Organic anion transporters (OAT) play essential roles in the body disposition of clinically important anionic drugs, including antiviral drugs, antitumor drugs, antibiotics, antihypertensives, and anti-inflammatories. We reported previously (Kuze, K., Graves, P., Leahy, A., Wilson, P., Stuhlmann, H., and You, G. (1999) J. Biol. Chem. 274, 1519 -1524) that tunicamycin, an inhibitor of asparagine-linked glycosylation, significantly inhibited organic anion transport in COS-7 cells expressing a mouse organic anion transporter (mOAT1), suggesting an important role of glycosylation in mOAT1 function. In the present study, we investigated the effect of disrupting putative glycosylation sites in mOAT1 as well as its human counterpart, hOAT1, by mutating asparagine to glutamine and assessing mutant transporters in HeLa cells. We showed that the putative glycosylation site Asp-39 in mOAT1 was not glycosylated but the corresponding site (Asp-39) in hOAT1 was glycosylated. Disrupting Asp-39 resulted in a complete loss of transport activity in both mOAT1 and hOAT1 without affecting their cell surface expression, suggesting that the loss of function is not because of deglycosylation of Asp-39 per se but rather is likely because of the change of this important amino acid critically involved in the substrate binding. Single replacement of asparagines at other sites had no effect on transport activity indicating that glycosylation at individual sites is not essential for OAT function. In contrast, a simultaneous replacement of all asparagines in both mOAT1 and hOAT1 impaired the trafficking of the transporters to the plasma membrane. In summary, we provided the evidence that 1) Asp-39 is crucially involved in substrate recognition of OAT1, 2) glycosylation at individual sites is not required for OAT1 function, and 3) glycosylation plays an important role in the targeting of OAT1 onto the plasma membrane. This study is the first molecular identification and characterization of glycosylation of OAT1 and may provide important insights into the structure-function relationships of the organic anion transporter family. Organic anion transporters (OAT)1 play essential roles in the body disposition of clinically important anionic drugs including anti-human immunodeficiency virus therapeutics, antitumor drugs, antibiotics, antihypertensives, and anti-inflammatories (1). Several OAT isoforms have been identified by us and others (2). OAT1 and -3 are predominantly expressed in the kidney and brain. In the kidney, these transporters utilize a tertiary transport mechanism to move organic anions across the basolateral membrane into the proximal tubule cells for subsequent exit/elimination across the apical membrane into urine. Through this tertiary transport mechanism, Na ϩ -K ϩ -ATPase maintains an inwardly directed (blood to cell) Na ϩ gradient. The Na ϩ gradient then drives a Na ϩ -dicarboxylate cotransporter, sustaining an outwardly directed dicarboxylate gradient that is utilized by a dicarboxylate/organic anion (OA) exchanger to move th...

We used a novel approach to evaluate how the addition/acquisition and processing/modification of N-linked oligosaccharides play a role in the functional maturation of human organic anion transporter hOAT4. Inhibition of acquisition of oligosaccharides in hOAT4 by mutating asparagine to glutamine and by tunicamycin treatment was combined with the expression of wild-type hOAT4 in a series of mutant Chinese hamster ovary (CHO)-Lec cells defective in the different steps of glycosylation processing. We showed that both the disruption of the glycosylation sites by mutagenesis and the inhibition of glycosylation by tunicamycin treatment resulted in a nonglycosylated hOAT4, which was unable to target to the cell surface. In contrast, hOAT4 synthesized in mutant CHO-Lec cells, carrying different structural forms of sugar moieties (mannose-rich in Lec1 cells, sialic acid-deficient in Lec2 cells, and sialic acid/galactosedeficient in Lec8 cells) were able to traffic to the cell surface. However, hOAT4 expressed in CHO-Lec1 cells had significantly lower binding affinity for its substrates compared with that expressed in parental CHO cells. This study provided novel information that addition/acquisition of oligosaccharides but not the processing of the added oligosaccharides participates in the membrane insertion of hOAT4. Processing of added oligosaccharides from mannose-rich type to complex type is important for enhancing the binding affinity of hOAT4 for its substrates. Glycosylation could therefore serve as a means to specifically regulate hOAT4 function in vivo.

BACKGROUND AND PURPOSE The human organic anion transporting polypeptide 1A2 (OATP1A2) is expressed in cells from several regions of the human body, including the kidney, cholangiocytes and the blood‐brain barrier, and mediates the cellular flux of various anionic substances, including drugs in clinical use. Several related mammalian transporters have been shown to be subject to post‐translational regulation, including kinase‐induced internalization. In the present study the role of protein kinase C (PKC) in the regulation of OATP1A2 was investigated in an in vitro cell model. EXPERIMENTAL APPROACH COS‐7 cells in which OATP1A2 was overexpressed were treated with the PKC‐specific activator (phorbol 12‐myristate 13‐acetate; PMA) and the PKC‐specific inhibitor (Go6976). The impact of these treatments on the function and regulation of OATP1A2 was determined. KEY RESULTS PKC activation decreased the transport function of OATP1A2 in a time‐ and concentration‐dependent manner. PMA (0.1 µM) decreased the Vmax of oestrone‐3‐sulphate uptake and decreased the cell surface expression of OATP1A2 immunoreactive protein; these effects of PMA were prevented by the PKC specific inhibitor Go6976. In further studies, PMA treatment accelerated the internalization of OATP1A2 but did not affect its recycling. The disruption of clathrine‐dependent endocytosis attenuated both the constitutive and PKC‐modulated internalization of OATP1A2. In contrast, blocking the caveolin‐dependent pathway was without effect. CONCLUSIONS AND IMPLICATIONS PKC regulates the transport function of OATP1A2 by modulating protein internalization; this effect of PKC is mediated in part by clathrine‐dependent pathways.

Our study provides the first evidence of PEPT2-mediated uptake of polymyxins and contributes to a better understanding of the accumulation of polymyxins in renal tubular cells.

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