Role of the Differentially Spliced Carboxyl Terminus in Thromboxane A2 Receptor Trafficking

IDENTIFICATION OF A DISTINCT MOTIF FOR TONIC INTERNALIZATION*

Jean-Luc ParentDagger §, Pascale Labrecque§, Moulay Driss RochdiDagger , and Jeffrey L. Benovic||

From the Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 and the Dagger  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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The thromboxane A2 receptor (TP) is a G protein-coupled receptor that is expressed as two alternatively spliced isoforms, alpha  (343 residues) and beta  (407 residues) that share the first 328 residues. We have previously shown that TPbeta , but not TPalpha , undergoes agonist-induced internalization in a dynamin-, GRK-, and arrestin-dependent manner. In the present report, we demonstrate that TPbeta , but not TPalpha , also undergoes tonic internalization. Tonic internalization of TPbeta 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 YX3phi motif (where X is any residue and phi  is a bulky hydrophobic residue) found in the proximal portion of the carboxyl-terminal tail of TPbeta was critical for tonic internalization but had no role in agonist-induced internalization. Interestingly, introduction of either a YX2phi or YX3phi motif in the carboxyl-terminal tail of TPalpha induced tonic internalization of this receptor. Additional analysis revealed that tonically internalized TPbeta undergoes recycling back to the cell surface suggesting that tonic internalization may play a role in maintaining an intracellular pool of TPbeta . Our data demonstrate the presence of distinct signals for tonic and agonist-induced internalization of TPbeta and represent the first report of a YX3phi motif involved in tonic internalization of a cell surface receptor.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 (YXXphi 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 YXXphi 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.

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, TPalpha (343 residues) and TPbeta (407 residues) that share the first 328 amino acids (12-14).

In a previous study, we demonstrated that TPbeta , but not TPalpha , undergoes agonist-induced internalization in a variety of cell types (15). Internalization of TPbeta 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 TPalpha and TPbeta 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 TPbeta was essential for agonist-promoted internalization. During the course of these studies, we observed that TPbeta , but not TPalpha , also undergoes tonic internalization. In the present study, we characterize the mechanisms involved in tonic internalization of TPbeta . These studies reveal that a YX3phi motif found in the proximal portion of the carboxyl-terminal tail of TPbeta is responsible for tonic internalization.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 TPalpha and TPbeta 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 TPbeta 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 TPalpha were obtained using the primers TXAF and alpha Q338Y (5'-CAGGAATTCTCACTGCAGCCCGGAGCGGTACGTGAGCTGGGG-'3) or alpha R339Y (5'-CAGGAATTCTCACTGCAGCCCGGAGTACTGCGTGAGCTG-'3). The TPalpha (R339Y-T342) and TPalpha (R339Y-L342A) mutants were generated with 5'-GACCTCGAGCTACTGCAGTGTCCCGGAGTACTGCGTGAG-'3 and 5'-GACCTCGAGCTACTGCGCCCCGGAGTACTGCGTGAGCTG-'3, respectively, using TPalpha (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.

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.


    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 TPalpha and TPbeta , epitope-tagged receptors were transiently expressed in HEK293 cells. Previous studies have shown that the agonist binding affinities of TPalpha and TPbeta 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).



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Fig. 1.   Schematic representation of the amino acid sequence of the TPalpha and TPbeta 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 TPalpha are represented with arrows.

During our immunofluorescence analysis of agonist-induced internalization of thromboxane A2 receptors, we noted that TPbeta 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 TPbeta to intracellular compartments following incubation at 37 °C whereas minimal internalization of TPalpha is observed. Quantitation of intracellular and total cell fluorescence revealed that ~60% of TPbeta was tonically redistributed to a subcellular compartment following a 1-h incubation at 37 °C. These observations confirm that TPbeta , but not TPalpha , undergoes significant tonic internalization. In contrast, when the cells are incubated for 1 h at 4 °C or 16 °C, TPalpha and TPbeta remain entirely at the cell surface. Interestingly, the inability of TPbeta to undergo tonic internalization at 16 °C is a property shared with agonist-induced internalization of TPbeta (data not shown) and the beta 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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Fig. 2.   Immunofluorescence analysis of TPalpha and TPbeta distribution in HEK293 cells. A, FLAG-tagged receptors were transiently transfected in HEK293 cells. Cells were incubated with the FLAG antibody at 4 °C prior to any other treatment to detect receptors that were present initially at the cell surface. Immunofluorescence detection was performed as described under "Experimental Procedures." Top panel, receptor distribution in cells expressing TPalpha (left) and TPbeta (right) when incubated at 4 °C. Middle panel, after a 1 h incubation at 16 °C. Bottom panel, after a 1 h incubation at 37 °C. B, HEK293 cells, transiently transfected with TPbeta , were labeled with FLAG antibody at 4 °C prior to incubation at 37 °C for 1 h. Tonic internalization of TPbeta in the presence or absence of the TP receptor antagonist SQ29548, the inhibitors of clathrin-coated pit formation NH4Cl and sucrose, or dynamin-K44A was analyzed by immunofluorescence and quantitated. Data represent the percentage of intracellular immunofluorescence relative to total immunofluorescence of individual cells. Immunofluorescence was measured using the NIH Image 1.62b7f software. Results shown represent the mean ± S.E. of three independent experiments, where immunofluorescence of at least ten cells was evaluated for each experiment. Refer to "Experimental Procedures" for details.

To verify that the internalization of TPbeta 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 TPbeta confirming that the receptor is undergoing tonic internalization (Fig. 2B). The same trafficking properties for TPalpha and TPbeta 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 TPalpha and TPbeta is a property of the receptor.

We previously demonstrated that agonist-induced internalization of TPbeta is dynamin-, GRK2-, and arrestin-dependent (15). Thus, we next determined the role of these proteins in tonic internalization of TPbeta . Immunofluorescence analysis of TPbeta 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 TPbeta as assessed by ELISA (data not shown). In contrast, dynamin-K44A had no effect on cell surface expression of TPalpha (data not shown). When cells were preincubated with inhibitors of clathrin-coated pit formation such as sucrose and NH4Cl, tonic internalization of TPbeta was also suppressed (Fig. 2B). Whereas these results demonstrate that tonic and agonist-induced internalization of TPbeta are both dynamin-dependent, tonically internalized TPbeta is targeted to clathrin-coated pits via a mechanism independent of GRKs and arrestins.

Because the carboxyl terminus of TPbeta 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 TPbeta . Comparison of this region of TPbeta (EYSGTIS) with the corresponding region of TPalpha (TQRSGLQ) suggests that Tyr-339 in TPbeta 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 TPbeta and characterized tonic and agonist-induced internalization. Indeed, TPbeta -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 TPbeta 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 TPbeta appeared to undergo normal tonic internalization, whereas I343A was completely inhibited (Fig. 4). None of these mutations affected agonist-induced internalization of TPbeta (Fig. 4C), suggesting that the motif for tonic internalization is distinct from the region required for agonist-induced trafficking of TPbeta (15). Thus, our data demonstrate that the YXXXI motif plays a critical role in tonic internalization of TPbeta . This sequence is closely related to the YXXphi 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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Fig. 3.   Tonic internalization of different carboxyl tail truncation mutants of TPbeta . HEK293 cells were transfected with amounts of TPbeta DNA yielding receptor expression of ~1 pmol/mg protein for each construct. Cells were labeled with the FLAG antibody at 4 °C prior to being incubated at 37 °C for 1 h. A, tonic internalization of each receptor construct was analyzed by immunofluorescence. B, quantitation of tonic internalization of the different receptor constructs was performed as described in Fig. 2.



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Fig. 4.   Identification of the residues involved in tonic internalization of TPbeta . Amino acids between residues 338 and 344, inclusively, were individually mutated to alanines. A, immunofluorescence analysis of the tonic internalization of each receptor construct. B, quantitation of intracellular fluorescence, as described in Fig. 2. C, agonist-induced internalization of the receptor mutants. HEK293 cells were transiently transfected with FLAG epitope-tagged receptors and the percentage of receptors remaining at the cell surface after 2 h of stimulation with 100 nM U46619 was measured by ELISA analysis, as described under "Experimental Procedures." The results represent the mean ± S.E. of three independent experiments, each done in triplicate.

To further clarify the importance of Tyr-339 in tonic internalization, we generated Q338Y and R339Y mutants in the carboxyl terminus of TPalpha . Interestingly, introduction of a Tyr at position 339 in TPalpha , creating a YXXphi motif, induced tonic internalization of TPalpha (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 YXXXphi motif similar to that found in TPbeta . This latter addition did not affect the internalization induced by the Tyr residue in R339Y, verifying that both YX2phi and YX3phi can function as efficient internalization motifs. Interestingly, a Q338Y mutant (also creating a YX3phi motif but one residue closer to the plasma membrane than in TPbeta and TPalpha (R339Y-T342)) did not induce tonic internalization of TPalpha . 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 TPalpha (data not shown). Our data suggest that both the distance between the Tyr and the hydrophobic residue and the position of the YX2-3phi 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 TPalpha . Moreover, mutation of residues within this motif in TPbeta did not affect tonic internalization (data not shown).



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Fig. 5.   Redistribution of receptors in HEK293 cells after introduction of a tyrosine residue at position 339 in the carboxyl tail of TPalpha . HEK293 cells were transfected with amounts of DNA yielding receptor expression of ~1 pmol/mg of protein for each TPalpha construct. A, redistribution of TPalpha mutants was assessed by immunofluorescence. B, intracellular fluorescence was evaluated as described in Fig. 2.

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 TPbeta , 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. TPbeta 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 TPbeta between the cell surface and an unidentified intracellular compartment, similar to what has been observed for the thrombin receptor (24). Thus, tonic internalization of TPbeta likely helps to maintain an intracellular pool of functional receptors that recycle to the cell surface to preserve agonist sensitivity.



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Fig. 6.   Tonically internalized TPbeta receptors recycle to the cell surface. FLAG epitope-tagged receptors were transiently transfected in HEK293 cells. A, cells were labeled with the FLAG antibody at 4 °C and incubated at 37 °C for 1 h to allow tonic internalization. B, the cell surface was then stripped of the remaining FLAG antibody with two quick washes with cold PBS/EDTA (1 mM). C, cells were then reincubated for 1 h at 37 °C to permit recycling of receptors to the cell surface (see "Experimental Procedures" for details). Results shown are representative of at least five independent experiments.

The YXXphi 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 YXXphi 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 YXXphi 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 XXXYXXphi 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 phi  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 XXXYXXphi signals; however, there was also considerable overlap in specificity (7). Although these studies did not characterize µ interaction with YXXXphi motifs, the YX3phi motif found in TPbeta 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 YXXphi motif of the CI mannose-6-phosphate receptor, EGF receptor, and CTLA-4. Similar to TPbeta , 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 YXXphi 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 YXXphi 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 TPalpha 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 (YX3phi ) 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 YX3phi motif in the receptor carboxyl tail appear to be important determinants in this process. In addition, secondary signals that function in concert with the YX3phi motif may influence the destination of the receptor. Further analysis of sequence and contextual requirements for the function of the YX3phi signal will be necessary to fully understand its specificity. Importantly, two adjacent sequences in the carboxyl terminus of TPbeta , the YX3phi 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 YX3phi 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 YX3phi 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 TPbeta , but not TPalpha , could undergo agonist-induced internalization (15). In the present report, we demonstrate that only TPbeta is capable of undergoing tonic internalization. Tonic internalization was attributed to a YX3phi 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 TPalpha and TPbeta 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.


    FOOTNOTES

* 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


    ABBREVIATIONS

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES


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