title: Oligomerization Study of Human Organic Anion Transporting Polypeptide 1B1.
abstract: Organic anion-transporting polypeptides play important roles in the uptake of various endogenous and exogenous compounds. It has been proposed that OATP family members, as membrane proteins, may form oligomers. However, oligomerization status of OATPs is still largely unclear. In the present study, HEK293 cells stably expressing OATP1B1 were generated to investigate the oligomerization status of the transporter. Chemical cross-linking and coimmunoprecipitation experiments revealed that OATP1B1 may form homo-oligomers, possibly through disulfide bonds. When wild-type OATP1B1 was coexpressed with a loss-of-function mutant W258A, cells showed reduced uptake of prototypic substrate estrone-3-sulfate (ES). Interestingly, such a coexpression did not affect OATP1B1 transport activity of high concentrations ES, implicating that oligomerization status may affect only the high affinity component of ES. OATP1B1 possesses three GXXXG motifs that have been associated with protein dimerization in other membrane proteins. When glycine residues were replaced with alanine, G219A and G393A showed drastically reduced uptake function. Further studies revealed that G219A has a similar association capability to that of the wild-type, while mutation at Gly393 may affect oligomerization status of the transporter. Kinetic analysis showed that both G219A and G393A have a dramatically reduced Vmaxfor ES uptake. Kmof G219A was increased while that of G393A exhibited a decreased value for high affinity component of ES binding. Our studies demonstrated that OATP1B1 may function as oligomers in the high affinity site of ES while acting as monomers for the low affinity binding component of the substrate.
authors.affiliation: College of Life Sciences, South China Agricultural University , Guangzhou 541642, China.
authors.forename: Chunxu
authors.initials: C
authors.lastname: Ni
authors.affiliation: College of Life Sciences, South China Agricultural University , Guangzhou 541642, China.
authors.forename: Xuan
authors.initials: X
authors.lastname: Yu
authors.affiliation: College of Life Sciences, South China Agricultural University , Guangzhou 541642, China.
authors.forename: Zihui
authors.initials: Z
authors.lastname: Fang
authors.affiliation: College of Life Sciences, South China Agricultural University , Guangzhou 541642, China.|Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms , Guangzhou 541642, China.
authors.forename: Jiujiu
authors.initials: J
authors.lastname: Huang
authors.affiliation: College of Life Sciences, South China Agricultural University , Guangzhou 541642, China.|Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms , Guangzhou 541642, China.
authors.forename: Mei
authors.identifier: 0000-0001-9219-2844
authors.initials: M
authors.lastname: Hong
mesh_terms: D001692:Biological Transport / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004970:Estrone / Q000031:analogs & derivatives / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D027381:Liver-Specific Organic Anion Transporter 1 / Q000378:metabolism*; D027361:Organic Anion Transporters / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D058105:Polymerization
chemical_list: D027381:Liver-Specific Organic Anion Transporter 1; D027361:Organic Anion Transporters; D010455:Peptides; C503999:SLCO1B1 protein, human; D004970:Estrone; C017296:estrone sulfate
keywords: GXXXG motif; drug transporter; oligomerization; organic anion transporting polypeptide; uptake function