Characterisation of the Cullin‐3 mutation that causes a severe form of familial hypertension and hyperkalaemia

Abstract

Deletion of exon 9 from Cullin-3 (CUL3, residues 403–459: CUL3Δ403–459) causes pseudohypoaldosteronism type IIE (PHA2E), a severe form of familial hyperkalaemia and hypertension (FHHt). CUL3 binds the RING protein RBX1 and various substrate adaptors to form Cullin-RING-ubiquitin-ligase complexes. Bound to KLHL3, CUL3-RBX1 ubiquitylates WNK kinases, promoting their ubiquitin-mediated proteasomal degradation. Since WNK kinases activate Na/Cl co-transporters to promote salt retention, CUL3 regulates blood pressure. Mutations in both KLHL3 and WNK kinases cause PHA2 by disrupting Cullin-RING-ligase formation. We report here that the PHA2E mutant, CUL3Δ403–459, is severely compromised in its ability to ubiquitylate WNKs, possibly due to altered structural flexibility. Instead, CUL3Δ403–459 auto-ubiquitylates and loses interaction with two important Cullin regulators: the COP9-signalosome and CAND1. A novel knock-in mouse model of CUL3WT/Δ403–459 closely recapitulates the human PHA2E phenotype. These mice also show changes in the arterial pulse waveform, suggesting a vascular contribution to their hypertension not reported in previous FHHt models. These findings may explain the severity of the FHHt phenotype caused by CUL3 mutations compared to those reported in KLHL3 or WNK kinases.

Mutant Cullin 3 causes familial hyperkalemic hypertension via dominant effects

“…The Sigmund group reported that WT CUL3/CUL3-Δ9 heterodimers are less stable than CUL3 WT/WT homodimers, and that CUL3-Δ9 may also sequester adapters (29). Finally, the Kurz group confirmed our finding with respect to inappropriate degradation of KLHL3 but also reported that CUL3-Δ9 displays autoubiquitination, leading to its own proteasomal degradation (30). A major limitation with most of these studies has been the reliance on in vitro approaches, but the Kurz group also generated a mouse model of FHHt by targeted deletion of exon 9 of the Cul3 gene.…”

“…CUL3-Δ9 protein was detectable in infected primary cells from CUL3-Het/Δ9 mice but not from CUL3-Het mice, with the size of CUL3-Δ9 confirmed using lysates from HEK293 cells transfected with plasmids encoding the transgene and Cre ( Figure 1F). CUL3-Δ9 was expressed at extremely low levels, consistent with its ability to initiate its own degradation via autoubiquitination (30). CUL3 has been proposed to be activated by covalent linkage of an 81-amino acid protein Neural precursor cell expressed developmentally downregulated protein 8 (Nedd8), a process called neddylation that is specific to Cullins (35).…”

“…CUL3 has been proposed to be activated by covalent linkage of an 81-amino acid protein Neural precursor cell expressed developmentally downregulated protein 8 (Nedd8), a process called neddylation that is specific to Cullins (35). Importantly, the region deleted in CUL3-Δ9 has been shown to interact with a large protein complex, the COP9 signalosome, which removes Nedd8 from Cullins (30). We found that CUL3-Δ9 displays a high degree of neddylation, consistent with its increased activity toward itself, KLHL3, and WNKs (28).…”

“…While it has been demonstrated that CUL3-Δ9 expression can cause a primary defect in the vasculature independently of possible secondary effects of hypertension (30,34), we sought to examine the possibility that increased activation of the Na…”

“…The mechanism by which CUL3-Δ9 leads to FHHt is controversial, but this is primarily related to the fact that most studies have been performed in vitro (28)(29)(30)34). A constitutive deletion of exon 9 in mice suggested that FHHt is due to haploinsufficiency resulting from degradation of CUL3-Δ9 following autoubiquitination (30).…”