0 1989 by The American Society for Biochemistry and Molecular Biology, Ine THE JOURNAL OF BIOLOGICAL CHEMISTRY . 264, No. 28, Issue of October 5, pp. 16496-16501. 1989

Printed in U.S.A.

Purification of the Thromboxane Aa/Prostaglandin Hz Receptor from Human Blood Platelets*

(Received for publication, May 17, 1989)

Fumitaka Ushikubi, Masatoshi Nakajima, Masakazu Hirata, Minoru OkumaS, Motohatsu Fujiwara, and Shuh Narumiyas From Department of Pharmacology and the $First Diuision, Department of Internal Medicine, Kyoto Uniuersity Faculty of Medicine, Yoshida, Sakyo-ku, Kyoto 606, Japan

5-145 (5Z-7-(3-endo-phenylsulfonylamino-(2.2.1.)- bicyclohept-2-exo-y1)heptenoic acid) is a potent and selective antagonist for thromboxane Az/prostaglandin H2 receptor. Using this compound as an immobilized ligand for affinity chromatography and [3HJS-145 as a radioligand, we have purified the thromboxane A,/ prostaglandin H2 receptor from the membranes of hu- man blood platelets. The purification procedures con- sisted of solubilization of the receptor with 3-[(3-cho- lamidopropyl)dimethylammonio]- 1-propanesulfonate (CHAPS), affinity chromatographies on columns of S- 145 affinity gel, wheat germ agglutinin agarose and red agarose, and repeated gel filtration high perform- ance liquid chromatography on a TSK gel G-3000SW column. On the second gel filtration high performance liquid chromatography, the r3H]S-145 binding activity was eluted as a symmetrical peak which overlapped exactly with a peak of ultraviolet absorption at 280 nm. By these procedures, the receptor was purified about 8700-fold from the solubilized extract with a recovery of 6%. The final preparation showed a broad protein band at M, 57,000 on sodium dodecyl sulfate- polyacrylamide gel electrophoresis and maximally bound 19.2 nmol of [’H]S-l45/mg protein with a Kd of 29.8 nM. The [3H]S-145 binding to the purified recep- tor was specifically displaced by several thromboxane A2/prostaglandin Hz analogues.

Thromboxane (TX)’ A,, a major arachidonate metabolite

*This work was supported in part by Grants-in-Aid 63115029, 63637007,63638509,63641006, and 63641513 for Scientific Research on Priority Areas from the Ministry of Education, Science, and Culture of Japan and by grants from the Cell Science Foundation, the CIBA-GEIGY Foundation for Promotion of Science, the Japan Intractable Diseases Research Foundation, and the Japanese Foun- dation on Metabolism and Diseases. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “acluertisernent” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

§To whom correspondence and reprint requests should be ad- dressed.

The abbreviations used are: TX, thromboxane; TXA,, thrombox- ane A*; CHAPS, 3-[(3-cholamidopropyI)dimethylammonio]-l-pro- panesulfonate; EDC, l-ethyl-3-(3-dimethylaminopropyl)carbodi- imide; HPLC, high performance liquid chromatography; ONO-3708, 9,11-dimethylmethano-11,12-methano-13,14-dihydro-13-aza-14-oxo- 15-cyclopentyl-16,17,18,19,2O-pentanor-15-epi-thromboxane A,; PG, prostaglandin; PTA-OH, 9,11-dimethylmethano-ll,l2-methano-16- (4-hydroxyphenyl)-13,lri-dihydro-l3-aza-15-cu~w-tetranor-thrombox- ane AP; SDS, sodium dodecyl sulfate; S-145,52-7-(3-endo-phenylsul- fonylaminobicyclo(2.2.l)hept-2-exo-yl)heptenoic acid; STA2, 9,11-epithio-11,12-methano-thromboxane AZ; U-46619, 15(S)-hy- droxy-11,9-epoxymethano-prosta-5Z,13E-dienoic acid WGA, wheat germ agglutinin; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid.

in platelets, is a potent stimulator of platelet aggregation and constrictor of vascular and respiratory smooth muscles (1, 2). Because of such potent biological activities and its formation in response to various stimuli, TXA, has been implicated as a mediator in many pathophysiological conditions such as myocardial infarction, stroke, and anaphylaxis (3). However, its mechanism of action has not been fully elucidated. Since TXA, is a very unstable compound ( tlh = about 30 s), several structural analogues have been developed for the analyses of its actions. Pharmacological studies using the analogues which either mimic or antagonize the TXA, actions have suggested the presence of a receptor for this very unstable compound (4-7). Following these studies, several groups de- veloped radiolabeled derivatives of these analogues and car- ried out radioligand binding experiments for this putative receptor. These include studies with [3HH]13-azaprostanoic acid (8), [3H]15(S)-9,11-epoxymethano-PGH2 (91, [‘251]PTA- OH (10, 11), and [3H]U-46619 (12, 13). These studies have revealed the existence of a class of binding sites in platelets which appears common to these analogues. The specificity of this binding to the TX and PG analogues has suggested that this class represents the putative TXA, receptor and that the PG endoperoxide, PGHz, shares this same receptor with TXA2. Halushka and his collaborators (14, 15) reported sol- ubilization of this putative TXA2/PGH2 receptor from platelet membranes and partially characterized it by gel permeation and density gradient centrifugation. However, isolation and further characterization of the receptor have not been re- ported so far, probably because the solubilized receptor quickly loses the binding affinity to the above ligands (14).

Recently, a series of compounds having a bicycloheptane ring and a sulfonamide linkage have been developed as new TXAl receptor antagonists (16, 17). The representative com- pound, S-145, is one of the most potent TXA, antagonists reported so far and exerts a strong persistent antagonism to TXAz actions in platelets, blood vessels, and airway smooth muscles of various species (18, 19). Based on this compound we have developed two new TXA, receptor radioligands, i.e. [3H]S-145 and [*251]I-S-145-OH, and found that these ligands bind to the solubilized receptor without significant loss of the binding affinities (20). Using [3H]S-145 as a radioligand and S-145 as an immobilized ligand for affinity chromatography, we have purified the TXA2/PGH2 receptor from the mem- branes of human blood platelets.

EXPERIMENTAL PROCEDURES

Materiak-[’H]S-145 (24.5 Ci/mmol) and S-145 were synthesized as described previously (16, 20) and kindly provided by Shionogi Research Laboratories, Osaka, Japan. ONO-3708, PGDz, PGEI, PGFZe, STA,, and TXBz were gifts from Ono Pharmaceuticals, Osaka, Japan. U-46619 was purchased from Cayman Chemicals. CHAPS was obtained from Dojin Chemical, Kumamoto, Japan. A TSK gel

16496

Purification of Platelet Thromboxane Az Receptor 16497

G-3000SW column was purchased from Toso, Tokyo, Japan. Red- Agarose (Matrex Red A,) was obtained from Amicon. Affi-Gel 102 and molecular weight calibration proteins for SDS-polyacrylamide gel electrophoresis were #obtained from Bio-Rad Laboratories. Asolec- tin was obtained from PLmerican Lecithin Company, Inc. N-Acetyl- D-glucosamine, Amido ELlack 10B, benzamidine hydrochloride, EDC hydrochloride, phenylmethylsulfonyl fluoride, polyethyleneimine, Sil- Best stain (a silver staining kit for polyacrylamide gel), and Tween 60 were obtained from INacalai Tesque, Inc., Kyoto, Japan. WGA- Agarose was purchased from Seikagaku Kogyo, Tokyo, Japan. PD-10 columns (prepacked Sephadex G-25 columns for rapid desalting and buffer exchange) were obtained from Pharmacia LKB Biotechnology Inc. GF/C filters were obtained from Whatman International, Maid- stone, United Kingdom. All other chemicals used were of reagent grade.

Human platelet concentrates out of date for clinical use were kindly supplied by Red Cross ]Blood Centers. The platelets were freed of contaminating blood cells by centrifuging at 120 X g for 15 min and then pelleted by centrifuging at 2,300 X g for 30 min. The platelet pellets were suspended in %o of the original volume in 5 mM Tris. HCl, pH 7.4, 5 mM EGI'A, 1 mM benzamidine hydrochloride, and 0.43 mM phenylmethylsulfonyl fluoride. The suspension was homog- enized by two strokes in a Potter-Elvehjem homogenizer. The ho- mogenate was centrifuged at 100,000 X g for 30 min. The pellets were again suspended in the above buffer, homogenized, and centrifuged as described. The second pellets were suspended in 20 mM Tris. HCl, pH 7.4, and 5 mM EGTA at a concentration of 10-15 mg protein/ml. This membrane fraction was stored at -80 "C until use.

Preparation of S-145 Affinity Gel (Fig. +-We immobilized S-145 by conjugating its carboxylic group with the amino group of Affi-Gel 102, because the modification of the carboxylic group of S-145 did not significantly alter its affinity to the TXAZ receptor (20). Affi-Gel 102 (14.5 pmol of active group/ml of gel), 90 ml, was washed thor- oughly by filtration with 10 volumes each of ethyl alcohol and distilled water and suspended in 100 ml of distilled water. The pH of the suspension was adjusted to pH 4.5 with dilute HCl. S-145, 10 g (26.5 mmol), was dissolved in 200 ml of dioxane and [3H]S-145 (5.5 X lo7 cpm, 2 nmol) was added as a tracer, The S-145 solution was mixed with 15 g (78.2 mmol) of EDC in 100 ml of distilled water at pH 4.5. With gentle stirring, this mixture was added dropwise to the gel suspension. The pH of the suspension was maintained at pH 4.5 by the addition of dilute HCl. The mixture was gently shaken for 30 h at room temperature. The gel was then washed successively with 500 ml of 50% dioxane, and 1 liter each of 0.1 M Tris.HCI, pH 8.2, distilled water, and 20% ethyl alcohol. By these procedures, 2.5 pmol of S-145 was incorporated to 1 ml of the gel. This S-145 affinity gel (S-145 Affi-Gel) was kept at 4 "C in 99.5% ethyl alcohol until use. Deterioration of the binding capacity was not observed during storage for several months under these conditions.

Binding Assays-TXA2 receptor was assayed as the [3H]S-145 binding activity. The standard mixture contained 20 mM Tris. HCl, pH 7.4, a sample, 20 nM f'H]S-145 (1 X lo6 cpm), and 0.5 mg/ml asolectin in a total volume of 200 pl. Incubation was performed at 37 "C for 30 min and terminated by the addition of 5 ml of 10 mM Tris. HCl, pH 7.4, precooled at 4 "C. The mixture was then filtered in U ~ C U O through a GF/C filter presoaked in 0.3% polyethyleneimine (21). After the filter was washed with 5 ml of the precooled Tris.HC1 buffer five times, the radioactivity on the filter was determined in a Triton-toluene scintillator on a Packard Minaxi model 4530 liquid scintillation system. Nonspecific binding determined as the binding in the presence of 20 p M Si-145 was usually less than 5% of the total binding, and only specific binding was shown in the following results. Scatchard analyses were carried out on the receptor preparations at each purification step. The samples were incubated with various concentrations (1.25-120 nM) of [3H]S-145, and the binding was determined as described above. The Bmax values were used as the receptor amount in the preparations. Association and dissociation of [3H]S-145 to and from the purified receptor were analyzed as de- scribed previously for the crude membranes (20).

Purification of the TXA, Receptor-Procedures have been devel-

FIG. 1. Chemical structure of S-145 affinity gel.

oped for the purification of the TXAZ receptor from human platelets in substantial quantities. Purification by these procedures has been employed several times with comparable results. All procedures were performed at 4 "C, unless otherwise specified.

Solubilization of the Receptor-CHAPS, 200 mM, and glycerol were added to 600 ml of the platelet membrane fraction (6000 mg of protein) to the final concentrations of 10 mM and 20% (w/v), respec- tively. The mixture was vortexed at room temperature for 2 min and centrifuged at 200,000 X g for 60 min. The supernatant, 700 ml, was recovered as the solubilized extract. Thirty one percent of the binding activity in the membranes was solubilized by these procedures.

Affinity Chromatographies on Columns of S-I45 Affi-Gel and WGA- Agarose-The solubilized extract was mixed with 180 ml of 20 mM Tris.HC1, pH 7.4, 10 mM CHAPS, 20% (w/v) glycerol, 2.5 M KCl, 5 mM EGTA, and 1 mg/ml asolectin. The solution was then mixed with 30 ml of S-145 Affi-Gel equilibrated with the buffer AF5 (20 mM Tris.HCI, pH 7.4, 10 mM CHAPS, 20% (w/v) glycerol, 0.5 M KCl, 1 mg/ml asolectin, and 0.2 mM EGTA). The mixture was gently agitated for 14 h and the gel was transferred to a glass column (1.5 cm, inner diameter, X 14 cm) and washed with 250 ml of buffer AF5. After washing, the outflow tube of the Affi-Gel column was connected to a WGA-agarose column (1.0 cm, inner diameter, X 8 cm, 6 ml), and elution was performed with 20 ml of buffer AF5 containing 0.1 mM S-145. The eluate was recycled through the two columns at the rate of 12 ml/h for 70 h. The columns were then separated and the WGA- agarose column was washed with 250 ml of buffer AF5. The receptor was eluted with 30 ml of buffer AF5 containing 0.3 M N-acetyl-D- glucosamine, and the active fractions were pooled (19 ml, Affi-Gel- WGA fraction).

Red-Agarose Column Chromatography-The Affi-Gel-WGA frac- tion, 18.2 ml, was applied to PD-10 columns for buffer exchange. The columns were preequilibrated with buffer AT0 (20 mM Tris.HC1, pH 7.4, 10 mM CHAPS, 20% (w/v) glycerol, 0.04% Tween 60, 0.2 mg/ml asolectin, and 0.2 mM EGTA), and the samples were eluted with the same buffer. The active fractions, 25.5 ml, were pooled and applied to a column of red-agarose (1.0 cm, inner diameter, X 3 cm, 3 ml). After the column was washed with buffer ATO, elution was performed with a linear gradient between 0 and 0.7 M KC1 in 100 ml of buffer ATO. The activity was eluted as a broad peak between 0.2 and 0.5 M KCl, and the active fractions were pooled (50 ml, the red-agarose fraction).

Repeated Gel Filtration HPLC on a TSK Gel G-3000s W Column- The red-agarose fraction was concentrated to about 0.25 ml with the aid of Diaflo PM-30 and Centricon 30 (Amicon) and applied to a TSK gel G-3000SW column connected to a Pharmacia fast protein liquid chromatography system (22). Elution was performed with buffer AT0 containing 0.5 M KC1 at a flow rate of 0.3 ml/min and monitored by absorbance at 280 nm. Fractions (0.5 ml) were collected, and the activity was eluted between fractions no. 25 and 39. The peak fractions (no. 28-33) were combined (3 ml, the first TSK gel fraction). This fraction, 2.5 ml, was concentrated again to 0.25 ml with the aid of Centricon 30 and injected onto a TSK gel G-3000SW column. Elution was performed as described above and the active fractions from no. 29 to 32 were pooled (2 ml, the second TSK gel fraction).

Electrophoresis and Protein Determinutiom-Protein samples for SDS-polyacrylamide gel electrophoresis were precipitated by the ad- dition of sodium deoxycholate and trichloroacetic acid as described previously (23). The precipitates were dissolved in 40 pl of Laemmli sample buffer with or without 100 mM dithiothreitol and subjected to SDS-polyacrylamide gel electrophoresis in a 10% gel (24). Proteins were silver-stained according to the method of Merril et al. (25) using Sll-Best stain. Protein amounts in the solubilized extract and the Affi-Gel-WGA fraction were determined by the use of Coomassie Brilliant Blue G-250 according to the method of Bradford (26), and those in the red-agarose and TSK gel fractions were determined by the method of Schaffner and Weissman (27) using Amido Black 10B.

RESULTS

Effect of Asolectin on the PHIS-145 Binding Activity in the Purified Preparations-We added asolectin to the binding assay mixture, because it restored the binding activity in the purified preparations which was otherwise quite low. As shown in Fig. 2, asolectin dose-dependently enhanced the binding activity of the receptor preparations. About 8-fold stimulation was achieved when it was added at 0.5 mg/ml to the Affi-Gel-WGA fraction. At the higher concentrations,

16498 Purification of Platelet Thromboxane AB Receptor

I ’

z f U 0 1 I I I

0 1 2 3

Asoleck Concentration (rng/ml)

FIG. 2. Effect of asolectin on the [‘H]S-145 binding to the purified receptor. The Affi-Gel-WGA fraction deprived of asolectin by gel filtration was incubated with 20 nM [3H]S-145 in 20 mM Tris. HC1, pH 7.4, in the presence of 0, 0.05, 0.3, 0.5, 0.7, 1.0, 1.5, 2.0, and 3.0 mg/ml of asolectin in a total volume of 200 pl. Incubation was carriedout at 37 “C for 30 min, and [3H]S-145 binding was determined as described under “Experimental Procedures.”

TABLE I Purification of the TXA2 receptor from human blood platelets

[3H]S-145 binding Fraction Protein T~~~~ specific ::ii Recovery

amount’ activity mg pmol pmoljmg -fold %

Solubilized ex- 2,886 6,395 2.22 1 100

Affi-Gel-WGA 0.399 1,425 3570 1,620 22 Red-Agarose 0.197 996 5060 2,300 16 First TSK gel 0.052 657 12,600 5,730 10 Second TSK gel 0.020 384 19,200 8,730 6

of each fraction.

tract

Determined from the Bmax values obtained by Scatchard analysis

however, this stimulation was decreased dose-dependently. We, therefore, included 0.5 mg/ml concentration asolectin in the following binding assays.

Purification of the T U 2 Receptor-A representative puri- fication of the TXA2 receptor from the membranes of human platelets is shown in Table I. About 8,700-fold purification was achieved from the solubilized extract with a recovery of 6%. On solubilization, 30-40% of the binding activity in the membranes was reproducibly solubilized with 10 mM CHAPS, but no enrichment of the activity in the solubilized extract was found. When the solubilized extract was incubated with the S-145 Affi-Gel as described under “Experimental Proce- dures,” the solubilized activity bound to the gel in a time- dependent manner; more than 70% of the activity was ad- sorbed in 8 h (Fig. 3). After the gel was washed, the activity was eluted with the buffer containing 0.1 mM S-145. Asolectin was added to the buffers, because, in addition to its effect on the binding as described, it improved recovery of the activity from the Affi-Gel column from as low as a few percent to about 50%. The elution of the activity from the Affi-Gel column required a large volume of the elution buffer contain- ing the cold ligand and caused dilution of the binding activity. We, therefore, saved it by connecting the WGA-agarose col- umn in sequence to the Affi-Gel column. As shown by a preliminary experiment in Fig. 4, the solubilized activity could be adsorbed onto this column and eluted with N-acetyl-D- glucosamine. Thus, in the present purification, the eluate was recycled through the two columns, and the activity eluted

0 4 8 12 16 20 A

24

Incubation Time (h)

FIG. 3. Time-dependent adsorption of the solubilized recep- tor to S-145 Affi-Gel. The solubilized extract was incubated with s-145 Affi-Gel as described under “Experimental Procedures.” At 0, 0.25, 0.5, 1, 2, 4, 8, and 24 h, 20 pl of aliquots were taken. The gel in the aliquots was spun down at 400 X g for 5 min. The [3H]S-145 binding activity (0) and protein amount (0) in the supernatants were determined and expressed as relative amounts to the starting mate- rial.

I

0.6 GlcNAc 9000

10 20 30 40 50

Fraction Number FIG. 4. WGA-Agarose column chromatography of the solu-

bilized extract. The solubilized extract (12.5 ml containing 11.0 mg of protein) was applied to a column of 2 ml of WGA-agarose equili- brated with 20 mM Tris. HCI, pH 7.4, containing 5 mM CHAPS and 2% (w/v) glycerol. After the column was washed with the equilibration buffer, the binding activity was eluted with the buffer containing 0.3 M N-acetyl-D-ghcosamine as indicated by the arrow. 0, absorbance at 280 nm; 0, [3H]S-145 binding activity.

from the Affi-Gel column was successively adsorbed to WGA- agarose. The activity collected on a WGA-agarose column after several cycles was then eluted with N-acetyl-D-giucosa- mine. This combined affinity chromatography purified the receptor about 1,600-fold with 22% recovery. The Affi-Gel- WGA fraction was then subjected to red-agarose column chromatography. We have screened several dye-linked aga- roses for purification of this receptor and found that red- and blue-agaroses are useful in this purification. Although the purification by this column chromatography was only 1.5 fold, this step was essential in excluding minor impurities which otherwise accompanied the final preparation. The red-agarose fraction was then concentrated and subjected to TSK gel HPLC. Small concentrations of Tween 60 and asolectin were added to the sample and the filtration buffer. Tween 60 improved separation and sharpened the peak of activity. Aso- lectin appeared to prevent aggregation of the molecules, be-

Purification of Platelet Thromboxane Az Receptor 16499

cause in its absence the binding activity was eluted at the void volume. The elution profile from the second TSK gel HPLC is shown in Fig. 5A. The activity was eluted as a symmetrical peak around fraction no. 30 which overlapped exactly with an ultraviolet absorption peak. SDS-polyacryl- amide gel electrophoresis of aliquots of each fraction revealed that elution of a protein of M , 57,000 corresponded with these peaks (Fig. 5B). This second TSK gel fraction was used as the final preparation.

Properties of the Purified Receptor-Samples from each purification step were analyzed by SDS-polyacrylamide gel electrophoresis as shown in Fig. 6. The final preparation (the second TSK gel fraction) showed a broad protein band follow- ing electrophoresis (lune 6), the molecular weight of which was estimated to be 57,000. The final preparation also showed a faint protein band migrating higher a t M , 85,000-90,000. As shown in Fig. 5B, this band came from the protein of that molecular weight which was eluted before the [‘H]S-145 bind- ing activity on TSK gel HPLC and appeared a contaminant

A

0 co cu .c. m lil 0.02

0.01

0 20 30 40 U

Fraction Number

B Fraction Number

97KDa ,

66KDa - 43KDa - 31KDa-

21KDa -- FIG. 5. Second gel filtration HPLC on a TSK gel G-3000SW

column. A, the first TSK gel fraction was concentrated to 0.25 ml wit,h the aid of Centricon 30 and reapplied to a TSK gel G-3000SW column connected to a Pharmacia fast protein liquid chromatography system as described under “Experimental Procedures.” Fractions (0.5 ml) were collected, and 7-pl aliquots of each fraction were used for the binding assays. The solid line represents the elution of absorbance at 280 nm, and the binding activity in each aliquot is shown by closed circles. An ultraviolet peak corresponding to fractions no. 34-44 was due to asolectin micelles in the preparation. B, aliquots (50 pl) of each fraction from this gel filtration HPLC were subjected to SDS- polyacrylamide gel electrophoresis in a 10% gel. Proteins were stained with the silver reagent as described under “Experimental Procedures.” A sharp band at M , 60,000 seen in all lanes of this figure is an artifact often seen in our silver staining method.

1 2 3 4 5 6

97KDa *

66KDa *

43KDa *

31KDa *

21KDa *

FIG. 6. SDS-polyacrylamide gel electrophoresis of the var- ious receptor preparations. Samples from various receptor prep- arations were dissolved in Laemmli buffer without dithiothreitol and subjected to SDS-polyacrylamide gel electrophoresis in a 10% gel a t a constant current of 36 mA. Proteins were stained with the silver reagent. Lane 2, the solubilized extract, 4.95 pg; lone 3, the Affi-Gel- WGA fraction, 1.05 pg; lane 4, the red-agarose fraction, 0.89 pg; lane 5, the first TSK gel fraction, 0.78 pg; lane 6, the second TSK gel fraction, 0.76 pg. Lane 1, molecular weight markers. An arrow indi- cates an M , 57,000 broad protein band.

0 25 50 75 100 125

C3H1S-145 (nM) FIG. 7. Saturation of [SH]S-145 binding to the purified

receptor. The purified receptor, 0.038 pg, was incubated with various concentrations of [‘H]S-145 either in the presence or absence of 20 p~ cold S-145, and the specific binding was determined as described under “Experimental Procedures.” Inset, Scatchard analysis of the [‘H]S-145 binding.

in the receptor preparation. The protein of M , 57,000 was, therefore, regarded to represent T U z receptor. This protein band was broader than those of the marker proteins, and this is probably due to the glycoprotein nature of this protein as evidenced by its behavior on the WGA-agarose column. Such broadness of protein bands was also noted for @-adrenergic receptor (28) and muscarinic receptor (29) and suggested to be caused by microheterogeneity in the carbohydrate residues. Reduction with 100 mM dithiothreitol did not change the electrophoretic mobility of this protein band (data not shown).

The purified receptor showed a time-dependent and revers- ible [3H]S-145 binding as in intact platelets and crude mem- branes (data not shown). As shown in Fig. 7, this [‘H]S-145 binding to the purified receptor was saturable around 100 nM of the ligand. Scatchard analysis of these data yielded a single straight line, suggesting a single class of binding sites in the

16500 Purification of Platelet Thromboxane AP Receptor I I I I I 1

9 8 7 6 5 4 3

Compounds (-log M)

FIG. 8. Competition for [aH]S-145 binding to the purified receptor by various compounds. The purified receptor, 0.023 pg, was incubated with 30 nM [3H]S-145 in the presence of various concentrations of compounds, and the specific binding was deter- mined as described under “Experimental Procedures.” The 100% binding represents the specific binding obtained without any compet- itor. a, S-145; 0, ONO-370% A, STA,; A, U-46619; V, PGDz; a, PGE1; 0, PGFz,; (3, TXBz.

preparation (Fig. 7, inset). The Kd and B,,, values were estimated to be 29.8 nM and 19.2 nmol/mg protein, respec- tively. The purified receptor was stable at -80 “C but lost 50% of its binding activity during storage at 4 “C for 7 days.

The specificity of [3H]S-145 binding was examined by com- petition of the binding with various compounds (Fig. 8). The binding was specifically displaced by various TXA2/PGH2 analogues such as S-145 and ONO-3708 (both antagonists) and STA, and U-46619 (both agonists) in a concentration- dependent manner to the level of the nonspecific binding. The ICso (concentration to inhibit the specific radioligand binding by 50%) values of these compounds were 42, 220, 1,750, and 40,000 nM for S-145, ONO-3708, STA2, and U- 46619, respectively. The K, values calculated from Ki = IC50/ (1 + [L] /Kd) in which [ L ] and Kd are the concentration and dissociation constant of the radioligand, respectively (30), were 21,110,870, and 19,900 nM for S-145,ONO-3708, ST&, and U-46619, respectively. These values were in good agree- ment with those observed for the solubilized receptor (20). On the other hand, the binding was not effectively competed by other PGs such as TXB2, PGD,, PGE1, and PGF?,. Thus, the specificity of the binding to the final preparation corre- lated well with that found in intact platelets and the crude membranes.

DISCUSSION

The present study describes purification of the [3H]S-145 binding protein from the membranes of human blood plate- lets. Several lines of evidence suggest that this purified protein is the platelet TXA2/PGH2 receptor. First, S-145 is a selective TXAz receptor antagonist in platelets as well as other systems (16-19), and S-145 binding in platelets has already been shown to correlate well with its antagonistic activities (18, 20). Second, only a single class of [3H]S-145 binding was detected in intact platelets and membranes, and a major portion of this binding activity was solubilized from the membranes (20). On the subsequent chromatographic steps, too, only a single peak of the activity was detected and purified. Third, the [3H]S-145 binding to the purified protein was specifically displaced by TXA2/PGHz analogues and not by other PGs. The affinities of the two antagonists, S-145 and ONO-3708, to the purified protein were not much differ-

ent from those observed in intact platelets. The agonists such as STA, and u-46619, on the other hand, showed much lower affinities for the purified protein compared to intact cells. As reported previously (14, 20), this decrease has been observed with membrane preparations and following receptor solubi- lization and could be attributed to separation of the transduc- ing component(s) from the receptor during these procedures. As discussed below, TXAz receptor has been suggested to couple with a GTP-binding protein. Modulation of receptor affinities to agonist ligands by GTP-binding proteins has already been reported for several types of receptors (see Ref. 31 for example). The final preparation possessed a specific activity of [3H]S-145 binding of 19.2 nmol/mg protein. As- suming that the molecular weight of this receptor protein is 57,000, the theoretical value for this specific activity is 17.5 nmol/mg protein. This suggests that the 57-kDa protein pu- rified here binds one ligand per molecule.

Actions of PGs and TX are believed to be mediated via the respective receptors (32). However, none of the receptors had ever been isolated and there remains many important ques- tions unanswered yet. One is the molecular structure of the prostanoid receptor. The availability of the purified receptor shown here now permits us to determine its partial amino acid sequences and to start cDNA cloning. We have already reported that cultured human erythroleukemia cells such as HEL and K-562 express the TXAz receptor analogous to the platelet receptor and can be used as an mRNA source for cloning of this receptor (33). Another issue concerning TXAz receptor is possible molecular heterogeneity of receptor sub- types found in platelets and smooth muscles. They have been reported to be different in response to several agonists (34, 35) and also different in transduction pathways. TXA2 ago- nists evoke a rise in the intracellular free calcium concentra- tion and stimulate phosphoinositide metabolism in platelets (12,36,37), and it is generally assumed that phosphoinositide metabolism comes first in TXA, actions in platelets. On the other hand, calcium channel blockers such as nifedipine and verapamil inhibit TXA2-induced contraction in isolated blood vessel preparations and TXAz has been presumed to somehow open a calcium channel in this tissue (38, 39). Whether such a difference is due to different structures of TXAz receptor subtypes or to the difference in transduction pathways be- tween the two tissues should be clarified by future study. AS for the transduction mechanisms, several groups (40-43) have reported that TXA2 agonists stimulate high affinity GTPase and induce phosphoinositide turnover in platelets and other cells and that these processes are insensitive to pertussis and cholera toxins. These findings have led them to conclude that TXA2 receptor couples with a GTP-binding protein which is distinct from G, and Gi/G,. However, the identity of the GTP- binding protein coupling the TXAz receptor to phosphoino- sitide turnover remains unknown. Recently, several novel GTP-binding proteins have been reported in platelets (44- 47), and the above issue will be examined by reconstitution experiment using the purified receptor and these GTP-bind- ing proteins. Another important issue is the cellular topogra- phy of the TXA, receptor. Immunohistochemical study with antibodies against the purified or recombinant receptor pro- tein will clarify its location in cells.

In conclusion, we have purified the platelet TXAZ receptor. This purification of the prostanoid receptor should help un- derstanding the mechanism of PG actions at the molecular level.

Acknowledgments-We are grateful to the Japanese Red Cross Society for their supply of outdated platelets for research purpose. We would like to express our thanks also to Dr. M. Narisada of the

Purification of Platelet Thromboxane A, Receptor 16501

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