DRUG TRANSPORT IN THE KIDNEY - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 476

15.3.2. Phosphorylation

All OAT isoforms have several sites for phosphorylation by protein kinase C (PKC)
in the large intracellular loop between the sixth and seventh transmembrane domains
(TMDs). Several studies have revealed that the activation of PKC decreases the
transport activity of OATs. 123-126 This inhibitory effect is also associated with altered
substrate selectivity. A reduced OAT-mediated transport activity is rescued by PKC
inhibitors. 123 , 124 Furthermore, recent studies have demonstrated the OAT1 activity
to be stimulated by epidermal growth factor (EGF) via mitogen-activated protein
kinases (MAPKs). 127 In addition to the sites for phosphorylation by PKC, OAT
isoforms have putative sites for phosphorylation by PKA, casein kinase II, or tyrosine
kinase. It is not clear whether these protein kinases are involved in the regulation of
transporter functions.
Similar to OATs, All OCT isoforms also have several sites for PKC phosphoryla-
tion. In HEK293 cells, the rOct1-mediated transport of 4-(4-(dimethylamino)styryl)-
N -methylpyridinium iodide (ASP) was stimulated by PKC, PKA, and tyrosine kinase
activators. 128 In contrast, ASP uptake into hOCT2-expressing HEK293 cells was not
affected by PKC activator, whereas it was slightly inhibited by PKA agonist. 129 OCT2
is constitutively activated by Ca 2 + /calmodulin complex. 129

15.3.3. Protein-Protein Interaction
Several renal apical transporters possess the PDZ motif at their C-terminus. 13 , 130
The PDZ motif is one of the important protein-protein interaction modules, and it
is composed of three amino acid residues: S/T-X- (where X is any amino acid and

is a hydrophobic residue). The renal apical organic anion transporters URAT1,
OAT4, PEPT2, and OCTN2 possess the PDZ motif at their C-terminus. Yeast two-
hybrid experiments revealed that these transporters interact with the multivalent PDZ
domain-containing proteins such as PDZK1 and NHERF1 via their C-terminal PDZ
motifs. 131-134 The coexpression of transporters and PDZK1 in HEK293 cells in-
creases their transport activity. This synergic effect is abolished when the C-terminal
PDZ motif deletion mutants of transporters are coexpressed with PDZK1. These re-
sults indicate that PDZK1 regulates transport activities via interaction with the PDZ
motif.

15.3.4. Glycosylation

The glycosylation sites in the first extracellular loop between first and second TMDs
are conserved in OATs. Tunicamycin, an inhibitor of asparagine-linked glycosylation,
inhibited PAH transport activity in mOat1-transfected COS7 cells. 135 Immunofluo-
rescence revealed that the mOat1 protein remained primarily in the intracellular com-
partment after tunicamycin treatment. 135 This study indicates that the glycosylation
of the mOat1 protein is necessary for proper trafficking of the protein to the plasma
membrane. Other experiments have demonstrated that disrupting Asp39 (one of the
glycosylated sites) in mice resulted in a complete loss of transport activity of OAT1