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

Abstract

OATP1B3 is a 12 transmembrane domain protein expressed at the basolateral membrane of human hepatocytes where it mediates the uptake of numerous drugs and endogenous compounds. Previous western blot results suggest the formation of OATP1B3 multimers. In order to better understand the function of OATP1B3 under normal physiological conditions, we investigated its oligomerization status. We transiently transfected OATP1B3 with a C-terminal His-, FLAG- or HA-tag in HEK293 cells and used co-immunoprecipitation and a Proximity Ligation Assay to detect interactions between the different constructs. All three constructs retained similar transport rates as wild-type OATP1B3. Immunofluorescence experiments indicated that in contrast to wild-type, His- and FLAG-tagged OATP1B3, where the C-terminal end is on the cytoplasmic side of the membrane, the C-terminal end of HA-tagged OATP1B3 is extracellular. After cross-linking, anti-FLAG antibodies were able to pull down FLAG-tagged OATP1B3 (positive control) and co-transfected His- or HA-tagged OATP1B3, demonstrating the formation of homo-oligomers and suggesting that the C-terminal part is not involved in oligomer formation. We confirmed co-localization of His- and FLAG-tagged OATP1B3 in transfected HEK293 cells with the Proximity Ligation Assay. Transport studies with a non-functional OATP1B3 mutant suggest that the individual subunits and not the whole oligomer are the functional units in the homo-oligomers. In addition, we also detected OATP1B3-FLAG co-localization with OATP1B1-His or NTCP-His, suggesting that OATP1B3 also hetero-oligomerizes with other transport proteins. Using the Proximity Ligation Assay with transporter specific antibodies, we demonstrate close association of OATP1B3 with NTCP in frozen human liver tissue. These findings demonstrate that OATP1B3 can form homo- and hetero-oligomers and suggest a potential co-regulation of the involved transporters.

The N-terminal region of organic anion transporting polypeptide 1B3 (OATP1B3) plays an essential role in regulating its plasma membrane trafficking

“…OATP1B3-WT, both cytoplasmic and surface fractions displayed multiple immunoreactive bands with near and above its calculated molecular weight (approximately 80 kD). The presence of multiple immunoreactive bands likely reflects varying extents of post-translational modifications and/or oligomerization as reported previously[19]. While the immunoreactive bands were readily detected in both cytoplasmic and surface fractions of cells transiently expressing OATP1B3-WT, the immunoreactive bands of OATP1B3-Δ28 were detected mainly in the cytoplasmic fractions, but barely detectible in the surface fractions (detectable only after a prolonged exposure, data not shown).…”

Rat Organic Anion Transport Protein 1A1 Interacts Directly With Organic Anion Transport Protein 1A4 Facilitating Its Maturation and Trafficking to the Hepatocyte Plasma Membrane

“…hOATP1B1, another transporter in human liver, has a C‐terminal sequence (ETHC), which is predicted to bind to the first PDZ binding domain of PDZK1 (http://pow.baderlab.org)), whereas hOATP1B3 has a C‐terminal sequence (AAAN), which is predicted not to bind to PDZK1 or other PDZ proteins. Of potential importance, data have been published suggesting that hOATP1B1 and hOATP1B3 may interact with each other when cotransfected into HEK293 cells . Although studies of the trafficking of these human OATPs is beyond the scope of the present study, the paradigms developed in this study will facilitate future elucidation of OATP interactions, trafficking, and function in human liver.…”

“…Of potential importance, data have been published suggesting that hOATP1B1 and hOATP1B3 may interact with each other when cotransfected into HEK293 cells. (40) Although studies of the trafficking of these human OATPs is beyond the scope of the present study, the paradigms developed in this study will facilitate future elucidation of OATP interactions, trafficking, and function in human liver.…”

Treatment with proteasome inhibitor bortezomib decreases organic anion transporting polypeptide (OATP) 1B3-mediated transport in a substrate-dependent manner

“…As shown in the supplemental results ( S7 Fig ), the higher molecular weight bands of FLAG-OATP1B1 and FLAG-OATP1B3 (~250 kD) from the whole cell lysate samples were prominent after boiling, while these bands were minimal without boiling. Recently, OATP1B3 was reported to be able to form homo-oligomers [ 47 ]. In this report, in the absence of crosslinking agent, OATP1B3 eluted from immunoprecipitation without boiling had a major molecular weight of >100 kD (monomer), while in the presence of crosslinking agent, homo-oligomer OATP1B3 was detected at higher molecular weight of >150 kD [ 47 ].…”

“…In this report, in the absence of crosslinking agent, OATP1B3 eluted from immunoprecipitation without boiling had a major molecular weight of >100 kD (monomer), while in the presence of crosslinking agent, homo-oligomer OATP1B3 was detected at higher molecular weight of >150 kD [ 47 ]. This report [ 47 ], together with the results shown in S7 Fig , suggest that the observed higher molecular weight FLAG-OATP1B1 and FLAG-OATP1B3 bands (~250 kD) in the immunoprecipitation samples ( Fig 1A and 1B , lanes 13 and 15) might be related to protein aggregation or oligomerization after boiling [ 45 , 46 ]. Second, in a previous report [ 48 ], treatment with tunicamycin, an inhibitor of protein N-linked glycosylation, markedly reduced the amount of high molecular weight band of OATP1B1.…”