Proc. Nati. Acad. Sci. USAVol. 75, No. 1, pp. 228-232, January 1978Biochemistry

Agonist-induced increase in apparent f3-adrenergic receptor size(hormone receptor/adenylate cyclase/ligand-induced molecular interconversions/guanine nucleotides)

LEE E. LIMBIRD AND ROBERT J. LEFKOWITZDepartments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

Communicated by James B. Wyngaarden, November 3,1977

ABSTRACT The properties of digitonin-solubilized ,B-ad-renergic receptors from frog erythrocyte membranes werestudied by gel exclusion chromatography on AcA 34 Ultragel.P-Adrenergic rece tor binding activity in these membranes canbe identified by both an agonist ligand, [3H~hydroxybenzyl-isoproterenol, and the antagonist ligands, [3H~dihydroalprenololand'251Iabeled hydroxybenzylpindolol. Occupancy of the ,-adrenergic receptors with the [3H]hydroxybenzylisoproterenolagonist prior to their solubilization from the membrane leadsto an increase in apparent receptor size. Alterations in the mo-lecular size of the receptor cannot be mimicked by occupancyof the binding site with the antagonist ligands. Exposure of frogerythrocyte membranes to [3H~hydroxybenzylisoproterenolagonist in the presence of 10 pM Gpp(NH)p, a guanyl nucleotideanalog that exerts multiple regulatory effects on the catechol-amine-sensitive adenylate cyclase [ATP pyrophosphate-lyase(cyclizing); EC 4.6.1.1] system, results in the elution of the[3H]hydroxybenzylisoproterenol radioligand in both the regioncharacteristic of the agonist-receptor complex and the regioncharacteristic of the antagonist-receptor complex.The precise molecular interactions responsible for the ago-

nist-induced increase in apparent f-adrenergic receptor site arestill unresolved. However, the low concentrations of agonist thatare capable of altering apparent receptor size and the sensitivityof this effect to guanyl nucleotides suggest that these phenom-ena may be intimately involved in eliciting the physiologicaleffects of jB-adrenergic catecholamines at the molecularlevel.

Several hormones and drugs, including ,B-adrenergic cate-cholamines, interact with cell surface receptors and subse-quently activate adenylate cyclase [ATP pyrophosphate-lyase(cycling); EC 4.6.1.1], thus raising intracellular cyclic AMPlevels (1). The first step in this sequence of biochemical eventscan be studied directly by using radiolabeled agents that retaintheir biological activity as tracers of drug-receptor interaction.For example, we have used the f0-adrenergic antagonist (-)-[3H]dihydroalprenolol ([3H]DHA) to delineate the propertiesof ,B-adrenergic receptor binding in several mammalian tissuesas well as in frog erythrocyte membranes, a useful model systemfor adenylate cyclase-coupled ,B-adrenergic receptors (2). An-other high affinity antagonist, 125I-labeled hydroxybenzyl-pindolol (125I-HYP) has also been shown to identify physio-logically relevant ,B-adrenergic receptors (3).

Although fl-adrenergic antagonist agents recognize the j0-adrenergic receptor with a high degree of specificity, they lacksome of the fundamental properties associated with agonistdrugs, namely, (i) stimulation of the adenylate cyclase enzyme,(tt) desensitization of the adenylate cyclase system concomitantwith a decrease in functional receptor number (4), and (iii)reduction in apparent receptor affinity in the presence ofguanyl nucleotides (5, 6). For these reasons a radiolabeledagonist, [3H]hydroxybenzylisoproterenol ([3H]HBI), was de-veloped to compare the characteristics of agonist and antagonist

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked"advertfsemevit" in accordance with 18 U. S. C. §1734 solely to indicatethis fact.

228

binding (7). An important difference between [3H]DHA (an-tagonist) binding and [3H]HBI (agonist) binding is the kineticsof radioligand dissociation from the 13-adrenergic receptor. Incontrast to the rapid and complete dissociation of [3H]DHAfrom frog erythrocyte membranes at physiological tempera-tures, [3H]HBI agonist binding is neither fully nor rapidly re-versible except in the presence of guanyl nucleotides (7, 8). Itis possible that the slow and incomplete dissociation of agonistbinding may be directly related to the unique capability ofagonist drugs to stimulate as well as to desensitize the adenylatecyclase-coupled receptor system (8).The manner in which receptor occupancy by agonist drugs

results in the activation of the adenylate cyclase enzyme is stillunknown. For this reason, the molecular events that accompanyoccupancy of the receptor by an agonist agent are of primaryinterest since they may be related to the mechanism of recep-tor-enzyme "coupling". In the present report, we describe anagonist-specific increase in the apparent molecular size of thefrog erythrocyte 0-adrenergic receptor induced by the bindingof radiolabeled [3H]HBI. Occupancy of the receptor with theantagonist [3H]DHA or 125I-HYP does not alter the apparentreceptor size, assessed by gel exclusion chromatography. Inaddition, if [3H]HBI binding to the membranes is carried outin the presence of the guanyl nucleotide analog, Gpp(NH)p,the [3H]HBI radioligand elutes both in the region characteristicof the agonist-receptor complex and the region characteristicof the antagonist-receptor complex. The possible molecularevents responsible for the agonist-induced increase in apparent3-adrenergic receptor size and their relationship to receptor-enzyme coupling in the adenylate cyclase system are dis-cussed.

MATERIALS AND METHODS

Materials. (-)[3H]Dihydroalprenolol, 33 or 48 Ci/mmol,and (h:)[3H]hydroxybenzylisoproterenol, 20-26 Ci/mmol, weretritiated at New England Nuclear, as described (7, 9). 1251-Labeled hydroxybenzylpindolol, 2000 Ci/mmol, was a gen-erous gift from Keith Crutcher and was prepared and purifiedas described by Maguire et al. (3). AcA 34 Ultragel resin wasfrom LKB Biochemicals. Sources of all other materials havepreviously been reported (10, 11).Membrane Preparation. Purified frog erythrocyte mem-

branes were prepared fresh for each experiment by reportedmethods, and were suspended in 75 mM Tris-HCl/25 mMMg9l2 at pH 7.5.Membranes were pretreated with [3H]HBI, [3H]DHA,

125I-HYP, or Gpp(NH)p for 30 min at 25°. Since pyrocatechol(0.7 mM) and ascorbic acid (4 mM) were included during the[3H]HBI preincubation phase to protect the radiolabeled agonistfrom degradation or oxidation, these agents were included in

Abbreviations: [3H]HBI, (+)[3H]hydroxybenzylisoproterenol; [3H]-DHA, (-)[3H]dihydroalprenolol; 125I-HYP, hydroxybenzylpindolol;Gpp(NH)p, guanyl-5'-yl imidodiphosphate.

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Proc. Natl. Acad. Sci. USA 75 (1978) 229

all preincubations to standardize the methodology. After thepreincubation phase, membranes were washed with ice-coldbuffer by centrifugation at 30,000 X g for 10 min.

Solubilization. fl-Adrenergic receptor binding and adenylatecyclase activity were solubilized by suspending the membranesin 50 mM Tris/5 mM EDTA at pH 7.5 at 40 containing 0.6%digitonin and 4.5-5.5 mg of membrane protein in 3 ml of dig-itonin buffer, as described (11, 12). This suspension was stirredon ice for 30 min and then centrifuged for 60 min at 105,000X g. The supernatant from this centrifugation represents the"solubilized preparation".Column Chromatography. The AcA 34 Ultragel resin was

chosen for gel filtration in these experiments because of its re-ported exclusion properties (4 X 105 daltons) in comparison withSepharose 6B (6 X 106 daltons), while it still allowed rapid flowconditions because of its polyacrylamide-agarose matrix.Predictably, the narrower molecular size fractionation rangeof AcA 34 resulted in considerably greater resolution of [3H]-HBI-induced alterations in fl-adrenergic receptor profiles whencompared with Sepharose 6B.

Solubilized preparations, concentrated 10- to 15-fold by ul-trafiltration through an Amicon PM-30 membrane, were ap-plied to AcA 34 Ultragel columns and eluted in the presenceof 0.1% digitonin/75 mM Tris/25 mM MgCl2/1 mM ethyleneglycol bis(fl-aminoethyl ether) N,N,N',N'-tetraacetic acid, 1mM 2-mercaptoethanol at pH 7.4 at 4°. The dimensions of theAcA 34 columns were 2.5 X 90 cm, and the flow rate wasmaintained at 18 ml/hr.We have refrained at present from calculating a corre-

sponding Stokes radius, R,, for the eluted receptor and enzymeactivities since, in contrast to our experience with Sepharose 6B,some of the soluble marker proteins used do not elute as pre-dicted. The most marked deviation is observed with ferritin,or apoferritin, and may be due to direct interactions of thisprotein with the Ultragel.

All elution profiles reported were observed in multiple ex-periments on three or four separate AcA 34 columns. Thus, thebehavior of some of the calibrating proteins and, more impor-tantly, the agonist-induced increase in apparent fl-adrenergicreceptor size described in Results are not a consequence ofabnormalities resulting from unusual packing of a particularcolumn.

Binding Assays. The methods used in this study for the as-sessment of "specific" fl-adrenergic receptor binding in par-ticulate preparations with [3H]DHA (12) and [3H]HBI (7) andin solubilized preparations with [3H]DHA (11) have beenpreviously reported.Adenylate Cyclase Assay. Adenylate cyclase activity was

assayed as described for particulate (10) and solubilized (11)preparations.

Proteins. Proteins were determined by the method of Lowryet al. (14), with bovine serum albumin as standard. When sol-ubilized material was assayed, 0.6% digitonin buffer was si-multaneously assayed as a blank.

RESULTSThe ,B-adrenergic receptor of frog erythrocyte membranes canbe solubilized by exposure to digitonin, a glycoside with milddetergent properties (12). Fig. 1 compares the elution profilesof the solubilized ,-adrenergic receptor derived from frogerythrocyte membranes pretreated with radiolabeled agonist(Fig. 1B) and antagonist (Fig. 1C) with the elution profile ofreceptor binding assayed in the column fractions in the absenceof membrane pretreatment (Fig. IA). When the membraneshad not been exposed to either ,B-adrenergic agonist or antag-onist ligands prior to solubilization, the ,B-adrenergic receptor

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FIG. 1. Gel filtration of digitonin'-solubilized ,B-adrenergic re-ceptors on AcA 34 Ultragel. (A) Elution profile of ,B-adrenergic re-ceptor binding activity solubilized from control frog erythrocytemembranes. The data plotted represent [3H]DHA binding detectedin the eluted fractions. The specific activity of [3H]DHA binding was0.34 pmiol/mg of protein in the soluble starting material (40 ml) priorto concentration by ultrafiltration. 3H]DHA, 33 Ci/mmol, was presentin the binding assay at 20 nM. (B) Elution profile of agonist [3H]HBIreceptor complex (-). Fvrog erythrocyte membranes were pretreatedwith 2 nM [3H]HBi. Specific [3H]HBI binding at the end of the 30-min preincubation at 26° was 68% of the total radioligand binding.An assessment of the elution profile of "nonspecific" [3H]HBI bindingwas made by chromatographing solubilized material derived frommembranes pretreated with 5.3 nM [3H]HBI in the presence of lOIuM(±)propranolol, (,&). (C) Elution profile of antagonist [3H]DHA re-ceptor complex (0). Membranes were pretreated with 10 nM [3H]-DHA. Specific [3H]DHA binding at the end of the 30-min preincu-bation at 25° was 87% of the total radioligand binding. Consistent withthe known reversibility of antagonist [3H]DHA binding, a significantportion of radioactivity added to the column was retrieved in the saltvolume. Dissociating radioligand probably accounts for the increasedradioactivity detected beginning at -250 ml. An assessment of"-nonspecific" [3H]DHA binding was made by chromatographingsolubilized material derived from membranes pretreated. with 10 nM[3H]DHA in the presence of 0.1 mM (-)isoproterenol (,&). The mo-lecular weights of the soluble marker proteins are: ferritin, 456,000;catalase, 250,000; IgG, 166,000; bovine serumn albumin (BSA), 67,000;and ovalbumin, 45,000.

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230 Biochemistry: Limbird and LefkowitzPr.NaiAcdSc.UA7(98

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FIG. 2. Elution profiles of membrane-labeled [3H]HBI-receptorcomplex (M& and specific [3H]DHA binding (0) in the AcA 34 Ultragelcolumn elu'ates. Frog erythrocyte membranes were pretreated with

0.8 nM [3H]HBI prior to solubilization. Specific [3HJHBI binding at

the end of the 30-mmn, 250 incubation was 85% of the total radioligand

binding. Specific [3H]DHA binding in the soluble preparation was

0.216 pmol/mg of protein prior to concentration by ultrafiltration.

[3H]DHA, 48 Ci/mmol, was present at 6 nM in the binding assay.

binding activity eluted from the AcA 34 column at a peakelution volume of 216 ml 4- 0 (n = 3) (Fig. 1A). In separate

control experiments we documented that this characteristic

elution profile was not altered when membranes were incu-

bated in the absence of #3-a'drenergic agents for 30 min at 250.

Incubation of membranes for 30 min at 250 in the presence of

the f3-adrenergic antagonist [3H]DHA resulted in the solubili-

zation of a [3H]DHA-membrane Iprotein complex which also

eluted in the same -region as the native receptor, VE = 213, 4

0.9 ml (n = 2) (Fig. 1C). This peak of radioactivity cannot be

attributed to nonspecific (ije., nonreceptor) radioligand bindingto membrane components since membrane pretreatment with

[3H]DHA in the presence of saturating unlabeled isoproterenol

concentrations resulted in a complete loss of this peak and the

[-SH]DHA was retrieved in the salt volume of the column, peakat -'-330 ml.

In contrast to the elution profile observed for the native or

antagonist-occupied fladrenergic receptor, Fig. lB demon-

strates that pretreatment of frog erythrocyte membranes with

the agonist [,3H]HBI prior to solubilization resulted in the elution

of a [3H]HBI-membrane protein complex at 192.5 4- 0.6 ml (n

=10). Since this peak of radioactivity was not observed when

membranes were preincubated with [,3H]HBI in the presence

of saturating concentrations of unlabeled propranolol, this peakwas not due to nonspecific radioligand binding to membrane

proteins. We refer to this peak eluting at 192 ml as the [,3H]-HBI-receptor complex.When a relatively low concentration of [3H]HBI was used

in the preincubation phase so that only a portion of the receptor

population was occupied by the agonist radioligand, the

adrenergic receptor binding activity of the unoccupied sites

could be detected in the eluted fractions with [3-H]DHA. The

fl-adrenergic receptors not occupied by agonist ligand prior to

solubilization appeared to elute independently of the [3H]HBIreceptor complex and in the same region as the native receptor

(VE = 216 ml) (Fig. 2). In a large series of experiments, we

observed that increasing the concentration of [-3H]HBI in the

preincubation phase reduced our ability to reliably quantitate

[3H]DHA binding sites in the eluted fractions. This was not

unexpected, since, as shown in Table 1, increasing the con-

centration of [3H]HBI present during the 30--mmn membrane

preincubation phase decreased the number of (3H]DHA

binding sites that can be subsequently assayed in the solubilized

Table 1. Effect of increasing concentrations of [3HJHBI agonistligand during membrane preincubation on the amount of [3H]-DHA binding detectable in subsequently prepared solubilized

material

Specific Specific[3H]HBI binding [3H]DHA binding

nM [3H]HBI after in solubilizedduring preincubation, preparations,

preincubation cpm/ml cpm/mg protein

Control 0 3907.90.8 1160 30241.4 1970 15962.3 6230 815.79.5 6920 847.3

Purified frog erythrocyte membranes, suspended in 75mM Tris/25mM Mg9l2/0.7 mM pyrocatechol/4 mM ascorbate at pH 7.5, wereincubated for 30 min at 250 in the presence of increasing concentra-tions of [3HJHBI agonist. Membranes were washed by centrifugationwith 10 volumes of ice-cold buffer. After solubilization, specificbinding activity was assayed in the 100,000 X g supernatant with[3HJDHA (7 nM, 48 Ci/mmol) and binding was corrected for amountof membrane protein solubilized.

material. We have also documented that unlabeled HBI be-haves identically to [3H]HBI in its ability to reduce the numberof receptor sites available for subsequent [H]DHA binding(data not shown). These observations are similar to data ob-tained in membrane preparations (8) that demonstrate thatincreased receptor occupancy by [3H]HBI results in a propor-tionate decrease in receptor sites that can be detected by[3H]DHA. The data from both the particulate and soluble sys-tems suggest that [3H]HBI and [3H]DHA are binding to thesame macromolecule on the frog erythrocyte. Thus, the dif-ferent elution profiles of the [3H]HBI-receptor complex andthe [3H]DHA-receptor complex from the'AcA 34 columnrepresent an [3H]HBI agonist-induced increase in apparentreceptor size rather than independent binding of [3H]HBI and[3H]DHA to two distinct populations of sites.

Since guanyl nucleotides have multiple regulatory effectson the catecholamine-sensitive adenylate cyclase system, weevaluated the effects of Gpp(NH)p on the agonist-induced in-crease in apparent f3-adrenergic receptor size. For these ex-periments, [3H]HBI was incubated with frog erythrocytemembranes for 30 min at 250 in the presence of 10 AMGpp(NH)p. Under these conditions, the amount of [3H]HBIspecifically bound to the membranes at the end of the prein-cubation phase was less than that observed in the absence ofGpp(NH)p, a consequence of the decreased affinity (8) andincreased reversibility (7, 8) of agonist binding in the presenceof guanine nucleotides. When the solubilized preparationsderived from these membranes w'ere applied to the AcA 34resin, the [3H]HBI ligand elutes in both the region characteristicof the agonist-receptor complex and the region characteristicof the antagonist-receptor complex (Fig. 3B). Since the datain Fig. 3B were obtained on an AcA 34 column different fromthat used in Fig. 1, the "agonist-receptor complex" and "an-tagonist-receptor complex" 'regions on this column are shownin Fig. 3A for comparison. The elution profiles in Fig. 3A wereobtained in a "double label" experiment in which frog eryth-rocyte membranes were exposed, sequentially, to [3H]HBI and125I-HYP prior to solubilization.

DISCUSSIONThe data demonstrate that interaction of the f3-adrenergic re-ceptors with an agonist ligand, [3H]HBI, results in a markedchange in the elution properties of the fl-adrenergic receptor

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Proc. Natl. Acad. Sci. USA 75 (1978) 231

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FractionFIG. 3. (A) Elution profile of agonist [3H]HBI-receptor complex

(@) and antagonist 1251-HYP-receptor complex (M) on AcA 34 Ul-tragel. Frog erythrocyte membranes were pretreated with 2 nM[3H]HBI for 30 min at 250, washed, and subsequently treated with13 pM 1251-HYP for 30 min at 250. Specific [3H]HBI binding was 9096and specific 1251-HYP binding was 7096 of total radioligand binding.(B) Elution profile of [3H]HBI prelabeled to membrane f3-adrenergicreceptors in the presence of Gpp(NH)p. [3H]HBI, 8 nM, andGpp(NH)p, 10 AtM, were incubated with frog erythrocyte membranesfor 30 min at 250. Specific [3H]HBI binding was 68% of the total ra-

dioligand binding at the end of the preincubation. The volume of thecolumn fractions in both A and B was 1.2 ml.

from a molecular sieving resin, consistent with an increase inapparent receptor size. This increase in molecular size is notobserved when the fl-adrenergic receptors are occupied by theradioligand antagonists [3H]DHA or 125I-HYP. Thus, the ob-served changes in 13-adrenergic receptor size are agonist specificand may relate to the molecular events responsible for elicitingthe physiological effects of activating agents, in particular,stimulation of adenylate cyclase.The markedly altered [3H]HBI elution profile observed when

[3H]HBI binding is carried out in the presence of Gpp(NH)pfurther substantiates the hypothesis that these observations aremanifestations of physiologically relevant biochemical events.Guanine nucleotides play an important regulatory role in sev-eral phenomena related to 3l-adrenergic receptor-adenylatecyclase "coupling" in frog erythrocyte membranes. For ex-

ample, at low substrate ATP levels, stimulation of adenylatecyclase by catecholamines is not observed in the absence ofguanyl nucleotide (8). At higher ATP concentrations,Gpp(NH)p dramatically enhances the efficacy of catechol-amine stimulation (15). In addition, Gpp(NH)p is able to reverseand prevent agonist-induced "receptor" desensitization studiedin isolated frog erythrocyte membranes (16). Furthermore,Gpp(NH)p-modulated changes in receptor affinity for ,8-ad-

renergic agents are directly related to the "coupling efficiency"of the agents (5, 6). The apparent affinity of full and partialagonists is reduced in the presence of guanyl nucleotides indirect proportion to their intrinsic activity in stimulating ade-nylate cyclase, while receptor affinity for antagonists is unaf-fected. This apparent decrease in receptor affinity for agonistagents may, in part, be due to yet another effect of Gpp(NH)pon this hormone sensitive system, i.e., the promotion of c-om-plete and rapid dissociation of otherwise slowly reversibleagonist binding (7, 8). The biphasic elution profile of the[3H]HBI-receptor complex after solubilization of membranespretreated with [3H]HBI in the presence of Gpp(NH)p isprobably a direct consequence of the effect of guanine nu-cleotides on the affinity and reversibility of agonist binding. Ourfeeling is that the larger apparent molecular size form of theagonist-receptor complex represents the slowly dissociable formof the receptor uniquely induced by agonists which may playa necessary role in enzyme stimulation. We thus postulate thatthe reversal of this agonist-induced increase in apparent mo-lecular size by Gpp(NH)p may represent a molecular con-comitant of the multiple regulatory effects of guanyl nucleo-tides on agonist-promoted physiological effects.The precise molecular events or components responsible for

the agonist-induced increase in apparent receptor size are stillspeculative. The earlier elution of the [3H]HBI-receptorcomplex from the AcA 34 column could be due to a variety ofphenomena, including (i)) receptor interaction with othermacromolecules composing the adenylate cyclase system, e.g.,the catalytic moiety and/or the putative nucleotide binding site(17), (ii) receptor-receptor association promoted by agonistbinding or, (iii) agonist-induced changes in receptor confor-mation resulting in greater molecular assymetry of the bindingsite.

'The test of the hypothesis that the agonist-induced increasein f3-adrenergic receptor size reflects a structural coupling ofthe receptor to adenylate cyclase will rely on the ability to sol-ubilize hormone-responsive adenylate cyclase from the frogerythrocyte membranes and stabilize the labile catalytic activitythroughout the chromatographic procedures. At present, ade-nylate cyclase can be detected in the column fractions onlywhen the membranes are pretreated with 10 ,gM Gpp(NH)por 10mM NaF prior to solubilization. After Gpp(NH)p or NaFpretreatment of membranes for 30 min at 250, adenylate cy-clase is no longer stimulated by catecholamine and the solubi-lized enzyme elutes prior to both native and agonist occupied0-adrenergic receptors [VE = 174 ml, Gpp(NH)p-activatedenzyme; VE = 166 ml, NaF-activated enzyme].

Agonist-induced receptor aggregation could also account forthe increase in apparent receptor size. It has previously beenpostulated that ligand-induced association of integral mem-brane proteins may result in conformational changes crucialfor function (18). For example, the clustering of cell surfacereceptors for antibodies and lectins is induced by the bindingof specific multivalent ligands (18). It is, however, conceivablethat univalent ligands such as ,B-adrenergic agonists might alsobe capable of inducing receptor-receptor association if bindingto the receptor enhanced the affinity of that receptor for otherreceptor molecules (19). There is considerable precedent insoluble enzyme systems for the association of multiple subunitssubsequent to interaction with univalent ligands (20, 21). Theonly evidence to date suggesting receptor-receptor associationin a hormone-sensitive system is the recent report that physio-logical concentrations of gonadotropin releasing hormone in-Aiduce the formation of multiple aggregates of bovine anteriorpituitary receptor protein (22). On the other hand, decreasesin apparent hormone receptor size have been observed in the

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232 Biochemistry: Limbird and Lefkowitz

binding of 125I-labeled glucagon to liver membranes (23) and'25I-labeled insulin to avian erythrocyte preparations (24). Inthe insulin system, it was suggested that the dissociation of themultimeric receptor complex is a molecular concomitant of thenegative cooperativity observed with insulin binding. Althoughnegatively cooperative interactions have also been observedamong the f3-adrenergic receptors in frog erythrocyte mem-branes (13, 25), this cooperativity is induced by both agonist andantagonist ligands. Thus, the agonist-specific increase in fl-adrenergic receptor size appears to be a molecular event in-dependent of the negative cooperativity in this system.The agonist-induced increase in apparent molecular size of

the f3-adrenergic receptor might also reflect a change in re-ceptor conformation to greater molecular asymmetry. Oneinteresting speculation is that agonist occupancy of the p3-ad-renergic receptor lengthens the transmembrane axis of thereceptor molecule, thus allowing its more facile interaction withthe adenylate cyclase enzyme, presumably located on the innerlamella of the biomembrane.

Finally, it should be noted that an implicit assumption in thepreceding discussion has been that the state of the receptor-agonist and receptor-antagonist complexes in the digitonin-solubilized preparations is essentially the same as that in theintact membrane. Alternatively, it is possible that both agonistand antagonist ligands cause alterations in receptor confor-mation or association, but that the agonist-induced alterationsmore effectively resist reversal in detergent solutions than dothe antagonist-receptor complexes.The observed increase in f3-adrenergic receptor size specif-

ically induced by occupancy of the receptor with low levels ofradiolabeled agonist is a demonstration of a molecular eventpotentially involved in eliciting catecholamine-stimulatedphysiological effects. The precise molecular interactions re-sponsible for the agonist-induced increase in the apparentmolecular size of the 3-adrenergic receptor need to beprobed.

We wish to acknowledge Dr. Jacqueline A. Reynolds for helpfuldiscussions and continued encouragement during the course of theseexperiments. The excellent technical assistance and cheerful companyof Ms. Anne Hickey are also gratefully appreciated. L.E.L. is a recipientof a Young Investigator Award, National Institutes of Health Grant21051. This work was supported by Grants HL 20339 and HL 16037from the Department of Health, Education, and Welfare and agrant-in-aid from the American Heart Association. R.J.L. is an Inves-tigator of the Howard Hughes Medical Institute.

1. Robison, G. A., Butcher, R. W. & Sutherland, E. W. (1971) CyclicAMP (Academic Press, New York).

2. Lefkowitz, R. J., Limbird, L. E., Mukherjee, C. & Caron, M. G.(1976) Biochim. Biophys. Acta. 457, 1-39.

3. Maguire, M. E., Wiklund, R. A., Anderson, H. J. & Gilman, A.G. (1976) J. Biol. Chem. 251, 1221-1231.

4. Mukherjee, C., Caron, M. G. & Lefkowitz, R. J. (1975) Proc. Nati.Acad. Sci. USA 72, 1945-1949.

5. Maguire, M. E., Van Arsdale, P. M. & Gilman, A. G. (1976) Mol.Pharmacol. 12, 335-339.

6. Lefkowitz, R. J., Mullikin, D. & Caron, M. G. (1976) J. Biol.Chem. 251, 4684-4692.

7. Lefkowitz, R. J. & Williams, L. T. (1977) Proc. Nati. Acad. Sci.USA 74, 515-519.

8. Williams, L. T. & Lefkowitz, R. J. (1977) J. Biol. Chem. 252,7207-7213.

9. Mukherjee, C., Caron, M. G., Coverstone, M. & Lefkowitz, R.J. (1975) J. Biol. Chem. 250,4869-4876.

10. Limbird, L. E. & Lefkowitz, R. J. (1976) Mol. Pharmacol. 12,559-567.

11. Limbird, L. E. & Lefkowitz, R. J. (1977) J. Biol. Chem. 252,799-802.

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

13. Limbird, L. E. & Lefkowitz, R. J. (1976) J. Biol. Chem. 251,5007-5014.

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

15. Lefkowitz, R. J. (1974) J. Biol. Chem. 249,6119-6124.16. Mukherjee, C. & Lefkowitz, R. J. (1976) Proc. Natl. Acad. Sci.

USA 73, 1494-1498.17. Pfeuffer, T. & Helmreich, J. M. (1975) J. Biol. Chem. 250,

867-876.18. Singer, S. J. & Nicholson, G. L. (1972) Science 175,720-731.19. Singer, S. J. (1976) in Surface Membrane Receptors, eds. Brad-

shaw, R. A., Frazier, W. A., Merrell, R. C., Gottlieb, D. I. &Hogue-Angeletti, R. A. (Plenum Press, New York), pp. 1-24.

20. Hammes, G. G. & Wu, C.-W. (1974) Annu. Rev. Blophys. Bioeng.3, 1-33.

21. Dunne, C. P. & Wood, W. A. (1975) Curr. Top. Cell. Regul. 9,65-101.

22. Zolman, J. & Valenta, L. (1976) "Abstract no. 52," in Proceedingof the Fifth International Congress of Endocrinology Abstracts,p. 21.

23. Klein, I., Fletcher, M. A. & Levey, G. S. (1973) J. Biol. Chem. 248,5552-5554.

24. Ginsberg, B. H., Kahn, C. R., Roth, J. & DeMeyts, P. (1976)Biochem. Blophys. Res. Commun. 73,1068-1074.

25. Limbird, L. E., DeMeyts, P. & Lefkowitz, R. J. (1975) Biochem.Biophys. Res. Commun. 64,1160-1168.

Proc. Natl. Acad. Sci. USA 75 (1978)

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