An Activating Deletion Variant in the Submembrane Region of Natriuretic Peptide Receptor-B Causes Tall Stature

Figures

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  Figure 1.. Photographs and radiographs of the feet and hand of the proband´s daughters. (A) Macrodactyly of the great toes of the eldest (upper panels) and youngest daughter (lower panels). The radiograph of the eldest daughter shows iatrogenic bone abnormalities on the distal part of metatarsal 1 after ablation of the pseudo-epiphyses (upper panel; white arrow). The pseudo-epiphyses in the first metatarsophalangeal joint and proximal interphalangeal joint of the youngest daughter are clearly distinguishable (lower panel; white arrows). (B) Left hand of the eldest daughter. The mid- and proximal phalanges of all fingers show pseudo-epiphyses (white square). Carpalia and metacarpals are normal.

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  Figure 2.. Growth curves of proband’s daughters. The black lines represent growth data of the daughters. The green band represent population reference data (from -2 SDS to +2 SDS). Red dot: bone age at time of measurement. TH: target height. (A) The growth curve of the eldest daughter shows progressive growth acceleration until the age of 4.5 years up to a height of +2 SDS. (B) The curve of the youngest daughter also seems to follow this pattern.

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  Figure 3.. Alignments of the amino acids 459–519 of NPR-B comparing the sequences of wild-type NPR-B across species, human NPR1 and mutant NPR-B. TMD represents the transmembrane domain and PKD the start of the protein kinase-homology domain. The black arrows label the 2 amino acids (Met482-Leu483) that are deleted in the cytosolic sub-membrane region of NPR-B in the proband (last line). For comparison, the sequence of the human natriuretic peptide receptor type A (NPR-A, also named guanylyl cyclase-A [GC-A]) is also included. Please note the high sequence conservation of the trans- and submembrane regions of NPR-B among species and between NPR-B and NPR-A, which suggests that the here studied deletion variant affects a functionally critical region. NPR, natriuretic peptide receptor.

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  Figure 4.. Cultured skin fibroblasts prepared from the proband have increased baseline cGMP levels and greater cGMP responses to CNP. (A) Western blot analysis of membrane NPR-B expression levels in fibroblasts cultured from a control donor and the proband. NPR-B expression was normalized to Na+/K+-ATPase expression. (B) Baseline intracellular cGMP levels of fibroblasts from the donor and the proband. (C) Effects of CNP on intracellular cGMP levels. Means ± SEM (n = 3); **P < 0.01. cGMP, cyclic guanosine monophosphate; CNP, C-type natriuretic peptide; NPR, natriuretic peptide receptor.

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  Figure 5.. HEK-293 cells expressing the mutant Met482-Leu483del NPR-B exhibit increased baseline cGMP levels and greater cGMP responses to CNP. (A) Western blot analysis of membrane NPR-B expression levels in HEK-293 cells transfected with cDNA encoding WT or mutant NPR-B (10 μg DNA/dish). Expression was normalized to Na+/K+-ATPase expression. (B) Baseline intracellular cGMP levels (-) and effects of CNP on intracellular cGMP levels of HEK-293 cells expressing WT or mutant NPR-B. (C and D) Guanylyl cyclase activity assays with crude membranes prepared from cells expressing WT or mutant NPR-B. Membranes were incubated with vehicle (-), CNP or detergent (1% Triton X-100). cGMP production was measured by radioimmunoassay. In D, values are presented as percent of the maximal Triton-induced activity (as 100%). Means ± SEM (n = 3 independent experiments); **P < 0.01. cGMP, cyclic guanosine monophosphate; CNP, C-type natriuretic peptide; NPR, natriuretic peptide receptor.

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  Figure 6.. The mutant Met482-Leu483del NPR-B has lower basal and CNP-stimulated cGMP-synthesis activity compared with the previously published mutant Arg655Cys NPR-B. (A) Membrane NPR-B expression levels and (B) CNP (0.1 nM)-induced cGMP formation in fibroblasts cultured from a control donor or from the probands with either variant. (C) Western blot analysis of membrane NPR-B expression levels in HEK-293 cells expressing the Met482-Leu483del or Arg655Cys NPR-B mutants. NPR-B expression was normalized to Na+/K+-ATPase expression. (D and E) Guanylyl cyclase activity assays demonstrate that the mutant Met482-Leu483del NPR-B exhibits lower baseline enzymatic activity and lower cGMP responses to CNP compared with the mutant Arg655Cys. Crude membranes prepared from HEK-293 cells expressing either mutant were incubated with vehicle (-), CNP or detergent (1% Triton X-100). cGMP production was measured by radioimmunoassay. (E) Values are expressed as percent of the maximal Triton-induced activity (as 100%). Means ± SEM (n = 3); **P < 0.01. cGMP, cyclic guanosine monophosphate; CNP, C-type natriuretic peptide; NPR, natriuretic peptide receptor.

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  Figure 7.. A theoretical 3D model of the trans- and cytosolic submembrane segments of WT and mutant NPR-B. Bioinformatic modelling predicted that Leu456 is the N-terminal start and Phe478 the end of the transmembrane region, which is consistent with the notion that basic residues at the C-term of transmembrane helices (here, residues Arg479 and Lys480) are important to vertically fix such helices in the lipid bilayer (17). Secondary structure prediction indicated an uninterrupted α-helical element comprising residues Leu456 to Trp492. (A) A theoretical 3D model of this helix was built for WT NPR-B (A, left helix) and mutant NPR-B (A, right helix; the deleted residues are marked with C-atoms colored in magenta). The deletion Met482-Leu483 results in a rotational rearrangement of the helix past the deletion site (in panel A, the right panel compares the WT and mutant helix, the rotation is marked). This in silico model suggests that in the mutant NPR-B the residues Glu484 to Trp492 are rotated by 120° clockwise and that the amphipathic structure of the helix is markedly changed. (B) Left panel: 3D model of the helix covering the transmembrane and cytoplasmic submembrane regions is shown for…