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As described above, the OATs are characterized by two large interconnecting loops, one between TMD 1/2 and another between TMD 6/7 (Fig. 4.1). The first large loop is extracellular and contains multiple consensus N‐glycosylation sites [32], the role of which remains unclear, although glycosylation of OAT1 was shown to be required for protein trafficking to the membrane [57]. The second large loop is intracellular and together with the intracellular carboxy terminus contains several potential phosphorylation sites for PKC, PKA [53], casein kinase II and tyrosine kinase [13], as well as for ubiquitination, another type of post‐translational modification (PTM). Once considered to be static at the cell membrane, OATs have recently been shown to undergo internalization and recycling to and from the cell surface constitutively or in response to stimuli [80–82]. As the levels of OATs at the cell surface are critical to their transport activity, the dynamic expression of OATs at the cell surface would improve the transporter’s ability to initiate trafficking in response to stimuli, thus providing efficient and fast fine‐tuning of OAT activity.

While most of the information discussed below refers to OAT1 and OAT3, the term OAT is used generically to emphasize concepts applicable to other OATs as well. Several PTMs are involved in the regulation of OAT trafficking. PTMs, the modifications on the target protein following synthesis, refer to the covalent addition of functional group(s) to the target proteins and include glycosylation, ubiquitination, phosphorylation, and many others [83, 84]. Most of the PTMs are catalyzed by specific enzymes that attach or remove functional group(s) and are dynamic and reversible. Through promoting and demoting the modifications, PTMs add complexity to the functional diversity of the proteome. Since different PTMs can modify target proteins individually or simultaneously through various mechanisms, the functional diversity of the target proteins exceeds their molecular diversity [85–87].

Ubiquitination, the addition of ubiquitin molecule(s) to the lysine residue(s) on the substrate protein, is a type of PTM that can occur in different conjugations, including monoubiquitination, multiubiquitination, and polyubiquitination. Monoubiquitination refers to the addition of a single ubiquitin molecule to a single lysine residue on the target protein, while multiubiquitination is the conjugation of several monoubiquitin molecules to multiple lysine residues on the target protein. Polyubiquitination refers to the conjugation of a polyubiquitin chain to the substrate. The polyubiquitin chain is formed between a lysine residue of one ubiquitin molecule and a glycine residue of another ubiquitin molecule [88, 89]. Ubiquitin itself has seven lysine residues, which includes K6, K11, K27, K29, K33, K48, and K63. Ubiquitination regulates the target proteins through altering their cellular location, stability, activity, and protein–protein interactions.

OAT ubiquitination is catalyzed by the ubiquitin ligases Nedd4‐1 or Nedd4‐2 and facilitates the internalization of OATs from the cell surface to the intracellular early endosomes. Once in the endosomes, OATs move to the proteolytic system for degradation or becomes deubiquitinated, resulting in OATs recycling back to the cell membrane. Nedd4‐2 is mainly involved in protein‐kinase‐regulated OAT ubiquitination, whereas Nedd4‐1 is largely involved in the constitutive OAT ubiquitination [80–82,90–92]. Several protein kinases have been reported to exert their regulation on OATs through phosphorylating Nedd4‐2 at different sites, which either weaken or strengthen the protein–protein interaction between OATs and Nedd4‐2. The strength of this interaction leads to a decreased or enhanced OAT ubiquitination and, ultimately, to stimulated or inhibited OAT transport activity. For example, the short‐term activation of PKC enhances Nedd4‐2 phosphorylation, and therefore OAT ubiquitination, which leads to accelerated OAT internalization from the cell surface to the intracellular endosomes. As a result, OAT expression at the cell surface and transport activity are reduced. The prolonged PKC/Nedd4‐2 activation results in enhanced OAT internalization from the cell surface to the intracellular endosomes and subsequent degradation in proteolytic systems. Overexpression of Nedd4‐2/C821A, a ligase‐dead mutant of Nedd4‐2, or the knockdown of endogenous Nedd4‐2 with Nedd4‐2‐specific siRNA evaded the PKC‐induced change in OAT ubiquitination, trafficking, and function in cultured cells [82, 91, 93]. On the other hand, serum and glucocorticoid‐regulated kinase 1 (Sgk1) phosphorylated Nedd4‐2 on Ser327 in cultured cells, which weakened the interaction between OATs and Nedd4‐2 and decreased OAT ubiquitination, leading to increased OAT transport activity [94]. Sgk2, an isoform of Sgk1, impaired the binding between OATs and Nedd4‐2 and decreased OAT ubiquitination, leading to elevated OAT cell surface expression and transport activity in cultured cells [95, 96]. In summary, PKC (negative regulation) and Sgk1/2 (positive regulation) exert opposite effects on OAT trafficking to the cell membrane and transport activity through phosphorylation of Nedd4‐2 at distinct sites. Nedd4‐2 serves as a central switch in these regulations.

Ubiquitination is a dynamic and reversible process. Deubiquitination is catalyzed by deubiquitinating enzymes (DUBs) and removes ubiquitin molecule(s) from the substrate protein [97, 98]. Deubiquitination and ubiquitination form a dynamic and opposing network, which is involved in a variety of physiological and pathological processes [99–102]; ~100 DUBs have been identified to date and are grouped into two classes: metalloproteases and cysteine proteases. Metalloproteases and cysteine proteases can be further subdivided into five subfamilies: ubiquitin‐specific proteases (USP), ubiquitin C‐terminal hydrolases (UCH), Jab1/Mpn/Mov34 (JAMM) enzymes, Machado‐Joseph domain proteases (MJD), and ovarian tumor proteases (OTU). Each of these subfamilies exhibits different structures and specificities toward distinct ubiquitin linkages [103–105]. USP8, a member of USP family, increased OAT cell surface expression and transporter activity in cultured cells, stemming from decreased OAT internalization, degradation, and ubiquitination. Knocking down the endogenous USP8 with USP8‐specific siRNA led to an increase in OAT ubiquitination, which correlated with reduced OAT transport activity [106]. In summary, ubiquitination and deubiquitination combine to help regulate OAT trafficking and transport activity (Fig. 4.2).

During the past few years, positive and negative crosstalk between different PTMs has been explored. Positive crosstalk is when one PTM serves as a signal for the modification of a second PTM, whereas negative crosstalk is when one PTM directly competes with another PTM or indirectly masks the recognition site for another PTM. The interplay between the PTMs that occur on the same type of amino acid residue(s) has attracted research attention because of its potential to regulate a wide array of cellular functions. One key example of negative crosstalk occurs between ubiquitination and SUMOylation, in which both ubiquitin and small ubiquitin‐like modifier (SUMO) are covalently attached to the lysine residue(s) of the target protein. Ubiquitin and SUMO can either conjugate to the same lysine residue(s) in the substrate protein in a competitive manner or conjugate to different lysine residues in the target protein. Under both circumstances, SUMOylation may preclude the ubiquitin‐mediated trafficking of the target protein [107]. It has been reported that the enhanced OAT SUMOylation induced by PKA activation occurs in parallel with a decrease in OAT ubiquitination, leading to an increased rate of OAT recycling and decreased rate of OAT degradation, without affecting the internalization rate of OATs. Therefore, SUMOylation and ubiquitination may coordinately regulate OAT trafficking and transport activity through negative crosstalk [108].

FIGURE 4.2 Post‐translational modifications of OATs. Ubiquitination and deubiquitination combine to regulate the expression of OATs at the cell surface, and thus their function. Ub: ubiquitin.

Several hormones and chemicals have been shown to regulate OAT trafficking through the protein kinases/Nedd4‐2 signaling pathway. Angiotensin II, an endogenous hormone, activated PKC/Nedd4‐2 pathway, which led to an increased rate of OAT internalization and therefore a reduction in OAT transport activity [109, 110]. AG490, a specific inhibitor of the Janus tyrosine kinase 2 (JAK2), reduced Nedd4‐2 phosphorylation at tyrosine residue(s), resulting in enhanced interaction between OAT and Nedd4‐2 and enhanced OAT ubiquitination, which led to a reduction in OAT cell surface expression and transport activity in cultured cells. Moreover, AG490 also increased the degradation rate of OATs. On the other hand, the inhibition effect of AG490 on OATs was diminished by knocking down the endogenous Nedd4‐2 using Nedd4‐2‐specific siRNA [111]. Dexamethasone, an upstream hormone of Sgk1, increased Nedd4‐2 phosphorylation, leading to stimulated OAT expression and transport activity in cultured cells [112]. Insulin, an endogenous hormone, phosphorylated Nedd4‐2 on Ser327 and impaired the interaction between OAT and Nedd4‐2, resulting in the upregulation of OAT expression and transport activity. Knocking down the endogenous Nedd4‐2 with Nedd4‐2‐specific siRNA diminished the upregulation on OATs induced by insulin [113].