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J. Biol. Chem., Vol. 279, Issue 11, 10020-10031, March 12, 2004

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Termination of Protease-activated Receptor-1 Signaling by -Arrestins Is Independent of Receptor Phosphorylation^*

Chii-Heui Chen, May M. Paing, and JoAnn Trejo¶

From the Department of Pharmacology, Cardiovascular Biology Center, Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7365

Received for publication, September 24, 2003 , and in revised form, December 16, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Protease-activated receptor 1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is the prototypic member of a family of protease-activated receptors. PAR1 is irreversibly proteolytically activated; thus, the magnitude and duration of thrombin cellular responses are determined primarily by mechanisms responsible for termination of receptor signaling. Both phosphorylation and -arrestins contribute to rapid desensitization of PAR1 signaling. However, the relative contribution of each of these pathways to the termination of PAR1 signaling is not known. Co-expression of PAR1 with -arrestin 1 (arr1) in COS-7 cells resulted in a marked inhibition of PAR1 signaling, whereas -arrestin 2 (arr2) was essentially inactive. Strikingly, signaling by a PAR1 cytoplasmic tail mutant defective in agonist-induced phosphorylation was also attenuated more effectively by arr1 compared with arr2. In contrast, both -arrestin isoforms were equally effective at desensitizing the substance P receptor, a classic reversibly activated GPCR. PAR1 coimmunoprecipitated arr1 in an agonist-dependent manner, whereas arr2 association was virtually undetectable. Remarkably, arr1 also interacted with phosphorylation defective PAR1 mutant, whereas arr2 did not. Moreover, constitutively active -arrestin mutants, arr1 R169E and arr2 R170E, that bind to activated receptor independent of phosphorylation failed to enhance either wild type or mutant PAR1 desensitization compared with normal versions of these proteins. In contrast, -arrestin mutants displayed enhanced activity at desensitizing the serotonin 5-hydroxytryptamine_2A receptor. Taken together, these results suggest that, in addition to PAR1 cytoplasmic tail phosphorylation itself, -arrestin binding independent of phosphorylation promotes desensitization of PAR1 signaling. These findings reveal a new level of complexity in the regulation of protease-activated GPCR signaling.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Thrombin, a coagulant protease, is generated at sites of vascular injury and produces a variety of cellular effects critical for hemostasis, thrombosis, and inflammatory and proliferative responses triggered by vascular damage (1, 2). Thrombin activates cells through at least three proteolytically activated G protein-coupled receptors: PAR1, ¹ -3, and -4 (3). The prototype of this family, PAR1, is activated by an unusual irreversible proteolytic mechanism in which thrombin binds to and cleaves the amino-terminal exodomain of the receptor. This cleavage generates a new amino terminus that functions as a tethered ligand by binding intramolecularly to the body of the receptor to cause transmembrane signaling (4–6). The synthetic peptide SFLLRN, which represents the newly formed amino terminus of the receptor, can activate PAR1 independent of thrombin and receptor cleavage. PAR1 is irreversibly activated; thus, the mechanisms that contribute to the termination of signaling are critical determinants of the magnitude and kinetics of the thrombin response in cells. Given the irreversible nature of PAR1 activation, we hypothesize that signal termination events are probably unique, since all other GPCRs are reversibly activated.

The molecular events responsible for GPCR desensitization and resensitization have been extensively studied using the ₂-adrenergic receptor (7, 8). In the classic paradigm, GPCRs are initially desensitized by rapid phosphorylation of activated receptors by G protein-coupled receptor kinases (GRKs) and other kinases. Receptor phosphorylation enhances the affinity of interaction with arrestins, and arrestin binding prevents receptor-G protein interaction, thereby uncoupling the receptor from signaling. Arrestins also interact with components of the endocytic machinery to facilitate recruitment of GPCRs to clathrin-coated pits and internalization from the plasma membrane (9, 10). Once internalized into endosomes, GPCRs dissociate from their ligands, become dephosphorylated, and then return to the cell surface in a state capable of responding to ligand. Thus, for most classic, reversibly activated GPCRs, signaling is terminated at the plasma membrane, and receptor trafficking is linked to resensitization of signaling.

Phosphorylation of activated PAR1 also appears to be important for rapid uncoupling from G protein signaling. Overexpression of either GRK3 or GRK5 enhances PAR1 phosphorylation and markedly inhibits inositol phosphate (IP) accumulation (11, 12). A PAR1 mutant in which all of the serines and threonines in the cytoplasmic tail (C-tail) are converted to alanines (S/T A) is neither extensively phosphorylated nor inhibited by GRK3 overexpression in multiple cell types (11, 13, 14). In addition, we recently found that arrestins are also critical for the termination of PAR1 signaling. Desensitization of PAR1-promoted phosphoinositide (PI) hydrolysis is significantly impaired in mouse embryonic fibroblasts lacking both arrestin isoforms, arrestin 2 and arrestin 3 (also termed -arrestin 1 and -arrestin 2), whereas PAR1 internalization remained intact (15). However, in both wild-type and -arrestin-deficient cells, phosphorylation of activated PAR1 is still necessary for internalization through clathrin-coated pits. Moreover, unlike classic GPCRs, proteolytically activated PAR1 is internalized and sorted rapidly to lysosomes, an event critical for termination of receptor signaling (16, 17). Thus, PAR1 defines a new class of GPCRs that utilize a phosphorylation-, clathrin-, and dynamin-dependent pathway for endocytosis that operates independent of -arrestins and receptor trafficking is linked to termination of signaling.

The precise function of arrestins in signal regulation of a GPCR such as PAR1 that does not use these molecules for internalization through clathrin-coated pits has not been examined. Moreover, the relative contribution of phosphorylation versus -arrestins to the termination of PAR1 signaling remains to be determined. In the present study, we used COS-7 cells to investigate the roles of phosphorylation and -arrestins in uncoupling PAR1 from G protein signaling. Our findings strongly suggest that -arrestins are able to bind and desensitize activated PAR1 independent of phosphorylation. Thus, these studies reveal a complex regulation of PAR1 signaling that involves both PAR1 C-tail phosphorylation and phosphorylation-independent binding of -arrestins.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Reagents and Antibodies—Human -thrombin was purchased from Enzyme Research Laboratories. Agonist peptide SFLLRN was synthesized as the carboxyl amide and purified by reverse phase high pressure liquid chromatography (UNC Peptide Facility, Chapel Hill, NC). Substance P peptide was purchased from Phoenix Pharmaceuticals. 2,5-Dimethoxy-4-iodophenylisopropylamine was from Sigma.

Monoclonal M1 and M2 anti-FLAG antibodies were from Sigma. Rabbit polyclonal anti--arrestin antibody A1CT was previously described (18) and generously provided by Robert J. Lefkowitz (Duke University). Anti-PAR1 rabbit polyclonal antibody was generated as previously described (19). Horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit secondary antibodies were from Bio-Rad.

cDNAs and Cell Lines—The cDNAs encoding FLAG-tagged PAR1 wild-type and C-tail phosphorylation site mutant (S/T A) were previously described (11). The PAR1 third intracellular loop (IC₃) mutants in which serine residues Ser²⁹⁷, Ser²⁹⁸, and Ser²⁹⁹ were converted to alanine (IC₃ S²⁹⁷SS²⁹⁹ mutant) were generated using the QuikChange^TM site-directed mutagenesis kit (Stratagene), specific mutations were confirmed by dideoxy sequencing. A plasmid encoding wild type substance P receptor containing an amino-terminal FLAG epitope was generated as described (17). cDNAs encoding untagged and FLAG-tagged -arrestins were gifts from Robert J. Lefkowitz (Duke University). Green fluorescent protein (GFP)-tagged -arrestins were obtained from Marc Caron (Duke University). Mutant arr1 R169E and arr2 R170E were kindly provided by Vsevolod V. Gurevich (Vanderbilt University) and have been previously described (20). The FLAG-tagged human 5-hydroxytryptamine_2A (5-HT_2A) serotonin receptor was generously provided by Bryan L. Roth (Case Western Reserve University). The plasmids encoding G_q wild type and GTPase-deficient, constitutively active Q205L mutant were generously provided by T. Kendall Harden (University of North Carolina, Chapel Hill, NC).

COS-7 cells were obtained from the American Type Culture Collection (Manassas, VA) and grown in DMEM supplemented with 10% fetal bovine serum, 4.5 mg/ml glucose, 100 units/ml penicillin, and 100 µg/ml streptomycin.

Transient Transfection—COS-7 cells were plated at 4 x 10⁴ cells/well in fibronectin-coated 24-well dishes (Falcon) and grown overnight. Cells were then transiently transfected with a total of 0.4 µg of either PAR1, PAR1 mutants, SPR, or 5-HT_2A receptor and either arr1, arr2, arr mutants, or pcDNA using LipofectAMINE reagent according to the manufacturer's instructions (Invitrogen). Cells plated at 2 x 10⁵ cells/well in 6-well dishes (Falcon) were grown overnight and transfected with a total of 2 µg of either PAR1 or PAR1 mutants and either arr1, arr2, or pcDNA using LipofectAMINE reagent.

Phosphoinositide Hydrolysis—COS-7 cells plated at 4 x 10⁴ cells/well of 24-well dishes were grown overnight, transiently transfected, and then labeled with 2 µCi/ml myo-[³H]inositol (American Radiolabeled Chemicals, Inc.) in serum-free DMEM containing 1 mg/ml bovine serum albumin for 18–24 h. Cells were washed with DMEM containing 1 mg/ml bovine serum albumin, 10 mM HEPES buffer, and 20 mM lithium chloride. Cells were then incubated in the absence or presence of either 10 nM -thrombin, 100 nM substance P, or 10 µM 2,5-dimethoxy-4-iodophenylisopropylamine diluted in DMEM containing lithium chloride for various times at 37 °C. Cell incubation medium was removed, and [³H]inositol phosphates ([³H]IPs) were extracted with 50 mM formic acid. Cell extracts were neutralized with 150 mM NH₄OH, and IPs were isolated by column chromatography as described (15). Scintillation counting was then used to quantitate IPs eluted in this assay.

Data Analysis—Data were analyzed using Prism 3.0 software, and statistical significance was determined using InStat 3.0 (GraphPAD, San Diego, CA). The initial rate of PAR1 desensitization was determined by quantifying the decrease in thrombin response over time. The data were normalized to the amount of [³H]IPs formed in untreated control cells for each time point.

Cell Surface ELISA—Transiently transfected COS-7 cells plated at 4 x 10⁴ cells/well in 24-well dishes were either left untreated or treated with 50 µM SFLLRN or 100 nM substance P for 30 min at 37 °C. Cells were fixed with 4% paraformaldehyde for 5 min at 4 °C and then incubated with M1 anti-FLAG antibody for 1 h at 25 °C in DMEM containing 1 mg/ml bovine serum albumin and 10 mM HEPES, pH 7.4. Cells were then washed and incubated with horseradish peroxidase-conjugated goat anti-mouse secondary antibody for 1 h at 25 °C. Cells were washed again and incubated with one-step 2,2'-azino-bis-3-ethyl-benz-thiazoline-6-sulfonic acid (Pierce) for 10–20 min at room temperature. An aliquot was removed, and the optical density was determined at 405 nm using a Molecular Devices SpectraMax Plus microplate reader.

Immunoblotting and Coimmunoprecipitation—Lysates were prepared as previously described (17) from cells plated at 4 x 10⁴ cells/well of 24-well dishes (Falcon) transiently transfected with PAR1 and various amounts of FLAG-tagged -arrestins. Protein concentrations were determined using BCA protein assay reagent (Pierce), and equivalent amounts of lysates were resolved by SDS-PAGE and transferred, and membranes were incubated with M1 anti-FLAG antibody (1:1000) overnight. To detect PAR1 and -arrestin association, COS-7 cells were plated at 2 x 10⁵ cells/well in a 6-well dish, transiently transfected with FLAG-tagged PAR1 and -arrestin cDNAs, and then stimulated with 50 µM SFLLRN for 2.5 min at 37 °C. Cells were lysed in 1% Triton X-100 lysis buffer containing 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM EDTA, 50 mM NaF, 10 mM sodium pyrophosphate, and 200 µM sodium orthovanadate containing protease inhibitors, and equivalent amounts of lysates were used for immunoprecipitation with M2 anti-FLAG antibody. Immunoprecipitates were resolved by SDS-PAGE, transferred, and immunoblotted with rabbit polyclonal anti--arrestin A1CT antibody (1:5000) or anti-PAR1 antibody (1:1000). Membranes were washed, incubated with species-specific secondary antibodies conjugated to horseradish peroxidase (1:10,000), and washed again. Immunoblots were developed with ECL-Plus (Amersham Biosciences), imaged by autoradiography, and quantitated by a Bio-Rad Fluor-S MultiImager.

Immunofluorescence Confocal Microscopy—Transiently transfected COS-7 cells were grown on fibronectin-coated glass coverslips (22 x 22 mm) and incubated with M1 anti-FLAG antibody for 1 h at 4 °C, washed, and exposed to agonist at 37 °C. Cells were fixed and then processed for immunofluorescence microscopy as described (15). Images were collected using an Fluoview 300 laser-scanning confocal imaging system (Olympus) configured with an IX70 fluorescent microscope fitted with a PlanApo x 60 oil objective (Olympus). The final composite image was created using Adobe Photoshop 6.0 (Adobe Systems).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
-Arrestin-mediated Desensitization of PAR1 Signaling Is Independent of Receptor Phosphorylation—PAR1 couples to G_q and stimulates PI hydrolysis through the activation of phospholipase C- (21). Thus, we sought to determine the roles of phosphorylation and -arrestins in PAR1 desensitization by measuring G_q activation of PI hydrolysis in COS-7 cells. COS-7 cells are known to express low levels of endogenous -arrestins (22). We initially compared the signaling properties of PAR1 wild type and a phosphorylation-defective mutant that lacks all potential C-tail phosphorylation sites (S/T A) and is insensitive to GRK-mediated desensitization in multiple cell types including COS-7 (11, 14). The concentration effect curves for thrombin at wild type and mutant PAR1 were determined by incubating cells labeled with myo-[³H]inositol and varying concentrations of thrombin for 5 min at 37 °C. The accumulation of [³H]IPs was then measured. The effective concentration of thrombin to stimulate a half-maximal response after 5 min was similar for both PAR1 wild type and S/T A mutant in these studies (Fig. 1A). However, activated PAR1 S/T A mutant caused an enhanced maximal signaling response compared with wild type receptor (Fig. 1A). These findings suggest that each activated PAR1 S/T A mutant coupled longer to PI hydrolysis before signaling was shut off.

View larger version (20K): [in this window] [in a new window]   FIG. 1. -Arrestins promote termination of PAR1 signaling independent of receptor phosphorylation. A, the concentration effect curves of -thrombin at PAR1 wild type and S/T A phosphorylation-defective mutant were determined in transiently transfected COS-7 cells labeled with myo-[3H]inositol after 5 min of agonist incubation. The data shown are the mean ± S.D. for triplicates in one experiment and are representative of at least three separate experiments. B and C, COS-7 cells transiently transfected with PAR1 wild type or S/T A mutant and either arr1, arr2, or pcDNA were labeled with myo-[3H]inositol and incubated in the absence or presence of 10 nM -thrombin for various times at 37 °C. The amounts of [3H]IPs accumulated were then measured. The data shown (mean ± S.D.; n = 3) are expressed as -fold increase over basal [3H]IPs of one experiment and are representative of six independent experiments. The initial level of receptor surface expression was similar for each transfection condition. The values (mean ± S.D.; n = 3) for PAR1 wild type and S/T A mutant surface expression co-transfected with either arr1, arr2, or pcDNA were 0.112 ± 0.005, 0.123 ± 0.002, or 0.124 ± 0.008 and 0.173 ± 0.011, 0.171 ± 0.005, or 0.181 ± 0.003, respectively. The insets confirm a similar amount of arr1 and arr2 expression in total cell lysates of an equivalent well.

To examine the contribution of phosphorylation versus -arrestin binding to PAR1 desensitization, we assessed signaling by the PAR1 S/T A phosphorylation-defective mutant in cells co-expressing either arr1 or arr2. In COS-7 cells expressing the PAR1 S/T A mutant, thrombin stimulated an 5-fold increase in PI hydrolysis (Fig. 1C), a response substantially greater than that observed with comparable amounts of wild type receptor in these same cells (Fig. 1B). Expression of arr2 failed to significantly decrease signaling by PAR1 S/T A mutant (Fig. 1C), similar to that observed with wild type receptor. In contrast, however, arr1 caused a marked 50% inhibition of PAR1 S/T A signaling (Fig. 1C), suggesting that arr1-mediated PAR1 uncoupling from G protein signaling is independent of phosphorylation.

We next examined whether the initial coupling of activated PAR1 to G_q-promoted PI hydrolysis was affected by either arr1 or arr2. PAR1 wild type or S/T A mutant was transiently co-expressed with either arr1 or arr2, and the capacity of receptor to promote IP accumulation was compared. The concentration effect curves for thrombin at wild type and mutant PAR1 co-expressed with either arr1, arr2, or vector was shown in Fig. 2. The EC₅₀ values for stimulation (5-min assay) of IP accumulation by thrombin were comparable in each transfection condition (Fig. 2, Table I). The maximal effect of 30 nM thrombin for stimulation of IP accumulation by PAR1 wild type and S/T A mutant co-expressed with either arr1, arr2, or vector was also similar (Fig. 2, Table I). Together, these findings imply that the initial coupling of activated PAR1 wild type and S/T A mutant to G protein-induced signaling response is not affected by arrestins.

View larger version (29K): [in this window] [in a new window]   FIG. 2. Activated PAR1 initial coupling to G protein-mediated PI hydrolysis is not affected by -arrestins. A and B, the concentration effect curves of -thrombin at PAR1 wild-type and S/T A mutant were determined in COS-7 cells transiently co-transfected with either arr1, arr2, or pcDNA. Cells labeled with myo-[3H]inositol were incubated with varying concentrations of -thrombin for 5 min at 37 °C, and [3H]IPs were then measured. The data shown are the mean ± S.D. for triplicates in one experiment and are representative of at least three separate experiments. The initial surface levels of PAR1 wild type and S/T A mutant in cells co-transfected with either arr1, arr2, or pcDNA were 0.336 ± 0.003, 0.335 ± 0.010, 0.345 ± 0.009 and 0.472 ± 0.008, 0.449 ± 0.005, or 0.459 ± 0.014, respectively. The amount of antibody binding to untransfected cells was less than 5% of that observed in transfected cells. The insets reveal a similar amount of arr1 and arr2 expression in total cell lysates from an equivalent well.

View this table: [in this window] [in a new window]   TABLE I Effect of -arrestins on relative efficacy and potency of thrombin at PAR1 wild type and S/TA mutant COS-7 cells transiently expressing PAR1 and S/TA mutant together with arr1, arr2, or vector were incubated with varying concentrations of -thrombin for 5 min at 37 °C, and [3H]inositol phosphates were then measured. The data are representative of three independent experiments (mean ± S.E.) performed in triplicate.

View larger version (28K): [in this window] [in a new window]   FIG. 3. Effect of -arrestins on initial rate of PAR1 wild type and S/TA mutant desensitization. A and B, COS-7 cells transiently expressing PAR1 wild type or S/T A mutant together with either arr1, arr2, or pcDNA labeled with myo-[3H]inositol were incubated with 10 nM -thrombin for 10 min at 37 °C. Lithium chloride was added after various times of agonist exposure, and the amounts of [3H]IPs formed were then measured. The data shown (mean ± S.D.; n = 3) are expressed as -fold increase over basal [3H]IPs determined at each time point of one experiment and are representative of three independent experiments. Each time point on the graph corresponds to the amount of PAR1 signaling activity remaining after various times of thrombin exposure. The insets indicate a comparable amount of arr1 and arr2 expression in total cell lysates from an equivalent well.

View larger version (26K): [in this window] [in a new window]   FIG. 4. Both of the -arrestin isoforms are equally effective at desensitizing SPR signaling. A, COS-7 cells transiently expressing SPR and either arr1, arr2, or pcDNA were labeled with myo-[3H]inositol and treated with or without 100 nM substance P for various times at 37 °C and processed as described above. The data (mean ± S.D.; n = 3) shown are expressed as -fold increase in [3H]IPs over basal and are representative of three or more experiments. The initial levels of surface SPR expression in cells co-transfected with arr1, arr2, or pcDNA were 0.148 ± 0.006, 0.160 ± 0.002, or 0.155 ± 0.002, respectively. The inset shows a comparable amount of arr1 and arr2 expression in total cell lysates from an equivalent well. B, COS-7 cells were co-transfected with Gq or Gq Q205L mutant plasmids and either arr1, arr2, or pcDNA vector, and [3H]IP accumulation was measured after a 30-min agonist incubation. The data are the mean ± S.D. for triplicate samples in one experiment and are representative of three separate experiments.

We next assessed thrombin-stimulated PI hydrolysis in cells expressing wild type and mutant PAR1 and varying amounts of the individual -arrestin isoforms to exclude the possibility that the differential effects of -arrestins on PAR1 signaling are due to differences in the levels of -arrestin expression. COS-7 cells transiently transfected with either PAR1 wild type or S/T A mutant and varying amounts of FLAG-tagged arr1 or FLAG-tagged arr2 were incubated in the absence or presence of agonist for 30 min at 37 °C. The generation of IPs was then measured, or cell lysates were prepared, and -arrestin expression was detected by immunoblotting. In the absence of -arrestin expression, an 2-fold and 4-fold increase in IP accumulation was detected in PAR1 wild type- and S/T A mutant-expressing cells following 30 min of agonist exposure, respectively (Fig. 5, A and B, lane 1). In cells expressing wild type PAR1 and maximum amounts of arr2, activated PAR1 signaling was modestly diminished by 20% (Fig. 5A), whereas arr1 caused a significantly greater 50% inhibition of agonist-stimulated signaling (Fig. 5A). In PAR1 S/T A mutant-expressing cells, agonist-stimulated PI hydrolysis was decreased more effectively by arr1 compared with arr2 (Fig. 5B), similar to the results observed with wild type receptor. However, both -arrestin isoforms were quite efficacious at attenuating thrombin-induced PI hydrolyis, suggesting that the PAR1 S/T A mutant is more sensitive than wild type receptor to -arrestins. Regardless, in cells expressing comparable amounts of arr1 and arr2, the arr1 isoform appears more effective than arr2 at terminating activated PAR1 signaling even in the absence of receptor phosphorylation.

View larger version (26K): [in this window] [in a new window]   FIG. 5. -Arrestin isoforms differentially regulate PAR1 signaling in an expression-dependent manner. A and B, COS-7 cells were transiently co-transfected with a constant 0.2 µg of PAR1 wild type or S/T A mutant and varying amounts of either FLAG-arr1, FLAG-arr2, or pcDNA vector equaling 0.2 µg such that the total plasmid amount equaled 0.4 µg. Cells were then labeled with myo-[3H]inositol and incubated in the absence or presence of 10 nM -thrombin for 30 min at 37 °C and processed as described above. The data are the mean ± S.D. of triplicates determined in one experiment and are representative of three or more individual experiments. The basal [3H]IPs determined from cells co-transfected with wild type PAR1 and either pcDNA, arr1, or arr2 were on average 230 ± 59, 226 ± 43, and 214 ± 44 cpm per well, respectively. PAR1 S/T A mutant co-transfected with either pcDNA, arr1, or arr2 yielded basal [3H]IPs of 354 ± 76, 227 ± 35, or 256 ± 33 cpm/well, respectively. Lysates were prepared from cells transfected exactly as described above and immunoblotted (IB) to detect -arrestin expression.

View larger version (19K): [in this window] [in a new window]   FIG. 6. -Arrestins fail to enhance agonist-induced PAR1 internalization. A and B, COS-7 cells transiently co-transfected with PAR1 wild type or S/T A mutant and either arr1, arr2, or pcDNA were incubated with or without 50 µM SFLLRN for 30 min at 37 °C. Cells were then fixed, and the amount of PAR1 remaining on the cell surface was measured by ELISA and used as an index for receptor internalization. The data are expressed as a percentage of the total amount of antibody bound to the cell surface of a transfected untreated control for each transfection condition. The data (mean ± S.D.) of triplicate samples of one experiment shown are representative of five experiments. C, transiently transfected COS-7 cells expressing SPR and either arr1, arr2, or pcDNA were incubated in the absence or presence of 100 nM substance P for 30 min at 37 °C. Cells were fixed, and the amount of receptor remaining on the cell surface was determined as described above. Data (mean ± S.D.; n = 3) are expressed as a percentage of total receptor measured in transfected untreated control cells and are representative of three separate experiments performed in triplicate.

Immunofluorescence confocal microscopy studies are consistent with a failure of -arrestins to enhance internalization of PAR1. COS-7 cells were transiently co-transfected with PAR1 wild type or S/T A mutant together with either GFP-tagged arr1 or GFP-arr2, and internalization of PAR1 was assessed by confocal microscopy. In the absence of agonist, both wild type and mutant PAR1 are localized predominantly to the cell surface (Fig. 7, A and B, top panels). However, a small fraction of unactivated receptor was found in an intracellular pool in both wild type- and mutant PAR1-expressing cells, consistent with tonic cycling of these receptors as previously reported (25). In cells expressing wild type PAR1, exposure to SFLLRN for 10 min at 37 °C caused substantial internalization of receptor into endocytic vesicles (Fig. 7A). A similar extent of agonist-induced PAR1 internalization was observed in both arr1- and arr2-expressing cells (Fig. 7A). In contrast, agonist failed to promote PAR1 S/T A mutant internalization, even in cells overexpressing arr1 and arr2 (Fig. 7B). These findings provide further support for an arrestin-independent internalization of PAR1 in COS-7 cells.

View larger version (177K): [in this window] [in a new window]   FIG. 7. Effect of -arrestins on agonist-induced internalization of PAR1 wild type and S/TA mutant examined by immunofluorescence microscopy. A and B, COS-7 cells transiently expressing PAR1 wild type or S/T A mutant together with pcDNA, GFP-arr1, or GFP-arr2 were incubated in the absence (Ctrl) or presence of 50 µM SFLLRN for 10 min at 37 °C. Cells were fixed, immunostained for PAR1 (top panels) and GFP--arrestin expression (bottom panels), and imaged by confocal microscopy. These images are representative of many cells examined in three different experiments. The scale bar represents 10 µm.

View larger version (36K): [in this window] [in a new window]   FIG. 8. Agonist-induced association of -arrestins with PAR1. A and B, COS-7 cells transiently expressing PAR1 wild type or S/T A mutant and either arr1, arr2, or pcDNA vector were incubated in the absence or presence of 50 µM SFLLRN for 2.5 min at 37 °C. Cells were lysed, and PAR1 was immunoprecipitated with M2 anti-FLAG antibody. Immunoprecipitates (IP) were resolved by SDS-PAGE and then immunoblotted (IB) for either -arrestins or PAR1 using rabbit polyclonal anti-arrestin A1CT antibody or anti-PAR1 antibody, respectively. The expression of -arrestins in total cell lysates was detected with anti-arrestin A1CT antibody. Similar findings were observed in three separate experiments. Results in the bar graphs represent the mean ± S.E. from three independent experiments and are shown as the -fold increase in arr associated with PAR1 compared with untreated control. The extent of arr1 associated with activated wild type PAR1 was significant (**, p < 0.01). Statistical analysis was determined using an unpaired t test.

View larger version (27K): [in this window] [in a new window]   FIG. 9. -Arrestin wild type and constitutively active mutants are equally effective at desensitizing PAR1 signaling. A and C, COS-7 cells transiently transfected with PAR1 wild type or S/T A mutant and either pcDNA, arr1, or arr1 R169E. Cells were labeled with myo-[3H]inositol and incubated in the absence or presence of 10 nM -thrombin for 30 min at 37 °C, and [3H]IPs were then measured. The data shown in bar graphs are the mean ± S.D. of one experiment with triplicate samples and are representative of six individual experiments. The initial levels of PAR1 wild type and S/T A mutant surface expression (mean ± S.D.; n = 3) co-transfected with pcDNA, arr1, or arr1 R169E were 0.139 ± 0.002, 0.131 ± 0.003, or 0.136 ± 0.004 and 0.153 ± 0.002, 0.152 ± 0.012, or 0.168 ± 0.004, respectively. The difference in PAR1 wild type or S/T A mutant signaling co-expressed with pcDNA compared with either arr1 or arr1 R169E was significant (**, p < 0.01). Statistical significance was determined using an unpaired t test. B and D, COS-7 cells transiently transfected with PAR1 wild type or S/T A mutant and either arr2 or arr2 R170E were labeled with myo-[3H]inositol and processed as described above. The initial levels of PAR1 wild type and S/T A mutant co-transfected with pcDNA, arr2 or arr2 R170E were 0.141 ± 0.012, 0.146 ± 0.013, or 0.144 ± 0.012 and 0.151 ± 0.003, 0.157 ± 0.004, or 0.147 ± 0.002, respectively. The data shown in bar graphs are the mean ± S.D. of one experiment with triplicate samples and are representative of six individual experiments.

Next, we examined the ability of wild type and mutant -arrestins to desensitize PAR1 S/TA mutant signaling. In cells expressing PAR1 S/TA mutant alone, a significant increase in basal signaling was consistently observed compared with cells expressing comparable amounts of wild type receptor (Fig. 9). These findings suggest that the PAR1 S/TA phosphorylation-defective mutant is at the least partially constitutive active. Interestingly, expression of either arr1 or arr1 R169E caused a significant 50% decrease in both basal and agonist-induced signaling by PAR1 S/TA mutant (Fig. 9C). These findings suggest that arr1 is able to uncouple activated PAR1 from signaling independent of phosphorylation. arr2 and arr2 R170E mutant also modestly decrease both basal and agonist-induced signaling by PAR1 S/TA mutant but were clearly less effective than arr1 (Fig. 9, C and D). Surprisingly, however, compared with wild type -arrestins, arr1 R169E and arr2 R170E fail to significantly attenuate signaling of either PAR1 wild type or S/TA phosphorylation-defective mutant.

A cluster of three serine residues residing in the third intracellular loop (IC₃) of PAR1 could potentially contribute to -arrestin binding and desensitization of PAR1 signaling. To assess whether these residues are important for termination of PAR1 signaling, the IC₃ serine residues (S²⁹⁷SS²⁹⁹) of both PAR1 wild type and S/T A mutant were mutated to alanines. COS-7 cells expressing PAR1 wild type or IC₃ S²⁹⁷SS²⁹⁹ mutant and either arr1 or arr1 R169E mutant were exposed to agonist for 30 min, and IP accumulation was assessed. The mutation of the IC₃ serine cluster failed to effect the ability of either arr1 or arr1 R169E mutant to terminate PAR1 signaling (Fig. 10A). Interestingly, mutation of the three serine residues in the IC₃ loop of PAR1 S/T A mutant also failed to effect desensitization of signaling by either arr1 or arr1 R169E (Fig. 10B). Both arr2 wild type and arr2 R170E mutant also failed to alter signaling by PAR1 wild type or S/T A mutant in which the IC₃ serine cluster was mutated (data not shown). Together, these findings support the distinct possibility that phosphorylation-independent -arrestin binding contributes to PAR1 desensitization.

View larger version (27K): [in this window] [in a new window]   FIG. 10. PAR1 wild type and IC3 S297SS299 mutants are regulated similarly by -arrestins. A and B, COS-7 cells transiently co-transfected with PAR1, PAR1 S/T A, PAR1 IC3 S297SS299 mutant, or PAR1 S/T A IC3 S297SS299 mutant and either pcDNA, arr1, or arr1 R169E were stimulated with 10 nM -thrombin for 30 min at 37 °C, and [3H]IP accumulation was determined. The bar graph results are the mean ± S.D.; n = 3 of one experiment representative of three independent experiments. The initial surface expression (mean ± S.D.; n = 3) of PAR1 wild type and IC3 S297SS299 mutant co-expressed with pcDNA, arr1, or arr1 R169E were 0.196 ± 0.002, 0.205 ± 0.026, or 0.216 ± 0.003 and 0.297 ± 0.006, 0. 316 ± 0.003, or 0.306 ± 0.010, respectively. PAR1 S/T A and S/T A IC3 S297SS299 mutant initial surface expressions were 0.107 ± 0.006, 0.118 ± 0.041, or 0.103 ± 0.002 and 0.095 ± 0.007, 0.084 ± 0.001, or 0.087 ± 0.009, respectively. The signaling responses by receptors co-expressed with pcDNA were significantly different (p < 0.01) compared with receptor responses in cells co-expressing arr1 or arr1 R169E in all cases. Statistical significance was determined using an unpaired t test.

View larger version (25K): [in this window] [in a new window]   FIG. 11. Constitutively active -arrestin mutants display enhanced ability at desensitizing the 5-HT2A receptor. A and B, COS-7 cells were transiently transfected with FLAG-tagged 5-HT2A receptor and either pcDNA, arr1, arr1 R169E, arr2, or arr2 R170E. Cells were then labeled with myo-[3H]inositol and incubated in the presence or absence of 10 µM 2,5-dimethoxy-4-iodophenylisopropylamine for 30 min at 37 °C, and [3H]IPs were then measured. The data are expressed as the mean ± S.D. of an experiment performed in triplicate and are representative of three independent experiments. The initial levels of 5-HT2A receptor expressed on the cell surface were similar in each transfection condition as assessed by cell surface ELISA. The data are shown as the mean ± S.D. of triplicates of one experiment representative of three independent experiments. The amount of antibody binding to untransfected cells (UT) is shown. Co-expression of either arr1 or arr2 caused a significant (p < 0.01) decrease in activated 5-HT2A signaling response compared with vector control. The expression of mutant arr1 R169E and arr2 R170E induced a further significant decrease (p < 0.01) in 5-HT2A signaling response compared with wild type -arrestins. Statistical analysis was performed with an unpaired t test.

	DISCUSSION

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