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).