influence of gz and gi2 transducer proteins in the affinity of opioid agonists to μ receptors

European Journal of Neuroscience, Vol. 10, pp. 2557–2564, 1998 © European Neuroscience Association

Influence of Gz and Gi2 transducer proteins in the affinityof opioid agonists to µ receptors

Javier Garzon, Marian Castro and Pilar Sanchez-BlazquezNeurofarmacologıa, Instituto Cajal, Consejo Superior de Investigaciones Cientıficas, C/Dr Arce 37, 28002 Madrid, Spain

Keywords: 125I-Tyr27 human β-endorphin, antibodies to Gα subunits, G-proteins, mouse brain, opioid binding

Abstract

The affinity displayed by different opioids to µ receptors (ORs) was determined in mouse brain membranesincubated with antibodies directed to Gα subunits of the guanine nucleotide-binding proteins Gi2 and Gz. Assayswere conducted with 10 pM 125I-Tyr27-β-endorphin in the presence of 300 nM N,N-diallyl-Tyr-(α-aminoisobutyricacid)2-Phe-Leu-OH (ICI-174 864), which prevented the binding of the iodinated neuropeptide to δ-ORs.Gpp(NH)p or the preincubation of mouse brain membranes with IgGs to Gi2α or Gzα subunits, promotedreductions in the affinity exhibited by the labelled probe. The potencies of β-endorphin, [D-Ala2,N-MePhe4,Gly-ol5]-enkephalin (DAMGO) and [D-Pen2,5]enkephalin (DPDPE) were reduced after impairing the coupling ofµ-ORs to Gi2 or Gz proteins. Morphine showed a loss of affinity towards the µ-OR after preincubation ofmembranes with IgGs to Gzα subunits. However, it retained its potency after treatment with the anti-Gi2α IgGs.Conversely, [D-Ala2, D-Leu5]enkephalin (DADLE) and [D-Ser2, Leu5] enkephalin-Thr6 (DSLET) showeddecreased affinity to µ-ORs after treatment with anti-Gi2α IgGs, with no noticeable change following the use ofIgGs to Gzα subunits. The affinity exhibited by the opioid antagonists naloxone, naltrexone, naloxonazine and[Cys2,Tyr3,Orn5,Pen7 amide]somatostatin analogue (CTOP) remained unchanged after either treatment.Therefore, the affinity exhibited by opioid agonists of µ-ORs, but not antagonists, depends on the nature of theG-protein coupled to these receptors.

Introduction

Our knowledge of the classes of G-proteins regulated by opioidreceptors in the production of pharmacological and physiologicaleffects has improved over the last few years. The opioid regulationof Gi/Go families was initially suggested after experiments withpertussis toxin inin vitro (Hsia et al., 1984) and inin vivo systems(Parentiet al., 1986; Sańchez-Blazquez & Garzoń, 1988, 1991). Theuse of antibodies directed to specific peptide sequences of Gαsubunits, as well as oligodeoxynucleotides hybridizing mRNA forGα subunits, has provided valuable information on the classes of G-proteins involved in a particular opioid effect. The productive couplingof δ-OR andµ-OR with Gi2, Gi3 and Go proteins has been established(Uedaet al., 1988, 1990; McKenzie & Milligan, 1990; Offermannset al., 1991; Roeriget al., 1992)

It has been convincingly documented that G receptors exhibitselectivity towards certain classes of G-proteins, e.g. kyotorphin(tyrosine–arginine) receptors (Uedaet al., 1989), muscarinic acetyl-choline receptors (Offermannset al., 1994), somatostatin receptors(Law et al., 1994) and dopamine receptors (Luiet al., 1994). Withrespect to opioid receptors, differences have been reported in the G-proteins regulated to produce, among other effects, antinociception.Theµ-OR is functionally coupled to Gi2 and Gz,δ-OR to Gi2 andGi3 andκ3-OR regulates Gi1 and Gi3 (Sańchez-Blazquez & Garzoń,1993; Sańchez-Blazquez et al., 1993, 1995; Raffaet al., 1994;Standifer et al., 1996). In membranes from mouse periaqueductal

Correspondence:Javier Garzoń. E-mail: [email protected]

Received 7 November 1997, revised 25 February 1998, accepted 26 March 1998

gray matter, agonists bindingµ- and δ-ORs promoted the release ofGi2α subunits and stimulated associated lowKm GTPase activity(Garzon et al., 1997a,b). Notably, preferential agonists ofµ-ORs alsoreleased Gzα subunits and stimulated their GTPase activity – an effectthat was poorly exhibited by agonists acting atδ-ORs (Garzońet al., 1997a,b).

Pharmacological studies have revealed that opioid agonists ofpeptide and non-peptide classes interact withµ-ORs in a differentmanner (Wardet al., 1986; Sańchez-Blazquez & Garzoń, 1988;Garzon & Sanchez-Blazquez, 1991). The isolation of the cDNAsencoding different subtypes of the opioid receptor (Kiefferet al.,1992; Evanset al., 1992; Chenet al., 1993; Thompsonet al., 1993)allowed the identification of the receptor domains involved in thebinding of different ligands. Studies with site-directed mutagenesisindicated differences in the binding profiles of agonists and antagon-ists. It was revealed that antagonists bind to larger and more diffusereceptor territories than those implicated in agonist binding. Moreover,small non-peptide ligands with agonist properties, such as sufentanylor morphine, bind to regions of theµ-OR partially distinct from thosebound by peptide agonists such as DAMGO (Xueet al., 1995; Fukudaet al., 1995a,b; Wanget al., 1995). These differences have also beendescribed for the binding of selective ligands atδ-ORs (Befortet al.,1996) andκ-ORs (Menget al., 1995).

The study of the supraspinal antinociceptive effect of opioids

2558 J. Garzoń et al.

mediated byµ-ORs has shown that the impairment of a single classof G-proteins brings about decreases in the potency of some, but notall, agonist. Antagonistic properties are therefore revealed (Sańchez-Blazquez & Garzoń, 1988; Garzoń et al., 1994). Interestingly, adifferent rank order of opioid potency has been obtained dependingon the class of G-protein impaired (Garzoń et al., 1994). In membranesfrom mouse periaqueductal gray matter,δ-OR-binding opioids exhibit-ing low to moderate capacity to activate a certain class of G-proteins(Gi2 or Gz) antagonized the potency of the ligands with higherefficacy for this effect (Garzoń et al., 1997a). These results wereinterpreted in terms of the efficacy of agonists to favour selectivelyreceptor-activation when coupled to one G-protein as opposed toanother (see Kenakin, 1995). This possibility has also been suggestedin certain expression systems: theDrosophila octopamine-tyraminereceptor in Chinese hamster ovary (CHO) cells (Robbet al., 1994),and the pituitary adenylyl cyclase activating polypeptide receptortransfected into LLCPK1 cells (Spengleret al., 1993). It is knownthat coupling of receptors to G-proteins is essential for increasingtheir affinity toward agonists, but not antagonists (DeLeanet al.,1980). However, the classes of Gα subunits are not homogeneous inthe peptide sequences that interact with G receptors. This fact, plusthe finding that not all ligands bind to identical domains in thereceptor, led to the proposal that agonists not only display differentefficacies in activating their receptors when coupled to different G-proteins, but that they might also bind these receptor-G-proteincomplexes with different affinities. Discernment of conformationalcoupled states of the receptor is therefore possible.

In the present study, interactions of theµ-OR with Gi2 and Gztransducer proteins were further examined. The affinity displayed bya series of opioids was determined under conditions which interferedwith the coupling of receptors to G-proteins. This was achieved byincubation of mouse brain membranes in the presence of specificantibodies directed to peptide sequences of Gi2α and Gzα subunits(McKenzie & Milligan, 1990; Sweeney & Dolphin, 1992; Georgoussiet al., 1993; Garzoń et al., 1997b). To label coupled states of theµ-OR to G-proteins and to minimize the labelling of uncoupled receptors,concentrations of an agonist far below its Kd are required. With thisin mind, 125I-Tyr27 humanβ-endorphin, which has a high specificactivity of radiolabelling, was selected as a probe. The results of thisinvestigation showed differences in the affinities displayed by agonistsat µ-ORs when coupled to Gi2 or Gz proteins. These observationscould be of potential interest in the understanding of G receptorsystems.

Materials and methods

Preparation of membranes from mouse brain

Albino, male, CD/1 mice (Charles River, Barcelona, Spain) weighing22–27 g were used to provide experimental tissue. Mice were killedby cervical dislocation. Brains were quickly removed and washed inice-cold 50 mM Tris–HCl, 0.32M sucrose, pH 7.5, at 4 °C. Wholebrains minus cerebella and medulla were then collected and homogen-ized using a Polytron homogenizer (model PT 10-35, Kinematica,Kriens-Luzern, Switzerland) at setting of 3 for 15 s and centrifugedat 1000g at 4 °C for 10 min (Sorvall RC5C, rotor SS-34 Newton,CT, USA). After the pellet was discarded, the supernatant wascentrifuged at 20 000g for 20 min. That pellet was resuspended inbuffer and centrifuged at 20 000g for an additional 20 min. The finalpellet (P2) was diluted in Tris buffer supplemented with a mixture ofprotease inhibitors (0.2 mM phenylmethylsulphonyl fluoride, 2µg/mLleupeptin, and 0.5µg/mL aprotinin) to a final protein concentration

© 1998 European Neuroscience Association,European Journal of Neuroscience, 10, 2557–2564

of about 2µg/µL. This was then divided into fractions and storedat – 70 °C until use.

Binding studies

On the day of the experiment the corresponding aliquot was thawedat 4 °C and brought to room temperature (22 °C) while stirring lightly.To facilitate the access of the IgGs to the target Gα subunits, themembranes were then incubated with 100µM guanosine 59 triphos-phate (GTP), 50 nM β-endorphin in the absence or presence of affinitypurified IgGs (preimmune or directed to Gi2α or Gzα subunits;typically 0.2µg affinity purified IgGs per 1µg membrane protein) at37 °C for 2 h. The concentrations of GTP and unlabelledβ-endorphin,as well as the temperature and interval of the preincubation weredetermined in pilot assays. To remove the unlabelledβ-endorphin,the preincubation mixture was diluted 1 : 15 with Tris buffer. Aftercentrifugation at 20 000g for 20 min, the pellets were resuspended,centrifuged again and finally used for binding assays. In thesecircumstances, the binding profile of iodinatedβ-endorphin to mem-branes preincubated with 50 nM β-endorphin plus 100µM GTP wasidentical to that observed to membranes preincubated in the absenceof these agents (not shown).125I-Tyr27 humanβ-endorphin, obtainedlyophilized, was reconstituted with 100µL of distilled water to give0.25% bovine serum albumin (BSA), 5% lactose, 0.2%L-cysteinehydrochloride, 10 mM citric acid, and 800 KIU/mL aprotinin. Thereconstituted peptide was divided into aliquots of 0.5µCi/5 µL andstored at –20 °C. Iodinated neuropeptide was recovered from theplastic vials (µ 95%) and diluted using binding incubation buffer.Four mL siliconized (Sigmacote from Sigma, St Louis, MO, USA)borosilicate glass tubes were used (adsorption of the iodinated peptidewas reduced to, 5%). For competition assays the incubation mixturesconsisted of 10 pM (µ60 000 c.p.m.)125I-Tyr27 humanβ-endorphinwith varying concentrations of non-labelled opioids (competitionassays of Fig. 1). To restrict the especific binding of iodinatedβ-endorphin to onlyµ-ORs, the following assays were conducted inthe presence of 300 nM ICI-174864: effect of Gpp(NH)p (Fig. 2),competition with varying opioid concentrations (Figs 3 and 4 anddata in Table 1), and saturation assays of125I-Tyr27 human β-endorphin used at concentrations ranging from 0.3 pM to 3 nM (Fig. 3).In all assays, the membrane suspension reached a final concentrationof 0.4 mg/mL, 0.2% BSA, 0.01% bacitracin, incubation volume wasmade up to 2 mL with 50 mM (final concentration) Tris–HCl buffer,pH 7.5. Non-specific binding was assessed in the presence of 1µM

unlabelledβ-endorphin. Samples were incubated in triplicate at 25 °Cfor 90 min in a shaking incubator and were filtered under vacuum(Harvester M-12R, Brandel Gaitherburg, MD, USA) through glassfibre disks (Whatman GF/B, Maidstone, UK) previously immersedfor 3 h in 5 mM Tris–HCl/0.3% polyethylenimine (Sigma), to minimizebinding to the filters. After filtration, the filters were washed threetimes with 4 mL of ice-cold 5 mM Tris–HCl buffer, pH 7.5. Filterswere placed in polyethylene counting vials and counted in a LKBCompugamma CS counter. Protein content was determined using themethod of Lowryet al. (1951).

Chemicals and antisera

(3-[125I]Iodotyrosyl27)-β-endorphin-(1–31) (human) (IM.162, 2000Ci/mmol) was obtained from Amersham (Buckinghamshire, UK).Humanβ-endorphin, (Cys2,Tyr3,Orn5,Pen7 Amide; Somatostatin Ana-log) (CTOP), [D-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAMGO), [D-Ala2,D-Leu5]enkephalin (DADLE), [D-Pen2,5]enkephalin (DPDPE),[D-Ala2]deltorphin II, [D-Ser2,Leu5]enkephalin-Thr6 (DSLET), [D-Thr2, Leu5]enkephalin-Thr6 (DTLET) and [D-Ala2]-Met-enkephalin-

Coupling ofµ receptors to Gz and Gi2 proteins 2559

FIG. 1. Competition curves of different opioids for125I-Tyr27 human β-endorphin specific binding. The labelled neuropeptide was incubated (10 pM

µ60 000 c.p.m.) with mouse brain membranes (minus cerebella and medulla)in the presence of varying concentrations of non-labelled opioids at 25 °C for90 min. The final protein concentration used wasµ 0.4 mg/mL. The resultsare plotted as the percentage of the maximum specific binding of the labelledopioid vs. – log of the molar concentration of the unlabelled opioid. Non-specific binding was assessed in the presence of 1µM unlabelledβ-endorphin.

amide (DAMA) were purchased from Peninsula Laboratories (SanCarlos, CA, USA). Morphine sulphate was acquired from Merck(Darmstadt, Germany).N,N-diallyl-Tyr-(α-aminoisobutyric acid)2-


Phe-Leu-OH (ICI-174 864) from CRB (Cambridge, UK), andnaloxone hydrochloride, naltrexone hydrochloride, guanosine 59-triphosphate sodium salt (GTP), 59-guanylylimidodiphosphate[Gpp(NH)p], leupeptin, aprotinin, bacitracin, phenylmethylsulphonylfluoride and BSA were purchased from Sigma Chemical Co. Naloxon-azine dihydrochloride and dextrorphanD-tartrate came from RBI(Natick, MA, USA) The antibodies used had been previously charac-terized (Sańchez-Blazquezet al., 1993; Garzoń et al., 1997a,b) andwere raised in New Zealand White rabbit (Biocentre, Barcelona,Spain). The antigenic sequences used were: Gi2α internal fragment[115–125: EEQGMLPEDLS] S/1, and the Gzα internal fragment[111–125: C-TGPAESKGEITPELL] W/1 of the cDNA-predictedsequence of these proteins (Jones & Reed, 1987; Matsuokaet al.,1990). IgGs from preimmune and immune sera were affinity-purifiedas described in Garzoń et al. (1995).

Results

Specific binding of 125I-Tyr27 human β-endorphin to µ- andδ-ORs in mouse brain membranes

Labelled antagonists are useful in the detection of changes in thebinding properties of agonists (Georgoussiet al., 1993). In particular,after massive uncoupling of receptors from G-proteins, they maintaintheir affinity toward receptors and permit detection of any decreasein affinity for the competing agonists, even when the specific bindingof labelled agonists is almost undetectable. Notwithstanding, agonistsare the only ligands able to discern neatly between coupled anduncoupled states of the receptor. Under appropriate experimentalconditions, the specific binding of labelled agonists can be practicallyrestricted to coupled receptors. This has been elegantly documentedfor δ-ORs expressed in recombinant baculovirus-infectedTrichoplusiain ‘High 5’ insect cells (Wehmeyer & Schulz, 1997). In these cellsthe agonist DPDPE was a poor competitor for the binding of thelabelled antagonist [3H]-naltrindole, but a good competitor for [3H]-DPDPE-binding. After adding the correct class of exogenous G-proteins, the apparent binding capacity, but not affinity, of [3H]-DPDPE increased as did its potential to compete for [3H]-naltrindolebinding; the antagonist now labelled a larger population of coupledvs. uncoupledδ-ORs. These observations indicate that agonists, butnot antagonists, display a very low affinity towards uncoupledreceptors and that changes in the availability of G-proteins havedirect repercussions on the binding properties of agonists.

The present study tried to discern whether the affinity displayedby an agonist changes after impairing the coupling of the receptor toa single class of G-proteins. It is known thatµ-ORs regulate a varietyof G-proteins in the cellular membrane (see Introduction). Thus, itwas necessary to have a labelled agonist able to tag the receptorcomplexed to Gi2 and Gz proteins with high but similar affinity,otherwise the labelling of one or even both complexes would bemasked by the signals fromµ-ORs coupled to non Gi2 or Gz proteins.This requisite guarantees that the uncoupling of theµ-ORs from oneof these two G-proteins will decrease the affinity displayed by thelabelled probe. The affinity of the radiolabelled agonist will be thatresulting from its binding toµ-ORs, but now coupled to the rest ofthe regulated G-proteins. In competition assays, only those unlabelledligands displaying high affinity to theµ-OR when coupled to theimpaired G-protein reflected reductions in their binding affinity.

The Gi2 and Gz proteins has been described as important for theeffects of the agonist humanβ-endorphin (Sańchez-Blazquezet al.,1995; Garzoń et al., 1997a,b). The iodinated form of this neuropeptidewas therefore selected. To define the specific binding of125I-Tyr27


TABLE 1 Affinity exhibited by opioid ligands toµ-ORs in membranes from mouse brain exposed to antibodies directed to Gi2α or Gzα subunits. Ki in nmol/Land 95% confidence limits were derived by computer modelling of curves for the competition of iodinatedβ-endorphin with each of the unlabelled ligands.Binding studies were performed in the presence of 10 pM 125I-Tyr27 humanβ-endorphin, 300 nM ICI-174 864 and increasing concentrations of the competingligand at 25 °C for 90 min. The data were analysed with the LIGAND programme (Munson & Rodbard, 1980). For each treatment (preimmune IgGs, IgGs toGi2α and to Gzα subunits) homologous (same labelled and unlabelled ligands) and heterologous competition curves (the unlabelled ligands competing for thespecific binding of the labelled prove) were included in the analysis. The one-site model showed a significantly better (P , 0.01) fit than did the two-site model.All assays were repeated either twice (antagonists, [D-Ala2]deltorphin II and DPDPE) or three times (the other ligands). Analyses always gave comparable results

Ki [nM]

Ligand Preimmune Anti Gi2α Anti Gzα

βh-Endorphin 0.3 (0.48–0.18) 0.91 (1.47–0.56)* 1.16 (1.74–0.77)*Morphine 1.40 (2.05–0.78) 2.01 (2.90–1.38) 8.91 (13.00–6.10)*DAMGO 0.92 (1.20–0.67) 5.21 (8.42–3.20)* 3.03 (5.41–1.66)*DADLE 7.84 (10.27–5.96) 19.8 (28.9–13.5)* 6.01 (7.92–4.51)DSLET 52.2 (70.9–38.2) 148 (222–98)* 65.3 (94.2–44.1)DPDPE 235 (380–145) 704 (1196–414)* 896 (1702–471)*[D-Ala2]Deltorphin II 679 (1222–377) 1340 (2412–744) 1239 (2108–728)Naloxone 1.22 (1.74–0.82) 1.64 (2.46–1.09) 1.84 (2.43–1.33)Naltrexone 0.42 (0.55–0.31) 0.31 (0.42–0.21) 0.42 (0.57–0.30)Naloxonazine 0.53 (0.64–0.38) 0.52 (0.69–0.39) 0.38 (0.52–0.27)CTOP 0.48 (0.67–0.34) 1.56 (2.23–1.09) 1.26 (1.73–0.91)

FIG. 2. Effect of antibodies directed to Gi2α and Gzα subunits and Gpp(NH)pon 125I-Tyr27 humanβ-endorphin specific binding toµ-ORs in mouse brainmembranes. Affinity-purified IgGs of anti-Gα or preimmune sera were usedat 0.2µg per 1µg membrane protein. The membranes were incubated with100µM GTP, 50 nM β-endorphin in the presence of preimmune IgGs or IgGsto Gi2α or Gzα subunits, at 37 °C for 2 h. After removal of unbound IgGs,GTP and the opioid agonist (see Materials and methods), pellets wereresuspended and binding assays conducted in the presence of concentrationsof Gpp(NH)p and 300 nM ICI-174 864. Ordinate: for every curve, 100%specific binding is that observed in the absence of Gpp(NH)p. Each point isthe mean6 SEM from three experiments with samples in triplicate.

human β-endorphin toµ-ORs, the competition curves of differentopioids were constructed. The labelled neuropeptide was used at afixed concentration of 10 pM, i.e. at least 30 times lower than its Kdfor theµ-OR (about 0.3 nM) (Garzon et al., 1995; Fig. 3, Table 1).Selective ligands ofδ-ORs, such as the agonists DTLET, DPDPE,[D-Ala2]deltorphin II (Sanchez-Blazquez & Garzoń, 1989; Zakiet al.,1996) and the antagonist ICI-174 864 (Cottonet al., 1984), showeda biphasic pattern with a plateau after inhibiting 10–15% of thespecific binding of125I-Tyr27 humanβ-endorphin-(1–31) (Fig. 1B).This characteristic was also exhibited by dextrorphan (Fig. 1C). The


selective agonists ofµ-ORs, DAMGO, morphine (Handaet al., 1981;Sanchez-Blazquez & Garzoń, 1989; Mattheset al., 1996) and theselective antagonist of this receptor, CTOP (Gulyaet al., 1986),produced the plateau when about 85% of the specific binding of thelabelled neuropeptide was abolished (Fig. 1A,C). These results revealthat binding of 10 pM 125I-Tyr27 humanβ-endorphin mainly bindsto µ-ORs although a small amount binds toδ-ORs. This was expectedasµ receptors are more abundant thanδ receptors in the mouse brainandβ-endorphin displays a higher affinity towardsµ receptors (Garzońet al., 1983; Sańchez-Blazquez & Garzoń, 1989). The binding of125I-Tyr27 humanβ-endorphin toδ-ORs was prevented in the presence of300 nM ICI-174 864. Saturation analysis of this binding toµ-ORsindicated a Kd of about 0.3 nM and a binding capacity ofµ 90 fmol/mg of membrane protein (present work; Garzoń et al., 1995).

Effect of antibodies to Gi2α and Gzα subunits and Gpp(NH)pon the specific binding of 125I-Tyr27 human β-endorphin toµ-ORs

The antibodies to Gα subunits used in the present study havepreviously demonstrated an ability to impair certain opioid-mediatedeffects, e.g. the stimulation of low Km GTPase activity in membranesfrom mouse periaqueductal gray matter (Garzoń et al., 1997b) or theproduction of supraspinal analgesia (Sańchez-Blazquezet al., 1993,1995; Garzoń et al., 1994). The IgGs were used at concentrationspreviously determined as effective inin vitro assays (Garzoń et al.,1997b). To test the effect of these antibodies on the coupling ofµ-ORs to Gi2 and Gz proteins, the membranes were incubated withagents able to release Gα subunits from the trimer Gαβγ, thusfacilitating the access of the IgGs. This was achieved by using 100µM

GTP, 50 nM β-endorphin in the absence or presence of IgGs to Gi2αor Gzα subunits at 37 °C for 2 h. After removal of the unbound IgGs,GTP and the opioid agonist (see Materials and methods), the pelletswere resuspended and binding assays conducted in the presence ofconcentrations of Gpp(NH)p ranging from 1µM to 1 mM and 300 nMICI-174 864. The specific binding exhibited by 10 pM 125I-Tyr27

humanβ-endorphin in presence of 300 nM ICI-174 864, to membranestreated with or without preimmune IgGs was practically identical(about 5800 c.p.m.). The poorly hydrolysed GTP analogue Gpp(NH)p,after exchanging with GDP at Gα subunits, promoted the uncoupling


FIG. 3. Specific binding of125I-Tyr27 humanβ-endorphin toµ-ORs in mousebrain membranes. The Scatchard plots show the iodinated neuropetide’sbinding to membranes preincubated in the absence or presence of preimmuneIgGs. Increasing concentrations of125I-Tyr27 humanβ-endorphin in thepresence of 300 nM ICI-174 864 were incubated withµ 0.4 mg/mL finalprotein concentration at 25 °C for 90 min. In the competition experiments(homologous) 10 pM 125I-Tyr27 humanβ-endorphin was incubated with 300 nM

ICI-174 864 and increasing concentrations of the unlabelled neuropeptideβ-endorphin. The Kd values were derived using the Ligand weighted non-linearleast squares regression programme (Munson & Rodbard, 1980). Details asin Fig. 2.

of µ-OR from G-proteins and produced a concentration-dependentimpairment of 10 pM 125I-Tyr27 humanβ-endorphin specific binding(Fig. 2). The use of antibodies to Gα subunits reduced the specificbinding of the iodinated neuropeptide from 5800 c.p.m. to about 3500c.p.m. In membranes previously exposed to anti-Gα IgGs, Gpp(NH)palso reduced the specific binding of the labelled probe (Fig. 2). Thus,the blockade of Gi2α or Gzα subunits did not prevent theµ-OR toremain coupled to the other classes of G-proteins.


FIG. 4. Heterologous competition curves of morphine, DAMGO and DADLEwith 125I-Tyr27 humanβ-endorphin specific binding. Details as in Figs 2 and 3.

Role of Gi2 and Gz transducer proteins in the affinityexhibited by opioids to µ-ORs in mouse brain membranes

Membranes from mouse brain (minus cerebella and medulla) wereused to determine the affinity exhibited by a series of opioids afterimpairing the coupling ofµ-ORs to either Gi2 or Gz proteins. Insaturation assays of125I-Tyr27 humanβ-endorphin performed in thepresence of 300 nM ICI-174 824, the affinity of the neuropeptide totheµ-OR did not change whether the membranes had been preincub-ated in the presence [0.39 (0.47–0.32)] or the absence of preimmuneIgGs [0.36 (0.43–0.30)nM; Kd are in nmol/L an 95% confidencelimits]. Saturation and homologous competition assays showed thatthe iodinated and the unlabelled forms ofβ-endorphin exhibitedthe same binding affinity toµ-opioid receptors (Fig. 3, Table 1).Competition assays were carried out using 10 pM of 125I-Tyr27 humanβ-endorphin in the presence of 300 nM ICI-174 824. Homologous(same labelled and unlabelled ligand) and heterologous curves (theunlabelled opioids competing for the specific binding of125I-Tyr27

humanβ-endorphin) were included in the analysis for estimation of


FIG. 5. G-protein-directed agonist affinity for theµ-OR. In the model, thisopioid receptor is coupled to two G-proteins (Gi2 and Gz). Each receptor-G-protein complex is bound by two different agonists (morphine and DADLE,or DSLET) but with reversed affinities.

Ki values (Munson & Rodbard, 1980). In this experimental paradigm,the affinity ofβ-endorphin toµ-ORs diminished after exposure of themembranes to either antisera (Fig. 3; Table 1). The specific bindingof the iodinated neuropeptide after treatment with antibodies to Gαsubunits was about 3500 c.p.m. The binding that remained in thepresence of 1µM non-labelled β-endorphin hardly reached 1000c.p.m. This ratio of specific vs. non-specific binding allowed theprecise determination of the Ki values of the competing opioids. Theimpairing activity promoted by the anti-Gα IgGs on the specificbinding of 125I-Tyr27 humanβ-endorphin was prevented after prein-cubating the affinity-purified IgGs with the immunogenic peptides(about 50µg of antigenic peptide for every 0.1µg IgG) for 1 h at25 °C, or following heating the IgGs at 100 °C for 10 min. In theabsence of the antibodies the antigenic peptides were devoid of effecton the binding of iodinatedβ-endorphin (not shown).

The changes in affinity promoted by the antibodies could not berelated to the described binding preferences of the opioid agoniststo µ- or δ-ORs. After treatment with IgGs to either Gi2α or Gzαsubunits, increased Ki values were observed for DAMGO, theselective ligand ofµ-ORs. Morphine, the preferential ligand ofµ-ORs (Sańchez-Blazquez & Garzoń, 1989; Mattheset al., 1996),recognized the action of the IgGs anti-Gzα subunits, but the changein Ki value observed after impairing Gi2α subunits did not reach thelevel of statistical significance (Fig. 4, Table 1). The opposite wasfound for DADLE and DSLET (preferential ligands ofδ-ORs). Onlythe antibody to Gi2α was effective in impairing the binding of theseligands toµ-ORs (Fig. 4, Table 1). As expected, DPDPE and [D-Ala2]deltorphin II, selective ligands ofδ-ORs displayed high Kivalues towards theµ-ORs. In brain membranes treated with eitherantibody, the affinity of DPDPE to this receptor appeared significantlydecreased. A tendency to increase Ki values was observed for [D-Ala2]deltorphin II, although not at the level required for statisticalsignificance (Table 1).

The Ki values exhibited by opioid antagonists such as, naloxone,naltrexone, naloxonazine and CTOP did not change after treatmentwith antibodies to the Gα subunits (Table 1). The low affinityexhibited by ICI-174 864 and dextrorphan toµ-ORs made it difficultto determine the Ki values of these compounds (see Fig. 1B,C)


Discussion

The specific binding of125I-Tyr27 humanβ-endorphin to mouse brainmembranes was effectively restricted toµ-ORs only. Theδ-OR-selective ligands exhibited successive inhibition of the labelled probebinding toδ-ORs andµ-ORs. A concentration of 300 nM ICI-174 864abolished the binding of the iodinatedβ-endorphin toδ-ORs (Garzońet al., 1995; present work). The concentration of 10 pM 125I-Tyr27

humanβ-endorphin was about 30 times lower than the Kd of thisligand towardµ-ORs in this tissue (present work; Garzoń et al.,1995), and facilitated the almost exclusive labelling of receptorscoupled to G-proteins.

The use of antibodies to Gα subunits in functional studies requiresthe labelling of the native protein and the subsequent impairment ofits assigned function. The antisera used are directed to peptidesequences of the helical domain of Gα subunits, which sufferconformational changes during nucleotide exchange (Noelet al.,1993; Lambrightet al., 1994). These antisera have proved useful forrecognizing the corresponding Gα subunits inin vitro studies (Garzońet al., 1997a,b). Moreover, the anti-Gα IgGs also impair the functionsmediated by their antigens inin vivo (Sanchez-Blazquezet al., 1993,1995; Garzoń et al., 1994) and inin vitro systems (Garzoń et al.,1997b). In contrast to other approaches, e.g. the use of pertussis toxinor guanosine 59 triphosphate analogues, antibodies provide selectivitytowards a single class of G-protein. In the mouse brain membranesof the present study, Gpp(NH)p non-specifically uncoupled thereceptors from the G-proteins, thus promoting a reduction of thebinding of the labelled probe toµ-ORs. In membranes previouslyexposed to anti-Gα IgGs, Gpp(NH)p also reduced the specific bindingof the labelled probe. Thus, after reducing the availability of oneclass of G-protein, theµ-ORs remained coupled to other classes thatalso undergo regulation by these receptors.

The changes detected in the binding of the agonists toµ-ORs afterinterfering with the coupling of these receptors to Gi2 or Gzproteins indicate that the transduction regulated by this receptor isheterogeneous. The affinity exhibited by pharmacological antagonistswas not influenced by the uncoupling of the receptors from G-proteins. Negative results must be analysed carefully as they mightreflect lack of effect of the antisera; however, the data from theagonists clearly indicated that the IgGs did reach their antigen targetsin the mouse brain membranes. Therefore, and in agreement with theidea of antagonists displaying similar affinities to coupled anduncoupled states of the receptor, the anti-Gα antibodies did not alterthe binding affinity of these agents. Decreases in the affinity ofagonists asβ-endorphin, DAMGO and DPDPE could be detectedafter promoting the uncoupling ofµ-ORs from either Gi2 or Gzproteins. This indicates that these agonists bind with similar affinityto this receptor when coupled to either class of G-protein. Changeswere only detected with the agonist morphine after uncouplingµ-ORs from Gz, but not from Gi2 proteins. The opposite was seen forDADLE and DSLET. The binding of both agonists only exhibitedreduced affinity after uncouplingµ-ORs from Gi2 proteins. Thissuggests that certain agonists have the capacity to bind with greateraffinity when the receptor is coupled to a particular type of G-protein (Fig. 5).

There is ample literature describing pleiotropic agonist responsesat a single receptor. Coupling to a single or to multiple G-proteinsmight account for this phenomenon. Differences in the activationprofiles of agonists can then be explained on the basis of heterogeneoustransduction and efficacy in the activation of all, or the most efficientlycoupled, G-proteins. However, in certain circumstances, agonistsacting on the same receptor show reversal of potency. This suggests


the existence of active agonist-specific receptor states (see Kenakin,1995). Accordingly, some agonists might promote one receptor-G-protein complex while others favour the association of the receptorwith a different G-protein. A few examples in which the receptormediates activation of two different signalling cascades have beenreported: the production of cyclic adenosine monophosphate (cAMP)and inositol phosphate mediated by splice variants of the pituitaryadenylyl cyclase activating polypeptide (Spengleret al., 1993), andthe attenuation of cAMP and Ca21 transients via different couplingmechanisms ofDrosophila octopamine-tyramine receptor expressedin CHO cells (Robbet al., 1994). When the receptor is coupled to acertain class of G-protein, a highly efficient agonist, following itsbinding to the same receptor when coupled to a different G-protein,should act as a partial agonist or even antagonist of an effect moreefficiently promoted by another agonist. This has already beendescribed in the production of antinociception for opioid agonistsat µ- or δ-ORs. The impairment of a single or several classes of G-proteins promotes the reversal of agonist potencies. Certain ligandseven exhibit antagonist properties (Sańchez-Blazquez & Garzoń,1988; Garzoń et al., 1994). After reducing the availability of Gi2proteins, DADLE antagonized the analgesic potency of morphine inmice. Conversely, the reduction of functional Gz proteins broughtabout the antagonism of DADLE-evoked antinociception by morphine(Garzon et al., 1994). Antagonism was also described inin vitrosystems studyingδ-OR-mediated activation of Gi2 or Gz proteins.In mouse periaqueductal gray membranes, DPDPE and [D-Ala2]del-torphin II, both agonists atδ-ORs, produced different levels ofG-protein-activation. DPDPE acted as a potent antagonist of [D-Ala2]deltorphin II on the activation of Gi2 or Gz proteins (Garzońet al., 1997a).

Certainly, the activity of the agonists depends directly on theaffinity and intrinsic efficacy towards their receptors. However, inlight of this and previous reports describing agonist-dependent bindingdomains of the receptor, and the capacity of receptors to discriminatebetween G-proteins (see Introduction), it would appear that not onlyagonist efficacy but also affinity depends on the nature of the G-protein coupled to the receptor.

AcknowledgementsThis work was supported by SAF95-0003 (J.G.) and FIS97/0506 (P.S.B.)

AbbreviationsCTOP Cys2,Tyr3,Orn5,Pen7 Amide; Somatostatin AnalogDADLE [D-Ala2,D-Leu5] enkephalin[D-Ala2]deltorphin II Tyr-D-Ala-Phe-Glu-Val-Val-Gly NH2DAMA [D-Ala 2]-Met-enkephalinamideDAMGO [D-Ala2,N-MePhe4,Gly-ol5] enkephalinDPDPE [D-Pen2,5] enkephalinDSLET [D-Ser2, Leu5] enkephalin-Thr6

DTLET [D-Thr2, Leu5] enkephalin-Thr6

G-protein guanine nucleotide-binding regulatory proteinGpp(NH)p 59-guanylylimidodiphosphateGTP guanosine 59 triphosphateICI-174864 N,N-diallyl-Tyr-(α-aminoisobutyric acid)2-Phe-Leu-OHOR opioid receptor

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influence of gz and gi2 transducer proteins in the affinity of opioid agonists to μ receptors

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