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cGMP induces the degradation of internalized NKCC2 in thick ascending limbs: role of the proteasome
Author(s) -
Ares Gustavo,
Ortiz Pablo
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.1067.7
Subject(s) - mg132 , leupeptin , ubiquitin , chemistry , internalization , microbiology and biotechnology , proteasome , proteasome inhibitor , medicine , biochemistry , biology , receptor , protease , enzyme , gene
NaCl reabsorption by the thick ascending limb (TAL) is mediated by the Na/K/2Cl cotransporter NKCC2. cGMP decreases NKCC2 activity by decreasing the amount of NKCC2 in the apical membrane. Furthermore, we found that internalized NKCC2 is degraded slowly (0.33%/min) but it is not known whether NKCC2 degradation is regulated. We hypothesized that internalized NKCC2 is degraded by the ubiquitin‐proteasome system in a process stimulated by cGMP. TAL surface proteins were biotinylated, allowed to internalize and biotin remaining in surface proteins stripped away. Internalized NKCC2 was measured by western blot. cGMP enhanced the rate of disappearance of internalized NKCC2 by 83 % and this was blocked by a proteasomal (MG132) but not lysosomal (leupeptin) inhibitor (Control: 0.29 ± 0.04%/min; cGMP: 0.53 ± 0.10%/min; cGMP + MG132: 0.10 ± 0.10%/min; cGMP + Leupeptin: 0.44 ± 0.06 %/min; p < 0.05). Ubiquitination of membrane proteins is involved in their degradation. We found that NKCC2 inmunoprecipitated with ubiquitin. Proteasome inhibition induced the accumulation of ubiquitin‐NKCC2 and this was enhanced by cGMP (MG132: 59 ± 14%, MG132+cGMP: 111 ± 25%; p<0.05). We concluded that internalized NKCC2 is degraded via the proteasome pathway in a process stimulated by cGMP. cGMP induced degradation of internalized NKCC2 may contribute to decreased NKCC2 trafficking to the apical membrane of TALs.