**3. The Physiological Functions of ABCG2, and Its Role in Multidrug Resistance** *3.1. The Physiological Roles of ABCG2*

As mentioned previously, ABCG2 is overexpressed in drug-resistant cell lines and tumors. Habitually, it is expressed at a relatively high level in cell types located at the entry and exit boundaries of the body, as well as in barrier tissues at the borders of sanctuary sites [34,35]. These include the epithelial cells of the gastrointestinal track, especially in small intestine enterocytes [36], the kidney tubular epithelial cells [37], hepatocytes [34], placental syncytiotrophoblasts [38], mammary alveolar epithelial cells (a part of the blood-milk barrier) [39], and brain capillary endothelial cells (a key element of the blood-brain barrier) [40,41]. In these polarized epithelial and endothelial cells, ABCG2 is localized to the apical plasma membrane domain. In addition to these cells constituting tissue barriers, ABCG2 is also expressed in various types of stem cells including hematopoietic stem cells [42], pluripotent stem cells [43,44], and cancer stem cells [45–48]. Interestingly, ABCG2 is also present in the membrane of red blood cells (RBCs) [34,49,50].

As is typical of a multidrug transporter, ABCG2 recognizes a vast variety of compounds as transported substrate molecules. These include uric acid in the first place, but also various endogenous conjugated hormones and metabolites, several hydrophobic and amphipathic drugs, as well as their conjugates [51–53]. This promiscuity and the tissue distribution detailed above delineate the physiological function of this transporter. In general, ABCG2—depending on its location—restricts the uptake or facilitates the excretion of potentially toxic or unwanted substances. Specifically, in the brain capillaries, ABCG2 restricts the passage of substances through the blood-brain barrier, whereas in the placenta, it protects the fetus from maternally derived toxins. For instance, ABCG2 restricts the maternal-fetal transfer of bile acids, which is especially important in expecting mothers with intrahepatic cholestasis of pregnancy, a frequent liver disease leading to augmented serum levels of bile acids [54,55]. In the small intestine, ABCG2 controls the absorption of various molecules and participates in extra-renal clearance of uric acid; in the kidney proximal tubules, it contributes to the elimination of unwanted toxins and metabolites, including uric acid. Impaired ABCG2-mediated urate transport may lead to gout or hyperuricemia, therefore, specific mutations and polymorphisms in ABCG2 are genetic risk factors for these conditions [37,56–58] to be discussed in detail in Section 6. Interestingly, a recent study reported unequal contribution of ABCG2 to renal and extra-renal clearance of uric acid [58]. In mammary alveolar epithelial cells, this transporter influences the milk composition. Endogenous substrates transported by ABCG2 through the blood-milk barrier include riboflavin (vitamin B2) and bile acids [59,60]. Certainly, vigilance is required for breast-feeding mothers, as various medications can be transported by ABCG2 into the milk [39,61]. The relevance of the Abcg2-mediated drug transport for the dairy industries is also self-evident [62,63].

In these physiological boundaries, ABCG2 accomplishes this 'bouncer duty' in a coordinated fashion together with the other MDR proteins, MDR1/ABCB1 and MRP1/ABCC1, exploiting their partially overlapping substrate recognition and specific subcellular localization. In polarized epithelial cells, ABCG2 is localized to the apical membrane ipsilaterally to MDR1/ABCB1 and contralaterally to MRP1/ABCC1, whereas in cerebral endothelial cells, all three major MDR proteins reside at the same side, i.e., the apical membrane [64]. Accordingly, ABCG2 along with other MDR proteins potentially alters the absorption, distribution, and excretion, as well as, consequently, the metabolism and toxicity (ADME-Tox properties) of pharmaceutical drugs. Especially important is the potential contribution of these transporters to drug–drug interactions, since modification of one (or more) of the MDR proteins by a drug may greatly influence the pharmacokinetics of another one. Therefore, the examination of drug interactions with MDR proteins, including ABCG2, is a requirement in preclinical drug development [53,65,66]. Interestingly, in mammary epithelial cells, the apically localized ABCG2 and the basolateral MRP4/ABCC4 counteract one another in bile acid transport [60].

The physiological role of ABCG2 in red blood cells and stem cells is enigmatic to some extent. Since phototoxic porphyrins, such as the plant-derived pheophoride A and the heme precursor protoporphyrin IX (PPIX), are noted substrates of ABCG2, its expression in the erythroid precursor cells and in mature RBCs may indicate its involvement in heme metabolism [50,67,68]. It is worth noting, however, that numerous membrane proteins without known function in RBCs are present in their membrane, i.e., the sterol transporter ABCA1 (http://rbcc.hegelab.org/, accessed on 9 February 2021) [69,70]. It is plausible that many of these membrane proteins can be just remnants from previous stages of cell differentiation and maturation. In various stem cell types, a protective role similar to that observed at the border of sanctuary sites has been proposed for ABCG2 [42–44]. Stem cells are poised between self-renewal and differentiation, and are thus exceptionally sensitive to environmental factors. ABCG2 can contribute to the stem cells' self-protective mechanisms. The presence of the transporter may, however, backfire in cancer stem cells, as they can provide tumors with drug-resistant cell populations.
