Introduction

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G-protein-coupled receptors (GPCRs) are heptahelical transmembrane proteins (Palczewski et al., 2000) devoted to cellular signal transduction. GPCRs are activated by numerous stimuli as varied as light, odorants, nucleotides, and proteins. Intracellularly, they are coupled to G-proteins that amplify the extracellular stimulus and convert it into an intracellular response. GPCRs acting through G-proteins can stimulate various second messenger systems, such as increase in intracellular Ca²⁺, activation of kinase cascades, and induction of gene transcription. The regulation of GPCR function and signaling is of paramount interest because GPCRs are involved in numerous pathologies and are obvious targets for therapeutic intervention.

One of the ways that GPCR function is regulated is via desensitization, which is an attenuated response of a GPCR upon repeated or constant stimulation by its agonist. One mechanism of desensitization is internalization, in which the receptor is translocated from the cell surface membrane to an intracellular compartment. The classical pathway for GPCR internalization is exemplified by the agonist-induced internalization of the ₂-adrenergic receptor (Lefkowitz, 1998). Thus, upon agonist stimulation, the ₂-adrenergic receptor is phosphorylated by a GPCR kinase that recruits -arrestin, which in turn initiates clathrin-dependent internalization. However, other mechanisms of GPCR internalization exist that, for example, involve an initial phosphorylation by protein kinase C (PKC) instead of GPCR kinase (Ferrari et al., 1999; Hipkin et al., 2000; Xiang et al., 2001). An important development as it concerns receptor internalization is the recognition that internalization is not the end of signaling. The process of internalization can activate signaling pathways, such as that of the mitogen-activated protein kinase (Pierce et al., 2000). Knowledge of the internalization of a given receptor is, therefore, important toward understanding its overall signaling potential.

The FP prostanoid receptors are GPCRs whose physiological agonist is prostaglandin F₂ (PGF₂). The FP receptors regulate diverse physiological processes, including inflammation and luteolysis. FP receptors consist of two isoforms called FP_A and FP_B, which were originally isolated and cloned from a sheep corpus luteum library (Pierce et al., 1997). These two isoforms are generated by alternative mRNA splicing that gives rise to differences in their intracellular carboxyl-terminal domain. Studies on these receptor isoforms have demonstrated that upon stimulation with PGF₂ more than one signaling pathway can be activated. Thus, stimulation of either FP receptor isoform by PGF₂ has been shown to activate both the G_q and rho signaling pathways (Pierce et al., 1999). In addition, it has recently been shown that the agonist stimulation of the FP_B, but not the FP_A, isoform can activate transcription through a -catenin-signaling pathway (Fujino and Regan, 2001). Additional differences between these isoforms have been shown with respect to their regulation by PKC in which the FP_A, but not the FP_B, isoform is subject to negative feedback by PKC (Fujino et al., 2000b). The mechanism of this negative feedback involved PKC-mediated phosphorylation of the carboxyl-terminal domain of the FP_A isoform, leading to an inhibition of stimulated inositol phosphate formation.

Besides signaling differences, the FP_A and FP_B prostanoid receptor isoforms may differ in their expression and localization, particularly in response to agonist exposure. In this regard, it has recently been shown that there are significant differences between the TP and TP thromboxane receptor isoforms with respect to agonist-induced receptor internalization (Parent et al., 1999). These isoforms, like the FP receptor isoforms, also represent carboxyl-terminal splice variants, and TP, the longer of the two, undergoes clathrin-dependent internalization, whereas TP does not. The present study was conducted to determine whether similar differences might exist between the FP receptor isoforms. We now report that the FP_A isoform undergoes a rapid agonist-induced internalization that requires PKC and involves clathrin, whereas the FP_B isoform undergoes a constitutive, agonist-independent internalization that does not involve either PKC or clathrin.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Materials. Dulbecco's modified Eagle's medium, bovine serum albumin, Opti-MEM, hygromycin B, geneticin, and gentamicin reagent solutions were obtained from Invitrogen (Carlsbad, CA). Gö 6976 and phorbol 12-myristyl 13-acetate (PMA) were purchased from Calbiochem (San Diego, CA). Clathrin heavy-chain antibodies and anti-phosphotyrosine (PY20) antibodies were obtained from BD Biosciences (San Jose, CA). Methanol, acetone, dimethyl sulfoxide, anti-FLAG M2 antibodies, Fab-specific anti-mouse IgG conjugated to fluorescein isothiocyanate, and sucrose were purchased from Sigma-Aldrich (St. Louis, MO). PGF₂ was obtained from the Cayman Chemical Company (Ann Arbor, MI). HEK-293 cells stably expressing either the FLAG-tagged FP_A or FLAG-tagged FP_B receptor isoforms were used in all the experiments (Fujino et al., 2000b). Cells were maintained at 37°C with 5% CO₂/95% air in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 250 µg/ml geneticin, 200 µg/ml hygromycin B, and 100 µg/ml gentamicin.

Whole Cell Labeling. The protocol for whole cell labeling was modified from that of Orsini and Benovic (1998). Approximately 100,000 cells expressing either the FLAG-tagged FP_A receptor isoform or the FLAG-tagged FP_B receptor isoform were split into tissue culture plates containing glass coverslips. On day 3, prior to the start of the experiments, the cells were examined by microscope to confirm cell density (Fujino et al., 2000a). To evaluate agonist-dependent internalization, the cells were treated either with a 1:500 dilution of anti-FLAG M2 antibodies diluted in Opti-MEM or with a 1:500 dilution of anti-FLAG M2 antibodies concurrent with 1 µM PGF₂ diluted in Opti-MEM for 10 min at 37°C. The tissue culture plates were then placed on ice, and the medium was aspirated. The cells were fixed and permeabilized with methanol/acetone (7:3) for 10 min at 20°C and blocked in BLOTTO (5% nonfat dry milk in Tris-buffered saline with 0.05% Triton X-100) at 37°C for 30 min. The glass coverslips were removed and placed on stoppers in a covered box, and 200 µl of a 1:500 dilution of secondary antibodies (Fab-specific anti-mouse IgG conjugated to fluorescein isothiocyanate) in BLOTTO were applied to each coverslip. The box was then gently shaken for 1 h at room temperature. The coverslips were then transferred to tissue culture plates, washed six times with antibody wash buffer leaving 2 ml of the last wash in each well, and placed at 37°C for 30 min. The coverslips were then mounted onto glass slides using p-phenylenediamine and Cytoseal (ProSciTech, Kelso, QLD, Australia). Images were obtained using a Leica TCS-4D scanning confocal microscope (Leica Microsystems, Inc., Deerfield, IL) using a 100× oil immersion objective and were processed using Adobe Photoshop (Adobe Systems Inc., Mountain View, CA). Experiments with PMA were done exactly as above using 10 µM PMA instead of PGF₂. Pretreatments with 100 nM Gö 6976 were done for 5 min prior to treatment with either vehicle, PGF₂, or PMA. Pretreatment with 0.4 M sucrose was for 15 min.

Immunoblot Analysis. FP_A and FP_B cells were cultured to 80% confluency in 10-cm tissue culture dishes. After treatment with 1 µM PGF₂ or vehicle, the cells were scraped and sonicated in a lysis buffer consisting of 20 mM Tris-HCl (pH 7.5), 10 mM EDTA, 2 mM EGTA, 2 mM phenylmethylsulfonyl fluoride, 0.1 mg/ml leupeptin, and 2 mM sodium vanadate. Samples were centrifuged (16,000g) for 15 min at 4°C, the supernatant (cytosolic fraction) was removed, and the pellet (particulate fraction) was solubilized with lysis buffer containing 0.05% Triton X-100 and centrifuged again to remove insoluble debris as previously described (Fujino and Regan, 2001). Protein concentrations were determined using a Bio-Rad assay kit (Bio-Rad Laboratories, Hercules, CA), and 30 µg of protein/sample was separated on 7.5% SDS-polyacrylamide gels. The proteins were then transferred to nitrocellulose membranes and blocked with 1.5% nonfat dry milk in Tris-buffered saline containing 0.1% polyoxyethylenesorbitan monolaurate (TBST) overnight at 4°C. The membranes were then incubated with anti-phosphotyrosine antibodies (1:1,000 dilution) for 1 h at room temperature with rotation. After three 15-min washes each with TBST, the membranes were incubated with anti-mouse secondary antibodies conjugated to horseradish peroxidase (1:10,000 dilution) for 1 h at room temperature with rotation. The membranes were washed and visualized by enhanced chemiluminescence (SuperSignal; Pierce, Rockford, IL). To examine the presence of clathrin heavy chain, the membranes were stripped in a buffer containing 2% SDS, 62.5 mM Tris-HCl (pH 6.8), and 100 mM -mercaptoethanol for 30 min at 65°C. The membranes were washed, blocked overnight at 4°C, and incubated with the clathrin heavy-chain antibodies (1:2,000 dilution) for 1 h at room temperature. The membranes were then washed, incubated with anti-mouse secondary antibodies, and exposed to film as above.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Agonist-Dependent Internalization of the FLAG-FP_A Contrasts with Constitutive Internalization of the FLAG-FP_B Isoform. Immunofluorescence microscopy was used to examine the internalization of the FP_A and FP_B isoforms in HEK-293 cells stably expressing the FLAG-tagged constructs of these receptors. Initial labeling of cell surface receptors was achieved according to a modified method of Orsini and Benovic (1998), in which live cells were exposed to either vehicle or 1 µM PGF₂ concurrently with anti-FLAG M2 antibodies for 10 min as detailed under Experimental Procedures. Figure 1 shows that, after treatment of cells with vehicle (top left panel), the FLAG-FP_A receptors are localized almost exclusively on the outer cell membrane, whereas the FLAG-FP_B receptors show a more punctate localization on both the outer membrane and intracellularly (Fig. 1, top right panel). Thus, FLAG-FP_B receptors undergo constitutive internalization even in the absence of PGF₂. After treatment with PGF₂, however, the localization of the FLAG-FP_A receptors shifts to a more punctate pattern with an increase in intracellular labeling reflecting receptor internalization (Fig. 1, bottom left panel). Agonist-induced internalization of the FLAG-FP_B receptors was difficult to assess because of the constitutive internalization, and treatment of FLAG-FP_B-expressing cells with PGF₂ does not produce any obvious changes in receptor localization (Fig. 1, bottom right panel).

View larger version (38K): [in this window] [in a new window]   Fig. 1.   Immunofluorescence localization of FLAG-tagged FPA and FPB prostanoid receptor isoforms after treatment with either vehicle or 1 µM PGF2 for 10 min at 37°C. HEK-293 cells stably expressing either the FLAG-FPA or FLAG-FPB isoforms were labeled concurrently with anti-FLAG M2 antibodies and examined by fluorescence microscopy as described under Experimental Procedures. Scale bars represent 10 µm. The data shown are representative of three independent experiments.

Inhibition of PKC by Gö 6976 Inhibits Agonist-Dependent Internalization of FLAG-FP_A Receptors but Has No Effect on the Localization of FLAG-FP_B Receptors. Gö 6976, a specific inhibitor of PKC, was used to study the role of PKC in the agonist-induced internalization of the FLAG-FP_A receptors and to determine whether PKC activity was required for the constitutive internalization of the FLAG-FP_B receptors. Cells were pretreated with 100 nM Gö 6976 for 5 min at 37°C and incubated with either vehicle or 1 µM PGF₂ concurrently with the anti-FLAG M2 antibodies for 10 min. As shown in the top panels of Fig. 2, pretreatment with Gö 6976 itself did not affect the localization of either the FLAG-FP_A or the FLAG-FP_B receptors in the vehicle-treated cells; i.e., the FLAG-FP_A receptors are still localized predominantly on the cell surface, whereas the FLAG-FP_B receptors show punctate labeling on both the cell surface and intracellularly (compare with top panels of Fig. 1). On the other hand, in cells that were treated with PGF₂, Gö 6976 pretreatment blocked the internalization of the FLAG-FP_A (Fig. 2, bottom left panel) but did not affect the localization of FLAG-FP_B receptors (Fig. 2, bottom right panel).

View larger version (90K): [in this window] [in a new window]   Fig. 2.   The effect of Gö 6976 on immunofluorescence localization of FLAG-FPA and FLAG-FPB prostanoid receptor isoforms after treatment with either vehicle or 1 µM PGF2 for 10 min at 37°C. HEK-293 cells stably expressing FLAG-FPA or FLAG-FPB isoforms were pretreated for 5 min with 100 nM Gö 6976, labeled concurrently with anti-FLAG M2 antibodies, and examined by fluorescence microscopy as described under Experimental Procedures. Scale bars represent 10 µm. The data shown are representative of three independent experiments.

Activation of PKC by PMA Induces FLAG-FP_A Receptor Internalization but Has No Effect on the Localization of FLAG-FP_B Receptors. Since inhibition of PKC by Gö 6976 blocked the PGF₂-induced internalization of FLAG-FP_A receptors, we decided to examine the effects of PMA, a direct activator of PKC, on the localization of FLAG-FP_A and FLAG-FP_B receptors, both alone and after pretreatment of the cells with Gö 6976. Cells were pretreated with either vehicle or 100 nM Gö 6976 for 5 min at 37°C and were stimulated with 10 µM PMA for 10 min concurrently with the anti-FLAG M2 antibodies at 37°C. They were then fixed and examined by fluorescence microscopy. As seen in the top panels of Fig. 3, treatment with PMA alone induces internalization of FLAG-FP_A receptors but does not influence the distribution of FLAG-FP_B receptors. However, when FLAG-FP_A cells were pretreated with Gö 6976, PMA-mediated internalization was blocked (Fig. 3, bottom left panel). Pretreatment of the FLAG-FP_B cells with Gö 6976, did not change the pattern of receptor localization compared with treatment with PMA alone (Fig. 3, bottom right panel).

View larger version (53K): [in this window] [in a new window]   Fig. 3.   Immunofluorescence localization of FLAG-tagged FPA and FPB prostanoid receptor isoforms after treatment with either vehicle or 10 µM PMA for 10 min at 37°C. HEK-293 cells stably expressing FLAG-FPA or FLAG-FPB isoforms were pretreated for 5 min with 100 nM Gö 6976, labeled concurrently with anti-FLAG M2 antibodies, and examined by fluorescence microscopy as described under Experimental Procedures. Scale bars represent 10 µm. The data shown are representative of three independent experiments.

Sucrose Blocks PGF₂ and PMA-Stimulated Internalization of FLAG-FP_A Receptors but Does Not Affect the Constitutive Internalization of FLAG-FP_B Receptors. Previous studies on GPCRs have indicated that agonist-dependent internalization is frequently a clathrin-dependent process. To test the role of clathrin in both the agonist-induced internalization of FLAG-FP_A receptors and the constitutive internalization of FLAG-FP_B receptors, we examined the effects of sucrose, a known inhibitor of clathrin-dependent internalization, on the immunofluorescent localization of these receptors. Cells stably expressing either FLAG-FP_A or FLAG-FP_B receptors were pretreated with 0.4 M sucrose for 15 min, followed by treatment with either vehicle, 1 µM PGF₂, or 10 µM PMA for 10 min concurrently with the anti-FLAG M2 antibodies at 37°C. As shown in the top panels of Fig. 4 sucrose pretreatment alone does not affect the localization of either FLAG-FP_A receptors or FLAG-FP_B receptors. Interestingly, however, sucrose pretreatment blocks internalization of FLAG-FP_A receptors but does not affect the localization of FLAG-FP_B receptors after stimulation with PGF₂ (Fig. 4, middle panels). Similarly, sucrose pretreatment blocks PMA-mediated internalization of FLAG-FP_A receptors but does not affect the localization of FLAG-FP_B receptors (Fig. 4, bottom panels).

View larger version (49K): [in this window] [in a new window]   Fig. 4.   The effect of sucrose on the immunofluorescence localization of FLAG-tagged FPA and FPB prostanoid receptor isoforms after treatment with either vehicle, 1 µM PGF2, or 10 µM PMA for 10 min at 37°C. HEK-293 cells stably expressing FLAG-FPA or FLAG-FPB isoforms were pretreated for 15 min with 0.4 M sucrose, labeled concurrently with anti-FLAG M2 antibodies, and examined by fluorescence microscopy as described under Experimental Procedures. Scale bars represent 10 µm. The data shown are representative of three independent experiments.

PGF₂ Stimulates Tyrosine Phosphorylation of a p200 Protein in FLAG-FP_A but Not in FLAG-FP_B Cells. Previous studies have shown that internalization of some GPCRs by a clathrin-dependent mechanism results in the activation of tyrosine kinases (Maudsley et al., 2000). To examine the potential for tyrosine phosphorylation of intracellular proteins following the internalization of FP receptors, cells stably expressing either FLAG-FP_A or FLAG-FP_B receptors were treated with 1 µM PGF₂ and subjected to immunoblotting with anti-phosphotyrosine antibodies as described under Experimental Procedures. As shown in Fig. 5, a band of approximately 200 kDa was tyrosine-phosphorylated in an agonist-dependent manner in FLAG-FP_A-expressing cells but not in the FLAG-FP_B-expressing cells. The size of this p200 tyrosine-phosphorylated protein is close to the known size of clathrin heavy chain (~180 kDa) and, therefore, these experiments were repeated, immunoblotting first with anti-phosphotyrosine antibodies, followed by stripping and reprobing with anti-clathrin heavy-chain antibodies. As shown in the top panel of Fig. 6, treatment of FLAG-FP_A cells with 1 µM PGF₂ for 10 min again resulted in agonist-induced tyrosine phosphorylation of the p200 protein (Fig. 6, arrow), which was not observed in FLAG-FP_B-expressing cells. Reprobing with anti-clathrin heavy-chain antibodies (Fig. 6, lower panel) shows that both the FLAG-FP_A cells and FLAG-FP_B cells contained similar amounts of clathrin heavy chain, which comigrated with the p200 tyrosine-phosphorylated protein shown in Fig. 6, top panel. To directly test whether this p200 protein might be clathrin heavy chain, cell lysates were immunoprecipitated with antibodies to clathrin heavy chain and immunoblotted with anti-phosphotyrosine antibodies. The results were inconclusive (data not shown) and, thus, it was not possible to confirm the exact identity of the p200 tyrosine-phosphorylated protein.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Time course of tyrosine phosphorylation of a p200 protein (arrow) in cells stably expressing either the FLAG-FPA or FLAG-FPB receptors after treatment with PGF2. Cells were incubated with 1 µM PGF2 for the indicated times, and particulate fractions were prepared and subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting (IB) with PY20 as described under Experimental Procedures. Position of the 118-kDa marker is indicated. The data shown are representative of three independent experiments.

View larger version (38K): [in this window] [in a new window]   Fig. 6.   Immunoblotting (IB) of cell lysates with PY20 (top panel) and with anti-clathrin heavy-chain (CHC) antibodies (bottom panel) after treatment of FLAG-FPA or FLAG-FPB-expressing cells with 1 µM PGF2 for 10 min. Particulate fractions were prepared from cell lysates and subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting as described under Experimental Procedures. The membranes were first probed with anti-phosphotyrosine antibodies and were stripped and reprobed with anti-clathrin heavy-chain antibodies. Position of the 118-kDa marker is indicated. The data shown are representative of three independent experiments.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The importance of the carboxyl terminus in receptor desensitization and internalization has been generally recognized for a number of GPCRs, although its role in the internalization of prostanoid receptors is not well established. This is particularly true of the FP receptors, in which alternate mRNA splicing gives rise to two isoforms that differ only in their carboxyl-terminal domains. These two isoforms, termed FP_A and FP_B, differ from one another in that the FP_B is essentially truncated by 46 amino acids compared with the FP_A. Contained within these 46 amino acids are four consensus sites for PKC, and we have previously shown that the FP_A isoform, but not the FP_B, is phosphorylated by PKC and subject to a rapid negative feedback involving PKC (Fujino et al., 2000b).

We have also shown in functional studies that the FP_A and FP_B receptor isoforms undergo a similar degree of desensitization but that the FP_A isoform resensitizes more rapidly than the FP_B (Fujino et al., 2000a). The potential role of receptor internalization in this process is unknown, and until now even the basic characteristics of the internalization of these isoforms have never been described. To address this issue, we FLAG-tagged the FP_A and FP_B isoforms and examined their localization by immunofluorescence microscopy with or without PGF₂ treatment. We found that even without PGF₂ treatment, the FP_B isoform undergoes a rapid constitutive internalization, whereas the FP_A isoform does not. In the presence of PGF₂, however, the FP_A isoform was internalized in a PKC- and clathrin-dependent manner. Because of constitutive internalization of the FP_B receptor isoform, we could not determine whether there was additional agonist-dependent internalization; however, we established that the constitutive internalization of the FP_B isoform is neither PKC- nor clathrin-dependent.

As noted above, PGF₂-induced internalization of the FP_A receptor was dependent upon PKC in that internalization was blocked when cells were pretreated with Gö 6976, an inhibitor of PKC. It would appear likely, therefore, that phosphorylation of the carboxyl terminus by PKC is required for the internalization of the FP_A isoform. This is further strengthened by the fact that direct activation of PKC by PMA could induce FP_A receptor internalization even in the absence of PGF₂. Since activation of PKC by PMA leads to internalization of the FP_A receptor, cells expressing the FP_A receptors are potential candidates for heterologous desensitization similar to that shown for the somatostatin receptor (Hipkin et al., 2000) and the opiate receptor (Xiang et al., 2001). On the other hand, the constitutive internalization of the FP_B isoform clearly did not require PKC because it was unaffected by treatments with either Gö 6976 or PMA. It thus appears that PKC has a major role in the internalization of the FP_A isoform, although it does not preclude a role for a G-protein receptor kinase. Dileucine- and tyrosine-based motifs have also been shown to play a role in phosphorylation-dependent, clathrin-mediated, receptor internalization (Mukherjee et al., 1997; Parent et al., 2001). Inspection of the unique carboxyl-terminal sequence of the FP_A isoform relative to the FP_B isoform did not reveal any such motifs that could explain the different internalization of these isoforms. Further mutagenesis studies, however, could shed light on whether a functionally similar motif is present as well as the specific PKC sites that are responsible for internalization.

Two lines of evidence indicate that the internalization of the FP_A receptor isoform involves a clathrin-dependent mechanism. Thus, both PGF₂- and PMA-induced internalizations of the FP_A isoform were blocked by hypertonic sucrose. Hypertonic sucrose is a well known inhibitor of clathrin-dependent receptor internalization and has been shown to inhibit the internalization of both GPCRs and tyrosine kinase receptors. Interestingly, the constitutive internalization of the FP_B isoform was not blocked by pretreatment with hypertonic sucrose, indicating that the internalization of the FP_B isoform does not involve a clathrin-dependent mechanism. A second line of evidence suggesting that clathrin is involved in the internalization of the FP_A isoform, but not the FP_B, is our observation of the possible tyrosine phosphorylation of clathrin heavy chain in FP_A-expressing cells, but not in FP_B-expressing cells, after treatment with PGF₂. Presently, we have not been able to confirm the tyrosine phosphorylation of clathrin heavy chain, and we do not know the kinase or pathway involved. However, tyrosine phosphorylation of clathrin heavy chain has been shown to occur after the agonist-induced internalization of the epidermal growth factor (EGF) receptor (Wilde et al., 1999). Furthermore, transactivation of the EGF receptor has been demonstrated following the activation of ₂-adrenergic receptors (Maudsley et al., 2000), which provides a potential mechanism linking GPCR activation to an EGF receptor-mediated tyrosine phosphorylation of clathrin heavy chain. Recent studies have also shown that the nonreceptor tyrosine kinase, c-src, is recruited to the ₂-adrenergic receptor during agonist-induced internalization (Miller et al., 2000), which could also mediate a tyrosine phosphorylation of clathrin heavy chain. Further work will hopefully provide answers to these possibilities.

In contrast to the FP_A isoform, the FP_B isoform showed a remarkable degree of spontaneous receptor internalization that occurred in the absence of agonist treatment. This constitutive internalization has also been observed for other GPCRs, most notably for truncation mutants of µ-opioid receptors and somatostatin-2 receptors (Segredo et al., 1997; Schwartkop et al., 1999). Truncation per se, however, is unlikely to be the basis for constitutive internalization as it has been recently shown that TP, the longer of the two carboxy-terminal splice variants of the thromboxane receptor, undergoes constitutive internalization, whereas TP, the shorter isoform, does not (Parent et al., 2001). This constitutive internalization of the TP isoform was associated with a specific amino acid motif that is not present, however, in the FP_B isoform. Another significant difference between the spontaneous internalization of the FP_B and TP receptor isoforms is that internalization of the TP isoform was blocked by sucrose, whereas internalization of FP_B isoform was not. Despite these differences, it is interesting that for both the TP and FP receptor subtypes there are two known carboxyl-terminal splice variants that differ from one another in their degrees of constitutive receptor internalization. Although the full biochemical consequences of the constitutive internalization of these receptor isoforms are presently unknown, they are probably relevant to the physiological functions of these isoforms.