Characterization of Plasma Membrane Localization and Phosphorylation Status of Organic Anion Transporting Polypeptide (OATP) 1B1 c.521 T>C Nonsynonymous Single-Nucleotide Polymorphism

Abstract

Purpose: Membrane transport protein organic anion transporting polypeptide (OATP) 1B1 mediates hepatic uptake of many drugs (e.g. statins). The OATP1B1 c.521T>C (p. V174A) polymorphism has reduced transport activity. Conflicting in vitro results exist regarding whether V174A-OATP1B1 has reduced plasma membrane localization; no such data has been reported in physiologically relevant human liver tissue. Other potential changes, such as phosphorylation, of the V174A-OATP1B1 protein have not been explored. Current studies characterized the plasma membrane localization of V174A-OATP1B1 in genotyped human liver tissue and cell culture and compared the phosphorylation status of V174A-and wild-type (WT)-OATP1B1.Methods: Localization of V174A-and WT-OATP1B1 were determined in OATP1B1 c.521T>C genotyped human liver tissue (n=79) by immunohistochemistry and in transporter overexpressing human embryonic kidney (HEK) 293 and HeLa cells by surface biotinylation and confocal microscopy. Phosphorylation and transport of OATP1B1 was determined using 32 P-orthophosphate labeling and [ 3 H]estradiol-17β-glucuronide accumulation, respectively.Results: All three methods demonstrated predominant plasma membrane localization of both V174A-and WT-OATP1B1 in human liver tissue and in cell culture. Compared to WT-OATP1B1, the V174A-OATP1B1 has significantly increased phosphorylation and reduced transport.

Genotyping of the OATP1B1 c. 521 T>C Polymorphism from the Formalin-Fixed Paraffin-Embedded (FFPE) Tissue Specimens: An Optimized Protocol

“…Similar as shown in Figure 2, using DNA (50 ng) from biopsy FFPE tissues in a 20 μl volume also yielded successful amplification of the 339 bp PCR product (data not shown). Sequencing of the expected 339 bp PCR products from different tissues was able to distinguish between all three genotypes, c. 521 TT, TC and CC (Figure 3 and also as shown in the original publication [Crowe et al , 2019]). The FFPE tissues from both surgical resection and biopsy specimens are all suitable for genotyping using the current method.…”

“…Also, this method optimized the amount of template DNA from FFPE tissue suitable for PCR. This protocol is reproducible and requires a small amount of tissue, even from biopsy specimens, to yield sufficient PCR products for Sanger-sequencing-based genotyping of OATP1B1 c. 521 T>C. This method has also been successfully used for genotyping of another polymorphism of OATP1B1 c. 388 A>G (Crowe et al , 2019).…”

Post-translational regulation of the major drug transporters in the families of organic anion transporters and organic anion–transporting polypeptides

“…The early investigations reported that the p.174Val>Ala variation was associated with a decreased membrane localization and a decreased Vmax value with no change in Km value (111,112). A recent study, however, reported different findings that the Val174Ala variation does not necessarily reduce the OATP1B1 level at the plasma membrane (113). Instead, the authors noted that Val174Ala variation is associated with a modest increase in protein phosphorylation.…”

Two common polymorphic variants of OATP4A1 as potential risk factors for colorectal cancer

“…Therefore, these results suggested that SNPs in SLCO genes may also alter the transport functionality of the OATPs. For example, a nonsynonymous SNP in OATP1B1 was previously identified to change its phosphorylation status, subcellular localization, and the ability to transport its substrate estradiol glucuronide (49).…”