Investigation of the Mechanisms of G Protein: Effector Coupling bythe Human and Mouse Prostacyclin ReceptorsIDENTIFICATION OF CRITICAL SPECIES-DEPENDENT DIFFERENCES*

Received for publication, April 8, 2002, and in revised form, May 10, 2002Published, JBC Papers in Press, May 16, 2002, DOI 10.1074/jbc.M203353200

Sinead M. Miggin 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

We recently identified a novel mechanism explaininghow the mouse (m) prostacyclin receptor (IP) couples toG�s, G�i, and G�q (Lawler, O. A., Miggin, S. M., andKinsella, B. T. (2001) J. Biol. Chem. 276, 33596–33607)whereby mIP coupling to G�i and G�q is dependent onits initial coupling to G�s and subsequent phosphoryla-tion by cAMP-dependent protein kinase A (PKA) onSer357. In the current study, the generality of that mech-anism was investigated by examining the G protein cou-pling specificity of the human (h) IP. The hIP efficientlycoupled to G�s/adenylyl cyclase and to G�q/phospho-lipase C activation but failed to couple to G�i. Couplingof the hIP to G�q, or indeed to G�s or G�i, was unaffectedby the PKA or protein kinase C (PKC) inhibitors H-89and GF 109203X, respectively. Thus, mIP and hIP exhibitessential differences in their coupling to G�i and in theirdependence on PKA in regulating their coupling to G�q.Analysis of their primary sequences revealed that thecritical PKA phosphorylation site within the mIP, atSer357, is replaced by a PKC site within the hIP, at Ser328.Conversion of the PKC site of the hIP to a PKA sitegenerated hIPQL325,326RP that efficiently coupled to G�sand to G�i and G�q; coupling of hIPQL325,326RP to G�i butnot to G�s or G�q was inhibited by H-89. Abolition of thePKC site of the hIP generated hIPS328A that efficientlycoupled to G�s and G�q but failed to couple to G�i.Finally, conversion of the PKA site at Ser357 within themIP to a PKC site generated mIPRP354,355QL that effi-ciently coupled to G�s but not to G�i or G�q. Collec-tively, our data highlight critical differences in signal-ing by the mIP and hIP that are regulated by theirdifferential phosphorylation by PKA and PKC togetherwith contextual sequence differences surrounding thosesites.

The prostanoid prostacyclin (prostaglandin I2) is the majorproduct of arachidonic acid metabolism in the vascular endo-thelium (1). It plays a key role in the local control of vascularhemostasis, acting as a potent inhibitor of platelet aggregationand as a vasodilator (2), and exhibits pro-inflammatory andanti-proliferative properties in vitro (3, 4). The actions of pros-tacyclin generally counteract those of thromboxane A2, and therelative levels of these two prostanoids regulate platelet/endo-

thelium/vascular smooth muscle interactions (5). Prostacyclinis abundantly produced during cardiac ischemia/reperfusion,conferring a cytoprotective effect (6), and, from studies carriedout in prostacyclin receptor-deficient mice, is known to exert aprotective effect on cardiomyocytes independent of its effects onplatelets and neutrophils (7). Perturbations in prostacyclinand/or prostacyclin receptor signaling have been implicated inthe pathogenesis of conditions such as ischemic heart disease,atherosclerosis, renal failure, and systemic and pregnancy-induced hypertension (5, 8–10).

Prostacyclin signals through activation of its cell surface Gprotein-coupled receptor (GPCR),1 termed the prostacyclin re-ceptor or IP (11). The IP is subject to post-translational modi-fications such as N-glycosylation and phosphorylation that playcentral roles in regulating receptor function (12, 13). The IPappears to be somewhat unique among GPCRs in that it isisoprenylated (14). Although isoprenylation is not required forligand binding or membrane localization, it is required forefficient IP:G protein coupling and may regulate agonist-induced IP internalization (14–16).

A number of independent studies have demonstrated that,although the IP primarily couples to activation of adenylylcyclase, mediating prostacyclin inhibition of platelet aggrega-tion and vascular tone (5, 17), it may also regulate a number ofother effector systems, perhaps in a tissue- and/or species-specific manner. In the rat medullary thick ascending limb, IPcouples to Gi, but not to Gs, suggesting inhibition rather thanactivation of adenylyl cyclase (18). Iloprost, a stable prostacy-clin analogue, can activate calcium-activated potassium chan-nels (KCa channels) in rat small arteries (19) and can stimulatethe opening of ATP-sensitive K� channels resulting in hyper-polarization and relaxation of canine carotid artery (20). Thecloned mouse (m) and human (h) IPs couple to both GS and toGq, leading to phospholipase C (PLC) activation and to mobili-zation of intracellular calcium (12–16, 21). More recently, wehave demonstrated that, in addition to its coupling to Gs, themIP also couples to both Gi, leading to inhibition of adenylylcyclase, and to Gq, leading to PLC activation, through a novel Gprotein switching mechanism (22). In this mechanism, mIPcoupling to both Gi and Gq are dependent upon its initialcouplingtoGs/adenylyl cyclaseactivationandconsequentcAMP-dependent protein kinase A (PKA) phosphorylation of Ser357

* This work was supported by grants (to B. T. K.) from The WellcomeTrust, The Irish Heart Foundation, Enterprise Ireland, and The HealthResearch Board of Ireland. The costs of publication of this article weredefrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

‡ To whom correspondence should be addressed. Tel.: 353-1-716-1507; Fax: 353-1-283-7211; E-mail: Therese.Kinsella@UCD.IE.

1 The abbreviations used are: GPCR, G protein-coupled receptor;C-tail, carboxyl-terminal tail; [Ca2�]i; intracellular calcium; HA, he-magglutinin; HEL, human erythroleukemia; IP, prostacyclin receptor;IP3, inositol 1,4,5-trisphosphate; PKA, protein kinase A; PKC, proteinkinase C; PLC, phospholipase C; PTx, pertussis toxin; FBS, fetal bovineserum; PBS, phosphate-buffered saline; MES, 4-morpholineethanesul-fonic acid; PVDF, polyvinylidene difluoride; HBS, HEPES-buffered sa-line; Fsk, forskolin; GTP�S, guanosine 5�-3-O-(thio)triphosphate.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 30, Issue of July 26, pp. 27053–27064, 2002© 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org 27053

This article has been withdrawn by the authors. The authors of the paper have become aware that some features had been duplicated in Figs. 7, C

and D, and 8G. As the original autoradiograms and scan images relating to the aforementioned figures are no longer available to investigate the

matter, the authors wish to withdraw the article in the interests of maintaining their publication standards, while also respecting the highest standards of transparency and reliability of their research and of the JBC.

Replica data sets for each of the figures in question that the authors state fully validate the findings and conclusions of the published article are

available, and, accordingly, a revised version of the manuscript with the replica data sets can be obtained by contacting the corresponding author.

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within the carboxyl-terminal cytoplasmic (C) tail of the mIP,thereby switching mIP coupling from GS to Gi and to Gq sig-naling (22).

Thus, it is evident that the IP is capable of coupling tomultiple G protein/effectors in a species- and/or tissue-specificmanner. However, the molecular basis of this species-specificcoupling has not been investigated in detail. Thus, in the pres-ent study, in view of the central roles of prostacyclin within thehuman vasculature, we sought to define the G protein couplingspecificity of the hIP. Moreover, given the central role of PKA-mediated phosphorylation of the mIP in determining its Gprotein specificity and mechanisms of signaling, we sought toinvestigate whether the hIP undergoes a similar G proteinswitching mechanism accounting for its patterns of G proteincoupling and intracellular signaling. Our data highlight criticaldifferences in the signaling of the mIP and hIP that are regu-lated by their differential phosphorylation by the second mes-senger-regulated kinases PKA and PKC together with sur-rounding contextual sequence differences within those kinaserecognition sites, thereby accounting for essential species-de-pendent differences in signaling by the mIP and hIP.

EXPERIMENTAL PROCEDURES

Materials

Cicaprost was obtained from Schering AG (Berlin, Germany). Ilo-prost ([3H]iloprost, 15.3 Ci/mmol) was purchased from Amersham Bio-sciences. Fura2/AM, D-myo-inositol 1,4,5-triphosphate, and its 3-deoxy-hexasodium salt (stable analogue of IP3) were purchased fromCalbiochem. [32P]Orthophosphate (8000–9000 Ci/mmol) was obtainedfrom PerkinElmer Life Sciences. [3H]IP3 (20–40 Ci/mmol) and[3H]cAMP (15–30 Ci/mmol) were purchased from American Radiola-beled Chemicals Inc. GF 109203X, H-89, and Pertussis toxin werepurchased from Calbiochem-Novabiochem. Polyvinylidene difluoridefilters, chemiluminescence Western blotting kit, and rat monoclonal3F10 anti-hemagglutinin (HA) antibody were purchased from RocheMolecular Biochemicals. Mouse monoclonal 101R anti-HA antibody wasobtained from BAbCO. Oligonucleotides were synthesized by GenosysBiotechnologies. A QuikChange site-directed mutagenesis kit was pur-chased from Stratagene.

Methods

Site-directed Mutagenesis of the hIP and mIP—All site-directed mu-tagenesis procedures were performed using the StratageneQuikChange site-directed mutagenesis kit. Conversion of Ser328 of thehIP to Ala328, herein designated hIPS328A, was performed using pHM-hIP (15) as template and mutator oligonucleotides: 5�-CTT TCC CAGCTC GCC GCC GGG AGG AGG GAC C-3� (sense primer) and 5�-G GTCCCT CCT CCC GGC GGC GAG CTG GGA AAG-3� (antisense primer;the sequence complimentary to mutator Ser (TCC) to Ala (GCC) codonis underlined). Conversion of Gln325 and Leu326 of the hIP to Arg325 andPro326, herein designated hIPQL325,326RP, was performed using pHM-hIPas template and oligonucleotides: 5�-CAG ACA CCC CTT TCC CGGCCC GCC TCC GGG AGG AG-3� (sense primer) and 5�-CT CCT CCCGGA GGC GGG CCG GGA AAG GGG TGT CTG-3� (an-tisense primer; the sequence complimentary to mutators Gln and Leu(CAG CTC) to Arg and Pro (CGG CCC) codons are underlined).Conversion of Arg354 and Pro355 of the mIP to Gln354 and Leu355, hereindesignated mIPRP354,355QL, was performed using pHM-mIP (14) astemplate and oligonucleotides: 5�-CAG GCG CCC CTT TCC CAA CTTGCA TCG GGG AGA AG-3� (sense primer) and 5�-CT TCT CCC CGATCG AAG TTG GGA AAG GGG CGC CTG-3� (antisense primer; thesequence complimentary to mutators Arg and Pro (AGA CCT) to Glnand Leu (CAA CTT) codon are underlined). All resulting plasmidspHM-hIPS328A, pHM-hIPQL325,326RP, and pHM-mIPRP354,355QL and mu-tations were verified by automated double-stranded DNA sequencingand encode hemagglutinin (HA) epitope-tagged forms of hIPS328A,hIPQL325,326RP, and mIPRP354,355QL, respectively.

Cell Culture and Transfections—Human erythroleukemia 92.1.7(HEL) cells and human embryonic kidney (HEK) 293 cells were ob-tained from the American Type Culture Collection and maintained at37 °C, 5% CO2. HEL cells were routinely cultured in RPMI 1640 me-dium, 10% fetal bovine serum (FBS). HEK 293 cells were cultured inminimal essential medium with Earle’s salts, 10% FBS. The plasmidpRK5:�ARK1-(495–689) encoding the carboxyl-terminal 495–689

amino acid residues of � adrenergic receptor kinase (ARK)1 has beenpreviously described (23, 22). The plasmids pCMV-G�q and pCMV-G�s

have been previously described (14, 22).HEK 293 cells were transfected with 10 �g of pADVA and 25 �g of

pCMV- or pHM-based vectors using the calcium phosphate/DNA co-precipitation procedure (24). For transient transfections, cells wereharvested 48 h after transfection. HEK.mIP and HEK.hIP cell linesoverexpressing HA epitope-tagged forms of the wild type mIP and hIP,respectively, have been previously described (22, 15). To create theHEK.mIPRP354,355QL, HEK.hIPQL325,326RP, and the HEK.hIPS328A stablecell lines, HEK 293 cells were transfected with 10 �g of ScaI-linearizedpADVA plus 25 �g of PvuI-linearized pHM:mIPRP354,355QL, pHM:hIPQL325,326RP, or pHM:hIPS328A, respectively. Forty eight hours posttransfection, G418 (0.8 mg/ml) selection was applied, and after �21days, G418-resistant colonies were selected and individual pure clonalstable cell lines/isolates were examined for IP expression by radioligandbinding.

Radioligand Binding Studies—Cells were harvested by centrifuga-tion at 500 � g at 4 °C for 5 min and washed three times with phos-phate-buffered saline (PBS). For membrane preparation, cells wereresuspended in homogenization buffer (25 mM Tris-HCl, pH 7.5, 0.25 M

sucrose, 10 mM MgCl2, 1 mM EDTA, 0.1 mM phenylmethylsulfonylfluoride), and membrane fractions were prepared by homogenizationfollowed by centrifugation (100,000 � g, 40 min at 4 °C). The pelletfraction (P100), representing crude membranes, was resuspended inresuspension buffer (10 mM MES-KOH, pH 6.0, 10 mM MnCl2, 1 mM

EDTA, 10 mM indomethacin). IP radioligand binding assays were car-ried out at 30 °C for 1 h, using 100 �g of membrane protein (P100), in100-�l reactions in the presence of 4 nM [3H]iloprost (15.3 Ci/mmol) aspreviously described (22). Protein determinations were carried outusing the Bradford assay (25).

Preparation of Platelets—Blood was drawn via venipuncture fromnormal human volunteers, who had not taken any medication for atleast 10 days, into syringes containing indomethacin (10 �M) and 3.8%sodium citrate (9:1 v/v) (final concentration, 0.38% sodium citrate). Theblood was centrifuged for 10 min at 160 � g; the platelet-rich plasmawas removed and recentrifuged for 10 min at 160 � g to removecontaminating red blood cells. Washed platelets were prepared, follow-ing the addition of 5 mM EDTA as anti-coagulant, by centrifuging theplatelet-rich plasma at 900 � g for 15 min and resuspending theplatelets in a modified Tyrode’s albumin buffer (26) containing 10 �M

indomethacin.Measurement of cAMP—cAMP assays were carried out as previously

described (14). Briefly, cells were harvested by scraping and washedthree times in ice-cold PBS. Cells (�1–2 � 106 cells) or washed humanplatelets (1.85 � 106 platelets/�l) were resuspended in 200 �l ofHEPES-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) con-taining 1 mM 3-isobutyl-1-methylxanthine and were preincubated at37 °C for 10 min. Thereafter, ligands (50 �l) were added and cells werestimulated at 37 °C for 10 min in the presence of the ligand (1 �M

cicaprost, 10 �M forskolin, or 1 �M cicaprost plus 10 �M forskolin). Forconcentration response studies, cells were stimulated with 10�12 to10�6 M cicaprost. As controls, cells were incubated in the presence of 50�l of HBS in the absence of ligand. To investigate the effect of pertussistoxin (PTx), cells were preincubated with PTx (50 ng/ml) for 16 h priorto stimulation with cicaprost plus forskolin. To investigate the effect ofprotein kinase inhibitors on cAMP generation, cells were preincubatedin the presence of GF 109203X (50 nM), H-89 (10 �M), or vehicle (HBS)at 37 °C for 10 min prior to stimulation with cicaprost plus forskolin. Inseparate experiments, to examine the effect of co-transfection of G�s oncAMP generation, HEK 293, HEK.hIP, HEK.hIPQL325,326RP,HEK.hIPS328A, and HEK.mIPRP354,355QL cells were transiently co-trans-fected with pCMV-G�s (25 �g/10-cm dish) plus pADVA (10 �g/10-cmdish). After 48 h, cells were harvested and stimulated with 1 �M cica-prost or vehicle (HBS).

In each case, cAMP reactions were terminated by heat inactivation at100 °C for 5 min and the level of cAMP produced was quantified usingthe cAMP binding protein assay (27). Levels of cAMP produced byligand-treated cells over basal stimulation, determined in the presenceof HBS, were expressed as pmol cAMP/mg cell protein � standard errorof the mean (S.E.). Results are expressed as -fold stimulation relative tobasal (-fold increase � S.E.). Data were analyzed using the unpairedStudent’s t test. p values of less than or equal to 0.05 were consideredto indicate a statistically significant difference.

Measurement of IP3 Levels—Intracellular IP3 levels were measuredas previously described (28). Briefly, cells were transiently co-trans-fected with pCMV-G�q (25 �g/10-cm dish) plus pADVA (10 �g/10-cm

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dish). After 48 h, cells were harvested, washed twice in ice-cold PBS andwere then resuspended at �5 � 106 cells/ml in HBS containing 10 mM

LiCl. Cells (200 �l) were then preincubated at 37 °C for 10 min. Toinvestigate the effect of the protein kinase inhibitors, H-89 (10 �M) orGF 109203X (50 nM) were added and the cells were incubated for 2 minat 37 °C, 5% CO2 prior to stimulation with cicaprost. Thereafter, cellswere stimulated for 2 min at 37 °C in the presence of cicaprost (1 �M) or,for concentration response studies, were stimulated with cicaprost 10�6

to 10�12 M. To determine basal IP3 levels, cells were incubated in thepresence of an equivalent volume (50 �l) of the vehicle HBS. The IP3

levels produced were determined using the IP3 binding protein assay(28). Levels of IP3 produced by ligand-stimulated cells over basal stim-ulation, in the presence of HBS, were expressed in picomoles of IP3/mgof cell protein � standard error (pmol/mg � S.E.), and results arepresented as -fold stimulation over basal (-fold increase � S.E.). Thedata presented are representative of four independent experiments,each performed in duplicate.

Measurement of Intracellular [Ca2�] Mobilization—Measurements of[Ca2�]i mobilization in Fura2/AM-preloaded cells was carried out es-sentially as previously described (24). Where appropriate, the PKA(H-89, 10 �M) or PKC (GF 109203X, 50 nM) inhibitors were added 2 minprior to stimulation with cicaprost (1 �M). Drugs and inhibitors, withstock solutions dissolved in ethanol or Me2SO, were diluted in modifiedCa2�/Mg2�-free Hanks’ buffered salt solution containing 20 mM

HEPES, pH 7.67, 0.1% bovine serum albumin plus 1 mM CaCl2 to theappropriate concentration such that addition of 20 �l of the diluteddrug/inhibitor to 2 ml of cells resulted in the correct working concen-tration. In separate experiments, to examine the effect of the G�� on[Ca2�]i mobilization, cells were transiently co-transfected with the plas-mid pRK5:�ARK1-(495–689) encoding amino acid residues 459–689from the C-tail of �ARK1 (25 �g/10-cm dish) plus pADVA (10 �g/10-cmdish). To investigate the effect of PTx on [Ca2�]i mobilization, cells werepreincubated with PTx (50 ng/ml) for 16 h prior to stimulation withcicaprost plus forskolin. For each [Ca2�]i measurement, calibration ofthe fluorescence signal was performed in 0.2% Triton X-100 to obtainthe maximal fluorescence (Rmax) and 1 mM EGTA to obtain the minimalfluorescence (Rmin). The ratio of the fluorescence at 340 and 380 nm isa measure of [Ca2�]i assuming a Kd of 225 nM Ca2� for Fura2/AM. Theresults presented in the figures are representative data from at leastfour independent experiments and are plotted as changes (�) in [Ca2�]i

mobilized as a function of time (seconds) upon ligand stimulation or,alternatively, were calculated as mean changes in [Ca2�]i mobilized(�[Ca2�]i � S.E.; n � 4).

Measurement of Agonist-mediated IP Phosphorylation in WholeCells—Agonist-mediated IP phosphorylations in whole HEK.hIP,HEK.hIPQL325,326RP, HEK.hIPS328A, HEK.mIP, and HEK.mIPRP354,355QL cells were carried out essentially as previously described(22). Briefly, cells were washed once in phosphate-free Dulbecco’s mod-ified Eagle’s medium containing 10% dialyzed FBS and were metabol-ically labeled for 1 h in the same media (1.5 ml per 10-cm dish) con-taining 100 �Ci/ml [32P]orthophosphate (8000–9000 Ci/mmol) at 37 °C,5% CO2. Where appropriate, H-89 (10 �M), GF 109203X (50 nM), or thevehicle HEPES-buffered saline (HBS) was added for the duration of thelabeling period. Thereafter, 1 �M cicaprost or vehicle HBS was added for10 min at 37 °C, 5% CO2. Metabolic labeling of cells was terminated bytransferring the dishes to ice and aspiration of the medium. Thereafter,cells were quickly washed once in ice-cold PBS (2 ml/dish) and werelysed with 0.6 ml of radioimmune precipitation buffer (50 mM Tris-HCl,pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40 (v/v), 0.5% sodiumdeoxycholate (w/v), 0.1% SDS (w/v) containing 10 mM sodium fluoride,25 mM sodium pyrophosphate, 10 mM ATP, 1 �g/ml leupeptin, 10 �g/mlsoybean trypsin inhibitor, 1 mM benzamidine hydrochloride, 0.5 mM

phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate). Following15-min incubation on ice, cells were harvested and disrupted by sequen-tially passing through hypodermic needles of decreasing bore size (20-,21-, 23-, and 26-gauge), and soluble cell lysates were harvested bycentrifugation for 15 min at 13,000 � g at room temperature. HAepitope-tagged IP receptors were immunoprecipitated using the an-ti-HA antibody (101R, 1:300 dilution) at room temperature for 2 hfollowed by the addition of 10 �l of protein G-Sepharose 4B (Sigma) andfurther incubation at room temperature for 1 h. Immune complexeswere collected by centrifugation at 13,000 � g at room temperature for5 min and were washed three times in 0.3 ml of radioimmune precipi-tation buffer and were 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, 60 �l). Samples wereboiled for 5 min and then loaded onto 10% polyacrylamide gels, ana-lyzed by SDS-PAGE, and thereafter electroblotted onto PVDF mem-

branes. Electroblots were then exposed to Eastman Kodak Co. X-OmatXAR film to detect 32P-labeled proteins. Thereafter, blots were subjectto PhosphorImage analysis, and the intensities of phosphorylation rel-ative to basal phosphorylation were determined and were expressed inarbitrary units of intensity relative to basal levels. In parallel experi-ments, cells were incubated under identical conditions in the absence of[32P]orthophosphate; hIP, hIPQL325,326RP, hIPS328A, mIP, andmIPRP354,355QL receptors were immunoprecipitated, and immunoblotswere screened using the anti-HA 3F10 horseradish peroxidase conju-gate (1:500) to check for quantitative recovery of each receptor type.Immunoreactive proteins were visualized using the chemiluminescencedetection system (22).

Data Analyses—Statistical analysis was carried out using the un-paired Student’s t test using the GraphPad Prism version 2.0 program(GraphPad Software Inc., San Diego, CA). p values of less than or equalto 0.05 were considered to indicate a statistically significant difference.Amino acid sequence alignments were carried out using the ClustalWsoftware (29), where sequences were aligned to show maximumhomology.

RESULTS

Human Prostacyclin Receptor:G protein Coupling to AdenylylCyclase and to Phospholipase C—Previous studies have dem-onstrated that, although the mIP couples to G�s, mediatingincreases in cAMP generation, it may also couple to G�i, lead-ing to inhibition of adenylyl cyclase and cAMP generation andto G�q-mediated PLC activation through a novel PKA-depend-ent mechanism (22). In the present study, we sought to inves-tigate the G protein coupling specificity of the hIP and themechanism of its regulation thereof.

To this end, the effect of the selective agonist cicaprost onIP-mediated cAMP generation in HEK.hIP cells (16), whichstably overexpress the human (h)IP (Table I), was investigatedand compared with that which occurred in HEK.mIP cells (14)stably overexpressing the mouse (m) IP (Table I). Stimulationof HEK.mIP cells (Fig. 1A, p 0.001) and HEK.hIP cells (Fig.1B, p 0.001) with cicaprost each resulted in significant in-creases in cAMP generation consistent with coupling of boththe mIP and the hIP to G�s. Although stimulation of both celltypes with forskolin resulted in significant, receptor-indepen-dent increases in cAMP generation (Fig. 1, A and B, p 0.001),cicaprost significantly inhibited forskolin-induced cAMP gen-eration in HEK.mIP cells, consistent with mIP coupling to G�i

(Fig. 1A, p 0.001). In contrast, in HEK.hIP cells, cicaprostfailed to inhibit (Fig. 1B, p � 0.05) but, rather, augmented (Fig.1B, p 0.05) forskolin-induced cAMP generation indicative ofhIP coupling to G�s, but not to G�i. Moreover, cicaprost showedefficient concentration-dependent increases in cAMP genera-tion in HEK.hIP cells, in the absence or presence of forskolin,throughout the range of cicaprost concentrations used (10�6 to10�12 M) but failed to reduce forskolin-induced cAMP genera-tion (data not shown). Furthermore, stimulation of hIP endog-enously expressed in human erythroleukemic 92.1.7 (HEL)cells and in human platelets exhibited efficient cicaprost-induced rises in cAMP generation in both the presence andabsence of forskolin, consistent with coupling of the hIP to G�s,

TABLE IRadioligand binding assays

Cell type [3H]Iloprost bounda

pmol/mg protein

HEK.hIP 3.02 � 0.30HEK.hIPQL325,326RP 2.98 � 0.38HEK.hIPS328A 2.88 � 0.14HEK.mIP 3.16 � 0.38HEK.mIPRP354,355QL 2.73 � 0.10HEK 293 0.012 � 0.002

a Radioligand binding assays were performed on membrane fractionsin the presence of 4 nM [3H]iloprost. Data are presented as the mean �S.E. (n � 3).

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but not to G�i or to inhibition of adenylyl cyclase activity (Fig.1, C and D, respectively). Whereas mIP coupling to G�i wasinhibited by the PKA inhibitor H-89 (Fig. 1A) (22), the ability ofthe hIP expressed in HEL cells or in human platelets (data notshown) or in HEK.hIP cells to couple to G�s or to G�i wasunaffected by H-89 (p � 0.94; Fig. 1E) or by the PKC inhibitorGF 109203X (p � 0.64, data not shown). These data indicatethat, although the mIP couples to both G�s and G�i through aPKA-dependent mechanism, the hIP couples to G�s but not toG�i.

To investigate the ability of the hIP to couple to G�q-medi-ated PLC activation, cicaprost-induced IP3 generation and con-comitant increases in [Ca2�]i mobilization were investigated

and were compared with that of the mIP. Stimulations ofHEK.hIP, HEL, and HEK.mIP cells each resulted in efficientcicaprost-induced rises in [Ca2�]i mobilization (Fig. 2, A–C,respectively), and increases in IP3 generation (Fig. 3A and datanot shown), indicative of both hIP and mIP coupling to G�q-mediated PLC activation. Although cicaprost-induced [Ca2�]imobilization (Fig. 2C, p � 0.24) and IP3 generation (22) by thecontrol HEK.mIP cells were unaffected by preincubation withGF 109203X, H-89 significantly impaired mIP-mediated[Ca2�]i mobilization (Fig. 2, C and E, p 0.0005) and IP3

generation (22). In contrast, neither cicaprost-induced [Ca2�]imobilization nor IP3 generation in HEK.hIP cells (Figs. 2A, 2D,and 3B) or in HEL cells (Fig. 2B and data not shown) were

FIG. 1. Analysis of cAMP generation by hIP expressed in HEK.hIP cells, HEL cells, and in human platelets. HEK.mIP cells (A),HEK.hIP (B and E), HEL (C), and platelets (D) were stimulated with either 1 �M cicaprost (Cic), 10 �M forskolin (Fsk), or 1 �M cicaprost plus 10�M forskolin (Cic � Fsk), where cells in A (Cic � Fsk � H-89) and in E were preincubated for 10 min with H-89 (10 �M) prior to stimulation. Ineach case, basal cAMP levels were determined by exposing the cells to the vehicle HBS under identical incubation conditions. Levels of cAMPproduced in ligand-stimulated cells relative to basal cAMP levels were expressed as -fold stimulation of basal (-fold increase in cAMP � S.E., n �4). *, p � 0.05; **, p � 0.01.

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affected by H-89 or by GF 109203X. Thus, although the hIP andmIP both couple to Gq-mediated PLC activation, they displayessential differences in their dependence on PKA and, hence, intheir mechanism of Gq coupling whereby hIP independentlycouples to G�q, whereas mIP coupling is dependent on its PKAphosphorylation and consequent switching from G�s to G�q.

To fully exclude the possibility that cicaprost-induced [Ca2�]imobilization might be mediated through Gi-derived G�� sub-units, the effect of PTx and the G�� sequestrant peptide,�ARK1459–689 (23) on [Ca2�]i mobilization was investigated.Neither preincubation of cells with PTx nor transient overex-

pression of �ARK1459–689 (23) significantly affected cicaprost-induced [Ca2�]i mobilization in HEK.hIP cells (p 0.35 andp 0.1, respectively) or in HEK.mIP cells (22). Overexpressionof the carboxyl-terminal residues of �ARK1 was confirmed byWestern blot analysis using anti-GRK 2 (Santa Cruz Biotech-nology, As #C-15, data not shown).

Role of Second Messenger Kinases in hIP:G protein Cou-pling—We have previously established that mIP coupling toboth G�i and G�q is dependent on its initial coupling to G�s andsubsequent cAMP-dependent PKA phosphorylation, whereSer357 within the C-tail region of mIP was identified as the

FIG. 2. Effect of protein kinase inhibitors on cicaprost-induced [Ca2�]i mobilization. A–C: HEK.hIP (A), HEL (B), and, as controls,HEK.mIP (C) cells were preincubated for 2 min with 10 �M H-89 (Cic � H-89) or 50 nM GF 109203X (Cic � GF) prior to stimulation with 1 �M

cicaprost. Data were calculated as changes in intracellular Ca2� mobilized and are expressed as a percentage (%) relative to cells stimulated withcicaprost alone (�[Ca2�]i � S.E., %). D–E, HEK.hIP (D) and HEK.mIP (E) cells were preincubated for 2 min with 10 �M H-89 (�H-89) prior tostimulation with 1 �M cicaprost. Data presented are representative profiles from at least three independent experiments and are plotted as changesin intracellular Ca2� mobilization (�[Ca2�]i, nM) as a function of time (seconds) where cicaprost was added at the times indicated by the arrows.Actual mean changes in [Ca2�]i mobilized by HEK.hIP and HEK.mIP cells were: D, �[Ca2�]i � 145 � 6.6 nM and 132 � 7.9 nM in the absence andpresence of H-89, respectively; E, �[Ca2�]i � 152 � 10.9 nM and 41 � 1.53 nM in the absence and presence of H-89, respectively. **, p � 0.01.

FIG. 3. Analysis of IP3 generation by HEK.hIP cells. HEK.hIP cells transiently co-transfected with G�q were stimulated with cicaprost(10�12 to 10�6 M), and levels of IP3 generation were measured (A). Alternatively, HEK.hIP cells transiently co-transfected with G�q (B) werestimulated with 1 �M cicaprost (Cic) or were preincubated for 5 min with H-89 (10 �M, Cic � H-89) or with GF 109203X (50 nM, Cic � GF) priorto stimulation with 1 �M cicaprost. In each case, basal IP3 levels were determined by exposing the cells to the vehicle HBS under identicalincubation conditions. Levels of IP3 produced in ligand-stimulated cells relative to vehicle-treated cells (basal IP3) were expressed as -foldstimulation of basal (-fold increase in IP3 � S.E., n � 4).

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target residue for PKA phosphorylation (22). Optimal align-ment of the primary sequences of the C-tail regions of the mIP(21) and the hIP (30, 31) and their computational analyses forputative phosphorylation sites (32) revealed that the pre-viously identified consensus target site for PKA phosphor-ylation occurring at Ser357 within the mIP, with the se-quence RPAS357GRR, is replaced by a consensus target site forPKC phosphorylation within the hIP, with the sequenceQLAS328GRR (Fig. 4). Thus, in view of the differential G pro-tein coupling specificities of the mIP and the hIP to G�i and thedifferential dependence on PKA in regulating their PLC acti-vation, we sought to determine whether alteration of the con-sensus PKC recognition sequence of the hIP surroundingSer328 may impact on its G protein coupling specificity andintracellular signaling. Thus, site-directed mutagenesis of hIPwas performed to generate hIPQL325,326RP, a variant of hIPwhereby the critical residues Gln325-Leu326, within the consen-sus PKC phosphorylation site (QLAS328GRR), were mutated toArg325-Pro326, thereby generating a putative PKA consensussite (RPAS328GRR), identical to that of the mIP in this region.

Initial characterization of HEK.hIPQL325,326RP cells, recom-binant HEK 293 cells stably overexpressing hIPQL325,326RP bysaturation radioligand binding confirmed high level receptorexpression (Table I). Consistent with its ability to couple toG�s, the hIPQL325,326RP exhibited concentration-dependent in-creases in cicaprost-induced cAMP generation (Fig. 5A, p 0.05), and transient co-transfection of HEK.hIPQL325,326RP cellswith pCMV-G�s resulted in a significant augmentation (1.70-fold, p 0.05) in cAMP generation. In contrast to that of thehIP, cicaprost significantly inhibited forskolin-induced cAMPgeneration in HEK.hIPQL325,326RP cells (Fig. 5B), indicative ofhIPQL325,326RP coupling to G�i. Furthermore, pretreatment ofHEK.hIPQL325,326RP cells with PTx abolished hIPQL325,326RP

coupling to G�i (Fig. 5B). Although preincubation ofHEK.hIPQL325,326RP cells with H-89 had no effect on cicaprost-induced cAMP generation, it blocked hIPQL325,326RP coupling toG�i and to inhibition of adenylyl cyclase (Fig. 5C). The PKCinhibitor GF 109203X had no effect on hIPQL325,326RP couplingto G�s or to G�i (data not shown).

Thereafter, the ability of hIPQL325,326RP to couple to G�q andto PLC activation was investigated. Stimulation ofHEK.hIPQL325,326RP cells with cicaprost led to significantincreases in IP3 generation (Fig. 5D, p 0.05) and [Ca2�]imobilization (Fig. 5E) at levels that were not significantlydifferent from those of the hIP. Moreover, neitherhIPQL325,326RP-mediated IP3 generation nor [Ca2�]i mobiliza-tion were affected by preincubation with H-89 (Fig. 5, D and E,p 0.61) or GF 109203X (Fig. 5D and data not shown, p 0.75). Thus, coupling of hIPQL325,326RP to G�q and PLC activa-tion is independent of PKA and PKC. Taken together, these

data suggest that by conversion of a putative PKC phosphoryl-ation site within the hIP to a putative PKA site within thehIPQL325,326RP facilitates hIP coupling to PTx-sensitive G�i butdoes not influence its ability to couple to G�s or to G�q.

To further explore the essential role of PKA in regulatinghIP, or more specifically hIPQL325,326RP, coupling to G�i, site-directed mutagenesis was used to generate hIPS328A, a variantof hIP whereby the critical phospho-targeted residue Ser328

was converted to Ala328, thereby destroying the putative phos-phorylation site. Initial characterization of HEK.hIPS328A cells,recombinant HEK 293 cells stably overexpressing hIPS328A, bysaturation radioligand binding confirmed high level receptorexpression (Table I). Consistent with its ability to couple toG�s, hIPS328A exhibited efficient, concentration-dependent in-creases in cAMP in response to cicaprost stimulation (Fig. 6A),which were not significantly different from that obtained forthe hIP (Fig. 1C). In addition, transient co-transfection ofHEK.hIPS328A cells with pCMV-G�s significantly augmentedcicaprost-induced cAMP generation by 2.07-fold (p 0.005). Incontrast to that of hIPQL325,326RP, cicaprost did not significantlyinhibit, but rather augmented, forskolin-induced cAMP gener-ation in HEK.hIPS328A cells (Fig. 6B), indicative of hIPS328A

coupling to G�s but not G�i. Moreover, neither PTx nor H-89affected cAMP generation by HEK.hIPS328A cells (Fig. 6, B andC, p 0.28).

Next, the ability of the hIPS328A to couple to G�q and to PLCactivation was investigated. Stimulation of HEK.hIPS328A cellswith cicaprost led to significant increases in IP3 generation(Fig. 6D, p 0.001) and [Ca2�]i mobilization (Fig. 6E) thatwere not significantly different from those of the wild type hIP(Figs. 2 and 3). Moreover, neither IP3 generation nor [Ca2�]imobilization were affected by preincubation with H-89 (Fig. 6,D and E, p 0.05) or GF 109203X (Fig. 6D and data not shown,p 0.85). Thus, taken together, these data suggest that thehIPS328A can couple to G�s and to G�q through a PKA- andPKC-independent mechanism but that hIPS328A may not cou-ple to G�i.

Whole cell phosphorylations, through metabolic labelingstudies, established that the hIP underwent cicaprost-inducedphosphorylation as evidenced by the detection of a broad radio-labeled band between the 46- and 66-kDa molecular size mark-ers in the immunoprecipitates from HEK.hIP cells (Fig. 7A,lane 2) but not from vehicle-treated HEK.hIP cells or fromnon-transfected HEK 283 cells (Fig. 7A, lanes 1 and 5, respec-tively). Consistent with previous reports (12, 13), cicaprost-induced hIP phosphorylation was unaffected by H-89 but wasinhibited by the PKC inhibitor GF 109203X (Fig. 7A, lanes 3and 4, respectively). Although the hIPQL325,326RP underwentcicaprost-induced phosphorylation, it was unaffected by GF109203X but was inhibited by the PKA inhibitor H-89 (Fig. 7B).

FIG. 4. Alignment of human and mouse prostacyclin receptor amino acid sequences. The deduced amino acid sequences of thecarboxyl-terminal cytoplasmic (C) tail regions of the hIP (residues 299–386) and mIP (residues 328–417), aligned to show maximum homologyusing the ClustalW software (29), are shown. The consensus PKC phosphorylation site within the hIP is underlined, and the putative phospho-target residue Ser328 is highlighted in boldface (32). The consensus PKA phosphorylation site within the hIP is underlined, and the putativephospho-target residue Ser357 is highlighted in boldface (32). Throughout the alignment, gaps, indicated by the dash symbol (-), were inserted tooptimize the alignment; identical amino acids are indicated by an asterisk; conservative substitutions are represented by a colon, and semi-conservative substitutions are indicated by a period. Sequences for the hIP and mIP are based on published sequences (21, 30, 31).

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On the other hand, the hIPS328A did not undergo significantcicaprost-induced phosphorylation (Fig. 7C). The identities ofthe broad radiolabeled band between 46 and 66 kDa wereconfirmed to be those of the immunoprecipitated HA-taggedhIP and its variant hIPQL325,326RP in parallel immunoprecipi-tations whereby efficient, quantitative recovery of each recep-tor type was detected by Western blot analysis using the an-ti-HA 3F10 peroxidase antibody (Fig. 7D, lanes 1 and 2,respectively). Moreover, quantitative recovery of hIPS328A fromHEK.hIPS328A cells confirmed that the absence of significantphosphorylation of hIPS328A was not attributed to reduced re-ceptor expression or due to reduced immunoprecipitation (Fig.7D, lane 3).

Role of Second Messenger Kinases in mIP:G protein Cou-pling—As previously stated, coupling of the mIP to both G�i

and to G�q is dependent on its initial coupling to G�s andconsequent PKA phosphorylation of mIP at Ser357 therebyswitching mIP coupling from G�s coupling to G�i and G�q

coupling (22). In the present study, we sought to fully explorethe role of PKA in mediating mIP switching by investigatingwhether alteration of the consensus PKA target site surround-ing Ser357 to a PKC target site would alter the G proteincoupling specificity of the mIP. Thus, mIPRP354,355QL a variant

of mIP was generated whereby Arg354-Pro355, within the PKAconsensus sequence RPAS357GRR, were converted to Gln354-Leu355, to generate a putative PKC consensus sequenceQLAS357GRR (32).

Characterization of HEK.mIPRP354,355QL cells, recombinantHEK 293 cells stably overexpressing mIPRP354,355QL, by satu-ration radioligand binding confirmed high level receptor ex-pression (Table I). Consistent with their ability to couple toG�s, both the mIPRP354,355QL (Fig. 8A) and mIP (Fig. 8C) ex-hibited efficient, cicaprost-induced increases in cAMP genera-tion and transient co-transfection of HEK.mIPRP354,355QL cellswith pCMV-G�s significantly augmented that cAMP genera-tion (2.29-fold augmentation, p � 0.017). In contrast to that ofHEK.mIP cells (Fig. 8C), stimulation of HEK.mIPRP354,355QL

cells with cicaprost failed to inhibit, but rather augmented,forskolin-induced cAMP generation by mIPRP354,355QL (Fig. 8A,p 0.05). Moreover, although PTx (Fig. 8C) and H-89 (Fig. 8D)abolished mIP coupling to G�i, cAMP generation byHEK.mIPRP354,355QL cells was not affected by PTx (Fig. 8A) orby H-89 (Fig. 8B, p 0.05) or by the PKC inhibitor GF 109203X(data not shown).

Next, the ability of the mIPRP354,355QL to couple to G�q

and PLC activation was investigated and was compared with

FIG. 5. Analysis of signaling by the hIPQL325,326RP. A–C, HEK.hIPQL325,326RP cells were stimulated with 10�12 to 10�6 M cicaprost at 37 °C for10 min (A). Alternatively, HEK.hIPQL325,326RP cells (B) were stimulated with either 1 �M cicaprost (Cic), 10 �M forskolin (Fsk), or 1 �M cicaprostplus 10 �M forskolin (Cic � Fsk), or were preincubated with PTx (50 ng/ml, 16 h) prior to stimulation with 1 �M cicaprost plus 10 �M forskolin (Cic� Fsk � PTx). Alternatively, HEK.hIPQL325,326RP cells were preincubated for 10 min with 10 �M H-89 (C) prior to stimulation with ligand asoutlined in B. In each case, basal cAMP levels were determined by exposing the cells to the vehicle HBS under identical incubation conditions.Levels of cAMP produced in ligand-stimulated cells relative to basal cAMP levels were expressed as -fold stimulation of basal (-fold increase incAMP � S.E., n � 4). D, HEK.hIPQL325,326RP cells transiently co-transfected with G�q were stimulated with 1 �M cicaprost (Cic) or werepreincubated for 5 min with H-89 (10 �M, Cic � H-89) or with GF 109203X (50 nM, Cic � GF) prior to stimulation with 1 �M cicaprost. In each case,basal IP3 levels were determined by exposing the cells to the vehicle HBS under identical incubation conditions. Levels of IP3 produced inligand-stimulated cells relative to vehicle-treated cells (basal IP3) were expressed as -fold stimulation of basal (-fold increase in IP3 � S.E., n � 4).E, HEK.hIPQL325,326RP cells were stimulated with1 �M cicaprost or were preincubated for 2 min with 10 �M H-89 (�H-89) prior to stimulation withcicaprost (1 �M). Data presented are representative profiles from at least four independent experiments and are plotted as changes in intracellularCa2� mobilization (�[Ca2�]i, nM) as a function of time (seconds) where cicaprost was added at the times indicated by the arrow. Actual meanchanges in [Ca2�]i mobilized by HEK.hIPQL325,326RP cells were: E, �[Ca2�]i � 126 � 3.3, 122 � 7.68, and 131 � 1.67 nM in the absence or in thepresence of either H-89 or GF 109203X (data not shown), respectively. (n � 4). *, p � 0.05; **, p � 0.01.

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FIG. 7. Cicaprost induced-phosphorylation of hIP, hIPQL325,326RP, and hIPS328A. A–C, HEK.hIP (A, lanes 1–4), HEK.hIPQL325,326RP (B),HEK.hIPS328A (C), or control HEK 293 (A, lane 5) cells, metabolically labeled with [32P]orthophosphate, were incubated for 10 min with the vehicleHBS (A–C, lane 1) or were stimulated for 10 min with 1 �M cicaprost (A, lanes 2 and 5; B–D, lane 2). Additionally, cells were preincubated with10 �M H-89 (A–C, lane 3) or 50 nM GF 109203X (A–C, lane 4) for 10 min prior to stimulation with 1 �M cicaprost for 10 min. Thereafter, HAepitope-tagged IP receptors were immunoprecipitated using the anti-HA antibody 101R. Immunoprecipitates were resolved by SDS-PAGE andelectroblotted onto PVDF membranes and were then exposed to X-Omat XAR-5 film (Kodak) for 8–10 days. Thereafter, blots were subject toPhosphorImager analysis, and the intensities of cicaprost-mediated hIP, hIPQL325,326RP, and hIPS328A phosphorylation relative to basal phospho-rylation, in the presence of HBS, were determined and expressed in arbitrary units as follows: hIP: 1 �M cicaprost, 9.48-fold; 1 �M cicaprost plus50 nM GF 109203X, 1.17-fold; 1 �M cicaprost plus 10 �M H-89, 9.77-fold; hIPQL325,326RP: 1 �M cicaprost, 8.46-fold; 1 �M cicaprost plus 50 nM GF109203X, 7.53-fold; 1 �M cicaprost plus 10 �M H-89, 2.31-fold; hIPS328A: 1 �M cicaprost, 1.36-fold; 1 �M cicaprost plus 50 nM GF 109203X, 1.9-fold;1 �M cicaprost plus 10 �M H-89, 1.09-fold; HEK 293 cells: 1 �M cicaprost, 1.20-fold. D, HEK.hIP, HEK.hIPQL325,326RP, HEK.hIPS328A, or control HEK293 cells (lanes 1–4, respectively) were subject to immunoprecipitation using the anti-HA antibody 101R, and immunoprecipitates were resolvedby SDS-PAGE and electroblotted onto PVDF membranes. Membranes were screened using the anti-HA 3F10 peroxidase-conjugated antibody, andimmunoreactive bands were visualized by chemiluminescence detection. The positions of the molecular mass markers (kDa) are indicated to theleft and right of A and D, respectively. Data presented are representative of three independent experiments.

FIG. 6. Analysis of signaling by the hIPS328A. A–C, HEK.hIPS328A cells were stimulated with 10�12 to 10�6 M cicaprost at 37 °C for 10 min (A).Alternatively, HEK.hIPS328A cells (B) were stimulated with either 1 �M cicaprost (Cic) and 10 �M forskolin (Fsk) or 1 �M cicaprost plus 10 �M

forskolin (Cic � Fsk) or were preincubated with PTx (50 ng/ml, 16 h) prior to stimulation with 1 �M cicaprost plus 10 �M forskolin (Cic � Fsk �PTx). Alternatively, HEK.hIPS328A cells were preincubated for 10 min with 10 �M H-89 (C) prior to stimulation with ligand as outlined in B. In eachcase, basal cAMP levels were determined by exposing the cells to the vehicle HBS under identical incubation conditions. Levels of cAMP producedin ligand-stimulated cells relative to basal cAMP levels were expressed as -fold stimulation of basal (-fold increase in cAMP � S.E., n � 4). D,HEK.hIPS328A cells transiently co-transfected with G�q were stimulated with 1 �M cicaprost (Cic) or were preincubated for 5 min with H-89 (10�M, Cic � H-89) or with GF 109203X (50 nM, Cic � GF) prior to stimulation with 1 �M cicaprost. In each case, basal IP3 levels were determinedby exposing the cells to the vehicle HBS under identical incubation conditions. Levels of IP3 produced in ligand-stimulated cells relative to vehicletreated cells (basal IP3) were expressed as -fold stimulation of basal (-fold increase in IP3 � S.E., n � 4). E, HEK.hIPS328A cells were stimulatedwith 1 �M cicaprost or were preincubated for 2 min with 10 �M H-89 (�H-89) prior to stimulation with cicaprost (1 �M). Data presented arerepresentative profiles from at least four independent experiments and are plotted as changes in intracellular Ca2� mobilization (�[Ca2�]i, nM) asa function of time (seconds) where cicaprost was added at the times indicated by the arrows. Actual mean changes in [Ca2�]i mobilized byHEK.hIPS328A cells were: �[Ca2�]i � 124 � 12.7, 127 � 16.3, and 120 � 115.3 nM in the absence or in the presence of either H-89 or GF 109203X(data not shown), respectively (n � 4) (E). *, p � 0.05.

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the mIP. Unlike that of the mIP, stimulation of HEK.mIPRP354,355QL cells with cicaprost did not result in significantincreases in cicaprost-induced IP3 generation (Fig. 8E, p 0.32) or in [Ca2�]i mobilization (Fig. 8F, p 0.50). Moreover,neither H-89 nor GF 109203X affected mIPRP354,355QL-

mediated IP3 generation (Fig. 8E) or [Ca2�]i mobilization (datanot shown) but inhibited mIP-mediated Gq coupling and PLCactivation (22). Whole cell phosphorylation studies establishedthat, unlike the mIP (22), the mIPRP354,355QL did not undergoPKA- or PKC-induced phosphorylation in response to cicaprost

FIG. 8. Analysis of signaling by mIPRP354,355QL. A–D, HEK.mIPRP354,355QL (A and B) or, as controls, HEK.mIP (C and D) cells were stimulatedwith either 1 �M cicaprost (Cic), 10 �M forskolin (Fsk), or 1 �M cicaprost plus 10 �M forskolin (Cic � Fsk), or were preincubated with PTx (50 ng/ml,16 h) prior to stimulation with 1 �M cicaprost plus 10 �M forskolin (Cic � Fsk � PTx). Additionally, HEK.mIPRP354,355QL and HEK.mIP cells werepreincubated for 10 min with 10 �M H-89 (B and D) prior to stimulation with ligand as outlined in A and C. In each case, basal cAMP levels weredetermined by exposing the cells to the vehicle HBS under identical incubation conditions. Levels of cAMP produced in ligand-stimulated cellsrelative to basal cAMP levels were expressed as -fold stimulation of basal (-fold increase in cAMP � S.E., n � 4). E, HEK.mIPRP354,355QL cellstransiently co-transfected with G�q were stimulated with 1 �M cicaprost (Cic) or were preincubated for 5 min with H-89 (10 �M, Cic � H-89) orwith GF 109203X (50 nM, Cic � GF) prior to stimulation with 1 �M cicaprost. Additionally, as a control, HEK.mIP cells transiently co-transfectedwith G�q were stimulated with 1 �M cicaprost (mIP � Cic). In each case, basal IP3 levels were determined by exposing the cells to the vehicle HBSunder identical incubation conditions. Levels of IP3 produced in ligand-stimulated cells relative to vehicle-treated cells (basal IP3) were expressedas -fold stimulation of basal (-fold increase in IP3 � S.E., n � 4). F, HEK.mIPRP354,355QL (mIPRP354,355QL) and, as controls, HEK.mIP (mIP) cells werestimulated with 1 �M cicaprost. Data presented are representative profiles from at least four independent experiments and are plotted as changesin intracellular Ca2� mobilization (�[Ca2�]i, nM) as a function of time (seconds) where cicaprost was added at the times indicated by the arrows.Actual mean changes in [Ca2�]i mobilized by HEK.mIPRP354,355QL were �[Ca2�]i � 21.7 � 4.4 nM and by HEK.mIP cells were 148 � 4.91 nM,respectively (n � 4). G, HEK.mIPRP354,355QL (lane 1) or control HEK 293 (lane 2) cells were subject to immunoprecipitation using the anti-HAantibody 101R, and immunoprecipitates were resolved by SDS-PAGE and electroblotted onto PVDF membranes. Membranes were screened usingthe anti-HA 3F10 peroxidase-conjugated antibody, and immunoreactive bands were visualized by chemiluminescence detection. The positions ofthe molecular mass markers (kDa) are indicated to the right of panel G. Data presented are representative of three independent experiments. *,p � 0.05; **, p � 0.01.

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stimulation; despite this, high levels of expression ofmIPRP354,355QL were confirmed by radioligand binding studies(Table I) and by the efficient, quantitative recovery of theHA-tagged mIPRP354,355QL in the immunoprecipitates fromHEK.mIPRP354,355QL cells but not from control HEK 293 cells(Fig. 8G).

Thus, taken together, these data highlight the essential roleof PKA phosphorylation of Ser357 of the mIP in independentlymediating its coupling to both G�i and to G�q, whereby con-version of critical determinants of the PKA recognition site tothat of a defined PKC phosphorylation site inhibits cicaprost-induced mIP phosphorylation and switching from G�s and,thereby, inhibits its coupling to G�i and to G�q.

DISCUSSION

Prostacyclin plays a central role in the local control of vas-cular hemostasis acting as an endothelium-derived inhibitor ofplatelet aggregation and as a vasodilator (5). Mice deficient inthe IP show increased susceptibility to thrombotic stimuli,exhibit diminished pain perception and inflammatory re-sponses (3, 33), and develop more pronounced hypertensionand vascular remodeling following hypoxic exposure relativewild type mice (34). Although IPs are thought to primarilycouple to adenylyl cyclase (5, 21), in certain species/cell typesthey are reported to couple to diverse effectors, including PLCactivation (12–14, 16, 21, 22) and/or inhibition of adenylylcyclase (18). Several GPCRs have been established to activatedual or, indeed, multiple G protein:effector systems (35–39),and a number of independent mechanisms have been proposed(40, 41). We have recently established that the mIP couples toboth activation and to inhibition of adenylyl cyclase, via G�s

and G�i, respectively, and to activation of PLC, via G�q (22).Through detailed mechanistic studies, we established that cou-pling to both G�i and G�q was dependent on initial mIP cou-pling to G�s and adenylyl cyclase activation and subsequentcAMP-dependent PKA phosphorylation of the mIP at Ser357

within its carboxyl-terminal (C)-tail region, thereby terminat-ing mIP coupling to, and association with, G�s and switchingmIP coupling to both G�i and to G�q (22). In the current study,we sought to test the generality of this model by examining theG protein coupling specificity and mechanisms of signaling ofthe hIP, comparing it to that of the mIP.

Stimulation of HEK.mIP, HEK.hIP, and HEL cells and hu-man platelets with cicaprost led to concentration-dependentincreases in cAMP generation, consistent with mIP and hIPcoupling to G�s. Whereas stimulation of the mIP with cicaprostinhibited forskolin (Fsk)-induced cAMP generation, consistentwith mIP coupling to G�i, stimulation of the hIP augmentedFsk-induced cAMP generation indicative of the hIP coupling toG�s, but not to G�i. Moreover, although PTx and H-89 abol-ished mIP coupling to G�i, cAMP generation by the hIP was notaffected by these agents.

Stimulation of HEK.mIP and HEK.hIP cells with cicaprostalso led to increases in IP3 generation and to mobilization of[Ca2�]i consistent with both mIP (14, 21, 22) and hIP (12, 15,16) coupling to PLC activation. Although stimulation of HELcells with cicaprost also yielded significant increases in IP3

generation and [Ca2�]i mobilization, hIPs expressed in humanplatelets did not couple to PLC activation (Ref. 42 and data notshown). These data are consistent with previous findings dem-onstrating that, although hIPs expressed in the more imma-ture megakaryoblastic cells, such as HEL and MEG-01 cells,retain the ability to couple to PLC activation, those expressedin the more mature platelet have lost that function, largelyowing to changes in the profile of G protein expression duringthe progression of megakaryocytopoiesis (43). Although cica-prost-induced IP3 generation and [Ca2�]i mobilization by the

mIP were inhibited by H-89 (22), neither IP3 generation nor[Ca2�]i mobilization by the hIP were affected by H-89. Hence,there are fundamental differences in signaling by the mIP andhIP, in that the mIP coupling to G�i and G�q is dependent on itsinitial coupling to G�s and subsequent PKA-mediated phospho-rylation and switching (Fig. 9A (22)); the hIP, on the otherhand, independently couples to G�s and G�q but does not un-dergo PKA phosphorylation and switching and does not coupleto G�i (Fig. 9B).

While the mIP and rIP exhibit extensive amino acid se-quence identity (94% identity), the hIP and mIP only share 73%overall identity, exhibiting greater divergence within their C-tail regions (66% identity). Alignment of the C-tail regions ofthe mIP (21) and the hIP (30–32) revealed that the previouslyidentified PKA site at Ser357 within the mIP (RPAS357GRR), isreplaced by a consensus target site for PKC within the hIP(QLAS328GRR) whereby the Arg-Pro (RP) versus Gln-Leu (QL)represent the only divergent residues and therefore act as thedeterminants of PKA versus PKC phosphorylation of the tar-geted Ser. Although the sequence RPAS357GRR within the mIPmay actually be predicted to act as both a PKA or PKC site (32,21), we have confirmed that this site acts as a PKA, but not aPKC, site, and as stated, cicaprost-induced PKA phosphoryla-tion of Ser357 mediates mIP switching from G�s to G�i and G�q

(22). Moreover, Ser328 of the hIP has been confirmed to be adirect target for cicaprost-induced PKC, but not PKA, phospho-rylation (12, 13). Thus, in view of the differential G proteincoupling specificities of the mIP and the hIP to G�i and theirdifferential dependence on PKA in regulating their PLC acti-vation, we investigated whether alteration of the determinantresidues within the consensus PKC recognition sequence of thehIP surrounding Ser328 might alter its G protein coupling spec-ificity and intracellular signaling. Thus, the hIPQL325,326RP wasgenerated whereby the consensus PKC phosphorylation sitewas mutated to a putative PKA site, identical to that of the mIPin this region.

The hIPQL325,326RP exhibited both G�s and G�i coupling,with G�i coupling inhibited by PTx and H-89. This receptoralso exhibited G�q coupling that was unaffected by H-89 or byGF 109203X or PTx (data not shown), consistent with theindependent coupling of hIPQL325,326RP to G�q and PLC activa-tion. Although the hIP underwent cicaprost-induced phospho-rylation, consistent with previous reports (12, 13), this phos-phorylation was unaffected by H-89 but was inhibited by GF109203X. Although the hIPQL325,326RP also underwent cica-prost-induced phosphorylation, it was unaffected by GF109203X but was inhibited by H-89. Hence, conversion of thePKC site within the hIP to that of a PKA site generatedhIPQL325,326RP, which can switch from G�s to G�i through aPKA-dependent mechanism (Fig. 9C), and implies that thecontextual sequence surrounding Ser328 in the hIPQL325,326RP

is essential for G�i coupling following its phosphorylation byPKA.

To further explore the essential requirement of the latterPKA phosphorylation at Ser328 in mediating hIPQL325,326RP

switching and coupling to G�i, hIPS328A was generated. Al-though the hIPS328A coupled to G�s, it failed to exhibit couplingto G�i. The hIPS328A also mediated increases in IP3 generationand [Ca2�]i mobilization consistent with its independent cou-pling to G�q/PLC activation. Whole cell phosphorylations es-tablished that, unlike the hIP or the hIPQL325,326RP, thehIPS328A did not undergo significant cicaprost-induced phos-phorylation. Hence, the hIPS328A independently couples to bothG�s and G�q, similar to the hIP, but does not couple to G�i.

As previously stated, coupling of the mIP to both G�i and toG�q is dependent on its initial coupling to G�s and consequent

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PKA phosphorylation of mIP at Ser357 thereby switching mIPfrom G�s coupling to G�i and G�q coupling (22). Herein, we alsoinvestigated whether alteration of the critical divergent resi-dues within the consensus PKA target site surrounding Ser357,which converts it to a PKC target site identical to that foundwithin the hIP, would alter the G protein coupling specificity ofthe mIP. Although the mIPRP354,355QL coupled to G�s, it failedto exhibit coupling to G�i. Additionally, the mIPRP354,355QL

failed to couple to G�q, and whole cell phosphorylations estab-lished that mIPRP354,355QL did not undergo measurable cica-prost-induced phosphorylation. Hence, conversion of the PKAsite to a PKC site within the mIP yielded mIPRP354,355QL thatcan independently couple to G�s but that does not undergoagonist-induced phosphorylation on Ser357 and, hence, cannotswitch coupling from G�s to G�i or to G�q (Fig. 9D).

Taken together, these data with the hIP and the mIP andtheir mutants have indicated the critical requirement for thedipeptide RP, as opposed to a QL, in the �3 and �2 positions inaddition to a phosphorylated Ser (representing �1) in deter-mining mIP coupling to G�i and G�q and in determining hIPcoupling to G�i, but not to G�q. Further experiments are re-

quired to dissect the importance of the contextual nature of theindividual residues within the dipeptide RP sequence and/orany knock-on or consequent structural changes in determiningthat G�i/G�q interaction and coupling. A similar type of PKA-dependent mechanism is involved in regulating the human �2

adrenergic receptor switching from G�s to G�i (40). In this case,a 14-amino acid peptide containing a PKA recognition sequenceRRSS within the third intracellular loop of the �2 adrenergicreceptor could stimulate GTP�S binding by G�s but not by G�i;on the other hand, the PKA-phosphorylated peptide showedweak G�s activation and strong G�i activation (44). Similar toour findings with the mIP sequence (22), although the RRSScould be predicted to act as both a PKA and PKC recognitionsite, it was preferentially phosphorylated by PKA and not byPKC (44).

In essence, these studies highlight critical differences in mIPand hIP:G protein coupling and intracellular signaling. Al-though the mIP can couple to G�s, and to G�i and G�q, couplingto the latter G protein:effector systems is dependent on PKA-mediated phosphorylation and switching (Fig. 9A). The hIP, onthe other hand, can independently couple to G�s and to G�q,

FIG. 9. Model of mIP and hIP coupling to G�s, G�i, and G�q. A, ligand-activated mIP stimulates: (i) G�s-mediated activation of AC, leadingto increases in cAMP generation and, in turn, activation of PKA. Activated PKA phosphorylates the mIP at Ser357 switching mIP coupling fromG�s to (ii) pertussis toxin (PTx)-sensitive G�i and inhibition of AC, and to (iii) G�q and activation of phosphatidyl inositol-specific PLC (PI-PLC)leading to increases in IP3 generation and mobilization of [Ca2�]i. B, ligand-activated hIP independently couples to (i) G�s-mediated activation ofAC and to (iii) G�q-mediated activation of PLC. The hIP does not couple to (ii) G�i. C, ligand-activated hIPQL325,326RP (hIPQL) independently couplesto (iii) G�q-mediated activation of PLC and to (i) G�s-mediated activation of AC, leading to increases in cAMP generation and, in turn, activationof PKA. Activated PKA phosphorylates the hIPQL325,326RP at Ser328 switching its coupling from G�s to (ii) G�i and inhibition of AC. D,ligand-activated mIPRP354,355QL (mIPRP) stimulates (i) G�s-mediated activation of AC, leading to increases in cAMP generation and, in turn,activation of PKA. Unlike the mIP, mIPRP354,355QL does not undergo PKA phosphorylation at Ser357 and, hence, cannot switch coupling from G�sto (ii) G�i or to (iii) G�q. The steps inhibited by H-89 are indicated in A–D.

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but cannot couple to G�i (Fig. 9B). mIP coupling to G�i, versusthat of hIP, is primarily regulated by a targeted cicaprost-induced PKA consensus sequence at Ser357 of the mIP, which isreplaced by a PKC consensus sequence in the hIP, at Ser328.Conversion of the PKC recognition site within the hIP to a PKAsite within hIPQL325,326RP facilitates its coupling to G�i (Fig.9C), therefore emphasizing the importance of the contextualnature of the Arg325-Pro326 residues in addition to phosphoryl-ation of Ser328 in regulating G�i coupling. mIP coupling to G�q

is also regulated by a targeted cicaprost-induced PKA phospho-rylation site at Ser357 and conversion of that PKA recognitionsequence from a PKA to a PKC site (in mIPRP354,355QL) abol-ished mIP coupling to G�i and G�q (Fig. 9D). The hIP inde-pendently couples to G�q and is not regulated by PKA or PKCphosphorylation of hIP at Ser328 as neither (a) conversion of thePKC site of the hIP to a PKA recognition sequence withinhIPQL325,326RP (Fig. 9C) nor (b) abolition of the PKC sitethrough site-directed mutagenesis within hIPS328A impairedthe ability of the hIP to couple to G�q/PLC activation. Thus, itappears that other sequence differences within the hIP, asopposed to the mIP, act as determinants in facilitating itsindependent coupling to G�q. Given the extent of sequencevariation that exists between the hIP and the mIP, particularlywithin their C-tail sequences and third intracellular loop re-gions, it is likely that these domains may also contain thesequence determinants of G�q interactions with the hIP.

These critical differences in mechanisms of intracellular sig-naling by the hIP and the mIP may confer important species-dependent differences to the cellular responses to prostacyclinunder both physiologic/pathophysiologic settings, and an ap-preciation of these mechanisms may offer a basis for the dif-ferential targeting of prostacyclin-signaling in certain diseasesettings. Finally, data presented herein greatly expands ourexisting knowledge of the general mechanisms of GPCR signal-ing, particularly in relation to their ability to couple to multipleG protein:effector systems.

Acknowledgments—We are very grateful to Daniel O’Mahony andPaul Engel for helpful discussion. We also thank Paul Byrne.

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Sinead M. Miggin and B. Therese KinsellaSPECIES-DEPENDENT DIFFERENCES

Mouse Prostacyclin Receptors: IDENTIFICATION OF CRITICAL Investigation of the Mechanisms of G Protein: Effector Coupling by the Human and

doi: 10.1074/jbc.M203353200 originally published online May 16, 20022002, 277:27053-27064.J. Biol. Chem. 

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Sinead M. Miggin and B. Therese KinsellaSPECIES-DEPENDENT DIFFERENCES

Mouse Prostacyclin Receptors: IDENTIFICATION OF CRITICAL Investigation of the Mechanisms of G Protein: Effector Coupling by the Human and

doi: 10.1074/jbc.M203353200 originally published online May 16, 20022002, 277:27053-27064.J. Biol. Chem. 

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VOLUME 277 (2002) PAGES 27053–27064DOI 10.1074/jbc.W118.004804

Investigation of the mechanisms of G protein: Effectorcoupling by the human and mouse prostacyclinreceptors. Identification of critical species-dependentdifferences.Sinead M. Miggin and B. Therese Kinsella

This article has been withdrawn by the authors. The authors of thepaper have become aware that some features had been duplicated inFigs. 7, C and D, and 8G. As the original autoradiograms and scanimages relating to the aforementioned figures are no longer available toinvestigate the matter, the authors wish to withdraw the article in theinterests of maintaining their publication standards, while also respect-ing the highest standards of transparency and reliability of their researchand of the JBC. Replica data sets for each of the figures in question thatthe authors state fully validate the findings and conclusions of the pub-lished article are available, and, accordingly, a revised version of themanuscript with the replica data sets can be obtained by contacting thecorresponding author.

WITHDRAWALS/RETRACTIONS

J. Biol. Chem. (2018) 293(31) 12285–12285 12285© 2018 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.