Proc. NatL Acad. Sci. USAVol. 79, pp. 2778-2782, May 1982Biochemistry

The fi-adrenergic receptor: Rapid purification and covalentlabeling by photoaffinity crosslinking

(HPLC/catecholamines/p-aminobenzylcarazolol/adenylate cyclase-coupled receptor/affinity chromatography)

ROBERT G. L. SHORR*, SARAH L. HEALDt, PETER W. JEFFSt, THOMAS N. LAvIN*,MARK W. STROHSACKER*, ROBERT J. LEFKOWITZ*, AND MARC G. CARON**Howard Hughes Medical Institute, Departments of Medicine and Biochemistry, Duke University Medical Center and tDepartment of Chemistry,Duke University, Durham, North Carolina 27710

Communicated by James B. Wyngaarden, January 13, 1982

ABSTRACT New procedures for the rapid purification andcovalent labeling of the (3-adrenergic receptors have been devel-oped that should greatly accelerate progress in the study of thesewidely distributed adenylate cyclase-coupled receptors. Chro-matography of solubilized receptor preparations on a Sepharose-alprenolol affinity gel followed by HPLC on steric exclusion col-umns lead to rapid (2 days) and high yield ("-30%) purification ofthe receptors from frog erythrocytes. The receptor obtained bythese rapid procedures appears to be composed entirely of58,000Mr subunit(s) and to be identical to that previously purified bymuch lengthier procedures [Shorr, R. G. L., Lefkowitz, R. J. &Caron, M. G. (1981)J. BioL Chem. 256, 5820-5826]. A novel, veryhigh affinity, specific B-adrenergic antagonist, p-aminobenzyl-carazolol, has also been synthesized. It can be radioiodinated totheoretical specific radioactivity with 125I (2,200 Ci/mmol). Thisradioligand, which possesses an arylamine moiety, may then becovalently incorporated into the receptor binding subunit (58,000Mr peptide) of the frog erythrocyte membranes by the use of thebifunctional photoactive crosslinker N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (SANAH). Covalent incorporationis blocked by various drugs with a strict ,B-adrenergic specificity.This suggests that the photoaffinity crosslinking approach may beuseful for labeling a variety of small molecule and neurotrans-mitter receptors when appropriate ligands can be synthesized.

Ultimately, understanding the way in which hormones and neu-rotransmitters modulate biological processes will require thepurification and characterization of the individual componentsof the hormone and drug sensitive biological systems. Of thesecomponents the receptors, which are involved in the initial spe-cific binding interaction with the biologically active ligands,have become the focus of great attention. Because of their widedistribution and close coupling to the adenylate cyclase system,the B-adrenergic receptors have been intensely studied. Re-cently, efforts to purify the ,3-adrenergic receptors have suc-ceeded and revealed that the ,3-adrenergic receptor of frogerythrocytes is composed of58,000 Mr subunit(s) (1). However,major obstacles have continued to impede progress in this area:(i) the length of time required for the preparation of pure re-ceptors, (ii) the low overall yield of pure receptor protein, and(iii) the unavailability of high specific activity, high affinity, co-valent labels that could be used to follow the receptor throughvarious stages of purification and subsequent characterization.

In this report we describe two significant developments thatshould accelerate the pace of research directed at characteriza-tion ofthe ,B-adrenergic receptor: (i) the development ofa rapid,high yield purification procedure based on the use of affinity

chromatography and gel exclusion HPLC, and (ii) the synthesisand validation of a novel, radioiodinated high affinity /3-adren-ergic receptor antagonist that can be incorporated covalentlyinto the f3-adrenergic receptor-binding subunit by the use ofphotoactive crosslinking reagents.

MATERIALS AND METHODSMaterials. [3H]Dihydroalprenolol, "2I-labeled hydroxyben-

zylpindolol, and Na' I (carrier-free) were from New EnglandNuclear. Cyanopindolol was from G. Engel of Sandoz (Basel,Switzerland) and was iodinated according to Engel et al. (2). 4-(2,3-Epoxypropoxy)-carbazole was a gift of Boehringer Mann-heim and p-nitro-a', a-dimethylphenethylamine hydrochloridewas synthesized as described (3). Other materials were from thesame sources as previously reported (4).

Preparation of Purified Erythrocyte Membranes. Purifiedfrog erythrocyte membranes were prepared as described (4)except that soybean trypsin inhibitor (5 ,ug/ml), bacitracin (100,ug/ml), benzamidine (1 mM), EDTA (0.1 mM), and phenyl-methylsulfonyl fluoride (0.01 mM) were included. Membraneswere used immediately or stored frozen at -90°C in 250 mMsucrose/25 mM Tris-HCV2 mM MgCl2/1 mM dithiothreitol,pH 7.4, at 4°C with protease inhibitors.

Solubilization of Receptor Activity and Receptor Assay. Pu-rified erythrocyte membranes were solubilized as described(4) by using digitonin (British Drug House, Poole, England) inthe presence of the above concentrations ofprotease inhibitors.Particulate material was removed by centrifugation at 240,000X g for 40 min in a Beckman Ti-45 rotor. Soluble receptor ac-tivity was then assayed by using [3H]dihydroalprenolol at sat-urating concentrations (16 nM) or l"I-labeled cyanopindolol at250 pM. In either case, bound ligand was determined by gelfiltration as described (4).

Purification of the Solubilized fi-Adrenergic Receptor byAffinity Chromatography and HPLC.. Digitonin-solubilizedpreparations (300-600 ml) obtained from 100-200 ml ofpurifiedmembranes were chromatographed on Sepharose-alprenolol asdescribed (5). Receptor activity was eluted with approximately1-1.5 column volumes of 0.1% digitonin/100mM NaCV10mMTris-HCl, pH 7.4, containing 40 ,uM (±)-alprenolol and theprotease inhibitors. Eluates were lyophilized, redissolved in1/10 the initial volume, concentrated to 2 ml by using anAmicon concentration cell with a PM 30 membrane, and chro-matographed on two Waters I-250 and one I-125 columns tan-

Abbreviations: PAMBC, p-aminobenzylcarazolol; PNBC, 4'-nitroben-zylcarazolol; IPAMBC, 3'-iodo-4'-aminobenzylcarazolol; SANAH, N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate; PAMBIC, 4'-aminobenzyl-3-iodocarazolol; IPAMBIC, 3'-iodo-4'-aminobenzyl-3-iodocarazolol.

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The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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dem-linked (Waters Associates, Bedford, MA). Alternatively,60-cm Toyosoda TSK-3000 (one) and TSK4000 (two) columnswere used in a tandem configuration. Receptor activity was as-sayed at each step of the purification by [3H]dihydroalprenololbinding after samples were desalted on Sephadex G-50 (affinitycolumn eluate) or for each fraction ofthe HPLC run. Both Seph-adex G-50 and HPLC on steric exclusion columns separate freealprenolol from receptor-bound alprenolol. When assays arethen carried out at saturating concentrations of, [3H]di-hydroalprenolol, the small amount of receptor-bound al-prenolol dissociates and does not interfere with the binding ofradioligand (5). Protein concentrations (HPLC) were monitoredby A2 or determined by the method of Lowry et aL (6) as mod-ified by Dolly and Barnard (7) using bovine serum albumin asstandard. Purified preparations of receptor were radiolabeledwith Na'"I by using chloramine-T (8) and were further sub-jected to NaDodSO4polyacrylamide gel electrophoresis or iso-electric focusing as described (1).

Synthesis, Characterization, and Labeling of p-Aminoben-zylcarazolol (PAMBC). PAMBC {(+)-1-(carbazol4-yloxy)-3-[1-(p-aminobenzyl)-2-isobutylamine]-2-propanol} was obtained ina two-step synthesis. 4-(2,3-Epoxypropoxy)carbazole was re-acted with p-nitro-a', a-dimethylphenethylamine in ethanol at659C under N2 for 72 hr and purified by liquid column chro-matography (silica). 2'-Nitro-benzylcarazolol (18% yield) and 4'-nitrobenzylcarazolol (PNBC; 72% yield) were isolated. PNBCwas then catalytically reduced in the presence of hydrogen with5% palladium on carbon in 2% HCVCH3OH to PAMBC.

In the iodination procedure, 6 tkg ofPAMBC in 20 mM HC1(1 ,ug/,lt; 15 nmol) was added to 24 Al ofsodium acetate buffer(0.5 M; pH 5.6) at room temperature. Na'25I, 20 Al in 0.1 MNaOH (-10 mCi; 1 Ci = 3.7 X 1010 becquerels; 4.5 nmol), wasthen added, followed by 6 ,ig of chloramine-T in H20 (1 ,ug/,l; 21 nmol). After 1 min, the reaction was stopped with 8 ,ugof sodium metabisulfite (1 ,kg/,41; 42 nmol). The reaction mix-ture was applied to a TLC plate (E. Merck, Darmstadt; pre-coated TLC plates, silica gel 60 F-254, 5 X 20 cm; 0.25-mmthickness) and developed in 20% CH30H/CHC1J1 mMphenol. TLC chromatograms were checked on a radioscanner(Berthold Series 6000) or by autoradiography. 3'-Iodo-4'-ami-nobenzylcarazolol (IPAMBC) was located at RF = 0.30 (44%yield) and eluted with 5 X 0.5 ml CHClJCH3CN/triethylam-ine (85:35:5). The eluate was concentrated immediately and leftin 1 ml of 1 mM phenoVethyl acetate under N2 in the dark at-20°C. Identification of reaction products was determined fol-lowing exactly the same procedures by using nonradioactiveiodine on a 105 larger scale. Products were separated and pu-rified by HPLC (0.46 X 25 cm Analtech 10-gm silica column;eluted with 10% CH3OH/CH2Cl2; flow rate 1.0 ml/min; mon-itored at 300 nm). The structures of the isolated products wereelucidated by 250 MHz 1H NMR and mass spectroscopy. Theknown nonradioactive IPAMBC was then demonstrated to co-migrate on TLC with the product of RF = 0.30. 4'-Aminoben-zyl-3-iodocarazolol (PAMBIC; RF = 0.25; 34% yield) and 3'-iodo-4'-aminobenzyl-3-iodocarazolol (IPAMBIC; RF = 0.38;10% yield) were also identified in both the synthesis of labeledand unlabeled products. All operations with the iodinated com-pounds were performed under dim fluorescent light.

RESULTSThe previously published purification procedure for the ,B-ad-renergic receptor involved a total of three affinity chromatog-raphy steps on a Sepharose-alprenolol gel as well as an addi-tional ion exchange step on DEAE-Sepharose (1). This procedurerequired several weeks to complete and yielded 4-8% of the

total receptor~activity. Therefore, to develop a rapid, high yieldprocedure we have adapted HPLC on steric exclusion columnsto the purification of the ,3adrenergic receptor. Fig. 1 dem-onstrates the profile obtained when alprenolol eluates of theaffinity columns that are purified approximately 100-fold frommembrane preparations (1) are chromatographed by usingHPLC. An approximate 25-fold further purification of the re-ceptor binding activity is achieved by this step (see Table 1).In addition, when fractions from the receptor binding peak arepooled, radioiodinated by chloramine-T and Nal25I, and sub-jected to one or two additional HPLC runs, a single peak ofradioactivity corresponding to receptor binding activity specif-ically prelabeled in the membranes prior to digitonin solubili-zation and HPLC is obtained (Fig. 2). Subsequent NaDodSO4/polyacrylamide gel electrophoresis of the purified "lI-labeledprotein reveals a single band at 58,000 Mr (Fig. 2 Inset). Thisis identical to the size of the receptor obtained by using thepreviously described purification procedures (1). Isoelectric fo-cusing of this material also reveals a single peak of radioiodin-ated protein at a pI of 5.8, identical to the pI of "2I-labeledhydroxybenzylpindolol prelabeled receptor or receptor puri-fied by the previously described procedures (1).

Table 1 presents a summary of the purification of receptoractivity by these procedures. As shown, after complete purifi-cation (i.e., affinity chromatography and two HPLC runs) a spe-cific activity of 11,800-14,400 pmol of binding sites per mgprotein is obtained. This represents a 6,500-fold purificationfrom initial detergent extracts and an 80,000-fold purificationfrom crude frog erythrocyte membranes.

Finally, one of the most convincing criteria for the identifi-cation of the isolated peptide with the ligand binding subunitof the receptor is the fact that the purified preparations bindadrenergic ligands with a typical 82 specificity as defined bycompetition experiments that use 125I-labeled cyanopindolol(2) (data not shown).

Thus, the protein purified by these procedures and com-posed entirely of 58,000 Mr subunit(s) can be shown to containthe ,B-adrenergic receptor binding site for both agonists andantagonists. In five such purification schemes an average of30%of the receptor binding activity present in the crude digitoninextracts could be recovered in the purified receptor prepara-tions. The total time required for the entire procedure de-scribed here-that is, the solubilization, affinity chromatogra-phy, and the sequential HPLC steps-was 2 days. This

-4

0'1

.ba<¢9:.-4

0L *--9 -.-

0 10 20 30 40 50 60 70Fraction

0.12

0

0.06 ll::O

FIG. 1. HPLC elution profile of partially purified P-adrenergicreceptor preparation. The concentrated eluate of a Sepharose-alpren-olol affinity gel was chromatographed on two Waters I-250 and one I-125 tandem-linked columns (total volume, 36 ml). The flow rate was1 ml/min and the mobile phase was 0.1% digitonin/100 mM Tris sul-fate, pH 7.2, at room temperature. Fractions of 300 Aul were collectedand the receptorwas located by [3H]dihydroalprenolol ([3H]DHA) bind-ing assay (W). Protein was monitored by A280 or Lowry assay or both(6, 7).

Biochemistry: Shorr et. al.

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x

Sl 70

10 50 x

30

10

0 0

0 10 20 30 40 50 60

Fraction

FIG. 2. HPLC elution profiles of radioiodinated, purified receptorand "MI-labeled hydroxybenz lpindolol labeled 3adrenergic receptor.*, A receptor preparation (1 I-protein) eluted from a Sepharose-al-prenolol gel was chromatographed as described in legend to Fig. 1. Thepeak of receptor activity was pooled and iodinated by the chloramine-T method (8), desalted on Sephadex G-50 to remove unreacted Na'25I,and chromatographed twice on two Waters 1-250 and one I-125 columnsin-tandem. (Inset) Pooled fractions of lMI-labeled P-adrenergic recep-

torpreparationwere electrophoresed on 12%homogeneousNaDodSO4/polyacrylamide slab gel according to the method of Laemmli (9). Sam-ples were denatured by boiling 3 min with 10% NaDodSO/5% 2-mer-captoethanol/12mM Tris HCl, pH 6.5, orby incubation-for 1 hr at 550Cwith 10% NaDodSO/2 mM dithiothreitol/12 mM Tris HCl, pH 6.5.*, A frog erythte membrane preparation in which receptors werelabeled with 12I-labeled hydroxybenzylpindol (125I-HYP), solubi-lized with 1% digitonin/100 mM NaCl/10 mM Tris-HCl, pH 7.2, atroom temperature, and chromatographed as described above.

represents a major improvement over our previously describedprocedures.To facilitate further characterization studies of the /&adren-

ergic receptor we developed a technique for covalently labelingthe receptor by photoaffinity crosslinking by using a high spe-

cific radioactivity, high affinity -adrenergic antagonist. Thestructures of this novel compound, PAMBC, and of the bi-functional photocrosslinker used (SANAH) (10) are shown inFig. 3.

This radioiodinated compound binds to sites in frog eryth-rocyte membranes with a specificity characteristic of the 13-ad-renergic receptor (data not shown). Saturation binding iso-

OH CH3 125iOCH2 -CH CH2 -NH C- CH2 NH2

CH3N~~~~~~~LN

H

125I-Labeled p-aminobenzylcarazolol

(125I-PAMBC)

M? 0 NO2

rN-O-C-CH2-CH2-CH2-CH2-CH2 -NH jN30o

N-Succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (SANAH)

FIG. 3. Structures of 125I-labeled PAMBC and the bifunctionalcrosslinking reagent SANAH.

therms with the radioiodinated compound revealed a singleclass of sites with an average Yd value of 4 pM (n = 2, deter-mined at a receptor concentration of 10-20 pM in the assay)(data not shown).

Fig. 4 shows the results obtained when frog erythrocytemembranes are labeled with '25I-PAMBC in the presence andabsence of competing adrenergic ligands, followed by cross-

linking with SANAH, photolysis, and NaDodSO4polyacryl-amide gel electrophoresis. A major band ofincorporated radio-activity migrating with an approximate molecular weight of58,000 and identical to that obtained with the purified proteinis observed. When labeling is performed in the presence of anexcess of j-adrenergic antagonist or agonist or, in the absenceof photolysis or crosslinker, no covalent incorporation is ob-tained. Thus, the photoaffinity crosslinking approach specifi-cally identifies the f-adrenergic binding site. As can be seen

in Fig. 4 several "protected" bands of higher molecular weightalso appear to be labeled by this approach. However, thesebands are not present when the crosslinking is performed withpartially purified receptor (Fig. 5) or when membrane samplesare denatured in the absence of heat.

Labeling of the receptor with 125I-PAMBC and crosslinkingin a preparation of /3-adrenergic receptor partially purified bya Sepharose-alprenolol affinity chromatography step (Fig. 5,first lane) leads to incorporation of radioactivity into a 58,000Mr band. Moreover, the results shown in Fig. 5 clearly indicate

Table 1. Summary of purification of 3-adrenergic receptor of frog erythrocytes byaffinity and HPLC

Yield at SpecificActivity, each Overall activity,* Purification, fold

Step pmol step, % yield, % pmol/mg Each step Overall

Detergent extract offrog erythrocytemembranes 396 100 100 1.9 1 1

Eluate of alprenololaffinity gel 245 .62 62 136 72 72

First HPLC 206 84 52 3,416 25 1,800Second HPLC 122 59 31 11,800 3.5 6,300

Typically, membranes from 200-300 ml of frog erythrocytes were solubilized with digitonin as described(l) The affinity chromatography and HPLC steps were performed as described in text. Protease inhibitorsat concentrations stated in text were included up to the first'HPLC step. The experiment shown is rep-resentative of five such experiments. The overall purification from crude frog erythrocyte membranes(0.15 0mol/mg)is 80,00-fold.* (-)[ H]Dihydroalprenolol bound.

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-0 125i-PAMBC '125i-PAR

Top--

94.000-0

U ___.e____

_ ______w__ _ __ _

43,000

67,000-*

43,000--*

30,000--

20,000>-20,000--

14,000O-

14,000--

FIG. 4. Photoaffinity crosslinking of l'2I-labeled PAMBC in frogerythrocyte membranes. Frog erythrocyte membranes (0.05 mg/ml)in 25 mM Tris-HCl/2 mM MgCl2, pH 7.4, at 25°C were incubated for1 hr with '25I-labeled PAMBC (100 pM) in the presence and absenceof 10 pM (+)-alprenolol or 1 mM (-)-isoproterenol. After incubation,an aliquot was filtered on siliconized GF/C glass fiber filters to assessreceptor specific binding. Remaining labeled membranes were washedtwice by centrifugation with 10mM sodium phosphate buffer atpH 7.4containing 0.1% bovine serum albumin and washed once with bufferalone to remove free ligand. Each pellet was then resuspended in 500W1 of 10 mM sodium phosphate buffer; 10 ,1 of SANAH (freshly dis-solved in dimethyl sulfoxide at 5 mM) was added, followed by incu-bation for 10 min at 25°C. The crosslinking reaction was then"quenched" by addition of 10 ,ul of a 1 M solution of glycine and themixture was exposed to a 450Whigh pressure mercury lamp (Hanovia)filtered through 6 mm of Pyrex for 150 sec. The samples were held12-15 cm from the light source and the light path through the samplewas 1 cm (500 Al in 12 x 75 mm Falcon 2052 polystyrene tubes). Afterphotolysis, samples were washed twice by centrifugation with 25 mMTris-HCl (pH 6.5), dissolved by sonication in 100 1d of 10% NaDodSO/2 mM N-ethylmaleimide, 10% glycerol, 25 mM Tris-HCl (pH 6.5), andincubated at 56°C for 20 min. Mercaptoethanol was then added to afinal concentration of 5% andthe incubation was continued for at least30 min. Samples were then loaded and electrophoresed on a 7.5-15%gradient pore NaDodSO4 gel according to the method of Laemmli (9).Arrows to the left of the figure denote the relative sizes of known io-dinated protein standards: 94,000, phosphorylase b; 67,000, albumin;43,000, ovalbumin; 30,000, carbonic anhydrase; 20,000, soybean tryp-sin inhibitor; 14,000, c-lactalbumin. 12 I incorporation into proteinswas revealed by exposing the dried gel with Kodak XAR film for 48hr at -90°C. Film was developed manually according to Kodak in-structions and with Kodak solutions. The gel shown here is repre-sentative of four such experiments.

that the specificity of the covalent labeling of the 58,000 M,peptide is typically 3-adrenergic. Both /-adrenergic agonistsand antagonists protect against covalent labeling (Figs. 4 and5) ofthe receptor. Strikingly, (-) isomers of both alprenolol and

-7

FIG. 5. Specificity of the photoaffinity crosslinking of '"I-PAMBCto a partially purified P-adrenergic receptor preparation ('25I-,8AR).Eluate of a Sepharose-alprenolol affinity gel (0.5 pmol) obtained asdescribed in text was incubated (4"C) with "700 pM of 1251-PAMBCin the presence and absence of the indicated drugs for 36 hr in 100 mMNaCl/0.1% digitonin/10 mM Tris HCl, pH 7.4. After incubation eachsample was chromatographed on a Sephadex G-50 column (0.6 x 12cm) that was equilibrated in 0.1% digitonin/10mM sodium phosphatebuffer atpH 6.5, and crosslinking with SANAH was performed exactlyas described in legend to Fig. 4. After photolysis samples were ly-ophilized, taken up in 100 ud of 10% NaDodSO4/10% glycerol/2 mMN-ethylmaleimide, and heated at 56TC for 20 min. Mercaptoethanolwas added to a final concentration of 5% and the incubation was con-tinued for at least 30 min. NaDodSO4/polyacrylamidegel electropho-resis and autoradiography were as described in Fig. 4. "I-Labeled pu-rified 13-adrenergic receptor ("15,000 cpm) obtained as described inFig. 2 was included in the gel for comparison. (The purified preparationused in this experiment contained a minor contaminating band at Mr-90,000.) Arrows to the left of the figure indicate the relative sizesof known iodinated standards (as in Fig. 4). Alp, alprenolol; Iso, iso-proterenol; Phento, phentolamine; Halo, haloperidol. The dried gel wasexposed for 72 hr and the film was developed as in Fig. 4.

isoproterenol protect more completely than do their respective(+) isomers, documenting the expected stereoselective prop-erties of binding of 125I-PAMBC to the ,B-adrenergic receptor(Fig. 5). Phentolamine, an a-adrenergic antagonist, and halo-peridol, a selective dopaminergic antagonist, are ineffective inblocking the covalent labeling of the 58,000 Mr peptide by 125I-PAMBC (Fig. 5).

DISCUSSIONWe describe the development of two new procedures thatshould greatly facilitate efforts to purify and characterize thewidely distributed adenylate cyclase-coupled ,B-adrenergic re-

ceptors for catecholamines. The first is a rapid procedure for

r.

94,000--

67,OO0->

43,000-0>

30,000--

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purification of receptors by sequential affinity and HPLC. Themethod takes only 2 days rather than 2 weeks and gives yieldsof 25-30% rather than 4-8%. The resulting product is a pure58,000 Mr protein comparable to that obtained by the muchlengthier procedures previously published (1). -This proteincontains the /3-adrenergic receptor as documented by ligandbinding studies.

In addition, we have developed a new, very high affinity ra-dioiodinated /3-adrenergic antagonist, PAMBC. The extraor-dinarily high affinity of the compound (3-5 pM) and its greatspecificity enable labeling of the receptor to be performed atvery low concentrations. As a result, highly specific labeling isobtained. Because this compound contains a free arylaminefunction, the bifunctional photoactive crosslinking reagentSANAH can be used to covalently incorporate the radioligandinto the P3-adrenergic receptor. The high specific radioactivityofthe radioiodinated compound permits labeling of the receptorin membranes under native conditions. In membranes 0.5-1.5%of the bound radioligand was incorporated (Fig. 4), whereas inpartially purified receptor preparations up to 2.5% of the spe-cifically bound ligand was covalently incorporated by-the cross-linking procedure (Fig. 5). The specificity of the labeling ob-tained is indicated by the complete and stereoselective inhibitionof incorporation by. either agonists or antagonists. Moreover,the covalently labeled protein migrates on NaDodSOJpoly-acrylamide gel electrophoresis as a 58,000 Mr peptide identicalto that purified by the previously described procedures.The use of photoaffinity crosslinking with neurotransmitters

or small hormone molecules such as catecholamines representsa novel approach to the characterization of receptor bindingsites. Affinity and photoaffinity crosslinking approaches havebeen used successfully with several larger polypeptide hor-mones. '"I-Labeled insulin has been specifically incorporatedinto at least two polypeptides of Mr ;135,000 and 90,000 (11)with this approach. Recently, Johnson et al. (12) and Rebois etaL (13) have shown that " I-labeled glucagon and -'"I-labeledhuman chorionic gonadotropin can be incorporated into pep-tides of-53,000 Mr and 100,000 Mr, respectively, in their targettissues by the use of bifunctional photosensitive crosslinkers.For labeling receptors for small molecules: this approach re-quires compounds that (i) possess high affinity, (ii) possess ap-propriate reactive groups, and (iii) can be nondestructively ra-diolabeled with iodine. Because catecholamines cannot bereadily radioiodinated or crosslinked into receptors, labeling oftheir receptors dictates the need for synthesis of novel com-pounds, generally antagonists of high affinity, that possess theabove properties. The synthesis and radiolabeling of p-amino-benzylcarazolol described here fulfill these important condi-tions and provide an initial example of the application of thephotoaffinity crosslinking approach to covalent labeling of asmall molecule or neurotransmitter receptor.

Another approach that has previously been used successfullyin the characterization of the ,B-adrenergic receptor is the syn-thesis of receptor-directed reagents that possess other function-ally reactive groups. Two such compounds-a tritiated bro-moacetylated derivative of alprenolol (14) and 3H-labeled p-azidobenzylcarazolol (3)-could be shown to covalently incor-porate. into the f-&adrenergic receptor subunit when partially

purified receptor preparations were labeled. However, a draw-back of tritiated affinity ligands is their relatively low specificradioactivity. Recently, Rashidbaidi and Ruoho (15) have de-scribed the use of I"I-labeled p-azidobenzylpindolol, a pho-toactive /3adrenergic antagonist, to label the /-adrenergic re-ceptor of duck erythrocytes. A major band of 45,000 Mr and aminor-band of 48,500 Mr appeared to be specifically labeled.The different size of the subunits of the ,3adrenergic receptorlabeled in that study is probably related to the different receptorsubtypes involved (,31 in duck, 82 in frog) or to species differ-ences (or both).

The availability of a 3-adrenergic antagonist of high affinityand specificity with a free arylamine group may also be usefulin preparing electron-dense probes for the B3-adrenergic recep-tor-such as ferritin-labeled derivatives of PAMBC for use inelectron microscopic studies.

In summary, the development of these rapid, high yield,purification procedures and the use of photaffinity crosslinkingtechniques to covalently label the binding site subunit shouldgreatly facilitate the characterization and purification of the (3-adrenergic receptor from a variety of tissues. Such methods willpermit comparison of the molecular properties of the varioussubtypes of 3-adrenergic receptors and the preparation of sig-nificant quantities of receptor sufficient for raising antibodiesand for reconstitution as well as other biochemical studies.

The authors thank Drs. Bartsh and Koch of Boehringer Mannheim(Federal Republic of Germany) for their generous gift of 4-(2,3-epoxy-propoxy)carbazole and Dr. L. Jirousek and Mr. M. Zdankiwiez of NewEngland Nuclear and Mr. Donald Harris of Waters Associates for help-ful discussions and suggestions. We thank Ms. Donna Addison and Ms.Lynn Tilley for preparing the manuscript. This work was supported inpart by Grant HL 16037 from the National Institutes of Health.

1. Shorr, R. G. L., Lefkowitz, R. J. & Caron, M. G. (1981)J. BioLChem. 256, 5820-5826.

2. Engel, G., Hoyer' D*, Berthold, R. & Wagner, H. (1981) Nau-nyn-Schmiedeberg's Arch. PharmacoL 317, 277-285.

3. Lavin, T. N., Heald, S. L., Jeffs, P. W., Shorr, R. G. L., Lef-kowitz, R. J. & Caron, M. G. (1981) J. BioL Chem. 256,11944-11950.

4. Caron, M. G. & Lefkowitz, R. J. (1976) J. Biol Chem. 251,2374-2384.

5. Caron, M. G., Srinivason, Y., Pitha, J., Kociolek, K. & Lefko-witz, R. J. (1979) J. BioL Chem. 254, 2923-2927.

6. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J.(1951)J. BioL Chem. 193, 265-275.

7. Dolly, J. 0. & Barnard, E. A. (1977) Biochemistry 16, 503-510.8. Greenwood, F. C., Hunter, W. M. & Glover, J. S. (1963)

Biochem.J. 89, 114-123.9. Laemmli, V. K. (1970) Nature (London) 222, 680-686.

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