoplasmic region. The allosteric interaction of ATP with the intracellular kinase homology domain (KHD) is hypothesized to derepress the carboxyl-terminal guanylyl cyclase catalytic domain, resulting in the synthesis of the second messenger, cyclic GMP. Here, we show that phosphorylation of the KHD is essential for receptor activation. Using a combination of phosphopeptide mapping techniques, we have identified six residues within the ATP-binding domain (S497, T500, S502, S506, S510, and T513) which are phosphorylated when NPR-A is expressed in HEK 293 cells. Mutation of any one of these Ser or Thr residues to Ala caused reductions in the receptor phosphorylation state, the number and pattern of phosphopeptides observed in tryptic maps, and ANP-dependent guanylyl cyclase activity. The reductions were not explained by decreases in NPR-A protein levels, as indicated by immunoblot analysis and determinations of cyclase activity in the presence of detergent. Conversion of Ser-497 to Ala resulted in the most dramatic decrease in cyclase activity (∼20% of wild-type activity), but conversion to an acidic residue (Glu), which mimics the charge of the phosphoserine moiety, had no effect. Simultaneous mutation of five of the phosphorylation sites to Ala resulted in a dephosphorylated receptor which was unresponsive to hormone and had potent dominant negative inhibitory activity. We conclude that phosphorylation of the KHD is absolutely required for hormone-dependent activation of NPR-A.

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A Constitutively “Phosphorylated” Guanylyl Cyclase-linked Atrial Natriuretic Peptide Receptor Mutant Is Resistant to Desensitization

Molecular Biology of the Cell ◽

10.1091/mbc.10.6.1811 ◽

1999 ◽

Vol 10 (6) ◽

pp. 1811-1820 ◽

Cited By ~ 45

Author(s):

Lincoln R. Potter ◽

Tony Hunter

Keyword(s):

Atrial Natriuretic Peptide ◽

Natriuretic Peptide ◽

Guanylyl Cyclase ◽

Phosphorylation Sites ◽

Natriuretic Peptide Receptor ◽

Wild Type ◽

Peptide Receptor ◽

Type Receptor ◽

Kinase Homology Domain ◽

Kinase Homology

Dephosphorylation of the natriuretic peptide receptor-A (NPR-A) is hypothesized to mediate its desensitization in response to atrial natriuretic peptide (ANP) binding. Recently, we identified six phosphorylation sites within the kinase homology domain of NPR-A and determined that the conversion of these residues to alanine abolished the ability of the receptor to be phosphorylated or to be activated by ANP and ATP. In an attempt to generate a form of NPR-A that mimics a fully phosphorylated receptor but that is resistant to dephosphorylation, we engineered a receptor variant (NPR-A-6E) containing glutamate substitutions at all six phosphorylation sites. Consistent with the known ability of negatively charged glutamate residues to substitute functionally, in some cases, for phosphorylated residues, we found that NPR-A-6E was activated 10-fold by ANP and ATP. As determined by guanylyl cyclase assays, the hormone-stimulated activity of the wild-type receptor declined over time in membrane preparations in vitro, and this loss was blocked by the serine/threonine protein phosphatase inhibitor microcystin. In contrast, the activity of NPR-A-6E was more linear with time and was unaffected by microcystin. The nonhydrolyzable ATP analogue adenosine 5′-(β,γ-imino)-triphosphate was half as effective as ATP in stimulating the wild-type receptor but was equally as potent in stimulating NPR-A-6E, suggesting that ATP is required to keep the wild-type but not 6E variant phosphorylated. Finally, the desensitization of NPR-A-6E in whole cells was markedly blunted compared with that of the wild-type receptor, consistent with its inability to shed the negative charge from its kinase homology domain via dephosphorylation. These data provide the first direct test of the requirement for dephosphorylation in guanylyl cyclase desensitization and they indicate that it is an essential component of this process.

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