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J. Biol. Chem., Vol. 275, Issue 27, 20412-20423, July 7, 2000

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The , but Not the , Isoform of the Human Thromboxane A₂ Receptor Is a Target for Prostacyclin-mediated Desensitization^*

Marie-Therese Walsh, John F. Foley, and B. Therese Kinsella^§

From the Department of Biochemistry, Conway Institute of Biomolecular and Biomedical Research, Merville House, University College Dublin, Belfield, Dublin 4, Ireland

Received for publication, September 27, 1999, and in revised form, March 17, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study, we examined the effects the prostacyclin receptor (IP) agonist cicaprost exhibited on U46619-mediated thromboxane A₂ receptor (TP) signaling in platelets and compared it to that which occurs in human embryonic kidney (HEK) 293 cells stably overexpressing the individual TP or TP isoforms. Consistent with previous studies, cicaprost abrogated U46619-mediated platelet aggregation and mobilization of intracellular calcium ([Ca²⁺]_i). In HEK 293 cells, signaling by TP, but not TP, was subject to IP-mediated desensitization in a protein kinase A-dependent, protein kinase C-independent manner. Desensitization of TP signaling was independent of the nature of the IP agonist used, the level of IP expression, or the subtype of G_q protein. Signaling by TP³²⁸, a truncated variant of TP devoid of the divergent residues of the TPs, or by TP^S329A, a site-directed mutant of TP, were insensitive to IP agonist activation. Whole cell phosphorylations established that TP, but not TP or TP^S329A, is subject to IP-mediated phosphorylation and that TP phosphorylation is inhibited by H-89. Thus, we conclude that TP, but not TP, is subject to cross-desensitization by IP mediated through direct protein kinase A phosphorylation at Ser³²⁹ and propose that TP may be the isoform physiologically relevant to TP:IP-mediated vascular hemostasis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The prostanoids thromboxane A₂ (TXA₂)¹ and prostacyclin play key, yet opposing, roles in the maintenance of vascular hemostasis (1). TXA₂, which is synthesized mainly by platelets, mediates platelet shape change and aggregation and constriction of vascular and bronchial smooth muscle, whereas prostacyclin, which is synthesized mainly by the vascular endothelium, is a potent inhibitor of platelet aggregation and induces vasodilation (2). TXA₂ may also induce prostacyclin release from endothelial cells in vivo (3). Perturbations in the levels of TXA₂ or prostacyclin, or their synthases or receptors, have been implicated in various cardiovascular disorders (4-8). However, the molecular mechanisms underlying the counter-regulation of TXA₂ and prostacyclin signaling are poorly understood.

Both TXA₂ and prostacyclin exert intracellular effects by interaction with specific members of the G protein-coupled receptor (GPCR) family, termed TP and IP, respectively (9, 10). There are two isoforms of TP in humans, termed TP and TP, as recommended by the International Union of Pharmacology classification on prostanoid receptors (11, 12). These receptors, which are identical for their first 328 amino acids and differ exclusively in their carboxyl-terminal cytoplasmic tail (C-tail) regions, arise due to alternative splicing in exon 3 of the TP gene (12, 13). The physiologic relevance for the existence of two receptors for TP is currently unknown. Wide cell and tissue distribution of the mRNA for both TP isoforms was recently confirmed by selective reverse transcription-PCR procedures (14). Isoform specific antibodies permitted detection of TP, but not TP, in human platelets, leading to the suggestion that TP may be the predominant isoform in platelets (15), despite the presence of mRNA for both isoforms in platelets (16). The major signaling pathway used by TP in vivo is G protein-dependent stimulation of the -isoforms of phospholipase C (PLC), resulting in increased intracellular concentrations of diacylglycerol and inositol 1,4,5-trisphosphate (IP₃) and mobilization of intracellular calcium ([Ca²⁺]_i) (17). Using a variety of in vitro approaches, various investigators have proposed that the platelet TPs might couple to the heterotrimeric G proteins G_q, G₁₂, G₁₃, G₁₆, and G_i2 (18-25). It was recently demonstrated that the cloned TP can functionally couple to both G_q and G₁₁ following stimulation with the selective TXA₂ mimetic U46619 and the isoprostane 8-epi-prostaglandin F₂ to mobilize [Ca²⁺]_i (26). Coupling to G₁₁ was more efficient than that to G_q. Both TP isoforms couple similarly to G₁₁ in stably transfected HEK 293 cells (27) but oppositely regulate adenylyl cyclase activity in transfected Chinese hamster ovary cells (16), suggesting a possible role for the C-tail in determining G protein specificity. Moreover, G_h, the novel high molecular weight G protein that may also function as a transglutaminase (28-31) can mediate agonist activation of TP, but not TP, leading to inositol phosphate production due to PLC activation (32).

A single receptor, termed IP, appears to mediate the actions of prostacyclin leading to activation of adenylyl cyclase via G_s and elevation of intracellular cAMP (33), a signaling system thought to be important in control of both vascular tone and platelet aggregation (34). However, IP may also couple to multiple G protein/effector systems including phosphoinositide turnover via a pertussis toxin insensitive G protein (35, 36). In human erythroleukemia cells, IP has even been proposed to differentially couple to both G_s and G_i (37). Iloprost, a stable carbacyclin analogue of prostacyclin, can stimulate opening of ATP-sensitive K⁺ channels, leading to hyperpolarization and relaxation of canine carotid artery (38). IP is unique among the family of GPCRs in that it undergoes posttranslational modification by carbon-15 farnesyl isoprene groups (39). This isoprenylation is absolutely required for receptor activation of adenylyl cyclase via G_S and for efficient coupling to PLC via G_q or G₁₁ (39).

A commonly observed phenomenon among GPCRs is desensitization, defined as reduced receptor responsiveness to repeated agonist challenge (40). GPCR desensitization consists of two key mechanisms, namely phosphorylation of the receptor by specific serine/threonine kinases and sequestration or internalization of receptors to intracellular vesicles where they are unavailable for interaction with G proteins. GPCRs can be subject to either homologous (41, 42) or heterologous (42-47) desensitization, mediated via phosphorylation by G protein-coupled receptor kinases or the second messenger-activated protein kinases, including cAMP-dependent PKA and PKC. Such desensitizations provide mechanisms for feedback regulatory loops following receptor activation and signaling and also for cross-talk between different second messenger systems (43). TP may be phosphorylated in vitro, in the third extracellular loop and the C-tail, by both PKA and PKC (48). Differences in the complement and distribution of serine (Ser) and threonine (Thr) residues within the divergent C-tails of TP and TP could affect their sensitivity to phosphorylation. Both TPs may be phosphorylated in response to stimulation with the TXA₂ mimetic U46619 in transfected HEK 293 cells (49), and recent studies indicate that TP but not TP undergoes agonist induced internalization (50). Like TP, IP is sensitive to desensitization by second messenger kinases following stimulation with the IP agonist iloprost, with a single PKC phosphorylation site being critical for its desensitization (51, 36).

Thus, both TP and IP are potentially vulnerable to "heterologous desensitization" by elements of intracellular cascades induced by activation of other receptors; for example, the IP:adenylyl cyclase system is essential to the control of platelet responses and may be manifested at different levels of the signaling system (52). Indeed, cross-talk occurs between TP and IP in human platelets, with prior U46619 stimulation enhancing iloprost-mediated generation of cAMP (52). Similarly, in the megakaryoblastic cell line MEG-01, TXA₂ mimetics U46619 and STA₂ dose-dependently augment subsequent iloprost-induced cAMP formation in a PKC-dependent manner (53). In view of the interplay between TXA₂ and prostacyclin in the maintenance of vascular homeostasis, we considered the potential influence that the intracellular signaling processes induced by IP may have on TP function. In particular, we examined the effect that the selective, high affinity IP agonists cicaprost and iloprost exhibited on U46619-mediated TP responses in platelets. More specifically, we investigated whether cross-talk between IP and TP signaling exists and considered whether such cross-talk may have differential impacts on signaling by the individual TP and TP isoforms. Our results indicate that TP, but not TP, appears to be a direct target for cross-talk between IP: TP responses. Furthermore, H-89, a selective inhibitor of PKA (54, 55), but not the PKC inhibitor GF 109203X (56), reduced IP-mediated desensitization of TP and platelet TP(s), whereas TP was insensitive to this desensitization pathway. Prior exposure of HEK.TP³²⁸ cells, stably overexpressing a variant of TP truncated at amino acid 328 at the point of divergence of TP and TP, to cicaprost or iloprost did not affect subsequent U46619-mediated TP signaling, implying that the C-tail region is a crucial determinant of heterologous desensitization of TP by IP-mediated signaling. TP and TP are predicted to contain 9 and 10 putative PKA sites, respectively; however, 8 are conserved between both isoforms, and thus, TP and TP contain 1 and 2 putative PKA sites, respectively, within their unique C-tail sequences. Thus, TP is predicted to contain a unique PKA consensus site within its divergent C-tail, where Ser³²⁹ represents the putative target residue for phosphorylation. U46619-mediated [Ca²⁺]_i mobilization by HEK.TP^S329A cells stably overexpressing a site-directed mutant of TP was insensitive to IP (cicaprost or iloprost)-mediated desensitization, confirming that Ser³²⁹ is a target for IP-mediated desensitization. Finally, whole cell phosphorylation assays established that TP, but not TP or TP^S329A, is subject to IP-mediated phosphorylation and that phosphorylation of TP is abrogated in the presence of H-89. Thus, taken together, our results establish that TP, but not TP, is subject to cross-desensitization by IP that is mediated through direct PKA phosphorylation of Ser³²⁹ and therefore imply that TP may be the isoform physiologically relevant to the maintenance of vascular hemostasis.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- The following chemicals were obtained from Cayman Chemical Co.: 5-heptenoic acid, 7-[6-(3-hydroxy-1-octenyl)-2-oxabicyclo [2,2,1]-hept-5-yl]-1R-[1,4,5(z),6(1E,3S*)]-9,11-dideoxy-9,11-methanoepoxy prostaglandin F₂ (U46619); 5-heptenoic acid, 7-(3-[[Z-[phenylamino carbonyl] hydrazino] methyl]-7-oxabicyclo [2.2.1] hept-2-yl-,[1S-[1,2(Z),3,4]] (SQ29,548); thromboxane B₂ enzyme immunology kit. G418, 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-2-oxy]-2-(2'-amino-5'-methylphenoxy)-ethane-N,N,N'N'-tetraacetic acid, pentaacetoxymethyl ester (Fura2/AM), D-myo-inositol 1,4,5-trisphosphate, 3-deoxyhexasodium salt (stable analogue of IP₃), and dibutyryl cAMP were purchased from Calbiochem. [³H]SQ29,548 (50.4 Ci/mmol) and [³²P]orthophosphate (8000-9000 Ci/mmol) were obtained from NEN Life Science Products. [³H]cAMP (15-30 Ci/mmol) and [³H]IP₃ (20-40 Ci/mmol) were obtained from American Radiolabeled Chemicals Inc. [³H]Iloprost (15.3 Ci/mmol) and iloprost were purchased from Amersham Pharmacia Biotech. Ultraspec total RNA isolation system was obtained from Biotecx Laboratories (Houston, TX); Moloney mouse leukemia virus reverse transcriptase, RNasin, deoxyribonucleotides, and Taq DNA polymerase were obtained from Promega. Expand High Fidelity® Taq DNA polymerase, Chemiluminescence Western blotting kit, polyvinylidene difluoride membrane, and rat monoclonal 3F10 anti-HA-horseradish peroxidase-conjugated antibody were obtained from Roche Molecular Biochemicals. Mouse monoclonal 101R anti-HA-peroxidase antibody (5-7 mg/ml) was obtained from Babco; horseradish peroxidase-conjugated goat anti-mouse secondary antibody was from Santa Cruz Biotechnology; protein G-Sepharose 4B Fast Flow was obtained from Sigma. All oligonucleotides were synthesized by Genosys Biotechnologies.

Subcloning and Site-directed Mutagenesis of TP and TP-- The plasmids pCMV5, pCMV:TXR (26), pcDNA3:TP, and pcDNA3:TP (27) have been previously described. To facilitate amino-terminal epitope tagging of proteins with the hemagglutinin (HA) epitope tag (57), cDNAs encoding TP and TP were subcloned in-frame into the HindIII-BamHI sites of the pHM6 (Roche Molecular Biochemicals) to generate pHM:TP and pHM:TP, respectively.

Deletion of the amino acids carboxyl to Arg³²⁸, at the point of divergence between TP and TP was achieved by conversion of Ser codon 329 to a stop codon (Ser³²⁹ TCG to stop³²⁹ TAA). Site-directed mutagenesis was performed by PCR mutagenesis using pCMV:TXR as template and oligonucleotides 5'-CTCTAAGCTTATG TGG CCC AAC GGC AGT-3' (sense primer; nucleotides +1 to +18 of TP sequences are underlined) and 5'-CTCTGGATCCTTATCTGGGCCGGGTGCTGAG-3' (antisense primer; sequences complementary to nucleotides + 967 to +984 of TP sequences are underlined, and the mutator in-frame stop codon is in boldface italics). The resulting PCR-amplified cDNA was subcloned into the HindIII-BamHI site of pcDNA3 (Invitrogen) to generate pcDNA3:TP³²⁸. Conversion of Ser³²⁹ of TP to Ala³²⁹ was performed by PCR mutagenesis using pCMV:TXR as template and oligonucleotides 5'-GAGAAGCTTG ATG TGG CCC AAC GGC AGT TCC-3' (sense primer; nucleotides +1 to +21 of TP sequences are underlined) and 5'-CTCT AAGCTT CTA CTG CAG CCC GGA GCG CTG CGT GAG CTG GGG CTG GAG GGA CAG CGC CCT GGG CCG GG-3' (antisense primer; nucleotides complementary to nucleotides + 974 to + 1032 of TP sequences are underlined, and the sequence complementary to mutator Ser (TCG) to Ala (GCG) codon is in boldface italics). PCR amplifications were performed using Expand High Fidelity® Taq DNA polymerase, and products were subcloned into the HindIII site of pHM6 to generate pHM:TP^S329A. The plasmids pcDNA3:TP³²⁸ and pHM:TP^S329A were verified by double-stranded DNA sequencing using Sequenase Version 2.0 (United States Biochemical Corp.). The plasmids pCMV:G₁₁, pCMV:Gq, and pcDNA3:mIP, coding for G₁₁, G_q, and mouse prostacyclin receptor (IP), respectively, have been described previously (26, 39).

Cell Culture and Transfections-- Human embryonic kidney (HEK) 293 cells were obtained from the American Type Culture Collection and were grown in minimal essential medium containing 10% fetal bovine serum.

Cells were transfected with 10 µg of pADVA (58) and 25 µg of pCMV-, pcDNA-, or pHM-based vectors using the calcium phosphate/DNA co-precipitation procedure (26). For transient transfections, cells were harvested 48 h after transfection. To create stable cell lines, HEK 293 cells were transfected with 10 µg of ScaI-linearized pADVA plus 25 µg of PvuI-linearized pcDNA- or pHM-based vectors. Forty-eight hours posttransfection, G418 (0.8 mg/ml) was added; after approximately 21 days, resistant colonies were selected, and clonal cell lines were expanded. In this way, HEK.TP³²⁸ (pcDNA3:TP³²⁸), HEK.HATP (pHM:TP), HEK.HATP (pHM:TP), and HEK.HATP^S329A (pHM:TP^S329A) stable cell lines were established using their respective plasmids (in parentheses). HEK.1 and HEK.3 stable cell lines overexpressing TP and TP, respectively, have been described (27). The HEK.10 cell line, which was established under identical conditions as HEK.1 cells and expresses similar levels of TP (K_d = 5.56 ± 0.98 nM; B_max 3.38 ± 0.08 pmol/mg of cell protein) and HEK.3 cells (K_d = 8.44 ± 1.44 nM; B_max 3.24 ± 0.33 pmol/mg of cell protein), was used in this study.

Preparation of Platelets-- Blood was drawn via venipuncture from normal human volunteers, who had not taken any medication for at least 10 days, into syringes containing indomethacin (10 µM) and 3.8% sodium citrate (9:1 v/v) (final concentration, 0.38% sodium citrate). The blood was centrifuged for 10 min at 160 × g; and the platelet-rich plasma was removed and recentrifuged for 10 min at 160 × g to remove contaminating red blood cells. Where necessary, platelet-poor plasma was prepared by spinning the remaining blood at 900 × g for 15 min. For aggregation studies, platelets in platelet-rich plasma were diluted to approximately 10⁸ platelets/ml in platelet-poor plasma; 0.5-ml aliquots were preincubated at 37 °C for 2 min before addition of the aggregating agent (1 µM U46619, 1 µM cicaprost) or vehicle, and the extent of aggregation was monitored by light transmission in a Biodata Pap 4 aggregometer. TXB₂ formation was routinely measured in platelet-rich plasma and platelet-poor plasma by enzyme immunoassay using a thromboxane B₂ EIA kit (Cayman Chemical Co.).

Calcium Measurements-- Measurements of intracellular calcium either in transfected HEK 293 cells or in platelets were made by monitoring the intensity of Fura2 fluorescence as described previously (26). Cells were stimulated with 1 µM U46619, 1 µM cicaprost, 1 µM iloprost unless otherwise specified. The PKA inhibitor H-89 or PKC inhibitor GF 109203X was added at times and concentrations specified in the figure legends. In all cases, the drug (agonists/kinase inhibitors in ethanol or DMSO) was diluted 1:1000 in vehicle (modified Ca²⁺/Mg²⁺-free Hank's buffered salt solution, containing 10 mM HEPES, pH 7.67, 0.1% bovine serum albumin) for HEK 293 cells and platelet resuspension buffer (10 mM HEPES, 145 mM NaCl, 5 mM KCl, 5.5 mM glucose, pH 7.4) for platelets, and 20 µl of the vehicle (containing an equivalent volume of the drug solvent) or drug in vehicle was added to 2 ml of cells; the vehicle had no effect on [Ca²⁺]_i mobilization by either TP isoform and had no effect on experimental data. The ratio of the fluorescence at 340 nm to that at 380 nm is a measure of [Ca²⁺]_i (59), assuming a K_d of 225 nM Ca²⁺ for Fura2/AM. The results presented in the figures are representative data from at least four independent experiments and are plotted as changes in intracellular Ca²⁺ mobilized as a function of time upon ligand stimulation.

Radioligand Binding Studies-- TP radioligand binding assays were carried out at 30 °C for 30 min in 100-µl reactions in the presence of 0-40 nM [³H]SQ29,548 for Scatchard analyses or in the presence of 20 nM [³H]SQ29,548 for saturation radioligand binding experiments (26). IP radioligand binding assays were carried out on nonfractionated HEK 293 cells, as described previously (39). Protein determinations were carried out using the Bradford assay (60).

Measurement of IP₃ Levels-- Measurement of IP₃ levels in HEK 293 cells was made on the basis of competition between unlabeled IP₃ and a fixed concentration of [³H]IP₃ for binding to an IP₃-binding protein derived from bovine adrenal glands, essentially as described by Godfrey (61). Briefly, cells were harvested by scraping, washed twice in ice-cold phosphate-buffered saline, and 2 × 10⁶ cells were resuspended in 200 µl of HEPES-buffered saline (HBS) (140 mM NaCl, 4.7 mM KCl, 2.2 mM CaCl₂, 1.2 mM KH₂PO₄, 11 mM glucose, 15 mM HEPES-NaOH, pH 7.4) supplemented with 10 mM LiCl. Cells were preequilibrated in this buffer at 37 °C for 10 min and stimulated for 1 min at 37 °C in the presence of cicaprost (1 µM) or U46619 (1 µM), in the presence of cicaprost (1 µM) for 1 min followed by U46619 (1 µM) for 1 min, or, to determine basal IP₃ levels in cells, in the presence of an equivalent volume (50 µl) of HBS vehicle. IP₃ extraction and quantification was determined by radioimmunoassay essentially as described by Godfrey (61). Levels of IP₃ produced by ligand-stimulated cells over basal stimulation, in the presence of HBS, were expressed in pmol of IP₃/10⁶ cells ± S.E. and as fold stimulation over basal (fold increase ± S.E.). The data presented are representative of four independent experiments, each performed in duplicate.

Measurement of cAMP-- cAMP produced was quantified by radioimmunoassay using the cAMP-binding protein from bovine adrenal medulla (62) as described previously (39). Levels of cAMP produced by cicaprost-stimulated cells over basal stimulation, in the presence of HBS, were expressed in pmol of cAMP/mg of cell protein ± S.E. and as fold stimulation over basal (fold increase ± S.E.). The data presented are representative of four independent experiments.

Reverse Transcriptase-Polymerase Chain Reaction-- Total RNA isolated from human erythroleukemia 92.1.7 or HEK 293 cells using the Ultraspec RNA isolation procedure was converted to first strand cDNA with Moloney murine leukemia virus reverse transcriptase, as described previously (14). Aliquots (3.5 µl) of each first strand cDNA were used as templates in PCRs (25 µl) using the following primers specific for the human IP cDNA: primer A, 5'-GCTCCCTGCCTCTCACGATCCGCTGCTTCACCC-3' (sense primer); and primer B, 5'-GTGGGGATCCAAGCTTTCAGCAGAGGGAGCAGGC-3' (antisense primer). Primers were designed to span across intron 2 of the human IP gene (63) to distinguish products derived from first strand cDNA from trace genomic DNA present in the total RNA.

Measurement of Agonist-mediated TP Phosphorylation-- Whole cell phosphorylation assays were performed essentially as previously (49) with certain modifications. Briefly, cells (2-3 × 10⁶ cells in 60-mm dishes) were washed once in phosphate-free Dulbecco's modified Eagle's medium, 10% dialyzed fetal bovine serum and were metabolically labeled for 60 min in the same medium (1.5 ml/60-mm dish) containing 100 µCi/ml [³²P]orthophosphate (8000-9000 Ci/mmol) at 37 °C, 5% CO₂. Where appropriate, kinase inhibitor (H-89, 10 µM) or vehicle was added during the labeling period. Thereafter, specific ligand or vehicle was added for 10 min. Reactions were terminated by transferring the dishes to ice and aspirating the labeling medium. Cells were washed once in ice-cold phosphate-buffered saline (2 ml/dish) and were lysed with 0.6 ml of radioimmune precipitation buffer (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40 (v/v), 0.5% sodium deoxycholate (w/v), 0.1% SDS (w/v) containing 10 mM sodium fluoride, 25 mM sodium pyrophosphate, 10 mM ATP, 1 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor, 1 mM benzamidine hydrochloride, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate. Following 15 min of incubation on ice, cells were harvested using a rubber policeman and disrupted by sequentially passing through hypodermic needles of decreasing bore size (G20, G21, G23, and G26), and soluble cell lysates were harvested by centrifugation for 15 min at 13,000 × g at room temperature. HA epitope TP receptors were immunoprecipitated using the anti-HA antibody (101R, 1:300 dilution) at room temperature for 2 h followed by the addition of 10 µl of protein G-Sepharose 4B (Sigma) and further incubation at room temperature for 1 h. Immune complexes were collected by centrifugation at 13,000 × g at room temperature for 5 min and were washed three times in 0.5 ml of radioimmune precipitation buffer and finally resuspended in 1× solubilization buffer (10%  mercaptoethanol (v/v), 2% SDS (w/v), 30% glycerol (v/v), 0.025% bromphenol blue (w/v), 50 mM Tris-HCl, pH 6.8; 40 µl). Samples were loaded without boiling onto 10% polyacrylamide gels, analyzed by SDS-polyacrylamide gel electrophoresis, and thereafter electroblotted onto polyvinylidene difluoride membranes, essentially as described previously (39). Electroblots were then exposed to Eastman Kodak Co. Xomat XAR film to detect ³²P-labeled proteins. Thereafter, blots were subject to phosphorimage analysis, and the intensities of phosphorylation relative to basal phosphorylation were determined and were expressed in arbitrary units of intensity relative to basal levels. In parallel experiments, cells were incubated under identical conditions in the absence of [³²P]orthophosphate; HA-TP receptors were immunoprecipitated from those cells, and immunoblots were screened using the anti-HA antibody to check for quantitative recovery of each receptor type. Thereafter, membranes were screened by immunoblot analysis using the anti-HA 3F10 horseradish peroxidase conjugate; immunoreactive proteins were visualized using the ECL detection system, as described by the manufacturer (Amersham Pharmacia Biotech).

Data Analyses-- Radioligand binding data were analyzed using GraphPad Prism V2.0 (GraphPad Software Inc.) to determine the K_d and B_max values. Statistical analyses were carried out using the unpaired Student's t test using the Statworks Analysis Package. p values of less than or equal to 0.05 were considered to indicate a statistically significant difference.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Differential Effects of Cicaprost on U46619 Signaling via TP and TP Isoforms-- To investigate the direct effect of IP signaling processes on those of TP, we employed the highly specific IP agonist cicaprost (64) and examined its signaling and its influence on U46619-induced intracellular Ca²⁺ mobilization ([Ca²⁺]_i) via the TP(s) expressed in human platelets and in HEK 293 cells overexpressing the individual TP and TP isoforms. Human platelets were preloaded with Fura2/AM and stimulated either with 1 µM U46619 (Fig. 1A) or with 1 µM cicaprost followed by 1 µM U46619 (Fig. 1B). Consistent with previous reports, the platelets exhibited efficient mobilization of [Ca²⁺]_i in response to 1 µM U46619 (Fig. 1A). Mobilization of [Ca²⁺]_i was abolished by prestimulation with cicaprost (Fig. 1B). Cicaprost, at 1 µM (Fig. 1B) or 10 µM (data not shown), failed to result in mobilization of [Ca²⁺]_i, indicating that the IP does not exhibit significant coupling to PLC isozymes in human platelets. Platelet aggregation studies indicated that although platelets aggregated irreversibly in response to 1 µM U46619, this aggregation was completely blocked by prior stimulation with 1 µM cicaprost (Fig. 1, C and D). To ensure that the observed effects in platelets were due to the addition of external ligands, rather than due to the production of endogenous cyclooxygenase-derived products, effective inhibition of platelet cyclooxygenase by 10 µM indomethacin was confirmed by routine measurement of levels of TXB₂ in platelet-rich plasma before and after external agonist activation (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Effect of cicaprost on U46619-mediated signaling in platelets. Platelets were preloaded with Fura2/AM and were stimulated with 1 µM U46619 (A) or 1 µM cicaprost followed by 1 µM U46619 (B); ligands were added at the times indicated by the arrows. The results presented are representative of at least four independent experiments and are plotted as changes in intracellular Ca2+ mobilized as a function of time following ligand stimulation. Actual changes in [Ca2+]i mobilization were as follows. A, 1 µM U46619, [Ca2+]i = 153.0 ± 26.9 nM. B, 1 µM cicaprost, [Ca2+]i = 0 nM; 1 µM U46619, [Ca2+]i = 0 nM. For aggregation studies, platelets were stimulated with 1 µM U46619 (C) or 1 µM cicaprost followed by 1 µM U46619 (D); ligands were added at the times indicated by the arrows, and aggregation of platelets was monitored using a Biodata Pap 4 aggregometer. The results presented are representative of at least four independent experiments and are plotted as percentage of aggregation as a function of time.

To assess whether the abrogation of U46619-induced [Ca²⁺]_i mobilization by cicaprost observed in human platelets may actually involve possible molecular cross-talk between IP activation and TP signaling and, if so, whether it may be targeted toward a specific TP isoform or both isoforms, we utilized HEK 293 cell lines stably expressing either TP (HEK.10) or TP (HEK.3) (27). TP signaling was assessed by measurement of [Ca²⁺]_i mobilization in Fura2/AM-loaded cells in response to U46619 (1 µM). In the case of both cell lines, for efficient mobilization of [Ca²⁺]_i in response to U46619, it was necessary to co-transfect the cells with the  subunit of a member of the G_q family of heterotrimeric G proteins (G₁₁, for example) (Fig. 2, A and E).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Analysis of prostacyclin receptor signaling in HEK.10 and HEK.3 cells. HEK.10 cells (A and B) or HEK.3 cells (E and F), transiently co-transfected with pCMV:G11, were preloaded with Fura2/AM and stimulated with either U46619 (1 µM) or cicaprost (1 µM) followed by U46619 (1 µM) as indicated. The ligands were added at the times indicated by the arrows. The results presented are representative of at least four independent experiments and are plotted as changes in intracellular Ca2+ mobilized as a function of time following ligand stimulation. Actual changes in [Ca2+]i mobilization were as follows. A, 1 µM U46619, [Ca2+]i = 40.3 ± 6.27 nM without G11; [Ca2+]i = 145 ± 12.6 nM with G11. B, 1 µM cicaprost, [Ca2+]i = 0 nM; 1 µM U46619, [Ca2+]i = 64.2 ± 10.6 nM. E, 1 µM U46619, [Ca2+]i = 38.7 ± 6.62 nM without G11; [Ca2+]i = 128.0 ± 29.2 nM with G11. F, 1 µM cicaprost, [Ca2+]i = 0 nM; 1 µM U46619, [Ca2+]i = 133 ± 29.6 nM. C and G, HEK.10 cells (C) and HEK.3 cells (G) were stimulated with 1 µM cicaprost or, as a control, with the vehicle HBS at 37 °C for 10 min. Levels of cAMP produced in ligand or vehicle treated cells were calculated and are presented as the mean value per mg of cell protein ± S.E., n = 4 (pmol of cAMP/mg of cells ± S.E.). D, agarose gel electrophoresis of the human prostacyclin receptor cDNA (405 base pairs) amplified from HEK 293 (lane 1) or human erythroleukemia 92.1.7 (lane 2) cell mRNA by reverse transcription-PCR. The negative control PCR, in which amplification primers without any template cDNA were added to the reaction, is shown in lane 3.

HEK.10 cells co-transfected with G₁₁ showed efficient mobilization of [Ca²⁺]_i in response to 1 µM U46619 (Fig. 2A). Cicaprost (1 µM) stimulated a 2-fold elevation of intracellular cAMP levels over vehicle-treated cells (Fig. 2C), thereby confirming the presence of IP in HEK.10 cells. The presence of mRNA encoding IP in HEK 293 cells was also confirmed by reverse transcription-PCR (Fig. 2D). Cicaprost at 1 µM (Fig. 2B) or 10 µM (data not shown) did not stimulate [Ca²⁺]_i mobilization in HEK.10 cells transiently co-transfected with G₁₁, indicating that the endogenous IP receptors present in these cells lack the ability to couple to PLC. However, following prior stimulation with 1 µM cicaprost, mobilization of [Ca²⁺]_i induced by U46619 was significantly reduced to 44.3 ± 7.3% of that generated by U46619 stimulation only (Fig. 2B, p < 0.001). This implies that, as in human platelets, activation of IP leads to desensitization of TP signaling in HEK 293 cells, confirming cross-talk between the cAMP signaling system induced by IP activation and the IP₃ dependent Ca²⁺ mobilization system induced by TP activation.

To investigate whether the "cross-talk" between the IP and TP signaling systems extended to TP, the effects of cicaprost on subsequent U46619-induced mobilization of [Ca²⁺]_i by HEK.3 cells was monitored. HEK.3 cells stimulated with 1 µM U46619 showed efficient mobilization of [Ca²⁺]_i, which was dependent on the presence of G₁₁ (Fig. 2E). Similar to that observed in platelets and HEK.10 cells, HEK.3 cells co-transfected with G₁₁ did not support [Ca²⁺]_i mobilization in response to cicaprost (Fig. 2F). However, in contrast to both platelets and HEK.10 cells, prior stimulation with 1 µM cicaprost showed no reduction in U46619-mediated mobilization of [Ca²⁺]_i (Fig. 2F). To determine whether the difference in IP-mediated desensitization of TP and TP could be accounted for by an inability of HEK.3 cells to support elevation in cAMP in response to cicaprost, the presence of functional endogenous IP receptors in HEK.3 cells was confirmed by analyzing cicaprost-mediated increases in cAMP formation (Fig. 2G). The observed elevation of cAMP levels was not significantly (p > 0.1) different between HEK.3 and HEK.10 cells (Fig. 2, C and G). The higher levels of cAMP elevation observed using 10 µM cicaprost were still insufficient to reduce subsequent U46619-induced [Ca²⁺]_i mobilization in HEK.3 cells (data not shown).

Similar to the results with cicaprost, prior exposure of HEK.10 cells with iloprost (1 µM) reduced subsequent U46619-mediated mobilization of [Ca²⁺]_i by TP to 58.4 ± 4.1% (Fig. 3A), whereas mobilization of [Ca²⁺]_i by TP was unaffected by iloprost (Fig. 3B). There was no significant difference in iloprost- or cicaprost-mediated desensitization of TP (p = 0.168). Moreover, whereas HEK 293 cells do contain endogenous IP (Table I), which is coupled to activation of adenylyl cyclase (Fig. 2, C and G), it is formally possible that the levels of endogenous IP expressed are not sufficiently high to mediate efficient desensitization of the TP isoforms, which might also account for the failure to observe desensitization of the TP isoform in these cells. Thus, to address this, HEK.10 or HEK.3 cells were transiently co-transfected with the cDNA encoding the mouse IP. Overexpression of IP was initially confirmed by saturation radioligand binding studies using [³H]iloprost (Table I). Thereafter, the effect of overexpression of IP on iloprost-mediated (Fig. 3C) or cicaprost-mediated (data not shown) desensitization of the TP isoforms was investigated. Similar to previous data involving endogenous IPs, prior stimulation of HEK.10 cells with iloprost significantly reduced U46619-mediated [Ca²⁺]_i mobilization by TP to 49.8 ± 5.78% (Fig. 3C), whereas iloprost had no effect on U46619-mediated [Ca²⁺]_i mobilization in HEK.3 cells (Fig. 3D). Moreover, overexpression of IP in HEK.10 cells did not significantly enhance iloprost-mediated (p = 0.29) or cicaprost-mediated (data not shown) desensitization of TP signaling, indicating that the endogenous levels of IPs expressed in HEK 293 cells are sufficient to mediate efficient desensitization of TP in those cells.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   IP-mediated desensitization of TP signaling is independent of IP agonist, the level of IP expression, and the Gq subunit. HEK.10 cells (A, C, and E) or HEK.3 cells (B, D, and F) were transiently co-transfected with pCMV:G11 (A-D), pcDNA3:mIP (C and D), or pCMV:Gq (E and F). After 48 h, cells were harvested, preloaded with Fura2/AM, and stimulated either with U46619 (1 µM) or with iloprost (1 µM) followed by U46619 (1 µM), as indicated. The ligands were added at the times indicated by the arrows. The results are representative of at least three independent experiments and are plotted as changes in intracellular Ca2+ mobilized as a function of time following ligand stimulation. Actual changes in [Ca2+]i mobilization were as follows. Panel A: (1 µM U46619, [Ca2+]i = 160 ± 10 nM); (1 µM iloprost, [Ca2+]i = 0 nM; 1 µM U46619, [Ca2+]i = 93.5 ± 6.5 nM). Panel B: (1 µM U46619, [Ca2+]i = 130 ± 10 nM); (1 µM iloprost, [Ca2+]i = 0 nM; 1 µM U46619, [Ca2+]i = 130 ± 18 nM). Panel C: 1 µM U46619, [Ca2+]i = 216 ± 4 nM); (1 µM iloprost, [Ca2+]i = 0 nM; 1 µM U46619, [Ca2+]i = 107 ± 12.5 nM). Panel D: (1 µM U46619, [Ca2+]i = 207 ± 21.4 nM). (1 µM iloprost, [Ca2+]i = 0 nM; 1 µM U46619, [Ca2+]i = 203 ± 8.82 nM). Panel E: (1 µM U46619, [Ca2+]i = 157 ± 3 nM); (1 µM iloprost, [Ca2+]i = 0 nM; 1 µM U46619, [Ca2+]i = 86.5 ± 3.5 nM). Panel F: (1 µM U46619, [Ca2+]i = 166 ± 4 nM); (1 µM iloprost, [Ca2+]i = 0 nM; 1 µM U46619, [Ca2+]i = 169 ± 1 nM).

To investigate whether IP-mediated desensitization of TP signaling in HEK 293 cells may be dependent on the nature of the coupling G protein, we extended our studies to investigate the effect of co-expression of the  subunit of G_q as a substitute to G₁₁. Prestimulation of cells with iloprost (Fig. 3E) or cicaprost (data not shown) reduced subsequent U46619-mediated [Ca²⁺]_i mobilization in HEK.10 cells to 55.1 ± 2.2% (Fig. 3E), whereas U46619-mediated [Ca²⁺]_i mobilization in HEK.3 cells was unaffected (Fig. 3F). Moreover, IP-mediated desensitization of TP was not significantly different in the presence of G_q compared with G₁₁ (p = 0.52). Thus, we conclude that TP but not TP is subject to IP-mediated desensitization in HEK 293 cells and that this desensitization is independent of the nature of the IP agonist, is independent of the level of IP expression, and is independent of the coupling G protein.

Differential Effects of Cicaprost on U46619-mediated IP₃ Generation via TP and TP Isoforms-- To further investigate the differential effects of IP activation on TP and TP signaling, U46619-induced IP₃ generation was measured in HEK.10 and HEK.3 cells in the presence or absence of prestimulation with cicaprost. Stimulation of HEK.10 and HEK.3 cells with U46619 (1 µM) resulted in 1.5- and 1.4-fold increases in IP₃ levels, respectively, comparable with previously reported data (16). However, preincubation of HEK.10 cells with cicaprost (1 µM) significantly (p = 0.024) reduced U46619-mediated IP₃ generation by TP (Fig. 4A). However, in contrast to HEK.10 cells, preincubation of HEK.3 cells with cicaprost (1 µM) did not significantly (p = 0.42) reduce U46619-mediated IP₃ generation by TP (Fig. 4B). Stimulation of HEK 293 cells with U46619 or HEK.10 and HEK.3 cells with cicaprost alone failed to generate any increase in IP₃, further indicating that endogenous IP receptors in HEK 293 cells do not couple to PLC.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Effect of cicaprost on U46619-mediated IP3 production in HEK.10 and HEK.3 cells. HEK 293 cells (control, A) and HEK.10 cells (A) or HEK.3 cells (B) transiently co-transfected with pCMV:G11 were stimulated at 37 °C for 1 min with 1 µM U46619 (U46619) or 1 µM cicaprost for 1 min followed by 1 µM U46619 for 1 min (U46619, cicaprost). In each case, basal IP3 levels were determined by exposing the cells to the vehicle HBS under identical conditions. Levels of IP3 produced in ligand-stimulated cells relative to vehicle treated cells (basal IP3) were expressed as fold stimulation of basal (fold increase in IP3 ± S.E.). The data presented are the mean values of four independent experiments, each carried out in duplicate.

H-89, an Inhibitor of PKA, Prevents Cicaprost-induced Inhibition of TP Signaling-- Second messenger protein kinases, such as PKA and PKC, have been implicated in heterologous desensitization and in mediating cross-talk between different G protein-coupled receptor signaling systems (42-47). Moreover, both IP and TP are subject to phosphorylation by these kinases (48, 49, 51). Thus, to investigate whether PKA and/or PKC is involved and provide a potential mechanism whereby IP activation cross-desensitizes TP signaling in platelets and HEK.10 cells, we used H-89, a specific inhibitor of PKA (54, 55), and GF 109203X, a specific PKC inhibitor (56). Preincubation of platelets with 50 nM GF 109203X for 2 min prior to agonist stimulation had no effect on cicaprost induced desensitization of U46619-mediated [Ca²⁺]_i (Fig. 5, A and B). On the other hand, pretreatment of platelets with 10 µM H-89 for 1 min prior to cicaprost (1 µM) stimulation completely restored subsequent U46619-mediated (1 µM) [Ca²⁺]_i mobilization to normal, precicaprost levels ([Ca²⁺]_i = 195 ± 45.9 nM; Fig. 5C). Similarly, in HEK.10 cells, whereas pretreatment with 50 nM GF 109203X for 2 min had no effect on cicaprost inhibition of U46619-induced [Ca²⁺]_i mobilization (Fig. 6, A and B), pretreatment with H-89 (10 µM, 1 min) significantly (p < 0.001) rescued cicaprost-induced inhibition of U46619-induced [Ca²⁺]_i mobilization from 45 to 86% (Fig. 6C). In HEK.3 cells, in which the U46619-induced changes in [Ca²⁺]_i mobilization are impervious to prestimulation by cicaprost, addition of GF 109203X or H-89 had no effect on signaling by TP (Fig. 6, D-F). These results indicate that the observed reduction of U46619-induced changes in [Ca²⁺]_i by cicaprost or iloprost in HEK.10 cells is largely due to activation of PKA and subsequent phosphorylation, either of TP directly or of some other element of its signaling pathway. Mobilization of [Ca²⁺]_i via TP is, on the other hand, unaffected by PKA in this cross-desensitization pathway.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Effect of protein kinase inhibitors on cicaprost-mediated desensitization of TP signaling in platelets. Platelets were preloaded with Fura2/AM and stimulated with 1 µM cicaprost followed by 1 µM U46619 (A). Alternatively, platelets were preincubated with 50 nM GF 109203X and then stimulated with 1 µM cicaprost followed by 1 µM U46619 (B) or were preincubated with 10 µM H-89 and then stimulated with 1 µM cicaprost followed by 1 µM U46619 (C). The ligands were added at the times indicated by the arrows. The results presented are representative of at least four independent experiments and are plotted as changes in intracellular Ca2+ mobilized as a function of time following ligand stimulation. Actual changes in [Ca2+]i mobilization were as follows. A, 1 µM cicaprost, [Ca2+]i = 0 nM; 1 µM U46619, [Ca2+]i = 0 nM. B, 1 µM cicaprost, [Ca2+]i = 0 nM; 1 µM U46619, [Ca2+]i = 0 nM. C, 1 µM cicaprost, [Ca2+]i = 0 nM; 1 µM U46619, [Ca2+]i = 195 ± 45.9 nM.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Effect of kinase inhibitors on cicaprost-mediated desensitization of TP signaling in HEK.10 and HEK.3 cells. HEK.10 (A-C) or HEK.3 (D-F), transiently co-transfected with pCMV:G11, were preloaded with Fura2/AM and stimulated with 1 µM cicaprost followed by 1 µM U46619 (A and D). Alternatively, cells were preincubated with 50 nM GF 109203X and then stimulated with 1 µM cicaprost followed by 1 µM U46619 (B and E) or were preincubated with 10 µM H-89 and then stimulated with 1 µM cicaprost followed by 1 µM U46619 (C and F). The ligands were added at the times indicated by the arrows. The results presented are representative of at least four independent experiments and are plotted as changes in intracellular Ca2+ mobilized (n = 4) as a function of time following ligand stimulation. Actual changes in [Ca2+]i mobilization were as follows: 1 µM U46619, [Ca2+]i = 114 ± 12.3 nM (data not shown). A, 1 µM cicaprost, [Ca2+]i = 0 nM; 1 µM U46619, [Ca2+]i = 50.0 ± 5.8 nM. B, 1 µM cicaprost, [Ca2+]i = 0 nM; 1 µM U46619, [Ca2+]i = 51.0 ± 11.5. C, 1 µM cicaprost, [Ca2+]i = 0 nM; 1 µM U46619, [Ca2+]i = 98.3 ± 11.3 nM. D, 1 µM cicaprost, [Ca2+]i = 0 nM; 1 µM U46619, [Ca2+]i = 117 ± 9.6 nM. E, 1 µM cicaprost, [Ca2+]i = 0 nM; 1 µM U46619, [Ca2+]i = 112 ± 3.1 nM. F, 1 µM cicaprost, [Ca2+]i = 0 nM; 1 µM U46619, [Ca2+]i = 110 ± 3.7 nM.

It has recently been reported that H-89 may act as antagonist of certain GPCRs, thereby calling into question its utility as a selective PKA inhibitor (65). Thus, to rule out the possibility that H-89 may act as an antagonist of the IP, cicaprost-mediated (1 µM) cAMP generation was measured in HEK 293 cells over expressing the IP in the absence and presence of 10 µM H-89. No significant difference (p > 0.84) was observed in cells stimulated in the absence (1 µM cicaprost; fold increase in cAMP = 22.2 ± 3.74) or presence (1 µM cicaprost, 10 µM H-89; fold increase in cAMP = 20.9 ± 4.71) of H-89, confirming that H-89 does not function as an antagonist of IP.

Cicaprost-induced Desensitization of TP Signaling Is Mediated by the TP C-tail-- In order to establish whether the C-tail of the TP contains the target regulatory site for phosphorylation by PKA, deletion mutagenesis was utilized to generate a truncated version of TP (TP³²⁸), which is devoid of C-tail sequences carboxyl-terminal to amino acid 328 at the point of divergence of TP and TP. A stable HEK 293 cell line overexpressing TP³²⁸ was established, and cells were characterized by Scatchard analysis using [³H]SQ29,548 as the specific radioligand. Values obtained for the affinity (K_d) and maximal binding (B_max) for TP³²⁸ (K_d = 6.99 ± 0.88 nM; B_max = 1.54 ± 0.28 pmol/mg; n = 5) compared well to values previously reported for the wild type TP and TP receptors (26). It is noteworthy that TP³²⁸ exhibited identical affinity for its ligand and retained the ability to mediate specific agonist induced intracellular signaling, albeit at somewhat reduced levels relative to those of the wild type TPs. Intracellular signaling by HEK.TP³²⁸ cells transiently co-transfected with G₁₁ was investigated by analyzing [Ca²⁺]_i mobilization and IP₃ generation in response to the TXA₂ mimetic U46619 and the effect of cicaprost on TP signaling was assessed. Stimulation of HEK.TP³²⁸ cells with U46619 (1 µM) led to mobilization of [Ca²⁺]_i (Fig. 7A), whereas stimulation with cicaprost (1 µM) did not (Fig. 7B). Unlike platelets or HEK.10 cells, prestimulation with cicaprost did not reduce subsequent U46619-induced [Ca²⁺]_i mobilization (Fig. 7B). Moreover, these effects were independent of the agonist used or the level of IP expression (Fig. 7, C and D; Table I). Similarly, U46619 stimulation of HEK.TP³²⁸ cells generated increases in IP₃ levels, which were not significantly reduced by prior stimulation with cicaprost (data not shown). These data confirm that the increased sensitivity to cicaprost or iloprost observed for TP, as opposed to TP and TP³²⁸, is due to unique elements in the C-tail of TP, most likely, given that pretreatment with H-89 alleviated cicaprost induced inhibition of TP signaling, at an important PKA-sensitive phosphorylation site(s).

View larger version (27K): [in this window] [in a new window]   Fig. 7.   Effect of IP agonists on U46619-mediated signaling by TP328. HEK.TP328 cells were transiently co-transfected either with pCMV:G11 (A and B) or with pCMV:G11 plus pcDNA3:mIP (C and D). After 48 h, cells were preloaded with Fura2/AM and were stimulated with 1 µM U46619 alone (A and C), with 1 µM cicaprost followed by 1 µM U46619 (B), or with 1 µM iloprost followed by 1 µM U46619 (D), as indicated. The ligands were added at the times indicated by the arrows. The results presented are representative of at least four independent experiments and are plotted as changes in intracellular Ca2+ mobilized as a function of time following ligand stimulation. Actual changes in [Ca2+]i mobilization were as follows. A, 1 µM U46619, [Ca2+]i = 55.1 ± 6.53 nM. B, 1 µM cicaprost, [Ca2+]i = 0 nM; 1 µM U46619, [Ca2+]i = 59.9 ± 8.23 nM. C, 1 µM U46619, [Ca2+]i = 95 ± 15 nM. D, 1 µM iloprost, [Ca2+]i = 0 nM; 1 µM U46619, [Ca2+]i = 104 ± 13.5 nM.

TP^S329A Is Not Subject to IP-mediated Desensitization-- Computational analysis of the amino acid sequence of the C-tail of TP for putative protein kinase phosphorylation sites using the PhosphoBase program for sequence analysis (66) identified the presence of a unique consensus PKA phosphorylation site within the sequence RPRSLSL, where Ser³²⁹ was identified as the target residue for phosphorylation. Thus, to investigate whether this consensus PKA phosphorylation site may represent a target site for IP-mediated desensitization of TP, the critical Ser³²⁹