MRP4

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MRP4 (multidrug resistance-associated protein 4)

Aliases: MOAT-B, MOATB
Gene name: ATP binding cassette subfamily C member 4 (ABCC4)

Summary

MRP4 is an ATP-dependent, unidirectional efflux transporter belonging to the C subfamily of the ABC protein superfamily. It is expressed in the kidney, blood-brain barrier (BBB), liver, and other tissues, and its localization to the basolateral or apical membranes is tissue-dependent. It plays an important role in alleviating the impact of cholestasis on hepatocytes by effluxing bile acids into the blood, and can be up-regulated in the liver in such instances. It may also be important in limiting CNS and hematopoietic cell exposure to xenobiotics, and as a transporter of cell signaling molecules. MRP4 has wide substrate specificity, including nucleoside analogs and antiviral drugs; however, reports of clinical DDIs due to MRP4 modulation are rare. MRP4 is highly polymorphic, and some of these polymorphisms may be clinically relevant. Current FDA and EMA guidances make no specific recommendations for MRP4. However, this transporter may be important where active renal secretion is suspected, or where changes in circulating levels of bile salts are observed.

Localization

MRP4 (recently reviewed in [1] is found in a variety of human tissues, with high levels of mRNA expression in the kidney and prostate, and lower levels in the liver, testis, ovary, lung, parotid gland, adrenal gland, and in various neurons and blood cells. MRP4 localizes to different membranes in different polarized cells. In the liver, choroid plexus, prostatic acinar cells, and the GIT, MRP4 is expressed at the basolateral membrane, while in the brain capillary endothelium and renal proximal tubule cells it is expressed at the apical membrane [2-6]. This means that, for instance, in the liver (where its abundance is relatively low) it effluxes its substrates into the bloodstream, whilst in the kidney (where it is one of the dominating ABC transporters [7]) it effluxes into the urine.

Function, physiology, and clinically significant polymorphisms

MRP4 with its 170-kDa molecular weight is the smallest among the MRP proteins. It comprises 12 putative membrane spanning helices and lacks the additional N-terminal helices found in MRP1, MRP2, MRP3, and MRP6 [8]. MRP4 mediates the transport of endogenous substrates including signaling molecules (cAMP/cGMP, eicosanoids), bile acids, urate, and conjugated steroid hormones (e.g. pregnenolone sulfate and dehydroepiandrosterone sulfate [9]). Clinical drug substrates include antivirals (adefovir, tenofovir), antibiotics (cephalosporins, benzylpenicillin), paracetamol conjugates, diuretics (furosemide, hydrochlorothiazide), the antihypertensive olmesartan, and cytotoxic agents (methotrexate, 6-thioguanine, 6-mercaptopurine, topotecan) [3, 10-12]. Inhibitors of MRP4 include non-steroidal inflammatory drugs, phosphodiesterase inhibitors, cardiovascular drugs, and flavonoids, among others [10, 13]. MRP4 plays a role in the renal excretion of organic anions and drugs [14], in the modulation of signaling pathways [15], and in the restriction of drug penetration through the blood-brain, blood-cerebrospinal fluid, and blood-testis barriers [16-18]. Basolaterally expressed MRP4 in gastric and intestinal epithelia is thought to be involved in the oral absorption of cephalosporin antibiotics, dasatinib, and potentially other orally administered drugs [5, 19].
Glutathione as a conjugation partner or co-transported substrate plays multiple and partially undefined roles in the transport mechanism of MRPs, especially of MRP2. MRP4 has long been known to require reduced glutathione for the transport of bile acids [20], but the MRP4-mediated extrusion of other substrates such as halobenzoquinones may also depend on GSH, possibly via conjugate formation [21].
Given its ability to transport important intra- and intercellular mediators such as cyclic nucleotides and eicosanoids, the physiological repertoire of MRP4 is thought to cover platelet aggregation, cell migration and proliferation, angiogenesis, and cardiomyocyte contraction. For the same reason, it is also implicated in the progression of multiple cancer types [10] including acute myeloid leukemia [22], ovarian cancer [23], and clear cell renal carcinoma [24], and the pharmacological modulation of MRP4-cAMP binding is being actively explored [25].
Along with many other xenobiotic transporters and metabolizing enzymes, the expression of MRP4 is regulated by the xenobiotic-responsive transcription factors AhR, CAR, PXR, and PPARα [18, 26]. In acute myeloid leukemia cells, MRP4 expression is also upregulated by histamine [27].
The rare PEL-negative blood group has recently been associated with a large biallelic deletion in the ABCC4 gene, resulting in an MRP4-null phenotype [28]. Cyclic nucleotide levels are normal in the red blood cells of PEL-negative individuals, suggesting compensation by other erythrocytic ABC transporters; however, platelet aggregation is impaired in the absence of functional MRP4. Albeit there is no evidence of diseases linked to single nucleotide polymorphisms in the ABCC4 gene, and an in vivo study showed no obvious abnormalities in Mrp4-null mice [29, 30], polymorphisms can alter the expression level and the transport rate of the protein, and may influence therapeutic outcomes and adverse events. Up to 2019, 12 variants with substantial clinical consequences had been identified; these SNPs affect both untranslated and coding regions of ABCC4 and result in altered transport of tenofovir, zidovudine, latanoprost, (Val)ganciclovir, methylated arsenic metabolites, bisphosphonates, cisplatin, furosemide, thioguanine drugs, methotrexate, imatinib, and cyclophosphamide [1].

Clinical significance

There are no specific citations of clinically relevant DDIs ascribed to this transporter, and there is limited information on its role in the clinical ADME of drugs. However, there is evidence of its role in the renal elimination of tenofovir, as the renal clearance of tenofovir was 15% lower and its AUC was 32% higher in ABCC4 3463G carriers compared with wild types in one study [31]. Since MRP4 also transports drugs which are used in HIV therapy, the efficacy of these drugs may be partially dependent on the expression of this transporter in T-cells [2, 8, 30, 32].
MRP4 transports bile acids in the presence of glutathione (GSH), and functions as a backup system for eliminating bile acids from hepatocytes. Although MRP4 expression in the liver is low, it can be induced by bile acids in cholestatic conditions [2, 14]; thus it plays an important compensatory role in protecting the liver from over-exposure to bile acids. The induction of MRP4 is FXR-independent and may instead be due to post-transcriptional regulation [33], as mRNA levels have been shown to remain unchanged [34].
MRP4 overexpression confers cellular resistance to nucleotide-base, nucleoside, and nucleotide analogues, as well as to certain tyrosine kinase inhibitors [35] and cytotoxic agents, therefore adversely affecting anticancer therapies. Overexpression of MRP4 confers doxorubicin resistance to osteosarcoma cells [36], and high MRP4 activity may be a determinant of resistance to arsenic-based chemotherapy regimens applied in leukaemia patients [37]. High MRP4 expression was also associated with diminished response to methotrexate in childhood acute lymphoblastic leukemia [38]. Therefore, selective MRP4 inhibitors are being developed to counter MRP4-mediated drug resistance, and some of them have markedly enhanced the sensitivity of MRP4-overexpressing HEK293 cells towards 6-mercaptopurine [39].
Since MRP4 transports arsenic metabolites such as dimethylarsenic acid, and polymorphic variants differ in their ability to do so, MRP4 SNPs potentially contribute to the risk of arsenic-induced toxicity and tumorigenesis [40]. SNPs in ABCC4 also affect dose tolerance of 6-mercaptopurine in pediatric acute lymphoblastic leukemia [41].
MRP4 protects hematopoietic cells, both healthy and leukemic, against cytarabine, and counters the myelosuppressive effect of prolonged beta-lactam use [42]. Conversely, the 3348A>G SNP in homozygous form predisposes to beta-lactam-induced neutropenia, and the 559G>T(G187W) nonsynonymous variant – which is fairly common in the Japanese population with a frequency of 12.5% – sensitizes cells to the active metabolite of irinotecan, SN-38 [43]. 

Regulatory requirements

Currently, neither the FDA nor the EMA guidance specifically recommends the study of MRP4 interactions for NCEs. This is probably due to the lack of clear clinical citations of MRP4-mediated DDIs. However, this transporter may be relevant to NCEs where active renal secretion is suspected, or where changes in circulating levels of bile salts are observed.

References

1.    Berthier, J., et al., Multidrug resistance-associated protein 4 in pharmacology: Overview of its contribution to pharmacokinetics, pharmacodynamics and pharmacogenetics. Life Sci, 2019. 231: p. 116540.
2.    Borst, P., C. de Wolf, and K. van de Wetering, Multidrug resistance-associated proteins 3, 4, and 5. Pflugers Arch., 2007. 453(5): p. 661-73. Epub 2006 Apr 4.
3.    Russel, F.G., J.B. Koenderink, and R. Masereeuw, Multidrug resistance protein 4 (MRP4/ABCC4): a versatile efflux transporter for drugs and signalling molecules. Trends Pharmacol Sci., 2008. 29(4): p. 200-7. Epub 2008 Mar 18.
4.    Ritter, C.A., et al., Cellular export of drugs and signaling molecules by the ATP-binding cassette transporters MRP4 (ABCC4) and MRP5 (ABCC5). Drug Metab Rev., 2005. 37(1): p. 253-78.
5.    de Waart, D.R., et al., Oral availability of cefadroxil depends on ABCC3 and ABCC4. Drug Metab Dispos, 2012. 40(3): p. 515-21.
6.    Lapczuk-Romanska, J., et al., Membrane Transporters in Human Parotid Gland-Targeted Proteomics Approach. Int J Mol Sci, 2019. 20(19).
7.    Oswald, S., et al., Protein Abundance of Clinically Relevant Drug Transporters in The Human Kidneys. Int J Mol Sci, 2019. 20(21).
8.    Borst, P., et al., A family of drug transporters: the multidrug resistance-associated proteins. J Natl Cancer Inst., 2000. 92(16): p. 1295-302.
9.    Grube, M., P. Hagen, and G. Jedlitschky, Neurosteroid Transport in the Brain: Role of ABC and SLC Transporters. Front Pharmacol, 2018. 9: p. 354.
10.    Wen, J., et al., The Pharmacological and Physiological Role of Multidrug-Resistant Protein 4. J Pharmacol Exp Ther, 2015. 354(3): p. 358-75.
11.    Zhao, X., et al., Involvement of human and canine MRP1 and MRP4 in benzylpenicillin transport. PLoS One, 2019. 14(11): p. e0225702.
12.    Koenderink, J.B., et al., Human multidrug resistance protein 4 (MRP4) is a cellular efflux transporter for paracetamol glutathione and cysteine conjugates. Arch Toxicol, 2020. 94(9): p. 3027-3032.
13.    Zhou, S.F., et al., Substrates and inhibitors of human multidrug resistance associated proteins and the implications in drug development. Curr Med Chem., 2008. 15(20): p. 1981-2039.
14.    Klaassen, C.D. and H. Lu, Xenobiotic transporters: ascribing function from gene knockout and mutation studies. Toxicol Sci., 2008. 101(2): p. 186-96. Epub 2007 Aug 13.
15.    Sassi, Y., et al., Multidrug resistance-associated protein 4 regulates cAMP-dependent signaling pathways and controls human and rat SMC proliferation. J Clin Invest., 2008. 118(8): p. 2747-57.
16.    Leggas, M., et al., Mrp4 confers resistance to topotecan and protects the brain from chemotherapy. Mol Cell Biol., 2004. 24(17): p. 7612-21.
17.    Kanamitsu, K., et al., Investigation of the Importance of Multidrug Resistance-Associated Protein 4 (Mrp4/Abcc4) in the Active Efflux of Anionic Drugs Across the Blood-Brain Barrier. J Pharm Sci, 2017. 106(9): p. 2566-2575.
18.    Whyte-Allman, S.K., et al., Xenobiotic Nuclear Receptors Pregnane X Receptor and Constitutive Androstane Receptor Regulate Antiretroviral Drug Efflux Transporters at the Blood-Testis Barrier. J Pharmacol Exp Ther, 2017. 363(3): p. 324-335.
19.    Furmanski, B.D., et al., Contribution of ABCC4-mediated gastric transport to the absorption and efficacy of dasatinib. Clin Cancer Res, 2013. 19(16): p. 4359-70.
20.    Rius, M., et al., Cotransport of reduced glutathione with bile salts by MRP4 (ABCC4) localized to the basolateral hepatocyte membrane. Hepatology, 2003. 38(2): p. 374-84.
21.    Li, J., et al., Multidrug Resistance Protein 4 (MRP4/ABCC4) Protects Cells from the Toxic Effects of Halobenzoquinones. Chem Res Toxicol, 2017. 30(10): p. 1815-1822.
22.    Copsel, S., et al., Multidrug resistance protein 4 (MRP4/ABCC4) regulates cAMP cellular levels and controls human leukemia cell proliferation and differentiation. J Biol Chem, 2011. 286(9): p. 6979-88.
23.    Jung, M., et al., ABCC4/MRP4 contributes to the aggressiveness of Myc-associated epithelial ovarian cancer. Int J Cancer, 2020. 147(8): p. 2225-2238.
24.    Colavita, J.P.M., et al., Multidrug resistance protein 4 (MRP4/ABCC4) is overexpressed in clear cell renal cell carcinoma (ccRCC) and is essential to regulate cell proliferation. Int J Biol Macromol, 2020. 161: p. 836-847.
25.    Yaneff, A., et al., MRP4/ABCC4 As a New Therapeutic Target: Meta-Analysis to Determine cAMP Binding Sites as a Tool for Drug Design. Curr Med Chem, 2019. 26(7): p. 1270-1307.
26.    Aleksunes, L.M. and C.D. Klaassen, Coordinated regulation of hepatic phase I and II drug-metabolizing genes and transporters using AhR-, CAR-, PXR-, PPARalpha-, and Nrf2-null mice. Drug Metab Dispos, 2012. 40(7): p. 1366-79.
27.    Rodriguez Gonzalez, A., et al., MRP4/ABCC4 expression is regulated by histamine in acute myeloid leukemia cells, determining cAMP efflux. FEBS J, 2020.
28.    Azouzi, S., et al., Lack of the multidrug transporter MRP4/ABCC4 defines the PEL-negative blood group and impairs platelet aggregation. Blood, 2020. 135(6): p. 441-448.
29.    Abla, N., et al., The human multidrug resistance protein 4 (MRP4, ABCC4): functional analysis of a highly polymorphic gene. J Pharmacol Exp Ther., 2008. 325(3): p. 859-68. Epub 2008 Mar 25.
30.    Belinsky, M.G., et al., Multidrug resistance protein 4 protects bone marrow, thymus, spleen, and intestine from nucleotide analogue-induced damage. Cancer Res., 2007. 67(1): p. 262-8.
31.    Kiser, J.J., et al., The effect of lopinavir/ritonavir on the renal clearance of tenofovir in HIV-infected patients. Clin Pharmacol Ther, 2008. 83(2): p. 265-72.
32.    Reid, G., et al., Characterization of the transport of nucleoside analog drugs by the human multidrug resistance proteins MRP4 and MRP5. Mol Pharmacol., 2003. 63(5): p. 1094-103.
33.    Wagner, M., et al., Role of farnesoid X receptor in determining hepatic ABC transporter expression and liver injury in bile duct-ligated mice. Gastroenterology., 2003. 125(3): p. 825-38.
34.    Denk, G.U., et al., Multidrug resistance-associated protein 4 is up-regulated in liver but down-regulated in kidney in obstructive cholestasis in the rat. J Hepatol., 2004. 40(4): p. 585-91.
35.    Macias, R.I.R., et al., Role of drug transporters in the sensitivity of acute myeloid leukemia to sorafenib. Oncotarget, 2018. 9(47): p. 28474-28485.
36.    He, Z., et al., The overexpression of MRP4 is related to multidrug resistance in osteosarcoma cells. J Cancer Res Ther, 2015. 11(1): p. 18-23.
37.    Yuan, B., et al., Multidrug resistance-associated protein 4 is a determinant of arsenite resistance. Oncol Rep, 2016. 35(1): p. 147-54.
38.    Jaramillo, A.C., et al., Ex vivo resistance in childhood acute lymphoblastic leukemia: Correlations between BCRP, MRP1, MRP4 and MRP5 ABC transporter expression and intracellular methotrexate polyglutamate accumulation. Leuk Res, 2019. 79: p. 45-51.
39.    Chen, Y., et al., Discovery of novel multidrug resistance protein 4 (MRP4) inhibitors as active agents reducing resistance to anticancer drug 6-Mercaptopurine (6-MP) by structure and ligand-based virtual screening. PLoS One, 2018. 13(10): p. e0205175.
40.    Banerjee, M., et al., Polymorphic variants of MRP4/ABCC4 differentially modulate the transport of methylated arsenic metabolites and physiological organic anions. Biochem Pharmacol, 2016. 120: p. 72-82.
41.    Tanaka, Y., et al., Multidrug resistance protein 4 (MRP4) polymorphisms impact the 6-mercaptopurine dose tolerance during maintenance therapy in Japanese childhood acute lymphoblastic leukemia. Pharmacogenomics J, 2015. 15(4): p. 380-4.
42.    Drenberg, C.D., et al., ABCC4 Is a Determinant of Cytarabine-Induced Cytotoxicity and Myelosuppression. Clin Transl Sci, 2016. 9(1): p. 51-9.
43.    Tsukamoto, M., et al., A Human ABC Transporter ABCC4 Gene SNP (rs11568658, 559 G > T, G187W) Reduces ABCC4-Dependent Drug Resistance. Cells, 2019. 8(1).