Organic anion transporting polypeptide 1B3 can form homodimers (1064.17)

Abstract

Organic Anion Transporting Polypeptide 1B3 (OATP1B3) is under normal physiological conditions mainly expressed at basolateral membrane of human hepatocytes where it is thought to mediate uptake of numerous drugs into the liver. Preliminary data from western blots indicated that OATP1B3 could form multimers. Therefore, we investigated if OATP1B3 indeed forms multimers and whether such multimer formation has any effect on transport function. OATP1B3 constructs with a His‐, a Flag‐ or an HA‐tag at the C‐terminal end were constructed and transiently transfected in HEK293 cells. Uptake of the OATP1B3 model substrates estradiol‐17β‐glucuronide (E17βG) and estrone‐3‐sulfate (E3S) was measured, and immunoprecipitation experiments were performed. Transport of E17βG and E3S into HEK293 cells transiently transfected with OATP1B3‐His, OATP1B3‐Flag or OATP1B3‐HA was comparable to HEK293 cells expressing wild‐type OATP1B3. Chemical cross‐linking experiment in HEK293 cells transiently transfected with OATP1B3‐His suggested the formation of OATP1B3 dimers. However, interactions with other proteins could not be excluded. Thus, HEK293 cells were transfected with a mixture of OATP1B3‐Flag and OATP1B3‐HA and immunoprecipitation with an anti‐HA antibody was performed. Anti‐HA antibodies were able to pull down OATP1B3‐HA as well as OATP1B3‐Flag, demonstrating that OATP1B3 indeed can form homodimers. Co‐transfection of wild‐type OATP1B3 with the nonfunctional OATP1B3 mutant K41C resulted in reduced but still detectable transport, suggesting that the dimerized OATP proteins function individually. In conclusion, OATP1B3 can form homodimers and the functional unit is the protein and not the dimer.Grant Funding Source: Supported by NIH Grant GM077336