(c~ 1984 by The American Society of Biological Chemists, Inc THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 259, No. 2, Issue of January 25, pp. 1344-1350, 1984

Printed in U S A .

The Fat Cell &Adrenergic Receptor PURIFICATION AND CHARACTERIZATION OF A MAMMALIAN @,-ADRENERGIC RECEPTOR*

(Received for publication, August 8, 1983)

Ana Cubero and Craig C. MalbonS From the DeDartment of Pharmacological Sciences. Health Sciences Center, The State University of New York a t Stony Brook, Stony Brook: New York‘ 11 794

I

The B1-adrenergic receptor of rat fat cells was effec- tively solubilized with digitonin and purified by affin- ity chromatography and steric exclusion high pressure liquid chromatography (HPLC). The purification strat- egy described permits an -24,000-fold purification of the &-adrenergic receptor of fat cells with an overall recovery of -70%. Purified receptor preparations demonstrate a specific activity for (-)[3H]dihydroal- prenolol binding of 12 nmol/mg of protein. The purified receptor was shown to migrate in steric exclusion HPLC as a Mr = 67,000 protein. Sodium dodecyl sul- fate-polyacrylamide gel electrophoresis of radioiodi- nated purified receptor revealed a single, major pep- tide of M, = 67,000. The binding of (-)13H]dihydroal- prenolol to purified receptor preparations displayed stereoselectivity and affinities for antagonists similar in nature to the membrane-bound and digitonin-solu- bilized O1-adrenergic receptor. In addition to the 1M, = 67,000 component, a M, = 140,000 form of the recep- tor was identified in HPLC runs of freshly prepared, affinity chromatographed receptor preparations that had not been frozen. This larger form of the receptor yielded binding activity of M, = 67,000 on sequential HPLC runs and was shown to contain the M, = 67,000 peptide. The &-receptor from this mammalian source, composed of a single M, = 67,000 peptide, is clearly quite distinct from the purified avian &-, amphibian &, and mammalian &adrenergic receptors described by others.

Catecholamines regulate a wide variety of biological func- tions. In addition to acting as neurotransmitters, catechol- amines regulate important metabolic functions such as lipol- ysis (1). Catecholamines stimulate cyclic AMP accumulation and lipolysis of rat fat cells via adrenergic receptors classified according to the scheme of Lands et al. (2) as PI. Direct radioligand binding studies have identified high affinity, p- adrenergic receptors on isolated fat cells (3) and plasma membranes prepared from these cells (4, 5 ) with properties consistent with those of a &-adrenergic receptor.

Due to the low abundance and relative instability of p- adrenergic receptors from mammalian tissues, most studies of the molecular properties of /3-adrenergic receptors have been confined to the &receptors of amphibian (6) and avian

* This work was supported by United States Public Health Grants AM 25410 and AM 30111. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adoertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported by Career Development Award KO4 AM-00786 from the National Institutes of Health.

- - ” ~- ~-

(7) erythrocytes. Turkey erythrocytes (8), like rat fat cells (3), possess &-adrenergic receptors that stimulate adenylate cy- clase. Shorr et al. (7 ) recently purified the &-adrenergic re- ceptor from turkey erythrocytes and reported that the radio- iodinated, purified receptor was composed of two polypeptides with M, = 40,000 and 45,000. In an effort to gain information on the molecular properties of mammalian &-adrenergic re- ceptors, we have purified and characterized the P-adrenergic receptor of rat fat cells. We chose rat fat cells for these studies because these cells can be isolated as a homogeneous popula- tion of cells free of contamination by other cell types (9), possess a well characterized &adrenergic receptor (4, 5 ) , and display a relatively high number of P-adrenergic receptors/ cell (3). In this paper, we report the large-scale purification of the &-adrenergic receptor of rat fat cells. The fat cell receptor displays molecular properties quite distinct from those reported by Shorr et al. ( 7 ) for the avian P1-adrenergic receptor.

EXPERIMENTAL PROCEDURES

Preparation of Fat Cells-White fat cells were obtained by enzy- matic digestion of parametrial adipose tissue according to the proce- dure of Rodbell (9), as previously described (3). Cells were prepared from pooled adipose tissue (200-400 g) from 100-200 rats (150-175 g (body weight) fed female Sprague-Dawley SD strain rats).

Preparation of Highly Purified Membranes-Several methodologi- cal obstacles were encountered in proceeding to a large-scale purifi- cation of the @-adrenergic receptor from fat cells. Large quantities of cells (0.3-1.0 kg packed cell weight) could be efficiently prepared from only relatively small (150-175 g body weight) rats due to the increased fragility of cells from older, more obese animals. The yield of highly purified membranes was also found to be highly dependent upon the procedure employed to homogenize the cells. Cell disruption using either a Brinkmann Polytron (PT-20) or nitrogen cavitation resulted in very poor yields of purified membranes. The use of a large- bore, glass mortar fitted with a Teflon pestle with a serrated edge to homogenize the cells provided the highest yield of purified membranes (0.25 mg of protein/g of fat cells). The specific activity of @-adrenergic receptors in the purified membranes was 0.7-1.3 pmol of receptor sites/mg of membrane protein. The method developed for the large- scale preparation of purified membranes from isolated rat fat cells is described below.

Isolated fat cells (200-400 g packed cell weight) were washed twice with 3-4 volumes of Krebs-Ringer phosphate buffer containing 1% bovine serum albumin (pH 7.4) and twice again with a buffer com- posed of 10 mM Tris-HCI (pH 7.4), 0.1 M sucrose, 1 mM disodium EDTA, and the following protease inhibitors: leupeptin (5 pglml), aprotinin (5 pg/ml), and phenylmethylsulfonyl fluoride (30 pM). The washed cells were collected by low-speed centrifugation, resuspended in one volume of the same Tris buffer, and homogenized at 22 “C by six strokes of a large-bore Potter-Elvehjem homogenizer (Arthur H. Thomas Co., Philadelphia, PA) fitted with a serrated-edged Teflon pestle. The homogenizer was driven by an Eberbach Con-Torque motor operating a t maximum speed. The homogenization must be performed a t 22 “C to minimize trappage of the plasma membranes in the coalescing fat cake. This homogenate was then processed according to the method described by McKeel and Jarett (10). Puri-

1344

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Purification of Fat Cell @-Adrenergic Receptor 1345

fied membranes collected from the sucrose density gradients were diluted with 5 volumes of 10 mM Tris-HC1 (pH 7.4), 1 mM EDTA buffer, and centrifuged at 48,400 X g for 30 min at 4 "C. The pellet was washed once by resuspension in an ice-cold buffer containing 50 mM Tris-HC1 (pH 8.0), 10 mM MgC12 (0.2 ml of buffer/g of fat cells) and collected by centrifugation a t 15,000 X g for 15 min a t 4 "C. The pellet was then resuspended in 50 mM Tris-HC1 (pH 7.4), 10 mM MgClz buffer (0.1 ml of buffer/g of fat cells), assayed for 0-adrenergic receptor content (see below) and immediately solubilized with digi- tonin. A single freeze-thaw cycle was found to reduce the amount of 6-adrenergic receptor binding of these membranes by 30-50%. The extraction with digitonin was therefore performed with freshly pre- pared membranes.

Preparation and Selection of Digitonin-Digitonin was routinely prepared as a 3% solution in a 10 mM Tris-HCl (pH 7.4), 90 mM NaCl buffer, which was then heated a t 95-100 "C for 30 min, and allowed to stand for 48 h a t 25 "C. The solution was centrifuged, and the resultant supernatant filtered through a 0.22-pm cartridge filter (Millipore). The filtered solution was diluted to 1% digitonin with the Tris-NaC1 buffer just prior to use for solubilizing the membranes. For use in HPLC' buffers, a 5% solution of digitonin was prepared in water following the procedure outlined above. The filtered solution was aliquoted, lyophilized and stored at -80 "C. The lyophilized digitonin was dissolved in the mobile phase buffer and filtered again just prior to use in HPLC separations.

The ability of various lots and grades of digitonin to solubilize receptor binding activity from fat cell membranes was found to be quite variable. All of the lots of digitonin, both purified (95-98% pure, from Sigma) and highly purified (>98% pure, from Gallard-Schles- inger) grades, solubilized 35-4074 of the protein from fat cell mem- branes when the extraction was performed with 1% digitonin. The amount of P-receptor binding activity solubilized by these various lots of digitonin ranged, however, from 0-4076 with the average being 15%. Two lots of purified digitonin, (lots 62F-0135 and 122F-0135 from Sigma) were identified which permitted solubilization of 35- 40% of the receptor binding activity. For large-scale purification of @-adrenergic receptors, it would seem most profitable to screen as wide a variety of lots and grades of digitonin as can be obtained in order to maximize the yield at this crucial first step.

Solubilization Procedure-Aliquots of highly purified membranes (approximately 50 mg of protein) were centrifuged at 15,000 X g for 15 min a t 4 "C and then resuspended in 5 ml of a buffer containing 10 mM Tris-HC1 (pH 7.4), 90 mM NaC1, 5 pg/ml of leupeptin, 5 pg/ ml of aprotinin, 30 p~ phenylmethylsulfonyl fluoride, and 1% digi- tonin. The mixture was homogenized with 6 strokes of the Potter- Elvehjem homogenizer, adjusted to a final volume of 20 ml with the same buffer, and stirred for 60 min in an ice bath. Following the extraction, the mixture was centrifuged at 100,000 X g for 60 min a t 4 "C to remove insoluble, particulate material. The digitonin extract was aspirated and diluted 1:l with digitonin-free buffer to reduce the final concentration of digitonin to 0.5%.

Radioligand Binding Assay-(-)[3H]Dihydr~alpren~lol binding to fat cell membranes was performed as described previously (5). Bind- ing of (-)[3H]dihydroalprenolol and [12'I]iodocyanopindolol to digi- tonin extracts of membranes and purified receptor preparations was performed according to the method of Vauquelin et al. (11) which employs polyethylene glycol to precipitate the receptor. The concen- tration of digitonin was reduced to or below 0.05% when binding studies using [1Z51]iodocyanopindolol were performed.

Preparation of Immobilized Alprenolol Matrices-(-)Alprenolo1 was linked to sodium thiosulfate derivatized Sepharose 4B as de- scribed by Caron et al. (12). The alprenolol-agarose affinity adsor- bents described by Vauquelin et al. (11) were also synthesized.

Affinity Chromatography Procedures-The 40-ml digitonin extract of fat cell membranes was loaded onto a column (1.6 X 70 cm) (Pharmacia) packed with 140 ml of the Sepharose 4B-alprenolol matrix. The loading was performed at 25 "C and the flow rate was adjusted to 35 ml/h. The column was equilibrated a t 4 "C and washed with 2 column volumes of a buffer composed of 10 mM Tris-HC1 (pH 7.4). 100 mM NaCl, 0.05% digitonin maintained at 4 "C. The column was then re-equilibrated to 25 "C and the &adrenergic receptors eluted with a 300-ml linear gradient of (-)alprenolo1 (0-100 p ~ ) made in the wash buffer described above. Twelve-ml fractions were collected into 30-ml Nalgene bottles maintained in an ice bath. Aliquots (0.5 "

The abbreviations used are: HPLC, high pressure liquid chro- matography; DHA, dihydroalprenolol.

ml) of each fraction were applied to a column (0.9 X 30 cm) (Phar- macia) packed with 19 ml of Sephadex G-50 (fine grade) equilibrated and run in 10 mM Tris-HC1 (pH 7.4), 100 mM NaCI, 0.05% digitonin a t 4 "C. The gel filtration chromatography was employed to reduce the concentration of alprenolol so that radioligand binding analysis could be performed. The P-adrenergic receptor activity eluted in the void volume of the G-50 column (9-10 ml) and could be recovered in a final volume of 2 ml.

High Pressure Size Exclusion Chromatography-P-Adrenergic re- ceptor activity from the affinity chromatography was concentrated by Amicon ultrafiltration and further purified by HPLC on macro- porous, spherical silica supports using a Beckman/Altex 342 system following the approach of Shorr et al. (13). The samples (0.1-0.5 ml) were chromatographed on two Beckman/Altex TSK-3000 (7.5 mm X 30 cm) and one Beckman/Altex TSK-2000 (7.5 mm X 30 cm) columns, tandem-linked. The mobile phase was composed of 0.1 M KH2P04/ K2HP0, (pH 7.0), 0.2 M NaC1, containing 0.2% digitonin, and was filtered through 0.22-pm filters (Millipore) just prior to use. Chro- matography was performed at 22 "C and at a flow rate of 0.5 ml/min. One-ml fractions were collected. Molecular weight standards were chromatographed by HPLC under identical condition and identified by measurement of absorbance a t 280 nm.

Radiolabeling of Purified P-Adrenergic Receptors-@-Adrenergic re- ceptor activity from the affinity chromatography and HPLC purifi- cation was radiolabeled with Na['2'1] using the chloramine-T method (14), except that the buffer present in the sample was used in the iodination reaction. Following the labeling, the samples were treated with 10% trichloroacetic acid and the protein collected by centrifu- gation. The pellets were washed twice with ether to remove any residual digitonin and then resuspended in Laemmli sample buffer (15). The proteins were separated by electrophoresis in the presence of sodium dodecyl sulfate on homogeneous, 10% acrylamide gels according to the method of Laemmli (15). The gels were fixed, stained, destained, and dried as described previously (16). The proteins were visualized by autoradiography of the dried gels using Kodak XAR-5 film.

Protein Determinations-Protein content of particulate mem- branes and extracts was assayed by the method of Lowry et al. (17), using bovine serum albumin as a standard. The relatively small amounts of protein present in fractions from the affinity matrix or HPLC necessitated the use of alternative approaches to establish the protein content. The three assays employed were (i) fluorometric assay of protein using fluorescamine as described by Bohlen et al. (18), (ii) radioiodination of samples of known protein content using Na[12sI] and chloramine-T (14) or lactoperoxidase (19) and determi- nation of protein recovery by liquid scintillation spectrometry, and (iii) measurement of absorbance a t 280 nm. Protein determinations were established independently by the use of at least two of these assays.

Molecular Weight Determinations-The relative molecular weights of samples separated by size exclusion HPLC was established using the following protein standards @Ir): cytochrome c (lZ,OOO), adenylate kinase (32,000), enolase (67,000), lactate dehydrogenase (140,000), and glutamate dehydrogenase (290,000) supplied in an HPLC cali- bration kit (No. 30180, U. S. Biochemical Corp., Cleveland, Ohio). The relative molecular weights of samples separated by polyacryl- amide gel electrophoresis in sodium dodecyl sulfate were established using the following prestained, "C-methylated, and "'I-iodinated protein standards ( M I : cytochrome c (12,300), P-lactoglobulin (18,4001, a-chymotrypsin (25,000), ovalbumin (43,000), bovine serum albumin (68,000), phosphorylase b (92,5001, and myosin (heavy-chain, 200,000).

Materials-Rats were purchased from Taconic Farms (German- town, NY). Crude bacterial collagenase (Clostridium hystolyticum, Type I, lot 42C287) was purchased from Worthington. All radiochem- icals were purchased from New England Nuclear. Lots of digitonin were purchased from Sigma and Gallard-Schlesinger Chemical Mfg. Corp. (Carle Place, NY). (-)Alprenolo1 was a generous gift from Dr. Enar Carlsson of A. B. Hassle (Molndal, Sweden). Reagents used in the synthesis of immobilized alprenolol supports were the highest grade commerically available and were purchased from Aldrich. Pre- stained and 14C-methylated protein standards were purchased from Bethesda Research Laboratories (Gaithersburg, MD). Protein stand- ards were also purchased from Bio-Rad and then radiolabeled with NalZ5I by the chloramine-T method. All other reagents were pur- chased from Sigma.

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1346 Purification of Fat Cell @-Adrenergic Receptor

RESULTS

The pharmacology of the @-adrenergic receptors solubilized from rat fat cell membranes by 1% digitonin was characterized using (-)["IDHA and direct radioligand binding assays un- der equilibrium conditions. Agonist competition for (-)[3H] DHA binding demonstrated a rank order of potencies indic- ative of a @*-adrenergic receptor (Fig. 1A). Both @-adrenergic agonists and antagonists competed for binding to the soluble receptor with a stereoselectivity that is characteristic of cate- cholamine binding to P-adrenergic receptors (Fig. 1B). This pharmacology of the P-adrenergic receptors solubilized from fat cell membranes agrees well with that of the receptors identified in isolated, intact fat cells ( 3 ) and the membranes prepared from these cells (4, 5 ) .

Affinity chromatography on (-)alprenolo1 immobilized to Sepharose 4R was employed as the initial step in the purifi- cat,ion of the @-adrenergic receptor of fat cells. Virtually all of the protein applied to the Sepharose 4B-alprenolol matrix failed to bind to the matrix or was released from the column during the wash procedure (Table I). @-Adrenergic receptor binding activity was eluted from the matrix using a linear gradient of (-)alprenolo1 (0-100 PM). The peak of receptor activity was found to elute at about 30 pM (-)alprenolo1 (Fig. 2 A ) . The major peak of protein bound to the matrix eluted at 60-65 FM (-)alprenolo1 (Fig. 2B). The fractions of the gra- dient that contained receptor binding represented 0.2% of the membrane protein applied to the affinity matrix and nearly twice the amount of P-adrenergic receptor binding activity measured in the initial digitonin extract (Table I). This ap- parent increase in receptor binding activity may result from a reduction in the digitonin from 0.5 to 0.05% in the affinity chromatography or from the removal of some component which interferes with the binding assay. During initial studies, we also compared this Sepharose 4B-alprenolol matrix to the affinity matrices developed by Vauquelin et al. (11) and found that the recovery of @-adrenergic receptors from the Sepha- rose-alprenolol matrix described by Caron et ai. (12) to be far

2 0 1 / /.a'

log,, (COMPETING LIGAND], hi

FIG. 1. Characterization of &adrenergic receptors solubi- lized from rat fat cell membranes with digitonin. Binding assays were performed in the presence of 40 nM (-)13H]DHA and the indicated drugs for 1 h at 22 "C. Data are expressed as the mean values from three separate experiments, each assayed in triplicate.

TABLE I Purification of the fat cell &adrenergic receptor

P-Adrenergic receptors were purified from digitonin extracts of fat cell membranes by affinity chromatography on Sepharose 4B-alpren- 0101 (see Fig. 2) and sequential HPLC on steric exclusion columns (see Fig. 3). Specific binding of (-)[3H]dihydroalprenolol was mea- sured at 40 nM radioligand in assays of purified membranes and digitonin extracts and at 100 nM in those of purified receptor prepa- rations. Details of each of these procedures and those used to establish protein content are described under "Experimenal Procedures." Data shown here are representative of 12 large-scale purifications.

Specific binding of

Sample Protein (-)[3H]DHA

Purification Total Yield 2;:;

Highlypurified fat 48 32.0 100 0.7

Digitonin extract 20 10.4 33 0.5 Sepharose-alprenolol 0.042 18.9 59 447.2 860

Steric exclusion 0.030 21.6 68 706.1 1,410

Steric exclusion 0.002 24.6 77 12,000.0 24,000

cell membranes

eluant

HPLC, first pass

HPLC, second pass

superior providing quantitative as compared to 10-15% recov- eries. Thus, by using the Sepahrose 4B-alprenolol matrix and gradient elution, we routinely obtained an 800-1000-fold en- richment in binding activity and quantitative recovery (Table I).

@-Adrenergic receptor binding activity eluted from the af- finity matrix was purified further by the use of steric exclusion HPLC. Fractions eluted from the Sepharose-alprenolol were frozen immediately upon collection while a small aliquot of the fraction was assayed for receptor activity. Fractions con- taining receptor were identified, thawed, pooled, and concen- trated 10-20-fold by ultrafiltration and injected immediately. The profile of @-adrenergic receptor binding activity in frac- tions from the HPLC is displayed in Fig. 3A. Receptor activity eluted as a single, symmetrical peak and co-chromatographed with the molecular weight marker protein enolase (M, = 67,000) (Fig. 3C). The HPLC fractions containing p-adrener- gic receptor binding represented 0.15% of the protein qf the initial digitonin extract and all of the receptor activity applied to the HPLC system. A 2-3-fold increase in the specific activity of the @-adrenergic receptors was achieved routinely by a single pass on the HPLC system (Table I). Receptor activity from the first pass on the HPLC was then concen- trated 6-IO-fold by ultrafiltration and subjected to an addi- tional HPLC run. A second pass on the HPLC enriched p- adrenergic receptor binding activity 10-20-fold and again provided quantitative recovery of the receptors (Table I). A constant specific activity of about 12 nmol of binding sites/ mg of protein was achieved by the combined use of affinity chromatography in tandem with steric exclusion HPLC (Ta- ble l).

The pharmacological properties of the purified fat cell @- adrenergic receptor were assessed by direct radioligand bind- ing assays using (-)[3H]DHA. A comparison of the affinities of the membrane-bound, digitonin-solubilized, and purified @- adrenergic receptor of fat cells is shown in Table 11. The purified receptor displayed stereoselective binding of both 0- adrenergic agonist and antagonist compounds. The affinity of the purified receptor for the agonist (-)isoproterenol was observed to be significantly reduced in the purified receptor

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Purification of Fat Cell @-Adrenergic Receptor 1347

FAT CELL BETA-ADRENERGIC RECEPTORS: ELUTION FROM SEPHAROSE- ALPRENOLOL

2 0 ' 4 L + 4 d d L + 0.2 0

60 120 180 240 300

120 f

I 3- A-

80 s 0 z LL W

a -1 a

40 L

0

ELUANT (mll FIG. 2. Sepharose 4B-alprenolol chromatography of digi-

tonin solubilized fat cell &adrenergic receptor activity. A, approximately 30-35 pmol of digitonin-solubilized receptor (40 ml) was applied (35 ml/h) to a column (140 ml) of Sepharose 4B-alpren- 0101 equilibrated with 0.05% digitonin, 100 mM NaC1, 10 m M Tris- HC1 (pH 7.4, 25 "C). After washing (0 "C) with 2 column volumes of the same buffer, adsorbed receptor was specifically eluted with a gradient (300 ml) of 0-100 p~ (-)alprenolol. Fractions (12 ml) were collected and an aliquot (500 pl) of each was desalted on Sephadex G-50 to remove the eluting ligand and permit receptor assay with ['HIDHA. B, profile of protein eluted from Sepharose 4B-alprenolol as monitored by absorbance a t 280 nm.

preparation. Similar data were obtained using ['251]iodocyano- pindolol as the radioligand.

An interesting result was obtained when affinity matrix- purified receptors were subjected to steric exclusion HPLC immediately following elution from the Sepharose-alprenolol without prior freezing. Two peaks of P-adrenergic receptor binding activity were observed in the HPLC profile (Fig. 3B). A large peak of receptor activity, designatedpeak I , was found to elute with a M , = 140,000, while a smaller peak of receptor activity, designated peak 11, was found to elute with a M , = 67,000 (Fig. 3C). As shown earlier, fractions containing recep- tors purified on the Sepharose-alprenolol that were frozen (-80 "C) prior to further purification displayed only the M, = 67,000 form of the receptor on HPLC runs using steric exclusion columns (Fig. 3A). When subjected to an additional HPLC run, the M , = 140,000 form of the receptor was ob- served to yield both the M, = 140,000 and 67,000 receptor forms. About one-third of the receptor activity of the M , = 140,000 form was recovered in peak ZI (Mr = 67,000) following a second pass on the HPLC system (data not shown).

To establish the nature of the purified P-adrenergic receptor of fat cells, fractions containing receptor activity from the affinity matrix and HPLC columns were radioiodinated and separated by gel electrophoresis in the presence of sodium dodecyl sulfate. Autoradiograms identified a M , = 67,000

1 STERIC EXCLUSION HPLC

a

- I 0 - 4 0

r

I

U I 0

I - ro Y

4-

@ 0 4-

x)

1. illLyy

I1

25 30

FRACTION NUMBER 20 25 30

0 \ 9 CY

0.5 0.7 0.9

0.03

0.02 0

2 m

0.01

0 lo6

I- z W LT

2 a U

104

FRACTION NUMBER R F

FIG. 3. Steric exclusion HPLC elution profile of soluble, partially purified &adrenergic receptors of rat fat cells. Frac- tions containing receptor activity eluted from the Sepharose 4B- alprenolol matrix were concentrated to 1 ml by ultrafiltration using an Amicon concentration cell fitted with a PM-30 membrane. After concentration, the receptor (500 pl) was chromatographed on two TSK-3000 (7.5 mm X 30 cm) and one TSK-2000 (7.5 mm X 30 cm) steric exclusion columns, tandem-linked. Determination of ['HIDHA binding was as described under "Experimental Procedures." In A, 0- adrenergic receptor activity was frozen prior to HPLC on steric exclusion columns. In B, the &adrenergic receptor activity was sub- jected to HPLC immediately upon elution from the Sepharose-al- prenolol matrix. In C, the molecular weight marker proteins cyto- chrome c ( C Y ) , adenylate kinase ( A K ) , enolase, lactate dehydrogenase (LDH) , and glutamate dehydrogenase (GDH) were subjected to HPLC under conditions identical to those employed to purify the 8- adrenergic receptor.

TABLE I1 Membrane-bound, digitonin-solubilized, and purified @-adrenergic

receptors of rat fat cells: affinities for adrenergic agents The affinity constant ( K d ) for the radioligand (-)[3H]dihydroal-

prenolol was established by direct equilibrium binding studies and Scatchard (37) analysis. The Kd for competing adrenergic agents was calculated from competition studies using the analysis of Cheng and Prusoff (38). A K d of 20 nM for the radioligand was employed in calculation of the affinity constants for the purified receptor. The concentration of radioligand was 20-22 nM for the competition studies reported below. The data are derived from three separate experiments in each case.

Ligand Membrane bound" Soluble Purified _ _ _ ~ ~ _ _ X.+ hM)

(-)Dihydroalprenolol 20 20 (-)Alprenolo1 20 20 20 (-)Propranolol 27 31 50 (+)Propranolol 400 1,000 >5,000b (-)Isoproterenol 400 300 1,750 (-)Norepinephrine 6,700 5,000 (-)Epinephrine 2,000 9,000 (+)Isoproterenol >100,000 5,000 >5,000b "Calculated from Malbon et al. (5).

Less than 50% inhibition was observed at 10 p~ of the competing ligand.

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1348 Purification of Fat Cell @-Adrenergic Receptor

peptide which eluted with P-adrenergic receptor activity from the Sepharose-alprenolol (Fig. 4, fraction 1 1 ) and co-migrated with receptor activity during steric exclusion HPLC runs (Fig. 5; compare with Fig. 3, A and B). The receptor preparations purified to a specific activity of 12 nmol of binding sites/mg of protein by affinity chromatography and two rounds of HPLC were found to contain predominantly this M, = 67,000 peptide and a variable but minor amount of peptides, M, = 50,000-55,000 (Fig. 5, HPLC). In some purified preparations, only the M, = 67,000 receptor peptide was observed (data not shown). These data demonstrate that the purified fat cell PI- adrenergic receptor is composed at a minimum of a binding subunit, the M, = 67,000. The M, = 140,000 form of the receptor was also shown to contain the M, = 67,000 peptide (Fig. 5, HPLC-1, fraction 24).

DISCUSSION

Elucidating the nature of the P-adrenergic receptor has been the focus of intense research. &Adrenergic receptors have been purified from amphibian erythrocytes (6) and ca- nine lung (20). Study of P,-adrenergic receptors in contrast, has been largely confined to a description of only the turkey erythrocyte receptor (7, 11, 21-23). In order to expand our knowledge of @-receptors, we undertook a large-scale purifi- cation of a mammalian @,-adrenergic receptor, the fat cell p- receptor. Rat fat cells, a homogeneous population of cells possessing a high abundance of &-adrenergic receptors, were ideally suited for this purpose.

@-Receptors were solubilized from fat cell membranes by digitonin and then purified in the presence of this plant sterol. The soluble receptor displayed binding characteristics similar to those observed for the receptor in the native cell membrane. Receptor preparations purified by affinity chromatography and sequential HPLC runs on steric exclusion columns dem-

. . . . .

-3 M,X 10

- 200

- 92.5

- 68.0

- 43.0 - 25.7

- 18.4

- 12.3

IO I I 1 2 13 14 15 16 M

F R A C T I O N N O . FIG. 4. Autoradiogram of fractions specifically eluted from

the Sepharose 4B-alprenolol matrix following radioiodina- tion and separation by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Aliquots of fractions eluted from Sepha- rose-alprenolol by the alprenolol gradient were radioiodinated and then subjected to polyacrylamide gel electrophoresis in sodium do- decyl sulfate. The autoradiogram is a 3-day exposure. The M, = 67,000 receptor peptide is identified by an arrow.

>

HPLC.1 HPLC.1 HPLC.2

I 8

- 92.5

- 45.0

- 31.0

- 21.5

' w ."

24 26 27 20 26 21 20

F R A C T I O N N O .

FIG. 5. Autoradiogram of fractions from sequential passes on HPLC steric exclusion columns after radioiodination and separation by gel electrophoresis in sodium dodecyl sulfate. Fractions from the affinity chromatography containing receptor ac- tivity were pooled, concentrated, and subjected to HPLC on steric exclusion columns as described in the legend to Fig. 3. @-Receptor activity identified after the first pass on HPLC was pooled, lyophi- lized, labeled with NaIz5I and chloramine-T, and separated by gel electrophoresis in sodium dodecyl sulfate. The autoradiogram is a 3- day exposure. 8-Receptor activity pooled from the first pass (fractions 26,27, and 28) was concentrated to less than 500 r l and subjected to an additional HPLC run. The fractions with @-adrenergic receptor activity were lyophilized, radioiodinated, and separated by gel elec- trophoresis. The autoradiogram is a 3-day exposure. The M, = 67,000 receptor peptide is identified by an arrow.

onstrate a specific activity of 12 nmol of binding sites/mg of protein. Autoradiograms of the radioiodinated, purified 8- receptor revealed a peptide that migrated during polyacryl- amide gel electrophoresis in sodium dodecyl sulfate with M, = 67,000. The specific activity of the purified receptor (12 nmol/mg of protein) approaches the theoretical value for a homogeneous receptor with a mass of 67,000 Da that is capable of binding 1 mol of ligand/mol of receptor. The co- migration of the M, = 67,000 peptide with purified receptor binding activity through affinity chromatography and two HPLC runs provides compelling evidence that the binding subunit of the fat cell &-adrenergic receptor is indeed this M, = 67,000 peptide. The purification strategy described in this communication permits the generation of microgram quan- tities of this essentially pure receptor from intact fat cells in less than 3 days with a greater than 70% recovery.

Purification of &adrenergic receptors has been a formidable task due to the extreme, low abundance of these membrane proteins. @*-Adrenergic receptors have been purified from the frog erythrocyte (7) and canine lung (20) and the binding

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Purification of Fat Cell @-Adrenergic Receptor 1349

subunits appear to be composed of M , = 58,000 and 52,000- 53,000 peptides, respectively. Using photoaffinity labeling techniques, the binding peptides of a variety of &adrenergic receptors have been identified. In the frog erythrocyte, the receptor peptides identified via photoaffinity labeling have been reported to be Mr = 58,000 (24, 25) and 60,000-67,000 (26). The nature of the binding peptides that have been photoaffinity labeled in mammalian lung tissue appears to be even more diverse. The binding peptides of &receptors have been reported to be M , = 50,000-55,000 in canine lung (20); 59,000 in bovine lung; (27) 64,000 in hamster lung (28), and 64,000, 53,000, and 44,000 in rat lung (28). The &receptor of S49 mouse lymphoma cells has been recently shown to be composed of M , = 65,000 and 55,000 peptides (29). Although metal-dependent proteolysis has been shown to be responsible for some of the receptor peptide heterogeneity (28), the nature and role of this extensive molecular heterogeneity observed in @?-receptor peptides remains to be established.

Less progress has been achieved in the description of the nature of @,-adrenergic receptors. Purification of P1-receptors has been reported only from the turkey erythrocyte (7, 22) and now the rat fat cell (present study). The binding peptides of the @-receptor purified from turkey erythrocytes have been reported to be Mr = 170,000, 33,000, and 30,000 (22) and 45,000 and 40,000 (7). Affinity and photoaffinity labeled pep- tides of @-receptors in turkey erythrocyte membranes have been reported by a variety of groups to be M , = 41,000 and 37,000 (30), 43,500 (26), 50,000 and 40,000 (31), 45,000 and 39,000 (25). Similar studies using membranes from pigeon erythrocytes and photoaffinity reagents revealed M , = 53,500, 46,000, and 45,000 receptor peptides (26). Proteolysis does not appear to be the basis for the appearance of two receptor binding peptides photolabeled in turkey erythrocyte mem- branes (32).

Polyacrylamide gel electrophoresis of radioiodinated, puri- fied &-adrenergic receptor from rat fat cells identified a predominant, M , = 67,000 peptide which was shown previ- ously to co-migrate with the M , = 67,000 receptor peptide of S49 mouse lymphoma cell membranes that is photoaffinity labeled by either p-azido-rn-[1251]iodobenzylcarazolol or ['"I] iodoazidobenzylpindolol(33). Our ability to label the P-recep- tor of rat fat cell membranes with either of these photoaffinity reagents has been limited and quite variable, even though we can consistently label the S49 mouse lymphoma receptor peptides with either reagent (33). Photoaffinity labeling of the cardiac @,-adrenergic receptor from left ventricle prepa- rations of rat, rabbit, human, canine, and porcine heart has been reported recently (32). Two peptides of M , = 62,000 (major component) and 55,000 (minor component) were iden- tified in these cardiac membranes (32). We too observe some minor bands of M , = 50,000 in autoradiograms of the purified receptor. The appearance of these bands was quite variable, but occurred most frequently upon storage of the purified receptor. It is likely that these bands are minor proteolytic fragments generated from the M , = 67,000 form. The use of membranes freshly prepared from viable, homogeneous cells and the use of metal chelators and protease inhibitors during purification of the fat cell ,&receptor appears to avoid major artefactual proteolysis of the receptor. The P1-adrenergic re- ceptor purified from this mammalian source (i.e. the fat cell) thus appears to be composed of a single Mr = 67,000 peptide and is therefore quite distinct from purified, turkey erythro- cyte D1-receptor composed of M , = 40,000 and 45,000 peptides. More detailed structural information (i.e amino acid analysis and sequence) will be required to determine the extent of homology between these &-adrenergic receptors.

The identification of purified P-receptor activity of M, = 140,000 by HPLC on steric exclusion columns is of interest. The molecular weight of digitonin-solubilized @-adrenergic receptors as calculated from hydrodynamic parameters has been reported in the range of 130,000-160,000 (20, 34, 35). A M , = 140,000 form of the fat cell P-receptor was observed only when receptors purified by affinity chromatography were im- mediately subjected to HPLC. The M , = 140,000 form was shown to contain the M, = 67,000 receptor peptide. There are several plausible explanations for these data. The M, = 140,000 form may represent the M , = 67,000 receptor peptide in association with a digitonin micelle, which has a micellar weight of 70,000 (36). The observation that the purified re- ceptor behaving on the exclusion columns as a M , = 67,000 protein migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a M, = 67,000 peptide suggests that deter- gent binding is not extensive. Unfortunately, it is not possible at present to precisely measure the amount of detergent bound to the purified receptor to directly address this point. An alternative explanation is that the Mr = 140,000 form of receptor may represent a homodimer, heterodimer, or some oligomeric structure containing the Mr = 67,000 receptor peptide. Target size analysis of P-adrenergic receptors of mammalian lung indicates a functional molecular weight for the binding moiety in situ of about 110,000 Da (27). On the basis of these and other data, it was proposed that the p2- receptor may be a homodimer of 59,000-Da subunits (27). Our data are compatible with this proposal. More detailed bio- chemical and biophysical characterization of the M , = 140,000 form of the receptor will be required, however, to establish the validity of this hypothesis.

To summarize, we have purified the &-adrenergic receptor of rat fat cells about 24,000-fold by employing affinity chro- matography and HPLC on steric exclusion columns. The purified receptor appears to be composed of a M, = 67,000 binding subunit that is quite distinct from &-receptor purified from turkey erythrocyte and &receptors purified from avian erythrocytes and mammalian lung. The ability to generate microgram quantities of purified receptor will undoubtably facilitate more detailed biochemical analyses of this interest- ing molecule.

Acknowledgments-We wish to thank Dr. Arnold Ruoho (Depart- ment of Pharmacology, University of Wisconsin Medical School) for his generous gift of ['251]iodoazidobenzylpindolol and for his assist- ance in the use of the reagent. We would also like to thank Dr. Marc Caron (Howard Hughes Medical Institute, Duke University) for his helpful discussions on the photoaffinity labeling studies, Barbara Yoza for her assistance with some of the early characterization studies, and June Moriarty for expert secretarial assistance.

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A Cubero and C C Malbonmammalian beta 1-adrenergic receptor.

The fat cell beta-adrenergic receptor. Purification and characterization of a

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