Homo- and hetero-dimeric architecture of the human liver Na⁺-dependent taurocholate co-transporting protein.
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.
Department of Metabolic Diseases, UMC Utrecht, Utrecht, The Netherlands.
Ingrid T G W
IT
Bijsmans
Rianne A M
RA
Bouwmeester
Joachim
J
Geyer
Klaas Nico
KN
Faber
Stan F J
SF
van de Graaf
D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D003432:Cross-Linking Reagents / Q000494:pharmacology; D006801:Humans; D008099:Liver / Q000378:metabolism*; D008954:Models, Biological; D029363:Organic Anion Transporters, Sodium-Dependent / Q000737:chemistry* / Q000378:metabolism*; D055503:Protein Multimerization / Q000187:drug effects / Q000502:physiology*; D020836:Protein Structure, Quaternary / Q000187:drug effects; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D021381:Protein Transport; D051381:Rats; D055571:Secretory Pathway / Q000502:physiology; D027981:Symporters / Q000737:chemistry* / Q000378:metabolism*; D013656:Taurocholic Acid / Q000378:metabolism
D001647:Bile Acids and Salts; D003432:Cross-Linking Reagents; D029363:Organic Anion Transporters, Sodium-Dependent; D021122:Protein Subunits; D027981:Symporters; C071908:sodium-bile acid cotransporter; D013656:Taurocholic Acid