Abstract
The sodium taurocholate cotransporting polypeptide (NTCP) is expressed at the basolateral membrane of hepatocytes, where it mediates the uptake of conjugated bile acids and forms the hepatocyte entry receptor for the hepatitis B and D virus. Here, we aimed to identify novel protein–protein interactions that could play a role in the regulation of NTCP. To this end, NTCP was precipitated from HA-tagged hNTCP-expressing HepG2 cells, and chloride channel CLIC-like 1 (CLCC1) and stomatin were identified as interacting proteins by mass spectrometry. Interaction was confirmed by co-immunoprecipitation. NTCP, CLCC1 and stomatin were found at the plasma membrane in lipid rafts, as demonstrated by a combination of immunofluorescence, cell surface biotinylation and isolation of detergent-resistant membranes. Neither CLCC1 overexpression nor its knockdown had an effect on NTCP function. However, both stomatin overexpression and knockdown increased NTCP-mediated taurocholate uptake while NTCP abundance at the plasma membrane was only increased in stomatin depleted cells. These findings identify stomatin as an interactor of NTCP and show that the interaction modulates bile salt transport.
Highlights
The sodium taurocholate cotransporting polypeptide (SLC10A1/NTCP) is a transmembrane glycoprotein expressed solely and at high level at the basolateral membrane of hepatocytes [1]
The second protein we identified is stomatin, a ubiquitously expressed integral membrane protein that is associated with the cytoplasmic face of the plasma membrane via its palmitoylation sites and a short hydrophobic hairpin region [15]
In order identify proteinsinteraction, which couldco-immunoprecipitation regulate NTCP function and its plasma membrane with localization via protein–protein interaction, co-immunoprecipitation assays were combined with mass mass spectrometry analysis
Summary
The sodium taurocholate cotransporting polypeptide (SLC10A1/NTCP) is a transmembrane glycoprotein expressed solely and at high level at the basolateral membrane of hepatocytes [1]. NTCP mediates uptake of conjugated bile acid from the portal vein into hepatocytes, playing an important role in enterohepatic circulation and intra-hepatic bile acid concentration [2,3]. Cholestasis leads to a decrease of NTCP expression and activity. This protective system, which reduces hepatocellular accumulation of bile acid, is mediated by at least two mechanisms. NTCP is repressed at the transcriptional level by the farnesoid X receptor (FXR), the main nuclear bile acid receptor [7]. Activity of FXR is subject to further fine-tuning by various mechanisms, including Sirtuin 1 (SIRT1)-dependent acetylation [8]
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