Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2020 Jan 30.
Published in final edited form as: Int J Pharm. 2018 Nov 16;555:165–174. doi: 10.1016/j.ijpharm.2018.11.040

The Mechanistic Links between Insulin and Human Organic Anion Transporter 4

Haoxun Wang 1, Jinghui Zhang 1, Guofeng You 1
PMCID: PMC6326839  NIHMSID: NIHMS1514331  PMID: 30453017

Abstract

Human organic anion transporter 4 (hOAT4) belongs to a class of organic anion transporters that exert critical function in the secretion, absorption, and distribution of numerous drugs in the body, such as anti-viral drugs, anti-cancer therapeutics, antibiotics, antihypertensive medicine, and anti-inflammatory drugs. hOAT4 is richly existent in the kidney and placenta. We previously established that serum- and glucocorticoid-inducible kinases (sgk) stimulate hOAT4 expression and transport activity by abrogating the inhibitory effect of a ubiquitin ligase Nedd4–2. Insulin is one of the upstream signaling molecules for sgk. We therefore investigated the effect of insulin on hOAT4 function. We showed that insulin stimulated hOAT4 expression and transport activity, and the action of insulin was abolished in cells overexpressing Nedd4–2-specific siRNA to knockdown the endogenous Nedd4–2. We further showed that insulin phosphorylated serine 327 on Nedd4–2 and weakened the interaction between hOAT4 and Nedd4–2. Interestingly, in cells overexpressing sgk2, the stimulatory effect of insulin on hOAT4 was diminished. In addition, the stimulatory effect of insulin on hOAT4 was blocked by wortmannin and buparlisib, two PI3K inhibitors. In conclusion, our study demonstrated that insulin stimulates hOAT4 expression and transport activity by abrogating the inhibition effect of Nedd4–2 on the transporter. Moreover, insulin regulates hOAT4 by competing with sgk2 rather than through sgk2.

Keywords: Organic Anion Transporter, Drug Transport, Regulation, Insulin, Serum and Glucocorticoid-Inducible Kinase, Ubiquitin Ligase

Graphical Abstract

graphic file with name nihms-1514331-f0011.jpg

1. Introduction

Human organic anion transporter 4 (hOAT4) belongs to a class of organic anion transporters, that exert critical function in the body secretion, absorption, and distribution of numerous drugs, such as anti-viral drugs, anti-cancer therapeutics, antibiotics, antihypertensive medicine, and anti-inflammatory drugs. The activity of these membrane proteins can be regulated at many levels such as transcriptional, translational, and posttranslational modulation (Ahn and Nigam, 2009; Dantzler and Wright, 2003; Srimaroeng et al., 2008; Terada and Inui, 2007; VanWert et al., 2010; You, 2002). OAT4-mediated transport was suggested to be bidirectional (Babu et al., 2002) and sodium-independent (Cha et al., 2000). hOAT4 is richly existent in the kidney and placenta (Cha et al., 2000). In the kidney, hOAT4 is expressed at the apical membrane of the proximal tubule cells and participates in renal excretion and reabsorption of endogenous substances in addition to numerous drugs and xenobiotics. In the placenta, hOAT4 is expressed at the basolateral membrane of syncytiotrophoblasts (Ugele et al., 2003). Placenta utilizes dehydroepiandrosterone sulfate (DHEAS), a precursor generated by the fetal adrenals, to synthesize estrogen. Buildup of extra DHEAS is linked with intrauterine growth retardation (Rabe et al., 1983). DHEAS is a hOAT4 substrate. Therefore, hOAT4 may exert a significant role in placental uptake of DHEAS for making estrogens and for protecting fetus from the DHEAS cytotoxicity. OAT4 has also been found to be expressed in choroid plexus epithelial cells and brain microvessel endothelial cells through RT-PCR assay (Kusch-Poddar et al., 2005; Xu et al., 2005). However, whether OAT4 protein is expressed in these cells are not known.

Due to such a critical role, delineating how hOAT4 is regulated has deep clinical significance. We formerly established that members of OAT family constitutively internalizes from and recycles back to plasma membrane and the transport activity of OAT can be modulated by changing the trafficking kinetics of these transporters (Zhang et al., 2008; Zhang et al., 2010; Zhang et al., 2012). An important event preceding OAT internalization is the ubiquitin conjugation to the transporter, catalyzed by a ubiquitin ligase Nedd4–2. Enhancement of Nedd4–2-dependent OAT ubiquitination leads to an acceleration of OAT internalization/removal from plasma membrane and subsequent degradation (Xu et al., 2017). As a result, OAT expression at the plasma membrane, and therefore OAT transport activity is reduced.

The serum- and glucocorticoid-inducible kinases (sgk) participate in governing many cellular processes including sodium Na+ balance, renal transport, and cell proliferation (Buse et al., 1999; Chen et al., 1999; Leong et al., 2003; Naray-Fejes-Toth et al., 1999; Rozansky et al., 2002; Waldegger et al., 1998). The family of the sgk protein kinases has three members: sgk1, sgk2 and sgk3. Sgk1 and sgk3 are richly present in every tissue, whereas sgk2 is expressed mainly in the liver, brain, kidney, and pancreas. We have recently demonstrated that sgk stimulated OAT transport activity by abrogating the inhibitory effect of Nedd4–2 on the transporter (Wang et al., 2016; Wang and You, 2017).

Insulin has been shown to regulate the cellular processes through multiple signaling pathways (Barros et al., 2009; Lee et al., 2007; Lungkaphin et al., 2014). One of the mediators for insulin is sgk. Therefore, in the current study, we investigated the effect of insulin on hOAT4. Interestingly, we discovered that insulin regulates hOAT4 by competing with sgk rather than through sgk.

2. Materials and Methods

2.1. Materials

[3H]-labeled estrone sulfate was acquired from PerkinElmer (Waltham, MA). COS-7 cells were acquired from American Type Culture Collection (Manassas, VA). Membrane-impermeable biotinylation reagent NHS-SS-biotin, streptavidin-agarose beads and protein G-agarose beads were acquired from Pierce (Rockford, IL). cDNA for human Nedd4–2 was kindly provided by Dr. Peter M. Snyder of the College of Medicine, University of Iowa (Iowa City, IA). cDNAs for mouse sgk2 (wild-type sgk2, inactive sgk2 (IN-sgk2) and constitutive active sgk2 (CA-sgk2) were kindly provided by Dr. Alan C. Pao from Department of Medicine, Stanford University (Stanford, CA). Mouse anti-Myc antibody (9E10) was acquired from Roche (Indianapolis, IN). Rabbit anti-Nedd4–2, was purchased from Abcam (Cambridge, MA). Rabbit anti-P-Nedd4–2 (Serine 327) antibody was acquired from Cell Signaling (Danvers, MA). Mouse anti‐β‐actin was acquired from Santa Cruz (Santa Cruz, CA). Nedd4–2-specific siRNA oligonucleotides (Silencer® Select, identification number s23570) and negative control siRNA oligonucleotides (Silencer® Select, catalog number 4390843) were acquired from Ambion (Grand Island, NY). Insulin and all other reagents were acquired from Sigma-Aldrich (St. Louis, MO).

2.2. Culturing of the Cells and cDNA Transfection

Parental COS-7 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum at 37 °C in 5% CO2. Transfection of cDNAs using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) was performed for 48 hours, following the manufacturer’s instructions. Cells stably expressing hOAT4 were kept in DMEM medium supplemented with 0.2mg/ml G418 (Invitrogen, Carlsbad, CA), 10% fetal bovine serum.

2.3. Transport Measurements

Uptake of [3H]-estrone sulfate (100 nM) was carried out, following standard protocol previously established in our lab (Wang et al., 2016). Uptake solution (phosphate-buffered saline with 1 mM CaCl2 and 1 mM MgCl2, pH 7.4 and 0.3μM [3H]-estrone sulfate) was added to 48-well plates in which cells were plated. The uptake process was terminated by removing the uptake solution and washing the cells with ice-cold PBS twice at indicated time points. 0.2 N NaOH was used for cell lysis and then 0.2 N HCl was used for neutralization. Liquid scintillation counter (Beckman LSC LS6500) was used for detection of [3H]-estrone sulfate. Uptake activity was expressed as a percentage of the uptake measured in control cells. Data were corrected for nonspecific background measured in mock cells (parental cells).

2.4. Cell Surface Biotinylation

The measurement of the expression level of hOAT4 at the plasma membrane was performed using a biotinylation strategy, following the standard protocol previously established in our lab (Wang et al., 2016). Sulfo-NHS-SS-biotin (0.5 mg/ml in phosphate-buffered saline with 1 mM CaCl2 and 1 mM MgCl2, pH 8.0) was added to 6-well plates in which cells were plated. After biotinylation and quenching of the unreacted sulfo -NHS-SS-biotin by rinsing with 100 mM glycine in PBS/CM, the cells were lysed on ice for 30 min. After 20 min centrifugation at 16000g at 4 °C, each sample supernatant of cell lysates was added to 40 µl of streptavidin-agarose beads to purify the cell membrane proteins. hOAT4 (tagged with Myc at its carboxyl terminus) was detected in the pool of surface proteins by SDS-PAGE and immunoblotting using an anti-Myc antibody 9E10 (Roche, Indianapolis, IN).

2.5. Immunoprecipitation

We followed the procedure previously established in our laboratory (Wang et al., 2016; Zhang et al., 2018). Cells were lysed with lysis buffer, containing 1% of proteinase inhibitor cocktail. For the experiment in which anti-Myc was used to pull-down hOAT4, the lyzed cells were precleared with protein G-agarose beads. Anti-Myc antibody (1:100) was incubated with protein G-agarose beads at 4 °C for 1.5 hours. The precleared cell lysates were then mixed with antibody-bound protein G-agarose beads with rotating at 4 °C overnight. For the experiment in which Anti-Flag M2 affinity gel (Sigma–Aldrich, St. Louis, MO) was used to pull-down Nedd4–2, the precleared cell lysates were mixed with Anti-Flag M2 affinity gel with rotating at 4 °C overnight. Proteins bound to the protein G-agarose beads or Anti-Flag M2 affinity gel were eluted with Urea buffer containing β-mecaptoethanol and examined by immunoblotting with appropriate antibodies.

2.6. Electrophoresis and Immunoblotting

We followed the procedure previously established in our laboratory (Wang et al., 2016). Protein samples were separated on 7.5% SDS-PAGE minigels and electroblotted on to PVDF membranes. The blots were incubated with 5% nonfat dry milk in PBS-0.05% Tween 20, washed, and incubated with appropriate primary antibodies, followed by horseradish peroxidase-conjugated secondary antibodies. The SuperSignal West Dura Extended Duration Substrate kit (Pierce, Rockford, IL) was used to detect the signals. The scanning densitometry with the FluorChem 8000 imaging system (Alpha Innotech Corp., San Leandro, CA) was used to quantify the nonsaturating, immunoreactive protein bands.

2.7. Data Analysis

We repeated each experiment a minimum of three times, and multiple experiments were used for statistical analysis. Statistical analysis was performed using Student’s paired t-tests between two groups or GraphPad Prism software (GraphPad Software Inc., San Diego, CA), one-way ANOVA, multiple comparisons among multiple treatments. A p-value of <0.05 was considered significant.

3. Results

3.1. Effect of insulin on hOAT4 transport activity

To examine the role of insulin in hOAT4 function, we treated hOAT4-expressing COS-7 cells with insulin, followed by the measurement of hOAT4-mediated uptake of [3H]-estrone sulfate (ES), a prototypical substrate for hOAT4. As shown in Fig. 1, insulin induced a dose-, and time-dependent rise in the uptake in compared with that in control cells with a ~ 180% stimulation at insulin concentration of 100 nM for 12 hr. treatment. To investigate the mechanism of insulin-induced stimulation of hOAT4 activity, we measured hOAT4-mediated uptake of [3H]-estrone sulfate at various substrate concentrations. Using an Eadie-Hofstee analysis (Fig. 2), we showed that treatment with insulin caused an increased maximal transport velocity Vmax of hOAT4 (129.77 ± 19.82 pmol·mg–1·3min–1 with control cells and 230.07 ± 22.21 pmol·mg–1·3min–1 with cells treated with insulin) without notable alteration in the substrate-binding affinity Km of the transporter (3.27 ± 0.30 µM with control cells and 3.45 ± 0.26 µM with cells treated with insulin).

Fig. 1. Effect of insulin on hOAT4 transport activity.

Fig. 1.

Open in a new tab

(a) Time-dependent stimulation of hOAT4 transport activity. hOAT4-expressing COS-7 cells were treated with insulin (100nM) for 2h, 4h, 6h and 12h. 3-min uptake of [3H]-estrone sulfate (0.3 µM) was then measured. Uptake activity was expressed as a percentage of the uptake measured in control cells. The data represent uptake into hOAT4-expressing cells minus uptake into mock cells (parental COS-7 cells). Values are mean ± SD (n = 3). *P<0.05. (b) Dose-dependent stimulation of hOAT4 transport activity. hOAT4-expressing COS-7 cells were treated with insulin at varies doses in 12h-treatment. 3-min uptake of [3H]-estrone sulfate (0.3 µM) was then measured. Uptake activity was expressed as a percentage of the uptake measured in control cells. The data represent uptake into hOAT4-expressing cells minus uptake into mock cells (parental COS-7 cells). Values are mean ± SD (n = 3). *P<0.05.

Fig. 2. Effect of insulin on the kinetics of hOAT4-mediated estrone sulfate transport.

Fig. 2.

Open in a new tab

COS-7 cells expressing hOAT4 were treated with the insulin (100nM, 12h), and initial uptake (3 min) of [3H]-estrone sulfate was measured at the concentration of 0.1–10 µM. The data represent uptake into hOAT4-transfected cells minus uptake into mock cells (parental COS-7 cells). Values are means ± SD (n = 3). V, velocity; S, substrate concentration.

3.2. Effect of insulin on hOAT4 Expression

Two possibilities could be responsible for a rise in the maximal transport velocity Vmax of hOAT4 shown in Fig. 2: either the number of the transporter at the plasma membrane could be increased or the transporter turnover rate could be enhanced. We carried out experiments that dissect these likelihoods by examining transporter expression both at the plasma membrane and in the total cell lysates. We showed that treatment with insulin led to an increase of hOAT4 expression at the plasma membrane (Fig. 3a), and in total cell lysates (Fig. 3c, top panel). Such a change in hOAT4 expression was not because of the overall perturbation of the cellular proteins since the expression of cellular protein marker β-actin was not affected under these situations (Fig. 3c, bottom panel).

Fig. 3. Effect of insulin on hOAT4 expression.

Fig. 3.

Open in a new tab

(a). Cell surface expression of hOAT4. hOAT4-expressing COS-7 cells were treated with the insulin (100nM, 12h). Cells were labeled with biotin. Biotinylated/cell surface proteins were separated with streptavidin beads, followed by immunoblotting (IB) with an anti-Myc antibody. Epitope Myc was tagged to hOAT4 for immune-detection. (b). Densitometry plot of results from Fig. 3a, as well as from other experiments. The values are mean ± SD (n = 3). *P<0.05. (c). Top panel: Total expression of hOAT4. hOAT4- expressing COS-7 cells were treated with the insulin (100nM, 12h). Cells were lysed, followed by immunoblotting (IB) with an anti-Myc antibody. Epitope Myc was tagged to hOAT4 for immune-detection. Bottom panel: the same blot from Fig. 3c was re-probed with anti β-actin antibody. β-actin is a cellular protein marker. (d). Densitometry plot of results from Fig. 3c, top panel as well as from other experiments. The values are mean ± SD (n = 3). *P<0.05.

3.3. Relationship between insulin and sgk2 on hOAT4 transport activity

One of the downstream mediators for insulin is sgk. We therefore investigated the relationship between insulin and sgk2 on hOAT4 transport activity. We transfected hOAT4-expressing cells with control vector, inactive form of sgk2 (IN-sgk2), wild-type of sgk2 (WT-sgk2), or the constitutive active form of sgk2 (CA-sgk2). Transfected cells were then treated with or without insulin (100nM, 12h), followed by [3H]-estrone sulfate uptake (3min, 0.3 µM). As shown in Fig. 4a, in the absence of insulin, hOAT4-mediated uptake was not significantly changed in IN-sgk2-transfected cells, was moderate in WT-sgk2-transfected cells, and was highest in CA-sgk2-transfected cells as compared to that in control cells, consistent with activity of the sgk2. However, when these cells were treated with insulin (Fig. 4b), we observed a maximum stimulation of hOAT4-mediated uptake in control or in IN-sgk2-transfected cells, suggesting that overexpression of IN-sgk2 failed to block insulin’s stimulatory effect. However, in cells transfected with WT-sgk2, the stimulatory effect of insulin became smaller as compared to that in cells transfected with IN-sgk2. In cells transfected with CA-sgk2, the stimulatory effect of insulin became diminished as compared to that in cells transfected with IN-sgk2. Similar results were observed in the cells overexpressing Nedd4–2 (Fig. 4c). These results indicate that the sgk2-stimulated hOAT4 uptake and insulin-stimulated uptake were not additive. When cells expressed highest level of sgk2 activity (CA-sgk2), insulin was unable to further stimulate hOAT4 transport activity.

Fig. 4. Relationship between insulin and sgk2 on hOAT4 transport activity.

Fig. 4.

Open in a new tab

(a) The effect of sgk2 on hOAT4 transport activity. COS-7 cells were transfected with hOAT4 and control vector, or with hOAT4 and the inactive form of sgk2 (IN-sgk2), or with hOAT4 and the wild-type of sgk2 (WT-sgk2), or with hOAT4 and the constitutive active form of sgk2 (CA-sgk2). After 48h transfection, cells were starved for 12h. [3H]-estrone sulfate uptake was then measured (3min, 0.3 µM). Uptake activity was expressed as a percentage of the uptake measured in control cells. The data represent uptake into hOAT4-expressing cells minus uptake into mock cells (parental COS-7 cells). Values are mean ± SD (n = 3). *P<0.05. (b) Relationship between insulin and sgk2 on hOAT4 transport activity. COS-7 cells were transfected with hOAT4 and control vector, or with hOAT4 and the inactive form of sgk2 (IN-sgk2), or with hOAT4 and the wild-type of sgk2 (WT-sgk2), or with hOAT4 and the constitutive active form of sgk2 (CA-sgk2). Transfected cells were then treated with or without insulin (100nM, 12h), followed by the measurement of [3H]-estrone sulfate uptake (3min, 0.3 µM). Uptake activity was expressed as a percentage of the uptake measured in control cells. The data represent uptake into hOAT4-expressing cells minus uptake into mock cells (parental COS-7 cells). Values are mean ± SD (n = 3). *P<0.05. NS: statistically not significant. (c) Relationship between insulin and sgk2 on hOAT4 transport activity in the cells overexpressing Nedd4–2. COS-7 cells were transfected with Nedd4–2, hOAT4 and the inactive form of sgk2 (IN-sgk2), or with Nedd4–2, hOAT4 and the constitutive active form of sgk2 (CA-sgk2). Transfected cells were then treated with or without insulin (100nM, 12h), followed by the measurement of [3H]-estrone sulfate uptake (3min, 0.3 µM). Uptake activity was expressed as a percentage of the uptake measured in control cells. The data represent uptake into hOAT4-expressing cells minus uptake into mock cells (parental COS-7 cells). Values are mean ± SD (n = 3). *P<0.05. NS: statistically not significant.

3.4. Effect of Nedd4–2 on insulin-stimulated hOAT4 expression

We previously demonstrated that the ubiquitination of OAT catalyzed by ubiquitin ligase Nedd4–2 leads to the internalization of the transporter from cell surface and subsequent degradation. In this experiment, we investigated the role of Nedd4–2 in insulin-stimulated hOAT4 expression (Fig. 5). In control cells, insulin significantly enhanced hOAT4 expression at the plasma membrane (Fig. 5a), whereas in cells transfected with Nedd4–2-specific siRNA to knock down the expression of endogenous Nedd4–2, insulin was unable to enhance hOAT4 expression at the plasma membrane (Fig. 5c). These results suggest that insulin stimulated hOAT4 expression through Nedd4–2.

Fig. 5. Role of Nedd4–2 in insulin-stimulated hOAT4 expression.

Fig. 5.

Open in a new tab

(a) COS-7 cells were co-transfected with hOAT4 and scrambled control siRNA. Transfected cells were treated with or without insulin (100nM) for 12h and then were labeled with biotin. Biotinylated/cell surface proteins were separated with streptavidin beads, followed by immunoblotting (IB) with an anti-Myc antibody. Epitope Myc was tagged to hOAT4 for immune-detection. (b). Densitometry plot of results from Fig. 5a, as well as from other experiments. The values are mean ± SD (n = 3). *P<0.05, (c) COS-7 cells were co-transfected with hOAT4 and Nedd4–2-specific siRNA. Transfected cells were treated with or without insulin (100nM) for 12h and then were labeled with biotin. Biotinylated/cell surface proteins were separated with streptavidin beads, followed by immunoblotting (IB) with an anti-Myc antibody. Epitope Myc was tagged to hOAT4 for immune-detection. (d). Densitometry plot of results from Fig. 5c, as well as from other experiments. The values are mean ± SD (n = 3). NS: statistically not significant.

3.5. Effect of Nedd4–2 on insulin-stimulated hOAT4 transport activity

We next investigated the role of Nedd4–2 in insulin-stimulated hOAT4 transport activity (Fig. 6). In control cells (Fig. 6a) and cells transfected with Nedd4–2 (Fig. 6b), insulin significantly enhanced hOAT4-mediated uptake of [3H]-estrone sulfate, whereas in cells transfected with Nedd4–2-specific siRNA to knock down the expression of endogenous Nedd4–2, insulin was unable to enhance hOAT4 transport activity (Fig. 6c), demonstrating that insulin stimulated hOAT4 transport activity through Nedd4–2.

Fig. 6. Role of Nedd4–2 in insulin-stimulated hOAT4 transport activity.

Fig. 6.

Open in a new tab

(a) COS-7 cells were co-transfected with hOAT4 and scrambled control siRNA. Transfected cells were treated with or without insulin (100nM) for 12h and then measured for the uptake of [3H]-estrone sulfate (3-min uptake and 0.3 μM estrone sulfate). The data represent uptake into hOAT4-transfected cells minus uptake into mock cells (parental COS-7 cells). Values are mean ± SD (n = 3). *P<0.05 (b) COS-7 cells were triple-transfected with hOAT4, Nedd4–2 and scrambled control siRNA. Transfected cells were treated with or without insulin (100nM) for 12h and then measured for the uptake of [3H]-estrone sulfate (3-min uptake and 0.3 μM estrone sulfate). The data represent uptake into hOAT4-transfected cells minus uptake into mock cells (parental COS-7 cells). Values are mean ± SD (n = 3). *P<0.05. (c) COS-7 cells were co-transfected with hOAT4 and Nedd4–2-specific siRNA. Transfected cells were treated with or without insulin (100nM) for 12h and then measured for the uptake of [3H]-estrone sulfate (3-min uptake and 0.3 μM estrone sulfate). The data represent uptake into hOAT4-transfected cells minus uptake into mock cells (parental COS-7 cells). Values are mean ± SD (n = 3). NS: statistically not significant.

3.6. Effect of insulin on the interaction of Nedd4–2 with hOAT4

Nedd4–2 regulates OAT ubiquitination by directly interacting with the transporter. We assessed the effect of insulin on the interaction of Nedd4–2 with hOAT4 through co-immunoprecipitation assay. hOAT4-expressing cells were transfected with Nedd4–2. Transfected cells were incubated with or without insulin. hOAT4 was then immunoprecipitated, followed by immunoblotting with anti-Nedd4–2 antibody to detect hOAT4-associated Nedd4–2. As shown in Fig. 7a top panel, in the absence of insulin, a significant quantity of Nedd4–2 was found in hOAT4 immunoprecipitates (lane 1), indicating a direct association between these two proteins. Yet, in cells treated with insulin (lane 2), the quantity of Nedd4–2 found in hOAT4 immunoprecipitates was much reduced. These data suggest that insulin hindered the interaction between hOAT4 and Nedd4–2. The difference in the amount of Nedd4–2 associated with hOAT4 was not due to the difference in the amount of hOAT4 pulled down because the same quantity of hOAT4 was immunoprecipitated under these conditions (Fig. 7a bottom panel). Similar results were obtained using a reciprocal approach, where Nedd4–2 was pulled down first, followed by immunoblotting with anti-Myc antibody (epitope Myc was tagged to hOAT4) to detect Nedd4–2-associated hOAT4 (Fig. 7c, top panel).

Fig. 7. Role of insulin in the interaction between Nedd4–2 and hOAT4.

Fig. 7.

Open in a new tab

(a) COS-7 cells were transfected with hOAT4 and Nedd4–2. Top panel: Transfected cells were treated with or without insulin (100nM, 12h) and then lysed, and hOAT4 was immunoprecipitated (IP) with anti-Myc antibody, followed by immunoblotting (IB) with the anti-Nedd4–2 antibody. Epitope Myc was tagged to hOAT4 for immune-detection. Bottom panel: The same immunoblot from Fig. 7a top panel was reprobed by anti-Myc antibody to detect the total amount of hOAT4 pulled down. (b) Densitometry plot of results from Fig. 7a as well as from other experiments. The values are mean ± SD (n = 3). *P<0.05. (c) Top panel: Transfected cells were treated with or without insulin (100nM, 12h) and then lysed, and Nedd4–2 (tagged with epitope Flag for immunodetection) was immunoprecipitated (IP) with anti-Flag M2 affinity gel, followed by immunoblotting (IB) with the anti-Myc antibody. Bottom panel: The same immunoblot from Fig. 7c top panel was reprobed by anti-Flag antibody to detect the total amount of Nedd4–2 pulled down. (d) Densitometry plot of results from Fig. 7c as well as from other experiments. The values are mean ± SD (n = 3). *P<0.05.

3.7. Effect of insulin on Nedd4–2 phosphorylation

We previously showed that sgk stimulates OAT activity by phosphorylating the amino acid residue serine 327 on Nedd4–2 (Wang and You, 2017). In this experiment, we examined whether insulin stimulates hOAT4 activity by phosphorylating Nedd4–2 at the same site. We transfected Nedd4–2 into hOAT4-expressing cells. The transfected cells were treated with or without insulin, followed by immunoblotting with phosphor-Nedd4–2 (Ser327)-specific antibody. As shown in Fig 8a, top panel, the phosphor-Nedd4–2-specific antibody detected a band at the size of Nedd4–2 in control cells, suggesting that Nedd4–2 was certainly phosphorylated at Ser327. The phosphorylation signal was enhanced by 70% in cells treated with insulin. The total expression of Nedd4–2 was not affected by insulin (Fig 8a, middle panel). Furthermore, the difference in the amount of Nedd4–2 phosphorylated was not due to an overall change in cellular proteins as the amount of cellular protein marker β-actin was not affected under this condition (Fig. 8a, bottom panel). Therefore, insulin enhanced the phosphorylation of Nedd4–2 at serine 327.

Fig. 8. Effect of insulin on hOAT4 phosphorylation.

Fig. 8.

Open in a new tab

(a) COS-7 cells were co-transfected with hOAT4 and Nedd4–2. Transfected cells were treated with or without insulin (100nM) and then lysed, followed by immunoblotting with the anti-Phospho-Nedd4–2 antibody (Top panel), anti-Nedd4–2 antibody (middle panel), or anti-β-actin antibody (bottom panel) respectively. β-actin is a cellular protein marker. (b). Densitometry plot of results from Fig. 8a, the top panel as well as from other experiments. The values are mean ± SD (n = 3). *P<0.05.

3.8. Effects of PI3K inhibitors on insulin-stimulated hOAT4-mediated estrone sulfate uptakes

PI3K has been shown to be the key regulator in insulin pathway (Avramoglu et al., 2006; Fry, 1994; Song et al., 2018) and PI3K inhibitors have been utilized to block insulin-mediated effect (MacKay et al., 2012; Zhou et al., 2014). To examine the effects of PI3K inhibitors on insulin-stimulated hOAT4-mediated estrone sulfate uptakes, we employed two selective PI3K inhibitors wortmannin and buparsilib. As shown in Fig. 9, both inhibitors significantly blocked the stimulatory effect of insulin on hOAT4 transport activity (wortmmanin or buparsilib alone did not show non-specific cytotoxicity). Together these results provide evidence that PI3K plays an essential role in the regulation of insulin stimulated hOAT4 activity.

Fig. 9. The effects of wortmannin and buparlisib on insulin stimulation of hOAT4-mediated transport.

Fig. 9.

Open in a new tab

hOAT4-expressing cells were treated with insulin (100nM, 12h) with or without PI3K inhibitors (wortmannin (100nM, 12h) or buparlisib (250nM, 12h)) or PI3K inhibitors alone, followed by measuring the uptake [3H] estrone sulfate (ES, 3min, 0.3 µM). Uptake activity was expressed as a percentage of the uptake determined in control cells. The data represent uptake into hOAT4-expressing cells minus uptake into mock cells. Values are mean ± SD (n = 3).*P<0.05.

4. Discussion

Organic anion transporters (OATs) is a major deciding factor of the effects of therapeutics and toxic chemicals. Thus, delineating the regulation of OATs at the molecular and cellular levels is clinically and pharmacologically important. In the present study, we investigated the mechanistic link between insulin and hOAT4-mediated drug transport.

Our present studies were performed in a heterologous cell system - COS-7 cells, which have been proven to be an excellent model system for investigation of the cloned organic anion transporter (Chioukh et al., 2014; Duan et al., 2010; Hagos et al., 2015; Zhang et al., 2008; Zhang et al., 2013). First, these cells are originated from the kidney. Examinations in such system have revealed other renal transport processes. Secondly, this cell line does not endogenously express OATs. Thus, expression of hOAT4 in these cells will permit us to characterize this transporter without the difficulty of being interfered by other organic anion transporters. Thirdly, the many signaling pathways are intact in these cells, providing a good exploratory model system for understanding the regulation of many transport processes. Lastly, the previous work revealed that the transport properties of OATs in these cells were consistent with those displayed in other systems. Our investigation in COS-7 cells will pave the path for the next stage of work concentrating on evaluating whether the similar mechanisms are operational in native epithelia.

Our lab has previously established that members of OAT family constitutively internalize from and recycle back to the plasma membrane and OAT transport activity can be modulated by changing the rates of either the internalization or the recycling of these transporters (Zhang et al., 2008; Zhang et al., 2010; Zhang et al., 2012). Ubiquitin conjugation (ubiquitination) of OAT, catalyzed by a ubiquitin ligase Nedd4–2 is an important mechanism which modulates the rate of OAT internalization. Enhancement of Nedd4–2-dependent OAT ubiquitination leads to an accelerated OAT internalization and degradation (Xu et al., 2016a; Xu et al., 2016b; Xu et al., 2017). As a result, OAT expression at the cell surface, and therefore OAT transport activity is decreased.

Nedd4–2 is under the control of many protein kinases (Debonneville et al., 2001; Garcia-Tardon et al., 2012; Lee et al., 2007; Snyder et al., 2004; Wang and You, 2017). It is possible that differential regulations of OAT by various protein kinases are exerted through the dynamic phosphorylation at different sites on Nedd4–2, in doing so, bringing about diverse conformational change in Nedd4–2 and altering its binding to OAT, which results in a change in OAT ubiquitination, trafficking and transport activity (Lee et al., 2007; Snyder et al., 2004; Wang and You, 2017). Our lab has recently demonstrated that the serum- and glucocorticoid-inducible kinases (sgks) stimulated OAT transport activity by selectively phosphorylating serine residue 327 on Nedd4–2 (Wang and You, 2017), which weakens the binding of Nedd4–2 to OAT, leading to a reduced ubiquitin-triggered OAT internalization and degradation (Wang et al., 2016; Wang and You, 2017). Here our results further indicate that insulin increases hOAT4 expression and activity by inducing phosphorylation of the same serine on Nedd4–2.

Insulin is a peptide hormone that regulates the activity of many membrane proteins (e.g. epithelial sodium channel (ENaC), Glucose Transporter type 4 (Glut4) and Na+/H+ exchanger 3) (Brewer et al., 2014; Klisic et al., 2002; Lee et al., 2007). The regulation of insulin in cellular processes could be through multiple signaling pathways. Several lines of evidence indicate that insulin was an upstream activator of sgks (Lee et al., 2007; Liu et al., 2014; Mansley et al., 2016). In the current study, we showed that insulin stimulated hOAT4 expression and transport activity (Figs. 1–3). We further showed that regulation of hOAT4 activity by insulin was mediated by ubiquitin ligase Nedd4–2. Knocking down endogenous Nedd4–2 by Nedd4–2-specific siRNA abolished the effect of insulin on hOAT4 (Figs. 5 and 6). The effect of insulin on hOAT4 expression and activity was paralleled with the phosphorylation of serine 327 on Nedd4–2 and a weakened association of Nedd4–2 to hOAT4 (Figs. 7 and 8). However, interestingly, the effect of insulin on hOAT4 was diminished in cells overexpressing sgk2 (Fig. 4), suggesting that the effect of insulin and sgk2 were not additive, and that insulin competes with sgk2, rather than acting through sgk2, in the regulation of hOAT4. PI3K has been shown to be the key regulator in insulin pathway (Avramoglu et al., 2006; Fry, 1994; Song et al., 2018). In our current study, we observed that two selective PI3K inhibitors wortmannin and buparlisib significantly blocked the stimulatory effect of insulin on hOAT4 transport activity (Fig. 9). PI3K has been reported to be a target for anti-tumor therapeutics. Wortmannin is a widely used model inhibitor for PI3K in both in vitro and in vivo studies (MacKay et al., 2012; Ng et al., 2000; Zhou et al., 2014). Buparlisib is currently under late-stage clinical development. Multiple clinical trials have been initiated to investigate the anti-tumor effect of Buparlisib in the patients with a variety of cancers (Criscitiello et al., 2018).

In addition to its role in the kidney, OAT4, specifically expressed in higher primates, is localized to the basolateral membrane of syncytiotrophoblast of the placenta and responsible for the elimination of steroid sulfates, xenobiotics, and clinically important drugs from the fetal compartment and therefore reducing the toxicity for the developing fetus. Gestational diabetes mellitus (GDM) is a condition in which a pregnant woman develops any degrees of glucose intolerance due to the lack of insulin. GDM has already become a major health concern with a growing prevalence. Further studies investigating the activity and expression of OAT4 in GDM placenta would be particularly interesting.

In conclusion, our current study demonstrated that insulin stimulates hOAT4 expression and transport activity through modulating the ubiquitin ligase Nedd4–2. Interestingly, the action of insulin is in competition with that of sgk2 rather than through sgk2 (Fig. 10).

Fig. 10. The Mechanistic Links between Insulin, sgk, and hOAT4.

Fig. 10.

Open in a new tab

U: ubiquitin. P: phosphorylation

Acknowledgement

This work was supported by grants (to Dr. Guofeng You) from National Institute of General Medical Sciences (R01-GM079123 and R01-GM097000).

We would like to thank Dr. Alan C. Pao for his generous gifts of mouse sgk2 plasmids (wild-type sgk2, inactive sgk2 (IN-sgk2) and constitutive active sgk2 (CA-sgk2)) and Dr. Peter M. Snyder for his generous gift of human Nedd4–2 plasmid.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Conflict of interest

The authors have declared that there is no conflict of interest.

References

  1. Ahn SY, Nigam SK, 2009. Toward a systems level understanding of organic anion and other multispecific drug transporters: a remote sensing and signaling hypothesis. Mol Pharmacol 76, 481–490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Avramoglu RK, Basciano H, Adeli K, 2006. Lipid and lipoprotein dysregulation in insulin resistant states. Clin Chim Acta 368, 1–19. [DOI] [PubMed] [Google Scholar]
  3. Babu E, Takeda M, Narikawa S, Kobayashi Y, Enomoto A, Tojo A, Cha SH, Sekine T, Sakthisekaran D, Endou H, 2002. Role of human organic anion transporter 4 in the transport of ochratoxin A. Biochim Biophys Acta 1590, 64–75. [DOI] [PubMed] [Google Scholar]
  4. Barros SA, Srimaroeng C, Perry JL, Walden R, Dembla-Rajpal N, Sweet DH, Pritchard JB, 2009. Activation of protein kinase Czeta increases OAT1 (SLC22A6)- and OAT3 (SLC22A8)-mediated transport. J Biol Chem 284, 2672–2679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brewer PD, Habtemichael EN, Romenskaia I, Mastick CC, Coster AC, 2014. Insulin-regulated Glut4 translocation: membrane protein trafficking with six distinctive steps. J Biol Chem 289, 17280–17298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Buse P, Tran SH, Luther E, Phu PT, Aponte GW, Firestone GL, 1999. Cell cycle and hormonal control of nuclear-cytoplasmic localization of the serum- and glucocorticoid-inducible protein kinase, Sgk, in mammary tumor cells. A novel convergence point of anti-proliferative and proliferative cell signaling pathways. J Biol Chem 274, 7253–7263. [DOI] [PubMed] [Google Scholar]
  7. Cha SH, Sekine T, Kusuhara H, Yu E, Kim JY, Kim DK, Sugiyama Y, Kanai Y, Endou H, 2000. Molecular cloning and characterization of multispecific organic anion transporter 4 expressed in the placenta. J Biol Chem 275, 4507–4512. [DOI] [PubMed] [Google Scholar]
  8. Chen SY, Bhargava A, Mastroberardino L, Meijer OC, Wang J, Buse P, Firestone GL, Verrey F, Pearce D, 1999. Epithelial sodium channel regulated by aldosterone-induced protein sgk. Proc Natl Acad Sci U S A 96, 2514–2519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chioukh R, Noel-Hudson MS, Ribes S, Fournier N, Becquemont L, Verstuyft C, 2014. Proton pump inhibitors inhibit methotrexate transport by renal basolateral organic anion transporter hOAT3. Drug Metab Dispos 42, 2041–2048. [DOI] [PubMed] [Google Scholar]
  10. Criscitiello C, Viale G, Curigliano G, Goldhirsch A, 2018. Profile of buparlisib and its potential in the treatment of breast cancer: evidence to date. Breast Cancer (Dove Med Press) 10, 23–29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dantzler WH, Wright SH, 2003. The molecular and cellular physiology of basolateral organic anion transport in mammalian renal tubules. Biochim Biophys Acta 1618, 185–193. [DOI] [PubMed] [Google Scholar]
  12. Debonneville C, Flores SY, Kamynina E, Plant PJ, Tauxe C, Thomas MA, Munster C, Chraibi A, Pratt JH, Horisberger JD, Pearce D, Loffing J, Staub O, 2001. Phosphorylation of Nedd4–2 by Sgk1 regulates epithelial Na(+) channel cell surface expression. EMBO J 20, 7052–7059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Duan P, Li S, You G, 2010. Angiotensin II inhibits activity of human organic anion transporter 3 through activation of protein kinase Calpha: accelerating endocytosis of the transporter. Eur J Pharmacol 627, 49–55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fry MJ, 1994. Structure, regulation and function of phosphoinositide 3-kinases. Biochim Biophys Acta 1226, 237–268. [DOI] [PubMed] [Google Scholar]
  15. Garcia-Tardon N, Gonzalez-Gonzalez IM, Martinez-Villarreal J, Fernandez-Sanchez E, Gimenez C, Zafra F, 2012. Protein kinase C (PKC)-promoted endocytosis of glutamate transporter GLT-1 requires ubiquitin ligase Nedd4–2-dependent ubiquitination but not phosphorylation. J Biol Chem 287, 19177–19187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hagos Y, Hundertmark P, Shnitsar V, Marada VV, Wulf G, Burckhardt G, 2015. Renal human organic anion transporter 3 increases the susceptibility of lymphoma cells to bendamustine uptake. Am J Physiol Renal Physiol 308, F330–338. [DOI] [PubMed] [Google Scholar]
  17. Klisic J, Hu MC, Nief V, Reyes L, Fuster D, Moe OW, Ambuhl PM, 2002. Insulin activates Na(+)/H(+) exchanger 3: biphasic response and glucocorticoid dependence. Am J Physiol Renal Physiol 283, F532–539. [DOI] [PubMed] [Google Scholar]
  18. Kusch-Poddar M, Drewe J, Fux I, Gutmann H, 2005. Evaluation of the immortalized human brain capillary endothelial cell line BB19 as a human cell culture model for the blood-brain barrier. Brain Res 1064, 21–31. [DOI] [PubMed] [Google Scholar]
  19. Lee IH, Dinudom A, Sanchez-Perez A, Kumar S, Cook DI, 2007. Akt mediates the effect of insulin on epithelial sodium channels by inhibiting Nedd4–2. J Biol Chem 282, 29866–29873. [DOI] [PubMed] [Google Scholar]
  20. Leong ML, Maiyar AC, Kim B, O’Keeffe BA, Firestone GL, 2003. Expression of the serum- and glucocorticoid-inducible protein kinase, Sgk, is a cell survival response to multiple types of environmental stress stimuli in mammary epithelial cells. J Biol Chem 278, 5871–5882. [DOI] [PubMed] [Google Scholar]
  21. Liu H, Yu J, Xia T, Xiao Y, Zhang Q, Liu B, Guo Y, Deng J, Deng Y, Chen S, Naray-Fejes-Toth A, Fejes-Toth G, Guo F, 2014. Hepatic serum- and glucocorticoid-regulated protein kinase 1 (SGK1) regulates insulin sensitivity in mice via extracellular-signal-regulated kinase 1/2 (ERK1/2). Biochem J 464, 281–289. [DOI] [PubMed] [Google Scholar]
  22. Lungkaphin A, Arjinajarn P, Pongchaidecha A, Srimaroeng C, Chatsudthipong L, Chatsudthipong V, 2014. Impaired insulin signaling affects renal organic anion transporter 3 (Oat3) function in streptozotocin-induced diabetic rats. PLoS One 9, e96236. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  23. MacKay KB, Lowenson JD, Clarke SG, 2012. Wortmannin reduces insulin signaling and death in seizure-prone Pcmt1−/− mice. PLoS One 7, e46719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mansley MK, Watt GB, Francis SL, Walker DJ, Land SC, Bailey MA, Wilson SM, 2016. Dexamethasone and insulin activate serum and glucocorticoid-inducible kinase 1 (SGK1) via different molecular mechanisms in cortical collecting duct cells. Physiol Rep 4, e12792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Naray-Fejes-Toth A, Canessa C, Cleaveland ES, Aldrich G, Fejes-Toth G, 1999. sgk is an aldosterone-induced kinase in the renal collecting duct. Effects on epithelial na+ channels. J Biol Chem 274, 16973–16978. [DOI] [PubMed] [Google Scholar]
  26. Ng SSW, Tsao MS, Chow S, Hedley DW, 2000. Inhibition of phosphatidylinositide 3-kinase enhances gemcitabine-induced apoptosis in human pancreatic cancer cells. Cancer Res 60, 5451–5455. [PubMed] [Google Scholar]
  27. Rabe T, Hosch R, Runnebaum B, 1983. Diagnosis of intrauterine fetal growth retardation (IUGR) and placental insufficiency by a dehydroepiandrosterone sulfate (DHAS) loading test. Biol Res Pregnancy Perinatol 4, 130–136. [PubMed] [Google Scholar]
  28. Rozansky DJ, Wang J, Doan N, Purdy T, Faulk T, Bhargava A, Dawson K, Pearce D, 2002. Hypotonic induction of SGK1 and Na+ transport in A6 cells. Am J Physiol Renal Physiol 283, F105–113. [DOI] [PubMed] [Google Scholar]
  29. Snyder PM, Olson DR, Kabra R, Zhou R, Steines JC, 2004. cAMP and serum and glucocorticoid-inducible kinase (SGK) regulate the epithelial Na(+) channel through convergent phosphorylation of Nedd4–2. J Biol Chem 279, 45753–45758. [DOI] [PubMed] [Google Scholar]
  30. Song C, Liu D, Yang S, Cheng L, Xing E, Chen Z, 2018. Sericin enhances the insulin-PI3K/AKT signaling pathway in the liver of a type 2 diabetes rat model. Exp Ther Med 16, 3345–3352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Srimaroeng C, Perry JL, Pritchard JB, 2008. Physiology, structure, and regulation of the cloned organic anion transporters. Xenobiotica 38, 889–935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Terada T, Inui K, 2007. Gene expression and regulation of drug transporters in the intestine and kidney. Biochem Pharmacol 73, 440–449. [DOI] [PubMed] [Google Scholar]
  33. Ugele B, St-Pierre MV, Pihusch M, Bahn A, Hantschmann P, 2003. Characterization and identification of steroid sulfate transporters of human placenta. Am J Physiol Endocrinol Metab 284, E390–398. [DOI] [PubMed] [Google Scholar]
  34. VanWert AL, Gionfriddo MR, Sweet DH, 2010. Organic anion transporters: discovery, pharmacology, regulation and roles in pathophysiology. Biopharm Drug Dispos 31, 1–71. [DOI] [PubMed] [Google Scholar]
  35. Waldegger S, Barth P, Forrest JN Jr., Greger R, Lang F, 1998. Cloning of sgk serine-threonine protein kinase from shark rectal gland - a gene induced by hypertonicity and secretagogues. Pflugers Arch 436, 575–580. [DOI] [PubMed] [Google Scholar]
  36. Wang H, Xu D, Toh MF, Pao AC, You G, 2016. Serum- and glucocorticoid-inducible kinase SGK2 regulates human organic anion transporters 4 via ubiquitin ligase Nedd4–2. Biochem Pharmacol 102, 120–129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wang H, You G, 2017. SGK1/Nedd4–2 signaling pathway regulates the activity of human organic anion transporters 3. Biopharm Drug Dispos 38, 449–457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Xu D, Wang H, You G, 2016a. An Essential Role of Nedd4–2 in the Ubiquitination, Expression, and Function of Organic Anion Transporter-3. Mol Pharm 13, 621–630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Xu D, Wang H, Zhang Q, You G, 2016b. Nedd4–2 but not Nedd4–1 is critical for protein kinase C-regulated ubiquitination, expression, and transport activity of human organic anion transporter 1. Am J Physiol Renal Physiol 310, F821–831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Xu D, Zhang J, Zhang Q, Fan Y, Liu C, You G, 2017. PKC/Nedd4–2 Signaling Pathway Regulates the Cell Surface Expression of Drug Transporter hOAT1. Drug Metab Dispos 45, 887–895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Xu G, Bhatnagar V, Wen G, Hamilton BA, Eraly SA, Nigam SK, 2005. Analyses of coding region polymorphisms in apical and basolateral human organic anion transporter (OAT) genes [OAT1 (NKT), OAT2, OAT3, OAT4, URAT (RST)]. Kidney Int 68, 1491–1499. [DOI] [PubMed] [Google Scholar]
  42. You G, 2002. Structure, function, and regulation of renal organic anion transporters. Med Res Rev 22, 602–616. [DOI] [PubMed] [Google Scholar]
  43. Zhang J, Liu C, You G, 2018. AG490, a JAK2-specific inhibitor, downregulates the expression and activity of organic anion transporter-3. J Pharmacol Sci 136, 142–148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Zhang Q, Hong M, Duan P, Pan Z, Ma J, You G, 2008. Organic anion transporter OAT1 undergoes constitutive and protein kinase C-regulated trafficking through a dynamin- and clathrin-dependent pathway. J Biol Chem 283, 32570–32579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Zhang Q, Li S, Patterson C, You G, 2013. Lysine 48-linked polyubiquitination of organic anion transporter-1 is essential for its protein kinase C-regulated endocytosis. Mol Pharmacol 83, 217–224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zhang Q, Pan Z, You G, 2010. Regulation of human organic anion transporter 4 by protein kinase C and NHERF-1: altering the endocytosis of the transporter. Pharm Res 27, 589–596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Zhang Q, Suh W, Pan Z, You G, 2012. Short-term and long-term effects of protein kinase C on the trafficking and stability of human organic anion transporter 3. Int J Biochem Mol Biol 3, 242–249. [PMC free article] [PubMed] [Google Scholar]
  48. Zhou X, Zhao Y, Xu L, Li X, Chen P, Zou H, Yu H, 2014. [Wortmannin and U0126 inhibit the promoting effect of insulin on differentiation of skeletal myoblasts in rats]. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi 30, 717–720. [PubMed] [Google Scholar]

RESOURCES