Is the Organic Anion Transporting Polypeptide (OATP) 1B3 palmitoylated and does palmitoylation affect protein-protein interactions with OATP1B1?

Abstract ID 129603 Poster Board 439 Background: The Organic Anion Transporting Polypeptides 1B1 and 1B3 (OATP1B1 & OATP1B3) are hepatic transporters that share 80% amino acid sequence homology and mediate the uptake of numerous therapeutics like antibiotics and statins. These transporters interact with one another through hetero-oligomerization and can regulate each other's function and surface expression when co-expressed. Various post-translational modifications, e.g. phosphorylation and glycosylation, have been shown to impact OATP1B1 and OATP1B3 function and localization. S-palmitoylation (also known as S-acylation or palmitoylation), is a reversible post-translational modification that adds palmitic acid (C16:0) to cysteine residues and can impact a protein's stability, function, localization, and protein-protein interactions. We previously demonstrated that OATP1B1 is S-palmitoylated on residue Cys24. Due to the high amino acid sequence homology between OATP1B1 and OATP1B3 and their known protein-protein interactions, we speculated that OATP1B3 is also palmitoylated. Aim: We hypothesized that OATP1B3 is palmitoylated at residue Cys24 and that this modification may impact the function, surface expression, and protein interaction between OATP1B1 and OATP1B3. Methods: OATP1B3 cysteine residues of interest were mutated via site-directed mutagenesis to alanine or serine residues. The mutants' ability to undergo S-palmitoylation was assessed using the acyl-resin assisted capture (RAC) assay and western blotting. Uptake of the model substrate cholecystokinin-8 (CCK-8) was measured to determine the functional changes in non-palmitoylated OATP1B3 when expressed alone or with OATP1B1. Surface biotinylation assays quantified both individual transporter expression and co-expression at the plasma membrane. Results: The acyl-RAC assay revealed that OATP1B3 is S-acylated at residue Cys24. OATP1B3-mediated uptake of 0.1 mM CCK-8 for 20 seconds was not altered in the non-palmitoylated mutants (OATP1B3-C24A and -C24S), but preliminary kinetic analysis suggests that the Vmax of these non-palmitoylated mutants is decreased. Co-expression of OATP1B3 with a non-palmitoylated OATP1B1 (OATP1B1-C24A) as well as co-expression of non-palmitoylated OATP1B3-C24A with OATP1B1-C24A resulted in increased uptake of CCK-8. Co-expression of OATP1B1-C24A with OATP1B3, OATP1B1 with OATP1B3-C24A, and OATP1B1-C24A with OATP1B3-C24A resulted in a decrease in surface expression of OATP1B3 when compared to co-expression of the wild-type proteins. Summary and Conclusions: Our results show that, like OATP1B1, OATP1B3 is palmitoylated at Cys24. Co-expression of non-palmitoylated OATP1B1 or OATP1B3 with wild-type proteins or mutants results in changes in their uptake function and surface expression. These findings suggest that palmitoylation regulates protein-protein interactions in these two hepatic drug transporters. Thus, pathophysiological states where palmitoylation is modified could impact drug targeting and disposition.