Membrane Trafficking of the Human Organic Anion-Transporting Polypeptide C (hOATPC)
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The human organic anion transporting polypeptide C (OATPC) is one of the major transport proteins involved in the enterohepatic circulation of bile salts and plays an important role in vectorial transport of organic anions and drugs across hepatocytes.
Materials and Methods
In this study, the effects of biological reagents on the membrane localization of OATPC were investigated by confocal microscopy and estrone-3-sulfate transport.
Our results demonstrated that the functional membrane expression of fluorescent chimera OATPC-GFP was achieved in non-polarized (COS7 and HEK293) and polarized (MDCK) cells. Both brefeldin A (a Golgi complex disruptor) and bafilomycin A1 (an inhibitor of vacuolar H+-ATPase) treatment significantly decreased the polarized membrane trafficking and markedly reduced the uptake of estrone-3-sulfate (∼40–90%) in OATPC-GFP transfected cells, suggesting that membrane sorting of hOATPC-GFP was mediated by Golgi complex and vacuolar H+-ATPase-related vesicle transport pathways. Treatment with 8-Br-cAMP (a cAMP analog) stimulated OATPC-GFP membrane localization and enhanced estrone-3-sulfate uptake by ∼20%. The protein kinase A (PKA) inhibitors (H89 and KT5720), but not a PKG inhibitor, blocked the polarized membrane expression of OATPC-GFP and reduced estrone-3-sulfate transport activity. The simultaneous treatment of cells with PKA activator/inhibitor and bafilomycin A1 demonstrated that bafilomycin A1 did not change the effects of 8-Br-cAMP and H89 on the membrane localization of OATPC-GFP compared with the use of 8-Br-cAMP and H89 alone.
These data suggest that a cAMP-PKA sensitive membrane sorting pathway for OATPC-GFP is independent of the vacuolar H+-ATPase associated (bafilomycin A1 sensitive) vesicle mediated membrane sorting pathway. In contrast, with combined treatment with brefeldin A, neither the PKA-activator (8-Br-cAMP) nor the inhibitor (H89) further altered the plasma membrane expression and transport activity of OATPC-GFP compared with brefeldin A treatment alone. These data suggest that the cAMP-PKA regulation of OATPC membrane expression involves the Golgi complex. When the Golgi apparatus was disrupted by brefeldin A treatment, the effects of cAMP-PKA on the Golgi-to-basolateral surface sorting process of OATPC was also diminished. In summary, the plasma membrane localization of human OATPC is mediated by Golgi complex and vacuolar H+-ATPase vesicle mediated membrane sorting pathways. cAMP-PKA regulates sorting process through the Golgi complex but not the vacuolar H+-ATPase associated vesicular pathway.
Key wordshuman OATPC membrane trafficking organic anion transporter protein kinase A
green fluorescent protein
GFP-fused human OATPC
human organic anion transporting polypeptide-C
protein kinase G inhibitor
The authors gratefully acknowledge Dr. Wen-Sheng Chen (Yale Liver Center Yale University School of Medicine New Haven CT) for the initial amplification of the human OATPC cDNA. This work was supported in part by the National Institutes of Health Grants 5R37HD020632-21 (to F. J. S.), DK 25636 (to J.L.B.), and the DK P30-34989 (to Yale Liver Center). Confocal laser scanning microscopy was performed at the MSSM-CLSM core facility supported with funding from the NIH-NCI shared resources grant (5R24 CA095823-04), NSF Major Research Instrumentation grant (DBI-9724504), and NIH shared instrumentation grant (1 S10 RR0 9145-01).
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