Rapid Modulation of the Organic Anion Transporting Polypeptide 2B1 (OATP2B1, SLCO2B1) Function by Protein Kinase C-mediated Internalization

Kathleen Köck,Anna Koenen,Bernd Giese,Martin Fraunholz,Karen May,Werner Siegmund,Elke Hammer,Uwe Völker,Gabriele Jedlitschky,Heyo K Kroemer,Markus Grube

doi:10.1074/jbc.m109.056457

Abstract

Members of the organic anion transporting polypeptide (OATP) family are involved in various pharmacological, pathophysiological, and physiological processes, such as hepatic drug uptake, progress of cancer, or transport of hormones. Although variability in expression and function of OATPs has been investigated in detail, data concerning regulation are rather limited. Here, we report a novel mechanism for rapid regulation of OATP2B1 mediated by protein kinase C (PKC) resulting in significant changes of transport activity. PKC activation by the phorbol ester (phorbol 12-myristate 13-acetate, PMA) resulted in increased phosphorylation of OATP2B1 as well as reduced OATP2B1 transport activity with a decrease in V(max) of E(1)S uptake (288 +/- 21 (control) versus 165 +/- 16 pmol/min/mg of protein (PMA)). This effect was sensitive to the PKC inhibitor bisindolylmaleimide I (BIM-I). Confocal microscopy, fluorescence-based internalization assay, and live-cell imaging using green fluorescent protein-tagged OATP2B1 revealed that transport inhibition was due to internalization of the transporter. Furthermore, colocalization with LAMP-2 and chloroquine-sensitive degradation of OATP2B1 suggest that the internalized protein is targeted to a lysosomal degradation pathway. With regard to the underlying mechanism inhibition of caveolin/lipid raft-mediated endocytosis failed to prevent OATP2B1 internalization, whereas inhibition of clathrin-mediated processes blocked OATP2B1 sequestration. However, small interfering RNA-mediated clathrin knock-down affected general trafficking of OATP2B1 and resulted in intracellular accumulation in the absence of PMA. In conclusion, our data demonstrate that OATP2B1 function is regulated by PKC-mediated, clathrin-dependent internalization and followed by lysosomal degradation. Furthermore, internalization could be shown in an ex vivo placenta perfusion. Our findings represent a new, rapid mechanism in regulation of human OATPs.

Highlights

Several single nucleotide polymorphisms have been identified within the liver-enriched OATP1B1 transporter with impact on transport function [1, 2]
This general finding for clathrin heavy chain (CHC) knock-down could be verified by Western blot analysis of the cell surface fraction as well as on a functional level (Fig. 6C). None of these treatments had an effect on E-cadherin, which was studied as a control plasma membrane protein. These findings indicated an involvement of CHC in trafficking of organic anion transporting polypeptide 2B1 (OATP2B1) they demonstrated that the knock-down is not useful to study the involvement of CHC in protein kinase C (PKC)-mediated internalization of OATP2B1
We describe a rapid regulation of the organic anion transporting polypeptide 2B1 (OATP2B1) by internalization following PKC activation

Summary

Introduction

Several single nucleotide polymorphisms have been identified within the liver-enriched OATP1B1 transporter with impact on transport function [1, 2]. Prolonged incubation periods up to 72 h resulted in a decline of internalization plays a role in the biological system, we intracellular OATP2B1 and enhanced plasma membrane ver- performed human placenta perfusion studies, a model system sus intracellular localization (supplemental Fig. S2). Western blot analysis correlated with the inhibition of transport activity and revealed a time-dependent decrease in the plasma membrane expression of OATP2B1 (Fig. 5A).

Results

Conclusion