From the 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
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ABSTRACT |
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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 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.
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 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 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.
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).
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.
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.
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.
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.
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.
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 H 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.
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 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- 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 ( 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
-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
-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
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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.
1 and
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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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.
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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.
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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.
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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).
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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).
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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.
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 H
resonances of amino acid residues in
-strand
or
-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
-strand or
-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 1 and
2, respectively. The NMR
spectra were processed and analyzed using the NMRPipe (13) and NMRView
(14) programs.
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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
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.
(Y is
Tyr, X is any amino acid, and
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
-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
-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 (
) 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
side chain and the aromatic ring of
tyrosine might be critical for stabilizing the type I
-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
-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).
) positions are unfavorable for the
-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 (
) 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
-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.
-turn unfavorable sequence, the Ntcp basolateral sorting may not
related to the clathrin-adaptor complex pathway as is the case with
many basolateral proteins.
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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.
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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
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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.
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