Human organic anion transporter hOAT1 forms homooligomers.
Human organic anion transporter hOAT1 belongs to a superfamily of organic anion transporters, which play critical roles in the body disposition of clinically important drugs, including anti-human immunodeficiency virus therapeutics, anti-tumor drugs, antibiotics, anti-hypertensives, and anti-inflammatories. To gain insight into the regulation of hOAT1, detailed information on its structural assembly is essential. In the present study, we investigate the quaternary structure of hOAT1 using combined approaches of chemical cross-linking, gel filtration chromatography, co-immunoprecipitation, cell surface biotinylation, and metabolic labeling. Chemical cross-linking of intact membrane proteins from LLC-PK1 cells stably expressing hOAT1 converted quantitatively hOAT1 monomer to putative trimer and higher order of oligomer, indicating that hOAT1 is present in the membrane as multimeric complexes. When co-expressed in LLC-PK1 cells, FLAG-tagged hOAT1 co-immunoprecipitated with myc-tagged hOAT1. The hOAT1 oligomer was also detected in gel filtration chromatography of total membranes from hOAT1-expressing LLC-PK1 cells. Cell surface biotinylation with membrane-impermeable reagents and metabolic labeling with [(35)S]methionine followed by immunoprecipitation showed that the oligomeric hOAT1 did not contain any other proteins. Taken together, this is the first study demonstrating that hOAT1 exists in the plasma membrane as a homooligomer, possibly trimer, and higher order of oligomer.
Hong
Mei
M
Department of Pharmaceutics, Rutgers, The State University of New Jersey, 08854, USA.
Xu
Wen
W
Yoshida
Takeshi
T
Tanaka
Kunihiko
K
Wolff
Donald J
DJ
Zhou
Fanfan
F
Inouye
Masayori
M
You
Guofeng
G
D000818:Animals; D001692:Biological Transport; D019921:Biotinylation; D002460:Cell Line; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000378:metabolism*; D002845:Chromatography; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D007668:Kidney / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry; D027362:Organic Anion Transport Protein 1 / Q000737:chemistry / Q000502:physiology*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D051381:Rats; D013552:Swine
D003432:Cross-Linking Reagents; D000939:Epitopes; D008565:Membrane Proteins; D027362:Organic Anion Transport Protein 1; D016271:Proto-Oncogene Proteins c-myc