Sorting of rat liver and ileal sodium-dependent bile  acid transporters in polarized epithelial cells

The rat ileal apical Na+-dependent bile acid transporter (ASBT) and the liver Na+-taurocholate cotransporting polypeptide (Ntcp) are members of a new family of anion transporters. These transport proteins share limited sequence homology and almost identical predicted secondary structures but are localized to the apical surface of ileal enterocytes and the sinusoidal surface of hepatocytes, respectively. Stably transfected Madin-Darby canine kidney (MDCK) cells appropriately localized wild-type ASBT and Ntcp apically and basolaterally as assessed by functional activity and immunocytochemical localization studies. Truncated and chimeric transporters were used to determine the functional importance of the cytoplasmic tail in bile acid transport activity and membrane localization. Two cDNAs were created encoding a truncated transporter in which the 56-amino-acid COOH-terminal tail of Ntcp was removed or substituted with an eight-amino-acid epitope FLAG. For both mutants there was some loss of fidelity in basolateral sorting in that ∼75% of each protein was delivered to the basolateral surface compared with ∼90% of the wild-type Ntcp protein. In contrast, deletion of the cytoplasmic tail of ASBT led to complete loss of transport activity and sorting to the apical membrane. An Ntcp chimera in which the 56-amino-acid COOH-terminal tail of Ntcp was replaced with the 40-amino-acid cytoplasmic tail of ASBT was largely redirected (82.4 ± 3.9%) to the apical domain of stably transfected MDCK cells, based on polarity of bile acid transport activity and localization by confocal immunofluorescence microscopy. These results indicate that a predominant signal for sorting of the Ntcp protein to the basolateral domain is located in a region outside of the cytoplasmic tail. These studies have further shown that a novel apical sorting signal is localized to the cytoplasmic tail of ASBT and that it is transferable and capable of redirecting a protein normally sorted to the basolateral surface to the apical domain of MDCK cells.

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The cytoplasmic tail of CD44 is required for basolateral localization in epithelial MDCK cells but does not mediate association with the detergent-insoluble cytoskeleton of fibroblasts.

The Journal of Cell Biology ◽

10.1083/jcb.121.6.1299 ◽

1993 ◽

Vol 121 (6) ◽

pp. 1299-1310 ◽

Cited By ~ 54

Author(s):

S J Neame ◽

C M Isacke

Keyword(s):

Epithelial Cells ◽

Mdck Cells ◽

Constitutive Expression ◽

Cytoplasmic Tail ◽

Apical Surface ◽

Wild Type ◽

Coated Pits ◽

Triton X ◽

Cortical Cytoskeleton ◽

Human Specific

A number of recent reports on the trafficking of receptor proteins in MDCK epithelial cells have provided evidence that delivery to the basolateral domain requires a specific targeting sequence and that deletion of this sequence results in constitutive expression on the apical surface. To date, these studies have concentrated on receptors which are competent for internalization via the clathrin coated pits. We have examined the localization of a resident plasma membrane protein by transfecting human CD44 into MDCK cells. Using human specific and cross-species reactive antibodies, we show that in MDCK cells both the endogenous and transfected wild-type CD44 are found on the basolateral surface where they are restricted to the lateral domain. Deletion of the CD44 cytoplasmic tail reduces the half life of this mutant protein and causes it to be expressed both on the apical surface and to a significant extent within the cell. We have also used biochemical and morphological analysis to investigate the interaction of CD44 with the cytoskeleton in detergent extracted cells. Strikingly different extraction results were obtained between epithelial and fibroblast cells. However, there is no difference in the Triton X-100 solubility of the transfected wild-type and tail-less CD44 in fibroblasts and both forms of the protein remain associated with the cortical cytoskeleton after Triton X-100 extraction. These results demonstrate that the sequence present in the cytoplasmic domain of CD44 responsible for its distribution in epithelial cells is functionally and spatially separate from the ability of this protein to associate with the cytoskeleton.

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475 Bile acid transport activity and intracellular distribution of PFIC2 mutants in MDCKII cells coexpressing NTCP and BSEP

Hepatology ◽

10.1016/s0270-9139(03)80517-7 ◽

2003 ◽

Vol 38 ◽

pp. 389-389 ◽

Cited By ~ 1

Author(s):

T KAGAWA ◽

K MOCHIZUKI ◽

M HARRIS ◽

N WATANABE ◽

T MINE ◽

...

Keyword(s):

Bile Acid ◽

Intracellular Distribution ◽

Acid Transport ◽

Transport Activity ◽

Bile Acid Transport ◽

Mdckii Cells

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Na(+)-dependent bile acid transport by hepatocytes is mediated by a protein similar to microsomal epoxide hydrolase

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1993.264.3.g528 ◽

1993 ◽

Vol 264 (3) ◽

pp. G528-G534 ◽

Cited By ~ 5

Author(s):

P. Von Dippe ◽

M. Amoui ◽

C. Alves ◽

D. Levy

Keyword(s):

Plasma Membrane ◽

Bile Acid ◽

Amino Acid ◽

Amino Acid Sequence ◽

Epoxide Hydrolase ◽

Sodium Dodecyl ◽

Microsomal Epoxide Hydrolase ◽

Acid Transport ◽

Bile Acid Transport ◽

Sodium Dependent

A protein mediating hepatocyte sodium-dependent bile acid transport across the sinusoidal plasma membrane has been purified by immunoprecipitation with monoclonal antibody (MAb) 25D-1, which specifically recognizes this protein on the surface of intact hepatocytes (Ananthanarayanan et al. J. Biol. Chem. 263: 8338-8343, 1988). The function of this protein was further established by proteoliposome reconstitution (von Dippe et al. J. Biol. Chem. 265: 14812-14816, 1990). NH2-terminal amino acid sequence analysis and amino acid composition revealed this protein to be closely related to the enzyme microsomal epoxide hydrolase (mEH). Both proteins exhibited the same elution times on a reverse-phase high-pressure liquid chromatography column, comigrated with an apparent molecular weight of 49,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and possessed identical isoelectric points of 8.2. The MAb was capable of immunoprecipitating chromatographically purified mEH, as well as a protein derived from the sinusoidal plasma membrane that exhibited mEH activity comparable to that of the protein isolated from the endoplasmic reticulum. The subtilisin fragmentation patterns derived from chromatographically purified mEH and the MAb-precipitated plasma membrane protein were also identical. Hydropathy profile analysis of the amino acid sequence of mEH suggested the presence of four transmembrane domains. The results of these studies indicate that a protein that is involved in mediating sodium-dependent bile acid transport is closely related to mEH.

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Mutations of the basic amino acid transporter gene associated with cystinuria

Biochemical Journal ◽

10.1042/bj3100951 ◽

1995 ◽

Vol 310 (3) ◽

pp. 951-955 ◽

Cited By ~ 30

Author(s):

K Miyamoto ◽

K Katai ◽

S Tatsumi ◽

K Sone ◽

H Segawa ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Transport ◽

Basic Amino Acid ◽

Amino Acid Transporter ◽

Regulatory Subunit ◽

Acid Transport ◽

Transport Activity ◽

Wild Type ◽

Neutral Amino Acid Transporter ◽

Acid Transporter

To investigate the function of a basic and neutral amino acid transporter-like protein (rBAT) which is a candidate gene for cystinuria, we analysed the rBAT gene in cystinuric patients. Patient 1 is a compound heterozygote with mutations in the rBAT gene causing a glutamine-to-lysine transition at amino acid 268, and a threonine-to-alanine transition at amino acid 341, who inherited these alleles from his mother (E268K) and father (T341A), respectively. Injection of T341A and E268K mutant cRNAs into oocytes decreased transport activity to 53.9% and 62.5% of control (L-cystine transport activity in oocytes injected with wild-type rBAT cRNA), respectively. Co-injection of E268K and T341A into oocytes strongly decreased amino acid transport activity to 28% of control. On the other hand, co-injection of wild-type and mutant rBAT did not decrease transport activity. Furthermore, immunological studies have demonstrated that the reduction of amino acid transport is not due to a decrease in the amount of rBAT protein expressed in oocyte membranes. These results indicate that mutations in the rBAT gene are crucial disease-causing lesions in cystinuria. In addition, co-injection experiments suggest that rBAT may function as a transport activator or regulatory subunit by homo- or hetero-multimer complex formation.

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Mutational Analysis of the GXXXG/A Motifs in the Human Na+/Taurocholate Co-Transporting Polypeptide NTCP on Its Bile Acid Transport Function and Hepatitis B/D Virus Receptor Function

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.699443 ◽

2021 ◽

Vol 8 ◽

Author(s):

Massimo Palatini ◽

Simon Franz Müller ◽

Kira Alessandra Alicia Theresa Lowjaga ◽

Saskia Noppes ◽

Jörg Alber ◽

...

Keyword(s):

Bile Acid ◽

Hepatitis B ◽

Mutational Analysis ◽

Hbv Infection ◽

Hek293 Cells ◽

Intact Protein ◽

Receptor Function ◽

Acid Transport ◽

Wild Type ◽

Bile Acid Transport

Homodimerization is essential for plasma membrane sorting of the liver bile acid transporter NTCP and its function as Hepatitis B/D Virus (HBV/HDV) receptor. However, the protein domains involved in NTCP dimerization are unknown. NTCP bears two potential GXXXG/A dimerization motifs in its transmembrane domains (TMDs) 2 and 7. The present study aimed to analyze the role of these GXXXG/A motifs for the sorting, function, and dimerization of NTCP. The NTCP mutants G60LXXXA64L (TMD2), G233LXXXG237L (TMD7) and a double mutant were generated and analyzed for their interaction with wild-type NTCP using a membrane-based yeast-two hybrid system (MYTH) and co-immunoprecipitation (co-IP). In the MYTH system, the TMD2 and TMD7 mutants showed significantly lower interaction with the wild-type NTCP. In transfected HEK293 cells, membrane expression and bile acid transport activity were slightly reduced for the TMD2 mutant but were completely abolished for the TMD7 and the TMD2/7 mutants, while co-IP experiments still showed intact protein-protein interactions. Susceptibility for in vitro HBV infection in transfected HepG2 cells was reduced to 50% for the TMD2 mutant, while the TMD7 mutant was not susceptible for HBV infection at all. We conclude that the GXXXG/A motifs in TMD2 and even more pronounced in TMD7 are important for proper folding and sorting of NTCP, and so indirectly affect glycosylation, homodimerization, and bile acid transport of NTCP, as well as its HBV/HDV receptor function.

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