Proc. Natl. Acad. Sci. USA Vol. 87, pp. 3180-3184, April 1990 Pharmacology

Chimeric opioid peptides: Tools for identifying opioid receptor types (dynorphin/dermorphin/deltorphin/monoclonal antibody/panning)

Guo-xi XIE*t, ATSUSHI MIYAJIMA*, TAKASHI YOKOTA*, KEN-ICHI ARAI*, AND AVRAM GOLDSTEINt *Department of Molecular Biology, DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA 94304; and tDepartment of Pharmacology, Stanford University, Stanford, CA 94305

Contributed by Avram Goldstein, January 23, 1990

ABSTRACT We synthesized several chimeric peptides in which the N-terminal nine residues of dynorphin-32, a peptide selective for the K opioid receptor, were replaced by opioid peptides selective for other opioid receptor types. Each chimeric peptide retained the high affminty and type selectivity characteristic of its N-terminal sequence. The common Cterminal two-thirds of the chimeric peptides served as an epitope recognized by the same monoclonal antibody. When bound to receptors on a cell surface or membrane preparation, these peptides could still bind specifically to the monoclonal antibody. These chimeric peptides should be useful for isolating ,A, 8, and c opioid receptors and for identifying opioid receptors on transfected cells in expression cloning procedures. The general approach using chimeric peptides should be applicable to other peptide receptors.

was assumed that the C-terminal amide group of dermorphin, deltorphins, and DSLET and the alcohol group of DAGO could be removed without affecting opioid binding. By analogy to dyn-32, which binds selectively to K opioid sites, DAGO-DYN and dermorphin-DYN should bind selectively to p.; deltorphins-DYN and DSLET-DYN should bind selectively to 8. mAbs 17.M and 39 should act as nonblocking antibodies to all these peptides. In the present study, we have demonstrated that the chimeric peptides do maintain the high affinities and type selectivities of their N-terminal sequences. mAbs 17.M and 39 bind to these peptides (as to dyn-32), even after the peptides are bound to receptors on brain membranes or on intact NG108-15 neuroblastoma-glioma hybrid cells.

Several peptide ligands selective for different opioid receptor types have been isolated from natural sources or synthesized. Among them [D-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAGO) (1) and dermorphin (2) have an optimal combination of high affinity and type selectivity for , binding sites. [D-Pen2, D-Pen5]enkephalin (DPDPE) (3) and deltorphin I and II (4) have similar properties with respect to 8 sites, as do dynorphin A (dyn A) (5) and its derivatives (6) for K sites. Each of these peptides has a subnanomolar dissociation constant at its preferred binding site, at least two orders of magnitude greater affinity than at its next-preferred binding site. For experiments on the expression cloning of opioid receptors we wished to develop a system in which peptide ligands and antibodies against the peptides could be used for affinity purification or for isolation of receptor-bearing cells. Dynorphin-32 (dyn-32) (Fig. 1), our model peptide, is, in effect, a fusion product of two peptides-the 17-residue dyn A at the N terminus and the 13-residue dynorphin B (dyn B) at the C terminus, connected by a "bridge sequence" LysArg. dyn-32 itself is a K opioid agonist (7). Of several monoclonal antibodies (mAbs) raised against dyn-32, two were useful in this study. mAb 17.M requires the bridge and C-terminal sequences; mAb 39 recognizes only the Cterminal domain (8). Opioid peptides require their N-terminal sequences for binding. The immediately adjacent residues are responsible for the binding-site selectivities, and further C-terminal extensions, in general, contribute little or nothing to opioid binding (9). Accordingly, and as documented later in this paper, mAbs 17.M and 39 did not block the K-receptor binding of dyn-32. Based on the above considerations and the structure of dyn-32, we synthesized several chimeric peptides with the sequences of DAGO, dermorphin, deltorphins and [DSer2,Leu5]enkephalin-Thr (DSLET) at the N termini, followed in every case by residues 10-32 of dyn-32 (Fig. 1). It

Peptides and mAbs. dyn-32 and all the chimeric peptides were synthesized on an Applied Biosystems 430A peptide synthesizer. Their purities and sequences were confirmed by HPLC analysis and by sequence analysis on the Applied Biosystems 477A protein/peptide sequencer. mAb 17.M was purified from mouse ascites fluid, by using a rat anti-mouse IgG antibody affinity column (Boehringer Mannheim). mAb 39 was produced in hybridoma cell culture in RPMI 1640 medium (J. R. Scientific, Woodland, CA) with fetal calf serum reduced gradually from 10% to 0%. The supernatant was collected by centrifuging (200 x g, 20C, 10 min) and concentrated on a Diaflo ultrafilter (YM type with 5000-Da limit, Amicon) under N2 pressure. Finally, the antibody was purified by immunoaffinity chromatography as above. Peptides and mAbs were labeled with 1251 by the chloramine-T method. 1251I-labeled peptides were purified on HPLC. 1251I-labeled IgG was purified on a Sephadex G-50 column and then on the rat anti-mouse IgG antibody affinity column. ELISA Assays. In antibody titration (dilution) assays, Microtiter plate (Dynatech) wells were coated with 100 ,ul of 0.1 M acetate buffer (pH 4.5) containing chimeric peptide at 5 ,ug/ml, incubated at 4°C overnight, and then washed three times with Dulbecco's phosphate-buffered saline (PBS) (GIBCO, containing calcium and magnesium)/0.05% Tween 20. One hundred microliters of PBS/0.1% bovine serum albumin (BSA) was added to the wells and incubated overnight at 40C to block uncoated sites. The plates were washed again with PBS/Tween. Different concentrations of mAb

MATERIALS AND METHODS

Abbreviations: BSA, bovine serum albumin; DAGO, [D-Ala2, N-MePhe4,Gly-ol5]enkephalin; Pen, penicillamine; DPDPE, [DPen2,D-Pen5]enkephalin; DSLET, [D-Ser2,Leu5]enkephalin-Thr; dyn,

dynorphin; dyn-32, dynorphin-32; KHB, Krebs-Hepes buffer; PBS, Dulbecco's phosphate-buffered saline; U50,488, trans-3,4-dichloro-

N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methanesulfonate; U69,593, (5a,7a,8,8)-(+)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro-(4,5)dec-8-yl]benzeneacetamide; mAb, monoclonal antibody. tTo whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 3180

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3181

Opioid Receptor Binding Assay. Guinea pig brain membranes prepared as described (10). About 50,000 cpm per tube (1-ml assay) of [3H]DAGO (47 Ci/mmol, NEN), [3H]DPDPE (28 Ci/mmol, NEN) and [3H]U69,593 (42 Ci/mmol, NEN) (U69,593 is (5a,7a,8,8)-(+)-N-methyl-N-[7-(1-pyrrolidinyl)1-oxaspiro-(4,5)dec-8-yl]benzeneacetamide) was used to label ,u, and K sites, respectively, of which 2000-3000 cpm was bound. Different concentrations of chimeric peptides were used to compete. A set of parallel tubes containing radioligands, competing peptides, and additionally 0.5 ,uM unlabeled DAGO or DPDPE (Peninsula Laboratories) or U50,488 (trans-

Tyr-Gly-Gly-Phe-Leu-Arg-Arg-lIe-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gin-Lys-ArgTyr-Gly-Gly-Phe-Leu-Arg-Arg-GIn-Phe-Lys-Val-Val-Thr

were

Dyn-32

Tyr-D-Ala-Gly-NMe-Phe-Gly-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln-Lys-Arg-Tyr-GlyGly-Phe-Leu-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr DAGO-DYN

8,

Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-Gly-Pro-Lys-Leu-Lys-Trp-Asp-Asn-GIn-Lys-ArgTyr-Gly-Gly-Phe-Leu-Arg-Arg-GIn-Phe-Lys-Val-Val-Thr Dermorphin-DYN

3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]ben-

Tyr-D-Ala-Phe-Asp-Val-Val-Gly-Phe-Leu-Thr-Pro-Lys-Leu-Lys-Trp-Asp-Asn-GlnLys-Arg-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr

zeneacetamide methanesulfonate; Upjohn) was used for subtraction of nonspecific binding (30-50o of total). The binding assays were carried out in Krebs-Hepes buffer (KHB) [118 mM NaCl/4.8 mM KCl/2.5 mM CaCl2/1.2 mM MgC12/25 mM Hepes (Hepes is N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid; Research Organics), pH adjusted to 7.4 with NaOH] in 96-tube Microtiter plates (1-ml capacity, Bio-Rad) in triplicate. After incubation (room temperature, 2 hr), samples were kept on ice for 10 min and then filtered on glass filter strips (Schleicher & Schuell, grade 32) on a 12-channel LKB harvester. After three washes with 5 ml of cold KHB, filters were cut, placed in scintillation vials, and counted. For the NG108-15 cells, the binding assay was performed on intact cells. Cells were cultured in Dulbecco's modified Eagle's medium (J. R. Scientific) containing 10% fetal calf serum (HyClone), vitamins (Sigma, biotin at 7.3 ,g/liter, lipoate at 200 ,g/liter, B12 at 10 mg/liter) and 100 ,uM hypoxanthine/0.4 ,uM aminopterin/16 ,uM thymidine (HAT; Sigma) at 37°C with 5% CO2. At half confluent density, cells were harvested, washed with KHB, and then resuspended in KHB/0.05% BSA. The 1-ml assay contained 105 cells. After incubation, cells were separated and washed three times with cold KHB/BSA by centrifugation (200 x g, 2°C, 5 min). Cell pellets were dissolved in scintillation solution and counted. Integrity of Chimeric Peptides in the Binding Assays. Peptide (100 nM, final concentration) containing =50,000 cpm of 125I-labeled peptide was incubated with a guinea pig brain membrane preparation or NG108-15 cells at room temperature for 2 hr. After chilling on ice for 5 min and centrifuging (10,000 x g, 2°C, 10 min), supernatants were transferred to new tubes and lyophilized; then they were redissolved in 200

Deltorphin I-DYN

Tyr-D-Ala-Phe-Glu-Val-Val-Gly-Phe-Leu-Thr-Pro-Lys-Leu-Lys-Trp-Asp-Asn-GInLys-Arg-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr Deltorphin Il-DYN

Tyr-D-Ser-Gly-Phe-Leu-Thr-Pro-Lys-Leu-Lys-Trp-Asp-Asn-GIn-Lys-Arg-Tyr-GlyGly-Phe-Leu-Arg-Arg-GIn-Phe-Lys-Val-Val-Thr DSLET-DYN

FIG. 1. Sequences of dyn-32 and synthetic chimeric peptides. N-terminal segment in boldface type represents unique sequence of each peptide; remaining C-terminal segment is common to all peptides.

17.M or 39 in PBS/BSA were added (100 per well) and incubated at room temperature for 2 hr. In peptide competition assays, the wells were coated only with dyn-32, and a fixed amount of mAb solution (1 ng per well in 100 jul of PBS/BSA) was added with different concentrations of chimeric peptides. After incubation, the plates were washed, and horseradish peroxidase-conjugated rabbit anti-serum against mouse IgG, IgA, and IgM (both heavy and light chains) (Zymed Laboratories, diluted 1/4000 with PBS/BSA, 100 Aul per well) was added, followed by incubation for 1 hr at room temperature. After washing, 100-Al substrate 2,2'azino-di-(3-ethylbenzthiazoline sulfonate) (ABTS) solution (Boehringer Mannheim) was added, followed by incubation for another 1 hr; then the plates were read at 405 nm on an ELISA reader. mAb

mAb 39

17.M

4

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3

2

2

LO 0

-

0

0

-7

-5

-6

-4

-3

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Log concentration of mAb,

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-3

-2

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0

Ag/well

0.3.

0.2

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0.11

0.11 n.L

-15

-4

-14

-13

-12 -11

-10

-9

-8

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Log concentration of peptide

-13

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-9

-8

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FIG. 2. (Top row) ELISA results for titration of mAbs 17.M and 39 binding to chimeric peptides. (Bottom row) Chimeric peptides competing for the binding of mAb 17.M and 39 (1 ng per well) to dyn-32. *, dyn-32; o, DAGO-DYN; *, dermorphinDYN; o, deltorphin I-DYN; *, deltorphin II-DYN; A, DSLETDYN; x, dyn A; +, /3-endorphin. Points are means of triplicates from single experiments.

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,pl of methanol/0. 1 M HCl (vol/vol, 1: 1) and injected onto a

1LBondapak C18 reversed-phase column (30-min linear gradi-

ent of 20-50% acetonitrile in 5 mM trifluoroacetic acid, 1.5 ml/min). Each fraction (0.6 ml) was collected, and radioactivity was determined on a y counter. Counts from fractions representing intact 1251I-labeled peptide peaks were compared with controls, which were peptides incubated without guinea pig brain membranes or with boiled membranes. Antibody-Peptide Sandwich Binding Assay. Different concentrations of chimeric peptides were incubated with guinea pig brain membranes in 1 ml KHB at room temperature for 1.5 hr followed by 30 min on ice. In another set of tubes 0.5 ItM DAGO, DPDPE, or U50,488 was included. After incubation, samples were washed three times with ice-cold KHB by centrifugation (10,000 x g, 20C, 20 min). Pellets were resuspended in 1 ml of cold KHB, and about 5 x 105 cpm of 125I-labeled mAb 17.M or 39 was added, followed by incubation on ice for 1 hr. After washing three times by centrifugation, pellets were transferred to new tubes, and radioactivity was determined. Assays without guinea pig brain membranes or without peptides served for background determination. Panning of NG108-15 Cells. We used a procedure modified from that described by Seed and Aruffo (11). Petri dishes (60-mm diameter) were coated with mAb 17.M (30 ,ug per dish in 3 ml of 50 mM Tris-HCI buffer, pH 9.5) for 2 hr and then washed three times with 0.15 M NaCl. Three milliliters of 0.1% BSA/PBS was added, followed by incubation at 4°C overnight. NG108-15 cells at half confluent density were detached from cell culture dishes by incubation with PBS

(GIBCO, without calcium and magnesium)/0.5 mM EDTA/ 0.02% sodium azide. After suspending once in KHB and centrifuging (200 x g, 2°C, 5 min), cells (2 x 105 per tube) were resuspended in 1 ml of KHB/0.1% BSA containing 100 nM dyn-32 or chimeric peptide. After 1.5-hr incubation at room temperature and 30 min on ice, cells were washed three times with cold KHB by centrifugation as above, then resuspended in 1 ml of PBS/0.5 mM EDTA/0.1% BSA and plated on the antibody-coated dishes. After 2 hr at room temperature, the dishes were washed gently three times with 3 ml of PBS. Cells remaining on the dishes were observed under the microscope. The specificity of a given peptide for panning the NG108-15 cells was tested by competition with type-selective opioid ligands.

RESULTS Antibody Recognition of Chimeric Peptides. Fig. 2 (top row) shows that mAbs 17.M and 39 recognized dyn-32 and all chimeric peptides but not dyn A. In the competition assays (Fig. 2, bottom row) all chimeric peptides were able to block the binding of mAbs 17.M and 39 to dyn-32; IC50 values were in the high picomolar to low nanomolar range. dyn A and 8-endorphin, which lack the C-terminal epitope, were ineffective. Opioid Receptor Binding Selectivities of Chimeric Peptides. DAGO-DYN and dermorphin-DYN exhibited typical high

0

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DSLET-DYN.

DA

Guinea. pig brain membranes

NG108-15 cells

FIG. 4. HPLC analysis of peptide degradation in receptorbinding assays with guinea pig brain membranes or NG108-15 cells. (a) -. . , Control 125I-labeled dyn-32 incubated with boiled membrane preparation; -, 1251I-labeled dyn-32 incubated with membranes. (b) Chimeric peptides; each bar represents the percentage area of intact 125I-labeled peptide HPLC peak compared with boiled membrane control (left) or cell-free control (right). DERM, dermorphin; DEL, deltorphin.

Pharmacology: Xie et aL

Proc. Natl. Acad. Sci. USA 87 (1990)

affinities and selectivities for Au sites in competing with 1U ligand [3H]DAGO (Fig. 3a), 8 ligand [3H]DPDPE (Fig. 3b), and K ligand [3H]U69,593 (Fig. 3c) in standard binding assays. Deltorphin I-DYN, deltorphin I-DYN, and DSLET-DYN had high affinity and selectivity for 8 sites, and the deltorphinDYN peptides did not compete for K binding even at 10 ILM concentration. dyn-32 displayed its well-known high affinity and selectivity for K sites. Integrity of Chimeric Peptides in Binding Assays. After 2-hr incubation with guinea pig brain membranes, only 23% of added dyn-32 remained intact in the supernatant; the remainder was degraded or formed complexes with membrane components that were eluted in different fractions on HPLC (Fig. 4a). All the chimeric peptides were degraded to about the same extent (Fig. 4b). In the binding assay with NG108-15 cells, 40-50% of the chimeric delta ligands were degraded (Fig. 4b). Antibody Recognizes Chimeric Peptides Bound to Receptors. mAbs 17.M and 39 were able to bind not only the free form of the chimeric peptides but also the receptor-bound form. Fig. 5 shows that the binding of 125I-labeled mAb 17.M to the peptide-receptor complex was dependent on peptide ligand concentration and was competitively reduced by a high concentration of the corresponding type of selective ligand. From the specific activity of radiolabeled IgG (41000 Ci/ mmol), the amount of membrane preparation, and the assumption that each IgG molecule binds two molecules of peptide, we could calculate the numbers of receptor sites that were saturated by ligand and recognized by antibody. The highest numbers found [pmol/g of brain (wet weight)] were 0.44 for ,u, 0.39 for 8, and 0.28 for K. Corresponding Bma,, values from standard ligand binding assays were 0.82, 1.1, and 2.8, respectively (10). To test whether the mAb was able to bind chimeric peptides that had already bound to receptors on cell surfaces, we carried out the experiment of panning NG108-15 cells on mAb 17.M-coated dishes. These cells express only 8 opioid receptors (12); under our culture and binding assay conditions Bm., 6

.a

5.

DAGO-DYN

b

5-

4.

was 410,000 sites per cell for [3H]DPDPE. If either the peptide ligand or antibody was omitted, very few cells were retained on the dish (data not shown). Typical panning results are shown in Fig. 6. Cells adherent to a plate coated with mAb 17.M after incubation with DAGO-DYN are shown before (Fig. 6a) and after (Fig. 6b) the wash step in the panning procedure; no significant adherence of cells to the dish was observed. The same negative result was seen when cells were incubated with dermorphin-DYN or dyn-32 (data not shown), consistent with the absence of A and K receptors from these cells. In contrast, after incubation with deltorphin I-DYN, most cells were retained on the antibody-coated dishes after the wash, even when a K or, ligand (e.g., DAGO, Fig. 6c) was present during the incubation. Similar results were obtained with deltorphin II-DYN or DSLET-DYN (data not shown). However, the positive panning result could be prevented by incubation with 0.5 ,uM DPDPE (Fig. 6d).

DISCUSSION To use peptide ligands and antibodies against the peptides for identifying and isolating peptide receptors, three important requirements should be realized: (i) The peptide should have high affinity and type-selectivity for a particular receptor type. (ii) The peptide should be long enough so that when bound to the receptor, sufficient free epitope still exists for antibody recognition. (iii) Binding of the antibody to the peptide should not affect binding of the peptide to the receptor. In the case of opioid receptors, most selective peptide ligands are relatively short, leaving little to protrude from the cell membrane after binding. And even for peptides with suitable length, raising a nonblocking antibody to each one separately would be a laborious procedure. We have simplified the approach by synthesizing chimeric peptides with high-affinity, type-selective sequences at the N terminus (which, in the case of opioid peptides, binds to the receptor), and we have placed at the C terminus a common peptide sequence containing the epitopes for either of two existing mAbs. As expected, these peptides possessed high

Dermorphin-DYN foa

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Deltorphin Il-DYN

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6

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FIG. 5. Binding of mAb 17.M to the chimeric peptide-

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1

0

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Concentration of chimeric peptide [nM]

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membrane receptor complexes. Points are means of triplicates from single experiments. ---, Total binding in the presence of 0.5 ,uM DAGO (a, b), DPDPE (c-e) or U50,488 (f); -, specific binding (difference plot).

3184

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d

FIG. 6. Panning of NG108-15 cells on mAb 17.M-coated dishes. (Top row) Cells were incubated with DAGO-DYN, and free ligand was removed by centrifuging; then cells were plated on antibodycoated dishes for 2 hr. (a) Before wash step in panning procedure. (b) After wash step. (Bottom row) Cells were incubated with deltorphin I-DYN together with another ligand (DAGO (c); DPDPE (d)) and then subjected to the wash step.

affinities and selectivities for the several types of opioid very similar to these properties of the original ligands. mAbs 17.M and 39 recognized these chimeric peptides and dyn-32 (against which they were raised) equally well. All the chimeric peptides could compete for binding of mAbs 17.M and 39 to dyn-32. The results from guinea pig brain membrane binding assays and from panning of NG108-15 cells indicate that after the chimeric peptides bound to opioid receptors through their N-terminal domains, their C-terminal extensions were long enough for antibody recognition. It has been known that the N-terminal 13 residues of the endogenous K ligand dyn A are necessary and sufficient for affinity and specificity (9). dyn A-analogue kappa ligand (DAKLI), a dyn A-derived peptide ligand designed on this principle, has high affinity and selectivity for K opioid receptors; and biotinylated DAKLI, even after it is bound to the receptors, can bind avidin (6). In the present study, the C-terminal amide group of dermorphin, deltorphins, and DSLET, as well as the alcohol group of DAGO were removed, and a 23-residue C-terminal extension was added. The maintenance of high affinity and typeselectivity by these synthetic chimeric peptides implies that their C-terminal sequences are not directly involved in receptor binding. Degradation of peptide ligands in receptor-binding assays can be tested by HPLC or equivalent analytical method. Goldstein et al. (6) demonstrated that dynorphin peptides, at low concentrations (