REGULATION OF DRUG TRANSPORTER ACTIVITY - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 523

In addition to affecting the ability of a transporter to bind its substrate, phosphory-
lation may also affect the ability of the transporter to interact with its accessory pro-
teins. It has been shown that the phosphorylation of multidrug resistance-associated
protein MRP2 has profound effects on the binding of this transporter with PDZ
proteins. 26 PDZ proteins contain specific domains that through binding to their tar-
geting molecules are known to play important roles in scaffolding, protein trafficking,
and the regulation of membrane transport activity. In MRP2, Ser-1542 forms part of
the consensus sequence for phosphorylation by PKC. It is shown that when Ser-
1542 was replaced by alanine (MRP2 S1542A), mimicking the dephosphorylation
state of MRP2, the binding of MRP2 to the PDZ protein EBP50 was much less than
that of the wild-type MRP2. In contrast, when Ser-1542 was replaced by glutamic
acid (MRP2 S1542E), mimicking the phosphorylation state of MRP2, the binding of
MRP2 to PDZ proteins EBP50 and IKEPP was much stronger than that of the wild
type.
In rat hepatocyte, the organic anion transport mediated by organic anion-
transporting polypeptide Oatp1 was down-regulated by extracellular ATP. 27 AT P w a s
speculated to exert its effect through P2Y purinergic receptor, which then leads to the
activation of intracellular signaling pathways. The down-regulation of Oatp1 func-
tion was also observed when hepatocytes were incubated with phosphatase inhibitors
okadaic acid and calyculin A. Exposure of hepatocytes to both extracellular ATP and
okadaic acid resulted in the phosphorylation of the transporter without affecting its
cell surface presentation, suggesting that loss of transport activity is not caused by
transporter internalization. Since Oatp1 functions as an organic anion exchanger, it
is hypothesized that addition of a negatively charged phosphate group to the inner
domain of the transporter may prevent this exchange from occurring.

17.5. DISULFIDE BONDS

A disulfide bond (SS bond), also called a disulfide bridge , is a strong covalent bond
formed by oxidation of two sulfhydryl groups ( SH) present in cysteine residue. In
eukaryotic cells, disulfide bonds are generally formed in the lumen of endoplasmic
reticulum (ER) but not in the cytosol. This is due to the oxidative environment of the
ER and the reducing environment of the cytosol, which is maintained by the high ratio
of oxidized glutathione to reduced glutathione. Thus, disulfide bonds are found only
in secretory proteins, lysosomal proteins, and the exoplasmic domains of membrane
proteins such as transporters. The formation of disulfide bonds can be reversed by
reducing conditions. These conditions may include the presence of agents with free
sulfhydryl groups such as dithiothreitol (DTT), -mercaptoethanol, or glutathione.

A disulfide bond that links two peptide chains together is called an intermolecular
disulfide bond , whereas a disulfide bond that links different parts of one peptide chain
is called an intramolecular disulfide bond . Disulfide bonds are very important to the
folding, subunit assembly, and functioning of proteins. The greater the number of
disulfide bonds, the less susceptible the protein is to denaturation by forces such as
detergents and heat.