Differential Internalization of the Prostaglandin F2 Receptor Isoforms: Role of Protein Kinase C and Clathrin

FP prostanoid receptors are G-protein-coupled receptors that mediate the actions of prostaglandin F2 (PGF2 ). Alternative mRNA splicing gives rise to two isoforms, FPA and FPB, which are identical except for their intracellular carboxyl termini. In this study, we examined the internalization of recombinant FLAGepitope-tagged FPA and FPB receptors that were stably expressed in human embryonic kidney-293 cells. Cell surface receptors on live cells were labeled with anti-FLAG antibodies either in the presence or absence of PGF2 and were examined by immunofluorescence microscopy. In the absence of PGF2 , FPA-expressing cells were labeled predominantly on the cell surface; however, FPB-expressing cells were labeled on both the cell surface and intracellularly, indicating constitutive internalization of the FPB isoform. After treatment with PGF2 , FPAexpressing cells were labeled intracellularly, reflecting receptor internalization, which could be mimicked with phorbol 12-myristyl 13-acetate (PMA), an activator of protein kinase C (PKC). Pretreatment of FPA-expressing cells with Gö 6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo5H-indolo[2,3-a]pyrrolo[3,4-c]carbozole], an inhibitor of PKC, blocked both PGF2 and PMA-induced receptor internalization. However, Gö 6976 did not block constitutive internalization of the FPB isoform, suggesting that the mechanisms of receptor internalization differ between the FPA and FPB isoforms. Furthermore, pretreatment with sucrose, an inhibitor of clathrin-dependent internalization, blocked PGF2 -induced internalization of the FPA isoform but did not block constitutive internalization of the FPB isoform. In conclusion, the FPA receptor isoform shows an agonist-induced internalization involving PKC and clathrin, whereas the FPB isoform undergoes agonist-independent internalization that does not involve PKC or clathrin. 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 Ca , 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 FPA and FPB, which were originally isolated and ABBREVIATIONS: GPCR, G-protein-coupled receptors; PKC, protein kinase C; PGF2 , prostaglandin F2 ; PMA, phorbol 12-myristyl 13-acetate; Gö 6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo[3,4-c]carbozole; HEK, human embryonic kidney; EGF, epidermal growth factor; PY20, anti-phosphotyrosine antibodies; FP, receptor for PGF2 ; TP, receptor for thromboxane A2; TP and TP , alternate mRNA splice variants of TP receptor. 0022-3565/02/3021-219–224$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 302, No. 1 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 4837/990535 JPET 302:219–224, 2002 Printed in U.S.A. 219 at A PE T Jornals on N ovem er 4, 2017 jpet.asjournals.org D ow nladed from 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 FPB, but not the FPA, isoform can activate transcription through a -cateninsignaling pathway (Fujino and Regan, 2001). Additional differences between these isoforms have been shown with respect to their regulation by PKC in which the FPA, but not the FPB, 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 FPA isoform, leading to an inhibition of stimulated inositol phosphate formation. Besides signaling differences, the FPA and FPB 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 clathrindependent 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 FPA isoform undergoes a rapid agonistinduced internalization that requires PKC and involves clathrin, whereas the FPB isoform undergoes a constitutive, agonist-independent internalization that does not involve either PKC or clathrin. Experimental Procedures 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, antiFLAG M2 antibodies, Fab-specific anti-mouse IgG conjugated to fluorescein isothiocyanate, and sucrose were purchased from SigmaAldrich (St. Louis, MO). PGF2 was obtained from the Cayman Chemical Company (Ann Arbor, MI). HEK-293 cells stably expressing either the FLAG-tagged FPA or FLAG-tagged FPB 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 g/ml geneticin, 200 g/ml hygromycin B, and 100 g/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 FPA receptor isoform or the FLAG-tagged FPB 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 M 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 Trisbuffered 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 l of a 1:500 dilution of secondary antibodies (Fabspecific 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 M 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. FPA and FPB cells were cultured to 80% confluency in 10-cm tissue culture dishes. After treatment with 1 M 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 g 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.