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Cloning and expression of SLC10A4, a putative organic anion transport protein.

ABSTRACT: AIM:To determine if novel bile acid transporters may be expressed in human tissues. METHODS:SLC10A1 (NTCP) was used as a probe to search the NCBI database for homology to previously uncharacterized ESTs. The homology search identified an EST (termed SLC10A4) that shares sequence identity with SLC10A1 and SLC10A2 (ASBT). We performed Northern blot analysis and RT-PCR to determine the tissue distribution of SLC10A4. SLC10A4 was cloned in frame with an epitope tag and overexpressed in CHO cells to determine cellular localization and functional analysis of bile acid uptake. RESULTS:Northern analysis revealed that SLC10A4 mRNA is ubiquitously expressed in human tissues with the highest levels of mRNA expression in brain, placenta, and liver. In SLC10A4-transfected CHO cells, immunoblotting analysis and immunofluorescence staining demonstrated a 49-kDa protein that is expressed at the plasma membrane and intracellular compartments. Functional analysis of SLC10A4 showed no significant taurocholate uptake in the presence of sodium when compared to untransfected CHO cells. CONCLUSION:To date, we have shown that this protein has no capacity to transport taurocholate relative to SLC10A1; however, given its ubiquitous tissue distribution, it may play a more active role in transporting other endogenous organic anions.

SUBMITTER: Splinter PL

PROVIDER: S-EPMC4087434 | biostudies-literature | 2006 Nov

REPOSITORIES: biostudies-literature

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Cloning and expression of SLC10A4, a putative organic anion transport protein.

Splinter Patrick-L PL Lazaridis Konstantinos-N KN Dawson Paul-A PA LaRusso Nicholas-F NF

World journal of gastroenterology 20061101 42

<h4>Aim</h4>To determine if novel bile acid transporters may be expressed in human tissues.<h4>Methods</h4>SLC10A1 (NTCP) was used as a probe to search the NCBI database for homology to previously uncharacterized ESTs. The homology search identified an EST (termed SLC10A4) that shares sequence identity with SLC10A1 and SLC10A2 (ASBT). We performed Northern blot analysis and RT-PCR to determine the tissue distribution of SLC10A4. SLC10A4 was cloned in frame with an epitope tag and overexpressed i ...[more]

PMID: 17106928

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Project description:Organic anion transport proteins (OATPs) on the basolateral surface of hepatocytes mediate uptake of a number of drugs and endogenous compounds. Previous studies showed that rat OATP1A1 (rOATP1A1) has a postsynaptic density protein, drosophila disc large tumor suppressor, zonula occludens-1 protein (PDZ) consensus binding motif at its C-terminus and binds to PDZ domain containing 1 (PDZK1), which is required for its cell-surface localization. PDZK1 associates with rOATP1A1-containing endocytic vesicles within cells, mediating recruitment of motor proteins required for microtubule-based trafficking to the plasma membrane. rOATP1A4 also traffics to the plasma membrane, although it lacks a PDZ binding consensus sequence. The current study was designed to test the hypothesis that trafficking of rOATP1A4 to the plasma membrane requires its direct interaction with rOATP1A1 resulting in a complex that traffics through the cell in common subcellular vesicles in which the cytosolic tail of rOATP1A1 is bound to PDZK1. We found that 74% of rOATP1A4-containing rat liver endocytic vesicles (n = 12,044) also contained rOATP1A1. Studies in transfected HEK293 cells showed surface localization of rOATP1A1 only when coexpressed with PDZK1 whereas rOATP1A4 required coexpression with rOATP1A1 and PDZK1. Studies in stably transfected HeLa cells that constitutively expressed PDZK1 showed that coexpression of rOATP1A4 with rOATP1A1 resulted in more rapid appearance of rOATP1A4 on the plasma membrane and faster maturation to its fully glycosylated form. Similar results were observed on immunofluorescence analysis of single cells. Immunoprecipitation of rat liver or transfected HeLa cell lysates with rOATP1A1 antibody specifically co-immunoprecipitated rOATP1A4 as determined by western blotting. Conclusion: These studies indicate that optimal rOATP1A4 trafficking to the cell surface is dependent upon coexpression and interaction with rOATP1A1. As rOATP1A1 binds to the chaperone protein, PDZK1, rOATP1A4 functionally hitchhikes through the cell with this complex.

Project description:The canalicular multispecific organic anion transporter (cMOAT), a member of the ATP-binding cassette transporter family, mediates the transport of a broad range of non-bile salt organic anions from liver into bile. cMOAT-deficient Wistar rats (TR-) are mutated in the gene encoding cMOAT, leading to defective hepatobiliary transport of a whole range of substrates, including bilirubin glucuronide. These mutants also have impaired hepatobiliary excretion of GSH and, as a result, the bile flow in these animals is reduced. In the present work we demonstrate a role for cMOAT in the excretion of GSH both in vivo and in vitro. Biliary GSH excretion in rats heterozygous for the cMOAT mutation (TR/tr) was decreased to 63% of controls (TR/TR) (114+/-24 versus 181+/-20 nmol/min per kg body weight). Madin-Darby canine kidney (MDCK) II cells stably expressing the human cMOAT protein displayed >10-fold increase in apical GSH excretion compared with wild-type MDCKII cells (141+/-6.1 pmol/min per mg of protein versus 13.2+/-1.3 pmol/min per mg of protein in wild-type MDCKII cells). Similarly, MDCKII cells expressing the human multidrug resistance protein 1 showed a 4-fold increase in GSH excretion across the basolateral membrane. In several independent cMOAT-transfectants, the level of GSH excretion correlated with the expression level of the protein. Furthermore, we have shown, in cMOAT-transfected cells, that GSH is a low-affinity substrate for the transporter and that its excretion is reduced upon ATP depletion. In membrane vesicles isolated from cMOAT-expressing MDCKII cells, ATP-dependent S-(2,4-dinitrophenyl)glutathione uptake is competitively inhibited by high concentrations of GSH (Ki approximately 20 mM). We concluded that cMOAT mediates low-affinity transport of GSH. However, since hepatocellular GSH concentrations are high (5-10 mM), cMOAT might serve an important physiological function in maintenance of bile flow in addition to hepatic GSH turnover.

Dataset Information

Cloning and expression of SLC10A4, a putative organic anion transport protein.

Publications

Cloning and expression of SLC10A4, a putative organic anion transport protein.

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