associate editor: paul a. insel drug transporters ......function in vitro, as well as drug...

1521-0081/67/3/656–680$25.00 http://dx.doi.org/10.1124/pr.115.010728PHARMACOLOGICAL REVIEWS Pharmacol Rev 67:656–680, July 2015Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics

ASSOCIATE EDITOR: PAUL A. INSEL

Drug Transporters and Na+/H+ Exchange RegulatoryFactor PSD-95/Drosophila Discs Large/ZO-1 Proteins

Dustin R. Walsh, Thomas D. Nolin, and Peter A. Friedman

Laboratory for G Protein–Coupled Receptor Biology, Department of Pharmacology and Chemical Biology, and Structural Biology,University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania (P.A.F.); and Center for Clinical Pharmaceutical Sciences,

Department of Pharmacy and Therapeutics, University of Pittsburgh School of Pharmacy, Pittsburgh, Pennsylvania (D.R.W., T.D.N.)

Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 656I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 657II. Drug Transporter Structure, Function, and Tissue Location . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 658

A. Solute Carrier Drug Transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6581. SLC22A Family.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 658

a. Organic cation transporters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 658b. Organic cation/carnitine transporters.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 660c. Organic anion transporters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 661

2. SLCO Family. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6613. Other Solute Carriers (SLC15A Family). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 663

B. ATP Binding-Cassette Drug Transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6641. Multidrug Resistance Protein 1 (ABCB1). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6642. Multidrug Resistance Proteins (ABCCs). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6653. Breast Cancer Resistance Protein (ABCG2). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 666

III. Tissue and Cellular Localization of PSD-95/Drosophila Discs Large/ZO-1 Proteins . . . . . . . . . . . . 667IV. Role of PSD-95/Drosophila Discs Large/ZO-1 Proteins in Regulation of Cell Membrane

Abundance, Localization, and Function of Drug Transporters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 669A. Solute Carrier Drug Transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 669

1. Organic Carnitine Transporters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6692. Organic Anion Transporters.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6703. Organic Anion-Transporting Polypeptides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6714. Peptide Transporters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 671

B. ATP Binding-Cassette Drug Transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6721. Multidrug Resistance Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 672

V. PSD-95/Drosophila Discs Large/ZO-1 Proteins and Drug Action in Disease States . . . . . . . . . . . . . 673A. Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 673B. Chronic Kidney Disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 674

VI. Conclusions and Future Directions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 675Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 676References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 676

Abstract——Drug transporters govern the absorption,distribution, and elimination of pharmacologicallyactive compounds. Members of the solute carrier andATP binding-cassette drug transporter family mediate

cellular drug uptake and efflux processes, thereby co-ordinating the vectorial movement of drugs acrossepithelial barriers. To exert their physiologic andpharmacological function in polarized epithelia, drug

Original research was supported the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases[Grants R01-DK054171 and R01-DK069998 (to P.A.F.)]; and the National Institutes of Health National Institute of General Medical Sciences[Grant R01-GM107122 (to T.D.N.)]. D.R.W. received support from the Department of Pharmacy and Therapeutics, University of PittsburghSchool of Pharmacy.

Address correspondence to: Peter A. Friedman, E1356 Biomedical Science Tower, 200 Lothrop St., Pittsburgh, PA 15261. E-mail:[email protected]

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transporters must be targeted and stabilized to appro-priate regions of the cell membrane (i.e., apical versusbasolateral). Despite the critical importance of drugtransporter membrane targeting, the mechanisms thatunderlie these processes are largely unknown. Severalclinically significant drug transporters possess arecognition sequence that binds to PSD-95/Drosophiladiscs large/ZO-1 (PDZ) proteins. PDZ proteins, such asthe Na+/H+ exchanger regulatory factor (NHERF) family,act to stabilize and organize membrane targeting ofmultiple transmembrane proteins, including manyclinically relevant drug transporters. These PDZ proteinsare normally abundant at apical membranes, wherethey tether membrane-delimited transporters. NHERF

expression is particularly high at the apical membranein polarized tissue such as intestinal, hepatic, and renalepithelia, tissues important to drug disposition. Severalrecent studies have highlighted NHERF proteins asdeterminants of drug transporter function secondary totheir role in controlling membrane abundance andlocalization. Mounting evidence strongly suggests thatNHERF proteins may have clinically significant roles inpharmacokinetics and pharmacodynamics of severalpharmacologically active compounds and may affectdrug action in cancer and chronic kidney disease. Forthese reasons, NHERF proteins represent a novel classof post-translational mediators of drug transport andnovel targets for new drug development.

I. Introduction

Plasma membrane transport proteins play a centralrole in mediating the disposition or pharmacokinetics,i.e., absorption, distribution, and elimination, of a broadspectrum of endogenous and exogenous compounds in-cluding pharmacologically active compounds and clini-cally used medications (Nigam, 2015). Such transportersare now acknowledged also as clinically significantdeterminants of drug response, i.e., pharmacodynamics.Although broadly expressed, drug transporters exhibithighest abundance in intestine, liver, and kidney,tissues important for drug disposition. Furthermore,drug transporters expressed at the blood-brain barrierand the placenta serve an additional protective role tominimize exposure of the brain and fetus, respectively,to toxic compounds.Solute carrier (SLC) and the ATP-binding cassette

(ABC) transporters form the two major superfamiliesof drug transporters. SLC transporters primarily medi-ate cellular uptake of drugs by facilitated diffusion orion-coupled secondary active transport. ABC trans-porters function as primary energy-dependent activetransporters and rely on the hydrolysis of ATP toenergize cellular drug efflux. Functionally, SLC andABC drug transporters work in concert to couple thevectorial movement of drugs and drug metabolitesacross epithelial barriers. The clinical impact of drugtransporters in pharmacotherapy highlights the needto fully characterize their regulatory mechanisms.Epithelial cells that form the intestinal, hepatic, and

renal barriers display characteristic apical-basolateralmembrane polarity. Drug transporters that are ex-pressed in these cells must be targeted and stabilizedat the appropriate membrane domain (i.e., apical orbasolateral) to elicit their physiologic and pharma-cologic function. A key role of drug transporters located

at epithelial barriers is to limit systemic drug exposure.Oral drug absorption is accomplished by an array ofdrug transporters that are distributed throughout theintestinal epithelium. In the liver, transporters coordi-nate the hepatobiliary disposition of drugs and serve aprincipal role in biliary drug excretion. Together, intes-tinal and hepatic drug transporters play a major role inmediating drug bioavailability. Drug transporters ex-pressed in renal proximal tubules move organic solutesacross the proximal tubule epithelium. Drug trans-porters in the kidney are significant contributors torenal drug elimination and are involved in both secre-tion and reabsorption into and from the tubular lumen.At all of these epithelial barriers, drug transporters arelocalized to specific membrane domains, which is criticalto their functional role in drug bioavailability, disposition,and response. Defining the mechanisms that determinedrug transporter abundance and membrane-delimitedlocalization is an emerging line of research.

PDZ proteins are named for the common structuraldomain shared with the postsynaptic density protein(PSD), Drosophila disc large tumor suppressor, andzonula occludens-1 protein (ZO-1). In addition to theirinvolvement in the assembly of multiprotein signalingcomplexes, PDZ domain–containing proteins also regu-late membrane abundance and the asymmetric sortingof proteins to specific membrane domains in polarizedepithelia (Shenolikar and Weinman, 2001; Shenolikaret al., 2002; Padanyi et al., 2010). PDZ proteins recognizeand bind to target proteins by specific internal andcarboxy-terminal amino acid sequences, referred toas PDZ ligands (Songyang et al., 1997; Stricker et al.,1997). PDZ domains are classified based on the struc-tural binding motif of the PDZ ligand with which theyinteract (Table 1). Class I PDZ proteins interact withligands terminating in the sequence -X-[S/T]-X-w, where

ABBREVIATIONS: ABC, ATP binding-cassette; BCRP, breast cancer resistance protein; CKD, chronic kidney disease; DJS, Dubin-Johnsonsyndrome; EBD, ezrin binding domain; ERM, ezrin/radixin/moesin/merlin; Gly-Sar, glycylsarcosine; MDR, multidrug resistance; MDR1,multidrug resistance protein 1; MRP, multidrug resistance-associated protein; NBD, nucleotide binding domain; NHE, Na+/H+ exchanger;NHERF, Na+/H+ exchange regulatory factor; OAT, organic anion transporter; OATP, organic anion-transporting polypeptide; OCT, organiccation transporter; OCTN, organic carnitine transporters; P450, cytochrome P450; PDZ, PSD-95/Drosophila discs large/ZO-1; PEPT, peptidetransporter; Pgp, P-glycoprotein; PKC, protein kinase C; PSD, postsynaptic density protein; PXE, pseudoxanthoma elasticum; PXR, pregnaneX receptor; SLC, solute carrier; TMD, transmembrane domain.

Drug Transporters and NHERF PDZ Proteins 657

X is promiscuous and w is a hydrophobic residue, gen-erally Leu, Ile, Val, or Met. Class II PDZ proteins preferligands terminating in -X-w-X-w and class III prefer aPDZ binding motif of the form -[D/E]-[K/R]-X-w. However,PDZ proteins may also recognize internal peptide frag-ments from target proteins. Residues up to 18-amino-acidsupstream from the carboxy terminus can participate intarget recognition (Mahon and Segre, 2004). Thesenoncanonical binding modes may also be important forestablishing target specificities. Several clinically sig-nificant SLC and ABC drug transporters harbor acarboxy-terminal PDZ ligand and therefore may bindPDZ proteins. Mounting evidence highlights the impactof PDZ proteins in regulating the membrane abundance,localization, and function of a growing number of drugtransporters (Karvar et al., 2014; Park et al., 2014;Zheng et al., 2014).The Na+/H+ exchanger regulatory factors (NHERFs)

are a family of PDZ proteins that also act as mediatorsof drug transporter function secondary to their role inorganizing drug transporter membrane abundance andlocalization. NHERF proteins alter drug transporterfunction in vitro, as well as drug absorption and dispo-sition in vivo (Park et al., 2014). NHERF proteins arehighly expressed at apical membranes of polarizedepithelial cells of the kidney, intestine, and liver, wherethey interact with numerous drug transporters fromboth SLC and ABC superfamilies (Kato et al., 2004,2006; Sugiura et al., 2011). The majority of reportedinteractions are mediated through canonical class IPDZ ligands, although interactions with drug trans-porters possessing class II and III PDZ ligands alsohave been described (Kato et al., 2004; Sugiura et al.,2008; Shimizu et al., 2011). Hence, NHERF proteinsmay have an indirect effect on drug response attribut-able to their influence on drug absorption and disposi-tion in these important pharmacological barriers. Overthe last decade, PDZ proteins have gained considerableattention as post-translational regulators of drug trans-porter function (Miyazaki et al., 2005; Kato et al., 2006;Noshiro et al., 2006; Sugiura et al., 2006, 2008, 2010,2011; Choi et al., 2011; Park et al., 2014; Wang et al.,2014; Zheng et al., 2014). To highlight these newfindings, we review the current knowledge of drugtransporter regulation by PDZ proteins and addressthe association of PDZ proteins with drug dispositionand response in select disease states.

II. Drug Transporter Structure, Function, andTissue Location

A. Solute Carrier Drug Transporters

Solute carrier (SLC) drug transporters are classifiedinto the SLC22A and SLCO gene families (uppercasedenotes human genes or gene products and lowercasedenotes rodent genes or gene products). The SLC22Agenes include the organic anion transporters (OATs),organic cation transporters (OCTs), and organic carni-tine transporters (OCTNs). The SLCO gene family con-sists of the organic anion-transporting polypeptides(OATPs). Members of both gene families are primarilyinvolved in cellular drug uptake and are abundantly ex-pressed at the epithelium in the liver, intestine, andkidney (Fig. 1) (Giacomini et al., 2010). Although mostSLC superfamily drug transporters display overlap-ping substrate specificities, OATs, OCTs, and OCTNstransport anions, cations, and zwitterions, respectively,whereas OATPs transport larger hydrophobic anions.

1. SLC22A Family. Transporter proteins encoded bythe SLC22A gene family include OCT1–3 (SLC22A1–3),OCTN1–2 (SLC22A4–5), OAT1–3 (SLC22A6–8), OAT4(SLC22A11), and URAT1 (SLC22A12). The prototypemembrane topology of the SLC22A family comprises12 a-helical transmembrane domains (TMDs), an in-tracellular N terminus, a large extracellular loop be-tween TMDs 1 and 2, which harbors several putativeN-glycosylation sites, a large intracellular loop betweenTMDs 6 and 7 that contains additional glycosylation andphosphorylation sites and an intracellular C terminus(Fig. 2). Tissue expression and membrane localization ofselected SLC22A drug transporters is summarized inFig. 1. SLC22A drug transporters and selected sub-strates are summarized in Table 2.

a. Organic cation transporters. OCT1 (SLC22A1),OCT2 (SLC22A2), and OCT3 (SLC22A3) mediate trans-port of organic cationic drugs such as metformin, hista-mine H2 receptor antagonists, and platinum-baseddrugs. OCT1–3 share three common functional charac-teristics. First, substrate transport is bidirectional; thatis, OCTs mediate both uptake and efflux processes.Second, transport occurs by passive facilitated diffusionthat is driven by the negatively charged intracellularmembrane voltage. The rate of substrate uptake andefflux therefore depends upon the electrochemicalgradient and is sensitive to changes in the membranepotential. Third, OCT-mediated transport is indepen-dent of sodium and pH. OCTs are expressed in thekidney, intestine, and liver, where they function pri-marily as basolateral uptake transporters. However,OCT3 may be expressed at both basolateral and apicalmembranes (Muller et al., 2005; Nies et al., 2009). Forexample, OCT3 is localized to basolateral/sinusoidalmembranes of hepatocytes and apical membranes ofenterocytes. OCT3 is expressed in renal proximal anddistal tubules, but additional studies are needed to

TABLE 1PDZ ligand recognition classification

Class C-Terminal Consensus Sequence of PDZ Liganda

I -X-[S/T]-X-wII -X- w -X-wIII -[D/E]-[K/R]-X-waX is permissive indicating any amino acid; S/T are serine and threonine, w

hydrophobic residue, typically L, I, V. D/E and K/R are aspartate/glutamate, andlysine/arginine, respectively.

658 Walsh et al.

define OCT3 membrane localization in the kidney(Wu et al., 2000b).OCTs exhibit broad selectivity. Common substrates

include hydrophilic, low molecular weight organic cat-ions (Table 2). OCT1 and OCT2 are drug transportersof emerging clinical significance because of their poten-tial influence on drug disposition and response. Forexample, OCT1 is an important factor in the pharma-cotherapy of diabetes and chronic myeloid leukemia(Grinfeld et al., 2013; Koren-Michowitz et al., 2014;Mahrooz et al., 2015). OCT1 is thought to be liverspecific; it is predominately expressed on basolateral/sinusoidal membranes of hepatocytes and mediatesdrug uptake (Gorboulev et al., 1997; Zhang et al., 1997).Although few endogenous OCT1 substrates have been

identified, evidence suggests that hepatic OCT1 maymediate the uptake of biogenic amines such as serotonin(Boxberger et al., 2014). Moreover, commonly prescribedmedications, such as diphenhydramine, fluoxetine,imatinib, and verapamil, possibly inhibit OCT1-dependentserotonin uptake, a finding highlighting the possibilitythat drugs may interfere with transport and eliminationof endogenous substrates at the transporter level. OCT2is found exclusively in the kidney at basolateral mem-branes of proximal tubule cells, where it facilitatessecretion of drugs such as cisplatin and oxaliplatin(Gorboulev et al., 1997; Urakami et al., 2002; Iwataet al., 2012). OCT3, in contrast to OCT1 and OCT2,displays a much broader tissue expression pattern. Inaddition to expression in the intestine, liver, and kidney,

Fig. 1. Selected drug transporters expressed at epithelial barriers of intestinal enterocytes, hepatocytes, and kidney proximal tubule cells. ABCtransporters are colored red, SLC in blue. ABC or SLC transporters harboring a PDZ ligand are additionally depicted with an inner dottedyellow circle.

Fig. 2. SLC drug transporter membrane topology. The prototype membrane topology of SLC22A family members (OCTs, OCTNs, OATs) is similar, asis SLCO for OATP family members. Blue cylinders represent predicted transmembrane helices. Putative N-glycosylation and phosphorylation sitesand important cysteine residues are shown as yellow, green, or red circles, respectively.


OCT3 mRNA levels can be detected in the placenta,heart, brain, and skeletal muscle (Hayer-Zillgen et al.,2002). It is not known why OCT3 localizes to apical mem-branes in enterocytes but to basolateral membranes inhepatocytes. It is also not understood why OCT3 iscapable of dual membrane localization that is tissuespecific but OCT1 and OCT2 are restricted to baso-lateral membranes. Of the three OCT isoforms, onlyOCT3 contains a C-terminal PDZ-ligand (-RSHL).Therefore, the membrane localization of OCT3 may bedetermined by tissue- and domain-specific localizationof PDZ proteins.b. Organic cation/carnitine transporters. The OCTN

subfamily consists of OCTN1 (SLC22A4) and OCTN2(SLC22A5). In addition to their physiologic role as up-take transporters for the zwitterion carnitine, membersof the OCTNs are known also to interact with drugssuch as levofloxacin, oxaliplatin, and cephaloridine(Hirano et al., 2008; Kano et al., 2009; Jong et al., 2011).Analogous to OCTs, OCTNs are able to perform bidirec-tional transport; however, there are subtle differences inthe transport mechanisms between OCTN1 and OCTN2.For example, OCTN1 can function as an organic cationuniporter or an H+/organic cation antiporter, whereasOCTN2 functions as a sodium/carnitine cotransporteror may perform sodium-independent cation transport(Tamai et al., 1998, 2004; Wu et al., 1999; Ohashi et al.,2001; Grundemann et al., 2005). Although broadly ex-pressed in multiple tissues, OCTN subfamily membersdisplay preferential tissue expression. For instance,OCTN1 is predominately expressed on apical mem-branes of renal proximal tubules but is found at much

lower levels in other tissues such as the liver, smallintestine, placenta, and brain (Tamai et al., 2000, 2004;Wu et al., 2000a). OCTN2 also is expressed on apicalmembranes of renal proximal tubule cells but itsexpression is not limited to polarized epithelia. It isfound in several organs including the heart, brain,liver, testis, and skeletal muscle (Tamai et al., 1998,2000). Because OCTN2 is believed to be the majorOCTN regulating serum carnitine concentrations (Nezuet al., 1999), its wider expression may have evolved toensure adequate carnitine absorption and distributionthroughout the body. Carnitine is required for fatty acidtransport in mitochondria for generation of metabolicenergy. Underscoring its vital nature, primary carnitinedeficiency, a disorder characterized by chronic muscleweakness, cardiomyopathy, hypoglycemia, and liverdysfunction, is caused by defects in OCTN2 activity(Tamai et al., 1998). The OCTN subfamily has beenlinked to other disease states as well. Several studiesimplicated OCTN1 and OCTN2 in the pathophysiologyof inflammatory bowel disease, diabetes, and cancer(Peltekova et al., 2004; Okabe et al., 2008; Martini et al.,2012; Muoio et al., 2012; Pochini et al., 2013). By virtueof their association with several diseases, OCTNs arestudied more in the context of pathophysiology and lessfor their potential pharmacological role as mediators ofdrug absorption and disposition. Nevertheless, OCTNsare involved in transport of organic cationic drugsand are potential sites for drug/carnitine interactions(Ganapathy et al., 2000). OCTN1 and OCTN2 mediatetransport of drugs such as antipsychotics, antihyper-tensives, antibiotics, and drugs used in the treatment of

TABLE 2Selected drug transporters

Transporter Superfamily Family Gene Name PDZ Ligand Selected Substrates

OCT1 SLC SLC22 SLC22A1 — Metformin, oxaliplatinOCT2 SLC SLC22 SLC22A2 — Metformin, ranitidine, variniclineOCT3 SLC SLC22 SLC22A3 R-S-H-L Histamine, epinephrineOCTN1 SLC SLC22 SLC22A4 L-T-A-F Quinidine, verapamil, ipratropium, ergothioneineOCTN2 SLC SLC22 SLC22A5 S-T-A-F Carnitine, amisulpride, cephaloridineOAT1 SLC SLC22 SLC22A6 — Lamivudine, acyclovir, ciprofloxacinOAT2 SLC SLC22 SLC22A7 — Bumetanide, erythromycin, paclitaxelOAT3 SLC SLC22 SLC22A8 — Furosemide, bumetanideOAT4 SLC SLC22 SLC22A11 S-T-S-L Bumetanide, urate, methotrexateURAT1 SLC SLC22 SLC22A12 S-T-Q-F UrateOATP1A2 SLC SLCO SLCO1A2 K-T-K-L Statins, fexofenadineOATP1B1 SLC SLCO SLCO1B1 — Statins, repaglinide, olmesartanOATP1B3 SLC SLCO SLCO1B3 — Statins, telmisartan, fexofenadineOATP1C1 SLC SLCO SLCO1C1 E-T-Q-L Triiodothyronine, thyroxineOATP2B1 SLC SLCO SLCO2B1 D-S-R-V Statins, fexofenadineOATP3A1 SLC SLCO SLCO3A1 E-S-V-L Benzylpenicillin, vasopressinOATP4A1 SLC SLCO SLCO4A1 Q-S-S-V Estradiol-17b-glucuronideOATP4C1 SLC SLCO SLCO4C1 — Digoxin, methotrexate, sitagliptinPEPT1 SLC SLC15 SLC15A1 Q-K-Q-M Cephalexin, valacyclovir, enalaprilPEPT2 SLC SLC15 SLC15A2 K-T-K-L Cephalexin, valacyclovir, enalaprilMDR1 (Pgp) ABC ABCB ABCB1 — Digoxin, loperamideBCRP ABC ABCG ABCG2 — Statins, methotrexateMRP1 ABC ABCC ABCC1 — Citalopram, methotrexate, Leukotriene C4MRP2 ABC ABCC ABCC2 S-T-K-F Olmesartan, methotrexate, etoposideMRP3 ABC ABCC ABCC3 — Estradiol-17b-glucuronideMRP4 ABC ABCC ABCC4 E-T-A-L Adefovir, topotecan, methotrexateMRP7 ABC ABCC ABCC10 D-T-R-L Docetaxel, paclitaxel, viscristine, vinblastine

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neurologic disorders (Ganapathy et al., 2000; Grubeet al., 2006; Urban et al., 2008; Nakamura et al., 2010;Jong et al., 2011). Both OCTN1 and OCTN2 containC-terminal PDZ ligands (-LTAF and -STAF, respec-tively). The role of PDZ proteins and OCTN membraneabundance and function is summarized in section IV.c. Organic anion transporters. OATsmediate the sys-

temic removal of organic anions including drugs, toxins,and endogenous metabolites. OAT1–3 (SLC22A6–8),OAT4 (SLC22A11), and URAT1 (SLC22A12) are deemedclinically significant human OAT isoforms due to theirprominent role in the kidney as mediators of renalsecretion and thus renal drug elimination (Giacominiet al., 2010; Burckhardt, 2012). Historically, OATs havebeen the targets of pharmacological strategies to extendlimited supplies of drugs. The classic example of OAT-targeted interventions of this manner is the utilizationof probenecid to conserve limited supplies of penicillinduring World War II. Probenecid, an OAT inhibitor,increases serum concentrations of penicillin by signifi-cantly reducing OAT-dependent renal secretion, thusextending the half-life of penicillin and optimizingtherapeutic efficacy by achieving concentrations abovethe minimum inhibitory concentration with a lower dose(Weiner et al., 1960). More recently, owing to its estab-lished inhibitory effects on OATs, probenecid has beensuggested as an adjuvant to other medications thathave been in short supply, such as the antiviral drugoseltamivir (Howton, 2006). Genetic variations encodingdifferent OAT isoforms are also associated with alteredtransporter activity and impaired renal handling ofOAT substrates (Bleasby et al., 2005; Xu et al., 2005;Erdman et al., 2006). For instance, genetic polymor-phisms in OAT3 are associated with reduced renalclearance of cefotaxime, a cephalosporin antibiotic (Yeeet al., 2013). Transcript variants of URAT1 may produceincreases or decreases in renal tubular uric acid reab-sorption, changes that result in hyperuricemia andhypouricemia, respectively (Wakida et al., 2005; Graessleret al., 2006).In contrast to the OCTs, OATs transport anions into

cells against the negatively charged electrochemicalgradient and therefore are energy dependent. OATstransport substrates by exchanging an intracellularmono- or divalent anion, typically a dicarboxylate, foruptake of an extracellular anion (Burckhardt, 2012).The energy expenditure that drives this process comesindirectly from the Na+/K+-ATPase that maintains in-tracellular sodium concentration at low levels andcreates a negative intracellular voltage. This favorableelectrochemical gradient drives sodium-coupled uptakeof dicarboxylate species by the sodium-dicarboxylatecotransporter (NaDC3). Together, the Na+/K+-ATPase,NaDC3, and OATs cooperate to facilitate the uptakeand removal of organic anions. OATs are heavily ex-pressed in the kidney, where they play a significant rolein the uptake step of renal drug accumulation. Among

the OAT family, OAT1 and OAT3 are the most exten-sively studied in the context of drug transport and havebeen shown to mediate the uptake of a wide variety ofstructurally diverse drug classes such as antihyperten-sive drugs, 3-hydroxy-3-methylglutaryl-co-enzyme A re-ductase inhibitors (statins), b-lactam antibiotics, H2receptor antagonists, antineoplastic drugs, nonsteroidalanti-inflammatory drugs, and antiviral drugs (Burckhardt,2012). However, OATs also transport multiple endog-enous compounds and play complex physiologic roles(Mandal and Mount, 2015; Nigam et al., 2015). Recentevidence suggests that OAT3 not only handles transportof drugs and drug metabolites, but also may play a rolein 1) pathways involved in the regulation of secondarymetabolites, such as prostaglandins, steroids, and uricacid; 2) the metabolism and handling of gut microbiomeproducts, as well as uremic toxins that accumulate inkidney disease; 3) handling of dietary flavonoids such asepicatechin; and 4) the tricarboxylic acid cycle and nu-cleotide and amino acid metabolism (Wu et al., 2013;Mandal and Mount, 2015; Nigam et al., 2015).

OAT1 and OAT3 are exclusively located at basolateralmembranes in renal proximal tubule cells. In the kidney,all members of the OAT family localize to basolateral mem-branes with the exception of OAT4 and URAT1. OAT4 isunique in that it is expressed only in humans and islocated at apical membranes of renal proximal tubulecells (Cha et al., 2000; Babu et al., 2002; Ekaratanawonget al., 2004). OAT4 is also expressed in the placenta but isrestricted to basolateral membranes of syncytiotropho-blasts (Cha et al., 2000). This distinctive tissue-specificpattern of OAT4 membrane localization is similar to thedual-membrane expression patterns of OCT3. LikeOCT3, OAT4 possesses a PDZ ligand (-STSL). Severalstudies suggest that multiple PDZ proteins may governthe membrane abundance and localization of OAT4 (Katoet al., 2004; Miyazaki et al., 2005; Zhou et al., 2008).

2. SLCO Family. The SLCO gene family encodes thehuman OATPs. These proteins transport various or-ganic amphiphilic compounds, the majority of which arelarge hydrophobic anions such as bile acids, steroid con-jugates, and anionic peptides, as well as several drugsincluding statins, angiotensin II receptor antagonists,and angiotensin-converting enzyme inhibitors (Chaet al., 2000; Babu et al., 2002; Ekaratanawong et al.,2004; Zhou et al., 2008). Eleven human OATPs havebeen identified to date. Selected OATPs discussed inthis manuscript are summarized in Table 2. There aresix known human OATP families. Classification of OATPsis based on their amino acid similarities. OATPs withinthe same family share$40% amino acid sequence identityand are assigned root names followed by a numericaldesignation (OATP1–6). Subfamily members share$60%amino acid sequence identity and are designated with aletter following the numerical family designation. Forinstance, OATP family 2 (OATP2) has two subfamilies(OATP2A and OATP2B). A numerical designation after


the subfamily heading is assigned to each member of asubfamily (OATP2A1 and OATP2B1). Size differences existacross members of the OATP family. OATPs have anaverage length of 710 amino acids but range from 643amino acids (OATP2A1) to 848 amino acids (OATP5A1).However, all OATPs share a similar predicted mem-brane topology that comprises 12 a-helical transmem-brane domains, intracellular N and C termini, andmultiple glycosylation sites within the second andfifth extracellular loop. The fifth extracellular loop islarger and contains multiple conserved cysteine resi-dues that participate in disulfide bonds (Fig. 2). Ingeneral, OATP-dependent transport involves bidirec-tional, sodium-independent exchange. OATPs couplethe cellular uptake of anions with efflux of intracellularsubstances such as bicarbonate, reduced glutathione, orlactate. In the case of OATP2B1, transport is pH sensitivebut only with selective solutes such as statins (Kobayashiet al., 2003; Nozawa et al., 2004; Varma et al., 2011). Theprecise mechanism of transport and how it relates to thephysiologic and pharmacological role of individual OATPsremains unclear.Tissue expression and membrane localization of

selected SLCO drug transporters is summarized inFig. 1. Some members of the OATP family display broadtissue expression. OATP1A2, OATP2A1, OATP2B1,OATP3A1, and OATP4A1 for instance are found inmultiple tissues including the kidney, placenta, skeletalmuscle, choroid plexus, and endothelial cells of theblood-brain barrier. Other OATPs exhibit a more re-stricted pattern. The major hepatic OATP isoforms in-clude OATP1B1 and OATP1B3; OATP4C1 predominatesin the kidney. Among the OATP family, OATP1B1,OATP1B3, OATP1A2, and OATP2B1 are consideredclinically important determinants of pharmacokineticsdue to their participation in transporting commonlyused medications such as statins (Giacomini et al.,2010). In particular, OATP1B1 and OATP1B3 medi-ate the hepatic uptake of drugs, whereas OATP1A2and OATP2B1 mediate intestinal drug absorption. Theextensive expression of OATPs in the intestine and liverlikely evolved as a protective mechanism to combat in-advertent ingestion of plants or foods containing toxins.An additional evolutionary advantage to OATP-dependentdetoxification comes in the form of cooperativity withintestinal and hepatic metabolizing enzymes. As ameans to provide protection against a wide spectrumof potentially harmful exogenous substances, intestinaland hepatic OATPs have adopted overlapping substratespecificity with intracellular cytochrome P450 (P450)enzymes. Together, OATPs cooperate with P450 en-zymes in the intestine and liver to reduce the systemicexposure of xenobiotics by exposing substrates tointracellular P450s for subsequent metabolism. Evi-dence of this cooperative functionality is observed con-cerning the hepatic clearance of erythromycin, a commonlyused probe to assess CYP3A function (Rivory et al.,

2001). Erythromycin uptake into hepatocytes is medi-ated by OATPs (Sun et al., 2010; Lancaster et al., 2012).OATPs facilitate the uptake of erythromycin from theportal vein and into hepatocytes where metabolism byCYP3A occurs (Franke et al., 2011). Therefore, hepaticclearance of erythromycin is a function of OATP-dependentuptake and CYP3A function. Although these examplesillustrate OATP-P450 cooperativity, they may notextend to other transporters and P450 proteins.

Although generally considered drug transporters,physiologic roles of OATPs also have been described.Members of the OATP family are involved in thetransport of bilirubin (OATP1B1 and OATP1B3) andthyroid hormones (OATP1C1) (Keppler, 2014; Mayerlet al., 2014). In the liver, bilirubin metabolism andelimination is accomplished, in part, by the coordinatedactions of OATPs and UDP glucuronosyltransferase-1family, polypeptide-1. Similar to the proposed mecha-nism of OATP-P450 cooperative functionality and drugmetabolism, hepatic OATP1B1 and OATP1B3 facilitatethe uptake of unconjugated bilirubin, which is followedby glucuronic acid conjugation catalyzed by intracellularUDP glucuronosyltransferase-1 family, polypeptide-1(Keppler, 2014). OATPs also may be involved in trans-porting endogenous substances that are otherwiseimpermeant and cannot traverse the cell membranepassively and, therefore, rely on transporters to entercells. OATP1C1 has been implicated recently in thetransport across the blood-brain barrier of thyroxine,the prohormone of triiodothyronine, a process thoughtto be important for proper brain development (Mayerlet al., 2014). The physiologic and pharmacological im-portance of renal OATPs, particularly the role ofOATP4C1, is not fully appreciated.

Similar to other SLC transporter families, mem-brane localization of OATP isoforms is not uniform andin some cases is tissue dependent. Although most arelocated on basolateral membranes, OATP1A2 is exclu-sive to apical membranes, whereas OATP2B1 displaysdual membrane localization. In the intestine, OATP1A2and OATP2B1 are located on the apical brush-bordermembrane, where they contribute to drug absorption. Inthe liver, OATP1B1, OATP1B3, and OATP2B1 are locatedon basolateral/sinusoidal membranes, where they maybe responsible for hepatic drug uptake (Kullak-Ublicket al., 2001; Kobayashi et al., 2003; Tamai, 2012).OATP1A2 is also found at apical membranes in endo-thelial cells of the blood-brain barrier, hepatocytes,and renal distal tubules (Lee et al., 2005). Analogousto the OCT and OAT families of drug transporters, someOATP isoforms harbor a C-terminal PDZ ligand and arebinding partners with PDZ proteins (Kato et al., 2004;Wang et al., 2005; Zheng et al., 2014). Multiple PDZproteins regulate the function of OATP1A2 (Zhenget al., 2014), which is discussed in section IV.

The OATP family is gaining considerable attentiondue to their central role in drug disposition and response

662 Walsh et al.

(Link et al., 2008; Shitara et al., 2013). Undoubtedly, thisrecognition is underscored by the importance of OATPson statin pharmacotherapy. Statins are orally adminis-trated medications and considered mainstay therapy forthe treatment of hypercholesterolemia. To elicit theirlipid-lowering effects, statins must be taken up intohepatocytes. Thus, OATPs are not only critical deter-minants of statin pharmacokinetics but also for deliveryto their site of action. Several OATPs are involved in theoral absorption and disposition of statins, particularlyOATP1B1 (Hirano et al., 2006; Romaine et al., 2010;Prueksaritanont et al., 2014). All statins in clinical useare substrates for OATP1B1 (Niemi et al., 2011). Fur-thermore, OATP1B1 polymorphisms have been shownto alter the pharmacokinetics and pharmacodynamicsof statins including pravastatin, atorvastatin, andsimvastatin (Oh et al., 2013; Meyer Zu Schwabedissenet al., 2015). OATP1A2 has also been recognized as im-portant for drug transport and disposition. Substratesfor OATP1A2 include statins, human immonodeficiencyvirus–protease inhibitors, and fexofenadine (Kullak-Ublicket al., 1998). Selected OATP substrates are listed inTable 2. By virtue of their expanding role in pharmaco-kinetics and pharmacodynamics, characterizing OATPregulatory mechanisms in health and disease remainsan area of focus.3. Other Solute Carriers (SLC15A Family). The

SLC15A gene family encodes two human peptide trans-porters, PEPT1 (SLC15A1) and PEPT2 (SLC15A2).PEPT1 and PEPT2 function primarily as intestinaland renal peptide transporters, respectively. BothPEPT1 and PEPT2 are sodium-independent symportersthat couple peptide transport with the movement of H+

down its electrochemical gradient. PEPT1 is a high-capacity, low-affinity transporter for di- and tripeptidesbut has negligible affinity for amino acids (Liang et al.,1995). PEPT1 is heavily expressed in the small intestine,where it localizes exclusively to the apical brush-bordermembranes of enterocytes (Liang et al., 1995). At theintestinal barrier, PEPT1 absorbs small peptides thatare generated upon enzymatic breakdown of endogenousor ingested protein. PEPT1 also plays an important phar-macological role as a mediator of oral drug absorptionand is a target for prodrug design (Hamman et al., 2005;Brandsch, 2013; Zhang et al., 2013). Hydrophilic drugcompounds that are taken orally often have poorbioavailability secondary to limited intestinal membranepermeability. This barrier to pharmacotherapy and lim-itation to drug design has been circumvented by ex-ploiting intestinal PEPT1. As a strategy to enhance theoral bioavailability of such compounds, prodrugs areformulated via amino acid esterification (Majumdar andMitra, 2006). Chemical modifications of this type thuspermit involvement of PEPT1 in drug uptake processesin the intestine. This approach has been used previously,most notably in the design of the nucleoside analogvalacyclovir (Jacobson, 1993). Valacyclovir, a PEPT1

substrate and amino acid ester derivative of the activedrug acyclovir, exhibits a 3- to 5-fold increase in bio-availability (Weller et al., 1993). Intestinal PEPT1 isalso involved in the absorption of other peptidomimeticdrug compounds and prodrugs, including b-lactam anti-biotics and angiotensin converting-enzyme inhibitors(Daniel and Adibi, 1993; Ganapathy et al., 1995; Sugawaraet al., 2000; Shu et al., 2001). PEPT2 is predominatelyexpressed in renal proximal tubules and localizes toapical membranes, where it is involved in reabsorptionof di- and tripeptides from the glomerular filtrate(Rubio-Aliaga et al., 2003). PEPT1 is also found atapical membranes in proximal tubules, where it may beinvolved in the reabsorption of peptides that are gener-ated by the action of brush border peptidases on filteredproteins. Although both PEPT isoforms are expressed inkidney, PEPT1 is expressed at much lower levels, andtherefore the relative contribution of PEPT1 to renaltubule reabsorption is likely less than that of PEPT2(Liang et al., 1995). Furthermore, in contrast to PEPT1,PEPT2 is a high-affinity low-capacity transporter. Thisclear distinction in transport kinetics was described byrevealing that PEPT1 and PEPT2 were responsible forthe low-affinity (Km of 1.2 mM) and high-affinity (Km of50 mM) transport of glycylsarcosine (Gly-Sar), respec-tively (Takahashi et al., 1998). It was later determinedin studies comparing the contribution of PEPT1 andPEPT2 to the renal reabsorption of b-lactam antibiotics,that PEPT2 mediates high-affinity transport of aminob-lactam antibiotics such as amoxicillin, cephalexin,and cefadroxil, which contain an a-amino group, andthat the involvement of PEPT1 is minimal in thisregard (Takahashi et al., 1998; Inui et al., 2000). Thesefindings strongly support that the pharmacological roleof PEPT2 in the kidney might be to participate inreabsorption of amino b-lactam antibiotics, as well asother pharmacologically active compounds with similarchemical structures. Taken together, the expressionpattern and transport kinetics of the PEPTs are notsurprising if consideration is given to their physiologicroles. The abundant expression of PEPT1, a high-capacitytransporter, at the apical membrane in enterocytes isstrategically placed to ensure adequate dietary proteinabsorption. Conversely, the expression of PEPT2, ahigh-affinity transporter with specific substrate affini-ties, at apical membranes in renal proximal tubulescells facilitates tight control of compounds that undergorenal elimination. The pharmacological implications ofPEPT1 and PEPT2 function may be to mediate theintestinal absorption and renal reabsorption of drugs,respectively.

Both PEPT isoforms diverge in their substrate affinityand transport capacity but share substrate specificity,transport mechanisms, and are exclusively located atapical membranes (Daniel and Adibi, 1993; Ganapathyet al., 1995; Liang et al., 1995; Leibach and Ganapathy,1996; Saito et al., 1996; Lin et al., 1999). The mechanism


for the exclusive apical localization of the PEPTs in theintestine and kidney is not clear. However, PEPT1(-QKQM) contains a class III PDZ ligand, whereasPEPT2 (-KTKL) contains a class I PDZ-ligand and bothPEPT1 and PEPT2 interact with PDZ proteins (Katoet al., 2004). Localization of PEPT1 at apical membranesin enterocytes from nherf32/2 mice is almost completelyabsent, indicating NHERF3 may play a role in PEPT1regulation (Sugiura et al., 2008). Although multiplePDZ proteins have been shown to influence PEPT2function, the role of PDZ proteins on PEPT1 functionis less understood (Kato et al., 2004; Noshiro et al.,2006; Boehmer et al., 2008). These observations suggestthat PDZ regulation of drug transporter membraneabundance may not solely depend on the presence ofa class I PDZ-ligand. This may indicate that PDZ-drugtransporter interactions are mediated through otherbinding motifs or facilitated by other cytoplasmic adapterproteins. A detailed analysis on the differences in PDZregulation of PEPT1 and PEPT2 in the intestine andkidney, respectively, will address this disparity and mayprovide important clues lending to the complexity ofPDZ function.

B. ATP Binding-Cassette Drug Transporters

The ATP binding cassette (ABC) superfamily repre-sents the largest family of membrane transporters,comprising 49 members divided into 7 subfamilies(ABCA–ABCG) (Table 2). Members of subfamilies B, C,and G are the best studied in the context of drug trans-port and disposition. Specifically, the multidrug resistanceprotein (MDR1, ABCB1), multidrug resistance-associatedproteins (MRPs, ABCCs), and breast cancer resis-tance protein (BCRP, ABCG2) are clinically significantmediators of multidrug resistance and response (Giacominiet al., 2010; Zamek-Gliszczynski et al., 2012; Hillgrenet al., 2013). Drug resistance is a clinical problem in thetreatment of many types of cancer. Resistance to severalanticancer agents is caused, in part, by increased ABCdrug transporter function (Kathawala et al., 2015). Intumors, ABC drug transporters actively extrude anti-cancer agents and prevent intracellular drug accumu-lation, resulting in poor drug response (Kim et al.,2015). In a similar manner, ABC drug transporters limitthe systemic exposure of drugs and drug metabolites bypreventing drug absorption and promoting drug elimi-nation at anatomic barriers such as intestinal, hepatic,and renal epithelia, tissues where ABC drug trans-porter expression is high. The mechanism of transport issimilar among all members in that they require ATPhydrolysis to energize cellular drug efflux. Most membersshare a membrane topology consisting of 12 hydrophobicTMDs that are divided into two distinct clusters, eachcontaining six TMDs, and each with its own nucleotide-binding domain (NBD) (Fig. 3). MRP1, MRP2, MRP3,MRP6, and MRP7 harbor an additional cluster consist-ing of five TMDs near the N terminus (Fig. 3) (Choi and

Yu, 2014). BCRP is unique among the ABC family inthat it contains only six TMDs and one NBD (Fig. 3).For that reason BCRP is considered a “half-transporter.”ABC drug transporters expressed in the intestine, liver,and kidney mediate the cellular efflux of a vast arrayof structurally diverse drugs from several drug classes,including anticancer drugs, antibiotics, statins, andhuman immonodeficiency virus protease inhibitors,among many others (Cascorbi, 2011; Keppler, 2011;Mao and Unadkat, 2015). ABC drug transporters arealso the molecular basis for clinically significant drug-drug interactions, which lead to changes in serum drugconcentrations and consequently drug response (Koniget al., 2013). To limit drug exposure by promoting drugelimination in polarized epithelia, ABC drug trans-porters must be targeted to the membrane domain thatcomplements this function, which in many cases is theapical membrane facing a luminal space. ABC drugtransporters facilitate drug extrusion into bile (fromhepatocytes), urine (from renal proximal tubule cells),and the intestinal lumen (from enterocytes). Tissueexpression and membrane localization of selected ABCdrug transporters is summarized in Fig. 1. ABC drugtransporters and a list of selected substrates are sum-marized in Table 2.

1. Multidrug Resistance Protein 1 (ABCB1). TheABCB1 gene encodes MDR1, commonly referred to asP-glycoprotein (Pgp). Pgp was one of the first membersof ABC drug transporters to be identified and has beenwell studied since it was first cloned and characterizednearly four decades ago (Juliano and Ling, 1976). Pgp isextensively distributed throughout the body with high-est expression in epithelial cells with excretory rolessuch as intestinal, hepatic, and renal epithelia (Thiebautet al., 1987). It is also expressed in epithelial cells liningthe pancreatic ducts, as well as in endothelial cells ofthe blood-brain barrier (Thiebaut et al., 1987; Beaulieuet al., 1997). Pgp specifically localizes to apical mem-branes. The location of Pgp at the luminal surfacesupports its physiologic role to protect susceptible organsfrom a broad array of exogenous compounds and envi-ronmental toxins, including an equally diverse list ofdrugs and drug metabolites (Padowski and Pollack,2010; Staud et al., 2010). Pgp actively extrudes intra-cellular substrates in an ATP-dependent manner andprevents tissue accumulation by promoting excretion.Pgp substrates include multiple drugs from structurallydiverse classes such as immunosuppressants, antibiot-ics, statins, b-blockers, anticancer drugs, and the cardiacglycoside digoxin, among many others (Fromm, 2004;Cascorbi and Haenisch, 2010; Cascorbi, 2011).

Pgp is limited to apical membranes. The mechanismof its specific membrane domain localization is unknown.Pgp does not harbor a C-terminal PDZ ligand, and thereis no evidence for interaction with PDZ proteins. Its ho-molog, the cholesterol efflux regulatory protein (ABCA1),contains a PDZ ligand (-ESYV) and is regulated by PDZ

664 Walsh et al.

proteins (Okuhira et al., 2010). Although Pgp appearsnot to interact with PDZ proteins directly, radixin, acytoskeletal adapter protein that interacts with PDZproteins, plays an important role in regulating intesti-nal Pgp localization and function (Yano et al., 2013).This implies that PDZ proteins may be involved in reg-ulating Pgp localization indirectly via interactions withradixin or other cytoskeletal adapter proteins such asezrin or moesin. Regulation of Pgp function secondary tomembrane abundance and localization is not well stud-ied and may be another example of higher order com-plexity regarding PDZ function.Pgp is widely acknowledged as a clinically significant

mediator of pharmacokinetics and pharmacodynamicsbecause of its high expression in tissues important fordrug disposition and well established list of substratesand inhibitors (Giacomini et al., 2010). Pgp is impli-cated in several clinically significant drug-drug inter-actions. The quindine-digoxin interaction is a classicexample of a Pgp mediated drug-drug interaction (Biggerand Leahey, 1982). The antiarrhythmic drug quinidine isan established Pgp inhibitor. When coadministered withthe cardiac glycoside digoxin, a Pgp substrate, patientsare at risk for digoxin-induced toxicity, including thedevelopment of cardiac arrhythmias (Gessman et al.,1983). This adverse drug event is partially attributed toan increase in the systemic exposure of digoxin secondaryto impaired Pgp-dependent renal elimination (De Lannoyet al., 1992). Other clinically significant Pgp interactionshave been documented (Aszalos, 2007). For these rea-sons, regulatory agencies such as the Food and DrugAdministration and European Medicines Agency suggest

that all new drug candidates be screened for in vitro Pgpsubstrate and inhibition liability.

2. Multidrug Resistance Proteins (ABCCs). TheMRP family of drug transporters is encoded by theABCC gene subfamily. The MRP family consists of 9proteins (MRP1–9). Analogous to other members ofthe ABC superfamily of drug transporters, MRPs medi-ate cellular drug efflux and play a central role in pro-tecting tissues from accumulation of toxic compounds.MRPs are also responsible for transporting several en-dogenous physiologic molecules, illustrating that theirfunction is not purely pharmacological. For example,MRP1 mediates the transport of reduced and oxidizedglutathione and the proinflammatory mediator cys-teinyl leukotriene C4 (Cole, 2014). The physiologic roleof MRP4 is to regulate intracellular concentrations ofcyclic nucleotides, such as cAMP and cGMP, by func-tioning as a nucleotide efflux transporter. MRP4 regu-lation of cAMPwas recently implicated in the mechanismof drug-induced diarrhea and in fibroblast cell migration(Sinha et al., 2013; Moon et al., 2015). In addition to theirpharmacological role as mediators of drug disposition,dysfunctional MRPs are responsible for the developmentof pathophysiological conditions. For example, sequencevariations that result in loss of functional MRP2 andMRP6 are the molecular basis for Dubin-Johnson syn-drome (DJS) and pseudoxanthoma elasticum (PXE),respectively (Kartenbeck et al., 1996; Toh et al., 1999;Bergen et al., 2007). DJS is characterized by dysfunc-tional hepatic secretion of conjugated bilirubin into thebile. The loss of functional MRP2 on the apical/canalicularmembrane of hepatocytes impairs biliary excretion of

Fig. 3. ABC drug transporter membrane topology. The membrane topology of ABC family drug transporters is not uniform but differs in the number ofTMDs and NBDs. Red cylinders represent predicted transmembrane helices, and NBDs are depicted as yellow ovals for the ABC proteins indicated onthe right, along with their corresponding human gene identification.


conjugated bilirubin, which results in elevated serumconcentrations of bilirubin and the development of DJS(Devgun et al., 2012). PXE is characterized by miner-alization and eventual degradation of elastic fibers inconnective tissue, which can lead to loss of vascular toneand premature arteriosclerosis (Li et al., 2009). Althoughthe molecular pathologies of PXE are still unclear,mutations in MRP6 cause PXE (Bergen et al., 2007).The physiologic MRP6 substrates that are involved inthe development of PXE remain unknown. In accor-dance with their functional role, MRPs are prominentlyexpressed at anatomic barriers, with high expression inthe intestine, liver, kidney, and blood-brain barrier.The MRPs display overlapping substrate specificity

for many endogenous and exogenous lipophilic organicanions. MRP drug substrates include statins, angioten-sin II receptor antagonists, antiviral drugs, and chemo-therapeutic agents (Keppler, 2011). Within the MRPfamily, MRP2 and MRP4 are deemed the most clinicallysignificant to drug development because of their estab-lished influence on drug disposition and expandinglist of drug substrates (Giacomini et al., 2010; Zamek-Gliszczynski et al., 2012; Hillgren et al., 2013). Severalcommonly used medications are substrates for MRP2dependent transport including methotrexate, olmesar-tan, and etoposide (Gerk and Vore, 2002). In the kidney,MRP4 plays a principal role in the renal luminal effluxof acyclic nucleotide reverse transcriptase inhibitorssuch as adefovir and tenofovir (Imaoka et al., 2007). Animportant clinical limitation to the usage of adefovir andtenofovir is the development of nephrotoxicity, which hasbeen proposed to be partially attributable to a reductionin MRP4-dependent renal secretion (Izzedine et al.,2005). MRP2 and MRP4 seem to be responsible fordrug transport, whereas other members of the MRPfamily are involved in transport of endogenous sub-strates. Although both MRP2 and MRP4 are expressedin the liver and kidney, MRP2 is considered the majorhepatic isoform, whereas MRP4 predominates in thekidney. Interestingly, both hepatic and renal MRP2localize to apical membranes, whereas kidney MRP4is found at apical membranes but at basolateral surfacesin hepatocytes. The mechanism responsible for the dif-ference in membrane localization of MRP4 in the liverversus the kidney is not known. Both MRP2 (-STKF)and MPR4 (-ETAL) contain C-terminal PDZ ligands.Multiple PDZ proteins regulate MRP2 and MPR4 func-tion secondary to preservation of membrane abundanceand organization of membrane localization (Hoqueet al., 2009; Karvar et al., 2014; Park et al., 2014). Inaddition, members of the ezrin/radixin/moesin/merlin(ERM) family have been shown to regulate MRP2 func-tion. Radixin selectively modulates the functional ex-pression of hepatic MRP2 but not other MRP isoforms(Kikuchi et al., 2002; He et al., 2012). Other studiesshow that both radixin and ezrin are required for MRP2apical membrane localization (Yang et al., 2007). The

role of radixin and ezrin in targeting MRP2 to apicalmembranes is believed to be secondary to preventinginternalization (Yang et al., 2007; Kojima et al., 2008;Rost et al., 2008; Saeki et al., 2011). Considering PDZproteins are known to interact with the ERM family,these observations may indicate that MRPs are subjectto tissue-specific PDZ regulation dictated by distinctpatterns of tissue expression and colocalization of PDZproteins with selected ERM proteins. Therefore, tissue-specific ERM proteins may influence the nonredundantphysiologic and pharmacological roles of PDZ proteinsin different tissues. Determining how PDZ proteinscooperate with cytoskeletal adapter proteins to regulatemembrane abundance, localization, and function of tar-get proteins in different tissues, such as drug trans-porters, may address these issues.

3. Breast Cancer Resistance Protein (ABCG2).Breast cancer resistance protein (BCRP) is the secondmember of the G subfamily of ABC drug transportersand is encoded by ABCG2. BCRP was originally iden-tified in several breast cancer cell lines, where it confersresistance to multiple anticancer drugs (Dietel et al.,1990; Taylor et al., 1991; Nakagawa et al., 1992; Futscheret al., 1994; Kellner et al., 1997). It was later cloned,characterized, and assigned to the G subfamily of ABCdrug transporters (Doyle et al., 1998). BCRP containsonly six TMDs and one NBD and thus is considered ahalf transporter because it diverges from the prototyp-ical membrane topology of other ABC drug transporters.Recent evidence shows that BCRP forms homodimers orhomooligomers in intact cells (Ni et al., 2010). Covalentlinkages mediated by cysteine residues in the third ex-tracellular loop are believed to be responsible for BCRPdimerization (Shigeta et al., 2010). However, dimeriza-tion is not a requirement for BCRP function (Kage et al.,2005; Shigeta et al., 2010). The expression pattern ofBCRP complements its role as an efflux drug trans-porter. BCRP is highly expressed at the luminal surfaceof intestinal and hepatic epithelia where it attenuatesintestinal drug absorption and promotes biliary drugexcretion (Hillgren et al., 2013). The highest expressionof BCRP is detected in placental tissue, specifically onapical syncytiotrophoblast membranes. Other tissueswith high expression of BCRP include the blood-brainbarrier, testis, and lactating mammary tissue (Janiet al., 2014). In the kidney, BCRP localizes to apicalmembranes of proximal tubule epithelial cells; however,its level of expression is less compared with the intestineand liver (Mao and Unadkat, 2015). BCRP transports abroad array of both endogenous and exogenous com-pounds, including several marketed drugs such asanticancer agents, nucleoside analogs, and statins. Alist of selected BCRP substrates is summarized inTable 2.

Recent clinical pharmacogenetic studies have em-phasized the importance of BCRP to drug dispositionand response, which has prompted the Food and Drug

666 Walsh et al.

Administration and European Medicines Agency to rec-ommend that all investigational new drugs be testedfor BCRP substrate and inhibitor liability, except forsubstrates that fall into biopharmaceutical classifica-tion system 1, drugs with high solubility and highpermeability. Loss of function BCRP polymorphismsthat result in decreased drug clearance and increaseddrug exposure and toxicity have been reported. For ex-ample, in carriers of BCRP polymorphisms, the sys-temic exposure of several drugs such as atorvastatin,rosuvastatin, sunitinib, and sulfasalazine is signifi-cantly increased (Mizuno et al., 2012; Giacomini et al.,2013; Lee et al., 2015). In the case of sunitinib, carriersof BCRP polymorphisms also experience a higher inci-dence of sunitinib-induced toxicity (Mizuno et al., 2012).In addition to altering pharmacokinetics and pharma-codynamics, BCRP polymorphisms are associated withdisease states such as Alzheimer’s disease and gout(Matsuo et al., 2011b; Feher et al., 2013; Takada et al.,2014). The association between BCRP and these diseasestates is hypothesized to be secondary to BCRP de-pendent handling of amyloid beta in Alzheimer’s diseaseand uric acid in patients with gout (Matsuo et al., 2011a,b;Abuznait and Kaddoumi, 2012).BCRP membrane location in polarized cells is re-

stricted to apical membranes. The mechanism thatunderlies the restricted apical membrane localization ofBCRP has yet to be defined. Despite the fact that BCRP(-KKYS) does not harbor a PDZ ligand, NHERF3 regu-lates intestinal BCRP function and plays an importantrole in localizing BCRP to apical membranes in enter-ocytes (Shimizu et al., 2011). NHERF3 also regulatesintestinal PEPT1, which harbors a class III PDZ ligand(-QKQM) (Sugiura et al., 2008; Shimizu et al., 2011).These observations suggest that the interaction be-tween NHERF3 and BCRP differs from those of otherdrug transporters, which interact with NHERFs via acanonical PDZ ligand. The mechanism of NHERF3regulation of BCRP may involve the presence of a non-canonical PDZ-binding motif. The NHERF3-interactingbinding sequence in BCRP is unclear. Because of its ex-tensive tissue distribution, expanding list of drug sub-strates, and association with altered pharmacokineticsand pathophysiology, BCRP has been recognized as a clin-ically relevant drug transporter of emerging importance.

III. Tissue and Cellular Localization of PSD-95/Drosophila Discs Large/ZO-1 Proteins

PDZ domains are the most abundant protein-proteininteraction modules in humans (Ponting et al., 1997;Feng and Zhang, 2009). Canonical PDZ domains consistof 80- to 90-amino-acid residues that form six b-stands(bA to bF) and two a-helices (aA and aB) that arearranged in a three-dimensional globular structure(Karthikeyan et al., 2001). Binding of a protein ligand tothe PDZ domain occurs within a hydrophobic binding

pocket that is created by the bB strand, the aA helix,and the loop connecting the aA and bB strands. Thiscarboxylate-binding loop contains a GLGF core motif ora related sequence such as GYGF, as is the case withthe NHERF family of PDZ proteins (Ponting et al.,1997). PDZ domains interact with target proteins viaspecific internal or carboxy-terminal amino acid resi-dues, commonly referred to as PDZ ligands. Carboxy-terminal PDZ ligands are a short stretch of amino acidsapproximately three to four residues in length, althoughupstream sequences affect affinity and specificity ofbinding. The amino acid sequence that comprises thePDZ ligand is conventionally numbered starting fromthe last amino acid at the extreme carboxy terminusand is assigned position zero (P0).

PDZ recognition motifs are grouped into threeclasses (class I–III) according to the consensus sequenceof the PDZ ligand for which they bind. Table 1 summa-rizes the classification of PDZ ligands. Binding specific-ity among the three classes is determined, in part, bythe interaction between the first amino acid residue inthe bB-helix of the PDZ domain and the amino acid atposition two (P2) of the PDZ ligand in the target protein.There are over 150 PDZ domain-containing proteins andmore than 250 distinct PDZ domains in the humanproteome. Individual PDZ proteins may contain multi-ple PDZ domains as well as other interaction modulessuch as ezrin-binding domains (EBDs). PDZ proteinslack intrinsic catalytic activity and therefore functionprimarily as scaffold proteins, where they execute arange of biologic functions that include establishing andmaintaining cellular polarity, assembling multiproteinsignaling complexes, and anchoring transmembraneproteins, including transporters, to the actin cytoskele-ton via interactions with the ERM family of adapters(Ponting et al., 1997; Wang et al., 2007, 2010, 2012;Georgescu et al., 2014; Zheng et al., 2014).

NHERF proteins are the most well studied PDZproteins that are expressed in polarized epithelia. TheNHERF family consists of four structurally relatedproteins (NHERF1–4) (Fig. 4). NHERF1 and NHERF2contain two tandem nonidentical PDZ domains and anEBD, whereas NHERF3 and NHERF4 have four PDZdomains but lack an EBD (Seidler et al., 2009). NHERFproteins play an important role in organizing signalingcomplexes, controlling apical membrane trafficking, andcoupling apical membrane transporters and receptorswith other PDZ targets (Wang et al., 2007; Klenk et al.,2010; Georgescu et al., 2014). Although NHERF iso-forms share these common functional characteristics,they perform distinct physiologic functions. For exam-ple, both NHERF1 and NHERF3 are important forphosphate homeostasis, yet in nherf12/2 and nherf32/2

mice, only nherf12/2 mice exhibit prominent phosphatewasting, suggesting that NHERF1 and NHERF3 playdifferent physiologic roles (Shenolikar et al., 2002;Kocher et al., 2003; Giral et al., 2011). NHERF proteins


also exhibit differential tissue expression. NHERF1 andNHERF2, for instance, are abundantly expressed andwidely distributed, with NHERF2 displaying a morerestricted tissue distribution compared with NHERF1.NHERF1 is highly expressed on the apical aspect ofpolarized epithelia in the kidney, liver, and placenta(Reczek et al., 1997). NHERF1 also is found in regions ofthe gastrointestinal tract (small intestine and colon),gastric parietal cells, and brain (Weinman et al., 1995;Reczek et al., 1997). Although NHERF2 is coexpressedwith NHERF1 in the kidney, its distribution within thekidney differs. NHERF1 is found in proximal tubules,whereas NHERF2 is expressed in the glomeruli, collect-ing duct, and throughout the renal vasculature and isabsent from the proximal tubule. The highest expres-sion of NHERF2 is in lung, including pulmonary alveoli.NHERF3 is expressed at the brush-border membrane ofproximal tubule cells, the intestinal epithelium, and isalso found in the liver (Custer et al., 1997; Kocher et al.,1998; Gisler et al., 2001). NHERF1 and NHERF3 pre-dominantly localize to the apical aspect of polarizedepithelia, whereas the localization of NHERF2 is vari-able, including both apical membranes and a diffusesubcellular cytosolic distribution among different tis-sues. NHERF4 represents the isoform with the mostrestricted tissue distribution. Significant levels ofNHERF4 are found in the kidney and gastrointestinaltract. Within these tissues, NHERF4 is localized to theapical surface and subapical regions throughout thecytoplasm (Gisler et al., 2001; Donowitz et al., 2005).NHERF proteins can form homo- and heterodimers

or undergo oligomerization. Dimerization is thought tobe mediated either by intermolecular interactionsbetween PDZ domains or by an intramolecular head-to-tail interaction between the carboxy terminus and aPDZ domain within the same protein, as is the case with

NHERF1 (Lau and Hall, 2001; Shenolikar et al., 2001;Morales et al., 2007). Head-to-tail dimerization ofNHERF1 is mediated by the interaction between thecarboxy terminus, which itself serves as a PDZ-ligand(-FSNL), with PDZ2 (Morales et al., 2007; Wang et al.,2012). Although the biologic significance of NHERF di-merization is not fully appreciated, a recent study showedthat heterodimerization of NHERF2 and NHERF3 isrequired for the inhibition of Na+/H+ exchanger-3 bycarbachol (Yang et al., 2014). The formation of a largescaffolding complex comprising multiple NHERF iso-forms because of dimerization may give rise to an elabo-rate network of cytoplasmic scaffold proteins. Formationof such a complex may dictate unique tissue-specificNHERF function. Elucidating the physiologic impor-tance of NHERF dimerization as it relates to functionmay aid in our understanding of the in vivo roles ofthe NHERF family.

Despite the structural similarities in PDZ domainsamong the NHERF family, target proteins that harborPDZ ligands, including drug transporters, display dif-ferent affinities for NHERF isoforms (Hoque and Cole,2008; Wang et al., 2010; Park et al., 2014). Such differ-ences in affinity may partially explain the nonredun-dant physiologic functions of the NHERF family, as wellas the fact that several tissues express multiple NHERFisoforms (Voltz et al., 2001). Additionally, because of thefact that NHERF proteins can form homo- and hetero-dimers, some NHERF isoforms may operate indepen-dently, whereas other physiologic processes may requireNHERF cooperativity. In the latter scenario, NHERFproteins would bind to target proteins directly via onePDZ domain, reserving the remaining PDZ domain(s) tointeract with other NHERF isoforms that in turn areresponsible for anchoring the entire multiprotein com-plex to the actin cytoskeleton through interactions with

Fig. 4. Schematic representation of human NHERF isoforms. PDZ domains are indicated by the light blue rectangular boxes and EBDs by dark blueboxes. The amino acid residues that constitute the core binding-motif are noted and shown as a purple bar indicating their location within therespective PDZ domain.

668 Walsh et al.

the ERM family. Further studies are needed to deter-mine the physiologic and potential pharmacologicalrelevance of NHERF dimerization, cooperativity, andregulation of drug transport.Given that NHERF expression parallels that of drug

transporters and that several drug transporters havebeen identified as NHERF binding partners, theseproteins constitute a novel class of post-translationalmediators of drug transport. For this reason, amongall PDZ proteins, the NHERF proteins are the mostextensively studied in the context of drug transporterregulation.

IV. Role of PSD-95/Drosophila Discs Large/ZO-1Proteins in Regulation of Cell MembraneAbundance, Localization, and Function of

Drug Transporters

The widely acknowledged clinical significance ofdrug transporters has led to a surge in studying theirregulatory mechanisms. Recently, the mechanismsthat underlie drug transporter membrane organizationand abundance have gained considerable interest. Tocoordinate the vectorial movement of drugs acrosspolarized epithelia, drug transporters must be sortedand stabilized to the appropriate membrane domain.By virtue of the fact that drug transporters displayunique tissue-specific apical or basolateral membranelocalization, domain organization is not random. If themechanisms that dictate membrane organization andstability are faulty, drug transporters may localize tothe wrongmembrane domain or display decreased mem-brane abundance. The consequences of faulty membranetargeting could result in an overall reduction in transporterfunction, which may then alter the pharmacokinet-ics and pharmacodynamics of drugs that use trans-porter pathways for elimination. The mechanism of

NHERF1-dependent renal phosphate excretion, sec-ondary to its role in stabilizing the sodium phosphatecotransporter-2a (NaPi-2a) to the apical membrane inrenal proximal tubule cells, best illustrates this. Inthe absence of NHERF1, the membrane abundanceof NaPi-2a decreases, preventing phosphate reab-sorption from the glomerular filtrate and thus pro-moting renal phosphate excretion (Shenolikar et al.,2002). Exploring NHERF in the setting of drug transportmay address disparities surrounding drug transporterfunction and drug response. Emerging evidence hasshown that PDZ proteins, namely the NHERF family,play a critical role in the localization, stabilization, andfunctional regulation of select SLC and ABC drugtransporters (Table 3) (Sugiura et al., 2011).

A. Solute Carrier Drug Transporters

1. Organic Carnitine Transporters. Although themolecular mechanisms of membrane targeting of theOCT and OCTN family are not well characterized,NHERF2, NHERF3, and NHERF4 have been shown tointeract with and/or directly regulate OCTN2 functionvia modulating membrane abundance (Kato et al.,2005; Sugiura et al., 2006, 2008; Watanabe et al., 2006).A pull-down study using recombinant C-terminal pro-teins identified an interaction between apically localizedOCTN1 and OCTN2 with both NHERF2 and NHERF3but not with OCT1 and OCT2, which in contrast, do notharbor PDZ ligands and exclusively localize to baso-lateral membranes (Kato et al., 2005). Additional studiesrevealed that the interaction required the presence ofthe last four amino acids of OCTN1 and OCTN2 (-LTAFand -STAF, respectively), both of which are class I PDZligands. The interaction of NHERF3 with the C terminusof OCTN2 was confirmed in a pull-down study usingkidney brush-border membrane vesicles. Subsequentimmunohistochemical analysis revealed that NHERF3

TABLE 3NHERF-dependent regulation of drug transporter function

Uppercase denotes human genes or gene products and lowercase denotes rodent genes or gene products.

Transporter NHERF Isoform ExperimentalModel SubstrateTransporterFunction Reference

OCTN2 NHERF3 NHERF4 HEK293 Carnitine Increase Kato et al., 2005Watanabe et al., 2006

Octn2 Nherf3 Nherf32/2 mice Carnitine Decrease Sugiura et al., 2008OAT4 NHERF1 NHERF3 HEK293 Estrone-3-sulfate Increase Miyazaki et al., 2005

LLC-PK1 Zhou et al., 2008URAT1 NHERF3 HEK293 Urate Increase Anzai et al., 2004OATP1A2 NHERF1 NHERF3 HEK293 Estrone-3-sulfate Increase Zheng et al., 2014Oatp1a1 Nherf3 Nherf32/2 mice Estrone-3-sulfate Increase Sugiura et al., 2010PEPT2 NHERF2 Xenopus oocytes Gly-Gly Increase Boehmer et al., 2008PEPT2 NHERF3 HEK293 Gly-Sar Increase Noshiro et al., 2006

Sugiura et al., 2006Pept1 Nherf3 Nherf32/2 mice Cephalexin Decrease Sugiura et al., 2008MRP2 NHERF1 WIF-B CMFDA Increase Karvar et al., 2014MRP4 NHERF1 HeLa 6-Mercaptopurine, 9-[2-(phosphonylmethoxy)

ethyl]-adenineDecrease Hoque and Cole, 2008

MRP4 NHERF3 HEK293 Adefovir Increase Park et al., 2014Mrp4 Nherf3 Nherf32/2 mice Adefovir Decrease Park et al., 2014Bcrp Nherf3 Nherf32/2 Cimetidine Decrease Shimizu et al., 2011

CMFDA, 5-chloromethylfluorescein diacetate.


and OCTN2 colocalize in mouse kidney brush bordermembranes. Furthermore, NHERF3 increased OCTN2mediated uptake of carnitine, a well established OCTN2probe substrate. This result could be explained by a6-fold increase in carnitine transport capacity in vitro.Modulation of OCTN2 function by NHERF3 was notobserved upon deletion of the last four amino acids ofOCTN2 (-STAF), supporting that these two proteinsinteract directly via the PDZ ligand of OCTN2 (Katoet al., 2005). An in vivo study performed in nherf32/2

mice found that absorption of carnitine was decreasedcompared with wild-type mice (Sugiura et al., 2008).Immunohistochemical analysis revealed that the apicallocalization of intestinal Octn2 was reduced in nherf32/2

mice compared with wild-type mice (Sugiura et al., 2008).Collectively, these studies suggest a role for NHERF3 inregulating OCTN2 membrane abundance and functionin the kidney and intestine. NHERF2 may have asimilar role.Coexpression of OCTN2 and NHERF4 in HEK293

cells also increased the uptake of carnitine (Watanabeet al., 2006). Subsequent kinetic analysis revealed a2-fold increase in transport capacity but a minimal effecton carnitine specificity for OCTN2, suggesting that theincrease in OCTN2 function was not due to changes incarnitine affinity but rather a greater number of OCTNsinvolved in transport. Furthermore, upon deletion of thelast four amino acids in OCTN2 (-STAF), the effect ofNHERF4 on OCTN2 was abolished (Watanabe et al.,2006), signifying that the mechanism is dependent onthe presence of the PDZ ligand. However, these re-sults were not observed when NHERF4 was coexpressedwith OCT3 or OCTN1, despite the fact that both OCT3and OCTN1 harbor class I PDZ ligands (-RSHL and-LTAF, respectively). These data indicate that NHERF4might operate by a mechanism that is reliant on struc-tural characteristics other than the existence of a PDZligand alone.Taken together, NHERF proteins may differentially

modulate OCTN function secondary to preservation ofmembrane abundance. These data also point to an un-characterized NHERF mechanism that operates in-dependent of a PDZ ligand. This hints to involvementof other cytoplasmic adaptor/scaffold proteins or non-canonical binding motifs affecting NHERF bindingaffinities and thus function. Given that OCTNs havedescribed roles in pharmacokinetics, pharmacodynam-ics, and pathophysiology, characterizing NHERF-mediated OCTN function may lead to a more detailedmechanistic explanation of the pharmacological andpathophysiological role of OCTNs.2. Organic Anion Transporters. The OATs play

clinically important roles in the disposition of severalpharmacologically active compounds as well as manyendogenous substances. OAT4 and URAT1 are highlyexpressed in the kidney and are exclusively found atapical membranes, whereas other renal OAT isoforms

(OAT1–3) are found at the basolateral membrane(Burckhardt, 2012). Among the OAT family, OAT4 andURAT1 contain class I PDZ ligands (-STSL and -STQF,respectively) and have been shown to be binding part-ners with NHERF1 and NHERF3. Evidence suggeststhat NHERF1 and NHERF3 play a role in regulatingOAT4 and URAT1 function (Anzai et al., 2004; Miyazakiet al., 2005; Zhou et al., 2008; Zhang et al., 2010a). Yeasttwo-hybrid studies and surface plasmon resonanceconfirmed OAT4 binds both NHERF1 and NHERF3 andthat the interaction is mediated by the C-terminalregion of OAT4 and the PDZ1 domain of NHERF1 andboth PDZ1 and PDZ4 domains in NHERF3 (Miyazakiet al., 2005). The association of OAT4 with NHERF1and NHERF3 depends on the presence of the C-terminalPDZ ligand of OAT4 (-STSL). Furthermore, association ofOAT4 with NHERF1 or NHERF3 enhanced transport ofestrone-3-sulfate, an established OAT4 probe substrate,in HEK293 cells. Upon deletion of the OAT4 PDZ ligand,this functional effect was abolished (Miyazaki et al.,2005). These data indicate that NHERF1 and NHERF3may affect OAT4 function through interactions medi-ated by specific PDZ domains.

The effect of NHERF proteins on OAT4 function maybe tissue specific (Zhou et al., 2008). In a comparison ofNHERF-mediated OAT4 function in LLC-PK1 versusBeWo cells, a kidney and placental cell line, respec-tively, NHERF1 and NHERF3 only enhanced OAT4-dependent estrone-3-sulfate uptake in LLC-PK1 cellsbut not BeWo cells. This suggests that the effect ofNHERF1 and NHERF3 may be cell or tissue specific.These results further support the hypothesis that NHERFproteins display tissue-specific function (Zhou et al., 2008).To evaluate the mechanism of NHERF1-mediated OAT4regulation, Zhang et al. (2010a) showed that proteinkinase C (PKC), which has previously been shown toinhibit NHERF1 function via phosphorylation, down-regulates OAT4 activity. Further studies in COS-7 cellsrevealed that NHERF1 attenuates OAT4 internalization(Zhang et al., 2010a). These studies demonstrate thatthe mechanism of NHERF1-mediated OAT4 regulationmay be secondary to preventing OAT4 internalization.

URAT1 has also been studied in the context of NHERF-mediated regulation. Similar to OAT4, the C-terminalPDZ ligand of URAT1 (-STQF) is required for interac-tion with NHERF3 and for NHERF3-dependent func-tional regulation (Anzai et al., 2004). In HEK293 cells,urate transport was increased by NHERF3, an effectthat was abolished upon deletion of the URAT1 C-terminalPDZ ligand. Kinetic analysis revealed that this effect wasdue to an increase in the capacity of urate transportrather than a change in urate specificity for URAT1.Subsequent coimmunoprecipitation studies showedthat URAT1 colocalizes with NHERF3 at the apicalbrush border membrane in renal proximal tubulecells and the presence of the URAT1 PDZ ligand isrequired for this interaction (Anzai et al., 2004).

670 Walsh et al.

Together, these studies highlight an important rolefor NHERF1 and NHERF3 in regulating OAT4 andURAT1 function. Although both transporters have beenproposed to play important pharmacological roles, theyalso have been shown to mediate the renal secretion andreabsorption of uric acid (Graessler et al., 2006; Sakiyamaet al., 2014). This implies that in addition to potentialeffects on renal drug secretion, NHERF proteins may alsoinfluence uric acid homeostasis and may have anunderlying role in the pathophysiology of gout.3. Organic Anion-Transporting Polypeptides. Five

members of the OATP family harbor a class I PDZligand at their C terminus (Table 2). Evolutionarily, theOATPs are poorly conserved and orthologs in rodentsmay not exist in humans, making findings from studiesperformed in animal models difficult to translate clin-ically. Although multiple studies indicate that NHERFproteins play a role in regulating rodent Oatps (Wanget al., 2005, 2014; Sugiura et al., 2010; Choi et al., 2011),there is little information regarding how NHERF pro-teins affect human OATP isoforms.OATP1A2 was the first identified human OATP

isoform and has since been well studied and found toplay an important role in the cellular uptake of avariety of drugs and endogenous substances. Recently,NHERF1 and NHERF3 were found to regulate OATP1A2function by modulating protein internalization andenhancing membrane stability. The influence of NHERF1and NHERF3 on OATP1A2 function was assessed usingHEK293 cells that coexpressed NHERF1 or NHERF3(Zheng et al., 2014). Similar to studies in OATs, estrone-3-sulfate uptake was used as a surrogate for OATP1A2function. Both NHERF1 and NHERF3 were found tosignificantly enhance E1S uptake. Overexpression ofNHERF1 or NHERF3 leads to an increase in OATP1A2membrane abundance and was proposed to be theunderlying basis for the observed increase in OATP1A2function. Subsequent studies revealed that the mecha-nism for the apparent increase in OATP1A2 membraneabundance was due to a decrease in its internalizationvia a clathrin-dependent, but caveolin-independent,manner (Zheng et al., 2014).NHERF proteins may have similar effects on the

other human OATPs that contain C-terminal PDZligands (Table 2). Particular attention should be givento OATP2B1 because of its established functional role inthe intestine as an uptake transporter for several drugsincluding fexofenadine and statins. OATP2B1 alsouniquely displays dual-membrane specificity, apical inenterocytes and basolateral in hepatocytes (Fig. 1). Thismay indicate that if NHERF proteins are involved inregulating OATP2B1, they may do so differently in theintestine than in the liver. Investigating the mechanismthat determines intestinal versus hepatic OATP2B1membrane targeting may provide clues on how NHERFproteins, or other PDZ proteins, function in differenttissues.

4. Peptide Transporters. PEPT1 and PEPT2 arerestricted to apical membranes in the intestine andkidney. Recent evidence shows that NHERF2 andNHERF3 interact with PEPT2 and enhance transportactivity by preserving PEPT2 membrane abundance(Kato et al., 2004; Noshiro et al., 2006; Sugiura et al.,2006; Boehmer et al., 2008). A preliminary screeningof several drug transporters and PDZ proteins identifieda potent interaction between PEPT2 and NHERF3(Kato et al., 2004). Studies using yeast two-hybridassays and surface plasmon resonance confirmed theinteraction between PEPT2 and NHERF3 is mediatedby the PDZ2 and PDZ3 domains (Noshiro et al., 2006).The influence of NHERF3 on PEPT2 transport wassubsequently assessed in HEK293 cells using Gly-Saruptake as a surrogate of PEPT2 function (Noshiro et al.,2006; Sugiura et al., 2006). Coexpression of NHERF3 inHEK293 cells augmented PEPT2 function and wasassociated with an increase in transport capacity.Coexpression with NHERF3 increased PEPT2 mem-brane abundance and was proposed to account for theincrease in function. Sugiura et al. (2006) later evalu-ated PEPT2 by assessing Gly-Sar uptake in HEK293cells stably expressing NHERF3 constructs with a mu-tation in the PDZ2 domain. Although HEK293 cellsoverexpressing the NHERF3 mutant displayed a de-crease in Gly-Sar uptake compared with those overex-pressed with wild-type NHERF3, the results were notsignificant. Identifying the effects of genetic poly-morphisms in NHERF proteins on drug transportmay help clarify which drug transporters are subject toNHERF regulation. Revealing which PDZ domains areimportant for drug transporter regulation also may pro-vide detail on the structural requirements of NHERFfunction.

Despite similarities in structure and domain organi-zation between NHERF1 and NHERF2, expressionstudies performed in Xenopus oocytes have shown thatNHERF2, but not NHERF1, enhance PEPT2membraneabundance and function. This is another example ofNHERF target-proteins displaying differential bindingaffinities for NHERF isoforms. The effect of NHERF2 onPEPT2 was not observed upon deletion of the C-terminalPDZ ligand of PEPT2 (-KTKL), suggesting that thePEPT2 PDZ ligand is a requirement for interaction withNHERF2 (Boehmer et al., 2008). Why NHERF1 doesnot produce similar effects, despite its similarity withNHERF2, is uncertain.

Collectively, these studies indicate that NHERFproteins modulate PEPT2 function and therefore mayplay important physiologic and pharmacological roles inoligopeptide and peptide-like drug transport in thekidney. However, several questions remain regard-ing the regulation of PEPT membrane targeting. Forinstance, it is unclear why PEPT2 preferentially bindsNHERF2 and NHERF3, but not NHERF1, especiallyconsidering NHERF1 and NHERF3 are highly expressed


in the kidney, where PEPT2 is also prominently ex-pressed. A closer inspection of PEPT2 regulation byNHERF proteins in the kidney may reveal cooperativeor opposing roles for NHERF1 and NHERF3. Thisuncertainty is an opportunity to flesh out details regard-ing regulation mediated by multiple NHERF isoforms.Alternatively, NHERFs might regulate PEPT transportindirectly. NHERF3 may regulate PEPT1 as described(Kato et al., 2006). PDZ proteins, such as NHERFs, mayinfluence drug transport indirectly by regulating pro-teins that are responsible for producing electrochemicalgradients necessary for dissipative transport. For ex-ample, PEPT1 utilizes an inwardly directed H+ gradientthat is produced by the Na+/H+ exchanger (NHE) toprovide the driving force for substrate uptake. NHERFsmay indirectly regulate PEPT1 by directly regulatingNHE or recruiting NHE in close proximity to PEPT1 toensure an adequate H+ gradient is achieved. Discover-ing the nature of the functional coupling of PEPT1 andNHE by NHERFs, as well as differences in NHERF-dependent PEPT membrane targeting, may broadenour understanding of NHERF action and drug trans-port. These studies will aid in understanding howPEPTs are organized at cell membranes and lead to newdiscoveries regarding how PDZ proteins, includingNHERF proteins, interact with each other to performnonredundant physiologic functions in different tissues.

B. ATP Binding-Cassette Drug Transporters

1. Multidrug Resistance Proteins. NHERF1 andNHERF3 have been reported to regulate the membraneabundance, localization, and function of MRP2 (-STKF)and MRP4 (-ETAL), both of which contain a class IC-terminal PDZ ligand. The initial discovery thatNHERF proteins interact with MRPs resulted fromstudies that focused on elucidating the cellular mecha-nisms responsible for multidrug resistance in cancerafter it was found that NHERF3 is upregulated inselected tumors of epithelial origin (Kocher et al., 1998,1999). This prompted an investigation of potentialNHERF3 binding partners. Expression studies andyeast two-hybrid assays performed in human carci-nomas confirmed an increase in NHERF3 mRNA andrevealed that NHERF3 interacts with the C-terminalportion of MRP2 (Kocher et al., 1999). Later, it wasrevealed that MRP2 interacts with all four NHERFisoforms (NHERF1–4) and that phosphorylation ofSer1542 in the MRP2 PDZ binding motif significantlyenhanced binding to NHERF1 and NHERF4; the func-tional consequence was not assessed (Hegedus et al.,2003). Because of these findings, others have investi-gated the role of intracellular kinases, such as PKC, andmembrane localization of MRP2. For example, specificPKC isoforms, such as conventional PKCa (cPKCa),novel PKCd (nPKCd), nPKC«, and atypical PKCz(aPKCz), appear to be involved in regulating hepaticMRP2 membrane recycling at the apical/canalicular

membrane (Beuers et al., 2003; Ito et al., 2005;Schonhoff et al., 2008; Stross et al., 2009; Park et al.,2012). It is still unclear if these PKC-mediated phos-phorylation events result from direct phosphorylation ofMRP2, NHERF proteins, or members of the ERM family,such as radixin, known to interact with and influenceMRP2 function (Suda et al., 2011, 2015). Investigatingphosphorylation events important for drug transporterregulation represents an emerging line of research thatintersects with the physiologic and pharmacological roleof PDZ proteins.

To describe furthe

associate editor: paul a. insel drug transporters ......function in vitro, as well as drug...

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