Structural and functional investigation of the organic anion transporting polypeptide 1B1 (OATP1B1)

• Main Author: Taylor-Wells, Jennina Charlotte
• Published: Oxford Brookes University 2013
• Subjects: 572.696
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.616570

Membrane transport proteins are the gatekeepers of the cell membrane, transporting compounds in and out of the cell. The organic anion transporting polypeptides (OATPs) are a family of transport proteins expressed in a variety of tissues, including absorptive I excretory cells of the liver and kidney. OATPs transport an array of endogenous and xenobiotic compounds including bile salts, thyroid hormones, statins and anti-cancer drugs. Therefore this protein family play an important role in cell homeostasis and drug disposition, and are implicated in disease and drug-drug interactions. However there is little information available regarding the structural properties of OATPs, which would reveal how the proteins fold and bind to substrates. To help further this understanding, the topology and signature sequence of the hepatocyte specific isoform OATPI B 1 have been investigated using a HEK293T cell model. OATP I B 1 was expressed in HEK293T cells and transport assayed using the substrate estrone-3-sulfate. Topology prediction programs were employed to map the transmembrane domains (TMs), which predicted a consensus of 12TMs with internal amino (N) and carboxyl (C) termini. The topology was studied experimentally using a FLAG epitope system, whereby the FLAG epitope was inserted into the putative extracellular/intracellular and terminal regions of the protein. Detection of FLAG antibody bind ing was quantified by Juminometry and visualised qualitatively using confocal microscopy. Results revealed that the pred icted internal N and C termini of the protein were external, in contrast to the predictions and current literature. A novel model has been generated from this data, encompassing these results and the current literature. All OATPs contain a conserved 13 amino acid signature sequence (D-X-RW­ (1,V)-GAWW-X-G-(F,L)-L) which spans the putative extracellular loop 3 and TM6, the function of which is not known. Six conservative mutations were made using site-directed mutagenesis; D251E, R253K, W254F, W258/259F and N261A. Kinetic, Iuminometric and immunofluorescence analysis revealed that all mutations except N261A reduced transport and expression on the membrane. The reduction in expression combined with the presence of structurally important arginine and tryptophan residues at the extracellular/TM interface indicate that this sequence may be important for membrane anchoring and/or protein fold ing. This knowledge of OATP structure and function is imperative to the understand ing the role of these proteins in the bind ing of endogenous and xenobiotic compounds.