Proc. Nati. Acad. Sci. USA Vol. 86, pp. 7321-7325, October 1989 Biochemistry Antagonists with negative intrinsic activity at 6 opioid receptors coupled to GTP-binding proteins (guanine nucleotide-binding regulatory proteins/GTPase/ternary complex)

TOMMASO COSTA* AND ALBERT HERZ Department of Neuropharmacology, Max-Planck-Institut fuer Psychiatrie, Am Klopferspitz 18a, Martinsried, Federal Republic of Germany Communicated by H. W. Kosterlitz, June 19, 1989 (received for review November 29, 1988)

ABSTRACT According to classical models of drug- nucleotides (11). The correctness of this model was recently receptor interactions, competitive antagonists share with ago- demonstrated by direct reconstitution ofpurified ,-adrenergic nists the ability to bind to a common site on the receptor receptors and the stimulatory G protein of the adenylate molecule. However, they are different from agonists, as they cyclase system (Gs) in liposomes (12, 13). Accordingly, the cannot trigger the "stimulus" that leads to biological re- intrinsic activities of receptor ligands represent their ability to sponses-i.e., they lack intrinsic activity. For those receptors stabilize the ternary complex and range from null values for whose signals are transduced to effector systems by GTP- antagonists, which passively occupy the binding site, to var- binding regulatory proteins (G proteins), a mechanistic equiv- ious degrees ofpositive values for partial and full agonists (11). alent of such a stimulus is an increased ability of agonist-bound At muscarinic, D2 dopaminergic, and A1 adenosine receptors, receptor to accelerate nucleotide exchange and thus GTPase however, guanine nucleotides exert more complex effects, activity on the G-protein molecule. Here we show that for a since agonist and antagonist binding to these receptors are member of this family of receptors (6 opioid receptors in reciprocally modulated by purine derivatives (14-16). To membranes of NG108-15 neuroblastoma-glioma cells), two accommodate these observations, an extension ofthe ternary- types ofcompetitive antagonists can be distinguished. One type complex model has been proposed that assumes that antago- has no intrinsic activity, since it neither stimulates nor inhibits nists can be "active" in promoting the dissociation ofreceptor the GTPase activity of G proteins and its apparent ainmity for from G protein (17). The implicit prediction of this extended the receptor is not altered by pertussis toxin-mediated uncou- model is that an antagonist may have negative intrinsic activity pling ofreceptor and G protein. The second type, however, can that should be apparent in its ability to inhibit the tonic inhibit GTPase and thus exhibits negative intrinsic activity; its activation of G proteins resulting from the spontaneous asso- affinity for receptors is increased following uncoupling from G ciation between "empty" receptor and G protein (precoupled proteins. The existence of antagonists with negative intrinsic complex). Reconstitution studies in liposomes have shown in activity may be a general feature of several classes of neuro- at least one case that an antagonist can inhibit the constitutive transmitters or hormone receptors and calls for a reevaluation activation ofG proteins by receptors in the absence ofagonist of biological effects produced by competitive antagonists. (13), but it is not clear whether such effects can occur in intact membranes. Here we show that for a 6-type opioid receptor in native membranes of NG108-15 cells (mouse neuroblastoma- Although ,u and 8 opioid receptors can be clearly distin- rat glioma hybrid cell line), two types of antagonists can be guished on a pharmacological basis (1), recent evidence (2, 3) distinguished: those that lack any, and those that have some indicates that these two types ofreceptors share the ability to negative intrinsic activity. interact with GTP-binding regulatory proteins (G proteins). In this respect, they belong to a large family of hormone and neurotransmitter receptors whose signals are transmitted to MATERIALS AND METHODS enzymes and ion channels across plasma membranes by GTPase Assays. Culturing of NG108-15 cells, membrane intervening G proteins (reviews in refs. 4-6). Activation of preparation, and GTPase assays were performed as de- one or more G proteins, which results in increase of GTPase scribed (18-20). The GTPase assay mixture (0.1 ml) con- activity, is the first detectable biochemical event that follows tained 50 mM Hepes/Tris (pH 7.5), 150 mM either NaCI or recognition of this group of receptors by agonists, regardless KCl, 10 mM MgSO4, 1 mM adenosine 5'-[,8,y-imidoltriphos- of the sort of signal that is actually propagated to effector phate (Li'), 0.5 mM ATP (Tris), 5 mM creatine phosphate molecules (5, 6). Receptor-mediated activation of G proteins (Tris), 2.5 units of creatine kinase, 2.5 mM cyclohexylam- involves the establishment of a ternary complex between monium phosphate, 0.2 mM dithiothreitol, 0.2 mM EGTA, ligand-occupied receptor and G protein, as suggested long 200 nM [y-32P]GTP (4-7 x 104 cpm per pmol), 5-8 ,ug of before the isolation ofG proteins. The findings (i) that guanine membrane proteins, and opioid ligands as indicated. Reac- nucleotides exert negative heterotropic effects on the affinity tions were started by the addition of membranes, allowed to of the receptor (7) only when the receptor is occupied by an proceed at 37°C for 10 min, and arrested by the addition of0.1 agonist (8, 9), (ii) that a receptor prelabeled by agonists can be ml of ice-cold 40 mM H3PO4. The amount of Pi released was solubilized in a higher molecular weight form than when determined using activated charcoal as described (18). High- prelabeled by antagonists (10), and (iii) that agonists but not antagonists display complex binding isotherms in the absence Abbreviations: G protein, GTP-binding regulatory protein; DADLE, of guanine nucleotides (11) were all explicable by a general [D-Ala2,D-Leu5]enkephalin; ICI 174864, [NN'-diallyl-Tyr1, model according to which agonists, but not antagonists, pro- Aib2'3]Leu5enkephalin; ICI 154129, [N,N'-diallyl-Tyr1, 4- mote the association of receptor to G protein in membranes (CH2S)Phe4,Leu5]enkephalin. The terms "neutral" and "negative" and that the resulting complex can be destabilized by guanine indicate antagonists having null and negative intrinsic activities, respectively. They are used for conciseness and not meant to suggest any new terminology. The publication costs of this article were defrayed in part by page charge *Present address: Laboratory of Theoretical and Physical Biology, payment. This article must therefore be hereby marked "advertisement" National Institute of Child Health and Human Development, NIH, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Building 10, Room 6 C 101, Bethesda, MD 20892. 7321 Downloaded by guest on September 26, 2021 7322 Biochemistry: Costa and Herz Proc. Natl. Acad. Sci. USA 86 (1989) affinity GTPase was determined following the subtraction of and thus not easy to characterize. We performed a detailed the cpm of Pi hydrolyzed in the presence of 50 AM GTP study on the ionic requirements for this effect (unpublished (low-affinity GTPase) (19). Low-affinity GTPase was not work). The addition of cyclohexylammonium ions (2.5-5 affected by any opioid ligand at any concentration. mM) to the reaction mixture slightly potentiated the inhibi- Pretreatment of Cells or Membranes. In some experiments tory effect of the antagonist. A pronounced effect was membranes or cells were preincubated with an antagonist to observed upon replacement of Na' by K+ (Fig. 1). Substi- free receptors from possible contaminating endogenous lig- tution of K+ for Na' (maintaining Cl- constant at 150 mM) and. Membranes (1 mg/ml) were preincubated with or with- produced an increase in basal GTPase activity, a decrease in out 100 ,M MR 2266 in 50 mM Hepes/Tris, pH 7.5/0.5 mM the net activity stimulated by the agonist, and a correspond- EGTA/0.5 mM dithiothreitol for 30 min at 4°C, diluted 5-fold ing increase in the inhibition by ICI 174864. Examination of with the same buffer, and centrifuged at 25,000 X g for 15 min the concentration-response curves of several antagonists for at 4°C. After three cycles of resuspension and centrifugation modulation of GTPase indicated that [N,N'-diallyl-Tyr1, in the same buffer they were assayed for GTPase in the qi(CH2S)Phe4,Leu5]enkephalin (ICI 154129), a close ana- presence and absence of agonist or antagonist (both 10 ,uM) was also effective in reducing basal as indicated. Confluent monlayers of NG108-15 cells were logue of ICI 174864, preincubated in growth medium (18) in the absence or pres- activity; naloxone produced a much smaller effect; the ben- ence of either MR 2266 (100 AM) or [D-Ala2,D-Leu5]- zomorphan antagonist MR 2266 had no inhibitory effect, enkephalin (DADLE, 100 nM) for 24 hr. Cells were har- whereas the oripavine antagonist diprenorphine was a partial vested, washed from the ligand as described (18), and frozen agonist (Fig. 1 a and b). Thus, three types of antagonists can as a pellet (-70°C) prior to membrane preparation and be distinguished: those exhibiting negative intrinsic activity GTPase assay. For N-ethylmaleimide treatment, membranes (ICI analogues), those with a partial negative effect, such as were centrifuged, to remove traces ofdithiothreitol present in naloxone, and those with null intrinsic activity, such as MR storage buffer (18), and resuspended (1-2 mg/ml) in 50 mM 2266. The relative intrinsic activity of agonists and antago- Tris/HCl (pH 7.5) containing various concentrations of nists was dependent on the type of cation present in the freshly prepared N-ethylmaleimide. Incubations lasted 30 reaction mixture. For example, diprenorphine was a weaker min at 4°C and were stopped by the addition of 10 mM partial agonist (30-40% of the maximal effect produced by dithiothreitol (final concentration). After centrifugation, the DADLE) in Na' than in K+ (maximal effect 70-80% of that membranes were resuspended and assayed for GTPase. For of DADLE). Antagonists were affected in an opposite but pertussis toxin treatment, confluent monolayers ofNG108-15 symmetrical manner: in K+, ICI 174864 produced a maximal cells were incubated with the indicated concentrations of inhibitory effect clearly larger than that of ICI 154129, toxin for 24 hr. The degree of ADP-ribosylation produced in whereas in Na' there was little difference in the maximal vivo was monitored by the decrease of pertussis toxin- catalyzed incorporation of [32P]ADP-ribose into a 40-kDa GTPase substrate of the membrane (20) and was in good agreement with the diminution of agonist-stimulated or antagonist- 22[ inhibited GTPase (data not shown). Inactivation of opioid receptors in intact cells with 8-chlornaltrexamine was per- E - formed as reported elsewhere (20). 0n 18 Radioligand Binding Studies. For equilibrium binding stud- E -L ies, membranes were prepared and [3H]diprenorphine (40 0 Ci/mmol, New England Nuclear; 1 Ci = 37 GBq) binding was E 14 assayed (18). The reaction mixture (2 ml) contained 50 mM Tris/HCl (pH 7.5), 10 mM MgCl2, 0.1 mM EDTA, 0 or 100 mM NaCl (as indicated), and 300 ,g of membrane proteins and was incubated 60 min at 25°C. Data were analyzed in Log LLigand] accordance with models for the binding to one or two forms C ~~~~~~~~~d 1.9 MR 2266 (pM) 0 A of the receptor based on mass action law, by the method of 0, 6 1.0 Munson and Rodbard (21). Statistical comparisons between CL 01 0.03 A models were also performed as described (21), with the level 0) 1.6 A3 of significance at P = 0.01. The experiments were done at 01 ~~~~~2~~ 0.8 ~2 least twice, with at least two different batches of membranes. The absolute level of GTPase activity varied as much as 0 1.C 2-fold, but the relative proportions of agonist and antagonist U- _-A - 0.6 effects on GTPase varied <10% between different batches of 1.c * ~~~~~Log(MR2266) Log (MR 2266) membranes. -9 -7 -5 -3 -8.5 -6.5 -4.5 Log COADLE] Log (IC1174) RESULTS FIG. 1. Effect ofopioid ligands on GTPase activity in membranes Effect of Opioid Antagonists on GTPase. Agonist-mediated of NG108-15 cells. (a and b) High-affinity GTPase was assayed in a activation of high-affinity GTPase activity in native mem- reaction mixture containing 150 mM NaCl (a) or KCI (b) and various branes for receptors that interact with the Gi/G0 group of G concentrations of opioid agonists and antagonists (U, DADLE; o, proteins is usually amplified in the presence of millimolar diprenorphine; o, MR 2266; *, naloxone; A, ICI 154129; A, ICI concentrations of NaCl (22, 23). Under these conditions, 174864). Basal activity is indicated by a broken line; points are means GTPase in membranes prepared from NG108-15 cells was of triplicate determinations. Data are representative of three addi- tional experiments performed with different batches of membranes. stimulated in the presence ofthe opioid agonist DADLE, and (c and d) Concentration-response curves for the stimulatory effect this stimulation was completely blocked in the presence of of the agonist DADLE, studied in 150 mM NaCl (c), and the the peptide opioid antagonist (N,N'-diallyl-Tyr',Aib2'3, inhibitory effect of ICI 174864, studies in 150 mM KCI (d), in the Leu5]enkephalin (ICI 174864; Aib, a-aminoisobutyric acid). presence of 0-1 ,uM MR 2266. Fractional response is the ratio of However, the antagonist also inhibited basal activity. The GTPase activity in the presence of ligand to that in its absence. inhibition of basal activity was reproducible but small (10%), (Insets) Schild plots (24) of the data. DR, dose ratio. Downloaded by guest on September 26, 2021 Biochemistry: Costa and Herz Proc. Natl. Acad. Sci. USA 86 (1989) 7323 inhibition produced by the two antagonists (Fig. 1 a and b). an effective way to covalently modify a critical cysteine The lack of effect of MR 2266 on GTPase (we observed only residue in the Gj/GO group of G proteins (27). These modi- a small stimulation in the presence of K+) is due to absence fications result in loss of their ability to interact with recep- of intrinsic activity (either positive or negative) and not lack tors (28, 29). Either N-ethymaleimide treatment (Fig. 3a) or of affinity for the receptor. In fact, increasing concentrations exposure to pertussis toxin (Fig. 3b) reduced in parallel of MR 2266 were able to produce parallel rightward shifts in agonist-mediated stimulation and antagonist-mediated inhi- the concentration-response curves of both the agonist (stud- bition of GTPase. Both manipulations also reduced "basal" ied in Na', Fig. ic) and the antagonist (examined in K+, Fig. activity, indicating that a substantial amount of this activity ld). Schild plots (24) obtained from these experiments (Insets reflects an activated state of the G protein, inasmuch as the of Fig. 1 c and d) yielded similar pA2 values (pA2 = negative intrinsic basal activity ofpurified G proteins is not altered by logarithm of the dissociation constant of the antagonist) for pertussis toxin (29). Alkylation of opioid receptors in intact MR 2266-mediated antagonism of DADLE (8.2 ± 0.1) and cells with the irreversible antagonist 13-chlornaltrexamine antagonism of ICI 174864 (7.9 ± 0.12). The effect ofMR 2266 also diminished the effects ofboth agonist and antagonist and was stereospecific: its inactive enantiomer MR 2267 blocked resulted in 20-25% reduction of basal activity (Fig. 3c). neither the effect of DADLE or that of ICI 174864 (data not Multiple Affinity Forms of the Receptor for Agonist and shown). Thus, a ligand devoid of intrinsic activity can an- Antagonist. Antagonists that neither promote nor oppose the tagonize in a similar fashion either the "positive" effect of a formation of the ternary complex between ligand-occupied full agonist or the "negative" effect of an antagonist. receptor and G protein do not discriminate between high- and Reversibility and Dependence ofthe Effect of Antagonists on low-affinity forms of receptor (11, 17) and their affinity is not Receptor Occupancy and Coupling. The question remained as changed by pertussis toxin treatment (29, 30). However, ifan to whether these findings could be explained in terms of true antagonist had greater affinity for the uncoupled form of the ability of some antagonists to reduce the probability of receptor than for the coupled form (17), its apparent binding spontaneous interactions between unoccupied receptors and affinity would conceivably be increased by toxin treatment. transduction protein, or whether they simply indicated that MR 2266 native membranes were contaminated by an endogenous The binding isotherms of DADLE, ICI 174864, and agonist. Indeed, opioid peptides are synthesized in small (Fig. 4 a-c) were obtained from competition for the binding amounts by the NG108-15 cell line (25). Fig. 2 shows that this sites labeled by [3H]diprenorphine and compared in mem- second explanation is unlikely. Preincubation of membranes branes prepared from cells that had been treated with per- with 100 ,uM MR 2266 (sufficient to prevent any binding of tussis toxin. Following toxin-mediated uncoupling, the ap- agonists to the receptor), followed by extensive washing, did parent affinity of the receptor was diminished for the agonist, not abolish the inhibitory effect of ICI 174864 (Fig. 2 Upper). increased for the "negative" antagonist, and unchanged for Likewise, in membranes prepared from cells that had been a grown in the presence of MR 2266 (100 ,uM, 24 hr), the effect 15 of ICI 174864 was comparable to that measured in control cells (Fig. 2 Lower). In contrast, in membranes obtained from cells that had been exposed to the agonist DADLE (100 nM, 24 hr) to desensitize and down-regulate opioid receptors (18, 10 26), neither agonists nor antagonists produced any effect on GTPase (Fig. 2 Lower). Exposure of cells to low doses of pertussis toxin, or membranes to low concentrations of N-ethylamaleimide, is 5 Log (N-ethylmaleimidel M PREINCUBATION OF MEMBRANES V- I b 20 E = BASAL 1 I nAnrAUL74 IiA..MlI(UpM) 'c 15 I r ,cl:1174 1 l0pM ) E 1) 0 E 10 Q.

- N1 -

T- Buffer only MR2266 (l00pM) x Log tPTX] ng ml-1 c i PREINCUBATION OF INTACT CELLS 16 0 12 a 0] I I a I 12 8 -r I 41L -8 -7 -6 -5 4. Log tCNA] M Control MR2266 (lOOpM) DADLE (1OOnM) FIG. 3. Effect ofN-ethylmaleimide (a), pertussis toxin (PTX) (b), FIG. 2. Effect of preexposure of opioid receptors to MR 2266 in or j3-chlornaltrexamine (CNA) (c) on GTPase measured in the isolated membranes (Upper) or intact cells (Lower) on the ability of absence (basal) or presence of agonist (DADLE) or antagonist (ICI ICI 174864 to inhibit basal GTPase. Assays were performed in the 174864). GTPase was assayed in 150 mM KCI (a and b) or NaCl (c). presence of 150 mM NaCl. Data are means of triplicate determinations (± SEM). Downloaded by guest on September 26, 2021 7324 Biochemistry: Costa and Herz Proc. Natl. Acad. Sci. USA 86 (1989)

B/Bo

1.0

0.8 \-CONTROL 0.6 PTX

0.4-

0.2

1.0 -10 -9 -8 -7 -6 -5 -4 Log EIC1174864J M 0.6 FIG. 5. Competition of ICI 174864 for the binding sites of [3H]diprenorphine in membranes obtained from cells treated with either pertussis toxin (PTX, A) or its diluent (control, *). Membranes 0.2 were prepared and assayed simultaneously. Binding was measured in 10 mM MgCl2 in the absence of NaCl. Specific binding in the absence I of ICI 174864 (BO) was 0.58 and 0.49 pmol/mg in control and -1 toxin-treated membranes, respectively. In control membranes there was a considerable improvement (P < 0.005) in goodness of fit with 1.0 MR 2266 a model involving two classes of sites (solid line) as compared to a AF model with one class of sites (dotted line). In toxin-treated mem- c branes a model involving one class of sites could not be rejected (P 0.6 = 0.12). Estimates (± SEM) of the percentage of receptors in the high-affinity (RH) and low-affinity (RL) forms and the corresponding dissociation constants (KH and KL) were as follows: RH = 53 9%, 0.2 RL = 47 8%; KH = 75 + 22 nM; KL = 1.05 0.23 ktM. In toxin-treated membranes the curve was consistent with a single affinity form with K = 63 + 5 nM. -10 -8 -6 in the membrane of a form of receptor tightly bound to an Log (Ligand] M agonist. In fact, the neutral antagonist MR 2266, which lacks FIG. 4. Binding isotherms of DADLE, ICI 174864, and MR 2266 intrinsic activity, could reverse the effects on GTPase ofboth for opioid receptors in membranes prepared from NG108-15 cells the agonist and the negative antagonist ICI 174864; from preincubated for 24 hr in the presence (A) or absence (i) of pertussis Schild plots it is apparent that MR 2266 has similar affinities toxin (10 ng/ml). Binding was measured (in the presence of 100 mM in each instance. This observation is incompatible with the NaCl) as competition for the binding sites labeled by [3H]- idea that basal GTPase activity results from a quasi-irre- diprenorphine (0.25 nM). Nonspecific binding (measured in the versible agonist-bound form of the receptor. Moreover, presence of 10 was gM diprenorphine) subtracted. B/Bo is the ratio washing ofintact cells or membranes after they were of specific binding in the presence of competing ligand to that in its exposed to a concentration absence. Bo was 0.29 and 0.11 pmol/mg of protein in control and saturating of neutral antagonist did not toxin-treated membranes, respectively. prevent inhibition of GTPase by the negative antagonist. The effect of the negative antagonist was abolished in parallel to the "neutral" antagonist. The leftward shift in the competi- that of the agonist when loss of receptor responsiveness was tion curve of ICI 174864 produced by pertussis toxin was caused either (i) by interfering with the receptor itself by more pronounced when binding was assayed in the absence agonist-mediated down-regulation or alkylation by an irre- ofNa' (see also Fig. 5), because, as reported previously (31), versible antagonist or (ii) by interfering with the ability ofthe this ion itself increases the affinity of this antagonist. When receptor to couple to G proteins by ADP-ribosylation by the binding isotherms of ICI 174864 obtained in untreated pertussis toxin or alkylation by N-ethylmaleimide. Collec- membrane were analyzed sequentially by models assuming tively, these data clearly indicate that the inhibitory effect of one or two distinct affinity forms of the receptor, the model ICI 174864 on GTPase does not result from reversal of involving two classes of sites provided a consistently better stimulation by contaminating endogenous agonists, but that fit (P < 0.005; Fig. 5). In contrast, the two-site model did not it requires a functional G protein able to interact with the improve significantly (P = 0.12) the goodness ofthe fit for the receptor and that it depends on receptor occupancy by the binding isotherms obtained in membranes that had been antagonist. In addition, the degree of negative intrinsic ac- ADP-ribosylated by pertussis toxin in vivo (Fig. 5). The tivity of antagonists for GTPase is correlated with an ability competition curves of MR 2266 were satisfactorily modeled of the antagonist to discriminate between the free and the by assuming a simple bimolecular interaction with a single G-protein-bound form ofthe receptor by having high and low class of sites for both the control and the toxin-treated affinities. Thus the data are best explained by, and provide membranes (data not shown). experimental support for, the model of Wregget and De Lean (17), which predicts that antagonists may be "active" by DISCUSSION hindering the ability of receptors to associate spontaneously The major finding of this study is that antogonists for a with G proteins in membranes. The negative intrinsic activity G-protein-coupled 6 opioid receptor can display a spectrum of ICI 174864 can also explain the notable discrepancy in of intrinsic activities that ranges from null to negative values, affinity for binding and potency in bioassays noted previously as determined from their abilities to inhibit basal GTPase for this antagonist (31-33). activity in intact membranes. This inhibitory effect of antag- A further suggestion arising from this study is that basal onists is neither due to contamination of membranes by GTPase activity in NG108-15 cell membranes is due to endogenous opioid agonists nor due to the putative existence stimulated activity, although it is not possible to decide Downloaded by guest on September 26, 2021 Biochemistry: Costa and Herz Proc. Natl. Acad. Sci. USA 86 (1989) 7325

whether this apparent activation results from a truly spon- ative antagonists become a general finding for these type of taneous interaction between empty receptors and G proteins receptors, the distinction among antagonists that have no or from another mechanism. Spontaneous activation of G intrinsic activity and those that have negative intrinsic ac- protein by the receptor in the absence of agonist occurs when tivity will not be ofpurely academic interest. Some biological P-adrenergic receptors and G. are reconstituted in liposomes; effects produced by these antagonists in vivo or in vitro may this tonic activity is stereospecifically reduced by the antag- not necessarily indicate tonic receptor stimulation by endog- onist alprenolol (13). Furthermore, D2 dopaminergic and enous neurotransmitters. Similarly, the receptor hypersen- a2-adrenergic receptors can be purified as tight complexes sitivity that often follows chronic exposure to antagonists with a G protein, and the GTPase activity of the complexes might not simply be the result of hindering endogenous can be stimulated by agonists (34, 35). However, the spon- agonistic imput to the receptor. taneous activity that results from the insertion of P-adren- ergic receptors and G protein in phospholipid vesicles largely Christine Gless performed these experiments with superb skill. We to this receptor are also grateful to Dr. R. Alan North (Oregon Health Sciences depends on the ability of thiols activate University) for stimulating discussions, Dr. David Rodbard (Nation- directly (36). We have shown that the inhibitory effect of al Institutes of Health) for helpful reviews of the manuscript, and antagonists is clearly detectable only when basal GTPase Ursula Bauerle for cell culture and artwork. This work was sup- activity is enhanced by replacing Na' with K+ in the reaction ported by the Deutsche Forschungsgemeinschaft, F.R.G. medium. It is noteworthy that under physiological conditions H. W. (1977) are to the intracellular milieu, where the 1. Lord, J. A. H., Waterfield, A. A., Hughes, J. & Kosterlitz, G proteins exposed Nature (London) 267, 495-499. concentration of K+ is high and that of Na' is low. Appel- 2. Milligan, G. & Klee, W. (1985) J. Biol. Chem. 260, 2057-2063. mans et al. (31) showed that Na' enhances the apparent 3. Ueda, H., Harada, H., Nozaki, M., Katada, T., Ui, M., Satoh, M. & affinity of ICI 174864 for 8 opioid receptors both in rat brain Takagi, H. (1988) Proc. Natl. Acad. Sci. USA 85, 7013-7017. and in NG108-15 cell membranes. The magnitude of the 4. Rodbell, M. (1980) Nature (London) 284, 17-22. was larger for 5. Gilman, A. G. (1987) Annu. Rev. Biochem. 56, 615-649. Na'-induced reduction of IC50 described (31) 6. Neer, E. J. & Clapham, D. E. (1988) Nature (London) 133, 129-134. ICI 174864 than for ICI 154129 and thus correlates with the 7. Rodbell, M., Krans, H. M. J., Pohl, S. & Birnbaumer, L. (1971) J. Biol. degree of negative intrinsic activity observed for these an- Chem. 246, 1872-1876. tagonists in the present study. This finding suggests that Na' 8. Maguire, M. E., Van Arsdale, P. M. & Gilman, A. G. (1976) Mol. selectively regulates the spontaneous association between Pharmacol. 12, 335-339. 9. Lefkowitz, R. J., Mullikin, D. & Caron, M. G. (1976) J. Biol. Chem. 251, receptor and regulatory G protein in native membranes and 4686-4692. that a larger proportion ofthe uncoupled form ofthe receptor, 10. Limbird, L. E. & Lefkowitz, R. J. (1978) Proc. Nat!. Acad. Sci. USA 75, which has high affinity for the negative antagonist, is avail- 228-232. in the membrane in the presence of Na'. 11. DeLean, A., Stadel, J. & Lefkowitz, R. J. (1980) J. Biol. Chem. 255, able 7108-7117. The existence ofantagonists with negative intrinsic activity 12. Asano, T., Pedersen, S. E., Scott, C. W. & Ross, E. M. (1984) Bio- is likely to be a general phenomenon that is not limited to the chemistry 23, 5460-5467. opioid receptors in this particular cell line. Guanine nucle- 13. Cerione, R. A., Codina, J., Benovic, J. L., Lefkowitz, R. J., Birn- effects on antagonist binding have been re- baumer, L. & Caron, M. G. (1984) Biochemistry 23, 4519-4525. otide-mediated 14. Burgisser, E. A., De Lean, A. & Lefkowitz, R. J. (1982) Proc. Natl. ported for muscarinic (14), D2 dopaminergic (15), and Al Acad. Sci. USA 79,1732-1736. adenosine (16) receptors. These observations strongly sug- 15. De Lean, A., Kilpatrick, B. F. & Caron, M. (1982) Mol. Pharmacol. 22, gest that antagonists with negative efficacy may exist for 290-297. most, if not all, G-protein-coupled receptors. This hypothesis 16. Green, R. D. (1984) J. Neurosci. 4, 2472-2476. 17. Wregget, K. A. & De Lean, A. (1984) Mol. Pharmacol. 26, 214-227. has several interesting implications. (i) The interpretation of 18. Vachon, L., Costa, T. & Herz, A. (1987) Mol. Pharmacol. 31, 159-168. antagonist affinities and their use for receptor classification 19. Cassel, D. & Selinger, Z. (1976) Biochim. Biophys. Acta 452, 538-551. are of great importance. If an antagonist has higher affinity 20. Costa, T., Klinz, F. J., Vachon, L. & Herz, A. (1988) Mol. Pharmacol. the form of the receptor that is not associated with G 34, 744-754. for 21. Munson, P. J. & Rodbard, D. (1980) Anal. Biochem. 107, 220-239. protein, its apparent dissociation constant would be expected 22. Kosky, G., Streaty, R. A. & Klee, W. (1982) J. Biol. Chem. 257, to vary depending on the proportion ofpreformed receptor-G 14035-14040. protein complexes in different membranes. Moreover, the 23. Aktories, K., Schultz, G. & Jakobs, K. H. (1981) Mol. Pharmacol. 21, of a negative antagonist with a full agonist may 336-342. competition 24. Arunlakshana, 0. & Schild, H. 0. (1959) Br. J. Pharmacol. 14, 48-58. be not a simply linear, for the two ligands will induce two 25. Glaser, T., Huebner, K. & Hamprecht, B. (1982) J. Neurochem. 39, distinct, although interconvertible, forms of the receptors 59-68. (17). Hence, if an antagonist has negative intrinsic activity, 26. Chang, K.-J., Eckel, R. W. & Blanchard, S. G. (1982) Nature (London) differences in pA2 values of that antagonist determined in 296, 446-448. 27. Winslow, J. W., Bradley, J. D., Smith, J. A. & Neer, E. J. (1987)J. Biol. different tissues and with different agonists would not un- Chem. 262, 4501-4507. equivocally reflect receptor heterogeneity. 28. Asano, T. & Ogasawara, N. (1986) Mol. Pharmacol. 29, 244-249. (ii) The interpretation of biological effects observed in the 29. Kurose, H., Katada, T., Haga, T., Haga, K., Ichiyama, A. & Ui, M. presence of an antagonist may be affected. The concept that (1986) J. Biol. Chem. 261, 6423-6428. 30. Haga, K., Haga, T. & Ichiyama, A. (1986) J. Biol. Chem. 261, 10133- an antagonist may have biological effects opposite to those of 10140. agonists because of an ability to stabilize an antipodal con- 31. Appelmans, N., Carroll, J. A., Rance, M. J., Simon, E. & Traynor, J. R. formation of the receptor is well established in the area of (1986) Neuropeptides 7, 139-143. receptor research; the 32. Corbett, A. D., Gillan, M. C. G., Kosterlitz, H. W., McKnight, A. T., y-aminobutyrate/benzodiazepine Paterson, S. J. & Robson, L. E. (1984) Br. J. Pharmacol. 83, 271-279. term "inverse agonism" has been proposed to describe such 33. Cotton, R., Kosterlitz, H. W., Paterson, S. J., Rance, M. J. & Traynor, an interaction (37). At G-protein-coupled receptors, biolog- J. R. (1985) Br. J. Pharmacol. 84, 927-932. ical effects observed with antagonists are usually interpreted 34. Senogles, S. E., Benovic, J. L., Amlaiky, N., Unson, C., Milligan, G., as result of their ability to block receptor activation Vinitsky, R., Spiegel, A. M. & Caron, M. G. (1987) J. Biol. Chem. 262, the 4860-4867. produced by an endogenous agonist. However, some of the 35. Regan, J. W., Nakata, H., De Marinis, R. M., Caron, M. & Leflowitz, biological effects of antagonists may possibly be due to R. J. (1986) J. Biol. Chem. 261, 3890-3894. negative rather than to zero efficacy. For instance, muscar- 36. Pedersen, S. E. & Ross, E. M. (1985) J. Biol. Chem. 260, 14150-14157. inic receptors in atrial cell membranes are coupled to K+ 37. Braestrup, C., Schmiechen, R., Neef, G., Nielsen, M. & Petersen, E. N. (1982) Science 216, 1241-1243. channels via a pertussis toxin-sensitive G protein (38) and can 38. Pfaffinger, P. J., Martin, J. M., Hunter, D. D., Nathanson, N. M. & depress basal K+-channel activity when occupied by the Hille, B. (1985) Nature (London) 317, 536-538. antagonist scopolamine (39). Should the occurrence of neg- 39. Soejima, M. & Noma, A. (1984) Pflugers Arch. 400, 424-431. Downloaded by guest on September 26, 2021