13-16 Two major types of signals have been described to regulate targeting and trafficking of a number of receptor proteins: (1) a tyrosine-based YXXØ sequence in which Ø is an amino acid with a bulky hydrophobic group; and (2) dileucine-based LL motifs in which the latter leucine may be replaced by a hydrophobic amino acid residue.16-19 Depending on the primary sequence context and the FK506 relative position of

the motif relative to the membrane, these motifs serve as plasma membrane targeting signals.20, 21 They are also used for efficient sorting to the endosomal system where the protein may be recycled or transported to the lysosome for degradation.22 Adaptin 2 (AP2) is a plasma membrane–localized clathrin adaptor whose subunits bind directly to tyrosine-based YxxØ or NPXY internalization motifs within cytoplasmic or transmembrane regions of proteins to mediate clathrin-dependent endocytosis.23-27 For example, the cytosolic domain of another ABC transporter, cystic fibrosis transmembrane regulator (CFTR) has numerous consensus endocytic motifs that regulate the clathrin-dependent endocytosis.14, 28 However, the presence

of discrete sorting signals in the cytosolic tail of BSEP has not been elucidated. In this study, we tested whether the C-terminus of human BSEP has a targeting/trafficking signal and whether it contributes to the cell surface expression of BSEP. We demonstrated Acalabrutinib nmr by domain swapping using the C-terminal region of BSEP attached to Tac (interleukin-2 receptor α [IL-2Rα]) that an internalization motif is present in BSEP. Tac is a cell surface type 1 transmembrane

protein that is targeted to the plasma membrane by default. Tac chimeras have been extensively used for the identification of trafficking signals because of the monomeric nature of the Tac protein and the existence of highly specific N-terminal antibodies to track the chimeric protein.29 We identified by mutagenesis analysis the exact motif in BSEP as a YYKLV sequence. By transferring this YYKLV motif directly to Tac, we further showed that this mafosfamide motif is sufficient for internalization. Finally, we demonstrated that this motif is functional in the full-length human BSEP. ABC, ATP-binding cassette; AP, adaptin; ATP, adenosine triphosphate; ATPase, adenosine triphosphatase; BSA, bovine serum albumin; BSEP/Bsep, bile salt export pump; cAMP, cyclic adenosine monophosphate; CFTR, cystic fibrosis transmembrane regulator; ELISA, enzyme-linked immunosorbent assay; ER, endoplasmic reticulum; GTPase, guanosine triphosphatase; HRP, horseradish peroxidase; IL-2R α, interleukin-2 receptor α; Leu, leucine; MDCK, Madin–Darby canine kidney; MDR1, multidrug resistant 1 P.