From the Department of Microbiology and Immunology, Kimmel Cancer
Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 and the Service de Rhumatologie, Centre de Recherche
Clinique, Université de Sherbrooke, Sherbrooke, Québec,
Canada
Received for publication, October 13, 2000, and in revised form, November 26, 2000
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ABSTRACT |
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The thromboxane A2 receptor
(TP) is a G protein-coupled receptor that is expressed as two
alternatively spliced isoforms, Cell surface receptors provide a primary mechanism by which cells
perceive their environment. Many cell surface receptors are dynamically
regulated and often undergo a process of endocytic sorting (1). For
some receptors (e.g. G protein-coupled and growth factor),
sorting is often initiated by hormone binding, whereas for others
(e.g. low density lipoprotein and transferrin), the
receptors undergo continuous or tonic internalization and recycling.
Recent studies have demonstrated that several
GPCRs1 including the CXCR4,
thyrotropin, M2 muscarinic, and thrombin receptors also
undergo tonic internalization (2-5). Although no particular motif
responsible for tonic internalization of GPCRs has been identified,
tyrosine-containing (YXX Thromboxane has been implicated in a number of cardiovascular,
bronchial, and kidney diseases (9, 10). It is produced by the
sequential metabolism of arachidonic acid by cyclooxygenase and
thromboxane synthase following activation of a variety of cell types
including platelets, macrophages, and vascular smooth muscle cells
(11). Thromboxane is a strong activator of platelet aggregation and
smooth muscle cell proliferation and mediates its effects via
interaction with a specific GPCR. The thromboxane A2
receptor (TP) is encoded by a single gene that is alternatively spliced
in the carboxyl terminus resulting in two variants, TP In a previous study, we demonstrated that TP Cell Culture and Expression Systems--
Human embryonic kidney
cells (HEK293) were maintained in Dulbecco's modified Eagle's Medium
(DMEM, Life Technologies, Inc.) supplemented with 10% fetal bovine
serum. P388D1, A431, Mia Paca (a human pancreatic carcinoma cell line
of epithelial morphology), COS-1 and CHO cells were obtained from ATCC
and grown as recommended by the supplier. Cells were kept in a
humidified atmosphere of 95% air, 5% CO2 at 37 °C.
Transfections were done with Fugene-6 (Roche Molecular Biochemicals)
following the manufacturer's recommendations.
Construction of Epitope-tagged Mutant TXA2
Receptors--
cDNAs for TP Agonist-induced Internalization Assays--
For quantitation of
receptor internalization, ELISA assays were performed as described
(15). Cells were plated at 6 × 105 cells per 60-mm
dish, transfected with 6 µg of DNA and split after 24 h into 6 wells of a 24-well tissue culture dish coated with 0.1 mg/ml
poly(L-lysine) (Sigma). After another 24 h, the cells
were washed once with phosphate buffered saline (PBS) and incubated in
DMEM at 37 °C for several minutes. The TP agonist U46619 was added
at a concentration of 100 nM in prewarmed DMEM to the
wells, the cells were incubated for 2 h at 37 °C, and reactions
were stopped by removing the medium and fixing the cells in 3.7%
formaldehyde in Tris-buffered saline (TBS) for 5 min at 22 °C. The
cells were washed three times with TBS and nonspecific binding was
blocked with TBS containing 1% bovine serum albumin (BSA) for 45 min
at room temperature. The first antibody (monoclonal HA 101R from Babco
or FLAG M1 from Sigma) was added at a dilution of 1:1000 in TBS/BSA for
1 h at 22 °C. The cells were washed three times with TBS and
then reblocked for 15 min at 22 °C. Incubation with goat
anti-mouse-conjugated alkaline phosphatase (Bio-Rad) diluted 1:1000 in
TBS/BSA was carried out for 1 h at 22 °C. The cells were washed
three times with TBS, and a colorimetric alkaline phosphatase substrate
was added. When adequate color change was reached, 100-µl samples
were taken for colorimetric readings. Cells transfected with pcDNA3
alone were studied concurrently to determine background, and all
experiments were done in triplicate.
Immunofluorescence Microscopy--
Cells (HEK293, P388D1, Mia
Paca, A431, and CHO) were grown in 35-mm dishes on coverslips and
transfected as described above with 2 µg of DNA/well. After 48 h, cells were incubated with FLAG M1 antibody (1:500 dilution) for
1 h at 4 °C in DMEM supplemented with 1% BSA and 1 mM CaCl2. Cells were washed twice with PBS
containing 1 mM CaCl2 and then treated with 100 nM U46619 for 1 h at 37 °C in DMEM with 20 mM HEPES, pH 7.4, 0.5% BSA, 1 mM
CaCl2. The cells were then fixed with 3.7%
formaldehyde/PBS for 15 min at 22 °C, washed with
PBS/CaCl2, and permeabilized with 0.05% Triton X-100/PBS/CaCl2 for 10 min at 22 °C. Nonspecific binding
was blocked with 0.05% Triton X-100/PBS/CaCl2 containing
5% nonfat dry milk for 30 min at 37 °C. Goat anti-mouse fluorescein
isothiocyanate (FITC)-conjugated secondary antibody (Molecular Probes)
was then added at a dilution of 1:150 for 1 h at 37 °C. The
cells were washed six times with permeabilization buffer with the last
wash left at 37 °C for 30 min. Finally, the cells were fixed with
3.7% formaldehyde as described. Coverslips were mounted using
Slow-Fade mounting medium (Molecular Probes) and examined by microscopy on a Nikon Eclipse E800 fluorescence microscope using a Plan Fluor 60×
objective under oil immersion. Cells expressing the lowest levels of
transfected proteins, but clearly above those of nonexpressing cells,
were chosen for view. Images were collected using the QED Camera
software and processed with Adobe Photoshop v. 3.0. The images were
then imported into the NIH Image 1.62b27f software and black and white
binary images were used for quantitation of fluorescence. Measurements
were made separately of areas corresponding to the intracellular
content (whole cell minus the plasma membrane) and to the whole cell.
The percentage of internalized receptors was then calculated as the
intracellular to whole cell fluorescence ratio × 100. Results
shown represent the mean ± S.E. of three independent experiments
where immunofluorescence of at least ten cells was evaluated in each experiment.
Recycling Studies--
The recovery of receptors at the cell
surface was evaluated using a modified version of the
immunofluorescence microscopy procedure described above. Following
internalization, cells were stripped of M1 antibody with two quick
washes of cold PBS, 1 mM EDTA. Cells were then washed with
PBS to remove residual EDTA and then reincubated for 1 h at
37 °C in DMEM with 20 mM HEPES, pH 7.4, 0.5% BSA, 1 mM CaCl2 to allow receptors to recycle back to
the cell surface. The cells were fixed with 3.7% formaldehyde/PBS and
treated as above for immunofluorescence analysis.
A schematic representation of the carboxyl terminus of the
two isoforms of the human thromboxane A2 receptor is shown
in Fig. 1. To investigate tonic
internalization of TP (343 residues) and
(407 residues) that share the first 328 residues. We have previously shown
that TP
, but not TP
, undergoes agonist-induced internalization in
a dynamin-, GRK-, and arrestin-dependent manner. In the
present report, we demonstrate that TP
, but not TP
, also
undergoes tonic internalization. Tonic internalization of TP
was
temperature- and dynamin-dependent and was inhibited by
sucrose and NH4Cl treatment but unaffected by wild-type or dominant-negative GRKs or arrestins. Truncation and site-directed mutagenesis revealed that a YX3
motif (where
X is any residue and
is a bulky hydrophobic residue)
found in the proximal portion of the carboxyl-terminal tail of
TP
was critical for tonic internalization but had no role in
agonist-induced internalization. Interestingly, introduction of either
a YX2
or YX3
motif in the carboxyl-terminal tail of TP
induced tonic
internalization of this receptor. Additional analysis revealed that
tonically internalized TP
undergoes recycling back to the cell
surface suggesting that tonic internalization may play a role in
maintaining an intracellular pool of TP
. Our data demonstrate
the presence of distinct signals for tonic and agonist-induced
internalization of TP
and represent the first report of a
YX3
motif involved in tonic internalization
of a cell surface receptor.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
and NPXY) and
dileucine motifs have been shown to be determinants for a number of
other receptor types (1). Various studies have demonstrated direct interaction between YXX
motifs and the µ chain of the
clathrin-associated proteins AP-1, AP-2 (Ref. 6 and references
therein), and AP-3 (7, 8), allowing the efficient targeting of
transmembrane proteins containing these motifs to clathrin-coated vesicles.
(343 residues) and TP
(407 residues) that share the first 328 amino acids
(12-14).
, but not TP
,
undergoes agonist-induced internalization in a variety of cell types
(15). Internalization of TP
was dynamin-, GRK-, and
arrestin-dependent in HEK293 cells, suggesting the
involvement of receptor phosphorylation and clathrin-coated pits in
this process. Additional characterization of the role of arrestins in
this process revealed that arrestin-3 coexpression promoted
agonist-induced internalization of both TP
and TP
but not of a
mutant truncated after residue 328. Analysis of various
carboxyl-terminal deletion mutants revealed that a region between
residues 355 and 366 in TP
was essential for agonist-promoted internalization. During the course of these studies, we observed that
TP
, but not TP
, also undergoes tonic internalization. In the
present study, we characterize the mechanisms involved in tonic
internalization of TP
. These studies reveal that a
YX3
motif found in the proximal portion of
the carboxyl-terminal tail of TP
is responsible for tonic internalization.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
and TP
were obtained by RT-PCR
and epitope-tagged receptors were constructed as previously described
(15). Mutant receptors were constructed by PCR using the Expand High Fidelity PCR System (Roche Molecular Biochemicals) following the manufacturer's recommendations. The S344Stop, L351Stop, G366Stop, and
D385Stop mutants have been previously described (15). An additional truncation mutant was created using the primers
TXAF (5'-GGAATTCATGTGGCCCAACGGCAGTTCCCTG-3') and L337Stop
(5'-CAGGAATTCTCACAGGCTGGGCCACAGAGTGAGACTCCG-3'). Mutants generated in
TP
were created using the primers TXAF, TXAB
(5'-CCGCTCGAGCATTCAATCCTTTCTGGACAGAGC-3') and the following: E338A,
5'-CTGTGGCCCAGCCTGGCGTACAGTGGCACGATC-'3; Y339A,
5'-TGGCCCAGCCTGGAGGCCAGTGGCACGATC-'3; S340A,
5'-CCCAGC CTGGAGTACGCTGGCACGATCTCGGCTCAC-'3; G341A,
5'-CTGGAGTACAGTGCCACGATCTCGGCTCACTGC-'3; T342A,
5'-GCAGTGAGCCGAGATCGCGCCACTGTACTCCAG-'3; I343A,
5'-GCAGTGAGCCGAGGCCGTGCCACTGTACTC-'3; S344A,
5'-GAGTACAGTGGCACGATCGCGGCTCACTGCAACCTC-'3; and their respective reverse complements. Mutants in TP
were obtained using the primers TXAF and
Q338Y
(5'-CAGGAATTCTCACTGCAGCCCGGAGCGGTACGTGAGCTGGGG-'3) or
R339Y
(5'-CAGGAATTCTCACTGCAGCCCGGAGTACTGCGTGAGCTG-'3). The TP
(R339Y-T342)
and TP
(R339Y-L342A) mutants were generated with
5'-GACCTCGAGCTACTGCAGTGTCCCGGAGTACTGCGTGAG-'3 and
5'-GACCTCGAGCTACTGCGCCCCGGAGTACTGCGTGAGCTG-'3, respectively,
using TP
(R339Y) cDNA as template. Wild-type and mutant
receptors were subcloned in pcDNA-HA- and pcDNA-FLAG vectors as
previously reported (15). All sequences were confirmed by dideoxy
sequencing at the Kimmel Cancer Center Nucleic Acid Facility.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
and TP
, epitope-tagged receptors were
transiently expressed in HEK293 cells. Previous studies have shown that
the agonist binding affinities of TP
and TP
are similar (14-17).
Moreover, addition of a FLAG epitope at the amino terminus of the
receptors did not alter ligand affinities, nor did it affect the
activation characteristics of the receptors as determined by their
respective EC50 values for agonist-promoted generation of
inositol phosphate (data not shown).
View larger version (6K):
[in a new window]
Fig. 1.
Schematic representation of the amino acid
sequence of the TP and TP
carboxyl terminus. Vertical lines illustrate the
sites where truncation mutations were made. The indicated residues were
individually mutated to alanines (bold, underlined) whereas
additional mutations that were introduced into TP
are represented
with arrows.
During our immunofluorescence analysis of agonist-induced
internalization of thromboxane A2 receptors, we noted that
TP could undergo tonic internalization (15). To further characterize this phenomenon, we performed a series of immunofluorescence studies on
cells transiently expressing FLAG-tagged TP receptors. Cells were
initially incubated with the FLAG antibody at 4 °C for 1 h,
washed, and then incubated at different temperatures so that tonic
receptor internalization could be followed. As shown in Fig.
2A, there is significant
redistribution of TP
to intracellular compartments following
incubation at 37 °C whereas minimal internalization of TP
is
observed. Quantitation of intracellular and total cell fluorescence
revealed that ~60% of TP
was tonically redistributed to a
subcellular compartment following a 1-h incubation at 37 °C. These
observations confirm that TP
, but not TP
, undergoes significant
tonic internalization. In contrast, when the cells are incubated for
1 h at 4 °C or 16 °C, TP
and TP
remain entirely at the
cell surface. Interestingly, the inability of TP
to undergo tonic
internalization at 16 °C is a property shared with agonist-induced internalization of TP
(data not shown) and the
2AR
(18), but quite distinct from tonic internalization of the transferrin
receptor which still occurs at 16 °C (data not shown) (18).
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To verify that the internalization of TP was not triggered by
endogenous production of thromboxane or by trace amounts of agonist in
the media, cells were pretreated with the TP antagonist SQ29548 (10 µM) or the cyclooxygenase inhibitor indomethacin (10 µM) for 15 min at 4 °C prior to incubation at
37 °C. These treatments had no effect on internalization of TP
confirming that the receptor is undergoing tonic internalization (Fig.
2B). The same trafficking properties for TP
and TP
were observed in P388D1 and A431 cells, which express endogenous TP
receptors, and Mia Paca, COS-1 and CHO cells, which lack endogenous TP
receptors, as well as in HEK293 cells stably expressing low levels of
thromboxane receptors (15) (data not shown). These results demonstrate
that the differential tonic internalization of TP
and TP
is a
property of the receptor.
We previously demonstrated that agonist-induced internalization
of TP is dynamin-, GRK2-, and arrestin-dependent (15). Thus, we next determined the role of these proteins in tonic
internalization of TP
. Immunofluorescence analysis of TP
redistribution in the presence of dominant-negative mutants of dynamin
(19), GRK2 (20), and arrestin-3 (21) was performed. Coexpression of
dynamin-K44A inhibited tonic internalization (Fig. 2B),
whereas GRK2-K220R and arrestin-3 (201) had no effect (data not
shown). Interestingly, coexpression of dynamin-K44A, but not GRK2-K220R
or arrestin-3 (201), also resulted in an ~2-fold higher cell
surface expression of TP
as assessed by ELISA (data not shown). In
contrast, dynamin-K44A had no effect on cell surface expression of
TP
(data not shown). When cells were preincubated with inhibitors of
clathrin-coated pit formation such as sucrose and NH4Cl,
tonic internalization of TP
was also suppressed (Fig.
2B). Whereas these results demonstrate that tonic and
agonist-induced internalization of TP
are both dynamin-dependent, tonically internalized TP
is targeted
to clathrin-coated pits via a mechanism independent of GRKs and arrestins.
Because the carboxyl terminus of TP appears critical in tonic
internalization of the receptor, we next determined whether this
function could be ascribed to any particular residues. Progressive deletion mutants were first used to address this question (Fig. 1). All
constructs were transiently transfected in HEK293 cells using
transfection conditions that yielded comparable levels of receptor
expression (~1 pmol/mg protein). The removal of up to 63 residues
from the carboxyl terminus (S344Stop) appeared to have no effect on
tonic internalization (Fig. 3), whereas
agonist-induced internalization was completely blocked (15). However,
truncation of an additional 7 amino acids (L337Stop) completely
abolished tonic internalization (Fig. 3). Thus, the region found
between residues 338 and 344 seems to play a critical role in tonic
internalization of TP
. Comparison of this region of TP
(EYSGTIS)
with the corresponding region of TP
(TQRSGLQ) suggests that Tyr-339
in TP
might be an important component of a tonic internalization
motif. A role for tyrosine-based internalization motifs in tonic
endocytosis of a variety of receptors has been demonstated (1, 6, 22). To test this hypothesis, we generated a Y339A mutant TP
and
characterized tonic and agonist-induced internalization. Indeed,
TP
-Y339A did not undergo any tonic internalization (Fig.
4), suggesting a critical role for
Tyr-339 in this process. Additional amino acids between residues 338 and 344 in TP
were then individually mutated to alanine in an
attempt to identify a motif for tonic internalization. The E338A,
S340A, G341A, T342A, and S344A mutants of TP
appeared to undergo
normal tonic internalization, whereas I343A was completely inhibited
(Fig. 4). None of these mutations affected agonist-induced internalization of TP
(Fig. 4C), suggesting that the
motif for tonic internalization is distinct from the region required
for agonist-induced trafficking of TP
(15). Thus, our data
demonstrate that the YXXXI motif plays a critical role in
tonic internalization of TP
. This sequence is closely related to the
YXX
motif identified as playing an important role in
tonic internalization of the transferrin receptor, T-cell receptor
(CD3), lgp-A/lamp-1, lgp-B/lamp-2, lysosomal acid phosphatase,
CI-mannose-6-phosphate receptor, polymeric Ig receptor, and TGN38
receptor (23).
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To further clarify the importance of Tyr-339 in tonic internalization,
we generated Q338Y and R339Y mutants in the carboxyl terminus of TP.
Interestingly, introduction of a Tyr at position 339 in TP
, creating
a YXX
motif, induced tonic internalization of TP
(Fig.
5). Mutation of Leu-342 to Ala in the
R339Y mutant inhibited tonic internalization, demonstrating the
importance of the hydrophobic residue in this process (Fig. 5). In an
effort to determine the importance of the spacing between the Tyr and hydrophobic residues, we introduced a Thr between Gly-341 and Leu-342
in the R339Y mutant (R339Y-T342) to create a YXXX
motif similar to that found in TP
. This latter addition did not affect the
internalization induced by the Tyr residue in R339Y, verifying that
both YX2
and YX3
can function as efficient internalization motifs. Interestingly, a
Q338Y mutant (also creating a YX3
motif but
one residue closer to the plasma membrane than in TP
and TP
(R339Y-T342)) did not induce tonic internalization of TP
. This
suggests that the position of the Tyr in the receptor carboxyl tail is
also an important determinant in this process. As expected, none of
these mutations conferred agonist-induced internalization of TP
(data not shown). Our data suggest that both the distance between the
Tyr and the hydrophobic residue and the position of the
YX2-3
motif in the receptor carboxyl tail
are important determinants in tonic internalization of the thromboxane
receptor. It is interesting to note that a YLGI peptide sequence found
in the second intracellular loop of both TP receptor isoforms is evidently not sufficient to induce tonic internalization because this
is not observed for TP
. Moreover, mutation of residues within this
motif in TP
did not affect tonic internalization (data not shown).
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Because tonic internalization has also been reported for other GPCRs
(2-5, 24), it is important to consider the biological role of this
process. It has been proposed that tonic internalization of the
thrombin receptor generates an intracellular pool of receptors that is
used to repopulate the cell surface with functional receptors (24). If
a similar role can be attributed to tonic internalization of TP, we
would expect that these receptors would recycle back to the cell
surface following tonic internalization. To address this issue, cell
surface receptors were labeled with M1 anti-FLAG antibody at 4 °C
and then allowed to undergo tonic internalization for 1 h at
37 °C. The cells were washed briefly with PBS/EDTA to strip the cell
surface antibody (which binds in a
Ca2+-dependent manner), reincubated at
37 °C, fixed, and then receptor distribution determined by
immunofluorescence. TP
was only detected intracellularly after cell
surface antibody was stripped with PBS/EDTA (Fig.
6, panel B). However,
following incubation at 37 °C, there was extensive redistribution of
the intracellular receptors to the cell surface (Fig. 6, panel
C). This recycling was not caused by new protein synthesis or to
transport of new receptors from intracellular stores because visualized
receptors originated from the initial labeling of cell surface
receptors with antibody. These data suggest that there is constant
recycling of the tonically internalized TP
between the cell surface
and an unidentified intracellular compartment, similar to what has been
observed for the thrombin receptor (24). Thus, tonic internalization of
TP
likely helps to maintain an intracellular pool of functional
receptors that recycle to the cell surface to preserve agonist
sensitivity.
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The YXX motif is one of the most extensively
characterized motifs within cytosolic domains involved in the targeting
of integral membrane proteins. Tyrosine-based sorting signals
conforming to the YXX
motif have been shown to interact
directly with the µ1, µ2, and µ3 subunits of the adaptor
complexes AP-1, AP-2, and AP-3, respectively (reviewed in Ref. 7). The
critical tyrosine does not need to be phosphorylated and, in fact, the
interaction of YXX
and µ may actually be reduced by
phosphorylation (25, 26). The AP-1 complex associates with the
trans-Golgi network and directs the transport of lysosomal enzymes to
endosomes, whereas the AP-2 complex associates with the plasma membrane
and directs the trafficking of cell surface proteins via
clathrin-coated pits. AP-3 is involved in the delivery of proteins to
lysosomes and lysosome-related organelles (27). Recent studies also
suggest that there is a fourth adaptor-related protein complex, AP-4,
that is associated with nonclathrin-coated vesicles in the region of
the trans-Golgi network (27, 28). The µ4 subunit of this complex
specifically interacts with a tyrosine-based sorting signal, suggesting
that AP-4 is also involved in the recognition and sorting of proteins with tyrosine-based motifs (27).
Ohno et al. (7) investigated the selectivity for interaction
of tyrosine-based sorting signals with µ1, µ2, µ3A, and µ3B subunits via screening of a combinatorial
XXXYXX library using the yeast two-hybrid
system. Their results revealed that there was no absolute requirement
for the presence of specific residues at any of the X or
positions. This contrasted with the critical tyrosine residue that
could not be substituted by any other residue without a dramatic
decrease in sorting activity (6, 7, 29-32) and binding affinity for µ subunits (7, 25, 26, 33, 34). It was shown that each µ subunit
exhibits a preference for certain XXXYXX
signals; however, there was also considerable overlap in specificity
(7). Although these studies did not characterize µ interaction with
YXXX
motifs, the YX3
motif
found in TP
displays a serine at position Y
3 and a glutamic acid
at Y
1, analogous to one of the specific sequences that binds to µ2
(SFEYQPL) (7). Similarly, the asialoglycoprotein receptor has a
threonine and a glutamic acid at the Y
3 and Y
1 positions,
respectively. A serine or threonine are also found at Y
3 of the
YXX
motif of the CI mannose-6-phosphate receptor, EGF
receptor, and CTLA-4. Similar to TP
, the CI mannose-6-phosphate
receptor, poly(Ig) receptor and HIV gp41 have a serine at position Y+1
whereas the CD mannose-6-phosphate receptor and furin have a glycine at
position Y+2. On the other hand, the µ3A subunit preference for
position Y
1 is also a glutamic acid (7). Amino acid differences in or
around the signal may possibly confer preferences for targeting to
particular cell compartments. A glycine preceding the Tyr in a
YXX
motif enhances targeting of lamp-1 and acid
phosphatase to lysosomes (6 and references therein). The position of
the motif within the cytoplasmic domain may also influence its activity (6). Indeed, displacement of the YXX
motif in lamp-1 by a single residue with respect to the transmembrane domain was reported to
disrupt lysosomal targeting (35), similar to our observations in the
TP
mutants. Similar findings were also reported when a Tyr was
inserted in the cytoplasmic terminus of influenza virus hemagglutinin
to generate an artificial internalization signal. In these studies,
internalization was dependent on the position of the Tyr relative to
the cell membrane indicating that the structural environment of the Tyr
was important (22).
Our results identified a distinct motif (YX3)
in the carboxyl terminus of a G protein-coupled receptor responsible
for tonic internalization. Both the distance between the Tyr and the
hydrophobic residue and the position of the
YX3
motif in the receptor carboxyl tail
appear to be important determinants in this process. In addition, secondary signals that function in concert with the
YX3
motif may influence the destination of
the receptor. Further analysis of sequence and contextual requirements
for the function of the YX3
signal will be
necessary to fully understand its specificity. Importantly, two
adjacent sequences in the carboxyl terminus of TP
, the
YX3
motif and the region between residues 355 and 366, seem to distinguish between tonic and agonist-induced
internalization, respectively. Molecular analysis of the interaction of
these sequences with their recognition molecules will provide
additional clues as to their role in receptor trafficking. For example,
it will be interesting to determine whether
YX3
and AP-2 directly interact and, if so,
which AP-2 subunit contributes to such interaction. Comparatively, the
region between residues 355 and 366 may dictate, at least in part, the
interaction of the receptor with arrestins (15). This would suggest
tight regulation of the factors involved. The
YX3
motif could be continuously available for
interaction with proteins involved in tonic internalization whereas
ligand occupancy of the receptor might expose the adjacent sequence to proteins involved in agonist-induced internalization.
In summary, our results demonstrate that the alternative splicing of
the carboxyl terminus of the thromboxane A2 receptor generates isoforms that show distinct trafficking characteristics. We
had previously shown that TP, but not TP
, could undergo
agonist-induced internalization (15). In the present report, we
demonstrate that only TP
is capable of undergoing tonic
internalization. Tonic internalization was attributed to a
YX3
motif, which is distinct from the
sequence required for agonist-promoted trafficking and is the first
such motif identified for tonic internalization of a GPCR. These
findings raise important questions concerning how trafficking
differences between TP
and TP
might contribute to mechanistic
differences in the desensitization, resensitization, and/or degradation
of these receptors as well as in their overall cellular physiology.
Similar studies on other GPCRs will help to further characterize the
signals, proteins, and cellular compartments involved in these processes.
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FOOTNOTES |
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* This research was supported by National Institutes of Health Grants GM44944 and GM47417 (to J. L. B.).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.
§ Both authors contributed equally to this work.
¶ Recipient of a postdoctoral fellowship from the Medical Research Council of Canada.
To whom correspondence should be addressed: Thomas Jefferson
University, 233 S. 10th St., Philadelphia, PA 19107. Tel.:
215-503-4607; Fax: 215-923-1098; E-mail:
benovic@lac.jci.tju.edu.
Published, JBC Papers in Press, December 8, 2000, DOI 10.1074/jbc.M009375200
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ABBREVIATIONS |
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The abbreviations used are: GPCR, G protein-coupled receptor; TP, thromboxane A2 receptor; GRK, G protein-coupled receptor kinase; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; HEK, human embryonic kidney; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; TBS, Tris-buffered saline; HA, hemagglutinin; PCR, polymerase chain reaction.
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REFERENCES |
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