Original Article
Molecular comparison of @2-adrenergic receptors from rat adrenocortical carcinoma and human blood platelet
Rama Kant Jaiswal1, Daniel R. Marshak2 and Rameshwar K. Sharma1
1Department of Biochemistry, University of Tennessee, Memphis, TN 38163, USA; 2Cold Spring Harbor Laboratories, Cold Spring Harbor, NY 11724, USA
Received 13 June 1988; accepted 18 August 1988
Key words: adrenergic receptors, @2-adrenergic receptor, purification
Summary
We have previously described a simple two-step purification technique to isolate @2-adrenergic receptors from the rat adrenocortical carcinoma (Jaiswal, R. K. and Sharma, R. K. (1985) Biochem. Biophys. Res. Commun. 130, 58-64). Utilizing this technique we have now achieved ~ 77 000-fold purification to apparent homogeneity of @2-adrenergic receptors from human platelets. We have compared the biochemical characteristics of these receptors with those from the rat, which were purified ~ 40000-fold to homogeneity.
The [125I] receptor proteins from two sources showed: (a) a single radioactive band with a Mr of 64000 as evidenced by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE); and (b) a single symmetrical peak with a pI of 4.2 by isoelectric focusing polyacrylamide gel electropho- resis. Both proteins showed typical @2-adrenergic binding characteristics with specific binding activities of 13.85 nmol/mg and 14.17 nmol/mg protein. These values are close to the theoretical binding activity of 15.6 nmol/mg protein for 1 mol of the ligand binding 1 mol of the receptor protein. These results attest to the purity of the receptors, to its Mr of 64000, and to its acidic nature. However, the peptide maps of the radioiodinated @2-adrenergic receptors from rat adrenocortical carcinoma and human blood platelets reveal some distinct differences which may relate to the differences in the pharmacological specificities between rodent and non- rodent @2-adrenergic receptors.
Abbreviations: PAC - p-aminoclonidine, PMSF - Phenylmethyl-sulfonylfluoride, DTT - Dithiothreitol, HPLC - High Performance Liquid Chromatography
Introduction
Rat adrenocortical carcinoma 494 cells [1, 2] contain extensively characterized @2-adrenergic receptors [3, 4] which are coupled positively to membrane guany- late cyclase and negatively to adenylate cyclase [5]. Because of the homogeneity of the cell type and the exclusive population of a-receptors as of @2- subtype, these cells are an excellent model as the
receptor-source and to elucidate the biochemical mechanism of @2-receptor mediated transmem- brane signal transduction. As a step in this direction we described a simple two-step purification tech- nique which was successfully applied for the first complete purification of the @2-receptors. The receptor purification was demonstrated by one - dimensional SDS-PAGE, high performance liquid chromatography and photoaffinity labeling studies
[6]. In the present study, by our simple technique, we have isolated and thoroughly characterized @2- adrenergic receptors from human platelets. We have compared the platelet and the tumor receptors by two-dimensional SDS-PAGE. Herein we show that the purified receptors from both rat tumor and hu- man platelets are indeed homogeneous and have a Mr = 64000. We have extended these studies in demonstrating that the @2-receptor protein is acidic in nature with a pI of 4.2. Further, in order to assess possible homologies between intraspecies receptor sources, we have compared the peptide maps gener- ated by partial proteolysis of @2-adrenergic receptor proteins obtained from these two sources by trypsin, a chymotrypsin and Staphylococcus aureus V8 pro- tease.
Experimental procedures
Materials
Yohimbine hydrochloride, epinephrine, norepine- phrine, TPCK-treated trypsin, a-chymotrypsin, S. aureus V-8 protease and DL-dithiothreitol were from Sigma Chemical Company; [3H]yohimbine (80.2 Ci/mmol), [3]para-aminoclonidine (40 ci/mmol), [3H]p-azidoclonidine (31 Ci/mmol), and carrier free Na125I from New England Nuclear; all of the rea- gents for SDS-PAGE and isoelectric focusing chro- matography were obtained from Bio-Rad and AH- Sepharose 4B from Pharmacia. Digitonin was pur- chased from Gallard-Schlesinger Chemical Corpo- ration. All other reagents were analytical grade from commercial sources. Out-dated platelet rich plasma from random donors was purchased from the Life Blood, Memphis, TN.
Preparation of sepharose-PAC affinity resin
Sepharose-PAC1 affinity resin was prepared as previously described [6].
Membrane preparation
Adrenocortical carcinoma membranes were pre-
pared as in [4] except that 100 uM phenylmethylsul- fonylfluoride and 5 mM EDTA were also present in the buffer. The membranes were used immediately for binding assay and for solubilization.
Outdated, platelet rich plasma, (20-25 units), were pooled and centrifuged at 100xg in a Sorval centrifuge using SS-34 rotor for 10 min to separate contaminated red blood cells. The supernatant was again centrifuged at 4000xg for 30 min and the pellet was resuspended in a buffer containing 50 mM Tris-HC1, pH 7.5, 150 mM NaCl and 20 mM EDTA, and were recentrifuged as before. The packed platelets were resuspended in a lysis buffer (5 mM Tris-HCI, 5 mMEDTA, 2 mM EGTA, 100 AM PMSF, pH 7.5) and were homogenized with five up and down strokes of a motor-driven Potter- Elvehjem homogenizer at a speed of 1000 rpm. The homogenate was kept on ice for 2 hours and then centrifuged for 40 min at 35000xg using a Sorval SS-34 rotor. The supernatant was pured off, and the pellet was resuspended in lysis buffer by homogeniz- ing again at low speed using Potter-Elvehjen homogenizer. The homogenate was centrifuged again at 35000xg as before and the pellet was resuspended in a buffer (50 mM Tris HCI, 10 mM MgCl2 0.1 mM EDTA and 100 uM PMSF, pH 7.8). The membrane suspension was used immediately or stored at -70℃.
Solubilization
The freshly prepared adrenocortical carcinoma membranes (1200 mg protein) suspension was cen- trifuged at 35 000 x g for 40 min in a Sorval SS-34 ro- tor and the pellets were resuspended in 325 ml of solubilization buffer (0.2% digitonin, 20 mM Tris- HC1, 15 mM EGTA, 1 mM DTT, 100 AM PMSF, pH 7.1) using a Brinkmann PT 10/20 homogenizer at setting 4. The suspension was stirred at 4℃ for 25 min and were then centrifuged at 48000xg for 40 min. The supernatent was used for binding assay and for affinity chromatography.
Platelet membranes (1 200 mg protein) were sus- pended in 300 ml of solubilization buffer (1% digitonin, 20 mM Tris-HCI, 15 mM EGTA, 1 mM DTT, 100 „M PMSF, pH 7.8) using a Brinkman PT 10/20 homogenizer set at position 4. The membrane
suspension was stirred at 4℃ for 1 hour and was then centrifuged for 40 min at 48 000 xg in a Sorval centrifuge. The supernatant containing @2- adrenergic receptors was then used for affinity chro- matography.
Affinity chromatography
A Sepharose-PAC affinity column (1.5x30 cm) was pre-equilibrated with 4 bed volumes of buffer A (50 mM Tris-HCI, 1 mM EDTA, 1.0 mM DTT, 100 µM PMSF, 0.2% digitonin, pH 7.2). An aliquot (300 ml) of soluble preparation was applied at a flow rate of 20 ml/hr. The column was washed with 200 ml buffer A, 200 ml of buffer A containing 20 mM NaCl and then again with 100 ml of buffer A. The flow rate was adjusted to 10 ml/hr and the column was eluted with 100 ml of buffer A contain- ing 100 µM phentolamine. Five-ml fractions were collected. All the operations were done at 4℃. A 2.5 ml aliquot of fractions eluted from the affinity matrix was subjected to gel filtration chromatogra- phy on a PD-10 column (Pharmacia) [6], to remove free phentolamine. Fractions containing the peak binding activity (20 ml) were pooled and concentrat- ed 10-15 fold by Amicon ultrafiltration using an Amicon stirred cell and a PM-30 membrane.
Gel permeation high performance liquid chromatography
Partially purified receptors from affinity column eluates were chromatographed on gel permeation column using one Zorbax GF-250 (DuPont) 9.5×250 mm column. The mobile phase containing 0.2% digitonin was prepared as described earlier [6] except it did not contain soya bean trypsin inhibitor and benzamidine. Samples (200 pl) were injected manually and chromatographed at a flow rate of 1 ml/min, fractions were collected every 15 seconds, and fractions containing receptor activity were pooled, concentrated and rechromatographed on a TSK 2000 (7.5 mm I.D. x 60 cm) column through a second HPLC run and assayed for the activity and protein.
Binding assays
The binding of [3H]yohimbine to membrane frac- tions were performed in 0.5 ml incubation mixture consisting of 0.1-0.2 mg of membrane preparation, 50 mM Tris-HC1 pH 7.4, 2 mM MgCl2, 2 mM EGTA and 10 nM [3H]yohimbine. Assays were con- ducted at 25℃ for 30 min. The incubation was ter- minated by adding 3.0 ml cold buffer and filtration through Whetman GF/C filters followed by 3 x 5 ml washes with cold buffer. Non-specific binding was determined in the presence of 10 AM yohimbine or phentolamine. For soluble receptor preparations, bound [3H]yohimbine was separated from free by filtration through polyethyleneamine treated glass fiber GF/B filter as described earlier [6]. The filters were dried and the radioactivity present in the filters was measured by liquid scintillation spectrometry.
Radioiodination of purified receptor preparation
Purified receptor preparations (0.1 ml) were mixed with 5 ul of Na125I (0.2 mCi) in 0.5 M sodium phosphate buffer (pH 7.4) and then with 10 ul of freshly prepared chloramine-T (1 mg/ml). After 30 seconds, 10 ul of sodium metabisulfite (3 mg/ml) was added to stop the reaction as described [7]. Fol- lowing the iodination, samples were chro- matographed on a Sephadex G-50 ; column (0.9x25 cm). The column was equilibrated and elut- ed with 0.05 M sodium phosphate buffer (pH 7.4) containing 0.2% digitonin. Fractions (0.5 ml) were collected, the aliquots (10 ul) were counted for radi- oactivity, and the void volume fractions were collect- ed and analyzed by SDS-PAGE.
Isoelectric focusing
For isoelectric focusing determination, the purified radioiodinated receptor protein (25 ul) was mixed with 9.5 M urea, 2% (w/v) Nonidet P-40, 5% ß- mercaptoethanol, and 2% ampholine [comprised of 1.6% Bio-Lyte (pH 5-7) and 0.4% Ampholine (pH 3.5-9.5)]. Aliquots (50 ul) were subjected to isoelectric focusing according to O’Farrel [8]. Isoe-
lectric focusing was performed using 12 cm tube gels of 0.5 cm diameter. The gels were cut into 3 mm pieces and counted for radioactivity. In other experi- ments the pH gradient of isoelectric focusing gels was determined by equilibrating the 3 mm segments of the tube gel in 1.0 ml degassed and deionized water in 10x75 mm glass test-tubes covered with Parafilm for 4-5 h with occasional vortexing. The pH was read by a microelectrode.
Two-dimensional SDS-slab gel electrophoresis
For two-dimensional gel electrophoresis of the radi- oiodinated receptor protein, the samples were pre- pared and run in the first dimension as described above. The tube gels were applied directly to the top of the SDS-polyacrylamide (10%) gel. The gel was overlaid with 1% agarose. The sample buffer con- taining 0.1% bromophenol was applied over the total length of the gel. The gels were then electrophoresed at 20 mA constant current until the bromo phenol reached the bottom of the gel. At the end of the run, the gels were stained, destained, dried, and autoradi- ographed using Kodak DEF-5 x-ray film. The au- toradiograms were developed at -70℃ for 3-4 days using an intensifying screen (DuPont-Gronex Light- ing Plus).
Partial proteolytic digestion of radioiodinated a2- adrenergic receptors
Partial proteolysis of purified radioiodinated @2- adrenergic receptors from human platelets and adrenocortical carcinoma membranes was per- formed using a modification of the method of Cleve- land et al. [9]. The radioiodinated @2-adrenergic receptors were solubilized in buffer containing 10% glycerol, 5% 2-mercaptoethanol and 3% SDS, and subjected to electrophoresis on 10% SDS- polyacrylamide flat gels (1.5 mm thick) in the first dimension. The gels were stained with Coomassie blue and destained. Gels were cut into 0.5 cm pieces and radioactivity was counted. The gel pieces con- taining peak radioactivity (25 000-30000 cpm) were placed in a 10 ml screw-top plastic test tube contain-
ing 5 ml of equilibration buffer (0.12 M Tris-HCI, 0.1% SDS, 1 mM Na2 EDTA pH 6.8). The tubes were gently agitated on a shaker. After one hour, the buffer was replaced with a further 5.0 ml of fresh buffer and the equilibration continued for a further hour. The buffer was drained off and the gel slices were placed (short end down) in the sample wells of an extra thick (3.0 mm) SDS-polyacrylamide gels (15%) with a 5 cm stacking gel (This is necessary in order to allow sufficient time for the protease to digest the proteins as well as to permit the efficient ‘stacking’ of the samples). The gel slices were co- vered with overlay buffer (0.12 M Tris HCI, 0.1% SDS, 1 mM Na2 EDTA, 20% glycerol, 0.01% (w/v) bromophenol blue pH 6.8) containing either 50 µg TPCK-treated trypsin, 50 µg «-chymotrypsin or 2 µg S. aureus V-8 protease. The samples were run overnight. The gels were fixed and autoradiography performed on the dried gels.
Protein determination
The protein content of particulate and soluble sam- ples were determined by the method of Bradford [10]. In the pure preparations, protein was deter- mined by the method of Fried et al. [11].
Amino acid analysis
Amino acid analysis was performed on samples hydrolyzed in vacuo for 20 hours at 105℃ in 6 N HCI containing 1% (v/v) phenol. The samples were dried, derivatized with phenyl isothiocyanate and analysed by HPLC using a modification of the procedure described by Bidlingmeyer et al., [12]. The elution buffer, containing 0.05% (v/v) triethylamine was adjusted to pH 6.5, and the column was operat- ed at 39℃. The chromatography was performed on a Hewlett-Packard 1090 HPLC with the following times gradient program: 0 min, 0% B; 0.5 min, 10% B; 1.0 min, 17% B; 2.0 min, 24% B; 10 min, 46% B; 10.5 min, 100% B. The last amino acid derivative [a, ebis (phenylthiocarbamyl) lysine] eluted at ap- proximately 9.8 min retention time. Absorbance was monitored at 269 nm and the results were quantitat-
ed by integration of the peak areas. The responses were linear in the range of 1000 pmol to 2 pmol with regression coefficients >0.99 for each amino acid derivatives. Standard amino acid mixtures were ana- lyzed before or after each sample to update the calibrations.
Results
The conditions promoting the best yield during de- tergent solubilization of @2-adrenergic receptors for adrenocortical carcinoma membranes were inves- tigated. These included the digitonin to protein ra- tio, percent digitonin solution and the final volume. Figure 1, shows how varying the digitonin to protein ratio affects the solubilization of membrane and of [3H]yohimbine binding activity. A ratio approach- ing 0.5 appeared optimal in term of specific binding, receptor solubilized and total receptor yield. Effec- tive solubilization and detection of receptors were achieved when the detergent/protein ratio was main-
[H] Yohimbine Binding (A)
60
-60
(%Solubi lized)
Protein ( % Solubilized)
40
40
20
20
0-5
1.0
1-5
2.0
2.5
Digitonin/ Protein
tained at 0.53/1.0 by weight. Solubilization under optimal conditions solubilized about 23 % of protein and released approximately 70% of the total binding activity. Increasing the detergent/protein ratio be- yond 0.5 led to a decrease in soluble binding activity which may reflect denaturation of the receptor.
The inhibition of [3H]yohimbine binding in these soluble receptor preparations showed a drug speci- ficity and order of potency characteristic of mem- brane bound @2-adrenergic receptor [6]. The appar- ent equilibrium dissociation constant (Kd) for [3H]yohimbine binding was 2.3±0.2 nM. Typically these solubilized preparations possessed 0.32-0.40 pmole ligand binding activity/mg protein.
Human blood platelets were solubilized with 1% digitonin buffer [13]. About 54% of the protein and 33% of the platelet receptors were solubilized. Solu- ble receptors from the platelets retained high yohim- bine affinity when bound ligand was separated from free ligand using the polyethyleneamine treated GF/B filters. The Kd of [3H]yohimbine binding in these preparation was 4.5±0.3 nM, which is in agreement of the reported values [6, 13]. The Bmax of these preparations was 187 fmol/mg protein.
We have previously described, a novel affinity res- in, Sepharose-PAC, which was utilized for the purification of @2-adrenergic receptors from adrenocortical carcinoma [6]. We have now used the same affinity resin as a major first step toward purification of @2-adrenergic receptor from human blood platelets. The digitonin extracts of adrenocor- tical carcinoma or blood platelets membranes were passed through a column of Sepharose-PAC resin at 4ºC. Virtually all of the non-receptor protein (>99%) present in the digitonin extract passed un- retarded through the column, and 75-80% of the solubilized receptors were adsorbed on the affinity resin. During the application of wash buffers (total- ing ~ 500 ml) about 10% of the initial binding ac- tivity eluted. A buffer containing 100 uM phentola- mine was then used to specifically elute the @2-adrenergic receptors. At least 44% of the @2- adrenergic receptor binding activity present in the digitonin extract was recovered at this step. The specific activity of [3H]yohimbine binding in pooled fractions eluted from the affinity matrix was 107 pmol/mg protein for adrenocortical carcinoma
A
.
SPECIFIC [+H) YOHIMBINE BOUND (*/%)
100
80
0
60
so
40
.
20
0
10
10
10
10
7
6
$
.4
”
107
-
J
[ ANTAGONIST]. molor
[ AGONISTI molar
@2-adrenergic receptor and 90 pmole/mg protein for bloodplatelets receptors. This represented 300- fold to 500-fold purification of the solubilized receptors.
Further evidence that the affinity-purified recep- tors retained the functional binding properties of the native @2-adrenergic receptor was obtained from competition experiments using [3H]yohimbine and appropriate @2-adrenergic competitors. Among the antagonists yohimbine was most potent in displac- ing the [3H]yohimbine (Fig. 2A). The degree of potency was yohimbine > phentolamine > prazo-
sine > propranolol. For agonist (Fig. 2B), the fol- lowing rank order of potencies was found: para aminoclonidine > (-) epinephrine > (+) epinephrine > isoprotrenolol.
The affinity chromatographed rat adrenocortical carcinoma and human blood platelet @2-adrenergic receptors were purified further by two cycles of HPLC on gel permeation columns [6]. Thirty-seven and forty-six percent of the receptors present after the first-HPLC cycle were recovered at the second HPLC cycle. Based upon the amount of [3H]yohim- bine binding activity and protein present, the specif-
| Step | Volume | Total binding activity | Total protein | Specificb activity | Overall purification |
|---|---|---|---|---|---|
| ml | pmol | mg | pmol/mg | -fold | |
| Soluble | 500 | 187 | 1000 | 0.180 | 1 |
| Affinity | 25 | 65 | 0.720 | 90.2 | 501 |
| 1st HPLC | 4 | 39 | 0.0048ª | 8125 | 45138 |
| 2nd HPLC | 2 | 18 | 0.0013ª | 13846 | 76922 |
a Protein was determined by the method of Fried et al. (1985) Anal Biochem 146, 271 - 276.
b [3H]Yohimbine bound.
Human blood platelets membrane (1840 mg protein) were solubilized and @2-adrenergic receptors were purified by affinity chromatog- raphy and sequential rounds of HPLC on gel permeasion column. All the data in this table are from the same experiment which is one of the three such experiments that have been done. Data are expressed as average result of a typical experiment.
ic activity of [3H]yohimbine binding in the pooled HPLC fractions were 14.1 and 13.8 nmol/mg protein for the adrenocortical carcinoma and blood platelets respectively. The theoretical specific activity for a receptor protein with a Mr of 64 000 is 15.6 nmol/mg protein assuming one ligand binding site per recep- tor molecule. From three different purification preparations, the average specific activities of the @2-adrenergic receptors were close to the theoretical value.
The pure @2-adrenergic receptors from rat adrenocortical carcinoma and human blood plate- lets membranes were radioiodinated, and subjected to electrophoresis on polyacrylamide gel in the pres- ence of SDS. Figure 3 shows an autoradiogram at the successive steps of purification. The pure protein migrated as a broad band with a Mr of 64000 (Lane 3). Ten or more protein bands were observed in the autoradiogram at the affinity-chromatography step
(Lane 1). The @2-adrenergic receptors from both sources at the first HPLC purification step were more than 60% pure. To determine if the Mr 64 000 protein which was obtained after second cycle of HPLC is indeed the @2-adrenergic receptor, the elu- ate from the affinity-chromatography of blood platelets was labeled with [3H]para azidoclonidine, an @2-adrenergic specific photoaffinity ligand. Para-azidoclonidine bound irreversibly to the recep- tor protein upon U.V. irradiation [14]. The labeled receptors were subjected to HPLC on a gel permea- tion column and the fractions were collected and counted for radioactivity. In parallel experiment un- labeled affinity eluate was subjected to the HPLC and the fractions containing [3H]yohimbine bind- ing activity were collected, concentrated and radi- oiodinated. The radioiodinated samples were sub- jected to electrophoresis on 10% SDS-poly acrylamide gels and radioautographed.
1 2 3 1 2 3
Mrx10
-3
94
67
43
30
21
PLATELET
TUMOR
64k
Y
A
20
B
[H]P-AZC. cpmx10-2
10
0
TO
20
30
40
FRACTION
50
60
70
The results show that the [3H]para-azidoclonidine labeled receptor and [125I]-iodinated protein elute at the identical position with a Mr of 64000. In the presence of non-radioactive para-aminoclonidine (1×10-4 M) no radioactivity peak was eluted, in- dicating the @2-specificity of the receptor. Similar experiments with rat adrenocortical carcinoma rev- ealed that the Mr 64000 protein contains a2-ligand binding activity [6]. These results demonstrate that the 64000-dalton platelet protein has the essential features of @2-receptor and is indistinguishable from the rat adrenocortical carcinoma membrane receptor.
To further assess the purity, the iodinated rat car-
cinoma and platelet receptors were subjected to two- dimensional polyacrylamide gel electrophoresis (Fig. 6) as described [8]. The autoradiograms show a single radioactive spot with identical protein migration in each case. Simultaneously, but in a sep- arate gel, the radioiodinated receptors from two tis- sue sources were isoelectric focused and the gels were fixed by staining and destaining. The gels were cut into 3 mm pieces and radioactivity and pH were de- termined (Fig. 5). The radioiodinated proteins fo- cused as a single radioactive peaks with an isoelectric point of 4.2. A reported [15] pI of 4.6 for the crude solubilized platelet receptor is in general agreement to our results.
In order to assess the degree of homology between rat adrenocortical carcinoma and human blood platelet @2-adrenergic receptors, the two dimension- al partial peptide mapping technique of Cleveland et al. [9] was employed. The purified receptor pro- teins were radioiodinated by the chloramine-T [7] and applied to a 10% SDS polyacrylamide gel. After electrophoresis, the gel was cut into 0.5 cm pieces and the pieces with peak radioactivity were put on another 15% SDS-polyacrylamide gel and overlaid with SDS buffer with various proteases as described in experimental procedure. The untreated @2- adrenergic receptors from blood platelets and tumor (Fig. 7, lanes 1) migrate as broad bands with Mr. centered around 64K. Partial proteolysis of the @2- adrenergic receptor from platelets by trypsin (Fig. 7, lane 2) gives rise to peptides of 43K, 27K, and 18K and a small peptide of Mr 14K; in the case of tumor (Fig. 7, lane 2) peptides of the same molecular weights were observed except that a peptide of Mr 40K is also observed. « Chymotrypsin treatment (Fig. 7, lanes 3) yielded fragments of Mr 40K, 23K, and 16K in the case of platelets and 40K, 30K, and 18K for tumor @2-adrenergic receptors. The diges- tion of @2-adrenergic receptors from tumor and platelets with S. aureus protease (Fig. 7, lanes 4) resulted in one broad band of peptide whose molecular weight centered around 20K, but the tumor resulted in an additional narrow band of Mr 40K. Variation of the exposure time for radioauto- grams revealed the reproducible fragmentation pat- tern.
The partial amino acid analysis of the @2-
6
7
12
7
PROTEIN ( cpm×10 )(-)
5
6
10
6
4
5
8
5
PH(-)
PROTEIN ( cpm
PH (0)
3
4
6
4
2
3
4
3
1
2
2
2
125
125
0
10
20
30
40
50
0
10
20
30
FRACTION
FRACTION
40
50
TUMOR
PLATELETS
adrenergic receptor from rat adrenocortical carcino- ma is shown in Table 3. In addition to those amino acids shown in the Table, there were several peaks of material that did not co-migrate with any of the stan- dard amino acids. One of these was Tris, some were probably amino sugars liberated during hydrolysis, and some may be artifacts of the digitonin. Valine and methionine could not be quantitated because
they were obscured by these unidentified peaks. The hydrolysate also had some charred material, suggest- ing that there was carbohydrate present.
Discussion
The present study indicates that our previously
| Step | Volume | Total binding activity | Total protein | Specificb activity | Overall purification |
|---|---|---|---|---|---|
| ml | pmol | mg | pmol/mg | -fold | |
| Soluble | 650 | 200 | 560 | 0.350 | 1 |
| Affinity | 20 | 88 | 0.812 | 107 | 305 |
| 1st HPLC | 4 | 46 | 0.006ª | 7666 | 21902 |
| 2nd HPLC | 2 | 17 | 0.0012ª | 14166 | 40474 |
a Protein was determined by the method of Fried et al. (1985) Anal Biochem 146, 271 - 276.
b [3H]Yohimbine bound.
Rat adrenocortical carcinoma membranes (2400 mg protein) were solubilized and @2-adrenergic receptors were purified by affinity chro- matography and sequential rounds on HPLC on gel permeasion columns. All the data in this table are from the same experiments which is one of three experiments that have been done. Data are expressed as average results of a typical experiments.
A
ISOELECTRIC FOCUSING
PH
415 DIMENSION
3.0
3.5
4:0
4:5
5.0
5.5
M.W
2nd DIMENSION
94 k
67k
SDS- PAGE
43k
30k
20k-
B
ISOELECTRIC FOCUSING
PH
DIMENSION
3.0
3.5
4:0
4:5
5.0
5.5
M.W
2nd DIMENSION
94 k
1
67k
SDS- PAGE
43k
30k
20k
described simple technique, which was utilized to isolate @2-receptors from the rat adrenocortical car- cinoma [6], is equally effective in purifying these receptors from human platelets. The complete recep-
tor purification is evidenced by one- and two- dimensional SDS-PAGE, by high performance liq- uid chromatography, by photoaffinity labeling tech- nique, by isoelectric focusing gel electrophoresis,
1 2 3 4 1 2 3
4
Mr =10~3
94
68
43
30
21
14
PLATELET
TUMOR
| Amino acid | @2-adrenergic receptor |
|---|---|
| mol % | |
| Aspartic acid | 9.6 |
| Glutamic acid | 6.2 |
| Serine | 5.8 |
| Glycine | 10.1 |
| Histidine | 4.5 |
| Arginine | 2.3 |
| Threonine | 1.8 |
| Alanine | 17.2 |
| Proline | 8.2 |
| Tyrosine | 5.5 |
| Isoleucine | 3.2 |
| Leucine | 5.0 |
| Phenylalanine | 3.5 |
| Lysine | 17.3 |
Purified @2-adrenergic receptors were prepared from rat adrenocortical carcinoma membrane as described in Table 2 and were hydrolyzed as described under ‘Experimental procedues’. Averages of two determinations are shown.
and by the stoichiometry of the ligand binding.
A ~70000-fold and 40000-fold purification was achieved from the human platelets and the tumor receptors, respectively. Both receptor proteins showed typical @2-adrenergic binding characteris- tics with specific binding activities of 13.85 nmol/mg and 14.17 nmol/mg protein. These values are close to the theoretical binding activity of 15.6 nmol/mg protein for 1 mol of the ligand bind- ing 1 mol of the receptor protein.
The purified @2-receptors from human platelets and the rat tumor were biochemically indistinguish- able: They revealed identical migration pattern by one-and two-dimensional SDS-PAGE, by high per- formance liquid chromatography and by isoelectric focusing electrophoresis. However, the autoradio- grams of partial proteolytic digests generated from radioiodinated @2-adrenergic receptors from these two sources revealed some distinct differences while sharing most peptide fragments in common. The non-homologous peptide fragments may account
for the differences in the pharmacologic properties between rodent and non-rodent @2-adrenergic receptor [16], whereas, the homologous fragments are responsible for a common ligand binding site and transmembrane signaling functions.
Although displaying some differences in peptide fragments, there are several common properties shared by the @2-adrenergic receptors of rat and hu- man platelets. Both have a subunit molecular mass of 64 kD. Isoelectric focusing studies reveal that both proteins are acidic, with a pI of 4.2. Recently two reports appeared describing the purification of @2-adrenergic receptor from porcine brain [17] and human blood platelets [18]. A third report appeared describing the cloning of human @2-adrenergic receptor [19]. Partial aminoacid composition does not indicate the acidity of the protein, however. This apparent discrepancy is most probably indicative of the posttranslational modification of the protein in- volving phosphorylation, adenylation, or glycosyla- tion. Indeed, some evidence for the apparent pres- ence of amino sugars and carbohydrate material in the receptor hydrolysate was found.
There are many tissues such as nerve terminals [20, 21], rat heart membrane [20], rat brain [21], hamster adipocytes [22] and human blood platelets [23, 24], which contain @2-adrenergic receptors, but, neither their biochemical nature nor their mechanism of sig- nal transduction is known. Limited studies have in- dicated that these receptors negatively regulate adenylate cyclase [25, 26] and a potentially exciting dual regulation of adenylate cyclase and membrane guanylate cyclase by @2-adrenergic signal transduc- tion has been found to be operative in the adrenocor- tical carcinoma cells [5]. We have recently purified the hormonally-dependent membrane guanylate cy- clase [27]. With our present demonstration that the @2-receptors from the rat adrenocortical carcinoma and human platelets are almost the same, we should be able to apply our simplified purification tech- nique to isolate a sufficient quantity of homogene- ous c2-receptors from the rat carcinoma model cell system. This not only should allow us to reconstitute the hormonally dependent receptor coupled guany- late cyclase system, but it will also help us to use the receptor for antibody production, and eventually for molecular cloning of the receptor gene.
Acknowledgement
This work was supported by the NIH grant NS- 23744 and NSF grant DCB-83-00500.
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Address for offprints: R. K. Sharma, The Cleveland Clinic Research Institute, Department of Brain and Vacular Research, 9500 Euclid Avenue, Cleveland, OH 44195, USA