Identification and Characterization of Palmitoylation Sites in the Organic Anion Transporting Polypeptide 1B1 (OATP1B1)

Abstract

<b>Abstract ID 20665</b> <b>Poster Board 180</b> <b>Background:</b> Organic Anion Transporting Polypeptide 1B1 (OATP1B1) is a hepatic transporter that mediates the uptake of numerous therapeutics like statins and antibiotics. Previous studies from our lab suggest that OATP1B1 is localized in membrane microdomains, also known as lipid rafts. Proteins in lipid rafts are frequently modified by S-acylation (also referred to as S-palmitoylation), a reversible post-translational modification that results in the addition of palmitic acid (C16:0) to cysteine residues. Using an acyl-resin-assisted capture (acyl-RAC) assay, we previously demonstrated that OATP1B1 contains S-acylated residues. We also demonstrated that 2-Bromopalmitate (2BP), a commonly used S-palmitoylation inhibitor, impacts OATP1B1 surface localization and function, suggesting that OATP1B1 is palmitoylated. Currently, nothing is known about specific palmitoylation sites in OATP1B1. <b>Aim:</b> We hypothesize that OATP1B1 is palmitoylated at various sites and that this may impact its function and surface expression. <b>Methods:</b> Cysteine palmitoylation was predicted using GPS-palm. Palmitoylation sites of OATP1B1 were also analyzed using mass spectrometry. Identified sites were mutated to alanine residues using site-directed mutagenesis. Transporter expression at the plasma membrane was quantified by a surface biotinylation assay. To assess the function, the uptake of the model substrate estradiol-17β-glucuronide was measured and then normalized for surface expression. The mutants were also analyzed using the acyl-RAC assay and western blotting. <b>Results:</b> Positions Cys24, Cys101, and Cys691 were predicted to be palmitoylated. Palmitoylation of Cys24 was confirmed using proteomics. In addition, mass spectrometry also identified N-palmitoylation at positions Lys19 and/or Lys20. Both OATP1B1 mutants C24A and C691A have an increase in surface expression as compared to wildtype OATP1B1. Normalizing for this surface-expression increase demonstrates that their overall uptake function is decreased compared to wildtype. Mutant C101A showed no changes in both surface expression and function. Interestingly, the acyl-RAC assay demonstrated that OATP1B1 loses its S-acylated residue only when Cys24 is mutated, suggesting that Cys24 is palmitoylated in OATP1B1. <b>Conclusions:</b> Our results show that OATP1B1 is S-palmitoylated at Cys24 and N-palmitoylated at Lys19/Lys20. Inhibition of this post-translational modification may alter the function and surface expression of this hepatic drug uptake transporter. Thus, pathophysiological states where palmitoylation is modified could impact drug targeting and disposition.