Ubiquitination of NKCC2 by the cullin-RING E3 ubiquitin ligase family in the thick ascending limb of the loop of Henle

Abstract

The Na/K/2Cl cotransporter (NKCC2) in the thick ascending limb of the loop of Henle (TAL) mediates NaCl reabsorption. Cyclic guanosine monophosphate (cGMP), the second messenger of nitric oxide and atrial natriuretic peptide, inhibits NKCC2 activity by stimulating NKCC2 ubiquitination and decreasing surface NKCC2 levels. Among the E3-ubiquitin ligase families, the Cullin-RING E3 ubiquitin ligase (CRL) family is the largest. Cullins are molecular scaffold proteins that recruit multiple subunits to form the CRL complex. We hypothesize that a CRL complex mediates the cGMP-dependent increase in NKCC2 ubiquitination in TALs. Cullin-1, -2, -3, -4A, and -5 express at the protein level, while the other members of the cullin family were expressed at the mRNA level in rats TAL. CRL complex activity is regulated by the neuronal precursor cell-expressed developmentally downregulated protein 8 (Nedd8) to cullins, process called neddylation. Inhibition of cullin neddylation blunted the cGMP-dependent increase in ubiquitinated NKCC2, while increasing the expression of cullin-1 by threefold, but this effect was not seen on other cullins. CRL complex activity is also regulated by cullin-associated Nedd8-dissociated 1 (CAND1). CAND1 binds to cullins and promotes the exchange of substrate-recognition proteins to target different proteins for ubiquitination. CAND1 inhibition exacerbated the cGMP-dependent increase in NKCC2 ubiquitination and decreased surface NKCC2 expression. Finally, cGMP increased neddylation of cullins. We conclude that the cGMP-dependent increase in NKCC2 ubiquitination is mediated by a CRL complex. To the best of our knowledge, this is the first evidence that a CRL complex mediates NKCC2 ubiquitination in native TALs.

Protein Quality Control of NKCC2 in Bartter Syndrome and Blood Pressure Regulation

“…NKCC2 is no exception, and therefore, besides ER and Golgi QC, NKCC2 is also subject to a quality control process at the plasma membrane. In an elegant study using native TAL cells, Ares, G. provided evidence that surface NKCC2 protein is constitutively ubiquitinated and regulated by the proteasome pathway, a process that is stimulated by cGMP [82]. This is of particular interest because cGMP is an important regulator of NaCl in the TAL by decreasing NKCC2 cell surface expression [83].…”

Treatments and Prevention of CVDs by Taking an Insight into the Mechanism of Cardiovascular Aging

“…Diuretics can reduce signs of fluid retention by increasing urine volume/speed and urinary sodium excretion, and they mainly contain five classes: thiazide, potassium-conserving diuretics, loop, osmotic, and carbonic anhydrase inhibitors [19]. They inhibit the Na K-2Cl co-transporter protein in the thick ascending limb of the Henry ring [20]. The diuretic response is related to a urine agent's capacity to prompt sodium excretion or diuresis once administered.…”