The RING finger- and PDZ domain-containing protein PDZRN3 controls localization of the Mg2+ regulator claudin-16 in renal tube epithelial cells

Abstract

Ion exchange in the renal tubules is fundamental to the maintenance of physiological ion levels. Claudin-16 (CLDN16) regulates the paracellular reabsorption of Mg in the thick ascending limb of Henle's loop in the kidney, with dephosphorylation of CLDN16 increasing its intracellular distribution and decreasing paracellular Mg permeability. CLDN16 is located in the tight junctions, but the mechanism regulating its localization is unclear. Using yeast two-hybrid systems, we found that CLDN16 binds to PDZRN3, a protein containing both RING-finger and PDZ domains. We also observed that the carboxyl terminus of the cytoplasmic CLDN16 region was required for PDZRN3 binding. PZDRN3 was mainly distributed in the cytosol of rat kidney cells and upon cell treatment with the protein kinase A inhibitor H-89, colocalized with CLDN16. H-89 also increased mono-ubiquitination and the association of CLDN16 with PDZRN3. Mono-ubiquitination levels of a K275A mutant were lower, and its association with PDZRN3 was reduced compared with wild-type (WT) CLDN16 and a K261A mutant, indicating that Lys-275 is the major ubiquitination site. An S217A mutant, a dephosphorylated form of CLDN16, localized to the cytosol along with PDZRN3 and the endosomal marker Rab7. PDZRN3 siRNA increased cell-surface localization of WT CLDN16 in H-89-treated cells or containing the S217A mutant and also suppressed CLDN16 endocytosis. Of note, H-89 decreased paracellular Mg flux in WT CLDN16 cells, and PDZRN3 siRNA increased Mg flux in the H-89-treated WT CLDN16 and S217A mutant cells. These results suggest that PDZRN3 mediates endocytosis of dephosphorylated CLDN16 and represents an important component of the CLDN16-trafficking machinery in the kidney.

Phosphorylation of tight junction transmembrane proteins: Many sites, much to do

“…41,42 This group found that phosphorylation at this site is required for interaction with syntaxin-8 and recycling to the membrane 42 and that dephosphorylated cldn16 was associated with the E3 ubiquitin ligase, PDZRN3, a protein containing both PDZ and ring finger domains. 43 Marunaka et al suggest this interaction was likely involved in the endocytosis of dephosphorylated cldn16; interestingly, PDZRN3 has also been implicated in regulation of the stability of endothelial cell junctions through targeting the multiPDZ protein MUPP1 for proteasomal degradation. 44 Cldn phosphorylation has also, although less frequently, been reported to play a role in removal from the tight junction.…”

PDZRN3 regulates differentiation of myoblasts into myotubes through transcriptional and posttranslational control of Id2

“…We previously cloned the complementary DNA (cDNA) of PDZRN3 from a human heart library with the use of a yeast two‐hybrid screen for proteins that bind to the PDZ domains of PSD‐95 (Ko et al, ). PDZRN3 is expressed in a variety of human tissues including heart, skeletal muscle, brain, and liver (Ko et al, ), and it has been implicated in various developmental processes including the differentiation of myoblasts (Ko et al, ), osteoblasts (Honda, Yamamoto, Ishii, & Inui, ), and adipocytes (Honda, Ishii, & Inui, ) as well as formation of neuromuscular junctions (Lu et al, ), neurogenesis (Andreazzoli et al, ), vascular morphogenesis (Sewduth et al, ), maintenance of the integrity of endothelial intercellular junctions (Sewduth et al, ), and regulation of paracellular Mg 2+ flux in renal tubular epithelial cells (Marunaka et al, ).…”

Rescue of tight junctional localization of a claudin-16 mutant D97S by antimalarial medicine primaquine in Madin-Darby canine kidney cells

“…These results indicated that primaquine may enhance stabilization of the D97S mutant in the TJs. The endocytosis of WT CLDN16 from the TJs to cytosolic compartment is regulated by a ubiquitination process 25 . The ubiquitination level of the D97S mutant was higher than that of WT CLDN16 (Fig.…”