Identification of Amino Acids Essential for Estrone-3-Sulfate Transport within Transmembrane Domain 2 of Organic Anion Transporting Polypeptide 1B1

Abstract

As an important structure in membrane proteins, transmembrane domains have been found to be crucial for properly targeting the protein to cell membrane as well as carrying out transport functions in transporters. Computer analysis of OATP sequences revealed transmembrane domain 2 (TM2) is among those transmembrane domains that have high amino acid identities within different family members. In the present study, we identify four amino acids (Asp70, Phe73, Glu74, and Gly76) that are essential for the transport function of OATP1B1, an OATP member that is specifically expressed in the human liver. A substitution of these four amino acids with alanine resulted in significantly reduced transport activity. Further mutagenesis showed the charged property of Asp70 and Glu74 is critical for proper function of the transporter protein. Comparison of the kinetic parameters indicated that Asp70 is likely to interact with the substrate while Glu74 may be involved in stabilizing the binding site through formation of a salt-bridge. The aromatic ring structure of Phe73 seems to play an important role because substitution of Phe73 with tyrosine, another amino acid with a similar structure, led to partially restored transport function. On the other hand, replacement of Gly76 with either alanine or valine could not recover the function of the transporter. Considering the nature of a transmembrane helix, we proposed that Gly76 may be important for maintaining the proper structure of the protein. Interestingly, when subjected to transport function analysis of higher concentration of esteone-3-sulfate (50 µM) that corresponds to the low affinity binding site of OATP1B1, mutants of Phe73, Glu74, and Gly76 all showed a transport function that is comparable to that of the wild-type, suggesting these amino acids may have less impact on the low affinity component of esteone-3-sulfate within OATP1B1, while Asp 70 seems to be involved in the interaction of both sites.

Cynomolgus Monkey as a Potential Model to Assess Drug Interactions Involving Hepatic Organic Anion Transporting Polypeptides: In Vitro, In Vivo, and In Vitro-to-In Vivo Extrapolation

“…Based on the results obtained with the three chosen substrates (E17bG, CCK-8, and E3S), it is concluded that cOATPs and hOATPs are qualitatively similar. Such a conclusion is partially supported by the fact that 4 amino acids (Asp70, Phe73, Glu74, and Gly76) located in the transmembrane domain 2 of OATP1B1 are essential to its transport function (Li et al, 2012), and a sequence alignment analysis showed that these four amino acid positions are identical between humans and cynomolgus monkeys for both OATP1B1 and OATP1B3. However, a more quantitative comparison (across different OATPs) requires expression data for each OATP protein in the individual cell lines.…”

Conserved Tryptophan Residues within Putative Transmembrane Domain 6 Affect Transport Function of Organic Anion Transporting Polypeptide 1B1

“…Amino acids within TMs have been shown to play important roles in substrate binding, maintenance of protein stability, and correct folding of proteins (Hong et al, 2004(Hong et al, , 2010Gui and Hagenbuch, 2009;Miyagawa et al, 2009;Li et al, 2012). Studies of single nucleotide polymorphisms have also pointed out that mutants located within TMs often result in functional changes (Kalliokoski and Niemi, 2009).…”

“…Cells in 48-well plates were used for transport measurement as previously described (Li et al, 2012) Cell-Surface Biotinylation and Western Blot. Cell-surface expression of OATP1B1 and its mutants was examined with the membrane-impermeable biotinylation reagent NHS-SS-biotin using a method described previously (Li et al, 2012).…”

“…Previous studies in our laboratory showed that four amino acids within TM2 of OATP1B1 are essential for its uptake of the prototypic substrate estrone-3-sulfate (E-3-S). Asp70 and Phe73 may interact with the substrate, while Glu74 and Gly76 are important in maintaining the proper structure of the transporter protein (Li et al, 2012). According to the computerbased hydropathy analysis, OATP1B1 as well as other OATP members are predicted to share a similar transmembrane domain organization of 12 transmembrane domains and a large extracellular loop 5 with conserved cysteine residues (Hagenbuch and Meier, 2003).…”

“…In a computer-generated model of OATP1B3, an OATP family member that has high homology compared with OATP1B1, it was proposed that TM1, 2, 4, and 5 of the N-terminal half and helices 7, 8, 10, and 11 of the Cterminal half of the uptake transporter face the pore that interacts with the substrate, while TM3, 6, 9, and 12 are largely embedded in the bilayer (Meier-Abt et al, 2005). In our previous study, a model of OATP1B1was generated using Escherichia coli glycerol-3-phosphate transporter (PDB ID 1PW4) as the template (Li et al, 2012). Similar with that of OATP1B3, our model implied that TM6 is mostly embedded within the lipid layer.…”