Journal of Neuroscience Research 31565-572 (1992)

Differential Coupling of Opioid Binding Sites to Guanosine Triphosphate Binding Regulatory Proteins in Subcellular Fractions of Rat Brain M. Szucs and C.J. Coscia Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary (M.S.); Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis (C.J.C.)

In this study, we present evidence for the occurrence of p, 6, and K opioid binding sites in synaptic plasma membranes (SPM) and microsomes of rat brain. Binding to all three opioid classes was inhibited by 5’-guanylylimidodiphosphate (Gpp[NH]p) in SPM, while microsomal sites proved to be insensitive to this GTP analog. Sensitivity was restored upon solubilization of microsomes with digitonin, suggesting that opioid receptors are physically separated from G proteins in this fraction. Modulation of microsomal binding by Na+ and M n + + was greater than that of SPM. Pertussis toxin-catalyzed adenosine diphosphate (ADP) ribosylation revealed the presence of G proteins with a-subunit molecular weights of 40 kDa in both subcellular fractions. Basal low K, GTPase activity in SPM was greater than in rnicrosomes. Etorphine elicited a concentration-dependent stimulation of guanosine triphosphatase (GTPase) activity in SPMs but not in microsomes, indicating functional coupling of opioid receptors to G protein in the former and an uncoupling in the latter. Microsomes from 3-day-old rat brain contained more p opioid sites and they were more sensitive to Gpp(NH)p inhibition than those in adults. These results are consistent with the hypothesis that opioid binding sites in adult microsomes are internalized and G protein uncoupled, while those in neonates are newly synthesized, coupled receptors.

third of the total receptors detected. Since these fractions were obtained by differential centrifugation, sucrose density gradient centrifugation techniques were adopted to confirm the presence of opioid binding in synaptosomes and microsomes (Terenius, 1973; Pert et al., 1974; Simantov et al., 1976; Smith and Loh, 1976; Leyert and Laduron, 1977; Glasel et al., 1980). Subsequently, Roth et al. (1981) initiated a detailed investigation of the subcellular localization of opioid receptors in rat brain. By the combination of differential and density gradient centrifugation, fractions highly enriched in SPM or endoplasmic reticulum and Golgi complexes were prepared as demonstrated by marker enzyme and electron microscopy studies. High-affinity opioid agonist and antagonist binding were found in both fractions (Roth et al., 1981, 1982; Roth and Coscia, 1984; Moudy et al., 1985). Since microsomal receptors comigrated on continuous sucrose density gradients with Golgi and endoplasmic reticulum marker enzymes, they may represent intracellular binding sites (Roth et al., 1982). A notable difference between the opioid binding sites in SPM and microsomes was that the latter displayed reduced sensitivity to guanine nucleotides (Roth et al., 1981; Roth and Coscia, 1984; Szucs and Coscia, 1989). Demonstration of guanine nucleotide-dependent modulation of opioid agonist binding is generally believed to reflect coupling of the receptor to G proteins, a class of membrane-bound, GTP-binding proteins that serve as transducers in several second messenger systems Key words: opioid receptor, subcellular localization, (for a review, see Gilman, 1986). G proteins appear to synaptic plasma membrane, microsome, G protein mediate the actions of opioids on adenylyl cyclase in rat

INTRODUCTION In their demonstration of opiate receptors in rat brain, Pert et al. (1973) found that mitochondrial-synaptosomal and microsomal fractions displayed substantial enrichment of binding, with the latter possessing one0 1992 Wiley-Liss, Inc.

Received March 14, 1991; revised September 24, 1991; accepted October 2, 1991. Address reprint requests to Carmine .I. Coscia, PhD, Department of Biochemistry and Molecular Biology, 1402 South Grand Blvd., St. Louis. MO 63104.

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brain as well as neuronal cell lines (Sharma et al., 1975; Blume et al., 1979; Law et al., 1981; Koski and Klee, 1981; Chen et al., 1988) as well as their effects on Ca' and K + ion channels (Werz and MacDonald, 1984; North et al., 1987; Chen et al., 1988). Biochemical, pharmacological, and neurophysiological studies suggest that many opioid actions involve pertussis toxin-sensitive G proteins, G, or Go (Kurose et a]., 1983; Costa et al., 1983; Abood et al., 1985; Hescheler et al., 1987; Ueda et a]., 1988; Nestler et al., 1989; Vogel et al., 1990). Naf and divalent cations (M+ +) likewise are required for opioid-dependent inhibition of adenylyl cyclase and have been implicated in guanosine triphosphate (GTP) interactions with Gi or Go (Koski and Klee, 1981; Law et al., 1981; Koski et al., 1982; Barchfeld and Medzihradsky, 1984; Werling et al., 1984). The binding of universal opioid agonists to receptors is also subject to regulation by Na' , M + + ,and guanine nucleotides (Simon et al., 1973; Pert and Snyder, 1974; Pasternak et al., 1975; Simantov et al., 1976; Blume, 1978; Childers and Snyder, 1978; Zajac and Roques, 1985). In most cases, Naf and Gpp(NH)p inhibit opioid agonist binding in unfracreverses this effect. tionated membranes, whereas M Recently, age-dependent differential effects of these agents on p, 6, and K opioid binding in unfractionated brain membranes have been described (Szucs et al., 1987, and references therein). Neonatal 6 opioid binding appeared to be more responsive to cations than their adult counterpart. For example, M + potentiated 6 binding (3H-DADLE with a p suppressor), and neonatal (P-5) brain sites were more sensitive to Mnf , Mg+ + , and Ca+ than those of older brain (P-10, P-21, and adult). In contrast, inhibition of p binding (3H-DAMGE) by M + appeared early in development and remained constant until adulthood. Inhibition of K binding (3H-EKC appeared after the with p and 6 suppressors) by M second or third week postnatally depending on the cation elicited a significant reversal of used. Moreover, Mn' Gpp(NH)p inhibition of 6 binding in the early neonatal period, exceeding that observed in the absence of cations. Inhibition of p and 6 binding by Na+ was greater and the Mn+ reversal of this effect was smaller in the first two postnatal weeks than in adults. Finally, kinetic studies in adult brain suggest ionic modulation of opioid receptor ligand interactions may occur before G protein coupling (Spain and Coscia, 1987). The aim of the present work was to determine whether other components of the signal transduction system that are known to operate in SPM exist in microsomes together with opioid binding sites. Here we report on the presence of K as well as p and 6 binding and demonstrate differences in their modulation by guanine nucleotides, Na+ and M n + + in rat forebrain microsomes compared with SPM. Pertussis toxin-sensitive G +

+

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proteins were identified in both fractions. Although microsomal intrinsic GTPase activity was also detected, its stimulation by opioids was not measurable. The results obtained suggest that some microsomal opioid receptors may be physically, but not functionally, uncoupled from G proteins.

MATERIALS AND METHODS Materials Chemicals were obtained from the following sources: enkephalin derivatives DAMGE (Tyr-D-AlaGly-MePhe-Gly-ol) and DADLE (Tyr-D-Ala-Gly-GlyD-Leu) (Bachem, Bubendorf, Switzerland); (+)ethylketocyclazocine (EKC) (Sterling Winthrop, Rennselaer, NY); levorphanol tartrate (Hoffman-La Roche, Nutley , NJ); naloxone (Endo Laboratories, Garden City, NY); 5'-guanylimidodiphosphate (Gpp[NH]p), creatine phosphate and creatine kinase (Boehringer Mannheim GmbH, or Sigma, St. Louis, MO); pertussis toxin (List Biol. Lab., Campbell, CA); [3H]DAMGE (60 Ci/mmol) and [3H]-DADLE (50 Ci/mmol) (Amersham, Arlington Heights, IL); [3H](+)EKC (19-23 Ci/mmol), [ Y - ~ ~ P ] Ci/mmol) GTP (30 Ci/mmol) and [ c x - ~ ~ P I N A(1,000 D (NEN, Dupont, Boston, MA); [3H]naloxone (57 Ci/ mmol) (Dr. G. Tbth, Szeged, Hungary; T6th et al., 1982). Other chemicals were purchased from Sigma. Subcellular Fractionation of Rat Brains Membrane fractions highly enriched in SPM or endoplasmic reticulum and Golgi complexes were prepared from rat forebrains by sucrose density gradient centrifugation as described elsewhere (Roth et al., 1981). Modifications in the protocol were the use of Tris buffer throughout and the omission of phenylmethylsulfonyl fluoride from buffered sucrose solutions. Brains of 3day-old (P-3) pups were subjected to subcellular fractionation following the same procedure as for adults. Our previous marker enzymes studies on adult (Roth et al., 1981) and neonatal (Bem et al., 1991) membranes established that cross contamination between SPMs and microsomes was minimal and cannot account for the microsomal opioid binding. Before utilization, SPM and microsomes from gradient fractions were diluted threefold with 50 mM Tris HCl, pH 7.4, pelleted at 100,OOOg for 1 hr and resuspended in the same buffer for binding assays, in K + phosphate buffer for adenosine diphosphate (ADP) ribosylation, or in 10 mM Tris HC1, pH 7.4, containing 0.6 mM EDTA for GTPase activity measurement. Binding Assays Opioid binding in membranes was measured with l3H1DAMGE (p), [3H]DADLE in the presence of 10 nM

Opioid Binding in Subcellular Fractions

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Fig. 1 . Modulation of p , 6 , and K opioid receptor binding in SPM and microsomes by 50 pM Gpp(NH)p (G), 100 mM NaCl (Na), and/or 0.5 mM MnCI, (Mn). Results are expressed as percent of control specific binding (binding in the absence of regulators) and are the means -+ SEM of at least four separate experiments, each performed in triplicate. Opioid binding was measured with 1 nM [3H]DAMGE( p ) , 1 nM [3H]DADLEin the presence of 10 nM DAMGE (S), and 3 nM [3H]EKCin the

presence of 100 nM DAMGE and 100 nM DADLE (K) at 25°C for 1 hr. Control specific binding (mean ? SEM fmol/mg protein) for SPM was p 125 & 7 , 6 64 16, and K 31 * 4 and for microsomes p 207 5 6, S 83 -I- 6, and K 52 5 9. Difference between SPM and microsomes A P < 0. I , A A P < 0.05. Reversal of Na+/Gpp(NH)pinhibition by MnCI, *P < 0.1, **P < 0.05.

DAMGE (6), and [3H]EKC in the presence of 100 nM DAMGE and 100 nM DADLE (K) as described previously (Szucs et al., 1987). Where appropriate, membranes (==1 mg protein) in a Tris buffer containing protease inhibitors were treated with 1% digitonin at 4°C for 30 min, then centrifuged at 100,OOOg for 1 hr to yield a solubilized preparation as described elsewhere (Simon et al., 1986). The supernatant was diluted tenfold, and an appropriate amount (100-200 p g protein) was incubated with [3H]naloxone or [3H]DAMGE. Solubilized samples were filtered on polyethyleneimine-treated Whatman GF/B filters (Burns et al., 1983). Binding assays were run in duplicate.

SPM and microsomes. ANOVAs were performed when evaluating different treatments on the same sites for a given subcellular fraction.

ADP Ribosylation and Low-K, GTPase Labeling of membranes with [c~-~*P]NAD in the presence of activated pertussis toxin was performed according to Wong et al. (1985), with modifications as reported by Szucs and Coscia (1990). GTPase assays were conducted in triplicate according to Barchfeld and Medzihradsky (1984) as described (Szucs and Coscia, 1990). Low-K, GTPase activity was calculated by taking the difference between values obtained in the absence and presence of 50 pM GTP, as suggested by Cassel and Selinger (1976). Statistical Analysis Means of data from three or more experiments were analyzed with the Student's t test when comparing

*

RESULTS The modulation of p, 6, and K opioid binding in subcellular fractions of rat brain by 50 pM Gpp(NH)p and/or 100 mM Na+ in the presence or absence of 0.5 mM Mn+ was assessed as shown in Figure 1. Na+ inhibited p and 6 binding of microsomes more than that of SPM. The effect of divalent cation alone on opioid binding has been described in a prior report (Szucs and Coscia, 1989). M n + + at the concentration used here was shown to attenuate K binding in SPM by 40% and in microsomes by 23%. In contrast, p binding in both SPM and microsomes was uninfluenced by M n + + , while 6 binding was slightly potentiated (10-20%). Here p and 6 microsomal binding was more sensitive to M n + + in the presence of Gpp(NH)p and/or Na+ than SPM binding. Mn+ partially antagonized the inhibitory effect of Gpp(NH)p in the absence and presence of Na+ . Little or no difference between SPM and microsomal K binding modulation was seen. Gpp(NH)p inhibited opioid agonist binding in SPM preparations similar to unfractionated membranes (Szucs et al., 1987, and references therein), whereas microsomes were relatively insensitive (Figs. l and 2). However, K binding was attenuated by Gpp(NH)p only +

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Fig. 2. Concentrationdependenceof Gpp(NH)peffect on [3H]DAMGEbinding in the absence (0) and in the presence (m) of 125 mM Naf in adult SPM (A), adult microsomes (B), and microsomes of 3-day-old rats (C). Also shown in A is the effect of 200 pM Gpp(NH)p in the absence (A) and in the presence (A)of Na+ in SPM of 3-day-old rats. Results are expressed as means k SEM of three or more experiments. * P < 0.05.

in the presence of Na+ , and both SPM and microsomal bindings were reduced by 50%. The inhibition of SPM binding by Gpp(NH)p was increased in the presence of Na+ additively for p and synergistically for 6 (Figs. 1 and 2). To explore further the “insensitivity” of microsoma1 p opioid binding sites to guanine nucleotides, the effect of 0.2-200 pM Gpp(NH)p in the absence and presence of Naf on [3H]DAMGE binding was measured (Fig. 2A,B). While dose-dependent inhibition was seen in SPM, and the effect of Gpp(NH)p Na+ was additive, microsomal p sites were not affected. Only 200 p M Gpp(NH)p in the presence of Na+ caused a slight inhibition of binding in microsomes. Conversely, p sites in the microsomal fraction of 3-day-old pups (P-3) displayed a dose-dependent inhibition by Gpp(NH)p in the absence and presence of NaC (Fig. 2C), the extent of which was similar to that seen in adult SPM. To determine whether G proteins that have been implicated in opioid signal transduction were present in microsomes, membranes were treated with [cx-~*P]NAD in the presence of pertussis toxin. Autoradiograms showed ADP ribosylation of G proteins with a molecular weight of about 40 kDa in both fractions (Fig. 3). On the basis of molecular weight and pertussis toxin sensitivity, this band corresponds to the a subunits of Gi and/or Go. Labeling of the 40 kDa protein appeared to be less in microsomes than in SPM on the basis of the intensity of bands on autoradiograms. Attempts to quantify G proteins further and to measure the extent of their coupling to opioid receptors are

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Fig. 3. Autoradiographicvisualization of [a-32P]ADP-ribosylated GTP binding proteins. SPM (HM) and microsomes (LM) (40 p g protein per lane) were incubated with 10 pM [a32P]NAD,2.5 pg preactivated pertussis toxin, 1 mM ATP, 100 pM GTP, 10 mM thymidine in K + phosphate buffer, pH 7.0, for 45 min at 37°C. Proteins were separated on 14% sodium dodecyl sulfate gels according to the procedure of Laemmli (1970). After staining with Coomassie brilliant blue, gels were dried and exposed to X-ray films. Positions of molecular weight standards are shown at left.

summarized in Table I. The basal low-K, GTPase activity of the SPM fraction was three times that of microsomes (32 2.7 [n = 31 vs. 10.7 5 0.7 pmol Pi/mg protein/min [n = 51, respectively). While the universal agonist etorphine increased the amount of Pi released by about 30% in SPM, it had little effect on microsomal GTPase activity. These results together with our binding data suggest that some opioid binding sites are not coupled to G proteins in microsomes.

*

Opioid Binding in Subcellular Fractions TABLE I. Low-K, GTPase Activity in Subcellular Fractions of Rat Brain* GTPase activity pmol Pi mg proteidmin SPM None Etorphine 10 p M 100 pM Microsomes None Etorphine 10 pM 100 ILM

Percent stimulation over basal

Both p and 6 agonist binding in microsomes were inhibited to a greater extent by Na+ than those in SPM (Figs. 1 and 2), consistent with our prior findings using less selective enkephalins as radioligands (Roth et al., 1981). Earlier we found that microsomal opioid sites were also more sensitive to M + + (Roth and Coscia, 1984; Szucs and Coscia, 1989). Moreover, Mn+ (0.250 mM) elicited a concentration-dependent inhibition of p and K binding and stimulated 6 binding in both fractions. Here we have demonstrated that Mn+ antagonized the inhibitory effect of Na+ in the absence and presence of Gpp(NH)p in both subcellular fractions. This is in good agreement with earlier findings in unfractionated membranes (Pasternak et al., 1975; Childers and Snyder, 1978) as well as subcellular fractions (Roth and Coscia, 1984). The most prominent feature of microsmal binding sites is their insensitivity to guanine nucleotides. Gpp(NH)p did not significantly affect either tritiated agonist binding (Figs. 1 and 2) or the ability of morphine to compete with the antagonist [3H]naloxone in microsomes (Fig. 4). Conversely, morphine displacement of naloxone in SPM is eliminated by Gpp(NH)p (Roth and Coscia, 1984). Moreover, p, 6, and K opioid receptors were all inhibited by Gpp(NH)p in SPM (the latter only in the presence of Na+). Pertussis toxin-mediated ADP ribosylation revealed the occurrence of G proteins with molecular weights of 40 kDa in both SPM and microsomes, with less labeling seen in the latter (Fig. 3). The presence of G protein was corroborated by measuring basal low-K, GTPase activity (Table I). The potent opioid agonist etorphine stimulated low-K, GTPase activity in SPM by about 3096, suggesting that opioid receptors are coupled to G proteins. These results agree well with our previous data for unfractionated membranes (Szucs and Coscia, 1990) and those of others (Barchfeld and Medzihradsky, 1984; Milligan et al., 1987). Only a slight stimulation over basal activity was measured with etorphine in microsomes. These results taken together with binding experiments suggest that some opioid receptors are not coupled to G proteins in microsomes . Our present results validate previous binding data obtained with less selective radioligands (Roth et al., 1981; Scheibe et al., 1984; Roth and Coscia, 1984; Spain et al., 1988). Consistent and even greater differences were detected for opioid 6 binding sites in a light membrane fraction from NG 108-15 cells that corresponds to microsomes (Sweat and Klee, 1985). Basal adenylyl cyclase and GTPase activities were greatly reduced and were less inhibited or were unaltered by opioid ligands in this fraction. We have discovered that neurohybrid cell light membrane 6 binding is almost totally insensitive to Gpp(NH)p (Belcheva et al., 1991). +

32 42 41

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*Data are values from one to five experiments, each performed in quadruplicate.

One possible explanation for the reduced GTP sensitivity of opioid binding in microsomes would be that these sites are physically segregated from G proteins rather than incapable of interacting due to some modification in their structure. To investigate this possibility, the microsomal fraction was solubilized with 1% digitonin, and morphine competition binding assays were performed with 3H-naloxone as radioligand in the presence and absence of 100 pM Gpp(NH)p. As is shown in Figure 4, Gpp(NH)p decreased the ability of the agonist morphine to compete with the antagonist [3H]naloxone to a greater extent in solubilized microsomes than in their membrane-bound form. Ki for morphine was 190 nM in the absence and 299 nM in the presence of Gpp(NH)p in control microsomes. The corresponding values were 190 nM and 1,200 nM, respectively, in solubilized microsomes. These results indicate that Gpp(NH)p sensitivity of agonist binding in microsomes is restored upon solubilization. Indeed, a dose-dependent Gpp(NH)p inhibition of [3H]DAMGE binding to solubilized microsomes was observed (data not shown).

DISCUSSION In the present study, we provide additional evidence for the existence of p, 6, and K opioid binding in microsomal fractions of rat brain. Site-specific assay conditions have been ensured either by using selective ligands or by suppressing cross reactivity to other sites. The examination of the effects of Gpp(NH)p in the presence of Mn+ and/or Na+ on these receptors suggests that there are fundamental differences between the guanine nucleotide regulation of opioid binding in microsomes and SPM and between the different classes of opioid receptors. +

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- a - 7 -6 -5 log [MORPHINE] , M

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Fig. 4. Effect of Gpp(NH)p on the ability of morphine to compete with [3H]naloxone.Microsomes before (A) or after (B) solubilization with 1% digitonin (as described in Materials and Methods) were incubated with 3 nM [3H]naloxoneand various concentrationsof morphine in the absence ( 0 ) or in the

presence ( 0 ) of 100 pM Gpp(NH)p for 1 hr at 4°C.Points represent the mean of triplicate determinations. Similar results were obtained in four independent experiments. Displacement curves were analyzed with the LIGAND program (Munson and Rodbard, 1980) to determine Ki values.

What is the significance of opioid binding sites and GTP regulatory proteins in microsomes? A possible explanation is that they are intracellular sites. Accordingly, they may be newly synthesized proteins en route to the surface of the cell and/or proteins that underwent internalization for the purpose of degradation, ligand internalization, or specific signal transduction. Indeed, Roth et al. (1981, 1982) demonstrated that the microsomal fraction is enriched in Golgi and endoplasmic reticulum marker enzymes, which comigrate with opioid binding sites during continuous sucrose density gradient centrifugation. We favor the hypothesis that internalized opioid receptors are uncoupled, while those that are newly synthesized are G protein-coupled, which may be assembled before they reach the cell surface. This interpretation is supported by our findings on intracellular opioid sites isolated from neonatal brain and cells with up-regulated opioid receptors (Belcheva et al., 1991), which also display a greater Gpp(NH)p sensitivity. We have also discovered that neonatal microsomes contain more G protein than their adult counterpart (Bem et al., 1991). This is in accordance with the greater Gpp(NH)p sensitivity observed for P-3 than adult microsomes (Fig. 2C) and may reflect the presence of greater amounts of newly synthesized receptors in neonates. Moreover, it has been shown by autoradiography that internalized opioid and cholinergic receptors undergoing retrograde axoplasmic flow are GTP insensitive, while newly synthesized receptors that are in transit from the soma toward the nerve terminal are GTP sensitive (Zarbin et al., 1990, and references therein). Of course, since only light microscopy

was used here, the highly improbable possibility of migration of receptors on the cell surface was not disproved. Evidence that opioid receptors may be internalized without G proteins in NG 108-15 cells has also been reported (Law et al., 1985). This is consistent with what has been reported for the down-regulation of P-adrenergic receptors (Benovic et al., 1988). Furthermore, a and P y subunits of Gi and G, have been found in subcellular fractions of hepatocytes, including endosomes and Golgi membranes (Ali et al., 1989). Opioids can increase or decrease G proteins depending on the state of maturation of the brain region (Attali and Vogel, 1989; Nestler et al., 1989; Vogel et al., 1990). The experiments presented here suggest that some opioid binding sites are uncoupled from G proteins in microsomes. One explanation is that the amount of G proteins in microsomes is insufficient to afford coupling with all opioid sites. Another possibility is that the receptor and/or the G proteins are covalently modified, e.g., phosphorylated (Benovic et al., 1988). Nevertheless, our finding that Gpp(NH)p sensitivity of opioid binding is restored upon solubilization suggests that opioid receptors and G proteins may have been compartmentalized separately and are thus physically uncoupled in microsomes. Upon solubilization, they become capable of undergoing functional coupling. Thus the possibility exists that brain microsomes contain a substantial proportion of coupled newly synthesized as well as uncoupled opioid binding sites. While the former seem to be prevalent in microsomes of 3-day-old rats, some opioid sites are not coupled to G proteins in their adult counterparts.

Opioid Binding in Subcellular Fractions

ACKNOWLEDGMENTS This was supported by grants from the National Science Foundation (BNS 88-09462 to C.J.C.), NIDA (DA 05412 to C.J.C.), and OTKA 895 from the Hungarian Academy of Sciences (to M.s.). The skillful technical assistance of Mrs. Ildiko Nemeth is greatly appreciated.

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