Differential Binding Domains of Peptide and Non-peptide Ligands in ...

Vol. 269, No. 48, Issue of December 2, pp. 30195-30199, 1994 Printed in U.S.A.

THEJOURNAL OF BroLoGlra CHEMISTRY D 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Differential Binding Domains of Peptide and Non-peptide Ligands in the Cloned RatK Opioid Receptor* (Received for publication, September 2, 1994, and in revised form, October 12, 1994)

Ji-Chun Xue, Chongguang Chen, Jinmin Zhu, Satya KunapuliS, J. Kim DeRiel9, Lei Yun, and Lee-Yuan Liu-Chenll From the Departments ofPh.armacology and $Physiology and SFels Znstitute for Molecular Biology and Cancer Research, Temple University School of Medicine, Philadelphia, Pennsylvania 19140 and the IDepartment of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, Indiana46202

This study was to identify specific regions Kin opioid re- putative transmembrane helices (TMHs)’ separated by intraceptors that accounted for binding selectivity of K ligands. and extracellular loops, characteristics of G protein-coupled Six chimeric~ J receptors K were constructedfrom cloned receptors. Comparisonof the amino acid sequences revealsthat rat K and p opioid receptors and transiently expressed in three opioid receptors display an overall -60% identity to each COS-1 cells.Allsixchimeric ~ J Kreceptorsbound r3H1 other.Sequences within TMHs are highly homologous; condiprenorphine with high affinities, indicating that these versely, the N-terminaldomain, C-terminal domain, second exaf- tracellular (e2) loop, and third extracellular (e3)loop are very chimeras retain opioid receptor conformation. Binding finities of three peptide ligands (dynorphin A, a-neo-endor- divergent. These divergent sequences may be critical in deterB) and three nonpeptide ligands (norphin, and dynorphin mining ligand selectivity of these receptors. The e2 loop of the binaltorphimine, U50,488H, and U69,593) forchimeras K receptor is unique, which contains 5 Asp residues and 3 Glu to those forp and K opioid residues. These acidic amino acids may be pivotal in the bindwere determined and compared receptors.The second extracellular loop and the adjoining ing of endogenous K peptides, i.e. the dynorphin family of pepC-terminalportionofthefourthtransmembrane helix tides, which are basic peptides. were essential for the high affinity of binding dynorphinA, Studies on G protein-coupled receptors indicate that while a-neo-endorphin, and dynorphinB to the K receptor. The binding domains of small molecule neurotransmitters reside third extracellular loop and the sixth and seventh trans-mostly in TMHs, extracellular loops are important for peptide membrane helicesplayed an important role in determining binding. For example, in p-adrenergic receptors, it has been K over thep the selectivityof nor-binaltorphimine for the shown that the agonist or antagonist bindingdomainlies receptor. U50,488H and U69,593 appeared to require the within the seven TMHs and most of the hydrophilic regions, whole K receptor except the second extracellular loop to attain high affinity binding. Thus, the K opioid receptor hasincluding the N- and C-terminal domains and extracellular differential binding domains for peptide and non-peptide loops appear to be dispensable for ligand binding (10, 11). In contrast, extracellular loops and TMHs of the neurokinin-1 ligands. receptor are involved in thebinding of substance P and related peptides (12, 13). Chimeric receptorsof closely related receptors have been very Opiates and opioid compounds act on receptors on cell mem- useful in delineation of ligand binding domains of receptors. For branes to produce their effects (1).The presence of at least example, studies on the ligand-binding characteristics of p,/& three types of opioid receptors (p, K , and 6) in the nervous chimeric receptors led t o the conclusion that the TMH 4 was system has been established on the basis of differential phar- largelyresponsible for versus p2 agonistbinding selectivity macological and binding properties and anatomical distribuand that theTMHs 6 and 7 were important for selectivity of PI tions (1). Inneuronal cells, activation of opioid receptors versus p2 antagonists (14). Chimericm,/m, muscarinic receptors couples via pertussis toxin-sensitive G proteins to various ef- have been useful in defining regions that are responsible for fectors including adenylate cyclase and K‘ and Ca” channels selective interaction with m, and m, antagonists (15). (for reviews, see Refs. 2 and 3). Many drugs act on all types of In this study, we investigated the regions in the K receptor opioid receptors, but there are selective ligands for each type, that are responsible for selective interaction with K ligands by The structural basis of the overlapping, yet distinct, ligand ~ examining bindingof these ligands to six chimeric F l j receptors. binding characteristics of opioid receptors has yet to be defined. Chimeric receptors were constructed from cloned rat p (RMOR) Following the cloning of 6 opioid receptors (4, 5), we (6, 7) as and K (RKOR) opioid receptors. Bindingofthree peptides (dynorwell as several other groups cloned p and K receptors (for re- phinA(1-171, a-neo-endorphin and dynorphin B) and threenonviews, see Refs. 8 and 9, and references therein). Elucidation of peptides (U50,488H, U69,593, and nor-binaltorphimine (norprimary structures makes possible it to define binding domains BNI)) was examined. Dynorphin A(1-171, a-neo-endorphin, and of ligands within receptors. Hydropathy analyses of deduced dynorphin B, three members of the prodynorphin peptide famamino acid sequences of these clones display the motif of seven ily, have high affinities and moderate selectivity for the K receptor (6,16).U50,488H and U69,593 are highly selective K opi* This work was supported by Grant DA 04745 from NIDA, National oid agonists (17, 18).nor-BNI is a highly selective K antagonist Institutes of Health. The costs of publication of this article were de- (19,201. Understanding of how these ligands are oriented inthe frayed in part by the payment of page charges. This article musttherefore be hereby marked “udvertisernent”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ’ The abbreviations used are: TMH, transmembranehelix; aa, amino 11 TO whom all correspondence and reprint requests should be ad- acid(s); e2 loop, the second extracellular loop; e3 loop, the third extradressed: Dept. of Pharmacology, Temple University Schoolof Medicine, cellular loop; i3 loop, the third intracellular loop; nor-BNI, nor-binal3420 N. Broad St., Philadelphia, PA 19140. Tel.: 215-707-4188; Fax: torphimine; nt, nucleotide(s1;PCR, polymerase chain reaction; RMOR, 215-707-7068. the rat p opioid receptor; RKOR, the rat K opioid receptor.

30195

30196

Peptide and Nonpeptide Binding Domains

of

K

Opioid Receptors

receptor would facilitate computerized modeling of the receptor structure.

41

50

EXPERIMENTAL PROCEDURES

91 1w

Materials [3HlDiprenorphine (35CYmmol) was obtained from Amersham Corp. Naloxone was a gift from DuPontMerck, and U50,488H and U69,593 were provided by Upjohn. Dynorphin A, a-neo-endorphin, and dynorphin B were purchased from Peninsula Laboratories (Belmont, CA); nor-BNI from RBI (Wayland, MA); the vectors pRdCMV and pcDNA3 from Invitrogen (San Diego, CA); Pfu DNA polymerase and the vector pBK-CMV from Stratagene (San Diego, CA). Construction of Chimeric ,uJ K Receptors Sixchimericreceptors,chimeras I, 11,111,IV, XI, and XI, were constructed from RMOR and RKOR. Schematic drawings are shown in Fig. 1 and Table 11. Nucleotide (nt) numbers of RMOR and RKOR are those of EMBL Data Bank accession numbers L13069 and L22536, respectively. Polymerase chain reaction (PCR) was carried out Pfu with DNA polymerase, and PCR cycle was 95 "C for 5 min, 50 "C for 5 min, and 72 "C for 2 min for 30 cycles. Fragments generated by PCR and transitional regions of each chimera were confirmed by nucleotide sequence determination with the methodof Sanger et al. (21). For chimera I (aminoacid (aa) ~1-184/p194-268/~263-380) and chimera I1 (aa p1-193/~185-262/~269-398),the fragments exchangedextended from the middle of the TMH 4 to the middleof the third intracellular (i3) loop. Chimera I was constructed on the basic structure of RKOR with the RMOR fragment (Leu'94 - S e P 8 ) ; chimera I1 had RMOR basic structure with the fragment of RKOR (Leuls5- Ser262) (Fig. 1 and Table 11). Chimera I-PCR was performed to generate a chimeric $K fragment according to the method of Horton et al. (22). PCR was performed on RKOR with primers RKOR n t 543-567 and RKOR n t 753-774RMOR n t 789-791 and on RMOR with RKOR n t 761-774EMOR n t 789-805 and RMOR n t 995-1019. Two fragments generated were subjected to overlap PCR to produce a chimeric $ K fragment, which was digested with HindIII andBum11 t o generate a 280-bp fragment,PCRKM1. For chimera I, the KpnIIHindIII fragmentof RKOR in pcDNA3 (nt 1-7171, PCRKM1, and the BamIIlNotI fragment of RKOR in pcDNA3 (nt 10072112) were ligated into KpnI andNot1 sites of pcDNA3. Chimera 11-PCR was performed with RKOR as the template and primers 1 K 2 (5'-CATTTGGATCCTGGCATCAGCTGTTG-3') (RKOR n t 765-790) and RKOR n t 995-1019 to create a BamHI site (underlined) with no change in the amino acid sequence. The PCR product was digested with BamHI and Ban11 t o generate PCRK2. The HindIIII BamHI fragment of RMOR in pRdCMV (nt 1-7851, PCRK2, and the BamIVHindIII fragment of RMOR in pRc/CMV (nt 1012-1437) were ligated into the HindIII site of pcDNA3. Correctly oriented recombinant was selected by DNA sequence. Chimera I11 (aa ~1-141/p151-398) and chimera IV (aa p1-150/~142380) were constructedby swapping the regions from the N terminus to the startof the TMH 3. A conserved unique AflIII site within the TMH 3 of both RKOR ( A ~ n ' ~ ~ / M e t ' ~ ~RMOR ) a n d( A ~ n ' ~ ~ / M e twas ' ~ ' ) used to generate fragmentsfor swapping (Fig.1 and Table 11). For chimera 111, the KpnVAflIII fragment of RKOR in pcDNA3 (nt 1-645) and theAflIIII XbaI fragment of RMOR in pRdCMV (nt 659-1437) were ligated into KpnI and XbaI sites of pcDNA3. For chimera IV, HindIIUAflIII fragment ofRMOR in pRdCMV (nt 1-658) and AflIIIIApaI fragment of RKOR in pcDNA3 (nt 6462112) were ligated into HindIII and ApaI sites of pcDNA3. Chimera XI (aa p1-268/~263-380)andchimera XI1 (aa ~1-2621 ~ 2 6 9 3 9 8were ) generatedby exchanging the regions from the middle of the i3 loop (Sera6z/Ar~s3 of RKOR and SeP68/Lys269 of RMOR) to the C terminus (Fig. 1and Table 11). For chimera X I , the HindIIIIBanII fragment of RMOR in pRdCMV (nt 1-1012) and the BanIVApaI fragment of RKOR-pcDNA3 (nt 1007-2112) wereligatedintoHzndIIIIApaI pcDNA3. PCR wasperformedtogenerate RKOR n t 797-1078. For chimera X I , the SacIIBglII fragment of RKOR in pcDNA3 (nt 1-9031, BglIIIBanII-treated PCR product (RKOR n t 904-1006) and the BanIIl HindIII fragment of RMOR in pRdCMV (nt 1013-1437) were ligated into SacIIHindIII-treated pBK-CMV. Dansient Expression of Wild e p e s a n d C h i m e r a s i n COS-1 Cells Wild types and chimeras were transfected into COS-1 cells as described (61, except that transfection was performed with DEAE-dextran-chloroquine method (23) at 6 8 pg of DNN100-mm dish. Cells

141

150

191 XX)

241 247

291 297

340

346 380 3%

FIG.1. Amino acid sequence comparison ofrat p and K opioid receptors and points of exchange ( A X ) for generation of chimeric receptors. Chimeras I (aa ~1-184/p194-268/~263-380)and I1 (aa p1-193/~185-262/p269-398) weregenerated by an exchange of fragments from point B to pointC. Chimeras I11 (aa ~1-141/p151-398) and IV (aa p1-150/~142-380) were constructedby swapping fragments from the N terminus to pointA. Chimera XI (aa p1-268/~263-380) and XI1 (aa ~1-262/p269-398)resulted from switchingfragments from point Ct o the C terminus. Dash indicates the same amino acid in thep receptor as in the K receptor. Dots represent gaps introduced for sequence alignment. Seven putative transmembrane helices (TMHs) are underlined. Amino acid residue numbers are indicated on both sides and in the vicinityof points B and C. were harvested 48-60 h following transfection, and membranes were prepared (6). Receptor Binding Experiments Opioid receptor binding was conducted with [3Hldiprenorphine according t o our published procedure (6).Saturation experiments were performedwitheightconcentrations of [3Hldiprenorphine(ranging from 0.05 to 5 nM). Competitive inhibitionof [3Hldiprenorphine binding was performed with 0.2-0.4 IIM [3Hldiprenorphine and 13 concentrations of the unlabeled drug. Naloxone (10 p ~ was ) used to define nonspecific binding. Binding data were analyzed with EBDA and LIGAND programs (24). Protein contents of membrane preparations were determined by the BCA method of Smith et al. (25). RESULTS

Chimeric p / K Opioid Receptors-For each chimera, DNA sequence of the fragment produced by PCR and those in the transitional regions between two receptorswere verified to ensure successful in-frame construction (Fig. 1 and Table 11). For chimeras I (aa ~1-184/p194-268/~263-380) and 11 (aa p1-193/~185-262/p269-398), the fragmentsexchanged contain the C-terminal halfof the TMH 4, the e2 loop, the TMH 5, and the N-terminal half of the i3 loop, due to consideration on feasibility of DNA manipulation. Since sequences within the TMH 5 and the N-terminal half of the i3 loop are highly homologous between p and K receptors(Fig. 11, the exchange essentially involves the e2 loop and the C-terminal portion of the TMH 4. Chimeras I11 (aa ~1-141/p151-398) and IV (aa pl-150/ ~ 1 4 2 - 3 8 0 )represent replacement of a segment of RKOR with that of RMOR from the middle of TMH 3 to theC terminus and from the N terminus to themiddle of TMH 3, respectively (Fig. 1 and Table 11). Substitution of a portion of RKOR with that of RMOR, from


of K

Opioid Receptors

30197

TABLEI Kd and B,, of fHldiprenorphine binding to rat p and K opioid receptors and p I K chimeric receptors expressed in COS-1 cells Data are shown as mean 2 S.E. of three independent experiments in duplicate. Kd

(nM)

B,,

RKOR

RMOR

I

I1

I11

IV

XI

XI1

0.26 20.03 13432274

0.35 eO.12 23025312

0.2220.07 5992128

0.1820.05 15792364

0.3020.14 35262302

0.78 20.31 11542178

0.4920.07 436247

0.1420.02 23852182

(fmoVmg protein)

the N terminus to themiddle of i3 loop and from the middle of i3 loop to the C terminus, formed chimeras XI (aa p1-2681 ~263-380) and XI1 (aa ~1-262/p269-398), respectively (Fig. 1 and Table 11). Binding of PHIDiprenorphine to Chimeric ,ul K Opioid Receptors-Saturation binding of [3H]diprenorphine to chimeras I, 11,111, IV, XI, and XI1 was performed and compared to p and K receptors. All six chimeric p / ~ receptors bound r3H]diprenorphine with high affinity, with Kd ranging from 0.14 to 0.78 nM, similar to p and K receptors (Table I). These results indicate that these chimeras retain opioid receptor conformation. However, there were differences in expression levels,with Elrnax ranging from 436 fmol/mg protein for chimera XI t o 3526 fmol/mg protein for chimera I11 (Table I). Remarkably, expression levels of the same chimera were fairly consistent from different transient expression experiments. In all competitive binding inhibition studies, we took this variation into consideration and adjusted receptor concentration to less than onetenth of the Kd value of L3H1diprenorphineto obtain valid estimates of K, values. Binding of Dynorphin A, Dynorphin B, and a-Neo-endorphin to p a n d K Opioid Receptors and Chimeric p / Receptors-To ~ determine the significance of the C-terminal portion of the TMH 4 and the e2 loop of RKOR in selective interactions of K ligands, we performed competitive inhibition of F3H]diprenorphine binding by dynorphin A, dynorphin B, and a-neo-endorphin to chimeras I and 11. K, values were determined andcompared to those of p and K opioid receptors (Fig. 2, A-C, and Table 11). All three peptides bound to RKOR with 25-55-fold higher affinities than theydid t o RMOR. Substitution of the e2 loop and the C-terminal portion of the TMH 4 of RKOR with that of RMOR (I) generated a receptor that had substantially decreased affinities for these three peptides, similar toRMOR. Conversely, substitution of the C-terminalportion of the TMH 4 and the e2loop of RMOR with that of RKOR (11)produced a receptor that had high affinities for these three peptides, similar to RKOR. These results indicate that the e2 loop and the C-terminal portion of the TMH 4 of RKOR are critical for high affinity binding of dynorphin peptides. Binding of U50,488H,U69,593, and nor-BNZto p and K Opioid Receptors and Chimeric p 1K Receptors-Competitive inhibition of [3Hldiprenorphine bindingby U50,488H, U69,593, and nor-BNI to I, 11,111, and IV as well as p and K opioid receptors were performed,and theirK, values were determined (Fig. 2, D-F, and Table 11). Chimera I bound U50,488H and U69,593 with high affinities, similar toRKOR. In contrast, chimeraI1 bound U50,488H and U69,593 with low affinities, similar t o RMOR. Thus, the loop of RKORis not C-terminal portion of the TMH 4 and the e2 important for the selectivity of U50,488H and U69,593, unlike dynorphin peptides (Fig. 2, D and E , and Table 11). Unexpectedly, U50,488H and U69,593 did not bind to either111or IV (K, > 10 p ~ ) Thus, . both regions from the N terminus to the middle of TMH 3 and from the middle of TMH 3 to the C terminus of RKOR are necessary for high affinity binding of these two Upjohn compounds. Binding of U50,488H to chimeras X I and XI1 was also examined. U50,488H did not bind XI (K,> 10 p ~ and had a much lower affinity for XI1 (K,= 99 nM) compared to

RKOR. These data indicate that a wide region ofRKOR appears t o be required for U50,488H and U69,593 binding. nor-BNI exhibitedsimilarlyhighaffinities for I, IV, and RKOR but bound t o 11,111, and RMOR with low affinities (Fig. 2 F and Table 11). Thus, the C-terminal portion of the TMH 4 and the e2 loop and the fragment from the N terminus to the middle of the TMH 3 are not importantfor selective interaction with nor-BNI. These data also indicate that the selectivity for nor-BNI may lie in two regions of the K receptor: 1) from the middle of the TMH 3 t o the middle of TMH 4 and/or 2) from the middle of the i3loop to the C terminus. Since the sequences of the former segments arehighly similar between p and K recept o be important for nor-BNI selectivity. tors, the latter appears We then examined binding of nor-BNI to chimeras XI and XII. Chimera XI bound nor-BNI with the same high affinity as RKOR. Chimera XI1 had a substantially decreased affinity for nor-BNI compared to RKOR. Therefore, the region from the middle of the i3 loop to the C terminus of the K receptor is important for high affinity binding of nor-BNI. We found that the C-terminal domains did not contribute t o ligand binding (data not shown). In addition, sequences within the i3 loop, most of TMH 6, and TMH 7 are very similar between p and K receptors. Selectivity of the K receptor for nor-BNI most likely lies in and around the e3loop. DISCUSSION

In this study, we have demonstrated that peptide and nonpeptide ligands bind t o different regionsof the K opioid receptor. The C-terminalportion of the TMH 4 and e2 loop are essential for highaffinitybinding of dynorphinpeptides but not for U50,488H, U69,593, and nor-BNI binding. The e3 loop and sequence around this loop appear to play an importantrole in the binding of nor-BNI. The high affinity binding of U50,488H and U69,593 requires a wider region that may include the whole receptor except the e2 loop. Thus, there is a clear distinction between nonpeptide and peptide ligands in the strucK opioid receptor that contribute tural determinants within the to their pharmacological selectivities. This is the first demonstration for opioid receptors of such distinction in binding domains of peptide and nonpeptide ligands. A clear difference in thebinding domains of nonpeptide and peptide ligands has been demonstrated for neurokinin 1 receptors. All four extracellular domains of the NK1 receptor contribute to the binding of peptide ligands (12, 13). On the other hand, thehelical packing of TMHs determine thespecies selectivity between the human and rat neurokinin1 receptors of non-peptide antagonists CP-96,345 and RP67,580, without affecting peptidebinding (26). His197within theTMH 5 bindsto CP-96,345 but not t o peptide agonists (27). One prerequisite for use of chimeric receptorsin such studies is that the chimera must retain the conformation of parent also a limitation of chimeric receptors to a large extent. This is receptor studies since only chimeras constructed from closely related receptors may be useful when TMHs are involved. This is tobe expected since for a certain receptor, the adjacent TMHs have been evolved to have minimal steric hindrance and most )favorable electrostatic interaction. When the receptor conformation is retained, anychanges in characteristics of receptors

30198


FIG.2. Competitive inhibition of ['Hldiprenorphine binding to chimeric p / receptors ~ as well as p and K opioid receptors by peptide and nonpeptide K selective ligands. Peptide ligands examined were dynorphin A ( A ) , a-neo-endorphin ( B ) , and dynorphin B (C); non-peptideligandswere U50,488 ( D l , U69,593 (E), andnor-BNI ( F ) . Note that competitive binding inhibition curves of eachligand(exceptdynorphin B) for chimeras are divided into two groups,thosesimilarto 1-1 receptorsand thosesimilar t o K receptors.Eachcurve representsone of thethreeexperiments performed. Symbols used are as follows: M, K receptor; 0, p receptor; 0, chimera I; 0, chimera 11; A, chimera 111; V, chimera W,x, chimera X I ; +, chimera XII. Dyn A, dynorphin A , Dyn B,dynorphin B; a-Neo, a-neo-endorphin.

of

K

Opioid Receptors

120

5 2 a .e

XI

o IM

u

5

$

2

60 4O

LOP Conc. iM)

b e Conr. OM)

Lag Conr. (#)

TABLEI1 K, values (in nM) of K compounds for rat j~ and K opioid receptors and chimericLI, / K receptors expressed in COS-1 cells Data are shown as mean * S.E. of three independent determinations in duplicate. ND, not determined.

wildtypes

chimeras

Ligands

RKOR

RMOR

I

I1

I11

IV

Dynorphin A 0.15L0.11 8.2k0.1 7.113.9 0.17+0.04 53212 0.43k0.14 0.42k0.05 2226 15+5 0.1340.22 68+15 0.44k0.05 a-Neo-endorphin Dynorphin B 1.8k0.4 47k13 1952 0.64k0.26 >10000 7.451.2 4.8L1.4 >10000 1423 >10000 >10000 >10000 U50,488 7.3f2.1 >10000 8.541.5 >10000 >10000 >10000 U69,593 Nor-BNI 0.21k0.03 24k11 0.31k0.15 24k0.6 24+5 0.45k0.13

XI

XI1

ND ND ND ND ND ND 99k27 >lo000 ND ND 0.15+0.06 28.3k4.7

can be attributed to modifications in primary structures of (Tyr-Gly-Gly-Phe-Leu-Arg) and a basic amino acid at the 7th receptors but not changes in secondary or tertiary structures. position (Arg in dynorphin A and dynorphin B, Lys in a-neoAn increase inbinding activity indicates theimportance of the endorphin). BesidesArg' and Arg7 or Lys7,there are three basic primary structuremodification, but a reduction in activity may amino acids in dynorphin A ( k g g , Lys", and Lys13), one in be due to changes in primary, secondary, or tertiary structures. a-neo-endorphin (Lys"), and one in dynorphin B(Lys"'). DynorOne salient featureof this studyis that within eachof the three phin A(1-17) and dynorphin A(1-13) have similaraffinities for pairs of chimeric FI/K opioid receptors, one is the"mirror image" the K receptor and similar activity profiles. Chavkin and Goldof the other. For peptides and nor-BNI, we have demonstrated stein (30) demonstrated thatLys13, Lysl', and Arg7 play impornot only reduction in binding in one of the pair, but also pres- tant roles in the potency of dynorphin A(1-13). In addition, ervation or acquisition of high affinity binding inthe other. By Lys" and Arg7have been found to be important for selectivity of doing so, we have strengthened our conclusion. dynorphin A(1-13) with the K receptor (30). Turcotteet al. (31) In thisstudy, all six chimeras bound [3Hldiprenorphine with synthesized dynorphin A( 1-13) analogs by single substitutions high affinity, similar to 1-1 and K receptors. Binding ofL3H1diof positions 1-11 with Ala and found that substitutions at Arg', prenorphine to all chimeras wascompletely blocked by nalox- Arg7, Ar?,and Lys" greatly lowered the potency of the moleone. These results indicate that these chimeras retain opioid cule, with Arg' and Arg7 being most important. Thus these receptor conformation to some extent. However, expression lev- basic amino acidsare importantfor the functions of dynorphin els varied from chimera to chimera. Variations in expression A. These amino acids most likely bind t o acidic amino acid level appear to be common among different clones. For ex- residues in the e2 loop. Which acidic residues are involved ample, RMOR was always expressed to a higher level than remains to be determined. Although basic amino acid residues RKOR in both transient and stable expression systems (Table are important for the binding of dynorphin A, Tyr' is essential I).' It is probably due to differences in secondary structures of for high affinity bindingto K receptors, as demonstratedby the cDNA and mRNA molecules. findings that dynorphin (2-17) and [Ala'ldynorphin A do not Dynorphin A(1-171, a-neo-endorphin, and dynorphin B are bind (6, 30, 31). According to the address-message division of derived from prodynorphin. The C-terminalhalf of the TMH 4 dynorphin A molecule (301, the e2 loop most likely represents and e2 loop of the K receptor are essential for high affinity the region that interacts with the "address" domain of the binding of these three peptides. Their K, values for RKOR de- dynorphin sequence, i.e. 5-13. The regions that interact with to those in the literature (28,the message domain of the dynorphin sequence, i.e. Tyr-Glytermined in this study are similar 29). They share the same 6 amino acids in the N-terminus Gly-Phe, remain to be determined. It is likely that TMHs are involved. Dynorphin B and a-neo-endorphin can be similarly divided into two domains. 'J.-C. Xue and L.-Y. Liu-Chen, unpublished observations

Peptide and Nonpeptide Binding Domains Among thesethree peptides, there are some differences. Dynorphin B hardly bound to chimera I11 with the Kt value greater than10 p ~ whereas , dynorphin Aand a-neo-endorphin still bound to chimera I11 with K, values below 0.1 p ~ These . results suggest thatsequences on the C-terminal side of position 7 contribute t o binding affinity. U50,488H and U69,593, sharing the samebasic structure of ~ arylacetamide, have similar binding profiles to these p / chimeric receptors. Their K, values for RKOR determined in this study are similar t o those reported previously for brain K receptors(18). It is likely that they bind t o similar multiple epitopes, which are scattered in several TMHs within the receptor. U50,488H and U69,593 bound to chimera I with high affinities, yet did not bind t o chimeras 11, 111, and IV (Kt > 10 p ~ ) U50,488H . bound t o XI1 with K, of 99 nM, yet did not bind XI ( K , > 10 PM). These results suggest that each of these epitopes is required for high affinity binding and alteration of any epitope abolishes high affinity binding. nor-BNI, a bivalent naltrexone derivative (191, has a very different binding profile from the two Upjohn compounds. The sequence in and around the loop e3 appears t o be important for the selectivity of nor-BNI. Portoghese et al. (32) demonstrated that the key structural features of the bimorphinans that contributed t o the K opioid receptor antagonist selectivity and potency were the presence of one or two free phenolic hydroxyl groups and an appropriate substituenton one of the basic nitrogens, e.g. N-allyl or N-cyclopropylmethyl group. In binding assays, BNI, having N-CH, in thepyrrole moiety instead of NH of nor-BNI, has similarly high affinities for K (KJ= 0.8 nM) and p (K, = 1.3 nM) receptors (20). In contrast, nor-BNI has higher affinity for K receptors and lower afflnity for p receptors, with K, values of 0.3 and 47 nM, respectively (20). Thus, NH in the pyrrole ring may play a n important role in its selectivity for the K receptor. In addition,bivalency is also important for selectivity for the K receptor sincenaltrexone andnaloxone have higher affinity for p than K receptors (32). Which part of the nor-BNI molecule interacts with the e3loop remains t o be determined. Obviously, there are other epitopes in the receptor that are important for the binding of nor-BNI. In conclusion, the C-terminal portion of the TMH 4 and the e2 loop of the K opioid receptor confers its selectivity for dynorphin family peptides but not for non-peptide ligands. The selectivity of the K opioid receptor for U50,488H and U69,593 may be conferred by multiple epitopes, presumably scattered in different TMHs. In contrast, the region accounted for the selectivity of nor-BNI was locatedin and around the loop e3 ofthe K opioid receptor. Thus, ligands are oriented differently in the receptor depending on their stereochemical features. Identification of regions responsible for selectivities of each K ligand, coupled with knowledge of structure-function relationship of

of

K

Opioid Receptors

30199

the ligand in the literature, permits further studies for identification of specific amino acid residues as binding epitopes. Elucidation of the molecular interactions inligand bindingwill be crucial for development of more selective drugs. Acknowledgments-We thank Dr. Barrie Ashby for valuable discussion and Y. Wang for help on the manuscript.

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