cGMP Induces Degradation of NKCC2 in the Thick Ascending Limb via the Ubiquitin-Proteasomal System.

cGMP Induces Degradation of NKCC2 in the Thick Ascending Limb via the Ubiquitin-Proteasomal System. Am J Physiol Renal Physiol. 2019 Feb 27;: Authors: Ares GR Abstract The thick ascending limb of Henle's loop (TAL) reabsorbs sodium chloride via the apical Na+/K+/2Cl- cotransporter (NKCC2). NKCC2 activity is regulated by surface NKCC2 levels. The second messenger cyclic guanosine monophosphate (cGMP) decreases NKCC2 activity by decreasing surface NKCC2 levels. We found that surface NKCC2 undergoes constitutive degradation. Therefore, we hypothesized that cGMP decreases NKCC2 levels by increasing NKCC2 ubiquitination and proteasomal degradation. We measured surface NKCC2 levels by biotinylation of surface proteins, immunoprecipitation of NKCC2 and ubiquitin in TALs. First, we found that inhibition of proteasomal degradation blunts the cGMP-dependent decrease in surface NKCC2 levels (vehicle = 100%; db-cGMP 500 µM = 70.3 ± 9.8%; MG132 20 µM = 97.7 ± 5.0%; db-cGMP + MG132 = 103.3 ± 3.4%; n = 5; p < 0.05). We then found that cGMP decreased the internalized NKCC2 pool and that this effect was prevented by inhibition of the proteasome but not the lysosome. Finally, we found that NKCC2 is constitutively ubiquitinated in TALs and that cGMP enhances the rate of NKCC2 ubiquitination (vehicle = 59 ± 14%, db-cGMP 500 µM = 111 ± 25%; n = 5; p < 0.05). We conclude that NKCC2 is constitutively ubiquitinated and that cGMP stimulates NKC...
Source: American Journal of Physiology. Renal Physiology - Category: Physiology Authors: Tags: Am J Physiol Renal Physiol Source Type: research