The Cardiac @-Adrenergic Receptor - Journal of Biological Chemistry

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(Received for publication, November 5, 1982). Gary L. Stiles$, Ruth H. StrasserB, Thomas N. Lavin, Larry R. JoneslI, Marc G. Caron, and Robert J. Lefkowitz.
THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 258, No. 13, Issue of duly 10, pp. 8443-8449.1963 Printed in U.S.A

The Cardiac@-AdrenergicReceptor STRUCTURAL SIMILARITIES OF @I AND LABELING*

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RECEPTOR SUBTYPES DEMONSTRATED BY PHOTOAFFINITY

(Received for publication, November 5,1982)

Gary L. Stiles$, Ruth H. StrasserB, ThomasN. Lavin, Larry R. JoneslI, Marc G . Caron, and RobertJ. Lefkowitz From the Howard Hughes Medical Institute ResearchLaboratories, Departments of Medicine (Cardiology) and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

§Supported by Grant13-Str. 241 1-1 from the Deutsche Forschungsgemeinschaft, Bonn. TIEstablishedInvestigator of the American Heart Association. Present address, Departments of Pharmacology and Medicine, Indiana University School of Medicine, 1100 W. Michigan St., Indianapolis, IN 46223.

EXPERIMENTALPROCEDURES

Materiak-[’251]I~docyan~pindolol was obtained from New England Nuclear or was synthesized according to the method of Engel et al. (8). p-Azid~-rn-[~~~I]iodobenzylcarazolol was obtained from New

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The @-adrenergic receptor photoaffinity ligand p - z.e. amino acid sequence,at theligand binding sitesof azido-rn-[1261]iodobenzylcarazolol has been used to CO- the receptorpeptide. valently label the fll and f12 adrenergic receptor binding subunits present in left ventricular myocardial membranes derived from mammalian (including human) Catecholamines exert profound effects on metabolic reguand nonmammalian species. Covalent incorporationof lation inmost mammalian tissues. A majorthrust of hormonethe photoaffinity ligand into membrane proteins was followed by sodium dodecyl sulfate-polyacrylamide gel receptorresearch has been the attempt to understand the electrophoresis. In thecase of the human, canine, por- structure,function,and physiological regulation of the pcine, rabbit, and rat left ventricle, ofall which contain adrenergic receptor-adenylate cyclase system. T o date, most predominantly or exclusively B,-adrenergic receptors, studies of @-adrenergic receptors aimedat probing their motwo peptides of M, = 62,000 (major component) and lecular properties have been performed with either erythroM, E 55,000 (minor component) were specifically la- cyte or cell culture model systems (1-3). This is in large part beled and visualized by autoradiography. Photoincor- due to the paucity and lability of 0-adrenergic receptors in poration into these two bands could be blocked with mammalian tissues such as the heart. The myocardium, howthe appropriate drugs to displaya Bl-adrenergic recep- ever, represents an organ system of immense interest both tor pharmacological specificity. Simultaneous sodium because of its dynamic regulation by catecholamines and the dodecyl sulfate-polyacrylamide gel electrophoresis of fact that altered sensitivity to catecholamines may well be samples from each species revealed that allof the M, = involved in many cardiovascular diseases in man. Moreover, 62,000 peptides co-migrated suggesting similarity in depending on thespecies, cardiac @-adrenergic receptors may the P,-adrenergic receptor binding subunit peptides in be of the or 8 2 subtype (4). all of these species. The minor componentM, % 55,000 A recently developed approach to studying the structureof appears tobe a proteolytic degradation productof the &adrenergic receptorsisphotoaffinity labelingwith lightM, = 62,000 peptide. Its formation could be decreased sensitive azide derivatives of potent @-adrenergicantagonists. by proteinase inhibitors. This suggests that the heterp-1251-aziogeneity of the labeling patternobserved in mammal- Several agents such as p-1251-azidobenzylpindolol, docyanopindolol, and p-‘251-azidobenzylcarazolol have been ian tissues inthis and previous studies may be the result developed (5-7). All appear to photoincorporate into P-adreof proteolyticdegradation of thereceptorsubunit nergic receptorpeptidesinmembranes from a variety of which occurs during membrane preparation. of frogventricular mem- tissues containing PI- and &adrenergic receptors. Peptides Photoaffinitylabeling branes which contain predominantly B2-adrenergic re-of varying size ( M , = 30,000-65,000) and heterogeneity have and P2 ceptors also revealed two peptides of M , s 62,000 been labeled but clear structural correlates of the @, receptor subtypes in mammalian tissues have not thus far (majorcomponent) and 55,000 (minorcomponent) with thepharmacological selectivityof a 82-adrenergic been clearly identified (5-7). receptor. Thesedata suggest marked similarities in the In the present studywe report two significant advances in Dl- and &adrenergic receptor binding subunits of dif- the application of photoaffinity labeling techniques to the ferent species and suggest that the pharmacological elucidation of the structureof mammalian @-adrenergic recepsubtype mightbe determined by the detailed structure, tors. Firstis the successful labeling of the receptors in cardiac tissue, a goal previously not achieved because of unacceptably tissue. * The casts of publication of this article were defrayed in part by high nonspecificbinding of photoaffinity probes in this the payment of page charges. This article must therefore be hereby Second is the documentation that, in a wide variety of species marked “aduettisement” in accordance with 18 U.S.C. Section 1734 (including man), the cardiac &adrenergic receptor binding solely to indicate this fact. site resides on a peptide of M, FZ 62,000 regardless of whether $Supported by Clinical Investigator Award HL01027 from the oz subtype. These findings have important National Institutes of Health. To whom reprint requests and corre- it is of the or spondence shouldbe addressed at, Box 3444, Department of Medicine, implications for the interpretation of photoaffinity labeling data previously acquired with submammalian systems. Duke University Medical Center, Durham, NC 27710.

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PhotoaffinityLabeling of Cardiac ,%Adrenergic Receptors

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England Nuclear and prepared according to the methods recently freshfor eachexperimentin 1 mM HCI/10-' M ascorbate. The published from this laboratory (7). ascorbate was present to inhibit oxidation of @-adrenergicagonists. Grass frogs (Ranapipiens) were obtained fromNASCO, Fort The photoaffinity ligand, at concentrations of 100-150 PM, was Atkinson, WI. Porcine hearts were obtained from Garrard's Country then added under dim fluorescent light. The stock solution of the Sausage Co., Durham, NC. Normal human myocardium was obtained radioligand was diluted in 10% ethanol, 5 mM HCI. The volume of from the organ donor program a t Duke University Medical Center. radioligand added represented - 2 4 % of the final incubation volume. Tissue was from normal young adults who had suffered traumatic The incubation was for 60 min a t 25 "C in the dark in Sorvall SS-34 braindeath. Adrenergic compounds werefrom sources previously polycarbonate tubes. Immediatelyfollowing incubation the tubes were described (9). Betaxolol was a generous gift from SyntheLabo, 37 Rue immersed in ice for 10 min in the dark. The tubes were then centride la Glaciere, Paris, France. IC1 118,551 was a generous gift from fuged a t 48,000 X g for 15 min in the dark. The pellets were resuslmperial Chemical Industries, Macclesfield, Cheshire, United Kingpended with a motor-driven Teflon pestle in ice-cold 20 mM Trisdom.Biochemical reagents weregenerally obtained fromSigma. HCI, 2 mM EDTA, (pH7.4 a t 25 "C) containingphospholipid vesicles, Premixed SDS'-polyacrylamide gel electrophoresis standards (phos- the preparation of which is described below. phorylase b, M, = 94,000; albumin, M, = 67,000; ovalhumin, M, = The resuspended membranes were sedimented a t 48,000 X g for 15 48,000; carhon anhydrase, M, = 30,000; soybean trypsin inhibitor,M. min and the procedure repeated once again. pellet This wassuspended = 20,000) were obtained from Pharmacia. Electrophoresis reagents in the above buffer (10 ml) withoutvesicles and photolyzed according were from Rio-Rad. X-ray film (XAR-5) and developing solutions to the procedure previously published (7). The 10 ml of membrane were from Kodak. Intensifying screens (Cronex Lightning Plus)were suspension were placed in a Petri dish for 90 s, 12 cm from a Hanovia from Dupont. 450W medium pressure mercury arc lamp filtered with 5 mm of Pyrex Membrane Preparations-Myocardial membranes were prepared glass. After photolysis, the suspension was sedimented a t 48,000 X g from fresh or frozen material according to the methodof Jones et al. and then suspended in SDS-PAGE sample buffer (see below). (10) for cardiac sarcolemmalvesicles. For porcine, canine, and human The phospholipid vesicles used for washing the membrane prepahearts only left ventricular tissue was used. In the case of the frog, rations were prepared as follows: 30 mg of L-tu-phosphatidylcholine the whole ventricle was used, and for rat and rabbit both left and from soybeans (Type 11-S obtained from Sigma) were suspended in 5 right ventricles wereutilized. All membranepreparations(except ml of 20 mM Tris-HCI, 2 mM EDTA, pH 7.4 a t 25 "C, and flushed canine) were performed in the presence of the following proteinase with Nf. The suspension was cooled on ice and then sonicated for 5 inhibitors:EDTA, 2 mM; phenylmethylsulfonyl fluoride, 0.5 mM; min with the small probe of a Sonic 300 dismembranator (Artex soybean trypsin inhibitor, 15 pg/ml; benzamidine, 10" M; and leu- System Corp.) at an outputof 35%. The solution was then diluted to peptin, 5 pg/ml, and pepstatin,7 pglml. This combination was chosen40 ml with the above buffer and centrifugedat 40,000 X g for 10 min. to include all four major classes of proteinase inhibitors-the serine The supernatant was then diluted to -300 ml with the above buffer proteinaseinhihitors,soyheantrypsininhibitorandbenzamidine, and used fresh for washing the membranes. combination serine and thiol proteinase inhibitors, phenylmethylsul- This washing procedure is different from that previously (7) refonyl fluoride and leupeptin, a metallo-dependent inhibitor, EDTA, ported from this laboratory. The change was necessitated when it was and a carhoxylproteinase inhibitor, pepstatin. Systematic optimizalound that the albumin hufler wash previously published produced tion of inhihitor concentration was performed for EDTA and the dramatic and deleterious effects on myocardial membranes such that concentration of the other proteinase inhibitors was selected based after two washes with the albumin buffer the myocardial membranes on literature values. The most important inhibitors for myocardial tissue appear to be EDTA, leupeptin, and pepstatin. Recent experiments in which proteinase inhibitors were added during canine heart MW membrane preparations demonstratea decreased proportion of M, = 5.5,000 labeled peptides. The final myocardial membrane preparations were suspended in 94K2.50 mM sucrose,10 mM histidine(pH 7.5 a t room temperature), frozen under liquid Nfr and stored at -90 "C until used. Purified canine and porcine membranes typically contained -500-700 fmol/ 67Kmg of protein, and frog, 900-1000 fmol/mg of protein of @-adrenergic receptor binding sites; while the human myocardial membranes contained -400 fmol/mg. 43KRaterythrocytemembranes were preparedeither as described previously (7) or with the following modifications. Whole rat blood was anticoagulated with heparin and 2 mM EDTA. The cells were then washed three times with ice-cold 150 mM NaCI, 3 mM EDTA, 30K+ pH 7.4, with intervening centrifugations at 600 X g. The cells then underwent hypotonic lysis with 10 volumes of 5 mM EDTA in the presence of the enzyme inhibitors, and left on ice for 10 min. The cell lysates were then dounce homogenized with 15 up and down strokes of a glass homogenizer. The lysates were then diluted 1:l with 20 mM Tris-HCI, 2 mM EDTA, pH 7.4, a t 25 "C with the above indicated proteinaseinhibitorsandcentrifuged a t 35,000 X g to pelletthe membranes. The supernatant was aspirated and the loosely packed pellet was gently rocked free of the dense pellet and decanted intoa clean centrifuge tube and resuspended in the same buffer with the proteinase inhibitors and recentrifuged. This procedure was repeated twice more and the white pellet was then resuspended in the same FIG. 1. Photoaffinity labeling and pharmacological specibuffer for photoaffinity labeling. Photoaffinity Labeling of Membrane Preparations with p-Azido-m- ficity of incorporation of ['*'I]pABC in canine myocardial membranes. Aliquots of the same membrane preparations used in ["sl]iodobenzyfcarazolol-Membranes, either fresh or frozen,were washed once with 20 mM Tris-HCI, 2 mM EDTA, pH 7.4, a t 25 "C the radioligand binding studieswere incubated with ['"I]pABC alone (control) or in the presence of the indicated concentration of comwith subsequent centrifugation a t 48,000 X g. Membranes were resuspended in 10 ml of the above buffer with the proteinase inhibitors peting ligand and photolabeled as indicatedunder"Experimental at the concentration indicated above to make a final receptor concen- Procedures." The samples were then solubilized as described under an acrylamide tration of -30-50 PM. T o each tube was added either H20/ascorbate "Experimental Procedures" and electrophoresed on 8% gel. The small arrows indicate the M, I 62,000 and 55,000 proteins. (lo-' M ) or competing ligand at the concentration noted such that 100 pl of solution were added to each tube.All ligands were made up The molecular weights (MW) shown X 1,000 ( K ) were determined with iodinated protein standards (3). The results shown above are ' The abbreviations used are: SDS, sodium dodecyl sulfate; PAGE, representative of three such experiments. (-)Pro, (-)-propranolol; polyacrylamide gel electrophoresis; CYP, iodocyanopindolol; pABC, (-)lso, (-)-isoproterenol;(-)Epi,epinephrine; ( - ) N E , (-)-norepip-azido-rn-['f51]iodobenzylcarazolol. nephrine; (+)l.so, (+)-isoproterenol.

Photoaffinity Labeling of Cardiac P-Adrenergic Receptors

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no longerexhibited anyspecific ['251]CYPor [1251]pABCbinding. This pharmacological selectivity of these agentsfor a @,-adrenergic might be related to the ability of albumin to delipidate membranes receptor. Stereoselectivity, a characteristic of the interaction although this mechanism was not documented. It should be noted of catecholamines with their receptors, is also demonstrated that this deleterious effect was specific for myocardial membranes since lipid vesicles or albumin washes could be employed in erythro- as (+)-isoproterenol is 1600-fold less potent than (-)-isoproterenol. Fig. 1 demonstrates the results of photolabeling of cyte or lung labeling experiments without any harmfuleffects. Radioligand Binding Assays-Radioligand binding assayswere per- the @-adrenergicreceptor in these membranes with ["'I]pABC formed a t 25 "C for 1 h using ['*'I]CYP as recently described (7). in the presence and absence of competing drugs. Following Bound ligand was separated from free with GF/C Whatman filters photoincorporation, the membranes were solubilized and suband washes ( 5 X 2.5 ml) with 75 mM Tris-HCI, 25 mM MgClz, pH 7.4, jected to SDS-polyacrylamide gel electrophoresis and autoduring vacuum filtration. Specific binding was determined by subradiography of the gel. The autoradiograph reveals incorpotracting the amountof radioactivity not competedfor by lo" M (-)isoproterenol from the total amount of radioactivity bound and usu- ration of the photoaffinity probe into two protected radioactive bands: a M,E 62,000 peptide (major component) and a ally amounted to 80-90% of total binding. Competition curve data were analyzed by computer-assisted tech- M,E 55,000 peptide (minor component) both of which display niques previously published and validated in this laboratory (11,12). all the pharmacological characteristics expected of a @'-adreSodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis-The nergic receptor. Several nonspecific bands of incorporated electrophoresis was performed according to the method of Laemmli radioactivity also appear on the gel autoradiogram but these (1.7)using homogeneous slab gels with the exception that samples were solubilized in 10% SDS, 10% glycerol, 5% 8-mercaptoethanol, bands could not be specifically protected. Densitometric scan25 mM Tris-HCI, pH 6.8, and denatured for45 min a t room temper- ning revealed the larger molecular weight band (M, 62,000) ature. Following electrophoresis the gels were dried using a Bio-Rad to be 4 times as intense as thelower molecular weight band gel dryer prior to autoradiographyat -80 "C. (M, 55,000). The incorporation of['*'I]pABC into either peptide is completely blocked by lo5M (-)-isoproterenol and RESULTS M propranolol but not by lo-' M (+)-isoproterenol (Fig.

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1).A t a concentration of 10"j M, (-)-norepinephrine is slightly more potentthan (-)-epinephrinein blocking ['*'I]pABC incorporation. It is evident from Fig. 1 thatan excellent correlation between the ability of agonists to compete for ['2511]CYPbinding in membranes (data not shown) and to block[I2'I]pABC incorporation intothe two peptides was observed. This suggests that thepopulation of @-adrenergicreceptors

B

MW A

94K0

Betoxolol

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IC1 118.551

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FIG. 2. The pharmacological subtype specificity of canine cardiac &adrenergic receptors by radioligand binding and photoaffinity labeling. A, competition curves of betaxolol (8, selective) and IC1 118,551 (& selective) with ['2sI]CYP in canine myocardial membranes. The radioligand binding assay was performed as described under "Experimental Procedures" with the indicated concentrations of betaxolol and IC1 118,551. The lines through the experimentally derived data points represent the computer-generated best fit based on the law of mass action (12). Binding in the absence of competitor was 16 p ~ B,. photoaffinity labeling and pharmacological (&adrenergic receptor subtype) specificity of incorporation of ['*'I]pABC in canine ventricular membranes. Aliquots of membranes were incubated with ["'IIpABC alone or in the presenceof the indicated concentrationof competing ligand and photolabeled as indicated under "Experimental Procedures." The samples were solubilized as indicated under "Experimental Procedures" and electrophoresed on an 8% acrylamide gel. The small arrows indicate theM, z 62,000 and 55,000 proteins. The molecular weight standards ( M W )are shown X 1,000 (K).The results shown above are representative of two similar experiments.

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The pharmacological characterization of the @-adrenergic receptors in the membrane preparations derived from canine left ventricular myocardium was performed by direct ligand binding with the &adrenergic antagonist ['2'II]iodocyanopindolol. Agonist and antagonist competition curves reveal that (-)-isoproterenol (E& = 67 nM) is more potent than (-)norepinephrine (ECso= 478 nM) which is slightly more potent than (-)-epinephrine (EGO= 1120 nM) consistent with the

PhotoaffinityLabeling Cardia. of

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FIG.4. Photoaffinity labeling of rat erythrocytes with [1251] pABC in the presence and absence of proteinase inhibitors. Aliquots of rat erythrocyte membranes prepared in the absence and presence of proteinaseinhibitors were incubated with ['*'I]pABC M (-)-isoproterenol and photolabeled alone or in the presence of as indicated under "Experimental Procedures." The samples were then solubilized as described under "Experimental Procedures" and electrophoresed on an 8% acrylamide gel. The molecular weights ( M W )shown X 1,000 ( K ) were determined with iodinated protein standards. The arrow head indicates the M. 62,000 peptide. Abbreviations are as in Fig. 1.

-

0101 is much more potent thanIC1 118,551and that assessed as by computer modeling, their curves are steep and uniphasic and model most appropriately to a single class of homogeneous receptors with high affinity (KD= 8.1 nM) for betaxolol and lower affinity for IC1 118,551 ( K n = 158 nM). These findings are thus consistent with the presence of a homogeneous population of &-adrenergic receptors in these membranes. It should be noted that a small component of p2Time of Storage Time of Storage adrenergic receptors ( 4 0 % ) might not be reproducibly detected (12). The autoradiograph in Fig. 2B reveals that betax0 days 30 days FIG.3. Effect of storage on the relative proportions of the 0101 is much more potent than IC1 118,551 in blocking phoAt M betaxolol can block M. E 62,000 and M. s 55,000 peptides. Canine membranes were toincorporation of["'IIpABC. incubated with ["'II]pABC alone or in the presence of the indicated by more than 50% the photoincorporation of ['2sI]pABC into concentration of IC1 118,551and photolabeled as under "Experimen- the two peptides M, = 62,000 and 55,000 as judged by the tal Procedures." The samples were then solubilized as under "Exper- decrease in intensity of thebands compared to control imental Procedures." One aliquot of membranes were electrophoresed on an 8% acrylamide gel. Another set of aliquots was stored frozen whereas at an equivalent concentration IC1 118,551 did not (-20 "C) for 30 days and then subjected to electrophoresis on an 9% decrease incorporation as could be predicted from the comacrylamide gel. Arrow heads indicate the 62,000 and 55,000 M,pep- petition dose response curves. Thus, there is striking agreeare shown X 1,000 (K). ment in the resultsobtained from antagonist competition tides. The molecular weight standards ( M W )

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being photolabeled with [I2'I]pABC has the samepharmacological characteristics as those receptors delineated by [1251] CYP binding in radioligand binding experiments. This correlation is important since the extent of photoincorporation of the ligand into these receptors is approximately 3%. The extent of incorporation was calculated from the amount of specifically bound ligand prior to SDS-PAGE uersus that measured from SDS-PAGE gel slices. This low yield of covalent incorporation into the receptor protein is common to most photoaffinity ligands and necessitates the demonstration that the small population of receptors being studied by the photoaffinitytechniquesaccurately reflects the entire receptor population (7). Accordingly, further characterization of the canine myocardial @-adrenergicreceptor was carried out by using the P-adrenergic receptor subtype selective antagonists IC1 118,551 and betaxolol (14). IC1 118,551 is a @, subtype selective ligand with high affinity for @,-adrenergic receptors in mammalian tissues (KI, z 3-5 nM) and a lower affinity for &-adrenergic receptors (Kn 200nM). Betaxolol, on the other hand, hasa high affinity for &-adrenergic receptors (KI, e 5 nM) and lower affinity for P,-adrenergic receptors (K,, = 200 nM). These drugs can be used to furtherdocument the subtype of &adrenergic receptor present in the canine cardiac membranesby performing competition binding curves with these subtype selective agents. Fig. 2A reveals the competitioncurves of these compounds with [I2'1]CYP in the canine sarcolemmal membranes and demonstrates that betax-

Photoaffinity Labeling of Cardiac P-Adrenergic Receptors

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FIG. 5. Photoaffinity labeling of porcine myocardial membranes with ['251]pABC. Aliquots of myocardial membranes were M (-)incubated with ['251]pABCalone or in the presence of isoproterenol and photolabeled as under "Experimental Procedures." The samples were then solubilized as described under "Experimental Procedures" and electrophoresed on an 8%acrylamide gel. The molecular weight standards ( M W )are shown X 1,000 ( K ) . The small arrow indicates the M. I 62,000 protein. This experiment is representative of five such experiments. Abbreviations are asin Fig. 1.

MW 94 K+

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FIG. 6. Photoaffinity labeling ofhuman left ventricular myocardial membranes with ['*'I]pABC. Aliquots of myocardial membranes were incubated with ['251]pABCalone or in the presence of the indicated concentration of competing ligand and photolabeled as under "Experimental Procedures." The samples were solubilized as indicated under "Experimental Procedures" and electrophoresed on an 8% acrylamide gel. The small arrow indicates the M,I62,000 protein. The molecular weight standards (MW)are shown X 1,000 ( K ) . Abbreviations are asin Fig. 1. This experiment is representative of three similar experiments.

not all of the smaller labeled peptides are always totally eliminated but their relative abundance is always reduced.A similar inhibition of proteolysis has recently been observed in rat lung [lzs1]pABClabeling experiments. These experiments demonstrated that thetwo lowerM,specifically labeledbands are generated from the upper M , band (16). Taken together these observations strongly support the conclusions that in canine left ventricular membranes the 55,000 M , band arises from the 62,000 M,band by proteolytic degradation and that the &-adrenergic receptor binding site resideson a M, P 62,000 polypeptide. To evaluate whether there is homology with other mammalian cardiac &adrenergic receptors, membranes from porcine left ventricle were similarly investigated. The agonist competition curves were similar to those found in the canine heart membranes (data not shown) in that they had the pharmacological specificityexpected of a B1-adrenergic receptor. The autoradiograph in Fig. 5 demonstrates the results obtained when porcine myocardial membranes were photolabeled with ['2sI]pABCin the presence and absence of M (-)-isoproterenol. Clearly, a peptide of M , = 62,000 appears to be covalently and specifically labeled by the @-adrenergic photoaffinity probe. These results suggest that thecardiac PIadrenergic receptor binding subunit of both the pig and dog heart may be very similar. In addition, it can be appreciated that the M , = 55,000 peptide is barely detectable in this preparation. Analysis of the autoradiograph by scanning densitometry revealed that the M, = 62,000 band was13-fold more abundant than thelower M , band. The human left ventricle contains predominantly &-adrenergic receptors (-86%) as we have previously shown (17).

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curves and the photoaffinity labeling technique. This finding further supportsthe contention that thepopulation of receptors studied by photoaffinity labeling accurately represents the entire8-adrenergic receptor population in the membranes. To investigate the possibility that the M , E 55,000 band might be a degradation product of the M , = 62,000 pept.ide, we attempted to both promote and inhibit the formation of the 55,000 M , band. When samples were photoaffinity labeled, solubilized inSDS, stored frozen, and then subjected to SDSPAGE after varying periods, it was found that the relative amount of the 55,000 M , peptide progressively increased as the amount of the 62,000 M,peptide decreased (see Fig. 3). Proteolysis, of a similar nature, occurring in SDSIP-mercaptoethanol-solubilizedpolypeptides has been describedby others (15). In addition, the use of myocardial tissue which was not rapidly frozen postmortem led to an increased quantity of the M , = 55,000 band. Previous work from this laboratory (7) demonstrated that in mammalian tissues such as the rat erythrocyte and lung, [12sI]pABC labeled several (two to three) peptides all of which demonstrated &adrenergic specificity. The reasonfor this heterogeneity was unknown but the two major possibilities considered were proteolysis or that the various peptides represented distinct PI- and &-adrenergic receptors. When the current mixture of proteinase inhibitors was applied to the rat erythrocyte system, it was found that theformation of the smaller peptides could be strikingly inhibited. This is demonstrated in Fig. 4. Without proteinase inhibitors, three isoproterenol "protected" peptides are seen ( M , = 62,000,53,000, and 45,000; lane 1).When the same experiment was performed in the presence of the proteinase inhibitors the lower bands were dramatically decreased (lam3). It should be noted that

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Photoaffinity Labeling

8448

of Cardiac &Adrenergic Receptors

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[Competing Ligand] M

FIG. 7. Characterization of frog myocardial &adrenergic receptors by radioligand binding and with ["'Ir]CYP in frog myocardial membranes. photoaffinity labeling. A, competition curves ofadrenergic ligands Radioligand binding assays and data analysis wereperformed as described under "Experimental Procedures." Abhreviations are as in Fig. 1. R, photoaffinity labeling and pharmacological specificity of incorporation of ["'I] pARC in frog myocardial membranes. Aliquots of myocardial membranes were incubated with ["'I]pARC alone (control) or in the presenceof the indicated concentrationof competing ligand and photolaheled as indicated under "Experimental Procedures." The samples were then solubilized and electrophoresed on an 8% acrylamide gel as indicated under "Experimental Procedures." The small arrou indicates the M , P 62,000 protein. The molecular weight standards ( M W ) are shown X 1,000 ( K ) .Abbreviations are as in Fig. 1. This experiment is representative of three similar experiments.

As can be seen in Fig. 6, the @-adrenergic receptor binding band is present. When myocardial membranes from rat and subunit of human myocardial membranes also appears to be rabbit hearts both of which contain predominantly &-adrepresent on a polypeptide of M, I 62,000 and has the phar- nergic receptors (4, 18) were photolabeled with ['"I]pABC, macological specificity of a &-adrenergic receptor with norboth demonstrated receptor binding subunits in the region of suggests epinephrine being slightly more potent than epinephrine in a M , = 62,000 peptide (data not shown). This further a striking similarityin the @-adrenergic binding subunit of f i l blocking [""IIpABC incorporation. This same agonist potency adrenergic receptors from various species as well as in and serieshas beenfoundpreviously inthislaboratoryusing radioligand binding techniques(17). In addition,when human @.-adrenergic receptors from various myocardial tissues. myocardial membranes were photolabeled in the presence of DISCUSSION the subtype selective antagonists betaxolol (Pi selective) and IC1 118,551 (8. selective) results similar to those in Fig. 2 Two major subtypes of &adrenergic receptors, 8, and Br, have been defined based on differingpharmacological profiles were obtained. This further substantiates that &-adrenergic receptorsarethepredominantsubtype in the human left and physiological responses. I t has been suggested that &receptors have evolved to function primarily as receptorsfor ventricle (data not shown). which they have In contrast to the porcine, canine, and human hearts, the the neurotransmitter norepinephrine (for (4). relatively high affinity) a t neuroeffector junctions while the frog heart contains predominantly &adrenergic receptors We therefore next sought to investigate the structure of the &adrenergicreceptorshave evolved as receptors for the An in&adrenergic receptor binding subunit from frog myocardial circulating adrenal medullary hormone epinephrine. tissue. As can be seen in Fig. 7A, the frog cardiac @-adrenergic teresting, important andpreviously unanswered question has which the structureof PI- and &adrenergic receptor has, by radioligand binding techniques, the pharma- been the extent to cological characteristics of a &-adrenergic receptor with epi- receptors might be similar or divergent. Two approaches to the delineation of the structure of 8nephrine (EC,, = 10.8 FM) being muchmore potent than norepinephrine ( E C , , = 101.0 PM) in competing with [1p51] adrenergic receptors have recently been utilized, namely, puCYP for occupancy of the &adrenergic receptor. In Fig. 7 B , rification and photoaffinity labeling (3, 5-7,19, 20). Previthe autoradiograph demonstrates that ["'II]pABC incorpora- ously, these techniques have been applied primarily to nonmammalian model systemssuchasamphibianandavian tion into a M , = 62,000 peptide is inhibited with the same agonist potency series as in the competition curves. Thus, erythrocytes which contain high concentrations of 8-adrenerlo-" M isoproterenol completely blocks photoincorporation of gic receptors. Very little information on the molecular struc[ ""IIpABC into the M,= 62,000 band while epinephrine a t a ture of mammalian el- and &adrenergic receptors has been concentration of 3 X M ismuch moreeffectivein pre- published mainly because of the relative paucity and lability in these tissues. In this paperwe venting photoincorporation than norepinephrineat the same of the @-adrenergic receptor concentration. Again, it is evident that a minor M,z 55,000 present the first direct identification of the @-adrenergicre-

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Photoaffinity Labeling-of Cardiac @-Adrenergic Receptors

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ceptor binding subunit in membranesderived from mamrnal- peptide as are mammalian@,-adrenergic receptors, but' mammalian &-adrenergic receptors also contain a similar peptide. ianandnonmammalian myocardial tissues(includingthe human heart) by photoaffinity labeling. The data provide It should be noted that attempts to demonstrate proteolysis as thecause of the two receptor binding subunitsphotolabeled strong evidence that cardiac pl- and &adrenergic receptor by [ '"I]pABC in turkey erythrocyte membranes(7) have been binding subunits have similar electrophoretic mobilities. We have found that the myocardial 8-adrenergic receptor unsuccessful and proteinase inhibitors do not reproducibly from six different species (five p1systems and onePz system) alter the relative proportion of the M , E 45,000 and the M , E are all composedprimarily, if not exclusively, of a Mr= 62,000 39,000 peptides in that system. 8,- and &adrenergic The data presented here indicate that peptide as determined by photoaffinity labelingwith [ I L 5 I ] pABC. All of these peptides co-migrate on SDS-PAGE. Fur- receptors of myocardial membranes (including human)reside thermore, the photoincorporation of [12sI]pABC into these on a 62,000 M, peptide which can be proteolyzed in situ to proteins displays all the appropriate pharmacological prop- yield smaller peptideswhich retain their p-adrenergic receptor erties expected of a &- or &adrenergic receptor subtype. This binding ability. Further work will be necessary to elucidate was demonstrated by the appropriate agonist and antagonist the structural differences which determine the unique pharpotenciesand stereospecificity forprotectingthe receptor macological specificity of the two adrenergicreceptor subpeptides from photolabeling. types. The fact that the band which contains the /&-adrenergic receptor subunit co-migrates with the P,-adrenergic receptor Acknowledgment-We would especially like to thank Lynn Tiller binding subunit of severalspeciessuggests that there are for her excellent help in transcribing this manuscript. homologies in the pl- and &adrenergic receptors. The similarities in the p, and p2 binding subunits (both M , E 62,000 REFERENCES peptides) suggest that the structuraldifferences which confer 1. Haga, T., Haga, K., and Gilman, A. G. (1977) J. Biol. Chem. 252, the pharmacological specificity of the individual P-adrenergic 5776-5782 receptor subtypes may be subtle. These structuraldifferences A. D. 2. Vauquelin, G., Geynet, P., Hanoune,J.,andStrosberg, remain unknown butlikely relate todifferences in amino acid (1977) Proc. Natl. Acad. Sei. I/. S. A. 74,3710-3714 sequence, secondary or tertiary protein structure. 3. Shorr, R. G . L., Lefkowitz, R. J., and Caron, M.G. (1981) J . Bid. Chem. 256,5820-5826 As noted under "Results," a M , = 55,000 peptide which can 4. Hancock, A. A., De Lean, A. L., and Lefkowitz, R. J . (1979) Mol. be observed in varying amounts in each of the heart prepaPharmacol. 16, 1-9 rations is thought to represent a degradation product of the 5. Rashidbaigi, A,, and Ruoho, A. E. (1982) Biochem. Biophys. Res. M , = 62,000 peptide.This is suggested by thefactthat Commun. 106, 139-148 following ['"IIpABC labeling, solubilization, and freezing, the 6. Burgermeister, W., Hekman, M., and Helmreich, E. J. M. (1982) relative amount of M , = 55,000 peptide increases with time, J. Biol. Chem. 257,5306-5311 7. Lavin, T. N., Nambi, P., Heald, S. L., Jeffs, P. W., Lefkowitz, R. at theexpense of the M,= 62,000 band. In addition,inclusion J., and Caron, M. G. (1982) J . Biol. Chem. 257, 12332-12340 of proteinase inhibitors (see "Experimental Procedures") de8. Engel, G., Hoyer, D., Berthold, R., and Wagner, H. (1981) Naucreased the relative amount of the M , = 55,000 peptide in nyn-Schmiedeberg's Arch. Pharmacol. 317, 277-285 many of the species examined. 9. Caron, M. G., and Lefkowitz, R. J. (1976) J. Biol. Chem. 251, Previous work from this laboratory (7), had indicated that 2374-2384 there is heterogeneity in the peptides whichcomprise the 10. Jones, L. R., Maddock, S. W., and Besch, H. R., Jr. (1980) J . Biol. Chem. 255,9971-9980 binding subunits of mammalian Dl- and &-adrenergic receptors. The adrenergic binding subunits were found togenerally 11. De Lean, A,, Munson, P. J . , andRodbard, D. (1978) Am. J . Physiol. 235, E97-E102 contain two to three peptides with an M , E 30,000-65,000. 12. De Lean, A., Hancock, A. A., and Lefkowitz, R. d . (1982) Mol. For example, by ["'II]pABC photoaffinity labeling therat Pharmacal. 2 1, 5-16 lung, which contains predominantly &adrenergic receptors, 13. Laemmli, U. K. (1970) Nature (Lond.)227, 680-685 was found to consist of three peptides ( M , = 62,000, 47,000, 14. Dickinson, K., Richardson, A,, and Nahorski, S. R. (1981) Mol. and 36,000) and the rat reticulocyte or erythrocyte, which Pharmacol. 1 9 , 194-204 contains exclusively &-adrenergic receptors, had three similar15. Pringle, J. R. (1970) Biochem. Biophys. Res. Commun. 39,46-52 peptides, all of which had thepharmacological properties of a 16. Benovic, J. L., Stiles, G. L., Lefkowitz, R. J., and Caron, M. G. (1983) Biochem. B i o ~ h v sRes. . Commun. 110. 504-511 &adrenergicreceptor. Whenraterythrocytemembranes .1 1 . Stiles, G. L., Taylor, S.,"and Lefkowitz, R. d . (1982) Circulation were prepared using our current mixtureof proteinase inhib66,II-207 itors, photoaffinity labeled, and subjected to SDS-PAGE, the 18. Brodde, 0 . E., Leifet, F. J., and Krehl, H. J. (1982) J . Cardiouasc. predominant band found on autoradiography was a M , Pharmacol. 4,34-43 62,000 peptide (Fig. 4). Similar findings have been demon- 19. Lavin, T. N., Heald, S. L., Jeffs, P. W., Shorr, R. G. L., Lefkowitz, M. G. (1981) J . Biol. Chem. 256, 11944R.J.,andCaron, strated in the rat lung (16). The previous finding of peptide 11950 heterogeneity by ['2sII]pABClabeling in mammalian systems 20. Shorr, R. G. L., Heald, S. L., Jeffs, P. W., Lavin, T. N., Strohsthus appears to be due to proteolysis. Thus, not only are acker, M. W., Lefkowitz, R. J., and Caron, M. G. (1982) Proc. amphibian &adrenergic receptors composed of a M , = 62,000 Natl. Acad. Sei. U. S. A. 79, 2778-2782

The cardiac beta-adrenergic receptor. Structural similarities of beta 1 and beta 2 receptor subtypes demonstrated by photoaffinity labeling. G L Stiles, R H Strasser, T N Lavin, L R Jones, M G Caron and R J Lefkowitz J. Biol. Chem. 1983, 258:8443-8449.

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