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Interaction of three regiospecific amino acid residues is required for OATP1B1 gain of OATP1B3 substrate specificity.

ABSTRACT: The human organic anion-transporting polypeptides OATP1B1 (SLCO1B1) and OATP1B3 (SLCO1B3) are liver-enriched membrane transporters of major importance to hepatic uptake of numerous endogenous compounds, including bile acids, steroid conjugates, hormones, and drugs, including the 3-hydroxy-3-methylglutaryl Co-A reductase inhibitor (statin) family of cholesterol-lowering compounds. Despite their remarkable substrate overlap, there are notable exceptions: in particular, the gastrointestinal peptide hormone cholecystokinin-8 (CCK-8) is a high affinity substrate for OATP1B3 but not OATP1B1. We utilized homologous recombination of linear DNA by E. coli to generate a library of cDNA containing monomer size chimeric OATP1B1-1B3 and OATP1B3-1B1 transporters with randomly distributed chimeric junctions to identify three discrete regions of the transporter involved in conferring CCK-8 transport activity. Site-directed mutagenesis of three key residues in OATP1B1 transmembrane helices 1 and 10, and extracellular loop 6, to the corresponding residues in OATP1B3, resulted in a gain of CCK-8 transport by OATP1B1. The residues appear specific to CCK-8, as the mutations did not affect transport of the shared OATP1B substrate atorvastatin or the OATP1B1-specific substrate estrone sulfate. Regions involved in gain of CCK-8 transport by OATP1B1, when mapped to the crystal structures of bacterial transporters from the major facilitator superfamily, are positioned to suggest these regions could readily interact with drug substrates. Accordingly, our data provide new insight into the molecular determinants of the substrate specificity of these hepatic uptake transporters with relevance to targeted drug design and prediction of drug-drug interactions.

SUBMITTER: DeGorter MK

PROVIDER: S-EPMC3319192 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Interaction of three regiospecific amino acid residues is required for OATP1B1 gain of OATP1B3 substrate specificity.

DeGorter Marianne K MK Ho Richard H RH Leake Brenda F BF Tirona Rommel G RG Kim Richard B RB

Molecular pharmaceutics 20120306 4

The human organic anion-transporting polypeptides OATP1B1 (SLCO1B1) and OATP1B3 (SLCO1B3) are liver-enriched membrane transporters of major importance to hepatic uptake of numerous endogenous compounds, including bile acids, steroid conjugates, hormones, and drugs, including the 3-hydroxy-3-methylglutaryl Co-A reductase inhibitor (statin) family of cholesterol-lowering compounds. Despite their remarkable substrate overlap, there are notable exceptions: in particular, the gastrointestinal peptide ...[more]

PMID: 22352740

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Project description:Influenza A viruses possess two glycoprotein spikes on the virion surface: hemagglutinin (HA), which binds to oligosaccharides containing terminal sialic acid, and neuraminidase (NA), which removes terminal sialic acid from oligosaccharides. Hence, the interplay between these receptor-binding and receptor-destroying functions assumes major importance in viral replication. In contrast to the well-characterized role of HA in host range restriction of influenza viruses, there is only limited information on the role of NA substrate specificity in viral replication among different animal species. We therefore investigated the substrate specificities of NA for linkages between N-acetyl sialic acid and galactose (NeuAcalpha2-3Gal and NeuAcalpha2-6Gal) and for different molecular species of sialic acids (N-acetyl and N-glycolyl sialic acids) in influenza A viruses isolated from human, avian, and pig hosts. Substrate specificity assays showed that all viruses had similar specificities for NeuAcalpha2-3Gal, while the activities for NeuAcalpha2-6Gal ranged from marginal, as represented by avian and early N2 human viruses, to high (although only one-third the activity for NeuAcalpha2-3Gal), as represented by swine and more recent N2 human viruses. Using site-specific mutagenesis, we identified in the earliest human virus with a detectable increase in NeuAcalpha2-6Gal specificity a change at position 275 (from isoleucine to valine) that enhanced the specificity for this substrate. Valine at position 275 was maintained in all later human viruses as well as swine viruses. A similar examination of N-glycolylneuraminic acid (NeuGc) specificity showed that avian viruses and most human viruses had low to moderate activity for this substrate, with the exception of most human viruses isolated between 1967 and 1969, whose NeuGc specificity was as high as that of swine viruses. The amino acid at position 431 was found to determine the level of NeuGc specificity of NA: lysine conferred high NeuGc specificity, while proline, glutamine, and glutamic acid were associated with lower NeuGc specificity. Both residues 275 and 431 lie close to the enzymatic active site but are not directly involved in the reaction mechanism. This finding suggests that the adaptation of NA to different substrates occurs by a mechanism of amino acid substitutions that subtly alter the conformation of NA in and around the active site to facilitate the binding of different species of sialic acid.

Project description:Organic anion transporting polypeptide (OATP) 1B3 is a drug transporter expressed at the basolateral membrane of human hepatocytes. Along with other transporters, including OATP1B1, Na+/taurocholate cotransporting polypeptide (NTCP), and organic cation transporter (OCT) 1, it is responsible for the uptake of endo- and xenobiotics into hepatocytes. Our previous studies demonstrated that OATP1B3 can form hetero-oligomers with OATP1B1 in human embryonic kidney 293T (HEK293) cells and with NTCP in both HEK293 cells and frozen human liver sections. To further characterize the hetero-oligomerization of OATP1B3, we investigated OCT1 as a potential interacting partner and determined the functional consequences of OATP1B3 hetero-oligomerization. We demonstrated interactions between OATP1B3 and OCT1 by coimmunoprecipitation with an anti-OATP1B3 antibody from human hepatocytes. In addition, we visualized the interaction using the proximity ligation assay in both HEK293 cells and in frozen human liver sections. We investigated the functional consequences of OATP1B3 hetero-oligomerization by measuring the OATP1B3 plasma membrane expression and the uptake of the OATP1B3 selective substrate cholecystokinin-8 (CCK-8) in the absence and presence of OATP1B1, NTCP, and OCT1. A significant decrease of OATP1B3 plasma membrane expression was observed after coexpression with OCT1, whereas coexpression with OATP1B1 or NTCP resulted in an increase of plasma membrane expression. With respect to transport, coexpression of OCT1 increased the apparent turnover rate of OATP1B3, whereas coexpression of OATP1B1 or NTCP decreased it. These findings demonstrated that coexpression of OATP1B3 with OATP1B1, NTCP, and OCT1 in HEK293 cells results in a transporter-dependent modification of OATP1B3-mediated CCK-8 transport and suggest that functional results obtained in single transporter overexpressing cell lines over- or underestimate OATP1B3 function in human hepatocytes. SIGNIFICANCE STATEMENT: Coexpression of organic anion transporting polypeptide (OATP) 1B3 with organic cation transporter (OCT) 1, Na+/taurocholate cotransporting polypeptide, or OATP1B1 in human embryonic kidney 293T cells affects its expression level and function. When OCT1 is knocked down in human hepatocytes, function of OATP1B3 goes up. These results suggest that protein-protein interactions can affect the expression and function of the involved proteins, and thus single transporter expression systems might lead to over- or underestimation of drug-drug interactions.

Dataset Information

Interaction of three regiospecific amino acid residues is required for OATP1B1 gain of OATP1B3 substrate specificity.

Publications

Interaction of three regiospecific amino acid residues is required for OATP1B1 gain of OATP1B3 substrate specificity.

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