The Rat Liver Na+/Bile Acid Cotransporter

IMPORTANCE OF THE CYTOPLASMIC TAIL TO FUNCTION AND PLASMA MEMBRANE TARGETING*

An-Qiang SunDagger §, Marco A. Arrese, Lei Zeng||, I'Kyori SwabyDagger , Ming-Ming Zhou||, and Frederick J. SuchyDagger **

From the Dagger  Department of Pediatrics and || Structural Biology Program, Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York, New York 10029-6574 and the  Department of Gastroenterology, Catholic University of Chile School of Medicine, Santiago 6510260, Chile

Received for publication, September 26, 2000, and in revised form, November 29, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To understand the potential functions of the cytoplasmic tail of Na+/taurocholate cotransporter (Ntcp) and to determine the basolateral sorting mechanisms for this transporter, green fluorescent protein-fused wild type and mutant rat Ntcps were constructed and the transport properties and cellular localization were assessed in transfected COS 7 and Madin-Darby canine kidney (MDCK) cells. Truncation of the 56-amino acid cytoplasmic tail demonstrates that the cytoplasmic tail of rat Ntcp is involved membrane delivery of this protein in nonpolarized and polarized cells and removal of the tail does not affect the bile acid transport function of Ntcp. Using site-directed mutagenesis, two tyrosine residues, Tyr-321 and Tyr-307, in the cytoplasmic tail of Ntcp have been identified as important for the basolateral sorting of rat Ntcp in transfected MDCK cells. Tyr-321 appears to be the major basolateral-sorting determinant, and Tyr-307 acts as a supporting determinant to ensure delivery of the transporter to the basolateral surface, especially at high levels of protein expression. When the two Tyr-based basolateral sorting motifs have been removed, the N-linked carbohydrate groups direct the tyrosine to alanine mutants to the apical surface of transfected MDCK cells. The major basolateral sorting determinant Tyr-321 is within a novel beta -turn unfavorable tetrapeptide Y321KAA, which has not been found in any naturally occurring basolateral sorting motifs. Two-dimensional NMR spectroscopy of a 24-mer peptide corresponding to the sequence from Tyr-307 to Thr-330 on the cytoplasmic tail of Ntcp confirms that both the Tyr-321 and Tyr-307 regions do not adopt any turn structure. Since the major motif YKAA contains a beta -turn unfavorable structure, the Ntcp basolateral sorting may not be related to the clathrin-adaptor complex pathway, as is the case for many basolateral proteins.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The process of vectorial transport of bile acids and other ions across hepatocytes is dependent upon the polarized distribution of specific transport mechanisms localized to the plasma membrane of these cells (1-3). Na+-dependent bile acid influx has been demonstrated across the sinusoidal (basolateral) membrane of hepatocyte (4). The cDNAs for liver Na+/taurocholate cotransporting polypeptide (Ntcp)1 have been identified and cloned from several species, including rat, mouse, and human (5-7). Rat liver Ntcp transports conjugated bile acids in a Na+-dependent fashion and is localized on the basolateral surface of hepatocytes (8, 9). This bile acid transporter is only expressed in differentiated mammalian hepatocytes in a developmentally regulated pattern (4). It is a glycoprotein with a seven-transmembrane structure that is similar to the protein superfamily of rhodopsin and the G protein-linked receptors (5, 10, 11). Rat Ntcp contains two N-linked carbohydrate sites at amino terminus and two potential Tyr-based sorting motifs at its carboxyl terminus (Y307-E-K-I and Y321-K-A-A) (5). These potential Tyr-based sorting motifs are conserved in the cytoplasmic tail of Ntcp from other species, such as the human and mouse. However, the mechanisms underlying the tissue-specific basolateral membrane sorting of the bile acid transporter are unknown.

Hagenbuch and Meier (7) have identified a short mouse liver Na+-dependent bile acid cotransporter (Ntcp2) in which the last 45 amino acid residues were missing compared with wild type (WT) Ntcp. Ntcp2 is presumed to be the result of alternative splicing. This potential alternative splicing structure (sites) on the genomic DNA was also found in Ntcp genes from rat and human liver. The physiological function of this tailless Ntcp isoform, Ntcp2, is unknown. Previous studies from our laboratory demonstrated that removing of the 56-amino acid cytoplasmic tail from rat Ntcp resulted in accumulation of the truncated form intracellularly and loss of the fidelity of basolateral membrane sorting in transfected Madin-Darby canine kidney (MDCK) cells (12).

The present studies were performed to understand the potential function of the cytoplasmic tail of Ntcp and to determine the basolateral sorting mechanisms for this transporter. Green fluorescent protein-fused wild type Ntcp and mutant rat Ntcps were constructed, and the function and cellular localization were assessed in transfected COS 7 and MDCK cells. The results from transport kinetics and substrate analog inhibition studies demonstrated that removal of the entire cytoplasmic tail from Ntcp did not change its bile acid transport function. Confocal microscopy and polarized taurocholate transport studies showed that, in contrast to WT Ntcp, most of the truncated Ntcp protein accumulated intracellularly in transfected COS 7 cells. Replacement of the Tyr-321 and Tyr-307 residues with alanines in the potential Tyr-based sorting motifs of cytoplasmic tail of Ntcp redirected the tyrosine to alanine (Y/A) mutant transporters to the apical domain of transfected MDCK cells. However, this apical surface localization of the Y/A mutant could be abolished by tunicamycin treatment. Two-dimensional NMR spectroscopy of a 24-mer peptide corresponding to the sequence from Tyr-307 to Thr-330 on the cytoplasmic tail of Ntcp confirms that both Tyr-321 and Tyr-307 regions do not adopt any turn structure. The above results suggest that 1) the 56-amino acid cytoplasmic tail of rat Ntcp is important for plasma membrane delivery, 2) both Tyr-321 and Tyr-307 residues mediated the specific basolateral surface sorting, 3) both Tyr-321 and Tyr-307 regions do not adopt any turn structure, and 4) N-linked carbohydrate groups can act as a apical sorting signal to direct the Y/A mutant transporter to the apical surface.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Construction of Mutant and Green Fluorescent Protein (GFP)-fused Chimeric cDNA-- Removal of the 56-amino acid cytoplasmic tail from rat hepatic Ntcp was done by using a PCR-based strategy to modify rat Ntcp coding sequence as described previously (12). PCR amplifications were carried out using a PTC-100TM Programmable Thermal Controller (MJ Research, Inc., Watertown, MA). Chimeric molecules that fuse the mutant or wild type rat Ntcp to GFP were made using similar PCR-based strategies. The full-length cDNA coding sequences of wild type or mutant rat Ntcp were used as templates. The PCR-generated mutant and wild type rat Ntcp cDNA products were subcloned into a green fluorescent protein vector, pEGFPN2 (CLONTECH, Palo Alto, CA), using standard techniques. These chimeras were made using a forward primer 5'-CGAAGCTTATGGAGGTGCACAACGTATCAGCC-3', which was designed to anneal to 5'-end coding sequence of rat Ntcp cDNA with an HindIII restriction site (bold type), and a reverse primer 5'- CCGGATCCCATTTGCCATCTGACCAGAATTCAGGCCATTAGGGG -3', containing codons that anneal to sequences 3' end region of rat Ntcp cDNA with a BamHI restriction site (bold type). For cytoplasmic tail truncated Ntcp, the same forward primer was used, whereas a reverse primer 5'-GGTGGATCCCGCACCGGAAGATAATGATGATGAG-3', which contained codons that anneal to the sequence A296 to C306 region of rat Ntcp cDNA with a BamHI restriction site (bolded) was used. These restriction sites were compatible with the pEGFP N2 polylinker. The PCR products were gel-purified, digested at sites incorporated at the ends of PCR products, and ligated directly to pEGFP N2 digested with the same restriction enzymes to produce chimeras. All plasmid constructs were checked for correct orientation by restriction digestion analysis. The positive clones containing the wild type or mutant cDNA inserts were verified by DNA cycle sequencing using a Perkin Elmer, GeneAmp 9600, ABI Prism 377 DNA Sequencer at the DNA Core Facility, Mount Sinai School of Medicine. These positive clones were used for further study.

The QuikChangeTM site-directed mutagenesis Kit (Stratagene, La Jolla, CA) was used to convert codons for Tyr-307 and Tyr-321 to alanine residues according to manufacturer's directions with minor modification. The GFP-fused Ntcp chimera was used as template. A Y307A substitution was made using a forward primer 5'-C1030TTCCGGTGCGCCGAGAAAATCAAGCCTCC1059-3', which annealed to the coding sequence in the Tyr-307 region, and a compatible reverse primer, in place of the codon for Tyr-307 was converted to alanine residue (underlined). A Y321A substitution was made using a forward primer 5'-G1063GACCAAACAAAAATTACCGCCAAAGCTGCTGCAACTG1100-3', which annealed to the coding sequence in the Tyr-321 region, and a compatible reverse primer, in place of the codon for Tyr-321 was converted to alanine residue (underlined). The constructs of Y307A and Y321A double substitutions were made using a Y307A mutated construct as template and using the same primer for Y321A substitution.

Cell Culture and Transfection-- COS 7 (SV40 transformed monkey kidney fibroblast) cells were maintained in complete Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% (v/v) fetal bovine serum, 50 units/ml penicillin, 50 µg/ml streptomycin, and 2 mM L-glutamine. COS 7 cells were transiently transfected with plasmids containing wild type or mutant rat Ntcp cDNA using LipofectinTM reagent (Life Technologies, Inc.) as described previously (12). Transfected cells were harvested 24-48 h later for bile acid transport and confocal microscopy analysis.

All MDCK II cells were grown in a humidified incubator at 37 °C under 5% CO2 atmosphere in complete minimum essential medium (Life Technologies, Inc.) that was supplemented with 10% (v/v) fetal bovine serum, 50 units/ml penicillin, 50 µg/ml streptomycin, and 2 mM L-glutamine. MDCK II cells were stably transfected with plasmids containing wild type or mutant rat Ntcp cDNAs using LipofectinTM reagent as described by the manufacture. Transfected cells were grown 10-15 days in media containing G418 (Geneticin (0.9 mg/ml), Life Technologies, Inc.). Thereafter, stably transfected colonies were selected. For polarity studies, monolayers of polarized MDCK epithelial cells were produced as described previously (12). Briefly, cells were seeded on Transwell filter inserts (0.4-µm pore size, PET track-etched membrane; Falcon, Franklin Lakes, NJ) at a density of ~1.5 × 105 cells/6.4-mm filter. Experiments were conducted 4-6 days after seeding. To enhance the gene expression, the stably transfected MDCK cells were preincubated in 10 mM Na+-butyrate for 15 h at 37 °C.

Northern Blots-- Northern blot hybridization was done according to standard technique. Total RNA was extracted from transfected COS 7 or MDCK cells and purified by TRIzol reagent (Life Technologies, Inc.). RNAs (10 µg/lane) were subjected to electrophoresis on a 1% agarose gel (containing 2.2 M formaldehyde, 40 mM MOPS, 10 mM Na+-acetate, 1 mM EDTA, pH 7.0), and blotted onto a GeneScreen membrane (PerkinElmer Life Sciences). Hybridization was carried out in 50% formamide buffer at 42 °C overnight with 32P-labeled full-length cDNA probe (1 × 107 cpm) encoding Ntcp protein, and detected by autoradiography.

Western Blots-- Total proteins extracted from transfected cells were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose. The Western blotting for wild type and cytoplasmic tail truncated rat Ntcp was carried out as described previously (12). GFP-fused chimeric proteins were detected using a rabbit GFP polyclonal antibody (IgG fraction, CLONTECH, Palo Alto, CA) at a dilution of 1:1000 and subsequently with horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma) at a dilution of 1:2000. Horseradish peroxidase activity was visualized by enzyme chemiluminescence (ECL) (Amersham Pharmacia Biotech). COS cell plasma membrane fractions were purified and separated by SDS-polyacrylamide gel electrophoresis as described previously (13). The wild type and truncated Ntcp proteins were detected by Westen blotting. The quantitation of the protein intensity was carried out by using Metamorph software (University Imaging Corp., West Chester, PA).

Bile Acid Influx Transport Assay-- Na+-dependent taurocholate (TC) influx and polarized transport analysis were performed as described previously (12).

Fluorescence Confocal Microscopy-- Indirect immunofluorescence microscopy for wild type and cytoplasmic tail truncated rat Ntcp was carried out as described previously (12). Confocal microscopy of GFP-fused chimeras was performed on a confluent monolayer of transfected cells cultured on glass coverslips. Glass coverslip-grown cells were rinsed three times with phosphate-buffered saline, fixed for 7 min in 100% methanol at -20 °C, and rinsed four times with phosphate-buffered saline, and then mounted with Aquamount (BDH Laboratory Supplies, Poole, United Kingdom). Fluorescence were examined with a Leica TCS-SP (UV) 4-channel confocal laser scanning microscope in the Imaging Core Facility Microscopy Center, Mount Sinai School of Medicine. The 488-nm wavelength line of an argon laser and the 568-nm wavelength line of a krypton laser were used. The cell monolayer was optically sectioned every 0.5 µm. Image resolution using a Leica 63× and/or 100× Neofluor objective and Leica TCS-SP software was 512 × 512 pixels.

NMR Spectroscopy-- All NMR spectra were acquired at 30 °C on a Bruker DRX-500 NMR spectrometer. The NMR sample of the Ntcp peptide of 10 mM was prepared in a 100 mM phosphate buffer of pH 6.5 in 90% H2O, 5% 2H2O, and 5% Me2SO-d6. The two-dimensional 1H/15N and 1H/13C heteronuclear single quantum coherence (HSQC) spectra were acquired using the nonisotope-enriched peptide with 96 and 1024 complex points in omega 1 and omega 2, respectively. The NMR spectra were processed and analyzed using the NMRPipe (14) and NMRView (15) programs. A 24-mer peptide, YEKIKPPKDQTKITYKAAATEDAT, corresponding to the sequence from Tyr-307 to Thr-330 on the cytoplasmic tail of rat Ntcp, was synthesized by the Howard Hughes Medical Institute Biopolymer/Keck Foundation Biotechnology Resource Laboratory (Yale University, New Haven, CT). The crude peptide was purified by reverse phase HPLC on a YMC C-18 column. The peak fractions were subjected to matrix-assisted laser desorption ionization mass spectrometry and analytical HPLC.

Tunicamycin Treatment-- Treatment of tunicamycin that abolishes the glycosylation of viral glycoproteins was performed as described previously (16). Briefly, the MDCK cells stably transfected with Tyr-321 and Tyr-307 both mutated rat Ntcp (YYAA) cDNA were incubated with 2 µg/ml tunicamycin for 15 h and followed by 12 µg/ml for 2 h at 37 °C. Following incubation, bile acid transport and fluorescence confocal microscopy analyses were utilized to verify the cell surface distribution of the mutant transporters. Inhibition of Ntcp glycosylation by tunicamycin treatment was confirmed by Western blotting.

Statistics Analysis-- Most of the results were expressed as mean value ± S.E. and examined by Student's t test. Results of different groups or categories were compared using the unpaired t test.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression of Truncated Rat Ntcp in COS 7 Cells-- Previous studies from our laboratory have demonstrated that removal of the 56-amino acid cytoplasmic tail from rat Ntcp resulted in a truncated protein that largely accumulated intracellularly in transfected MDCK cells (12). Moreover, a tailless Ntcp isoform, Ntcp2, has been isolated from mouse liver, but the function and cellular localization of this protein has not been defined (7). Thus, the cytoplasmic tail of Ntcp (Fig. 1A) may contain membrane sorting information and may be of functional importance. To understand further the possible functional role of the cytoplasmic tail of Ntcp, which would be relevant to a potential physiologic role for Ntcp2, the transport properties and cellular localization of a truncated mutant rat liver Ntcp were examined in transfected COS 7 cells. Northern blot analysis was performed with total RNA isolated from COS 7 cells transduced with plasmids containing cDNAs encoding for either the wild type or truncated Ntcp. Probing with a 32P-labeled Ntcp cDNA detected a single message of about 1.7 kilobase pairs in transfected COS 7 cells (data not shown). No hybridization was observed with total RNA from nontransfected COS 7 cells. mRNA from the truncated mutant (NL) was slightly smaller than that of the wild type Ntcp (data not shown). Bile acid transport studies showed that [3H]taurocholate influx was stimulated in both WT and truncated Ntcp cDNA-transfected COS 7 cells in the presence of a sodium but not a choline-containing buffer (Fig. 1B). However, the truncated transporter (NL) had much lower transport activity compared with the wild type transporter (Fig. 1B). These results suggested two possibilities: 1) the cytoplasmic tail of Ntcp was essential for bile acid translocation across the membrane, or 2) the cytoplasmic tail provided a signal for plasma membrane localization. To address these questions, indirect immunofluorescence microscopy was done on confluent monolayers of transfected COS 7 cells. When these cells were viewed enface, the WT Ntcp was predominantly located on the plasma membrane (Fig. 1C). In contrast, in cells expressing the truncated Ntcp (NL), most of the protein accumulated intracellularly, probably in the endoplasmic reticulum and Golgi apparatus. Immunoblotting of plasma membrane proteins isolated from transfected COS 7 cells confirmed that only ~10% of the truncated Ntcp (NL) was sorted to the plasma membrane compared with wild type Ntcp (Fig. 1D).



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Fig. 1.   Expression of truncated rat Ntcp in transfected COS 7 cells. Panel A, amino acid sequence of rat Ntcp cytoplasmic tail. Panel B, sodium dependence of taurocholate uptake in COS 7 cells transfected with wild type and truncated Ntcp. COS-7 cells were either untransfected or transfected with WT Ntcp or cytoplasmic tail truncated construct (NL). TC influx was measured with 10 µM [3H]TC in the presence of buffers containing Na+ or choline (116 mM) at 37 °C for 10 min. Data are presented as picomoles/mg of protein/min and represent the mean value ± S.E. of three independent experiments performed in triplicate. Panel C, immunofluorescence localization of transfected COS 7 cells grown on coverslips. The COS 7 cells were transiently transfected with WT or cytoplasmic tail truncated construct (NL), respectively. Transfected COS 7 cells were cultured on glass coverslips. An antibody against the NH2 terminus of rat Ntcp was used according to the methods described previously (12). Selected images were analyzed for immunofluorescence distribution of COS 7 cells nontransfected (left panel), transfected with wild type Ntcp (central panel), or cytoplasmic tail truncated Ntcp (NL) (right panel). Panel D, quantitation of the steady state surface distribution of wild type and truncated Ntcp in transfected COS 7 cells. The inset panel depicts a Western blot of plasma membrane fractions from COS 7 cells transfected with wild type and cytoplasmic tail truncated Ntcp (NL). The quantitation of the protein intensity was carried out by using Metamorph software.

Kinetic Analysis of Taurocholate Uptake-- To verify further the relationship between the cytoplasmic tail of Ntcp and its importance to transport function, the kinetics of taurocholate uptake was analyzed in transfected COS 7 cells. In these cells, equilibrium for the transport process was achieved between 10 and 15 min at 37 °C. No significant uptake was observed in the presence of a sodium gradient at 0 °C or with a choline gradient at both 37° and 0 °C (data not shown). The kinetics of bile acid uptake by wild type and truncated rat Ntcp in transfected COS 7 cells were measured in primary cultures 72 h after transfection. As shown in Fig. 2, in the presence of a sodium gradient, [3H]taurocholate uptake increased with substrate concentration and exhibited saturable kinetics. WT Ntcp exhibited [3H]taurocholate uptake with an apparent Km of 30.4 ± 6.6 µM and a Vmax of 393.4 ± 47.0 pmol/mg of protein/min in transfected COS 7 cells. The transport process mediated by the truncated Ntcp showed a similar Km value of 43.8 ± 3.2 µM, but a markedly reduced Vmax of 32.4 ± 3.9 pmol/mg/min. This value was only 10% of that observed with WT Ntcp. These results indicate that removal of the cytoplasmic tail does not significantly alter the substrate binding affinity of the transporter. However, the lower Vmax is likely related to a markedly reduced amount of the protein reaching the plasma membrane.



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Fig. 2.   Saturation kinetics for taurocholate uptake into COS 7 cells transiently transfected with wild type and cytoplasmic tail truncated construct (NL). Apparent Km values for TC influx were determined at 37 °C in transport buffer containing 116 mM NaCl by monitoring the initial rates for accumulation of the indicated concentrations of [3H]TC. Curves represent the best fit to the Michaelis-Menten equation with Km(app) values of 30.4 ± 9.6 and 43.8 ± 13.2 µM for the wild type and truncated Ntcp (NL), respectively. The Vmax values are 393.4 ± 47.0 and 32.4 ± 3.9 pmol/mg of protein/min for the WT and truncated Ntcp (NL), respectively.

To further characterize the importance of the cytoplasmic tail of Ntcp to bile acid transport, studies were done to examine the effects of several bile acid analogues and organic anions on taurocholate transport by transfected COS 7 cells. The COS 7 cells were incubated in the presence or absence of 100 µM unlabeled bile acids or other organic anion competitors. Taurocholate uptake in the absence of a competitor was set at 100% and all values measured relative to this level of activity. Fig. 3 demonstrates that the competitive inhibitors (cholate ~ 45%, taurodeoxycholate ~ 30%, taurochenodeoxycholate ~ 25%, and probenecid ~ 100%), and the noncompetitive inhibitor bromosulfophthalein (~ 15%) revealed similar effects on the initial rate of taurocholate uptake in COS 7 cells expressing either the wild type or truncated transporter. These results indicate that the cytoplasmic tail is not important for substrate binding specificity.



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Fig. 3.   Comparison of the effects of substrate analogs on taurocholate uptake in COS 7 cells transfected with wild type and cytoplasmic tail truncated Ntcp. Uptake of 10 µM [3H]taurocholate (10 min) was assessed in the presence of a 116 mM NaCl with and without the addition of unlabeled substrate analogs to the incubation media. The final concentration of each analog was 100 µM. Taurocholate uptake of wild type Ntcp in the presence of 116 mM sodium was set at 100%, and all values were graphed relative to this level. Data represent mean ± S.E. (bars) of triplicate determinations from three different cell-culture preparation. C, cholate; TDC, taurodeoxycholate; TCDC, tauro-chenodeoxycholate; Pro, probenecid; BSP, bromo-sulfthalein.

Effects of Protein Kinase A on Taurocholate Uptake-- Previous studies indicate that hepatic sodium taurocholate cotransport is stimulated by dibutyryl cyclic AMP (Bt2cAMP) via protein kinase A (17). Experiments were then done to compare the activation of transport activity by Bt2cAMP in COS 7 cells expressing either the wild type or truncated Ntcp. Bt2cAMP stimulated a significant increase (about 24%, p < 0.05) in taurocholate uptake by transfected cells expressing wild type Ntcp (Fig. 4). In contrast, 100 µM Bt2cAMP did not stimulate taurocholate uptake in COS 7 cells expressing the truncated Ntcp. These results indicate that, as suggested previously, the cytoplasmic tail may be important for regulation of Ntcp membrane localization via protein kinase A stimulation.



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Fig. 4.   Comparison of the effects of dibutyryl cAMP treatment on taurocholate uptake in COS 7 cells transfected with wild type and truncated transporters. The transfected COS-7 cells were pre-incubated with (+) or without (-) 100 µM Bt2cAMP (DiBcAMP) for 10 min following which TC uptake was measured. Taurocholate uptake in the absence of Bt2cAMP was set at 100% control. Each bar represents mean ± S.E. of triplicate determinations from three cell-culture preparations. (for wild type Ntcp,Bt2cAMP/+Bt2cAMP, p < 0.05).

Construction and Expression of Green Fluorescent Protein-fused Wild Type and Y/A Mutant Ntcp-GFP in COS 7 Cells-- To determine the importance of two potential Tyr-based sorting motifs in the cytoplasmic tail of Ntcp, a series of mutations were created in these sequences which potentially represent signals for sorting to the basolateral membrane. Three mutants were made by converting Tyr-307 (Y307A-GFP), Tyr-321 (Y321A-GFP), and both Tyr-307 and Tyr-321 (YYAA-GFP) to alanines by site directed mutagenesis. These mutated transporters, as well as wild type Ntcp, were fused with green fluorescent protein at the amino terminus to follow the intracellular localization of these proteins in transfected COS 7 and MDCK II cell lines. Northern and Western blotting were used to verify the cellular expression of the GFP-fused proteins.

First, the bile acid transport activity and cell surface expression of these chimeric proteins were examined in transfected COS 7 cells. Fig. 5 shows that GFP-fused Ntcp is functionally similar to the wild type protein in its capacity for sodium-dependent transport. Similar to the truncated Ntcp (NL), the GFP-fused truncated Ntcp chimera (NL-GFP) had a markedly reduced initial transport rate (data not shown). However, all of the chimeras containing point mutations, Y307A-GFP, Y321A-GFP, and YYAA-GFP, demonstrated transport activity similar to the wild type Ntcp-GFP (Fig. 5). To confirm the steady state surface expression of the GFP-fused transporter chimeras, transfected COS 7 cells were cultured on glass coverslips and then examined by fluorescence confocal microscopy. When viewed enface, Ntcp-GFP and all of the three Y/A mutants were detected on the plasma membrane of transfected COS 7 cells (Fig. 6). In contrast, in cells expressing NL-GFP, fluorescence was predominantly localized to the cytosolic portion of the cell near the nucleus (Fig. 6). These studies confirm that, similar to wild type Ntcp, the GFP-fused Y/A mutants were targeted to the plasma membrane of transfected COS 7 cells.



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Fig. 5.   Comparison of taurocholate uptake in COS 7 cells transfected with GFP-fused wild type and Y/A mutant transporters. COS 7 cells expressing WT and mutant Ntcp proteins were grown on 12-well plates. The Na+-dependent taurocholate uptake was measured by incubating cells in 10 µM [3H]taurocholate (with Na+ buffer) at 37 °C for 10 min. Data are presented as picomoles/mg of protein/min and represent the mean value ± S.E. of three independent experiments performed in triplicate. COS 7 cells were transfected with Ntcp-GFP, Y307A-GFP, Y321A-GFP, and YYAA-GFP.



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Fig. 6.   Effects of Y/A mutation of potential Tyr-based signal motifs on membrane localization in transfected COS 7 cells. Transfected COS 7 cells were grown on glass coverslips and fixed with methanol at -20 °C for 7 min. Selected images were analyzed for fluorescence distribution of COS 7 cells nontransfected (left top panel) and transfected with pEGFPN2 vector, Ntcp-GFP, cytoplasmic tail truncated Ntcp-GFP (NL-GFP) (right top panel), and Y/A mutated transporters (Y321A-GFP, YYAA-GFP, and Y307A-GFP, lower panel).

Expression of Green Fluorescent Protein-fused Wild Type and Mutant Ntcp in MDCK II Cells-- To examine whether the transporters with point mutations of the possible tyrosine-based sorting signals were sorted to the basolateral domain, the Y/A mutant chimeras were stably expressed in MDCK II cells. Fluorescent images both enface and in x-z cross-section were then gathered using laser scanning confocal microscopy. To further confirm the localization of these proteins within this cell line, bile acid uptake was measured across the apical and basolateral membrane domains of stably transfected MDCK cells, which were grown to confluence on permeable Transwell filter inserts. When membrane localization of the Y307A-GFP mutant was examined by both fluorescence confocal microscopy and polarized bile acid uptake, the Y307A-GFP protein was predominantly localized to the basolateral surface of MDCK cells when cells expressed the protein at a lower level (e.g. without sodium butyrate treatment) (Fig. 7). At higher levels of protein expression stimulated by sodium butyrate treatment, the Y307A-GFP protein was randomly distributed to both apical and basolateral surface of stably transfected MDCK cells (Fig. 7). In contrast, the other chimeric proteins, the Y321A-GFP with mutation of Tyr-321 and the YYAA-GFP with mutations of both tyrosine motifs showed predominantly apical domain localization in stably transfected MDCK cells (Fig. 8) with or without sodium butyrate treatment. These results suggest that the Tyr-321 signal motif is the major basolateral sorting determinant and that Tyr-307 signal motif acts as a supporting signaling mechanism, which is functional at high levels of protein expression to ensure delivery of the transporter to the basolateral surface.



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Fig. 7.   Effects of Tyr-307 mutation on steady state basolateral localization of GFP-fused Ntcp. Panel A, polarity of Na+-dependent taurocholate uptake was performed on a confluent monolayer of stably transfected MDCK cells cultured on permeable Transwell filter inserts. The MDCK cells stably transfected with Y307A-GFP were preincubated with (+) or without (-) 10 mM Na+-butyrate for 15 h at 37 °C. Taurocholate influx was measured from upper chamber (apical) or low chamber (basolateral) with 10 µM [3H]taurocholate in the presence of Na+ buffers at 37 °C for 10 min. Panel B, fluorescence confocal microscopic localization of Y307A-GFP expressed in stably transfected MDCK cells. Fluorescence photomicrographs were performed on the confluent monolayer of stably transfected cells cultured on glass coverslips until confluent. Confocal enface and x-z cross-sectional photomicrographs were of MDCK cells stably transfected with Y307A-GFP and were incubated with (+) or without (-) 10 mM Na+-butyrate for 15 h at 37 °C before fixed with methanol at -20 °C for 7 min.



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Fig. 8.   Effects of Tyr-321 and Tyr-321/307 double mutations on the steady state basolateral localization of rat Ntcp. Panel A, polarity of Na+-dependent taurocholate uptake was performed on a confluent monolayer of stably transfected MDCK cells cultured on permeable Transwell filter inserts. The MDCK cells stably transfected with Y307A-GFP or YYAA-GFP were preincubated with 10 mM Na+-butyrate for 15 h at 37 °C. Taurocholate influx was measured from upper chamber (apical) or low chamber (basolateral) with 10 µM [3H]taurocholate in the presence of Na+ buffers at 37 °C for 10 min. Panel B, fluorescence confocal microscopic localization of Y321A-GFP or YYAA-GFP expressed in stably transfected MDCK cells. Fluorescence photomicrographs were performed on the confluent monolayer of stably transfected cells cultured on glass coverslips until confluent. Confocal enface and x-z cross-sectional photomicrographs were of MDCK cells stably transfected with Y321A-GFP or YYAA-GFP and were incubated with 10 mM Na+-butyrate for 15 h at 37 °C before fixed with methanol at -20 °C for 7 min.

Two-dimensional NMR Secondary Structure Analysis of Synthetic Peptide Corresponding to 24-mer Basolateral Sorting Signal-- To determine whether the Tyr-based basolateral sorting motif region on the cytoplasmic tail of Ntcp assumes any unique conformation, NMR structural analysis of a 24-mer peptide, YEKIKPPKDQTKITYKAAATEDAT, corresponding to the sequence from Tyr-307 to Thr-330 on the cytoplasmic tail of this protein was conducted. The two-dimensional 1H/15N correlation spectrum (Fig. 9A) shows that the backbone amide resonances of the peptide exhibit very narrow chemical shift dispersion along the proton dimension (less than 1 ppm) between 7.7 and 8.4 ppm. These results suggest that the peptide residues are most likely to be in a random coil structure. In addition, the two-dimensional 1H/13C HSQC spectrum of the peptide (Fig. 9B) also reveals that Halpha resonances of the amino acid residues all reside in a range between 3.5 and 4.7 ppm, which is below the residual water signal at 4.76 ppm in the spectrum. It is known that Halpha resonances of amino acid residues in beta -strand or beta -turn would appear in the range above the water chemical shift (18), i.e. greater than 4.76 ppm; thus, it is clear that this peptide does not contain beta -strand or beta -turn conformation.



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Fig. 9.   Two-dimensional NMR spectrum of a 24-mer peptide corresponding to the potential tyrosine-based basolateral sorting motifs from the cytoplasmic tail of rat Ntcp. The NMR sample of the Ntcp peptide of 10 mM was prepared in a 100 mM phosphate buffer of pH 6.5 in 90% H2O, 5% 2H2O, and 5% Me2SO-d6. The two-dimensional 1H/15N (A) and 1H/13C (B) HSQC spectra were acquired using the nonisotope-enriched peptide with 96 and 1024 complex points in omega 1 and omega 2, respectively. The NMR spectra were processed and analyzed using the NMRPipe (13) and NMRView (14) programs.

Effects of N-Linked Glycosylation on Membrane Localization of the Y/A Mutant Transporters-- Previous studies have demonstrated that N-linked carbohydrate groups may act as an apical sorting signal (19, 20). This raises the question as to whether N-linked carbohydrates in the Ntcp molecule can direct the apical localization of the Y/A mutant of Ntcp. Therefore, the effect of glycosylation of Ntcp on apical sorting of Y/A mutant was examined by inhibiting glycosylation of Ntcp with tunicamycin. Two potential sites for glycosylation have previously been defined on the amino terminus of Ntcp (5). After treatment of tunicamycin, the transport of bile acids occurred randomly across the apical and basolateral surfaces of MDCK cells stably transfected with the Y/A mutant (YYAA-GFP) (Fig. 10). Confocal microscopy confirmed localization of the mutant transporters to both membrane domains. These results indicate that when basolateral sorting signals are removed from the cytoplasmic tail of Ntcp, the N-linked carbohydrate groups become predominant as a sorting signal and are able to redirect the Y/A mutant protein to the apical surface of MDCK cells.



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Fig. 10.   Effect of tunicamycin treatments on the apical localization of the YYAA mutant transporter chimera. Polarized taurocholate transport assay was assessed on confluent monolayer of MDCK cells stably transfected with mutant chimeras grown on permeable Transwell inserts. The stably transfected MDCK cells were incubated with 2 µg/ml tunicamycin for 15 h and followed by 12 µg/ml for 2 h at 37 °C. Following incubation, bile acid transport activity was assayed at 10 µM [3H]TC, at 37 °C for 10 min.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The purpose of these studies was to determine the importance of the cytoplasmic tail of Ntcp for transport activity and for targeting of the protein to the basolateral domain of the plasma membrane. Removal of the cytoplasmic tail of Ntcp led to a marked decrease in Vmax for the transport process, which was largely a result of accumulation of the truncated protein intracellularly. Our studies also demonstrated that truncation of the cytoplasmic tail of Ntcp did not change its substrate binding affinity and specificity for transport of bile acids and other organic anions. All of these results are of importance since a truncated form of the transporter, Ntcp2, has been cloned from mouse liver (7). This protein with a shortened COOH terminus also mediates sodium-dependent bile acid transport but its functional importance and cellular localization are unknown. The current studies would indicate that this protein may be located within the cell where it may contribute to intracellular transport of bile acids.

Previous studies have shown that treatment of hepatocytes with cAMP increased Na+-dependent bile acid transport by increasing the amount of Ntcp in basolateral membranes but not in liver homogenates (17, 21, 22). Ntcp content in an endosomal fraction was reduced, suggesting that cAMP increases the Vmax for bile acid transport at least in part by translocating Ntcp from endosomes to plasma membranes. Immunoblot analysis with phosphoamino antibodies showed that Ntcp is a serine/threonine and not a tyrosine phosphoprotein and that cAMP inhibited both serine and threonine phosphorylation (21, 22). Thus, cAMP-mediated dephosphorylation of Ntcp leads to increased retention of the transporter in the plasma membrane. Our results now show that truncation of the cytoplasmic tail of Ntcp abolished the increase in cAMP stimulation of transport activity in the fraction of the protein still sorted to the plasma membrane. This result indicates that the cytoplasmic tail of Ntcp may contain this important phosphorylation site.

Protein basolateral sorting signals, such as Tyr-based and dileucine motifs and surface loop structures, have been described for a variety of proteins, such as virus glycoproteins (23), cell adhesion molecules (24, 25), and many receptors (26, 27). Most of these basolateral signals are located in the cytoplasmic tails of these proteins. In many cases, basolateral localization signals are colinear with internalization signals (28-30). However, it has been shown that the two sorting processes are differentially sensitive to various point mutations and not all of the tyrosine residues are involved in these processes (31-33). For example, the Tyr-based signal in the cytoplasmic tail of VSV G protein is an efficient cytoplasmic basolateral-targeting signal but does not serve as a internalization signal (23). The 17-juxtamembrane cytoplasmic residues of the polymeric immunoglobulin receptor contain an autonomous basolateral-targeting signal that does not mediate rapid endocytosis (34). In the case of the transferrin receptor, the single cytoplasmic Tyr is important for endocytosis, but is not necessary for basolateral targeting (29, 35). Moreover, the Tyr-dependent motifs can be interpreted differentially in various polarized epithelial cell types. The H,K-ATPase beta  subunit, which contains a tyrosine-based motif in its cytoplasmic tail, was restricted to the basolateral membrane in MDCK cells, but was localized to the apical membrane in LLC-PK1 cells (36). Multiple signal motifs have also been identified, e.g. three endocytosis motifs, a tyrosine (YKGL765) motif, a leucine-isoleucine (LI760) motif, and a phenylalanine (Phe790) signal, in the furin cytoplasmic domain (37). The endocytosis of furin may occur via an AP-2/clathrin-dependent pathway. All of these data suggest that multiple pathways mediate the basolateral membrane sorting, and different adaptors may involve these processes.

In the case of the Tyr-based sorting signal, a crucial tetrapeptide element of this targeting motif has be represented by the sequence Y-X-X-phi (Y is Tyr, X is any amino acid, and phi  is an amino acid with a bulky hydrophobic side chain (such as leucine, isoleucine, phenylalanine, methionine, valine)) (32, 38). The structure predictions have indicated that the Tyr-based sorting motif, as well as other non-Tyr-based basolateral sorting motifs, contains an apparent beta -turn structure (34, 39, 40). Data from two-dimensional NMR analysis of synthetic peptides containing known Tyr-based internalization motifs showed that the tyrosine residue lies in most cases within a conserved structural feature, a tight turn, with the tyrosine within the first or last (i.e. fourth) position of the turn (20, 41). These results suggest that the beta -turns may be a common feature of basolateral signals, including both those that do and do not overlap with endocytosis signals. The ability of a particular signal to function as a basolateral signal and/or endocytosis signal might then be determined by the presence and location of specific side-chains (32, 34). Mutagenesis studies, such as alanine scanning, demonstrated that the tyrosine and Tyr+3 (phi ) residues are very critical for its function (38, 42). The structure of wild type peptide of LVLR reveals a very close apposition of the side chains, suggesting that an interaction between the phi  side chain and the aromatic ring of tyrosine might be critical for stabilizing the type I beta -turn (41). In general, the presence of hydrophobic residues at the X positions was unfavorable for the turn structure and sorting function. This conclusion is supported by mutagenesis studies (31) and the low frequency of hydrophobic residues at those positions in most naturally occurring YXXØ signals (see Table II in Ref. 32). The preference for polar or charged residues could indicate a participation of those residues in interactions with like groups on the surface of the adaptor subunit (32). The structural analysis and binding studies demonstrated that the beta -turn structure is important for the binding with clathrin-adaptor complexes for the membrane targeting and recycling of the proteins by endocytosis (19, 31, 32, 41).

Two Tyr-based sorting motifs, Y321-K-A-A and Y307-E-K-I, are found in the cytoplasmic tail of rat Ntcp (see Fig. 1A). Our results demonstrate that, in contrast to the removal of the cytoplasmic tail, mutation at Tyr-321 and Tyr-307 did not change the targeting of Ntcp to the plasma membrane in nonpolarized COS 7 and polarized MDCK cells. However, both Tyr-321 and Tyr-307 are critical for the polarized basolateral membrane sorting of Ntcp in polarized MDCK cells. Tyr-321 appears to be the major determinant for the basolateral sorting of Ntcp. Replacement of Tyr-321 with alanine residue redirected the Y/A mutant protein to the apical surface of transfected MDCK cells. Tyr-307 may act as a supporting basolateral sorting determinant to ensure delivery of the transporter to the basolateral surface, especially at high levels of protein expression. However, unlike all of the virus glycoproteins and receptors reported previously, these two Tyr-based signal motifs of Ntcp have novel amino acid components and location. Tyr-321 motif has triple alanine residues on the position of Y+2, +3, and +4. The double alanines at Y+2 and +3 (phi ) positions are unfavorable for the beta -turn structure and have not been found in any naturally occurring basolateral sorting motifs so far. Mutagenesis studies for other proteins also demonstrated that replacement of the amino acid residues at Y+2 and Y+3 (phi ) positions with alanine residues could abolish or decrease the sorting signal function (23, 31, 38, 42, 43). On the other hand, the Tyr-307 motif has a standard Tyr-based sorting motif structure and is located on the boundary of the plasma membrane, but appears to play only a minor role on the basolateral sorting of Ntcp. In a previous study, the transferrin receptor internalization signal YXRF was shown to retain its activity only when separated by at least seven residues from the transmembrane region (44). However, the study of Aroeti et al. (34) suggests that the cytoplasmic residues R655-H-R657 can be quite close to the plasma membrane, only two amino acids away, and still mediate basolateral sorting of the pIgR. All of these data suggest that Ntcp basolateral targeting motif, specially the Y321-K-A-A, may not have a beta -turn structure and does not take the clathrin-adaptor complex pathway. Two-dimensional NMR spectroscopy of the 24-mer peptide corresponding to the sequence containing the Tyr-321 and Tyr-307 basolateral sorting motifs confirms that both Tyr-321 and Tyr-307 region do not adopt any turn structure. It is notable that the carbohydrate groups have been reported as an apical sorting signal in glycoproteins (19, 20). When the basolateral sorting signals are mutated, the apical signal may become functionally active and direct the protein to the apical surface of cells. Our results support this possibility. When the basolateral sorting motifs are removed from cytoplasmic tail of rat Ntcp, the N-linked carbohydrate groups in the amino terminus become functionally active as an apical sorting determinant and redirect these Y/A mutant transporters to the apical surface of stably transfected MDCK cells.

In summary, our results demonstrate that the cytoplasmic tail of rat Ntcp is involved membrane delivery of this protein in nonpolarized and polarized cells. Both Tyr-321 and Tyr-307 are important for the basolateral sorting of rat Ntcp in transfected MDCK cells and these two motifs do not adopt any turn structure. When the two Tyr-based basolateral sorting motifs have been removed, the N-linked carbohydrate groups act as an apical determinant directing the Y/A mutants to the apical surface of transfected MDCK cells. Since the major basolateral sorting determinant Y321-K-A-A contains a beta -turn unfavorable sequence, the Ntcp basolateral sorting may not related to the clathrin-adaptor complex pathway as is the case with many basolateral proteins.


    ACKNOWLEDGEMENTS

We thank Rachita Salkar for assistance with initiation of this work. Confocal laser scanning microscopy was performed at the MSSM-CLSM core facility, supported with funding from National Institutes of Health Shared Instrumentation Grant 1 S10 RR0 9145-01 and National Science Foundation Major Research Instrumentation Grant DBI-9724504.


    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant HD20632 (to F. J. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence may be addressed: Dept. of Pediatrics, Box 1664, Mount Sinai Medical School, One Gustave L. Levy Pl., New York, NY 10029-6574. Tel.: 212-241-2366; Fax: 212-426-1972; E-mail: sun_an-qiang@smtplink.mssm.edu.

** To whom correspondence may be addressed: Dept. of Pediatrics, Box 1198, Mount Sinai Medical School, One Gustave L. Levy Pl., New York, NY 10029-6574. Tel.: 212-241-6933; Fax: 212-241-6933; E-mail: frederick_suchy@smtplink.mssm.edu.

Published, JBC Papers in Press, December 8, 2000, DOI 10.1074/jbc.M008797200


    ABBREVIATIONS

The abbreviations used are: Ntcp, rat liver Na+/taurocholate cotransporting polypeptide; NL, 56 amino acid residues from carboxyl terminus truncated rat Ntcp; GFP, green fluorescent protein; COS 7, SV40-transformed monkey kidney fibroblast cells; MDCK, Madin-Darby canine kidney; Ntcp-GFP, GFP-fused rat Ntcp; Y307A-GFP, a mutant rat Ntcp in which the tyrosine 307 residue was replaced with alanine and fused with green fluorescent protein; Y321A-GFP, a mutant rat Ntcp in which the tyrosine-321 residue was replaced with alanine and fused with green fluorescent protein; YYAA-GFP, a mutant rat Ntcp in which both tyrosine 307 and tyrosine 321 residues were replaced with alanines and fused with green fluorescent protein; TC, taurocholate; HPLC, high performance liquid chromatography; Bt2cAMP, dibutyryl cyclic AMP; WT, wild type; PCR, polymerase chain reaction; HSQC, heteronuclear single quantum coherence; MOPS, 4-morpholinepropanesulfonic acid.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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