A monoclonal antibody capable of modulating opioid binding to rat neural membranes

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc.

VOl. ,260, No. 29, Issue of December 15, pp. 15655-15661,1985 Printed in U.S.A.

A Monoclonal Antibody Capable of Modulating Opioid Binding to Rat Neural Membranes*

(Received for publication, June 7, 1985)

Jean M. Bidlack$ and R. Rex Dentong From the Center for Brain Research, The University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642

A monoclonal antibody capable of inhibiting opioid binding to rat neural membranes has been produced. Spleen cells from a BALB/c mouse, immunized with a partially purified opioid receptor complex, were fused with P3-X63.Ag8.653.3 myeloma cells. The cell line OR-689.2.4 secreted an IgM that was capable of partially inhibiting opioid binding to rat neural membranes under equilibrium binding conditions, while not affecting the binding of nonopioid ligands. Control mouse immunoglobulins and heat-denatured OR- 689.2.4 did not inhibit opioid binding to membranes. The purified immunoglobulin inhibited the binding of [3H]dihydromorphine in a titrable, saturable, and re- versible manner, as well as the binding of the 6-lig- and [3H][~-Ala2,~-Leu6]enkephalin, the K-ligand [3H] ethylketocyclazocine, and 'H-labeled antagonists. In addition to blocking the binding of opioids to membranes, the immunoglobulin could also displace bound [3H]dihydromorphine from neural membranes. The 12'I-labeled immunoglobulin specifically bound to neural membranes with a & of 1.3 n M and a maximal number of binding sites of 41.8 fmo1/0.25 mg of membrane protein. In a titrable manner, the immunoglobulin precipitated opioid binding sites from a solubilized preparation of neural membranes. When OR-689.2.4 conjugated to Sepharose was incubated with the partially purified opioid receptor complex, labeled with "'I, a 35,000-dalton protein was specifically bound by the immunoglobulin. This antibody provides a tool for probing the multiple opioid binding sites.

~~ ~~~ ~

The molecular basis for the different types of opioid receptor has remained elusive despite a considerable amount of work in this area. Both pharmacological (1,2) and biochemical (3,4) studies have indicated the presence of multiple types of opioid receptor in the central nervous system. "Binding sites are currently regarded as the opioid binding sites that have a high affinity for morphine and [~-Ala~,MePhe~,Gly-ol~]enkephalin (5, 6). &Binding sites are characterized by having the highest affinity for [~-Ala~,~-Leu']enkephalin and the &peptides [~-SeP,Leu~,Thr']enkephalin (7) and [~-Pen',~-Pen'] enkephalin (where Pen represents penicillamine) (8). K-Opioid receptors have the highest affinity for benzomorphans and dynorphin (9, 10). Due to the lack of high selectivity of the

* This work was supported by United States Public Health Service Grants DA03742 and DA00464 and a grant from the Genesee Valley Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom reprint requests should be addressed. Present address: Division of Toxicology, The University of Roch-

ester, School of Medicine and Der tistry, Rochester, NY 14642.

alkaloids and peptides used to study these binding sites, the exact delineation of the different types of opioid receptors has not been possible.

Monoclonal antibodies are highly specific probes for various regions of a protein molecule and have been used to elucidate the structure and function of many receptor systems. The molecular structure of the acetylcholine receptor has been extensively studied with monospecific immunoglobulins against determinants of various subunits (11-13) or against the cholinergic binding site (14, 15). Monoclonal antibodies have been used in the purification of the nicotinic cholinergic receptor (16) and the P-adrenergic receptor (17). Information on the structure and function of the thyrotropin receptor (18), the estrogen receptor (19, 20), and brain a-adrenergic receptors (21) has been obtained using monospecific immunoglobulins. A monoclonal antibody directed against the nerve growth factor receptor on PC12 cells is capable of stimulating the binding of nerve growth factor to PC12 cells (22).

The advantage that most researchers in other receptor systems had in generating monoclonal antibodies, specific for the receptor, was that the structure and the number of different types of receptor were known prior to initiating monoclonal antibody studies. With the opioid receptor system from the central nervous system, neither the receptor structure nor the number of unique opioid receptors has been elucidated. As a consequence, obtaining a monoclonal antibody directed against an opioid receptor has been more difficult. The only way to screen for a monoclonal antibody to the opioid receptor is to search for an immunoglobulin that inhibits opioid binding and/or is capable of precipitating opioid binding sites from a solubilized preparation. This study describes the production of a monoclonal IgM that will inhibit opioid binding to rat neural membranes and is capable of precipitating opioid binding sites from a solubilized preparation of neural membranes.

EXPERIMENTAL PROCEDURES Generating Monoclonal Antibodies to the Opioid Receptor--BALB/

c mice were immunized with a partially purified opioid receptor complex (23, 24). This receptor complex, obtained from a 14p-bromoacetamidomorphine affinity column (25), consisted mainly of three proteins with molecular weights of 43,000, 35,000, and 23,000 (26). Spleen cells from a mouse serum whose partially inhibited opioid binding to rat neural membranes were fused with the P3- X63.Ag8.653.3 myeloma cell line (23, 24, 27). Culture supernatants were initially screened for the production of an immunoglobulin that reacted with any component from the partially purified opioid receptor complex by radioimmunoassay using the '=I-labeled receptor complex. Antigen-antibody complexes were precipitated with goat anti-mouse immunoglobulins and normal mouse serum. Cell lines secreting an immunoglobulin to any component of the antigen were twice cloned by limiting dilution and bulk-cultured (23, 24). Culture supernatants from these cell lines were tested for their ability to inhibit opioid binding to rat neural membranes. The immunoglobulin OR-689.2.4 was obtained in a pure form by culturing cells for 48 h in protein-free medium. Culture supernatant was then harvested, buff-

15655

15656 A Monoclonal Antibody Interactive with an Opioid Receptor ered with 50 mM Tris, pH 7.5, and concentrated on an Amicon PM- 30 membrane. The immunoglobulin was then dialyzed against 50 mM Tris, pH 7.5. Samples of pure immunoglobulin, verified by SDS1- polyacrylamide gel electrophoresis, were filtered through a 0.2-pm filter and stored at -70 "C. Protein concentration was determined by the method of Bradford (28) using bovine serum albumin as the standard.

Measuring the Effect of OR-689.2.4 on the Binding of Opioid and Nonopwid Ligands to Rat Neural Membranes-Neural membranes from male Sprague-Dawley rats were prepared and washed at 37 "C for 30 min as previously described (29). Unless otherwise stated, opioid binding to neural membranes was measured in the following manner. In a final volume of 1 ml, 0.25 mg of neural membrane protein was incubated with culture supernatant, which had been dialyzed against 50 mM Tris, pH 7.5, or with purified antibody at 25 "C for 30 min. To measure nonspecific binding, 10 pM unlabeled ligand was added. The 3H-labeled ligand was then added and the incubation continued for 60 min. Bound radioactivity was determined by filtering samples through Whatman GF/B glass fiber filters. The filters were washed with 10 ml of cold 50 mM Tris, pH 7.5, followed by liquid scintillation spectrometry in 10 ml of Liquiscint scintillation fluid. All results are reported as specific binding, the difference between binding in the presence and absence of 10 p~ unlabeled ligand. Controls consisted of the addition of mouse y-globulins, the monoclonal IgM from the mouse myeloma cell line MOPC 104E or OR-689.2.4 that had been heated at 90 "C for 30 min.

The binding of nonopioid ligands to rat neural membranes was measured in a similar method used for the opioid ligands. Membranes were incubated with 100 pg of OR-689.2.4 for 30 min at 25 "C. Displacing ligands and 3H-labeled ligands were then added and the incubation was continued for 60 min. Samples were then filtered through GF/B glass fiber filters. The @-adrenergic ligand (-)-[3H] dihydroalprenolol at a concentration of 1 nM was displaced with 10 p~ (-)-alprenolol. The binding of 1 nM (-)-[3H]nicotine to membranes was measured at 0 "C with 10 p~ (-)-nicotine as the displacing ligand. For nicotine binding assays, the GF/B glass fiber filters were soaked in 0.5% polyethyleneimine. For the opioid, @-adrenergic, and nicotine ligands, membrane protein was at a concentration of 0.25 mg/ml in a final volume of 1 ml. Because of the large number of [3H] flunitrazepam and (-)-[3H]quinuclidinyl benzilate binding sites, membrane protein was used at a concentration of 50 pg/ml for the [3H]flunitrazepam binding studies and at a concentration of 50 pg/5 ml for the muscarinic cholinergic ligand. [3H]Flunitrazepam binding was measured at 0 "C with 20 p~ clonazepam as the displacing ligand. Nonspecific binding of (-)-[3H]quinuclidinyl benzilate was measured by the inclusion of 10 pM atropine.

Measuring the Ability of OR-689.2.4 to Displace Bound PHIDHM from Membranes-The ability to the antibody to displace [3H]DHM from membranes was examined by incubating membrane protein with varying concentrations of [3H]DHM at 25 "C for 60 min. The antibody was then added, and the incubation was continued for an additional 60 min prior to filtering the samples on GF/B filters. In studies designed to examine the ability of the antibody to block the binding of [3H]DHM, the reverse protocol was followed. Membranes were first incubated with 100 pg of OR-689.2.4 at 25 "C for 60 min. Varying concentrations of [3H]DHM were then added, and the incubation was continued for 60 min.

Reversibility of Inhibition of PHIDHM Binding by OR-689.2.4- To measure the reversibility of the inhibition in opioid binding by OR- 689.2.4, 0.25 mg of membrane protein was incubated in Microfuge tubes at 25 "C for 30 min with 100 pg of OR-689.2.4 in 0.5 ml of 50 mM Tris, pH 7.5. Membranes were centrifuged at 12,000 X g in a Microfuge for 4 min. The pellets were resuspended in 0.5 ml of 50 mM Tris, pH 7.5, and were incubated at 0 "C for 30 min. The wash step was then repeated. The binding of 1 nM t3H]DHM to the membranes was measured at 25 "C in a final volume of 1 ml. The binding of 1 nM [3H]DHM to repeatedly washed membranes that were not incubated with antibody did not vary by more than 4% from control samples.

Binding of '251-Labeled OR-689.2.4 to Neural Membranes-The purified monoclonal IgM was labeled with lZ5I by the chloramine-T procedure (30) to a specific activity of 400 cpm/fmol. In a final volume of 0.25 ml, 0.25 mg of neural membrane protein was incubated

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The abbreviations used are: SDS, sodium dodecyl sulfate; DHM, dihydromorphine; DADLE, [D-Ala2,D-Leus]enkephalin; EKC, ethylketocyclazocine, PBS, phosphate-buffered saline; PEG, polyethylene glycol.

in 50 mM Tris, pH 7.5, with 0.1-20 nM lZ5I-labeled OR-689.2.4 in polypropylene Microfuge tubes. Nonspecific binding was measured by the inclusion of 400 nM unlabeled immunoglobulin. After incubating for 1 h at 25 "C, 1 ml of cold 50 mM Tris, pH 7.5, was added, and the samples were centrifuged at 12,000 X g for 5 min. The supernatant was removed, and the surface of the pellet was washed with 1 ml of cold 50 mM Tris, pH 7.5. The pellets were counted in a y-counter.

Immunoprecipitating Opioid Binding Sites from a Solubilized Prep- aration of Neural Membranes-Opioid receptors were solubilized from rat neural membranes by Triton X-100, followed by the removal of the detergent (31). To test the ability of the purified immunoglobulin OR-689.2.4 to precipitate the receptor from a solubilized preparation, the following assay was employed. The immunoglobulin was added to 250 pg of solubilized neural membrane protein in 50 mM Tris, pH 7.5, or in PBS to a volume of 450 pl. After incubating at 4 "C for 16 h, 50 pl of goat anti-mouse immunoglobulins conjugated to agarose was added and the samples shaken at 4 "C for 4 h. After centrifugation at 12,000 X g for 2 min, the supernatant was removed and opioid binding to the supernatant was measured using a PEG binding assay (32). Solubilized membrane protein at a concentration of 0.1 mg/ml was incubated in 50 mM Tris, pH 7.5, with 10 pM displacing ligand at 37 "C for 15 min. The 3H-labeled ligand was added and the incubation continued for 15 min. The tubes were chilled on ice, and 0.4 ml of cold bovine y-globulin (10 mg/ml) was added, followed by the addition of 0.8 ml of cold 30% PEG (M, -6000). The samples were filtered on GF/B glass fiber filters, and the filters were washed with 10 ml of 7.5% PEG in 50 mM Tris, pH 7.5.

Identifying the Protein That OR-689.2.4 Is Directed against-To determine which protein(s) OR-689.2.4 was directed against, the IgM was conjugated to CNBr-activated Sepharose 4B at a concentration of 5 mg of IgM/1 ml of gel in 0.1 M NaHC03, pH 8.3, containing 0.5 M NaCl. The partially purified receptor complex was labeled with '%I using a chloramine-T procedure (30). In 1 ml of PBS containing 1% fetal bovine serum and 0.1% Triton X-100, 25 ng of lz5I-1abeled receptor complex was incubated with 50 pl of OR-689.2.4-Sepharose at 4 "C with continuous shaking for 16 h. Mouse y-globulins conjugated to Sepharose served as the control. The Sepharose gel was centrifuged at 12,000 X g for 2 min. The gel was subsequently washed five times with PBS containing 1% fetal bovine serum and 0.1% Triton X-100, followed by two washes with 1 ml of HzO. Bound protein was eluted from the gel by incubating the Sepharose at 25 "C for 1 h in 2% SDS. Sample buffer containing mercaptoethanol was added to the eluted fractions, which were then separated on a SDS- 12% polyacrylamide Laemmli slab gel (33). The gel was fixed in 10% acetic acid and 1% glycerol for 30 min. Subsequently, the gel was dried and exposed to Kodak DEF-5 x-ray film for 1-4 days.

Materials-Ligands purchased from New England Nuclear in- cluded [3H]DADLE (43.6 Ci/mmol), t3H]EKC (19.9 Ci/mmol), (-)- [3H]nicotine (68.6 Ci/mmol), and [3H]flunitrazepam (76.9 Ci/mmol). [3H]DHM (65 Ci/mmol), [3H]naloxone (43 Ci/mmol), [3H]diprenorphine (34 Ci/mmol), (-)-[3H]quinuclidiny1 benzilate (56 Ci/mmol), and (-)-[3H]dihydroalprenolol (95.7 Ci/mmol) were obtained from Amersham Corp. The following unlabeled opioid ligands were used. Morphine and cyclazocine were supplied by the National Institute on Drug Abuse, and DADLE was from Peninsula Laboratories (San Carlos, CA). Mouse y-globulins and goat anti-mouse immunoglobulin were obtained from Cooper Biomedical, Inc. (Malvern, PA). The mouse monoclonal IgM MOPC 104E was obtained from Litton Bio- netics, Inc. (Kensington, MD). Goat anti-mouse immunoglobulins conjugated to agarose and CNBr-activated Sepharose 4B, PEG (Mr -6000), and polyethyleneimine were purchased from Sigma. Liques- cint scintillation fluid was purchased from National Diagnostics (Somerville, NJ). Male Sprague-Dawley rats (180 g) were purchased from Charles River Breeding Laboratories.

RESULTS

Testing for an Antibody That Would Inhibit Opioid Binding to Rat Neural Membranes-From 1042 cell lines tested, 32 cell lines secreted an immunoglobulin that interacted with some component of the lZ5I-labeled receptor complex, and two of these immunoglobulins had an effect on opioid binding to rat neural membranes (23). Under equilibrium conditions, dialyzed culture supernatant from the cell line OR-689.2.4 inhibited the binding of 2 nM [3H]DHM to rat neural membranes in a titrable and saturable manner as shown in Fig. 1.

A Monoclonal Antibody Interactive with an Opioid Receptor 15657

OR-689.2.4

MOPC 104E

+ 0 0.2 0.4 0.6 0.8

CULTURE SUPERNATANT (ml)

FIG. 1. The effect of culture supernatant from the cell line OR-689.2.4 and the mouse monoclonal IgM MOPC 104E on the binding of 2 nM [aH]DHM to rat neural membranes. In a final volume of 1 ml, culture supernatant that had been dialyzed against 50 mM Tris, pH 7.5, was incubated with 0.25 mg of membrane protein/ml at 37 "C for 30 min. Buffer or 10 pM morphine was added, and the incubation was continued for 15 min. rH]DHM at a concentration of 2 nM was then added and the incubation continued at 37 "C for 15 min. After chilling on ice, the samples were filtered through Whatman GF/B filters. Controls consisted of culture medium and culture supernatant from an unrelated cell line and the monoclonal IgM MOPC 104E added to culture medium at a concentration of 100 pg/ml. None of these controls had any significant effect on the binding of opioids to rat neural membranes. Closed circles represent the inhibition obtained with OR-689.2.4. Open circles represent the inhibition obtained with the mouse monoclonal IgM MOPC 104E. Points represent the means of triplicate samples, which differed from each other by less than 6%, from a representative experiment. The experiment was replicated three times with similar results.

TABLE I Effect of immunoglobulins on the binding of 1 nM

dihydromorphine to rat neural membranes Rat neural membranes, 0.25 mg/ml, were incubated with 100 pg of

immunoglobulin for 30 min at 25 "C. Binding was measured as described under "Experimental Procedures." Results are expressed as the means & S.E. for three separate experiments.

Immunoglobulin Total binding S~ecific binding Inhibition

cpm cpm % None 2550 + 53 2080 k 52

Mouse y-globulins 2410 -t 144 1960 f 122 6-C4 MOPC 104E, IgM 2310 f 47 1920 f 102 8 f 5 OR-689.2.4,90 "C for 2300 f 37 1900 & 97 9 + 4

OR-689.2.4 1610 zk 38 1270 f 16 39 -c 2

30 min

The degree of inhibition observed with a certain volume of culture supernatant was dependent on the cell density and the time in culture. The control mouse IgM MOPC 104E, at a concentration of 100 pg/ml added to culture medium, did not significantly affect binding.

The IgM secreted from the OR-689.2.4 cell line was obtained in a pure form by culturing cells in a protein-free medium. Table I shows that 100 pg of OR-689.2.4 inhibited the binding of 1 nM [3H]DHM to 0.25 mg of rat neural membrane protein by 39%. Under identical conditions, 100 pg of mouse y-globulins or the mouse monoclonal IgM MOPC 104E did not have a significant effect on the binding of 1 nM [3H]DHM to membranes. When OR-689.2.4 was incubated at 90 "C for 30 min, the IgM was no longer capable of inhibiting opioid binding to membranes.

Table I1 shows the effect of 100 pg of OR-689.2.4 on the

binding of nonopioid ligands to rat neural membranes. In these experiments, the immunoglobulin inhibited the binding of 1 nM [3H]DHM by 36%, while the binding of 1 nM (-)- [3H]dihydroalprenolo1, 1 nM (-)-[3H]nicotine, 1 nM 13H]flunitrazepam, and 0.1 nM ( -)-[3H]quinuclidinyl benzilate did not vary significantly from control values. These results demonstrate the specificity of the immunoglobulin for opioid binding sites.

Fig. 2A shows the titration of OR-689.2.4 in its ability to inhibit 2 nM [3H]DHM binding to 0.25 mg of rat neural membrane protein. Maximal inhibition was obtained at 110 pg of immunoglobulin, a concentration of 110 nM. The concentration of membranes also affected the degree of binding inhibition, as shown in Fig. 2B. Under these conditions, 100 pg of OR-689.2.4 inhibited up to 40% of the binding of 2 nM [3H]DHM. The per cent inhibition of binding decreased with increasing membrane protein. The remainder of the studies described used 0.25 mg of membrane protein and 100 pg of OR-689.2.4. This ratio of antibody to opioid binding sites gave optimal results with regard to suffkient number of opioid binding sites present to obtain reproducible binding data and a reasonable amount of antibody in each experiment.

Ability of OR-689.2.4 to Inhibit the Binding of Opioid Li- gands to Rat Neural Membranes--Fig. 3 summarizes the ability of 100 pg of OR-689.2.4 to inhibit opioid binding to neural membranes using the ligands [3H]DHM, [3H]DADLE, and [3H]EKC at concentrations from 0.1 to 2 nM. From 0.1 to 0.5 nM [3H]DHM, the per cent inhibition fell gradually from 45 to 35%. The per cent inhibition remained essentially constant from 0.5 to 2.0 nM [3H]DHM. The ability of OR-689.2.4 to inhibit [3H]DADLE and [3H]EKC binding to rat neural membranes is different than for the p-ligand. With increasing concentrations of either 3H-labeled ligand, the per cent inhibition fell gradually from 50% at 0.1 nM to 15% at 2.0 nM.

The antibody was also capable of inhibiting opioid antagonist binding to rat neural membranes as is shown in Table 111. When OR-689.2.4 was incubated with neural membranes and varying concentrations of [3H]diprenorphine or [3H]naloxone, the immunoglobulin was capable of inhibiting 20-27% of the binding of [3H]diprenorphine and [3H]naloxone over a wide range of ligand concentrations. The antibody inhibited agonist binding preferentially over antagonist. This sidedness of inhibition may be a result of the fact that agonists used in these studies have greater preference for certain types of opioid receptors than do the antagonists.

Reversibility of Inhibition of 1 nM pH]DHM Binding by OR-689.2.4-Membranes were incubated with 100 pg of OR- 689.2.4 for 30 min and then were repeatedly washed by centrifugation and resuspension in 50 mM Tris, pH 7.5. Greater than 50% of the inhibition of [3H]DHM binding can be reversed by one wash of the membranes. Successive washing steps completely reversed the inhibition caused by the immunoglobulin.

Ability of OR-689.2.4 to Displace PHIDHM from Mem- branes-In these displacement studies, membranes at a concentration of 0.25 mg of protein/ml were equilibrated with varying concentrations of [3H]DHM in the presence or absence of 10 p~ morphine at 25 "C for 60 min. The immunoglobulin at a concentration of 100 pg/ml was added and incubation continued for 60 min. The samples were then filtered through glass fiber filters. As can be seen in Fig. 4, the immunoglobulin was effective in partially displacing [3H] DHM from membranes. The top trace shows the binding inhibition obtained when membranes were first equilibrated with OR-689.2.4 for 60 min, and then varying concentrations of [3H]DHM were added for an additional 60 min of incuba-

15658 A Monoclonal Antibody Interactive with an Opioid Receptor TABLE I1

Effect of OR-689.2.4 on the binding of nonopioid ligands to rat neural membranes Binding assays were performed as described under “Experimental Procedures.” Rat neural membranes were

incubated with and without 100 pg of OR-689.2.4 at 25 “C for 30 min prior to the initiation of the binding assay. Results are expressed as the means f S.E. for three separate experiments.

3H-Ligand Total binding Specific binding Change

cpm cpm %

2010 f 26 [3H]Dihydromorphine, 1 nM

Control 2490 +. 86 OR-689.2.4 1650 f 148 1290 f 115

5700 f 27 3890 f 126 5300 f 45

[3H]Dihydroalprenolo1, 1 nM -36 f 4

Control OR-689.2.4

[3H]Nicotine, 1 nM 3710 f 146 -5 f 4

Control 1000 f 29 820 .t 22 OR-689.2.4 1020 f 30 850 f 12 +3 f 2

[3H]Flunitrazepam, 1 nM Control 4600 f 40 OR-689.2.4

4470 f 125 4169 f 172 -7 f 4 4300 f 165

3400 f 265 3260 f 256 3240 f 190 3090 f 152 -5 f 5

[3H]Quinuclidinyl benzilate, 0.1 nM Control OR-689.2.4

W * A 30

0

30 -

0 125 250 375 500 loco MEMBRANE PROTEIN, ,ug

FIG. 2. Per cent of [3H]DHM binding inhibited with varying concentrations of antibody and membrane protein. A, neural membranes at a protein concentration of 0.25 mg/ml in 50 mM Tris, pH 7.5, were incubated with varying concentrations of pure immunoglobulin OR-689.2.4; B, OR-689.2.4 at a concentration of 100 pg/ ml was incubated with varying concentrations of membrane protein. Membranes were incubated with the purified immunoglobulin at 25 “C for 30 min. The binding of 2 nM [3H]DHM to membranes was measured at 25 “C as described under “Experimental Procedures.” Points represent the means of three separate experiments that differed from each other by less than 10%.

tion. The antibody was able to block 35-45% of the [3H]DHM binding sites. The affinity of this antibody for the receptor is sufficiently high to be able to displace opioids in addition to preventing them from binding. At low concentrations of [3H] DHM, the antibody displaced bound [3H]DHM almost as effectively as it could block its binding to the membranes. As the concentration of [3H]DHM increased, the ability of the immunoglobulin to displace bound [3H]DHM decreased. However, even at 1.6 nM [3H]DHM, the IgM displaced at half the number of binding sites that it could block at this concentration of ligand.

50-

40-

5 30- 9 5

20- &

’“1

=H- DHM A I H - E K C

0 1 I I I I 0 0.5 J.0 1.5 2.0

I H-LIGAND], nM

FIG. 3. Per cent inhibition of 3H-labeled opioid binding by OR-689.2.4 at varying ligand concentrations. Membranes at a protein concentration of 0.25 mg/ml were incubated with 100 pg of OR-689.2.4 for 30 min at 25 “C. Opioid binding was measured as describedunder “Experimental Procedures.” [3H]DHM, [3H]DADLE, and [3H]EKC were added at concentrations ranging from 0.1 to 2.0 nM. Displacing ligands were 10 p~ morphine, 10 p~ DADLE, and 10 pM cyclazocine, respectively. The per cent inhibition by the antibody is plotted as a function of the 3H-labeled ligand concentration. The points represent the means for six separate experiments which differed from each other by less than 8%.

Binding of 1251-Labeled OR-689.2.4 to Membranes-Fig. 5 shows a Scatchard plot of the binding of the 1251-labeled antibody to neural membranes. Membranes were incubated with the labeled antibody for 1 h at 25 “C. Under these conditions, the membranes bound the IgM with a Kd value of 1.3 nM. The Scatchard plot was linear, indicative of a single binding site, and the maximal number of binding sites was 41.8 fmo1/0.25 mg of membrane protein.

Ability of OR-689.2.4 to Precipitate the Opioid Receptor from a Solubilized Preparation-One of the best methods for ascer- taining if an antibody is interacting directly with a receptor is to determine if the receptor can be precipitated from a solubilized preparation by the immunoglobulin. Solubilized neural membranes were incubated with varying concentrations of OR-689.2.4 for 16 h. The receptor-immunoglobulin complex was brought out of solution by the addition of goat

A Monoclonal Antibody Interactive with an Opioid Receptor 15659

TABLE I11 Effect of OR-689.2.4 on the binding of r3H/diprenorphine and PHI

naloxone to rat neural membranes Rat neural membranes, 0.25 mg of protein/ml, were incubated with

100 pg of OR-689.2.4 for 30 min at 25 "C. Binding was measured as described under "Experimental Procedures." Results are expressed as the means f S.E. for three separate experiments.

3H-Ligand 'H-Ligand concentration Inhibition

nM % [3H]Diprenorphine 0.05 23 f 4

0.10 27 f 3 0.20 20 f 6 0.80 20 f 4

[3H]Naloxone 0.40 22 & 2 0.80 20 * 3 3.20 24 f 3 6.40 22 f 3

50 I I I I

0 BLOCK 0 DISPLACE ..

401 1 1

' O t 1 ob

I I I 0.4 0.8 1.2

E3H- DHMI , nM 1.6

FIG. 4. Ability of OR-689.2.4 to block and displace [3H] DHM from neural membranes. Closed circles represent the samples incubated under the blocking conditions. Rat neural membranes at a protein concentration of 0.25 mg/ml were incubated with 100 pg of OR-689.2.4 at 25 "C for 60 min. Displacing ligands, 10 p M morphine and [3H]DHM at concentrations varying from 0.1 to 1.6 nM, were added, and the incubation was continued for an additional 60 min. Open circles represent the samples incubated under the displacing conditions. In these experiments membranes were first equilibrated with displacing ligand and varying concentrations of [3H]DHM at 25 "C for 60 min. The antibody was then added, and the incubation was continued for an additional 60 min prior to filtration. Points represent the mean inhibition of [3H]DHM binding for three experiments which differed from each other by less than 8%.

anti-mouse immunoglobulins conjugated to agarose. The binding of 4 nM [3H]naloxone to the supernatant fraction showed the ability of the immunoglobulin to precipitate the receptor in a titrable manner (Fig. 6). Mouse y-globulins and the IgM MOPC 104E served as controls and did not decrease [3H]naloxone binding to the solubilized preparation. Up to 48% of the total number of [3H]naloxone binding sites in 250 pg of a solubilized preparation were precipitated by 50 pg of OR-689.2.4, a concentration of 40 nM. The immunoglobulin appeared to have a greater affinity for the solubilized receptor than the membrane-bound receptor.

Identifying the Protein That OR-689.2.4 Is Directed against-In order to identify which protein the antibody was directed against, the partially purified receptor complex was labeled with lZ5I. The lZ5I-labeled receptor complex was incubated with immunoglobulin OR-689.2.4 that had been conjugated to Sepharose. The specifically bound protein was eluted from the antibody-Sepharose column and analyzed by SDS- polyacrylamide gel electrophoresis and autoradiography. As can be seen in Fig. 7, the immunoglobulin appears to be

36

4 -

0 6 12 18 24 30 36 fmoles fZ511 OR-689.2.4 BOUND

FIG. 5. Scatchard analysis of the binding of 1261-labeled OR- 689.2.4 to neural membranes. In a final volume of 250 pl, 0.25 mg of membrane protein was incubated for 1 h at 25 "C with '9- labeled OR-689.2.4 a t concentrations ranging from 0.1 to 20 nM. Nonspecific binding was measured by the inclusion of 400 nM unlabeled OR-689.2.4. Just prior to centrifugation at 12,000 X g for 5 min, 1 ml of cold 50 mM Tris, pH 7.5, was added to the samples. The Scatchard plot gave a K d value of 1.3 nM and a B,, value of 41.8 fmo1/0.25 mg of membrane protein. The experiment was repeated four times.

r- &

T

&! oq 25 50 75 100 125 150

OR-689.2.4, pg FIG. 6. Per cent of [3H]naloxone binding sites precipitated

from a solubilized preparation of rat neural membranes by varying concentrations of OR-689.2.4. Rat neural membranes were solubilized as described under "Experimental Procedures." Vary- ing concentrations of OR-689.2.4 were added to 250 pg of solubilized membrane protein in PBS in a volume of 450 pl. After incubating at 4 "C for 16 h, 50 p1 of goat anti-mouse immunoglobulin conjugated to agarose was added and the samples shaken at 4 "C for 4 h. After centrifugation at 12,000 X g for 2 min, the supernatant was removed and the binding of 4 nM [3H]naloxone to the supernatant was measured using a PEG binding assay. Points represent the mean & S.E. for three separate experiments.

interacting with the 35,000-dalton protein. Lane A of Fig. 7 shows the lZ5I-labeled receptor complex, consisting mainly of three proteins with molecular weights of 43,000, 35,000 and 23,000. This receptor complex was used as the antigen. The antibody recognized and bound the 35,000-dalton protein, as is shown in Lane B. Mouse y-globulins conjugated to Sepha- rose served as the control for nonspecific binding (Lane C ) .

15660 A Monoclonal Antibody Interactive with an Opioid Receptor

A B C FIG. 7. Identifying the protein that OR-689.2.4 is directed

against. The opioid receptor complex that was used as the antigen was labeled with 1251. In 1 ml of PBS containing 1% fetal bovine serum and 0.1% Triton X-100, 25 ng of 1261-labeled receptor complex was incubated with shaking at 4 "C for 16 h with 50 pl of OR-689.2.4 conjugated to Sepharose, or as a control, mouse y-globulins conjugated to Sepharose. After centrifuging and washing the gel five times with PBS containing 1% fetal bovine serum and twice with H20, bound protein was eluted from the gel with 2% SDS. The eluted fractions were separated on a SDS-12% polyacrylamide slab gel. The gel was dried and subsequently exposed to Kodak DEF-5 x-ray film. Lane A is the autoradiograph of the '%I-labeled receptor complex. Lane B is the autoradiograph of the protein eluted from the Sepharose gel conjugated with OR-689.2.4 Lane C is the autoradiograph of the proteins eluted from the Sepharose gel conjugated with mouse y- globulins.

DISCUSSION

Elucidating the molecular properties of the opioid receptor has proven to be a difficult task. Monoclonal antibodies to the receptor may prove to be a method for identifying, puri- fying, and characterizing the multiple opioid receptors. The proteins that were used as the antigen were derived from a 14-bromoacetamidomorphine affinity column (25, 26). The fraction consisted mainly of three proteins with molecular weights of 43,000, 35,000, and 23,000. The maximal opioid binding obtained with this fraction was 80 pmol/mg of protein. If one assumes that 1 mol of opioid receptor bound at least 1 mol of ligand, the maximal binding obtained with a pure preparation should be in the range of nmol of opioid bound per mg of pure receptor. The lower than expected activity of the material derived from the affinity column may be explained on the basis either that the protein complex. was highly active but impure or that the complex was largely inactivated during purification. The same three proteins were specifically eluted from an antagonist affinity column when 14-chloroacetylnaltrexone was used as the affinity ligand, a finding that suggests the three proteins are associated with the receptor (32). Similar results were obtained from material prepared from the agonist or antagonist affinity column. By employing the protein complex derived from the affinity column, the task of preparing monoclonal antibodies would be considerably less than that required for solubilized or membrane preparations. An affinity column prepared with the

monoclonal antibody should aid in the isolation of the opioid receptor(s).

The ability of this IgM to inhibit opioid binding to neural membranes is a specific effect. Neither culture medium nor culture supernatant from unrelated cell lines inhibited the binding of [3H]DHM to neural membranes. Mouse 7-globulins or the mouse monoclonal IgM MOPC 104E did not have any significant effect on the binding of opioids to membranes. When OR-689.2.4 was heated to 90 "C for 30 min, the immunoglobulin was no longer capable of inhibiting binding. In an attempt to ascertain that the immunoglobulin was exerting a specific effect on opioid binding sites, the effect of the immunoglobulin on the binding of @-adrenergic, diazepam, nicotine, and muscarinic cholinergic ligands was examined. Since the IgM did not have any significant effect on the binding of these nonopioid ligands, it appeared to specifically interact with an opioid binding protein. Further evidence for its specificity derives from the fact the antibody displaces bound [3H]DHM.

The affinity of this antibody, 0.77 nM", is sufficiently high to perform equilibrium binding experiments. All of the described studies were performed using equilibrium binding conditions and a binding assay protocol that has been used by many investigators. In early experiments, membranes were preincubated with the antibody at 37 "C for 30 min prior to membrane binding assays. Experiments with the purified immunoglobulin demonstrated that incubating membranes with the immunoglobulin at 25 "C yielded the same results as a 37 "C incubation. As described in the legend to Fig. 2, it was necessary to optimize the ratio of opioid binding sites to amount of antibody in order to avoid a great excess of antigen. The inability of the antibody to produce complete inhibition may be explained on the basis that the antibody only recog- nizes a restricted population of [3H]DHM binding sites. In the binding of 2 nM [3H]DHM to membranes, the actual number of unique receptors binding [3H]DHM is not known. An alternative explanation is that the IgM, with a molecular weight of 980,000, may not be accessible to buried binding sites.

The fact that at concentrations of 3H-labeled ligand less than 0.5 nM the three ligands were inhibited virtually equally suggests the possibility that the antibody is acting at a com- mon high affinity site, such as the proposed pl site (34). Alternatively, the antibody may be acting at a p-site, which is known to also bind DADLE and EKC (35). Binding experiments using ligands more specific for the different types of opioid receptor will address this question as well as whether the antibody is acting competitively at the binding site or noncompetitively, producing a conformational change in the receptor. The fact that the antibody preferentially inhibited [3H]DHM binding to a greater degree than the binding of [3H]DADLE, [3H]EKC, or the antagonists [3H]diprenorphine or [3H]naloxone suggests that it is possible to generate monoclonal antibodies that are capable of discriminating among the multiple opioid binding sites. The fact that a higher per cent of inhibition of antagonist binding was not obtained with the immunoglobulin may result from the fact that [3H]diprenorphine is equipotent at all types of opioid binding sites, while [3H]naloxone, which has a slight preference for p-sites, is not very specific.

In a titrable and saturable manner, the immunoglobulin was capable of precipitating half of the [3H]naloxone binding sites from a solubilized preparation. Since saturation in the number of binding sites precipitated was reached before 100% of the sites were precipitated, the antibody was probably interacting with a restricted population of opioid receptors.

A ~ o ~ c ~ ~ l A n t i ~ d y Inter~ctive with an Opioid Receptor 15661

The results from both immunoaffinity chromatography and 10. Chavkin, c., James, 1. F., and Goldstein, A. (1982) Science 215 , SDS-polyacrylamide gel electrophoresis demonstrate that the 413-415 immunoglobulin is interacting with a 35,000-dalton protein. 11. Tzartos, S. J,, and Lindstrom, J. (1980) Proc. Natl. Acad. Sci. U.

Initially, we tried UnsuccesSfullY to identify which Protein the 12. &flick, W. J., Tzams, $., and Lindstrom, J. (1981) Biochemistry immunoglobulin interacted with by a Western blot technique 20,2173-2180 (36). Since SDS denatured the antigenic site, it would appear 13. Conti-Tronconi, B., Tzartos, S., and Lindstmm, J. (1981) Bio- that the immunoglob~in requires a conformation~ly re- chemistry 20,2181-2191 &icted site for interaction. Sensitivity to denaturing agents 14. Mochly-Rosen, D-, and FuChs- s- (19B1) 3 w c h e m ~ t ~ 20? 5920- has been observed for monoclonal antibodies to the acetyl- 15. Watters, D., and Maelicke, A. (1983) Biochemistry 22, 1811- 5924

choline receptor (37). 1819 Monoclonal antibodies will provide a powerful tool for 16. Lennon, V. A., Thompson, M., and Chen, J. (1980) J. Bwl. Chem.

studies of the structure and function of the multiple opioid 255,4395-4398

S. A. 77,755-759

receptors. The availability of a monoclonal antibody directed 17. Frazer, c. M., and Venter, J. c. (1980) Proc. Natl. Acad. sci. u. against an Opioid binding protein will. us to 'Ompare 18. Yavin, E,, Yavb, Z., Schneider, M. D., and Kahn, L. D. (1981)

S. A. 77,7034-7038

the b i n ~ ~ g properties of the opioid receptors from different species and different tissues and to further delineate the rob 19. Greene, G. L., Fit&, F. w., and Jensen, E. V. (1980) proe. Natl.

Proc. Natl. Acad. Sci. U. S. A. 78,3180-3184

of the multiple opioid receptors. Acad. Sci. U. S. A. 77,157-161 20. Moncharmont, B., Su, J. L., and Parikh, L. (1982) Biochemistry

Ackmwledgments-We would like to thank Dr. Edith M. Lord for 21. Dausse, J.-P., and Diop, L. (1983) Eur. J. Phurmacol. 9 5 , 135- the use of her tissue culture facilities, Lee W. Harwell for excellent 137 technical assistance, and Drs. L. G. Abood, S. Banerjee, W. Hoss, 22. Chandler, C. E., Parsons, L. M., Hosang, M., and Shooter, E. M. and C. Kelbgg for gifts of nonopioid ligands. (1984) J. Bwl. Chem. 259,6882-6889

23. Bidlack, J. M., Denton, R. R., and HarweU, L. W. (1983) Life Sci.

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A monoclonal antibody capable of modulating opioid binding to rat neural membranes