PURIFICATION AND BIOCHEMICAL CHARACTERIZATION OF 2-ADRENERGIC RECEPTOR FROM THE RAT ADRENOCORTICAL CARCINOMA1
Rama Kant Jaiswal and Rameshwar K. Sharma
Laboratories of Basic Hormone Research, Department of Biochemistry, University of Tennessee Center for the Health Sciences, Memphis, Tennessee 38163
Received May 16, 1985
The az-adrenergic receptor was purified from rat adrenocortical carcinoma 494 by an affinity chromatographic step using a novel para-aminoclonidine- sepharose resin followed by a gel-permeation high performance liquid chromatographic step. The iodinated receptor protein was homogeneous as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by high performance liquid chromatography. Both SDS-PAGE and high performance liquid chromatographic studies revealed that Mr of the protein was 64,000, suggesting the monomeric nature of the receptor protein. The purified protein showed the typical binding characteristics of 2-adrenergic receptor. @ 1985 Academic Press, Inc.
Rat adrenocortical 494 tumor cells (1,2) possess a2-adrenergic recep- tors that have been extensively characterized in both their membrane-bound (3,4) and solubilized forms (4). Because of the homogeneity of the cell type and the relative ease of obtaining the tumor tissue in large quantities, we have elected to attempt purification of the “2-receptor from this tissue with the ultimate purpose of obtaining sufficient material for structure/func- tion analysis and eventually for cloning the @2-receptor gene.
Here we report a relatively simple and straightforward procedure for purifying the receptor in two steps involving affinity chromatography and gel-permeation chromatography on HPLC. The purified material showed a single-band by SDS-PAGE with a Mr of 64,000 and has the necessary specific binding characteristics to suggest that it represents the @2-adrenergic receptor.
1 - This investigation was supported by Grant CA-16091 from the National Cancer Institute and Grant PCM80-0873 from the National Science Foundation.
Abbreviations: PAC, para-aminoclonidine; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography.
0006-291X/85 $1.50
MATERIALS AND METHODS
Yohimbine hydrochloride, epinephrine and norepinephrine were purchased from Sigma; [3H]yohimbine (80.2 Ci/mmol), [3H]PAC2 (40 Ci/mmol), [3H]azido- clonidine (31 Ci/mmol) and carrier free Na125I from New England Nuclear; all of the reagents for SDS-PAGE were obtained from Bio Rad; AH-Sepharose 4B and SDS-PAGE molecular weight standards were from Pharmacia. Digitonin was purchased from Fisher Scientific Company. All other reagents were analytical grade from commercial sources.
Rat adrenocortical carcinoma 494, a spontaneously occurring tumor discovered in (1) and maintained in our laboratory (2) was used for the membrane prepara- tion and binding studies.
Preparation of Sepharose-PAC Affinity Resin - Succinic anhydride solution (1 mmol/ml gel) was added to the AH-Sepharose-4B (1 g) suspension in 4 ml water, the pH was adjusted to 6.0 by the addition of 20% NaOH and the mixture was shaken at 4º for 12 hrs. The gel was filtered, washed successively with 0.1 M NaOH (250 ml) and water (1 L), suspended in 2 ml deionized water (pH 4.5) and 2 ml of an aqueous solution of PAC (0.1 mmol/ml gel) was added. The pH was adjusted to 4.5 - 5.0. To the reaction mixture 1-ethy1-3-(3-dimethyl- amino-propyl)carbodiimide hydrochloride was added to a final concentration of 0.1 M and the mixture shaken for 72 hrs. The coupled gel was washed alterna- tively with 0.1 M acetate buffer (pH 4.0) in 0.5 M NaCl and with 0.1 M bicarbonate buffer (pH 8.0) in 0.5 M NaCl and then extensively with deionized water.
Membrane Preparation - Adrenocortical carcinoma membranes were prepared as in (4) except that 10 ug/ml soyabean trypsin inhibitor, 200 u M benzamidine hydro- chloride and 30 uM phenylmethylsulfonylfluoride were also present in buffer.
Solubilization of an-Adrenergic Receptors - To the freshly prepared pellet, 1% digitonin solution in 50 mM Tris-HC1 buffer pH 7.5 containing protease inhibitors was added. “Gentle” homogenization using a hand-turned glass- homogenizer to a homogeneous suspension was followed by “gentle” stirring at 4º for 60 minutes with a magnetic stirring bar. The suspension was centri- fuged at 105,000 x g for 30 min. The freshly prepared supernatant (solubi- lized receptors) was immediately used for the binding studies or for further purification of receptors.
Binding Assays - The binding assays were performed as described earlier (3,4) unless otherwise stated. Briefly, [3H]PAC (10 nM) and membranes (200-300 ug) were incubated at 25º for 40 min in a total volume of 500 ul incubation buffer (10 mM MgC12, 50 mM Tris-HC1 [pH 7.5]) with or without competing agonists or antagonists. Incubation was terminated by diluting the incuba- tion mixture with 5 ml buffer followed by immediate filtration through Whatman GF/C glass fiber filter which was then washed with cold 3 x 5 ml incubation buffer. In the case of solubilized membranes, the reaction was stopped by adding 4 ml cold buffer. The unbound ligand was removed by immediate filtration through Whatman GF/B filters pretreated with 0.6% polyethyleneamine (5). The filter was washed four times with 5 ml cold buffer, dried and counted for radioactivity in Omnifluor/toluene scintil- lation mixture.
HPLC - Zorbax GF-250 (DuPont), a gel-filtration column, was used to purify the receptor protein. Mobile phase containing detergent was prepared by boiling 0.2% digitonin solution in deionized water. The solution was kept at room temperature for 24 hrs and any insoluble material was removed by filtra- tion through Whatman No. 1 filter paper. The solution was then kept in a refrigerator for 3 days and again any insoluble material was removed by filtration. The solution was finally filtered through a millipore membrane
(0.45 u) just before use. This “digitonin solution” was used to prepare 50 mM Tris S04 (pH 7.0) solution containing protease inhibitors (10 u g/ml soyabean trypsin inhibitors 200 uM Benzamidine hydrochloride and 30 u M phenyl- methyl sulfonyl fluoride) and was used for the HPLC analysis of the receptors. Amicon concentrated receptor samples (0.2 ml) were injected and chromatographed at a flow rate of 1 ml/min on a IBM LC9533 Ternary Gradient liquid chromatograph. Fractions (200- u 1) were collected and assayed for binding activity and the active fractions were pooled and concentrated.
Photoaffinity Labeling - Affinity purified receptors (2 ml) were incubated with [3H]para-azidoclonidine (16 nM; 0.06 u Ci/pmol) with or without non- radioactive para-aminoclonidine (1 x 10-4M) in a aluminum wrapped beaker for 16 hours at 4°; 0.5 ml of cold buffer comprised of 50 mM Tris-HC1 containing 0.1% digitonin was added and the reaction mixture was irradiated for 15 min with ultraviolet light (Mineralite, UVSL-25). The unbound ligand was separated by passage of the sample through a PD-10 column; a 3.5 ml fraction was collected after discarding the initial 2.5 ml effluent. After concentration, the photoaffinity labeled receptors were analyzed by HPLC and SDS-PAGE for determination of the molecular weight and subunit composition.
Radioiodination of a2-Adrenergic Receptor - HPLC purified receptor after concentration by Amicon, was iodinated by chloramine T according to the method of Hunter and Greenwood (6). 125I-receptor and unreacted Na125I were separated by filtration on a Sephadex G-25 column presaturated with 1% bovine serum albumin in 0.05 M sodium phosphate buffer, pH 7.5.
Polyacrylamide Gel Electrophoresis - The iodinated- or photoaffinity labeled receptors were analyzed by SDS-PAGE using gels with the composition of 10% acrylamide containing 0.1% SDS and 0.3% bis acrylamide (7,8). The nonradio- active receptors were stained with 0.05% Coomassie brilliant blue and destained with 7.5% acetic acid.
The gel containing the iodinated sample was analyzed by autoradiography (8). The gel containing the photoaffinity labeled sample was cut into 2 mm slices, dissolved in 2.0 ml of 30% hydrogen peroxide at 65°, and mixed with 7 ml Scintiverse. Radioactivity was determined by liquid scintillation spectrometry.
Protein Determination - Protein was determined by the method of Bradford (9) using the Bio-Rad reagent and bovine serum albumin as a standard. In the samples where the protein concentration was too low to be measured by the Bradford method (9), it was approximated by the HPLC detection system (254 nm) using bovine serum albumin (monomer) as a standard.
RESULTS AND DISCUSSION
Solubilized receptors (300-350 mg protein) were applied to the top of the affinity column (4 ml of packed gel). Most of the protein (> 99.5%) did not bind to the resin and was in the “flow through” fraction. The column was extensively washed with detergent containing buffer (50 mM Tris-HC1 (pH 7.5), 2 mM MgC12, 5 mM NaC1, 0.2% digitonin and protease inhibitors as described earlier) at 4º until the absorbance at 280 nm returned to the baseline. Subsequently, the gel was eluted with the above detergent buffer containing 100 uM phentolamine; 3 ml fractions were collected. The receptor in most cases was eluted in the first two fractions. Phentolamine was removed from the receptor by either chromatography on a PD-10 column or by extensive
dialysis against the detergent buffer containing 0.02% of digitonin instead of 0.2%. Appropriate aliquots (100- to 200-u1) were assayed for @ 2-adrener- gic binding affinity using [3H]PAC. Those fractions possessing @ 2-bind- ing characteristics were pooled and concentrated 15-20-fold by Amicon ultrafiltration through a PM-30 membrane.
The affinity purified receptors showed typical binding characteristics of @2-adrenergic receptors. Fig. 1 shows the displacement of [3H]para-amino- clonidine binding from the receptor by various adrenergic agonist and antago- nists. Among the antagonists phentolamine was most potent in displacing the para-aminoclonidine (Fig. 1). There was no binding affinity for the @1-antagonist prazosin (10) or for WB4101 (11) (data not shown).
HPLC was employed as a final purification procedure. A single symmetri- cal protein peak was observed (Fig. 2A). When the HPLC-purified receptor protein was radioiodinated and subjected to SDS-PAGE, a single band corre- sponding to Mr = 64,000 was obtained (Fig. 3). These results demonstrated that Mr 64,000 @2-receptor protein is homogeneous.
ABSORBANCE (254nm) (-)
£0.02
4
[3H]PAC BOUND (p mol)
Mr=64 K
3
0.01
10-0)
2
1
[H]PAC BOUND (% MAXIMUM)
100
00-0-0-00000-0-000-0-000000-
20000
80
20
[H]P-AZC, cpm x 10-2
60
15
40
10
20
5
1
10 -11 10-9 10-7 10-5 10-3
2
0
10
20
30
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50
LIGAND. M
FRACTION NUMBER
Mr x 10-3
TOP
94
68
64
43
31
21
DYE
Since the protein concentration, as assessed by the Bradford method (9), was below the detection limit, it was approximated by HPLC using bovine serum albumin (monomer) was a standard. The specific receptor activity by this method was in excess of 3875 pmol/mg, representing 23,500-fold purification of the @2-adrenergic receptor as compared with the specific receptor activity of the solubilized membranes (Table 1). It is noteworthy that this specific activity is 5-fold lower than theoretically possible if one assumes
| Step | Activity pmo1 | Overall Yield % | Specific Activity pmo1/mg | Purification |
|---|---|---|---|---|
| Digitonin Extract | 58 | 100 | 0.165 | 1 |
| Eluate of PAC Affinity | 22 | 38 | 150 | 901 |
| HPLC | 11 | 19 | 3875 | 23500 |
Solubilized receptors (300-350 mg protein) were applied on 4.0 ml of the affinity resin. The resin was washed with 0.2% digitonin containing 5 mM NaC1, 50 mM Tris-HC1 (pH 7.5), 10 ug/ml soyabean trypsin inhibitor, benzami- dine HC1 (200 uM) and phenylmethylsulfonylfluoride (30 u M), and the receptors were eluted in the same buffer containing 0.1 mM phentolamine at 4º. Eluated fractions containing receptor activity were pooled, concentrated, and chromatographed on PD-10 columns to remove receptor-bound phentolamine. The receptor activity was assayed by [3H]para-aminoclonidine binding.
that one mole of ligand binds one mole of receptor. Probably, this deficit in the level of purification reflects the gross inaccuracy of the protein determinations in the final product. In addition, it is possible that we were unable to remove the residual bound phentolamine from the affinity purified receptors, resulting in the gross underestimation of the receptor-bound ligand.
Due to the limited size of the sample, we could not carry out the detailed binding kinetics of the homogeneous receptors. Therefore, selective experiments were performed to scrutinize the @2-characteristics of the affinity purified receptors.
The HPLC purified receptors were photoaffinity labeled with [3H]para- azidoclonidine, a specific a2-photoaffinity ligand (12), and then analyzed by high performance liquid chromatography. The radioactive peak corresponded to the Mr = 64,000 protein (Fig 2B). In the presence of nonradioactive PAC (1 x 10-4M), no radioactivity was observed in the Mr 64,000 protein, indicating the @2-specificity of the receptors. When the photoaffinity labeled receptor was subjected to SDS-PAGE, the radioactive peak again corresponded with Mr = 64,000 protein (Fig. 4); there was no radioactivity in the protein sample which was photolyzed in the presence of nonradioactive PAC (Fig 4). All these results demonstrated the authenticity of Mr 64,000 protein as @2-adrenergic receptor protein.
Purification steps of the @2-adrenergic receptor involved the introduc- tion of protease inhibitors in all the buffers. This was done to overcome any possible proteolysis of the protein.
Regan et al., recently reported (14) the partial purification of @2-adrenergic receptor protein from human platelets. Using [3H]phenoxy- benzamine as a labeling probe for the @2-receptor, they demonstrated by SDS-PAGE that the radioactivity was specifically bound to a protein having Mr = 61,000, indicating that it was the receptor protein.
(3H )P-AZIDO CLONIDINE. com
200
94K
67K
30K
150
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FRACTION NUMBER
Although many tissues such as nerve terminals (15,16), rat heart membranes (17), rat brain (18), hamster adipocytes (19), and human platelets (20-23) contain @2-adrenergic receptors, the biological function of these receptors in cellular regulation is not clearly defined. It appears that these receptors are negatively coupled (21,22) with adenylate cyclase and positively (23) with the adrenocortical carcinoma guanylate cyclase. We anticipate that the simplified purification method of @ 2-adrenergic recep- tors described herein will allow one to isolate on a large scale quantities of the pure @2-receptor that could be used for reconstitution, antibody production, and eventually molecular cloning of the receptor gene.
ACKNOWLEDGEMENTS
We thank Dr. Martin Rodbell, NIH, for the critical review of this manuscript, Anita Hardeman for the excellent typing of this manuscript, and Boehringer Ingelheim for the generous gift of para-aminoclonidine.
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