LAUSR

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.