An Essential Role of Nedd4-2 in the Ubiquitination, Expression, and Function of Organic Anion Transporter-3

Abstract

Organic anion transporter-3 (OAT3) is a member of the organic anion transporter family that mediates the body disposition of a diverse array of clinically important drugs. We previously demonstrated that activation of protein kinase C (PKC) inhibits OAT3 transport activity by accelerating OAT3 internalization from cell surface into intracellular compartments. In the current study, we established that PKC-induced inhibition of OAT3 transport activity in monkey kidney COS-7 cells and in human kidney HEK293 cells occurred through an enhanced OAT3 ubiquitination, a process catalyzed by an E3 ubiquitin-protein ligase Nedd4-2 (neural precursor cell expressed, developmentally down-regulated 4-2). Overexpression of Nedd4-2 enhanced OAT3 ubiquitination, decreased OAT3 expression at the cell surface, and inhibited OAT3 transport activity. In contrast, overexpression of the ubiquitin ligase-dead mutant Nedd4-2/C821A or siRNA knockdown of endogenous Nedd4-2 had opposite effects on OAT3. Furthermore, immunoprecipitation experiments conducted both in culture cells and with rat kidney slices showed that there was a physical interaction between OAT3 and Nedd4-2. In conclusion, our results provided the first evidence that Nedd4-2 is an important regulator for OAT3 ubiquitination, expression and transport activity.

SGK1/Nedd4–2 signaling pathway regulates the activity of human organic anion transporters 3

“…Our laboratory recently demonstrated (Xu et al, ; Xu et al, ) that Nedd4–2 directly binds to OATs and catalyses the attachment of ubiquitin molecules to these transporters, which leads to the endocytosis/internalization of OATs from the plasma membrane to intracellular endosomes and subsequent degradation. Consequently, the amount of OATs at the plasma membrane is reduced and their transport activity is decreased (Xu et al, ; Xu et al, ; and our unpublished results). In exploring the relationship among sgk1, Nedd4–2 and hOAT3, it was found that sgk1 phosphorylated Nedd4–2 (Figure ) weakened the interaction between Nedd4–2 and hOAT3 (Figure ) and decreased hOAT3 ubiquitination (Figure ).…”

“…Our laboratory recently demonstrated (Xu et al, ; Xu et al, ) that Nedd4–2, a ubiquitin ligase, binds to OAT and inhibits OAT transport activity by promoting the conjugation of ubiquitin to OAT, which leads to OAT internalization from the plasma membrane and subsequent degradation (Xu et al, ; Xu et al, ; and our unpublished results). This experiment examined the relationship among sgk1, Nedd4–2 and hOAT3 using a co‐immunoprecipitation strategy.…”

“…Nedd4–2 is a key regulator of many membrane proteins such as sodium channel ENaC, potassium channel hERG and the amino acid transporter EAAT2 (Boehmer et al, ; Lamothe & Zhang, ; Snyder, Olson, & Thomas, ). Our laboratory recently demonstrated (Xu et al, ; Xu et al, ) that Nedd4–2 directly binds to OATs and catalyses the attachment of ubiquitin molecules to these transporters, which leads to the endocytosis/internalization of OATs from the plasma membrane to intracellular endosomes and subsequent degradation. Consequently, the amount of OATs at the plasma membrane is reduced and their transport activity is decreased (Xu et al, ; Xu et al, ; and our unpublished results).…”

“…cDNA for human Nedd4–2 was obtained as a generous gift from Dr Peter M. Snyder of the College of Medicine, University of Iowa (Iowa City, IA). The Nedd4–2 ligase‐dead mutant Nedd4–2/C821A was generated in our laboratory by site‐directed mutagenesis as described previously (Xu et al, , Xu et al, ). cDNAs for mouse Flag‐tagged sgk1s (wild‐type sgk1, constitutively‐active sgk1/S422D and kinase‐dead sgk/K127 M) were generously provided by Dr Alan C. Pao from the Department of Medicine, Stanford University, (Stanford, CA).…”

“…The ubiquitin attached onto the transporter can be recognized by the internalization machinery and therefore triggers the internalization step. The ubiquitination of OAT is a reaction, catalysed by an E3 ubiquitin ligase Nedd4–2 (Xu, Wang, & You, ; Xu, Wang, Zhang, & You, ). It was shown that Nedd4–2 inhibits OAT transport activity by enhancing OAT ubiquitination, and internalization, and therefore decreases OAT expression at the plasma membrane.…”