THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1984 by The American Society of Biological Chemists, Inc.

Val. 259. No. 4, Issue of February 25, pp. 2579-2587,1984 Printed in U. S. A.

Photoaffinity Labeling of Mammalian al-Adrenergic Receptors IDENTIFICATION OF THE LIGAND BINDING SUBUNIT WITH A HIGH AFFINITY RADIOIODINATED PROBE*

(Received for publication, September 12, 1983)

L. M. Fredrik Leeb-LundbergS, Kenneth E. J. Dickinson, Sarah L. Heald, Jar1 E. S. WikbergQT, Per-Otto HagenQ, John F. DeBernardisll, Marty WinnII, David L. ArendsenlJ, Robert J. Lefkowitz, and Marc G. Caron From the Howard Hughes Medical Institute, Departments of Medicine (Cardiology), Biochemistry, and Physiology, §Artherosclerosis Research Laboratories, Department of Surgery, Duke University Medical Center, Durham, North Carolina 27710 and the [[Division of Cardiovascular Research, Abbott Laboratories, Abbott Park, North Chicago, Illinois 60064

We have synthesized and characterized a novel high affinity radioiodinated al-adrenergic receptor pho- toaffinity probe, 4-amino-6,7-dimethoxy-2-[4-[5-(4- azido - 3 - [ 1251]iodophenyl)pentanoyl] - 1 - piperazinyll quinazoline. In the absence of light, this ligand binds with high affinity (KO = 130 PM) in a reversible and saturable manner to sites in rat hepatic plasma mem- branes. The binding is stereoselective and competi- tively inhibited by adrenergic agonists and antagonists with an al-adrenergic specificity. Upon photolysis, this ligand incorporates irreversibly into plasma mem- branes prepared from several mammalian tissues in- cluding rat liver, rat, guinea pig, and rabbit spleen, rabbit lung, and rabbit aorta vascular smooth muscle cells, also with typical crl-adrenergic specificity. Au- toradiograms of such membrane samples subjected to sodium dodecyl sulfate-polyacrylamide gel electropho- resis reveal a major specifically labeled polypeptide at M, = 78,000-85,000, depending on the tissue used, in addition to some lower molecular weight peptides. Pro- tease inhibitors, in particular EDTA, a metalloprotease inhibitor, dramatically increases the predominance of the M, = 78,000-85,000 polypeptide while attenuating the labeling of the lower molecular weight bands. This new high affinity radioiodinated photoaffinity probe should be of great value for the molecular characteri- zation of the al-adrenergic receptor.

Hormone receptors mediating the physiological responses to catecholamines can be divided into two main groups, a- and @-adrenergic, with two main subtypes of each (a, and a2 and /3, and p2) having been recognized. These different groups of receptors can be distinguished by their distinct affinities for various agonists and antagonists (1). The biochemical characterization of these receptors is presently in an early stage of development. At this time, more knowledge has been gained about the 8-adrenergic receptors than the a-adrenergic receptors. The p-adrenergic receptor has been isolated and purified from several tissues (2-5). In addition, the develop- ment of highly specific radioactive photoaffinity probes has

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported by United States Public Health Service Postdoctoral Training Grant T32 MH15177. 1 Supported by Grant 04X-05957 from the Swedish Medical Re-

search Council.

enabled investigators easily and rapidly to gain insight into the molecular structure of these receptors even in crude membrane preparations (6-10).

Early attempts at determining the molecular size of the a,- adrenergic receptor with [3H]phenoxybenzamine, an irrevers- ible ligand of low specific radioactivity, notoriously poor se- lectivity, and relatively low receptor affinity, have given con- flicting results (11-13). Recently, the synthesis and charac- teristics of a nonradioactive photoaffinity label with low af- finity (IC: = 85 nM) for the a,-adrenergic receptor have been reported (14). However, to date, no results have appeared regarding the use of this probe for identifying the a,-adrener- gic receptor. Accordingly, we felt it was important to establish a more secure and reproducible approach to the study of the molecular structure of this receptor. An approach similar to that used for the @-adrenergic receptor (6-10) was envisaged to elucidate the molecular structure of the a,-adrenergic re- ceptor. In the present study we describe the chemical synthe- sis and characterization of a novel a,-adrenergic receptor antagonist, lZ5I-APDQ,’ and its application as a receptor probe for identifying a,-adrenergic receptors in a variety of mam- malian tissues. Our results indicate that this probe is vastly superior to [3H]phen~xyben~amine, the only previously avail- able irreversible a,-adrenergic receptor agent, in terms of receptor affinity, selectivity, and specific activity. Thus, this receptor probe should prove highly useful for characterization of the molecular structure of the a,-adrenergic receptor.

EXPERIMENTAL PROCEDURES~

Materials I2’I-HEAT and carrier-free Na(IZ5I) were obtained from New Eng-

land Nuclear. Rats (Sprague-Dawley) were from Charles River Breed-

’ The abbreviations used are: ‘251-APDQ, 4-amino-6,7-dimethoxy- 2-[4-[5-(4-azido-3-[‘25I]iodophenyl)pentanoyl]-l-piperazinyl]quina- zoline; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel elec- trophoresis; PMSF, phenylmethylsulfonyl fluoride; BSA, bovine serum albumin; STI, soybean trypsin inhibitor; ’=I-HEAT, [‘251]iodo- 2- [~-(4-hydroxyphenyl)-ethylaminomethyl]tetralone; lZ5I-A55453, 4-amino-6,7-dimethoxy-2-[4-[5-(4-amino-3-[’25I]iodophenyl)penta- noyll-1-piperazinyl]quinazoline.

Portions of this paper (including part of “Experimental Proce- dures”) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 83M-5118, cite the authors, and include a check or money order for $2.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

2579

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

2580 al-Receptor Photoaffinity Label

ing Laboratories, Wilmington, Mass. New Zealand White rabbits were from a local supplier. Premixed SDS-PAGE standards (phos- phorylase b, M, = 94,000; BSA, M, = 67,000; ovalbumin, M, = 43,000; carbonic anhydrase, M, = 30,000; STI, M, = 20,100; a-lactalbumin, M, = 14,000) were from Pharmacia, Uppsala, Sweden, and were iodinated by the chloramine-T method of Greenwood et al. (15). Electrophoresis reagents were from Bio-Rad. SDS was from BDH Chemicals, Ltd., Poole, England. X-ray film (XAR-5) and developing solutions were from Kodak. Intensifying screens (Cronex Lightning Plus) were from DuPont. BSA (fatty acid free) was from Boehringer Mannheim. All adrenergic compounds were from sources previously described (16). Other biochemicals were from Sigma. Chemicals were usually from Aldrich and all solvents and reagents were of the highest grade available.

Methods

Synthetic and Analytical Methods for APDQ, "'1-APDQ, and Their Precursors-The procedures used in the synthesis of APDQ and '%I- APDQ are schematized in Fig. 1. The actual details of the chemical synthesis and characterization of the nonradioactive compounds is presented in the supplement to this paper (see Miniprint).

Preparation of 1251-A55453-A55453 (6 pg in 6 p1 of CHIOH, 11 nmol) was added to 1.0 M sodium acetate buffer (24 pl, pH 5.6) at room temperature. Carrier-free N a ( q (10 mCi, - 17 Ci/mg in 0.1 N NaOH, 4.5 nmol) was then added followed by chloramine-T (6 pg in 6 p1 of H20, 21 nmol). After 1 min; the reaction was halted with sodium metabisulfite (8 pg in 8 p1 of H20, 42 nmol) and the mixture made basic by the addition of 1 N NaOH (4 pl). Approximately 1-2 p1 of the reaction mixture was applied to a TLC plate (Scientific Products Silica Gel 60 F251,5 X 20 cm) and co-spotted with I-A55453. The remainder of the reaction mixture was applied across two TLC plates (5 X 20 cm). Both were developed in 15% CH30H/CH2C12, 1 mM phenol and were visualized by autoradiography. A single radio- active product which co-migrated with I-A55453 was observed from the analytical TLC (RF = 0.48, where A55453 RF = 0.38). From the preparative TLC, the band of silica containing lZ5I-A55453 was re- moved with a spatula and eluted with CHC13/CH3CN/triethylamine (8535:5, 5 X 0.5 ml). The eluate was filtered, concentrated immedi- ately, and stored in ethyl acetate, 1 mM phenol at -20 "C. The isolated lZ5I-A55453 was shown to co-migrate on TLC with I-A55453 using several solvent systems (1% CH30H/EtOAc, 0.01% triethylamine, RF = 0.10; 5% CH30H/CH2C12, 0.01% triethylamine, RF = 0.13; 15% CH30H/CH2C12, 0.01% triethylamine, RF = 0.48; 25% CH,OH/ CH,C12, 0.01% triethylamine, RF = 0.56). Due to the total separation of the radiolabeled product from A55453 and the use of carrier free Na(lZ5I), specific activity of 2175 Ci/mmol was assumed.

Conversion of 1251-A55453 to '251-APf)Q-'251-A55453 (2 mCi, 0.9 nmol) in ethyl acetate, 1 mM phenol was concentrated to dryness in a polypropylene microfuge tube and then reconstituted with 6 N acetic acid (10 pl ) . The solution was diluted with H20 (IO pl) and then treated with sodium nitrite (5 pg in 1 p1 of H20, 72 nmol). After 2 min, sodium azide (5 pg in 1 pl of H20, 77 nmol) was added. The reaction was halted after 5 min by neutralization with concentrated NH,OH (8 pl). Approximately 1 pl of the reaction mixture was applied to a TLC plate (as above) and co-spotted with I-APDQ while the remainder of the reaction mixture was applied across 4 cm of a single TLC plate. The reaction vial was rinsed with ethyl acetate and this was also applied to the preparative TLC. Both were developed and visualized as above using the same method for isolating the product. A single major product, lZ5I-APDQ, was observed from the analytical TLC and this was shown to co-migrate with I-APDQ both in the crude reaction mixture and as a purified product using several solvent systems (1% CH30H/EtOAc, 0.01% triethylamine, RF = 0.13; 5% CH30H/CH2C12, 0.01% triethylamine, RF = 0.21; 15% CH,OH/ CH2C12, 0.01% triethylamine, RF = 0.56; 25% CH30H/CH,C12, 0.01% triethylamine, RF = 0.60). In 15% CH30H/CH2C12, 0.01% triethylam- ine, APDQ has an R F = 0.52 close to that of "'1-APDQ, however, note that separation of '=I-APDQ from APDQ is not essential since initially A55453 can be completely separated from lZ5I-A55453. "'1- APDQ was stored in ethyl acetate, 1 mM phenol at -20 "C and was assumed to have a specific activity of 2175 Ci/mmol.

Membrane Preparations-Rat hepatic membranes were prepared as described previously (17) except that 10 pg/ml of STI, 200 pg/ml of bacitracin, 5 pg/ml of leupeptin, 100 p~ PMSF, and 5 mM EDTA were included to prevent proteolysis. The membranes were stored at a concentration of approximately 10 mg/ml in the protease inhibitor mixture buffered with 50 mM Tris-HCI, pH 7.5, at -80 "C until used.

Rat, guinea pig, and rabbit spleen and rabbit lung were dissected out of animals killed by cervical dislocation. The tissues were placed in ice-cold 10 mM NaHC03, 5 mM EDTA, pH 7.5, containing protease inhibitors (200 pg/ml of bacitracin, 10 pg/ml of STI, 10 pg/ml of leupeptin, 100 p M PMSF). Each tissue was diced with scissors and homogenized in 10 volumes of the above buffer by 3 X 10-s passes using a tissue disrupter (Brinkman Polytron). The homogenate was then centrifuged at 10,000 rpm (12,000 X g) for 10 min at 4 "C (Sorvall SS-34 rotor). The pellet was discarded and the supernatant passed through two layers of cheesecloth. The passthrough was centrifuged at 19,000 rpm (40,000 X g) for 30 min at 4 "C in the above rotor. The pellet was treated with 10 volumes of 0.6 M KCI, 20 mM imidazole, 5 mM EDTA, pH 7.0, and then washed twice in 50 mM Tris-HC1,5 mM EDTA, pH 7.5, including the protease inhibitors. The membranes were stored at a concentration of approximately 5 mg/ml in the above buffer at -80°C until used.

A vascular smooth muscle cell line derived from the medial layer of the thoracic aorta of a male New Zealand White rabbit was also used. Culture conditions were as described previously (18) and cells used in these studies were between the 20th and 30th passage. At the time of membrane preparation, the cells were harvested and prepared as previously described (18) except that all solutions, in addition, contained 10 pg/ml of STI, 200 pg/ml of bacitracin, 5 pg/ml of leupeptin, 100 p~ PMSF, and 1.5-5 mM EDTA. The final membranes were suspended in 225 mM NaCI, 250 mM sucrose, 50 mM Tris-HC1, 1.5 mM EDTA, pH 7.5, with the protease inhibitors to a protein concentration of 0.3-0.4 mg/ml and stored at -80 'C until used.

Radioligand Binding Assays

1251-APDQ-Prior to assay, the membranes were thawed and di- luted 10-50-fold in buffer containing 150 mM NaCI, 5 mM EDTA, 50 mM Tris-HC1, pH 7.5. Assays were performed in polypropylene tubes (75 X 12 mm) in a total volume of 0.1 ml and incubated for 90 min at 25 "C in the dark. Assays were initiated by the addition of 25 p1 of *=I-APDQ (previously diluted into 1% ethanol, 3 mM HCI) to a mixture of 50 pl of diluted membranes and 25 pl of buffer or competing agent. The final concentration of receptor was approximately 35 PM. Incubations were terminated by a 50-fold dilution with ice-cold buffer (50 mM Tris-HC1, pH 7.5) containing 0.2% BSA and rapid filtration on Whatman GF/C filters. The trapped membranes were washed with 20 ml of the above BSA containing buffer. The filters were then counted for radioactivity in a Packard Auto-Gamma 800 gamma counter at a counting efficiency of 75%.

Due to the rather hydrophobic nature of the ligand, the specific binding is significantly improved when low concentrations of BSA are included in the washing buffer used during the filtration of the binding assays. Under optimal conditions, the specific binding of lZ5I-

APDQ is approximately 60%. Under photolyzing conditions, nonspe- cific binding increases only slightly (IO-15%), while leaving the equilibrium binding parameters unaltered (results not shown).

1251-HEAT-Binding assays were performed as described in Ref. 16. Specific binding was defined as that binding displaced by 10 p~ prazosin.

Protein was determined according to the method of Lowry et al. (19). Data were analyzed by nonlinear least squares computer curve fitting as previously described (20).

Membrane-labeling Experiments-Membranes (1-2 mg of protein) were incubated in Sorvall SS-34 polycarbonate tubes in a total volume of 25 ml of buffer (150 mM NaCI, 5 mM EDTA, 50 mM Tris-HCI, pH 7.5,5-10 pM receptor concentration) including approximately 50-120 PM diluted radioactive ligand and either buffer or competing agent. The receptor concentration was kept 5-10-fold lower in the mem- brane-labeling assays compared to that in reversible binding assays to minimize any nonspecific binding. This protocol did not alter any of the ligand binding characteristics. Incubation was carried out for 60-90 min at 25 "C in the dark. The incubation mixture was then diluted to 40 ml with buffer, including 0.5% BSA, and centrifuged for a maximum of 10 min at 40,000 X g. The membranes were then homogenized in the BSA containing buffer and recentrifuged for the same length of time at 40,000 X g. The membranes were then resuspended in 15 ml of buffer lacking BSA and photolyzed in a Petri dish for 90 s, 12 cm from a Hanovia 450W medium pressure mercury arc lamp filtered with 5 mm of Pyrex glass. Photolysis can also be accomplished with a hand-held mineral light (254 nm). After photo- lysis, the membranes were pelleted at 40,000 X g for 15 min. The membranes were then solubilized for 30 min at 25 "C in SDS-PAGE

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

cq-Receptor Photoaffinity Label 2581

buffer (10% SDS, 10% glycerol, 5% P-mercaptoethanol, 25 mM Tris- HC1, pH 6.8).

SDS-Polyacrylamide Gel Electrophoresis-SDS-solubilized mem- branes (100-300 pg of membrane protein/lane) were electrophoresed on polyacrylamide slab gels according to the method of Laemmli (21) a t a constant current of 3 mA/lane. Upon completion of the electro- phoresis, the gel was dried using a Hoefer Scientific Instruments (Model SE540) slab gel drier and autoradiographed at -80 "C with Kodak XAR-5 films. No change in the electrophoretic pattern was apparent when photolabeling incubations or SDS-solubilized mem- branes had been supplemented with protease inhibitors.

RESULTS

Synthesis of APDQ and lZ5I-APDQ-The synthesis of 4- amino-6,7-dimethoxy-2-[4-[5-(4-azidophenyl)pentanoyl]-1- piperaziny1)quinazoline (henceforth referred to as APDQ) and its '251-labeled analogue is summarized in Fig. 1. The structure of APDQ was designed after that of the highly selective al- adrenergic receptor antagonist prazosin to maximize the bind- ing affinity and selectivity of the ligand for the al-adrenergic receptor.

The piperazinyl quinazoline ring system was afforded in high yield in two synthetic steps. 4-Amino-2-chloro-6,7-di- methoxyquinazoline was first substituted with N-benzylpi- perazine, and the N-benzyl protecting group was subsequently quantitatively removed by catalytic reduction. The free ali- phatic amine was then reacted with an acid chloride generated from 5-(4-nitrophenyl)pentanoic acid and PC13. The nitro- phenyl group was then catalytically reduced to an aryl amine to yield A55453.

The radioactively labeled photoaffinity ligand, lZ5I-APDQ, was readily prepared from A55453 in two synthetic steps. The aryl amine was first radioiodinated using chloramine-T and carrier-free Na(lZ5I) and then quantitatively converted to the

corresponding azide by diazotization (NaNOZ, AcOH) and reaction with sodium azide. Products were isolated by TLC, visualized by autoradiography and shown to co-migrate with fully characterized nonradioactive I-A55453 and I-APDQ in several solvent systems (see "Experimental Procedures"). Due to the use of carrier-free Na(lZ5I) and the complete purification of lZ5I-A55453 and lZ5I-APDQ, a theoretical spe- cific activity of 2175 Ci/mmol was assumed. APDQ, I-A55453, and I-APDQ were also prepared to investigate synthetic pro- cedures and to characterize structural features of these com- pounds by spectroscopy. These findings are described in the Miniprint.

Characterization of APDQ and '251-APDQ-Characteriza- tion of the piperazinyl quinazoline derivatives described in the previous section (see Miniprint) was most readily obtained from analysis of their 250 MHz 'H NMR spectra.

The quinazoline ring system yields clearly distinguishable singlet signals for the aromatic protons at 66.95 and 6.80 and the methoxyl groups at 63.98 and 3.94. The four substituted phenyl group gives the clearly recognizable symmetrical pat- tern of an AA'BB' system with the 3,5-proton signals of the 4-aminophenyl ring (66.97 and 6.62) occurring at a pro- nounced upfield shift in comparison to the shift observed from these proton signals for the 4-azidophenyl group (67.16 and 6.93). With the introduction of iodine to the 3-position of the phenyl group the AA'BB' patterns were clearly modi- fied to an AMX system with a marked downfield shift of the proton ortho to the iodine I-A55453: 67.47, 6.96, 6.67 and I- APDQ: 67.61, 7.20, 7.03). This series of compounds also yielded electron-induced mass spectral fragmentation pat- terns consistent with their proposed structures (see Mini- print). The calculated molecular weights were in excellent

Nn2

I II

APDQand 12'I-APDQ FIG. 1. Chemical synthesis of

NHz HCI NHz *HCl

P m

n2 ; pd/c

A 5 5 4 5 3 [I"I] A 5 5 4 5 3

APDO

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

al-Receptor Photoaffinity Label

I I - Total Blndrng *--m Nonspeclflc Blndtng

600 -

12511 APDQ ( p ~ )

FIG. 2. Saturation binding isotherm of '"I-APDQ in rat hepatic plasma membranes. Increasing concentrations of radioio- dinated ligand were incubated with rat hepatic plasma membranes (8 fig of protein/O.l ml of assay) and the binding assays were performed as described under "Methods." Total binding (O), nonspecific binding (H) as described by the addition of M prazosin, and specific binding (0) are shown. Each point is the average of triplicate deter- minations. The computer drawn curve represents the best fit to the experimental data (20) (KD = 162 pM, B,, = 391 fmol/mg of protein). Inset: Scatchard analysis of specific binding of "'I-APDQ (& = 267 PM, B,. = 456 fmol/mg of protein). The experiment was repeated four times with comparable results.

agreement with those determined experimentally from the high resolution analysis of the M+ ion.

Characteristics of Binding of lZ5I-APDQ to a,-Adrenergic Receptors-As shown in Fig. 2, in the absence of light lZ5I- APDQ binds in a saturable and reversible (results not shown) manner to hepatic plasma membranes with a dissociation constant of 130 f 56 PM (mean f SD; n = 4) to an apparently homogeneous population of binding sites present at a concen- tration of 385 +. 55 fmol/mg of membrane protein (mean f SD; n = 4). This number of binding sites is in good agreement with the number of binding sites identified with [3H]prazosin (22) and lZ5I-HEAT3 in the same membrane preparation.

Reversible binding of lZ5I-APDQ is competed for by several agents with a typical al-adrenergic receptor specificity and stereoselectivity. Thus, the potent @,-adrenergic antagonist prazosin competes for the binding with an affinity approxi- mately 5,000- and 20,000-fold greater than that of the non- selective a-adrenergic antagonist phentolamine and the selec- tive a2-adrenergic antagonist yohimbine, respectively (Fig. 3A). The at-adrenergic selectivity of APDQ can also be con- firmed by the low potency (Ki - 1,000 nM) of this compound in competing for [3H]yohimbine binding to the wadrenergic receptor of human platelets (results not shown). As shown in Fig. 3B, equimolar concentrations of the a-adrenergic agonists (-)epinephrine and (-)norepinephrine inhibit lZ5I-APDQ binding to a similar extent, and stereoselectivity of the inter- action of the receptor with these ligands can be shown by the approximately lo-fold lower potency of (+)epinephrine com- pared to (-)epinephrine. &Adrenergic receptor selectivity is excluded by the low potency of (-)isoproterenol (Fig. 3B). These results further ,document the a-adrenergic nature of the interaction of lZ5I-APDQ with the binding sites in rat liver plasma membranes. The rank order of potency of a series of adrenergic agonists and antagonists in competing for lZ5I-

L. M. F. Leeb-Lundberg, K. E. J. Dickinson, J. E. S. Wikberg, R. J. Lefkowitz, and M. G. Caron, unpublished observations.

APDQ binding agrees well with that for the binding of the two available radioactively labeled a,-adrenergic receptor se- lective ligands lZ5I-HEAT (Fig. 4) and [3H]prazosin (results not shown). All agents tested yielded steep, uniphasic com- petition curves such as those shown in Fig. 3, A and B, again indicating the presence of an apparently homogeneous popu- lation of binding sites. Thus, the above described results clearly document that under nonphotolyzing conditions, lZ5I- APDQ binds to al-adrenergic receptors in a competitive man- ner.

Covalent Photoincorporatwn of lZ5I-APDQ into al-Adrener- gic Receptors-To assess the usefulness of this probe in iden- tifying and labeling a,-adrenergic receptors, we investigated several mammalian tissues in which we and others have found high amounts of a,-adrenergic receptors. The tissues studied were rat liver (22), rat, guinea pig, and rabbit (23) spleen, rabbit lung, and cultured rabbit aorta vascular smooth muscle cells (18). The number of al-adrenergic receptors in these tissues as assessed by prazosin (10 pM)-displaceable lZ5I- HEAT binding, ranged from approximately 110 fmol/mg of protein in rabbit lung to approximately 400 fmol/mg of pro- tein in rat liver.

Fig. 5 shows the result obtained when rat hepatic plasma membranes are incubated with lZ5I-APDQ photolyzed and then subjected to SDS-polyacrylamide gel electrophoresis. Under these conditions (first lane, control; second lane, in the presence of 0.1 p~ prazosin) lZ5I-APDQ incorporates cova- lently into a major protected band at M, = 80,000. Moreover, two intermediate bands appear at M, = 52,000 and 42,000 and a few minor bands at M , = 32,000-16,000; all bands are either fully or partially protected by 0.1 p~ prazosin. In addition to the pattern described above, an unprotected band at M, = 59,000 is also labeled (Fig. 5 ) . This band does not appear to be receptor specific since it is not protected by prazosin or any other compound. Furthermore, this band does not mask a specifically labeled band, since, when the radioiodinated analogue of the precursor of lZ5I-APDQ, the 4-aminophenyl compound 1251-A55453, is covalently incorporated by photoaf- finity cross-linking, this band is not present on the gels, while the same bands labeled specifically by lZ51-APDQ are still ob~erved.~ Several unsuccessful attempts were made to block the incorporation of lZ5I-APDQ into this band. The efficiency of incorporation of lZ5I-APDQ into the major protected bands ranges from 10 to 15% of the ligand which was specifically bound. This efficiency agrees well with the values for the efficiency of incorporation of many azide derivatives into various enzymes and other hormone receptors.

In order to establish this photoaffinity probe as a specific a,-adrenergic receptor active site label, it is important that the incorporation into various membrane peptides occurs with a typical a,-adrenergic specificity. Such a specificity of incor- poration into all labeled peptides except the M, = 59,000 peptide from rat hepatic membranes is shown in Fig. 5. Thus, at a concentration of 0.1 p ~ , prazosin inhibits photoincorpor- ation of lZ5I-APDQ more efficiently than phentolamine, which in turn is more efficient than the az-adrenergic receptor antagonist yohimbine. The a-adrenergic nature of the labeled peptides is also documented by the fact that, at a 30 p M concentration, the effect of (-)epinephrine (-Inorepineph- rine > (+)epinephrine >> (-)isoproterenol. A possible argu- ment can be raised that the differential inhibition of incor- poration by the various adrenergic ligands may be due to scavenging of the radioiodinated arylazide by these ligands.

K. E. J. Dickinson L. M. F. Leeb-Lundberg, S. L. Heald, J. E. s. Wikberg, J. F. DeBernardis, M. Winn, D. L. Arendsen, M. G. Caron, and R. J. Lefkowitz, unpublished results.

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

al-Receptor Photoaffinity Label FIG. 3. Competition of "'I-

APDQ with adrenergic ligands in

2583

rat hepatic plasma membranes. A constant concentration of '251-APDQ (50-100 pM) was incubated with increas- ing concentrations of adrenergic antag- onists ( A ) and agonists ( B ) as shown and the assays performed as described under "Methods." '251-APDQ Bound refers to specific binding of '=I-APDQ to plasma membranes (8 pg of protein/O.l ml of assay) as determined in the presence of

M prazosin. 100% Specific binding represents approximately 120 fmol/mg of protein of '=I-APDQ binding. The lines through the experimental points represent the computer generated best fit to the data (20) of a single represent- ative experiment performed twice.

a x- 7 - - Slope = 1.003

' 6-

(-) Phenylephrmc 0

4 I I I I I

IO 9 8 7 6 5 4 3 -log (Ki Drug) M

FIG. 4. Correlation plot of log K, for various adrenergic agents on the binding of I2'I-APDQ and I2'I-HEAT in rat hepatic plasma membranes. Binding assays using hepatic mem- branes were performed as described in the legend to Fig. 3 and under "Methods." Ordinate: K, values using 'Z51-APDQ. Abscissa: Ki values using '=I-HEAT. IC, values were determined by fitting individual competition curves to a nonlinear least squares computer program (20), and Ki values were calculated using the Cheng and Prusoff equation (28). Values represent the mean of a t least two experiments performed in triplicate and the mean S.E. was < 10%.

However, the differential effect of the stereoisomers of epi- nephrine strongly refutes such a possibility. Similarly, the intensity of the nonspecifically labeled peptide a t M , = 59,000 did not change in any of the assays within a specific experi- ment.

Spleen, primarily a smooth muscle containing tissue, from several mammalian species was found to have a reasonable number (2 150 fmol/mg of protein) of al-adrenergic receptors as assessed by "'I-HEAT binding and by specific photoincor- poration of 1251-APDQ. Fig. 6 (first lane, control), shows the electrophoretic pattern obtained when photolabeled rat spleen membrane proteins are subjected to SDS-PAGE. A major labeled broad band appears a t MI = 79,000, with two minor labeled bands at MI = 42,000 and 35,000. In Fig. 6, four left lanes, al-adrenergic specificity of incorporation into the M , = 79,000 peptide is demonstrated by the effectiveness of both prazosin (0.1 pM) and (-)epinephrine (100 pM) in blocking incorporation and by the reduced effectiveness of yohimbine (0.1 p ~ ) . A similar order of efficacy is seen for the inhibition of incorporation into the peptides a t M, = 42,000 and 35,000.

&-"A I-lNE - I-JEpa * - 4 l+:Ep,

80 - - I-JIIO

60 -

40 -

20 -

I I I I I I I

O " 8 7 6 5 4 3 2 -LOG,, [ADRENERGIC AGENT] M

RAT LIVER

MW

67K 94K "I - 43K 4

30K - 20K -.I

""

FIG. 5. Photoaffinity labeling and pharmacological speci- ficity of incorporation of I2'I-APDQ into rat hepatic plasma membranes. Hepatic membranes were incubated with '*'I-APDQ (50-100 PM) alone or with prazosin, 0.1 pM (Pra); phentolamine, 0.1 p~ (Phe); yohimbine, 0.1 pM (Yoh); (-)epinephrine, 30 pM ((-)Epi); (+)epinephrine, 30 p M ((+)Epi); (-)norepinephrine, 30 pM ( ( - )NE; or (-)isoproterenol, 30 PM ((-)lso), washed, photolyzed, and electro- phoresed on 10% gels as desribed under "Methods." The results shown are identical with two other experiments. Arrows represent the mo- lecular weight ( M W ) of known proteins used as standards and shown x 1000 ( K ) .

However, unlike the M, = 79,000 band, the inhibition is only partial. This partial inhibition appears to be a common prop- erty of a t least the MI = 42,000 peptide from some of the tissues investigated. Fig. 6 (fifth lane, control), shows the electrophoretic pattern of photolabeled guinea pig spleen membranes. Three peptides of M , = 79,000,53,000, and 42,000 become radiolabeled. As shown in Fig. 6 (four right lanes) an al-adrenergic specificity of incorporation can only be ascribed to the M , = 79,000 peptide, as assessed by the same criteria as described above for photolabeling of rat spleen membranes.

A similar pattern of labeling is observed when spleen mem- branes from rabbit, a species evolutionarily different from the more closely related rat and guinea pig, is assayed for pho- toincorporation of '*'I-APDQ. Fig. 7 (first lane, control),

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

2584 q-Receptor Photoaffinity Label RAT SPLEEN G. P I G S P L E E N

"W

94K -S

67K -J

43K 4

MW A

94K +

67K - 43K -

94K - 67K - 43K -

30K - 30K - 30K -I

20K 4

14Kd

' p M " p M '

FIG. 6. Photoaffinity labeling and pharmacological speci- ficity of incorporation of '2"I-APDQ into rat spleen and guinea pig spleen membranes. Rat spleen and guinea pig spleen mem- branes were incubated with IZ5I-APDQ (50-100 PM) in the absence or presence of the agents indicated, washed, photolyzed, and electropho- resed on 10% gels as described under "Methods." The results are identical with two other experiments. Abbreviations are as described in the legend to Fig. 5.

RABBIT SPLEEN RABBIT LUNG

- MW

94K - 67K - 43K - 30K - 20K-

FIG. 7. Photoaffinity labeling and pharmacological speci- ficity of incorporation of 12"I-APDQ into rabbit spleen and rabbit lung membranes. Rabbit spleen and rabbit lung membranes were incubated with '*'I-APDQ (50-100 PM) in the absence and presence of the agents indicated, washed, photolyzed, and electropho- resed on 10% gels as described under "Methods." The results are identical with two other experiments. Abbreviations are as described in the legend to Fig. 5.

shows that in these membranes, incorporation occurs primar- ily into two peptides of M , = 78,000 and 39,000. Using the same pharmacological criteria as described above, al-adrener- gic specificity can only be ascribed to the M , = 78,000 band (Fig. 7, left four lanes). The same pattern of labeling as in

20K -c 20K - ,T T 7.7- 7-,T

a0,, , , ;.e ;.e 4 90 $0

$ < < < < , o p J 3 L$ r' +o ,T,T T T T,T

I-100 p M 4 & .2. $2 FIG. 8. Photoaffinity labeling and pharmacological speci-

ficity of incorporation of 12"I-APDQ into rabbit aorta vascular smooth muscle cell membranes. Vascular smooth muscle cell membranes were incubated with '=I-APDQ (100-200 PM) in the absence or presence of adrenergic agonists (100 PM) ( A ) and antago- nist (0.1 PM) (E), washed, photolyzed, and electrophoresed on 10% gels as described under "Methods." The results are identical with two other experiments. Abbreviations are: (-)norepinephrine ( ( - )Ne) , (+)norepinephrine ( (+ )Ne) , (-)epinephrine ( ( - )Epi ) , (-)isoproter- enol ((-)Iso), yohimbine (yoh), prazosin (Pra), phentolamine (Phent), and as described in the legend to Fig. 5.

l"O.1 p M 4

TABLE I Molecular weight estimates of al-adrenergic receptor peptides as

determined by SDS-PAGE All entries refer to the present study with '=I-APDQ unless oth-

erwise indicated.

Species Tissue Peptides identified

Affinitv labeline Purification Rat Cerebral cortex 79,000 (25)

Liver 80,000,52,000," 59,000b (27) 42,000"

16,000"

(12)'

32,000," 20,000,"

80,000,58,000

43,000' (13)

35,000" Spleen 79,000,42,000,"

Guinea pig Spleen 79,000 Rabbit Aorta vascular 85,000

smooth muscle cell (culture)

Cerebral cortex 78,O0Od Lung 79,000 Spleen 78,000, 39,000"

a Denotes minor components. Determined with radioiodinated protein.

e Determined with [3H]phenoxybenzamine. Unpublished observation.

rabbit spleen is also seen in rabbit lung (Fig. 7, right four lanes), where a M , = 79,000 peptide appears to be the only peptide of al-adrenergic receptor nature.

Finally, photoincorporation of '*'I-APDQ was assayed in membrane preparations of vascular smooth muscle cells orig- inating from rabbit aorta. In these membranes, and as shown in Fig. 8, A and B, the photoaffinity probe labels almost exclusively a peptide of M , = 85,000, with minor peptides of M , = 65,000 and 42,000 occasionally labeled. In agreement with all other tissues studied, incorporation of the label occurs with a a,-adrenergic specificity. Thus, at a concentration of 10 PM (Fig. 8A), inhibition of labeling of the M , = 85,000

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

cul-Receptor Photoaffinity Label 2585

peptide by adrenergic agonists follows a rank order of efficacy: (-)norepinephrine (-)epinephrine > (+)norepinephrine > (-)isoproterenol. Similarly, Fig. 8B shows that the inhibitory efficacy of several a-adrenergic antagonists, a t a concentra- tion of 0.1 VM, followed the order: HEAT prazosin > phentolamine > yohimbine.

The above results show that upon photolysis, I2‘II-APDQ irreversibly incorporates into specific a,-adrenergic receptor peptides in several mammalian species. Table I lists the molecular sizes of @,-adrenergic receptor peptides labeled by ”‘1-APDQ in various tissues. Also included in this list are the sizes of a,-adrenergic receptor peptides identified by other investigators either by purification (27) or by [‘Hlphenoxy- benzamine (12, 13).

Endogenous Proteolysis: Cause of Apparent Heterogeneity Revealed by Photoaffinity Labeling-Substantial heterogene- ity of a,-adrenergic receptor labeling is apparent in tissues like rat liver. The presence of lower molecular weight al- adrenergic receptor-like peptides may result from the presence of multiple types of receptors, multiple subunits of a single receptor, or simply be due to proteolytic degradation of the MI = 80,000 peptide. In order to investigate the issue of whether the lower molecular weight peptides may have origi- nated via proteolytic degradation from the peptide a t M, = 80,000, rat hepatic plasma membranes prepared either in the presence or absence of a series of protease inhibitors were photolabeled with ”‘1-APDQ (see “Methods”). As indicated in Fig. 9, protease inhibitors have substantial effects on the SDS-polyacrylamide gel electrophoresis pattern of photola- beled membrane peptides. Lanes 1 and 2 show the pattern of labeled samples obtained with rat hepatic membranes pre- pared in the absence of protease inhibitors. Based on the total radioactivity incorporated into “protectable” peptides (as- sessed by densitometric scanning of autoradiograms) the ratio of the MI = 80,000:52,000:42,000:32,000-16,000 peptides was

m m MW -

94K- .

67 K“-l

30K-

20K“

I 2 3 4 5 6 FIG. 9. Effect of protease inhibitors on the photoaffinity

labeling pattern of ‘2”I-APDQ in rat hepatic plasma mem- branes. Rat hepatic membranes were prepared as described under “Methods” either in the absence (lanes 1 and 2) or in the presence (lanes 3 and 4 ) of 10 pg/ml of STI, 200 pg/ml of bacitracin, 5 pg/ml of leupeptin, 100 p~ PMSF, and 5 mM EDTA or (lanes 5 and 6 ) 10 pg/ml of STI, 200 pg/ml of bacitracin, 5 pg/ml of leupeptin, 100 p~ PMSF. Membranes were incubated with [‘“IIJAPDQ (50-100 PM) alone (lanes I. 3, and 5) and in the presence of lo-’ M prazosin (lanes 2, 4, and 6) , washed, photolyzed, and electrophoresed as described under “Methods.” Occasionally, a faint specifically labeled band appeared a t M, > 90,000. This band is not present on the gel presented in Fig. 5 and appears to be the result of slight variations in sample preparation. Identical results were obtained in two other experiments. Abbreviations are as described in the legend to Fig. 5.

- MW

94K - 67K -

43K - 30K - 20K -

1 2 3 4 FIG. 10. Effect of protease inhibitors on the photoaffinity

labeling pattern of ‘*“I-APDQ in rabbit aorta vascular smooth muscle cell membranes. Vascular smooth muscle cell membranes were prepared as described under “Methods” either in the presence (lanes I and 2) or in the absence (lanes 3 and 4 ) of the protease inhibitors used in Fig. 9, lanes 3 and 4. Membranes were incubated with [Iz5I]APDQ ( 5 0 - 1 0 0 ~ ~ ) alone (lanes 1 and3) and in the presence of 100 p~ phentolamine (lanes 2 and 4) , washed, photolyzed, and electrophoresed on 24-cm 8% gels as described under “Methods.” In order to achieve better separation, the dye front was run off the end of the gel and the electrophoresis was then continued for 1 h. The results are identical with two other experiments. Abbreviations are as described in the legend to Fig. 5.

12:69:15:3. On the other hand, as shown in lanes 3 and 4, when the membranes were prepared in the presence of pro- tease inhibitors (as described under “Experimental Proce- dures”), the ratio changed to74:912:3. The predominance of the M , = 80,000 peptide under these conditions strongly suggests proteolytic conversion of the M, = 80,000 to these lower molecular weight peptides. Lanes 5 and 6 show that when EDTA is excluded from the mixture of protease inhib- itors, the electrophoretic pattern appears identical with that in the absence of any protease inhibitors. These results sug- gest that metalloproteases in the hepatic plasma membrane preparation may be responsible for the appearance of these lower M, peptides. In the absence of EDTA, specifically labeled material could sometimes be observed aggregated on top of the running gel. However, this apparent aggregation which is sensitive to EDTA and therefore appears to be dependent on divalent cations cannot explain the disappear- ance of the M, = 80,000 peptide since the M, = 52,000 peptide was proportionally increased in this sample. The aggregation was not seen in any of the other tissues investigated (see below).

The issue of a,-adrenergic receptor proteolysis was also investigated in vascular smooth muscle cells. Fig. 10 shows that in the presence of protease inhibitors (lanes 1 and Z), the photolabeled receptor peptide migrates on SDS-PAGE vir-

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

2586 al-Receptor Photoaffinity Label

tually as a single broad band centered at M, = 85,000. In the absence of protease inhibitors (lams 3 and 4), on the other hand, labeled material migrates as an even broader band centered around M, = 65,000, suggesting the proteolytic gen- eration of a series of peptide fragments of progressively smaller size. At first, this pattern appears quite different from that obtained with rat hepatic membranes (see Fig. 9). How- ever, careful examination of the gel pattern reveals that the smallest peptides generated are of molecular weights (Mr = 52,000-55,000) similar to that for the major proteolytic prod- uct in rat liver. In addition, minor amounts of a M , = 65,000 peptide can frequently be seen also in rat hepatic membranes as well as in other tissues.

DISCUSSION

Our results document the effectiveness of 9 - A P D Q for photoaffinity labeling mammalian a,-adrenergic receptor pep- tides. As noted, lz5T-APDQ has all the desirable features of a photoaffinity ligand, including high specific radioactivity and high affinity and selectivity for the receptor. Thus, this new probe should greatly facilitate work on the structure of the a,-adrenergic receptor.

These results also serve to clarify much of the current controversy which has arisen from previous attempts to label irreversibly the subunits of the a,-adrenergic receptor. The only ligand available to date has been [3H]phenoxybenzamine. As mentioned earlier, this ligand lacks many desirable fea- tures necessary for successful labeling of the receptor in crude membrane preparations from various tissues. [3H]Phenoxy- benzamine not only has relatively low affinity for the a,- adrenergic receptor, but its notoriously low specificity allows it to interact with several other hormone and neurotransmit- ter receptors. Thus, unless membrane preparations containing very high concentrations of a,-adrenergic receptors are used, this probe may be of limited use.

Recently, our laboratory has developed a highly specific arylazide photoaffinity probe for &adrenergic receptors. This photoaffinity probe, p-azido-rn-['251]iodobenzylcarazolol, was modeled after the potent @-adrenergic antagonist carazolol (24). Introduction of an arylamine moiety into the carazolol molecule not only preserves both affinity and specificity for @-adrenergic receptors, but in addition also allows the com- pound to be radioiodinated (24). Furthermore, after conver- sion of the arylamine to the arylazide, the compound can be irreversibly photoincorporated into the &adrenergic receptor (8-10, 24). A similar approach was taken to the design of a highly specific photoaffinity probe for the a,-adrenergic re- ceptor. The design of lZ5I-APDQ is based on the structure of the highly selective al-adrenergic antagonist prazosin. Re- placement of the furan ring in prazosin with a 4-amino-3- ['251]iodophenylbutanyl moiety yields the immediate precur- sor ('251-A55453) of '251-APDQ. '"I-A55453 interacts with rat hepatic membranes with characteristics similar to those of [3H]prazosin, including high affinity and high specific bind- ing! However, lZ5I-A55453 has the definite advantage of higher specific radioactivity (2175 Ci/mmol). Conversion of the arylamine in '251-A55453 to an arylazide yields a com- pound (lZ5I-APDQ) which retains a,-adrenergic specificity. Thus, under nonphotolyzing conditions, lZ5I-APDQ binds in a reversible manner to rat hepatic plasma membranes with a maximal number of binding sites of 385 fmol/mg of protein and with a dissociation constant of 130 PM; the values of both equilibrium binding parameters are almost identical with those of [3H]prazosin binding in the same membrane prepa- ration (22). Similarly, lZ5I-APDQ binding is competed for by

several adrenergic agonists and antagonists with a typical a,- adrenergic specificity.

The primary advantage of lZ5I-APDQ over all other selective @,-adrenergic receptor ligands, however, is that under photo- lyzing conditions, this probe can be irreversibly incorporated into the receptor with high efficiency. lZ5I-APDQ covalently incorporates into a,-adrenergic receptor peptides in several mammalian tissues. The largest and major al-adrenergic re- ceptor peptide labeled in rat liver membranes under condi- tions where proteolysis is attenuated is that at M, = 80,000. A receptor peptide of similar size is also the primary target for lZ51-APDQ in all other tissues investigated. In a recent preliminary paper, we reported that lZ5I-APDQ also incorpo- rates into a peptide of similar size (MI = 79,000) in rat cerebral cortex membranes (25). Minor differences in the size of this peptide in the various tissues may be due to differences in carbohydrate composition. However, it is readily apparent from our results that the native a,-adrenergic receptor binding site resides on a peptide subunit of M , = 78,000-85,000 in many mammalian tissues. This peptide corresponds to the larger of the two peptides labeled by [3H]phenoxybenzamine in rat liver reported by Kunos et al. (12). Unlike lZ5I-APDQ, the low specificity and specific radioactivity of [3H]phenoxy- benzamine has not allowed this ligand to be used for identi- fication of a,-adrenergic receptor peptides in any other tissue.

A lower molecular weight less prominent peptide of M , = 52,000-55,000 with a,-adrenergic characteristics is always present in rat liver. Photoaffinity labeling of rat liver mem- branes prepared in the presence and absence of protease inhibitors clearly reveals that this peptide appears to be generated from the M , = 80,000 peptide by proteolytic diges- tion. The same phenomenon most probably accounts also for the minor labeled peptides at M , = 65,000 and < 52,000. EDTA is the most efficient protease inhibitor tested, suggest- ing that a metalloprotease is responsible for the proteolytic activity. Interestingly, this situation appears to be similar to that recently discovered for mammalian p-adrenergic recep- tors in several tissues (26). The presence of small amounts of the MI = 52,000-55,000 peptide, even in the presence of EDTA, further underscores the intense proteolytic activity in rat liver, and may correlate with the overall metabolic activity of the tissue studied. The a,-adrenergic receptor from the apparently homogeneous cell population of rabbit aorta smooth muscle is also prone to proteolysis by endogenous metalloproteases. Interestingly, the proteolytic products gen- erated from the M , = 85,000 peptide in this tissue appear on gels as a very broad band centered around M, = 65,000. A peptide of the same size is often present also in rat hepatic membranes. In vascular smooth muscle, the M, = 65,000 peptide appears to be the major proteolytic product. In rat liver, on the other hand, the M , = 65,000 peptide appears to be either only a minor proteolytic product or an intermediate in the processing of the M, = 80,000 peptide into the M, = 52,000-55,000 peptide. These findings suggest differences be- tween the two receptors in the access of peptide sequences susceptible to cleavage by endogenous proteolytic enzymes.

The peptide at M, = 52,000-55,000 observed in our rat liver preparation probably corresponds to the peptide of M , = 58,000 labeled with [3H]phenoxybenzamine by Kunos et ai. (12) and the soluble peptide purified by Graham et al. (27) from the same tissue. As mentioned above, our data strongly suggest that this peptide is a proteolytic fragment of the native M, = 80,000 peptide. Nonetheless, this peptide contains an intact a,-adrenergic ligand binding site since it is labeled by the photoaffinity probe in membranes. This fact could account for its purification by biospecific affinity chromatog-

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

al-Receptor Photoaffinity Label 2587

raphy methods as reported by Graham et al. (27). Similarly, the putative proteolytic fragment a t M, = 42,000 observed in many of the preparations reported here may correspond to the peptide of M, = 43,000 labeled with [3H]phenoxybenza- mine by Guellaen et al. (13) in rat liver membranes.

In summary, we have described the synthesis and charac- terization of a novel high affinity high specific activity radio- iodinated selective al-adrenergic receptor photoaffinity probe. This probe labels an al-adrenergic receptor peptide of M, = 78,000-85,000 in several different mammalian tissues. Smaller ligand binding peptides are likely generated from the native M , = 78,000-85,000 receptor peptide by endogenous proteol- ysis. This new probe should greatly facilitate the molecular characterization of the al-adrenergic receptor.

Acknowledgments-We wish to thank Lynn Tiller for preparation of the manuscript and Diane F. Sawyer and Marjorie L. Akers for expert technical assistance.

REFERENCES 1. Hoffman, B. B., and Lefkowitz, R.J. (1980) Annu. Reu. Pharma-

col. Toricol. 20, 581-608 2. Shorr, R. G. L., Lefkowitz, R. J., and Caron, M. G. (1981) J. Biol.

Chem. 256,5820-5826 3. Shorr, R. G. L., Heald, S. L., Jeffs, P. W., Lavin, T. N., Strohs-

acker, M. W., Lefkowitz, R. J., and Caron, M. G. (1982) Proc. Natl. A d . Sei. U. S. A. 79 , 2778-2782

4. Shorr, R. G. I., Strohsacker, M. W., Lavin, T. N., Lefkowitz, R. J., and Caron, M.G. (1982) J. Biol. Chem. 257,12341-12350

5. Homcy, C. J., Rockson, S. G., Countaway, J., and Egan, D. A. (1983) Biochemistry 22,660-668

6. Rashidbaigi, A., and Ruoho, A. E. (1981) Proe. Natl. Acad. Sei. U. S. A. 78, 1609-1613

7. Burgermeister, W., Hekman, M., and Helmreich, E. J. M. (1982) J. Biol. Chem. 257,5306-5311

8. Lavin, T. N., Heald, S. L., Jeffs, P. W., Shorr, R. G. L., Lefkowitz, R. J., and Caron, M. G. (1982) J. BioLChem. 256,11944-11950

9. Lavin, T. N., Nambi, P., Heald, S. L., Jeffs, P. W., Lefkowitz, R.

J., and Caron, M. G. (1982) J. Biol. Chem. 257,12332-12340 10. Stiles, G. L., Strasser, R. H., Lavin, T. N., Jones, L. R., Caron,

M. G., and Lefkowitz, R. J. (1983) J. Biol. Chem. 258 , 8443- 8449

11. Guellaen, G., Aggerbeck, M., and Hanoune, J. (1979) J. Biol. Chem. 254,10761-10768

12. Kunos, G., Kan, W. H., Greguski, K., and Venter, J. C. (1983) J. Bid. Chem. 258,326-332

13. Guellaen, G., Goodhardt, M., Barouki, R., and Hanoune, J . (1982) Biochem. Phnrmacol. 3 1, 2817-2820

14. Hess, H.-J., Graham, R. M., and Homcy, C. J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80,2102-2106

15. Greenwood, F. C., Hunter, W. M., and Glover, J . S. (1963) Biochem. J. 89,114-120

16. Wikberg, J. E. S., Lefkowitz, R. J., and Caron, M. G. (1983) Biochem. Phnrmacol., 32 , 3171-3178

17. Clarke, W. R., Jones, L. R., and Lefkowitz, R. J. (1978) J. Biol. Chem. 253,5975-5979

18. Wikberg, J. E. S., Akers, M., Caron, M. G., and Hagen, P.-0. (1983) Life Sei., 33 , 1409-1417

19. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193,265-275

20. De Lean, A,, Hancock, A. A., and Lefkowitz, R. J. (1982) Mol. Phurmacol. 2 1 , 5-16

21. Laemmli, U. K. (1970) Nature (Lond.) 227,680-685 22. Hoffman, B. B., Dukes, D., and Lefkowitz, R. J. (1982) Lite Sci.

23. Hamilton, C. A., and Reid, J . L. (1981) J. Cardiouasc. Phurmacol.

24. Heald, S. L., Jeffs, P. W., Lavin, T. N., Nambi, P., Lefkowitz, R. J., and Caron, M. G. (1983) J. Med. Chem. 26,832-838

25. Leeb-Lundberg, L. M. F., Dickinson, K. E. J., Heald, S. L., Wikberg, J. E. S., DeBernardis, J. F., Winn, M., Arendsen, D. L., Lefkowitz, R. J., and Caron, M. G. (1983) Biochem. Biophys. Res. Commun., 115,946-951

26. Benovic, J. L., Stiles, G. L., Lefkowitz, R. J., and Caron, M. G. (1983) Biochim. Biophys. Res. Commun. 110,504-511

27. Graham, R. M., Hess, H.-J., and Homcy, C. J. (1982) J. Biol. Chem. 257 , 15174-15181

28,265-272

3,977-985

28. Cheng, Y. C., and Prusoff, W. H. (1973) Biochem. Pharmacol. 22,3099-3108 by guest on A

pril 7, 2018http://w

ww

.jbc.org/D

ownloaded from

DeBernardis, M Winn, D L Arendsen, R J Lefkowitz and M G CaronL M Leeb-Lundberg, K E Dickinson, S L Heald, J E Wikberg, P O Hagen, J Fof the ligand binding subunit with a high affinity radioiodinated probe.

Photoaffinity labeling of mammalian alpha 1-adrenergic receptors. Identification

1984, 259:2579-2587.J. Biol. Chem. 

  http://www.jbc.org/content/259/4/2579Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/259/4/2579.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on April 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from