Mutation in an Adaptor Protein PDZKI Affects Transport Activity of Organic Cation Transporter OCTNs and Oligopeptide Transporter PEPT2

doi:10.2133/dmpk.21.375

Drug Metabolism and Pharmacokinetics

Volume 21, Issue 5, 2006, Pages 375-383

https://doi.org/10.2133/dmpk.21.375 Get rights and content

Summary:

Genetic polymorphisms in xenobiotic transporters have recently been clarified to be associated with change in drug distribution and disposition. To expand on recent identification of direct interaction and functional regulation of several transporters by a PDZ (PSD95, Dig and ZO1) domain containing protein PDZKI, the effect of mutation in PDZKI on transport activity and subcellular localization of organic cation/carnitine transporters OCTNI and OCTN2, and oligopeptide transporter PEPT2 was examined in the present study. HEK293 cells stably expressing a mutant transcript PDZK1-E195K (HEK293/PDZK1-E195K) were constructed, followed by transient transfection of cDNA for each transporter. Uptake of tetraethylammonium by OCTNI was much higher in HEK293/PDZKI cells, compared with that in the parent HEK293 cells, the uptake in HEK293/PDZK1-E195K cells showing middle range between the two values. Such difference in transport activity was accounted for the difference in transport capacity, with minimal change in affinity of OCTN1 to the substrate or other compounds. The similar difference among HEK293/PDZK1, HEK293/PDZK1-E195K and HEK293 cells was also observed in transport property of OCTN2 and PEPT2, whereas the difference was not so remarkable in each transporter with the last four amino acids deleted, that has much lower interaction potential with PDZK1. Immunohistochemical analysis indicated that OCTN1 was colocalized with PDZK1 on cell-surface, whereas colocalization with PDZK1-E195K was partially observed in cytoplasmic region. These results suggest a novel hypothesis that mutation in PDZK1 potentially changes transport property of various types of xenobiotic transporters by affecting their subcellular localization, possibly leading to change in disposition of various types of substrate drugs.

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Probing the Architecture of a Multi-PDZ Domain Protein: Structure of PDZK1 in Solution
2018, Structure
The scaffolding protein PDZK1 has been associated with the regulation of membrane transporters. It contains four conserved PDZ domains, which typically recognize a 3–5-residue long motif at the C terminus of the binding partner. The atomic structures of the individual domains are available but their spatial arrangement in the full-length context influencing the binding properties remained elusive. Here we report a systematic study of full-length PDZK1 and deletion constructs using small-angle X-ray scattering, complemented with biochemical and functional studies on PDZK1 binding to known membrane protein partners. A hybrid modeling approach utilizing multiple scattering datasets yielded a well-defined, extended, asymmetric L-shaped domain organization of PDZK1 in contrast to a flexible “beads-on-string” model predicted by bioinformatics analysis. The linker regions of PDZK1 appear to play a central role in the arrangement of the four domains underlying the importance of studying scaffolding proteins in their full-length context.
The scaffold protein PDZK1 modulates expression and function of the organic anion transporting polypeptide 2B1
2018, European Journal of Pharmaceutical Sciences
Citation Excerpt :
One might hypothesize that the lack of modulation is due the fact that BeWo cells are not polarizing in culture. In this regard it is of importance to note that most in vitro studies on the role of scaffold proteins have been performed in non-polarized cells such as HEK293, or HeLa cells (Anzai et al., 2004; Kato et al., 2005; Miyazaki et al., 2005; Park et al., 2014; Sugiura et al., 2006a; Zheng et al., 2014). In our study we tested both polarized (MDCKII) and non-polarized cells (HeLa), and observed the influence of PDZK1 on OATP2B1 in both cell lines.
The protein family of Organic Anion Transporting Polypeptides (OATPs) summarizes various transporters known to facilitate cellular uptake of xenobiotics. One member of this family is OATP2B1. This transporter is ubiquitously expressed and possesses a PDZ-binding motif at the C-terminus. PDZK1 (PDZ domain-containing 1) is a scaffold protein that influences function of different membrane proteins by sorting/stabilization of their membrane localization. It was aim of the herein reported study to investigate whether there is an interaction between OATP2B1 and PDZK1, and to further characterize its impact on transport function. At first expression of both OATP2B1 and PDZK1 was evaluated in liver, kidney and intestine. Based on the existence of a C-terminal PDZ-class I binding motif in OATP2B1 and the co-expression in all tested tissues an interaction was likely. Testing the influence of PDZK1 on OATP2B1 transport function revealed enhanced transport capacity for estrone 3-sulfate, thereby suggesting a change in OATP2B1 amount in the membrane. This assumption was validated by Western blot analysis. Finally, deletion of the C-terminal PDZ-binding motif in OATP2B1 lowered the impact of PDZK1 on transport function. Taken together, we report an interaction of PDZK1 with OATP2B1, which influences localization and function of the transporter. Changes in PDZK1 expression may therefore be one factor contributing to interindividual differences in OATP2B1 mediated pharmacokinetic processes.
Adaptor protein is a new therapeutic target in chronic kidney disease
2017, Kidney International
Ergothinoneine, derived from food, has powerful antioxidant effects. In this issue, Shinozaki et al. showed a novel kidney-intestine interaction, mediated by the postsynaptic density 95/disk-large/ZO-1 domain–containing protein (PDZK1)–organic cation transporter 1 (OCTN1)-ergothinoneine axis. In chronic kidney disease, decreased PDZK1 disturbed the localization of OCTN1 on the intestine, resulting in decreased serum ergothinoneine, which canceled the compensatory expression of OCTN1 in the kidney. Because PDZK1 regulates the function of several transporters, targeting PDZK1 may be a new therapeutic target in chronic kidney disease.
Impairment of the carnitine/organic cation transporter 1–ergothioneine axis is mediated by intestinal transporter dysfunction in chronic kidney disease
2017, Kidney International
Citation Excerpt :
OCTN1 was localized on the surface of HEK293 cells that stably expressed PDZK1. In contrast, OCTN1 was localized to the cytoplasm in HEK293 cells transfected with mutant PDZK, and these cells showed decreased transport activity.27 In PDZK1-KO mice, transporters with PDZ binding motifs showed reduced activity.28,29
Carnitine/organic cation transporter 1 (OCTN1) is a specific transporter of the food-derived antioxidant ergothioneine. Ergothioneine is absorbed by intestinal OCTN1, distributed through the bloodstream, and incorporated into each organ by OCTN1. OCTN1 expression is upregulated in injured tissues, and promotes ergothioneine uptake to reduce further damage caused by oxidative stress. However, the role of the OCTN1–ergothioneine axis in kidney–intestine cross-talk and chronic kidney disease (CKD) progression remains unclear. Here we assessed ergothioneine uptake via intestinal OCTN1 and confirmed the expression of OCTN1. The ability of OCTN1 to absorb ergothioneine was diminished in mice with CKD. In combination with OCTN1 dysfunction, OCTN1 localization on the intestinal apical cellular membrane was disturbed in mice with CKD. Proteomic analysis, RT-PCR, Western blotting, and immunohistochemistry revealed that PDZ (PSD95, Dlg, and ZO1), a PDZK1 domain-containing protein that regulates the localization of transporters, was decreased in mice with CKD. Decreased intestinal ergothioneine uptake from food decreased ergothioneine levels in the blood of mice with CKD. Despite increased OCTN1 expression and ergothioneine uptake into the kidneys of mice with CKD, ergothioneine levels did not increase. To identify the role of the OCTN1-ergothioneine axis in CKD, we evaluated kidney damage and oxidative stress in OCTN1-knockout mice with CKD and found that kidney fibrosis worsened. Oxidative stress indicators were increased in OCTN1-knockout mice. Moreover, ergothioneine levels in the blood of patients with CKD decreased, which were restored after kidney transplantation. Thus, a novel inter-organ interaction mediated by transporters is associated with CKD progression.
Organic cation transporter Octn1-mediated uptake of food-derived antioxidant ergothioneine into infiltrating macrophages during intestinal inflammation in mice
2015, Drug Metabolism and Pharmacokinetics
Citation Excerpt :
The isolated LPMCs were resuspended with transport buffer (125 mM NaCl, 4.8 mM KCl, 5.6 mM d-glucose, 1.2 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, and 25 mM HEPES, pH 7.4). The uptake experiment was then performed by the silicon-oil layer method as described previously [28]. The uptake of [3H]ERGO was expressed as the distribution volume determined as the amount of cell-associated radioactivity divided by the radioactivity concentration in the medium.
OCTN1/SLC22A4 is expressed on apical membranes of small intestine, and is involved in gastrointestinal absorption of its substrates, including the food-derived antioxidant ergothioneine (ERGO). ERGO concentration in circulating blood of patients with inflammatory bowel disease (Crohn's disease) is lower than that in healthy volunteers; thus, circulating ERGO is a potential diagnostic marker, although the mechanisms underlying low ERGO concentration in patients are unknown. Here, we focused on intestinal macrophages, which infiltrate sites of inflammation, and examined possible first-pass uptake of ERGO by macrophages. ERGO concentration in blood was lower in mice with dextran sodium sulfate (DSS)-induced colitis than in controls. On the other hand, expression of octn1 gene product and ERGO concentration in intestinal tissues of DSS-treated mice were higher than in controls. Interestingly, lamina propria mononuclear cells (LPMCs) isolated from DSS-treated mice contained ERGO and showed [³H]ERGO uptake and Octn1 expression, whereas ERGO was undetectable in LPMCs of control mice. Functional expression of OCTN1 was also confirmed in LPS-stimulated human macrophage-like cell line, THP-1. In conclusion, OCTN1 is functionally expressed on activated intestinal macrophages, and ERGO uptake into these immune cells could contribute at least in part to the altered disposition of ERGO in intestinal inflammation.
Proton-coupled oligopeptide transporter family SLC15: Physiological, pharmacological and pathological implications
2013, Molecular Aspects of Medicine
Mammalian members of the proton-coupled oligopeptide transporter family (SLC15) are integral membrane proteins that mediate the cellular uptake of di/tripeptides and peptide-like drugs. The driving force for uphill electrogenic symport is the chemical gradient and membrane potential which favors proton uptake into the cell along with the peptide/mimetic substrate. The peptide transporters are responsible for the absorption and conservation of dietary protein digestion products in the intestine and kidney, respectively, and in maintaining homeostasis of neuropeptides in the brain. They are also responsible for the absorption and disposition of a number of pharmacologically important compounds including some aminocephalosporins, angiotensin-converting enzyme inhibitors, antiviral prodrugs, and others. In this review, we provide updated information on the structure–function of PepT1 (SLC15A1), PepT2 (SLC15A2), PhT1 (SLC15A4) and PhT2 (SLC15A3), and their expression and localization in key tissues. Moreover, mammalian peptide transporters are discussed in regard to pharmacogenomic and regulatory implications on host pharmacology and disease, and as potential targets for drug delivery. Significant emphasis is placed on the evolving role of these peptide transporters as elucidated by studies using genetically modified animals. Whenever possible, the relevance of drug–drug interactions and regulatory mechanisms are evaluated using in vivo studies.

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Regular ArticleMutation in an Adaptor Protein PDZKI Affects Transport Activity of Organic Cation Transporter OCTNs and Oligopeptide Transporter PEPT2

Summary:

Kidney Int.

J. Biol. Chem.

J. Biol. Chem.

Structure determination and mutagenesis of the Erbin PDZ domain. J. Biol. Chem.

PEBS Lett.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Screening of the interaction between xenobiotic transporters and PDZ proteins

Pharm. Res.

PDZK1 directly regulates the function of organic cation/carnitine transporter OCTN2

Mol. Pharmacol.

Modulation of renal apical organic anion transporter 4 function by two PDZ domain-containing proteins

J. Am. Soc. Nephrol.

Protein/protein interactions (PDZ) in proximal tubules

J. Membr. Biol.

Regular Article
Mutation in an Adaptor Protein PDZKI Affects Transport Activity of Organic Cation Transporter OCTNs and Oligopeptide Transporter PEPT2