REGULATION OF DRUG TRANSPORTER ACTIVITY - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 520

sugars, the first step in the glycosylation process, crucial for OAT activity, but modifi-
cation of added sugars, the processing step, has also been proven to be important. With
the use of mutant Chinese hamster ovary (CHO)-Lec cells lacking various enzymes
required for glycosylation processing, it was shown that processing of glycosylation
from a mannose-rich type to a complex type is associated with an increased affinity
of OAT4 for its substrate. 4
In contrast to the members of OAT family, where disruption of N-glycosylation
at individual sites has no effect on transport activity, elimination of glycosylation at
individual sites of organic cation transporter OCT2 showed a differential effect on
its transport function. 10 Removal of the glycosylation site at position 112 of OCT2
impaired the trafficking of OCT2 to the plasma membrane, removal of glycosylation
site at position 96 reduced the turnover number of the transporter, whereas removal
each of the three glycosylation sites at positions 71, 96, and 112 all increased the
affinity of the transporter for its substrate.
P-Glycoproteins are also heavily glycosylated plasma membrane proteins. Eval-
uation of the significance of N-glycosylation of human P-glycoprotein (MDR1) 11
revealed that transfection of cDNA encoding a N-glycosylation-deficient P-
glycoprotein yielded drug-resistant clones with a much lower frequency than did
transfection of wild-type cDNA, suggesting that N-glycosylation may prevent
P-glycoprotein from ending up or getting stuck in the wrong subcellular compart-
ments, and may improve the efficiency of P-glycoprotein routing or sorting.
The functional significance of N-glycosylation was also investigated with a
naturally occurring glycosylation-defective mutant of organic anion-transporting
polypeptide OATP1A2 (also known as human OATP-A or OATP1). 12 Genotypic
analyses of subjects from various ethnic populations identified six nonsynonymous
mutants within the coding region. One of the variants, A404T, has a mutation at a gly-
cosylation site (N135I). In vitro assessment revealed that the A404T variant had a shift
in the apparent molecular size, indicating an alteration in glycosylation status. This
variant also had a markedly reduced capacity for mediating the cellular uptake of all the
OATP1A2 substrate tested. Cell surface biotinylation and immunofluorescence confo-
cal microscopy suggested that altered plasma membrane expression of the transporter
might contribute to reduced transport activity associated with the A404T variant. 12

17.3. UBIQUITINATION

Ubiquitination is a three-step process. In the first step, ubiquitin, an 8-kDa polypep-
tide, is activated by a ubiquitin-activating enzyme. The activated ubiquitin is subse-
quently transferred to an ubiquitin carrier protein. Finally, ubiquitin-protein ligase
catalyzes the covalent binding of ubiquitin to the target protein. Ubiquitination of
cellular proteins usually serves to tag them for rapid degradation and can therefore
modulate their stability and activity (Figure 17.2). Cells degrade proteins through
two major systems, the proteasome and the lysosome. The proteasome is involved
in the degradation of most cytosolic and nuclear proteins as well as some membrane
proteins 13 − 15 and removes misfolded or misaggregated proteins in the endoplasmic