A Monoclonal Antibody Capable of Modulating Opioid Binding to Rat ...

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

THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc.

A Monoclonal Antibody Capableof Modulating OpioidBinding 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, Schoolof Medicine and Dentistry, Rochester, New York 14642

A monoclonal antibody capable of inhibiting opioid alkaloids and peptides used to study these binding sites, the binding to rat neural membranes has been produced. exact delineation of the different types of opioid receptors has Spleen cells from a BALB/c mouse, immunizedwith a not been possible. partially purified opioid receptor complex, were fused Monoclonal antibodies are highly specific probes for various with P3-X63.Ag8.653.3 myeloma cells. The cell line regions of a protein molecule and have been usedto elucidate OR-689.2.4 secreted an IgM that was capable of par- the structure and function of many receptor systems. The tially inhibitingopioidbinding to ratneuralmemmolecular structure of the acetylcholine receptor has been branes under equilibrium binding conditions, while not extensively studied with monospecific immunoglobulins affecting thebindingofnonopioidligands.Control against determinants of various subunits (11-13) or against OR- the cholinergic binding site (14, 15). Monoclonal antibodies mouse immunoglobulins and heat-denatured 689.2.4 did not inhibit opioid binding to membranes. have been usedin thepurification of the nicotinic cholinergic The purified immunoglobulin inhibited the binding of [3H]dihydromorphinein a titrable, saturable, and re- receptor (16) and theP-adrenergic receptor (17). Information on the structure and function of the thyrotropin receptor (18), versible manner, as well as the binding of the 6-ligthe K-ligand [3H] the estrogen receptor (19, 20), and brain a-adrenergic recepand [3H][~-Ala2,~-Leu6]enkephalin, ethylketocyclazocine,and'H-labeledantagonists. In tors (21) has been obtained using monospecific immunoglobaddition to blocking the binding of opioids to mem- ulins. A monoclonal antibody directed against the nerve capable of stimulating branes, the immunoglobulin couldalso displace bound growth factor receptor on PC12 cells is the binding of nerve growth factor to PC12 cells(22). [3H]dihydromorphinefromneuralmembranes.The The advantage that most researchers in other receptor 12'I-labeled immunoglobulin specifically bound to neural membraneswith a & of 1.3 n M and a maximal systems had ingenerating monoclonal antibodies, specific for number ofbinding sites of 41.8 fmo1/0.25 mg of mem- the receptor, was that thestructure and thenumber of differbrane protein. Ina titrable manner, the immunoglob- ent types of receptor were knownprior to initiating monocloulin precipitated opioid binding sites from a solubilizednal antibody studies. With the opioid receptor system from preparation of neural membranes. When OR-689.2.4 the centralnervous system, neither the receptor structure nor conjugated to Sepharose was incubated with the par- the number of unique opioid receptors has been elucidated. tially purified opioid receptor complex, labeled with As a consequence, obtaining a monoclonal antibody directed "'I, a 35,000-dalton protein was specifically bound by against an opioid receptor has been more difficult. The only the immunoglobulin.This antibody provides a tool for way to screen for a monoclonal antibody to the opioid receptor probing the multiple opioid binding sites. 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 proThe molecular basis for the different types of opioid recep- duction of a monoclonal IgM that will inhibit opioid binding tor has remained elusive despite a considerable amount of to ratneural membranes and is capable of precipitating opioid work in thisarea. Both pharmacological (1,2) andbiochemical binding sites from a solubilized preparation of neural mem(3,4) studies have indicated the presence of multiple types of branes. opioid receptor in thecentral nervous system. "Binding sites EXPERIMENTAL PROCEDURES are currently regarded as theopioid binding sites that have a Generating Monoclonal Antibodies to the Opioid Receptor--BALB/ high affinity for morphine and [~-Ala~,MePhe~,Gly-ol~]enkec micewere immunized with a partially purified opioid receptor phalin (5, 6). &Binding sites are characterized by having the complex (23, 24). This receptor complex, obtained from a 14p-brohighest affinity for [~-Ala~,~-Leu']enkephalin and the&pep- moacetamidomorphine affinity column (25), consisted mainly of three tides [~-SeP,Leu~,Thr']enkephalin (7) and [~-Pen',~-Pen'] proteins with molecular weights of 43,000, 35,000, and 23,000 (26). Spleen cells from a mouse serum whose partially inhibited opioid enkephalin (where Pen represents penicillamine) (8).K-Opioid binding torat neural membranes were fused with the P3receptors have the highest affinity for benzomorphans and X63.Ag8.653.3 myeloma cell line (23, 24, 27). Culture supernatants dynorphin (9, 10). Due to the lack of high selectivity of the were initially screened for the production of an immunoglobulin that ~~

~~~

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

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

A Monoclonal Antibody Interactive

15656

ered with 50 mM Tris, pH 7.5, and concentrated on an Amicon PM30 membrane. The immunoglobulin was then dialyzed against 50 mM Tris, pH 7.5. Samples of pure immunoglobulin, verified by SDS1polyacrylamide 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 culturesupernatant, 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 pgof 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 @-adrenergicligand (-)-[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 thedisplacing ligand. For nicotine binding assays, the GF/Bglass 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 1ml. 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]flunitrazepambinding studies and ata concentration of 50 pg/5 ml for the muscarinic cholinergic ligand. [3H]Flunitrazepam binding was measured at 0 "C with 20 p~ clonazepam as thedisplacing ligand. Nonspecific binding of (-)-[3H]quinuclidinyl benzilate was measured by the inclusion of 10 p M atropine. Measuring the Ability of OR-689.2.4 to Displace Bound PHIDHM from Membranes-The ability to theantibody 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 theincubation was continued for 60 min. Reversibility of Inhibition of PHIDHM Binding by OR-689.2.4To measure the reversibility of the inhibition in opioid binding by OR689.2.4, 0.25 mgof 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-LabeledOR-689.2.4to Neural Membranes-The purified monoclonal IgM was labeled with lZ5Iby 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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~~~

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

with an

Opioid Receptor

in 50 mM Tris, pH 7.5, with 0.1-20 nM lZ5I-labeledOR-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, pH7.5, 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, pH7.5. The pellets were counted in a y-counter. Immunoprecipitating Opioid Binding Sites from a SolubilizedPreparation of Neural Membranes-Opioid receptors were solubilizedfrom 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 toa 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 thesamples 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 supernatantwas 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 p M 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, pH7.5. 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, pH8.3, 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 wassubsequently washed five times with PBS containing 1% fetal bovine serum and 0.1% Triton X-100, followedbytwo washes with 1 ml ofHzO. 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 SDS12%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-rayfilm for 1-4 days. Materials-Ligands purchased from New England Nuclear included [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 weresupplied 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 Bionetics, Inc. (Kensington, MD). Goat anti-mouse immunoglobulins conjugated to agarose and CNBr-activated Sepharose 4B, PEG (Mr -6000), and polyethyleneimine were purchased from Sigma. Liquescint 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 WouldInhibit 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 ratneuralmembranes (23). Underequilibrium conditions, dialyzedculturesupernatantfrom the cell line OR-689.2.4 inhibited the binding of2 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

binding of nonopioid ligands to rat neural membranes. In these experiments, the immunoglobulin inhibited the binding OR-689.2.4 of 1 nM [3H]DHM by 36%, while the binding of 1 nM (-)[3H]dihydroalprenolo1,1nM (-)-[3H]nicotine, 1nM 13H]flunitrazepam, and 0.1 nM (-)-[3H]quinuclidinyl benzilate did not vary significantly from control values. These results demonstrate thespecificityof 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 mgof 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 MOPC 104E 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 0.8 0 0.2 0.4 0.6 increasing membrane protein. The remainder of the studies CULTURE SUPERNATANT (ml) FIG.1. The effect of culture supernatant from thecell line described used 0.25 mg of membrane protein and 100 pg of OR-689.2.4 and the mouse monoclonal IgM MOPC 104E on OR-689.2.4. This ratioof antibody to opioid binding sites gave the bindingof 2 nM [aH]DHM to rat neural membranes. In a optimal results with regard to suffkient number of opioid final volume of 1 ml, culture supernatant that had been dialyzed binding sites present to obtain reproducible binding data and against 50 mM Tris, pH 7.5, was incubated with 0.25 mg ofmembrane a reasonable amount of antibody in each experiment. protein/ml at 37 "Cfor 30 min. Buffer or 10 pM morphine was added, Ability of OR-689.2.4 to Inhibit the Binding of Opioid Liand theincubation was continued for 15 min. rH]DHM at a concengands to Rat Neural Membranes--Fig. 3 summarizesthe abiltration of 2 nM was then added and theincubation continued at 37 "C for 15 min. After chilling on ice, the samples were filtered through ity of 100 pg of OR-689.2.4 to inhibit opioid binding to neural Whatman GF/B filters. Controls consisted of culture medium and membranes using the ligands [3H]DHM, [3H]DADLE, and culture supernatant from an unrelated cell line and the monoclonal [3H]EKCat concentrations from 0.1 to 2 nM. From 0.1 to 0.5 IgM MOPC 104E added to culture medium at a concentration of 100 nM [3H]DHM,the per cent inhibition fell gradually from 45 pg/ml. None of these controls had anysignificant effect on the binding to 35%. The per cent inhibition remained essentially constant of opioids to rat neural membranes. Closed circles represent the inhibition obtained with OR-689.2.4. Open circles represent the inhi- from 0.5 to 2.0 nM [3H]DHM. The ability of OR-689.2.4 to bition obtained with the mouse monoclonal IgM MOPC 104E. Points inhibit [3H]DADLEand [3H]EKCbinding to ratneural memrepresent the means of triplicate samples, which differed from each branes is different than for the p-ligand. With increasing other by less than 6%, from a representative experiment. The exper- concentrations of either 3H-labeled ligand,the per cent inhiiment was replicated three times with similar results. bition fell gradually from 50%at 0.1 nM to 15% at 2.0 nM. The antibody was also capable of inhibiting opioid antagoTABLE I nist binding to rat neural membranes as is shown in Table Effect of immunoglobulinson the binding of 1 nM 111. When OR-689.2.4 was incubated with neural membranes dihydromorphine to rat neural membranes and varying concentrations of [3H]diprenorphineor [3H]naRat neural membranes, 0.25 mg/ml, were incubated with 100 pgof immunoglobulin for 30 min at 25"C. Binding was measured as loxone, the immunoglobulin was capable of inhibiting 20-27% described under "Experimental Procedures." Results are expressed as of the binding of [3H]diprenorphineand [3H]naloxone over a wide range of ligand concentrations. The antibody inhibited the means & S.E.for three separate experiments. agonist binding preferentially over antagonist. This sidedness Immunoglobulin Total binding S~ecificbinding Inhibition of inhibition may be a result of the fact that agonists used in cpm cpm % these studies have greater preference for certain types of None 2550 + 53 2080 k 52 opioid receptors than do the antagonists. 1270 f 16 39 -c 2 OR-689.2.4 1610 zk 38 Reversibility of Inhibition of 1 nM pH]DHM Binding by 2410 -t 144 1960 f 122 6-C4 Mouse y-globulins MOPC 104E, IgM 2310 f 47 8f5 1920 f 102 OR-689.2.4-Membranes were incubated with 100 pg of OROR-689.2.4,90 "C for 2300 f 37 9+4 1900 & 97 689.2.4 for 30 min and then were repeatedly washed by cen30 min trifugation and resuspension in 50 mM Tris, pH 7.5. Greater than 50% of the inhibition of [3H]DHM binding can be The degree of inhibition observed with a certain volume of reversed by one wash of the membranes. Successive washing culture supernatant was dependent on the cell density and steps completely reversed the inhibition caused by the imthe time in culture. The control mouse IgM MOPC 104E, at munoglobulin. Ability ofOR-689.2.4to Displace PHIDHM from Mema concentration of 100 pg/ml added to culture medium, did branes-In these displacement studies, membranes at a connot significantly affect binding. The IgM secreted from the OR-689.2.4 cell line was ob- centration of 0.25 mg of protein/ml were equilibrated with tained in a pure form by culturing cells in a protein-free varying concentrations of [3H]DHM in the presence or abmedium. Table I shows that 100 pg of OR-689.2.4 inhibited sence of 10 p~ morphine at 25 "C for 60 min. The immunothe binding of 1 nM [3H]DHM to 0.25 mgof rat neural globulin at a concentration of 100 pg/mlwasadded and membrane protein by 39%. Under identical conditions, 100 incubation continued for 60 min. The samples were then pg of mousey-globulins or the mouse monoclonalIgM MOPC filtered through glass fiber filters. As can be seen in Fig. 4, 104E did not have a significant effect on the binding of 1nM the immunoglobulin was effectivein partially displacing [3H] [3H]DHMto membranes. When OR-689.2.4 was incubated at DHM from membranes. The top trace shows the binding 90 "C for 30 min, the IgM wasno longer capable of inhibiting inhibition obtained when membranes were first equilibrated opioid binding to membranes. with OR-689.2.4 for 60 min, and thenvarying concentrations Table I1 shows the effect of 100 pg of OR-689.2.4 on the of [3H]DHMwere added for an additional 60 min of incuba-

+

A Monoclonal Antibody Interactive withan Opioid Receptor

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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 theinitiation of the binding assay. Results are expressed as themeans f S.E. for three separate experiments. 3H-Ligand

[3H]Dihydromorphine, 1 nM Control OR-689.2.4 [3H]Dihydroalprenolo1,1nM Control OR-689.2.4 [3H]Nicotine, 1 nM Control OR-689.2.4 [3H]Flunitrazepam,1nM Control OR-689.2.4 [3H]Quinuclidinylbenzilate, 0.1 nM Control OR-689.2.4

W

Total binding

Specific binding

Change

cpm

cpm

%

2490 +. 86 1650 f 148

2010 f 26 1290 f 115

-36 f 4

5700 f 27 5300 f 45

3890 f 126 3710 f 146

-5 f 4

1000 f 29 1020 f 30

820 .t 22 850 f 12

+3 f 2

4600 f 40 4300 f 165

4470 f 125 4169 f 172

-7 f 4

3400 f3260 265 3240 f 190

f 256 3090 f 152

-5 f 5

* A

30

=H- DHM A IH-EKC

50-

40-

55

30-

9 20-

&

0 30 -

’“1 01

0

I

0.5

I

I

J.0

1.5

I

2.0

I H-LIGAND], nM

FIG. 3. Per cent inhibition of 3H-labeled opioid binding by OR-689.2.4at varying ligandconcentrations. 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, 0 125 250 375 500 loco and [3H]EKC were added at concentrations ranging from 0.1 to 2.0 MEMBRANE PROTEIN, ,ug nM. Displacing ligands were 10 p~ morphine, 10 p~ DADLE, and 10 FIG. 2. Per cent of [3H]DHM binding inhibited with varying p M cyclazocine,respectively. The per cent inhibition by the antibody concentrations of antibody and membrane protein. A, neural is plotted as a function of the 3H-labeled ligand concentration. The membranes at a protein concentration of 0.25 mg/ml in 50 mM Tris, points represent the means for six separate experiments which difpH 7.5, were incubated with varying concentrations of pure immu- fered from each other by less than 8%. noglobulin 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 Binding of 1251-Labeled OR-689.2.4 to Membranes-Fig. 5 25 “C for 30 min. The binding of 2 nM [3H]DHM to membranes was shows a Scatchard plot of the binding of the 1251-labeled measured at 25 “C as described under “Experimental Procedures.” antibody to neural membranes. Membranes were incubated Points represent the means of three separate experiments that difwith the labeled antibody for 1 h at 25 “C. Under these fered 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 themembranes. 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 itcould block at this concentration of ligand.

conditions, the membranes bound the IgM with a Kdvalue 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 SolubilizedPreparation-One of the best methods for ascertaining 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

15659

A Monoclonal Antibody Interactive with anOpioid Receptor TABLE I11 Effect of OR-689.2.4on 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. 'H-Ligand concentration

3H-Ligand

Inhibition %

nM

[3H]Diprenorphine

[3H]Naloxone

50

401

.

I

22

0.05 0.10 20 0.20 0.80

23 f 4 27 f 3 f6 20 f 4

0.40 20 0.80 24 3.20 22 6.40

&2

*3 f3 f3 I

I

4-

I

0

BLOCK 0 DISPLACE 0

11

'Ot

ob

1.2

0.8

E3H- DHMI

1

1.6

12

18

24

30

36

r-

, nM

&

FIG. 4. Ability of OR-689.2.4 to block and displace [3H] DHM from neural membranes. Closed circles represent the samples incubated underthe 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 immunoglobulinsconjugated to agarose. The binding of 4 nM [3H]naloxoneto 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]naloxonebinding to the solubilized preparation. Up to 48% of the totalnumber of [3H]naloxonebinding 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-labeledreceptor 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 SDSpolyacrylamide gel electrophoresis and autoradiography. As can be seen in Fig. 7, the immunoglobulin appears to be

6

fmoles fZ511 OR-689.2.4 BOUND FIG. 5. Scatchard analysis of the binding of 1261-labeled OR689.2.4 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 '9labeled OR-689.2.4 at 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 12,000X 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.

I

I

I

0.4

36

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 ofOR-689.2.4. Rat neural membranes were solubilized as described under "Experimental Procedures." Varying 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 supernatantwas removed and thebinding of 4 nM [3H]naloxoneto the supernatantwas 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-labeledreceptor 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 conjugatedto Sepharose servedas thecontrol for nonspecificbinding (Lane C ) .

15660

A Monoclonal Antibody Interactive

with an

Opioid Receptor

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 wasno longer capable of inhibiting binding. In an attempt to ascertain that theimmunoglobulin wasexerting 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 A B C by many investigators. In early experiments, membranes were FIG. 7. Identifying the protein that OR-689.2.4 is directed against. The opioid receptor complex that was used as the antigen preincubated with the antibody at 37 "C for 30 min prior to was labeled with 1251. In 1 ml of PBS containing 1% fetal bovine membrane binding assays. Experiments with the purified serum and 0.1% Triton X-100, 25 ng of 1261-labeledreceptor complex immunoglobulin demonstrated that incubating membranes was incubated with shaking a t 4 "C for 16 h with 50 pl of OR-689.2.4 with the immunoglobulin at 25 "Cyielded the same results as conjugated to Sepharose, or as a control, mouse y-globulins conju- a 37 "C incubation. As described in thelegend to Fig. 2, it was gated to Sepharose. After centrifuging and washing the gel fivetimes with PBS containing 1%fetal bovine serum and twice with H20, necessary to optimize the ratio of opioid binding sites to bound protein was eluted from the gel with 2% SDS. The eluted amount of antibody in order to avoid a great excess of antigen. fractions were separated on a SDS-12% polyacrylamide slab gel. The The inability of the antibody to produce complete inhibition gel was dried and subsequently exposed to Kodak DEF-5 x-ray film. may be explained on the basis that the antibody only recogLane A is the autoradiograph of the '%I-labeled receptor complex. nizes a restricted population of [3H]DHM binding sites. In Lane B is the autoradiograph of the protein elutedfrom the Sepharose gel conjugated with OR-689.2.4 Lane C is the autoradiograph of the the binding of 2 nM [3H]DHM to membranes, the actual proteins eluted from the Sepharose gel conjugated with mouse y- number of unique receptors binding [3H]DHMis not known. An alternative explanation is that theIgM, with a molecular globulins. weight of 980,000, may not be accessible to buried binding sites. DISCUSSION The fact that at concentrations of 3H-labeled ligand less Elucidating the molecular properties of the opioid receptor than 0.5 nM the three ligands were inhibited virtually equally has proven to be a difficult task. Monoclonal antibodies to suggests the possibility that theantibody is acting at a comthe receptor may prove to be a method for identifying, puri- mon high affinity site, such as the proposed pl site (34). fying, and characterizing the multiple opioid receptors. The Alternatively, the antibody may be acting at a p-site, which proteins that were used as the antigen were derived from a is known to also bind DADLE and EKC (35). Binding exper14-bromoacetamidomorphine affinity column (25,26). The iments using ligands more specific for the different types of fraction consisted mainly of three proteins with molecular opioid receptor will address this question as well as whether weights of 43,000, 35,000, and 23,000. The maximal opioid the antibody is acting competitively at the binding site or binding obtained with this fraction was 80 pmol/mg of pro- noncompetitively, producing a conformational change in the tein. If one assumes that 1 mol of opioid receptor bound at receptor. The fact that the antibody preferentially inhibited least 1 mol of ligand, the maximal binding obtained with a [3H]DHM binding to a greater degree than the binding of pure preparation should be in the range of nmol of opioid [3H]DADLE,[3H]EKC,or the antagonists [3H]diprenorphine bound per mgof pure receptor. The lower than expected or [3H]naloxonesuggests that itis possible to generate monoactivity of the material derived from the affinity column may clonal antibodies that are capable of discriminating among be explained on the basis either that theprotein complex. was the multiple opioid binding sites. The fact that a higher per highly active but impure or that the complex was largely cent of inhibition of antagonist binding was not obtained with inactivated during purification. The same three proteinswere the immunoglobulin mayresult from the fact that [3H]diprenspecifically eluted from an antagonist affinity column when orphine is equipotent at all types of opioid binding sites, while 14-chloroacetylnaltrexone was used as the affinity ligand, a [3H]naloxone,which has a slight preference for p-sites, is not finding that suggests the three proteins are associated with very specific. the receptor (32). Similar results were obtained from material In a titrable and saturable manner, the immunoglobulin prepared from the agonist or antagonist affinity column. By was capable of precipitating half of the [3H]naloxonebinding employing the protein complex derived from the affinity col- sites from a solubilized preparation. Since saturation in the umn, the task of preparing monoclonal antibodies would be number of binding sites precipitated was reached before 100% considerably less than that required for solubilized or mem- of the sites were precipitated, the antibody wasprobably brane preparations. An affinity column prepared with the interacting with a restricted population of opioid receptors.

A~

o

~A n t ci ~ d Inter~ctive y~ ~ with l an Opioid Receptor

15661

The results from both immunoaffinity chromatography and 10. Chavkin, c.,James, 1. F., and Goldstein, A. (1982) Science 2 1 5 , SDS-polyacrylamide gel electrophoresis demonstrate that the 413-415 11. Tzartos, S. J,, and Lindstrom, J. (1980) Proc. Natl. Acad. Sci. U. immunoglobulin is interacting with a 35,000-dalton protein. S. A. 77,755-759 Initially, we tried UnsuccesSfullY to identifywhich Proteinthe 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 antigenicsite, it would appear 13. Conti-Tronconi, B., Tzartos, S., and Lindstmm, J. (1981) Biochemistry 20,2181-2191 that the immunoglob~in requires a conformation~lyre&icted site for interaction. Sensitivity to denaturing agents 14. Mochly-Rosen, D-, and FuChs- s-(19B1) 3 w c h e m ~ 20? t ~ 59205924 has beenobservedformonoclonalantibodies to the acetyl- 15. Watters, D., and Maelicke, A. (1983) Biochemistry 22, 1811choline 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 thestructureandfunctionof the multipleopioid 255,4395-4398 receptors. The availability of a monoclonal antibody directed 17. Frazer, c. M., and Venter, J. c. (1980) Proc. Natl. Acad. sci. u. S. A. 77,7034-7038 against an Opioid binding protein will. us to 'Ompare 18. Yavin, E,, Yavb, Z., Schneider, M. D., and Kahn, L. D. (1981) the b i n ~ properties ~ g of the opioid receptorsfrom different Proc. Natl. Acad. Sci. U. S. A. 78,3180-3184 species and differenttissues and to further delineate the rob 19. Greene, G.L.,Fit&, F. w., and Jensen, E. V. (1980)proe.Natl. multiple ofopioid receptors. the Sci. Acad. U. S. A. 77,157-161 20. Moncharmont, B., Su, J. L., and Parikh, L. (1982) Biochemistry

21,6916-6921 21. Dausse, J.-P., and Diop, L. (1983) Eur. J. Phurmacol. 9 5 , 135137 22. Chandler, C. E., Parsons, L. M., Hosang, M., and Shooter, E. M. (1984) J. Bwl. Chem. 259,6882-6889 23. Bidlack, J. M., Denton, R. R., and HarweU, L. W. (1983)Life Sci. 33,151-154 REFERENCES 24. Harwell, L. W., Bolognino, M., Bidlack, J. M., Knapp, R. J., and Lord, E. M. (1984) J. Immunol. Methods 66,59-67 '. Martin, w*R*p Eades, c. G . ~Thompson9 J' A.i Hupp'err R. E*? 25. Archer, S., Seyed-Mozaffari,A., Osei-Gyimah,P., Bidlack, J. M., and Gilbert, P. E. (1976) J. Pharmacol. Exp. T h r . 1 9 7 , 517and Abood, L. G. (1983) J. Med. Chem. 26,1775-1777 532 26. Bidlack, J. M.,Abood,L.G., Osei-Gyimah, P., and Archer, S. 2. Cowan, A. (1981) Life Sci. 28,1559-1570 (1981) Proc. Natl. Acad. Sci. U. 8.A. 78,636-639 3. Lord, J. A- H., Waterfield, A., Hughes, J.7 and Kosterlitz, w. 27. Bidlack, J. M., and Denton, R. R. (19%) Neuropp&&s 5 , 225(1976) in Opiates and Endogenous Opioid Peptides (Kosterlitz, 228 H. W., ed) PP. 275-280, E l s ~ i e r / N o ~ h - H o l ~Biomedical nd 28. Bradford, M. M. (1976) ~ ~B k hl m . 72, . 248-254 Press, Amsterdam 29. Pasternak, G. W., Wilson, H. A., and Snyder, S. H. (1975) Mol. 4. Gillan, M. G. C., and Kosterlitz, H. W. (1982) Br. J. Pharmacol. Pharmacol. 11,340-331 77,461-469 30. McConhahey, P. J., and Dixon, F. J. (1966) Int. Arch. Allergy 5. Chang, K.-J., Hazum, E., and Cuatrecasas, P. (1980) Proc. Natl. Appl. Immunol. 29,185-189 Acad. Sci. U. S. A. 77,4469-4473 31. Bidlack, J. M., and Abood, G.L. (1980) Life Sci. 27,331-340 6. Handa, B. K., Lane, A. C., Lord, J. A. H.,Morgan, B. A-, b n c e , 32. Bidlack, J. M., Abood, L. G., Munemitsu, S. M., Archer, S., Gala, M.J., and Smith,C. F. C. (1981) Eur. J. Phurmacol. 70,531D., and Kreilick, R. W. (1982) Adu. Bhchem. Psychophurmacol. 540 33,301-309 7. David, M., Moisand, C., Meunier, J.-C., Morgat, J.-L., G a d , G., 33. Laemmli, U. K., and Favre, M. (1973) J. Mol. Bid. 80,575-599 and Roques, B. P. (1982) Eur. J. Phurmaw~.78,385-387 , L., Recht, L. D., and Pasternak, G. W. (1984) Mol. 34. N i s ~ m u r aS. 8. Mosberg, H. I., Hurst, R., Hruby, V. J., Gee, K., Y ~ ~ u rH.a , P ~ m a c o l26,29-37 . I., Galligan, J. J., and Burks, T. F. (1983) Proc. Natl. Acad. Sci. 35. Pfeiffer, A., and Herz, A. (1982) Mol. P h a r m o L 21, 26G271 U. S. A. 80, 5871-5874 36. Towbin, H., Staehelin, T., and Gordon, J. (1979) Prac. Natl. Acad. 9. Pasternak, G. (1980) Proc:Natl. Acad. Sci. U. S. A. 7 7 , 3691Sei. U. S. A. 76,4350-4354 37. Lindstrom, J. (1983) Neurosci. Comment. 1,139-156 3694

Ackmwledgments-We would like to thankDr. Edith M. Lord for the use of her tissue culture facilities, Lee W. Harwell for excellent technical assistance, and Drs. L.G.Abood, S. Banerjee, W. Hoss, and C. Kelbgg for gifts of nonopioid ligands.