Homo- and hetero-dimeric architecture of the human liver Na+-dependent taurocholate co-transporting protein

Abstract

The NTCP (Na⁺-taurocholate co-transporting protein)/SLC10A [solute carrier family 10 (Nav/bile acid co-transporter family)] 1 is tightly controlled to ensure hepatic bile salt uptake while preventing toxic bile salt accumulation. Many transport proteins require oligomerization for their activity and regulation. This is not yet established for bile salt transporters. The present study was conducted to elucidate the oligomeric state of NTCP. Chemical cross-linking revealed the presence of NTCP dimers in rat liver membranes and U2OS cells stably expressing NTCP. Co-immunoprecipitation of tagged NTCP proteins revealed a physical interaction between subunits. The C-terminus of NTCP was not required for subunit interaction, but was essential for exit from the ER (endoplasmic reticulum). NTCP without its C-terminus (NTCP Y307X) retained full-length wtNTCP (wild-type NTCP) in the ER in a dominant fashion, suggesting that dimerization occurs early in the secretory pathway. FRET (fluorescence resonance energy transfer) using fluorescently labelled subunits further demonstrated that dimerization persists at the plasma membrane. NTCP belongs to the SLC10A protein family which consists of seven members. NTCP co-localized in U2OS cells with SLC10A4 and SLC10A6, but not with SLC10A3, SLC10A5 or SLC10A7. SLC10A4 and SLC10A6 co-immunoprecipitated with NTCP, demonstrating that heteromeric complexes can be formed between SLC10A family members in vitro. Expression of SLC10A4 and NTCP Y307X resulted in a reduction of NTCP abundance at the plasma membrane and NTCP-mediated taurocholate uptake, whereas expression of SLC10A6 or NTCP E257N, an inactive mutant, did not affect NTCP function. In conclusion, NTCP adopts a dimeric structure in which individual subunits are functional. Bile salt uptake is influenced by heterodimerization when this impairs NTCP plasma membrane trafficking.

The Hepatitis B Virus Receptor

“…The central role of NTCP in hepatic uptake of conjugated bile acids was recently demonstrated using NTCP knockout mice, which also lost hepatic binding of the HBV preS1 domain (Slijepcevic et al 2015). Crosslinking and biochemical studies indicate that NTCP may exist as a dimer (Bijsmans et al 2012), although this hypothesis requires further verification. NTCP is complex glycosylated with four potential N-glycosylation sites, two of which (N5 and N11) have been confirmed (Hallen et al 2002).…”

Organic anion transporting polypeptide 1B3 can form homo- and hetero-oligomers

“…Furthermore, there is the potential for protein-protein interactions that could influence transporter function, similar to what has been shown for drug-metabolizing enzymes [18]. It has been demonstrated that some rat transporters expressed in hepatocytes, including NTCP [19] and OCT1, can form homo-oligomers [20]. In addition, a recent study showed that human OATP1B1 may form and function as oligomers [21].…”

Characterization of cognitive impairments and neurotransmitter changes in a novel transgenic mouse lacking Slc10a4

“…Although homology analysis has shown that SLC10A4 belongs to the family sodium-bile acid cotransporters, experiments in rat and human cells either failed to confirm transport activity for bile acids or related molecules (Splinter et al, 2006; Geyer et al, 2008), or reported only low bile-acid transport activity for SLC10A4 (Bijsmans et al, 2012; Abe et al, 2013). The first evidence for a function of SLC10A4 in the brain was reported from the Kullander group (Zelano et al, 2013).…”