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 was assumed that the C-terminal amide group ofdermorphin, which the N-terminal nine residues of dynorphin-32, a peptide deltorphins, and DSLET and the alcohol group of DAGO selective for the K opioid receptor, were replaced by opioid could be removed without affecting opioid binding. By anal- peptides selective for other opioid receptor types. Each chi- ogy to dyn-32, which binds selectively to K opioid sites, meric peptide retained the high affminty and type selectivity DAGO-DYN and dermorphin-DYN should bind selectively characteristic of its N-terminal sequence. The common C- to p.; deltorphins-DYN and DSLET-DYN should bind selec- terminal two-thirds of the chimeric peptides served as an tively to 8. mAbs 17.M and 39 should act as nonblocking epitope recognized by the same monoclonal antibody. When antibodies to all these peptides. bound to receptors on a cell surface or membrane preparation, In the present study, we have demonstrated that the these peptides could still bind specifically to the monoclonal chimeric peptides do maintain the high affinities and type antibody. These chimeric peptides should be useful for isolating selectivities of their N-terminal sequences. mAbs 17.M and ,A, 8, and c opioid receptors and for identifying opioid recep- 39 bind to these peptides (as to dyn-32), even after the tors on transfected cells in expression cloning procedures. The peptides are bound to receptors on brain membranes or on general approach using chimeric peptides should be applicable intact NG108-15 neuroblastoma-glioma hybrid cells. to other peptide receptors. MATERIALS AND METHODS Several peptide ligands selective for different opioid receptor Peptides and mAbs. dyn-32 and all the chimeric peptides types have been isolated from natural sources or synthesized. were synthesized on an Applied Biosystems 430A peptide Among them [D-Ala2,N-MePhe4,Gly-ol5]enkephalin synthesizer. Their purities and sequences were confirmed by (DAGO) (1) and dermorphin (2) have an optimal combination HPLC analysis and by sequence analysis on the Applied ofhigh affinity and type selectivity for , binding sites. [D-Pen2, Biosystems 477A protein/peptide sequencer. D-Pen5]enkephalin (DPDPE) (3) and deltorphin I and II (4) mAb 17.M was purified from mouse ascites fluid, by using have similar properties with respect to 8 sites, as do dynorphin a rat anti-mouse IgG antibody affinity column (Boehringer A (dyn A) (5) and its derivatives (6) for K sites. Each of these Mannheim). mAb 39 was produced in hybridoma cell culture peptides has a subnanomolar dissociation constant at its in RPMI 1640 medium (J. R. Scientific, Woodland, CA) with preferred binding site, at least two orders ofmagnitude greater fetal calf serum reduced gradually from 10% to 0%. The affinity than at its next-preferred binding site. supernatant was collected by centrifuging (200 x g, 20C, 10 For experiments on the expression cloning of opioid re- min) and concentrated on a Diaflo ultrafilter (YM type with ceptors we wished to develop a system in which peptide 5000-Da limit, Amicon) under N2 pressure. Finally, the ligands and antibodies against the peptides could be used for antibody was purified by immunoaffinity chromatography as affinity purification or for isolation of receptor-bearing cells. above. Dynorphin-32 (dyn-32) (Fig. 1), our model peptide, is, in Peptides and mAbs were labeled with 1251 by the chloram- effect, a fusion product of two peptides-the 17-residue dyn ine-T method. 1251I-labeled peptides were purified on HPLC. A at the N terminus and the 13-residue dynorphin B (dyn B) 1251I-labeled IgG was purified on a Sephadex G-50 column and at the C terminus, connected by a "bridge sequence" Lys- then on the rat anti-mouse IgG antibody affinity column. Arg. dyn-32 itself is a K opioid agonist (7). Of several ELISA Assays. In antibody titration (dilution) assays, Mi- monoclonal antibodies (mAbs) raised against dyn-32, two crotiter plate (Dynatech) wells were coated with 100 ,ul of0.1 were useful in this study. mAb 17.M requires the bridge and M acetate buffer (pH 4.5) containing chimeric peptide at 5 C-terminal sequences; mAb 39 recognizes only the C- ,ug/ml, incubated at 4°C overnight, and then washed three terminal domain (8). Opioid peptides require their N-terminal times with Dulbecco's phosphate-buffered saline (PBS) sequences for binding. The immediately adjacent residues are (GIBCO, containing calcium and magnesium)/0.05% Tween responsible for the binding-site selectivities, and further 20. One hundred microliters of PBS/0.1% bovine serum C-terminal extensions, in general, contribute little or nothing albumin (BSA) was added to the wells and incubated over- to opioid binding (9). Accordingly, and as documented later night at 40C to block uncoated sites. The plates were washed in this paper, mAbs 17.M and 39 did not block the K-receptor again with PBS/Tween. Different concentrations of mAb binding of dyn-32. Based on the above considerations and the structure of Abbreviations: BSA, bovine serum albumin; DAGO, [D-Ala2, dyn-32, we synthesized several chimeric peptides with the N-MePhe4,Gly-ol5]enkephalin; Pen, penicillamine; DPDPE, [D- sequences of DAGO, dermorphin, deltorphins and [D- Pen2,D-Pen5]enkephalin; DSLET, [D-Ser2,Leu5]enkephalin-Thr; dyn, Ser2,Leu5]enkephalin-Thr (DSLET) at the N termini, fol- dynorphin; dyn-32, dynorphin-32; KHB, Krebs-Hepes buffer; PBS, lowed in every case by residues 10-32 of dyn-32 (Fig. 1). It Dulbecco's phosphate-buffered saline; U50,488, trans-3,4-dichloro- N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide meth- anesulfonate; U69,593, (5a,7a,8,8)-(+)-N-methyl-N-[7-(1-pyrrol- The publication costs of this article were defrayed in part by page charge idinyl)-1-oxaspiro-(4,5)dec-8-yl]benzeneacetamide; mAb, monoclonal payment. This article must therefore be hereby marked "advertisement" antibody. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 3180 Downloaded by guest on October 1, 2021 Pharmacology: Xie et al. Proc. Natl. Acad. Sci. USA 87 (1990) 3181 Tyr-Gly-Gly-Phe-Leu-Arg-Arg-lIe-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gin-Lys-Arg- Opioid Receptor Binding Assay. Guinea pig brain membranes Tyr-Gly-Gly-Phe-Leu-Arg-Arg-GIn-Phe-Lys-Val-Val-Thr were prepared as described (10). About 50,000 cpm per tube Dyn-32 (1-ml assay) of [3H]DAGO (47 Ci/mmol, NEN), [3H]DPDPE (28 Ci/mmol, NEN) and [3H]U69,593 (42 Ci/mmol, NEN) Tyr-D-Ala-Gly-NMe-Phe-Gly-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln-Lys-Arg-Tyr-Gly- Gly-Phe-Leu-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr (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 DAGO-DYN ,u, 8, and K sites, respectively, of which 2000-3000 cpm was bound. Different concentrations ofchimeric peptides were used Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-Gly-Pro-Lys-Leu-Lys-Trp-Asp-Asn-GIn-Lys-Arg- Tyr-Gly-Gly-Phe-Leu-Arg-Arg-GIn-Phe-Lys-Val-Val-Thr to compete. A set of parallel tubes containing radioligands, competing peptides, and additionally 0.5 ,uM unlabeled DAGO Dermorphin-DYN or DPDPE (Peninsula Laboratories) or U50,488 (trans- Tyr-D-Ala-Phe-Asp-Val-Val-Gly-Phe-Leu-Thr-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln- 3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]ben- Lys-Arg-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr zeneacetamide methanesulfonate; Upjohn) was used for sub- Deltorphin I-DYN traction of nonspecific binding (30-50o of total). The binding assays were carried out in Krebs-Hepes buffer (KHB) [118 mM Tyr-D-Ala-Phe-Glu-Val-Val-Gly-Phe-Leu-Thr-Pro-Lys-Leu-Lys-Trp-Asp-Asn-GIn- NaCl/4.8 mM KCl/2.5 mM CaCl2/1.2 mM MgC12/25 mM Lys-Arg-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr Hepes (Hepes is N-2-hydroxyethyl piperazine-N'-2-ethane- Deltorphin Il-DYN sulfonic acid; Research Organics), pH adjusted to 7.4 with NaOH] in 96-tube Microtiter plates (1-ml capacity, Bio-Rad) in Tyr-D-Ser-Gly-Phe-Leu-Thr-Pro-Lys-Leu-Lys-Trp-Asp-Asn-GIn-Lys-Arg-Tyr-Gly- triplicate. After incubation (room temperature, 2 hr), samples Gly-Phe-Leu-Arg-Arg-GIn-Phe-Lys-Val-Val-Thr were kept on ice for 10 min and then filtered on glass filter strips DSLET-DYN (Schleicher & Schuell, grade 32) on a 12-channel LKB har- vester. After three washes with 5 ml of cold KHB, filters were FIG. 1. Sequences of dyn-32 and synthetic chimeric peptides. cut, placed in scintillation vials, and counted. N-terminal segment in boldface type represents unique sequence of For the NG108-15 cells, the binding assay was performed each peptide; remaining C-terminal segment is common to all pep- on intact cells. Cells were cultured in Dulbecco's modified tides. Eagle's medium (J.