**5. Endocytosis and Membrane Recycling of Canalicular ABC Transporters**

Small GTPases, protein kinases, class V myosins and adaptor proteins have been identified as molecular players in the regulation of canalicular ABC transporter endocytosis and recycling [3]. The existence of ABCB11 intracytoplasmic reservoirs is well known [28,29], but the nature of those compartments long remained uncharacterized until a YFP-tagged ABCB11 was detected in the SAC, a RAB11-positive compartment. Indeed, RAB11 and myosin VB (MYO5B) are established regulators of the recycling of several proteins from the SAC to the plasma membrane [58]. ABCB11 continuously cycles between the canalicular membrane of hepatocytes and the SAC [59]. This constant exchange allows tight regulation of ABCB11 availability at the bile canaliculi. The perturbation of actin cytoskeleton or microtubules inhibits this traffic [59]. These results were corroborated as ABCB11 apical targeting is considerably slowed down in WIF-B9 cells expressing RAB11 or MYO5B-dominant negative constructs [60]. In the presence of a mutated or truncated MYO5B, ABCC2 displays an intracellular localization in RAB8- and RAB11-positive compartments, suggesting defects in canalicular transporter recycling [61]. Recently, mutations in the *MYO5B* gene, identified in patients, have been associated with a progressive familial intrahepatic cholestasis (PFIC)-like phenotype, further proposed as PFIC6 [61,62].

The ERM protein family has also been involved in the endocytic process of several ABC transporters. Coimmunoprecipitation performed with human liver lysates highlighted the interaction between ABCC2 and ezrin, and additional experiments showed that ezrin phosphorylation on its Thr567 regulates this interaction, thus further controlling the amounts of ABCC2 present at the plasma membrane [63].

Several isoforms of PKC, as well as PKA, PI3K, Pim-1 or Fyn kinases, play a role in the regulation of ABCB1, ABCC2 and ABCB11 membrane targeting or endocytosis [33,39,41,42,64–66]. Cantore and colleagues have reported that the Src family kinase Fyn induces ABCC2 and ABCB11 retrieval from the canalicular membrane by increasing cortactin phosphorylation [66]. Schonhoff and colleagues observed that taurolithocholate-activated PKCε phosphorylates and activates myristoylated alanine-rich C-kinase substrate (MARCKS), a membrane-bound F-actin crosslinking protein [67]. MARCKS is a crucial regulator of molecular interactions and cytoskeletal reorganization. In a nonphosphorylated state, MARCKS is associated with the cytosolic leaflet of the plasma membrane and can serve as a stabilizer for transmembrane proteins, whereas after phosphorylation, MARCKS is released in the cytosol, where it can interact with other proteins [68].. MARCKS has been

shown to regulate the endocytosis of ABCC2 and ABCB1 [67,69]. Indeed, in colon carcinoma cells, MARCKS expression has been associated with the reduced export function of ABCB1 [69].

The hematopoietic cell-specific Lyn substrate 1 associated protein X-1 (HAX-1) is a small protein abundantly expressed in the liver, regulating cortical actin organization. This protein has been identified as an interactor of the linker domain of ABCB1, ABCB4 and ABCB11 [70]. Through this interaction, HAX-1 has been proposed to stabilize ABCB11 at the plasma membrane [70]. However, the role of HAX-1 in other canalicular ABC transporter endocytosis has not been further investigated.

A tyrosine motif has been identified in the ABCB11 cytoplasmic tail [71] along with one of its partners, the clathrin adaptor protein complex 2 (AP2) [72]. AP2 is localized at the plasma membrane and binds tyrosine-based internalization motifs of proteins, including ABCB11, thus allowing its internalization from the canalicular membrane through clathrin-dependent endocytosis [72]. The ubiquitination of ABCB11 and ABCC2 has also been shown to be essential for clathrin-mediated endocytosis and degradation of these transporters [73].

Hormones and intracellular signaling molecules also play a role in canalicular ABC transporter internalization. In a model of estradiol-induced cholestasis, authors showed that following treatment with estradiol-17β-d-glucuronide (E217G), ABCB11 and ABCC2 are relocalized from canalicular membranes to intracytoplasmic compartments. The same group demonstrated later that ABCB11 and ABCC2 endocytosis is mediated by PKC, which is activated by E217G, and that PKC inhibitors prevent the internalization of both transporters after treatment with estradiol [74,75]. In the same model, Zuchetti and colleagues established that glucagon and an adrenaline analog mediate cAMP activation, thus preventing ABCB11 and ABCC2 membrane retrieval [76]. Moreover, they showed that this E217G-induced endocytosis is AP2- and clathrin-dependent [77]. It has also been suggested that lipopolysaccharides act as signals for ABCC2 and ABCB11 endocytosis as their canalicular expression is reduced, with no mRNA decrease in an in vitro cholestatic model [78].
