©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Location of Regions of the Opioid Receptor Involved in Selective Agonist Binding (*)

(Received for publication, July 11, 1994; and in revised form, December 2, 1994)

Kazuhiko Fukuda (§) Shigehisa Kato Kenjiro Mori

From the Department of Anesthesia, Kyoto University Hospital, Kyoto 606-01, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To elucidate which portions of the opioid receptor molecules are involved in the ligand selectivity, we have expressed chimeric receptors between the rat - and µ-opioid receptors from cDNAs and analyzed their ligand binding properties. We demonstrate that the major binding determinant for the -selective enkephalin-related peptide, [D-Pen^2,D-Pen^5]enkephalin, resides within the region comprising the transmembrane segments V-VII and the intervening loop regions. On the other hand, the region spanning from the intracellular loop I to the amino-terminal half of the transmembrane segment III is shown to be involved in determining high-affinity binding of the µ-selective enkephalin-related peptides, [D-Ala^2,MePhe^4,Gly-ol^5]enkephalin and [D-Ala^2,MePhe^4,Met-ol^5]enkephalin, whereas the major determinant for binding of the µ-selective alkaloids, morphine and codeine, is demonstrated to exist in the region spanning the transmembrane segments V-VII. These results indicate that distinct regions of the opioid receptor determine the selectivity for the - and the µ-selective enkephalin-related peptides and that the binding determinant for the µ-selective alkaloids is distinct from that for the µ-selective enkephalin-related peptides.


INTRODUCTION

The opioid receptors exhibit a widespread distribution throughout central nervous system and mediate actions of opioid analgesics and endogenous opioid peptides(1) . Pharmacologically, the opioid receptor has been classified into at least three types (µ, , and kappa) on the basis of their difference in apparent affinity for ligands(1, 2) .

Recently, the cDNAs encoding the µ-, - and kappa-opioid receptors have been cloned(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) . Analysis of the deduced amino acid sequences showed that these receptors possess seven putative transmembrane segments (TM I-VII), (^1)which is the major characteristic structural feature of G-protein-coupled receptors(16) , and have high amino acid sequence identities (60%) to each other. The present investigation has been designed to localize the region of the opioid receptor molecule responsible for type-selective binding of opioid agonists. For this purpose, chimeric opioid receptors with different combinations of the rat - and µ-receptors have been produced from cDNAs in COS-7 cells and analyzed for their agonist binding properties. The results obtained indicate that high-affinity binding of the - and the µ-selective enkephalin-related peptides is determined by distinct domains of the opioid receptor molecule. Furthermore, the binding determinant for the µ-selective enkephalin-related peptides is indicated to be present in the domain distinct from that for the µ-selective alkaloids.


EXPERIMENTAL PROCEDURES

Construction of cDNAs Encoding Chimeric Receptors

Chimeric /µ cDNAs were constructed by exchange of restriction fragments from the cDNAs encoding the rat - and µ-opioid receptors(7) . The restriction sites used and their positions in the deduced amino acid sequences of the -receptor/µ-receptor, respectively, were as follows (Fig. 1): AccI, at Tyr/Tyr in intracellular loop (ICL) I; NspI, at Met/Met in TM III; BglII, at Lys/Lys at the junction of extracellular loop (ECL) II and TM V; ScaI, at Val/Val in TM VII. The NspI sites are present in both the wild-type - and µ-receptor cDNAs and the BglII site exists in the wild-type -receptor cDNA. The other restriction sites used were introduced by oligonucleotide-directed mutagenesis (Amersham Corp.) without amino acid substitutions. All the constructs were confirmed by sequencing and by restriction endonuclease analysis. The entire protein-coding sequences of the wild-type and chimeric cDNAs were then inserted into the HindIII site of an expression vector pKCRH2 (17) in the same orientation with respect to SV40 early promoter.


Figure 1: Proposed transmembrane topography of the rat opioid receptor. The model is based on the amino acid sequences of the rat - and µ-opioid receptors deduced from the cDNA sequences (7) . The number of amino acid residues (circles) is based on the µ-receptor sequence; the sequence gaps introduced into the -receptor sequence to align the two polypeptides are indicated by striped circles and the insertions by arrows with the number of inserted amino acid residues (aa). Closed circles indicate the amino acid residues identical between the aligned sequences of the - and µ-receptors, and open circles indicate the non-identical amino acid residues. Positions of the restriction sites used to construct chimeric receptor cDNAs are indicated by thick lines with the names of the restriction enzymes.



Transfection and Ligand Binding Assay

COS-7 cells cultured in monolayers in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum were transfected by the calcium phosphate method (18) with expression vectors containing the wild-type or chimeric cDNAs. After 48-72-h incubation at 37 °C, crude membrane was prepared from the transfected cells. Cells were washed twice with phosphate-buffered saline and homogenized with a Dounce homogenizer in 50 mM Tris-HCl, pH 7.5. The homogenate was centrifuged at 1,000 times g for 10 min and the precipitate suspended in the same buffer, homogenized, and centrifuged at 1,000 times g for 10 min. The two supernatants were combined and centrifuged at 20,000 times g for 30 min. The pellet was suspended in the same buffer and used for binding assays. [^3H]Ethylketocyclazocine (EKC) binding reaction was performed with membrane preparations (20-50 µg of protein) in 0.2 ml of 50 mM Tris-HCl, pH 7.5, at 37 °C for 1 h. After incubation, the samples were collected on GF/B filters (Whatman) and washed with 10 ml of 50 mM Tris-HCl, pH 7.5. The filters were then counted for radioactivity. The apparent dissociation constants (K(d)) for opioid ligands were obtained by displacement of [^3H]EKC binding by unlabeled ligands in the presence of 10 nM [^3H]EKC and calculation by the equation K(d) = IC/(1 + [EKC]/K(d)*)(19) , where IC is concentration of the unlabeled ligand producing a 50% reduction in the [^3H]EKC binding and K(d)* is the apparent dissociation constant for [^3H]EKC estimated by Scatchard analysis. All determinations were performed in duplicate. The nonspecific binding was determined as the binding of [^3H]EKC in the presence of 1 mM naloxone.


RESULTS

Ligand Binding Properties of the Wild-type Opioid Receptors

Crude membrane preparations from COS-7 cells transfected with the expression plasmids containing the cDNAs for the rat - or µ-opioid receptor were subjected to ligand binding studies. The K(d) values for EKC of the - and µ-receptors obtained by Scatchard analysis were 9.2 nM and 5.4 nM, respectively (Table 1). Binding affinities to the other opioid ligands were assessed by displacement of [^3H]EKC binding (Table 1, Fig. 3Fig. 4Fig. 5Fig. 6Fig. 7). An enkephalin-related peptide [D-Pen^2,D-Pen^5]enkephalin (DPDPE) showed more than two orders of magnitude higher binding affinity for the -receptor (K(d) = 19 nM) compared with the µ-receptor (K(d) > 3,000 nM). In contrast, the binding affinities of the µ-receptor to an enkephalin-related peptide [D-Ala^2,MePhe^4,Gly-ol^5]enkephalin (DAGO) and an opioid alkaloid morphine (K(d) = 15 nM and 23 nM, respectively) were higher than those of the -receptor (K(d) = 650 nM and 390 nM, respectively). These results generally agree with our data for the - and µ-receptors stably expressed from the cloned cDNAs in Chinese hamster ovary cells(7) . Furthermore, another enkephalin-related peptide [D-Ala^2,MePhe^4,Met-ol^5]enkephalin (DAMO) (20) and another opioid alkaloid codeine also showed binding affinities for the µ-receptor higher than those for the -receptor.




Figure 3: Displacement of [^3H]EKC binding to membrane preparations from COS-7 cells transfected with cDNAs by DPDPE. Membrane was prepared from COS-7 cells expressing the wild-type - and µ-receptors and chimeric receptors. Competitive ligand binding assay in the presence of increasing concentrations of DPDPE was performed as described under ``Experimental Procedures.'' The chimeric receptors analyzed include AB series (A), BA series (B), C series (C), and D series (D). Values for 100 and 0% were determined by measurements in the absence of unlabeled ligands and in the presence of 1 mM naloxone, respectively. Each curve shown is representative of three to six experiments.




Figure 4: Displacement of [^3H]EKC binding to the wild-type and chimeric receptors by DAGO. Binding affinities to DAGO were measured as described in the legend to Fig. 3. The chimeric receptors analyzed include AB series (A), BA series (B), C series (C), and D series (D). Each curve shown is representative of three to five experiments.




Figure 5: Displacement of [^3H]EKC binding to the wild-type and chimeric receptors by DAMO. Binding affinities to DAMO were assessed as described in the legend to Fig. 3. The chimeric receptors analyzed are AB series (A) and BA series (B). Each curve shown is representative of three or four experiments.




Figure 6: Displacement of [^3H]EKC binding to the wild-type and chimeric receptors by morphine. Binding affinities to morphine were assessed as described in the legend to Fig. 3. The chimeric receptors analyzed include AB series (A), BA series (B), C series (C), and D series (D). Each curve shown is representative of three to six experiments.




Figure 7: Displacement of [^3H]EKC binding to the wild-type and chimeric receptors by codeine. Binding affinities to codeine were measured as described in the legend to Fig. 3. The chimeric receptors analyzed are AB series (A) and BA series (B). Each curve shown is representative of three experiments.



Construction of Chimeric Receptors

cDNAs encoding chimeric receptors between the rat - and µ-receptors were constructed by exchange of DNA restriction fragments by the use of the pre-existing restriction sites or new sites created by oligonucleotide-directed mutagenesis without alteration of the amino acid sequence (Fig. 1). Fig. 2schematically shows the structures of chimeric receptors in which corresponding portions of the rat - and µ-receptors are replaced with each other. We constructed four series of chimeric receptors by substituting the structure of the µ-receptor for that of the -receptor. Progressive substitutions from the carboxyl terminus resulted in the AB series chimeras, whereas the amino-terminal substitutions resulted in the BA series chimeras. In the C series chimeras, internal regions of the -receptor are replaced with the corresponding regions of the µ-receptor. The D series chimeras are mirror images of those of the C series chimeras, in that internal regions of the µ-receptor are replaced with the homologous regions of the -receptor. Chimeric receptors expressed transiently from cDNAs in COS-7 cells were analyzed by the ligand binding assay using [^3H]EKC as a radioligand. The expression levels of the chimeric receptors C2 and D1 were too low to be further analyzed. All chimeric receptors except C2 and D1 were shown to bind EKC with K(d) values comparable with those of the wild-type - and µ-receptors, ranging from 2.0 to 12.6 nM (Table 1), suggesting that general structural integrity of the chimeric receptors was not impaired.


Figure 2: Schematic representation of the structures of the wild-type - and µ-receptors and chimeric receptors. Thin lines indicate the amino acid sequences derived from the -receptor; thick lines indicate sequences derived from the µ-receptor. The chimeric receptors are classified into AB, BA, C, and D series. The compositions of the individual chimeric receptors are as follows (the numbers in parentheses indicate amino acid numbers(7) ; the junctional sequences common to the - and µ-receptors are represented by amino acid numbers of the -receptor): AB1, (1-79) and µ(99-398); AB2, (1-139) and µ(159-398); AB3, (1-218) and µ(238-398); AB4, (1-330) and µ(349-398); BA1, µ(1-92) and (74-372); BA2, µ(1-144) and (126-372); BA3, µ(1-232) and (214-372); BA4, µ(1-330) and (313-372); C1, (1-79), µ(99-144) and (126-372); C2, (1-139), µ(159-232) and (214-372); C3, (1-218), µ(238-330) and (313-372); C4, (1-79), µ(99-232) and (214-372); C5, (1-139), µ(159-330) and (313-372); D1, µ(1-92), (74-139) and µ(159-398); D2, µ(1-144), (126-218) and µ(238-398); D3, µ(1-232), (214-330) and µ(349-398); D4, µ(1-92), (74-218) and µ(238-398); D5, µ(1-144), (126-330) and µ(349-398).



Determinant for Selectivity to DPDPE

First, we tested the AB and BA series chimeras for their binding affinities to DPDPE (Table 1; Fig. 3, A and B). Progressive replacement of the amino acid sequence of the -receptor with that of the µ-receptor from the amino-terminal extracellular region to the amino-terminal half of TM III produced little change in the affinity to DPDPE (BA1 and BA2), suggesting that this portion contributes little to the high-affinity DPDPE binding of the -receptor. However, further substitution from the carboxyl-terminal half of TM III to ECL II (BA3) increased the K(d) value for DPDPE 6-fold. By further replacement of the region spanning TM V-VII (BA4), the affinity to DPDPE decreased more than 30-fold, resulting in an affinity as low as that of the wild-type µ-receptor. Replacement of the carboxyl-terminal cytoplasmic region of the -receptor with the corresponding region of the µ-receptor (AB4) resulted in a 7-fold increase in the K(d) value for DPDPE. Further replacement of the region spanning TM V-VII (AB3) increased the K(d) value more than 20-fold to be comparable with that of the wild-type µ-receptor. The K(d) value did not change significantly by further substitution of the µ-receptor sequence for the -receptor sequence (AB2 and AB1). These results suggest that the region spanning TM V-VII of the -receptor contains the major determinant for selectivity to DPDPE and that the region spanning from the carboxyl-terminal half of TM III to ECL II and the carboxyl-terminal cytoplasmic region also partly contribute to high-affinity binding of DPDPE.

To confirm this conclusion, we tested the C and D series chimeras (Table 1; Fig. 3, C and D). The affinities to DPDPE of C3, C5, D2, and D4, chimeric receptors possessing the region spanning TM V-VII derived from the µ-receptor, were much lower than that of the wild-type -receptor and similar as that of the wild-type µ-receptor. Replacement of the region spanning TM V-VII of the µ-receptor with the corresponding sequence of the -receptor (D3) increased the affinity to DPDPE more than 12-fold. These results support the view that the region spanning TM V-VII contains the major determinant for the high-affinity binding to DPDPE of the -receptor. However, the K(d) value of D3 was still 13-fold higher than that of the wild-type -receptor. Further replacement of the region spanning from the carboxyl-terminal half of TM III to ECL II (D5) or the carboxyl-terminal cytoplasmic region (BA3) resulted in 2-4-fold increase in the affinity to DPDPE, confirming the small contribution of these regions to the high-affinity DPDPE binding. The affinities to DPDPE of C1 and C4 were comparable with those of BA2 and BA3, respectively. This again indicates that the region spanning from the carboxyl-terminal half of TM III to ECL II of the -receptor partly contributes to the high-affinity DPDPE binding and further excludes contribution of the amino-terminal extracellular region and TM I.

Determinant for Selectivity to µ-Selective Enkephalin-related Peptides

The AB and BA series chimeras were tested for their binding affinities to DAGO (Table 1; Fig. 4, A and B). Replacement of the sequence of the amino-terminal extracellular region and TM I of the -receptor with that of the µ-receptor (BA1) caused little change in K(d) value for DAGO, suggesting that this region contributes little to the high-affinity DAGO binding of the µ-receptor. However, the affinity to DAGO was increased 30-fold by further replacement of the region spanning from ICL I to the amino-terminal half of TM III (BA2), resulting in the affinity nearly identical with that of the wild-type µ-receptor. The chimeric receptors with further replacement (BA3 and BA4) showed affinities to DAGO indistinguishable from that of the wild-type µ-receptor. Similarly, progressive replacement of the -receptor sequence with the µ-receptor sequence from the carboxyl-terminal cytoplasmic region to the carboxyl-terminal half of TM III (AB4, AB3, and AB2) did not significantly increase the affinity to DAGO. Further substitution of the region spanning from ICL I to the amino-terminal half of TM III (AB1) increased the affinity more than 20-fold, resulting in the affinity to DAGO similar as that of the wild-type µ-receptor. The results obtained for another µ-selective enkephalin-related peptide DAMO (Table 1; Fig. 5) resembled those for DAGO. The affinity to DAMO of AB1 was comparable with that of the wild-type µ-receptor, whereas the affinities of AB2, AB3, and AB4 were comparable with or lower than that of the -receptor. Similarly, in the BA series chimeras, the most remarkable change in the affinity to DAMO was found between BA1 and BA2. These results suggest that the region spanning from ICL I to the amino-terminal half of TM III determine the high-affinity binding of the µ-selective enkephalin-related peptides.

To test whether this region is sufficient to define the binding determinant for DAGO, the C and D series chimeras were examined (Table 1; Fig. 4, C and D). The chimeric receptor C1, in which the region of the -receptor spanning from ICL I to the amino-terminal half of TM III is replaced with that of the µ-receptor, showed an affinity to DAGO comparable with that of the wild-type µ-receptor, indicating that this region is sufficient to define the binding determinant for DAGO. Consistently with this conclusion, C4, D2, D3, and D5, chimeric receptors containing the region spanning from ICL I to the amino-terminal half of TM III derived from the µ-receptor, exhibited affinities to DAGO comparable with that of the wild-type µ-receptor, whereas C3, C5, and D4, chimeric receptors possessing this region derived from the -receptor, showed affinities to DAGO as low as that of the wild-type -receptor.

Determinant for Selectivity to µ-Selective Alkaloids

The results obtained by competitive binding assays using unlabeled morphine are shown in Table 1and Fig. 6. In the AB series chimeras, the K(d) value for morphine of AB1 was comparable with that of the wild-type µ-receptor, suggesting that the amino-terminal extracellular region and TM I contribute little to the high-affinity morphine binding of the µ-receptor. In contrast, the binding affinity to morphine of AB4 was as low as that of the wild-type -receptor. The most dramatic change in the affinity to morphine was found between AB3 and AB4. Similarly, replacement of the amino-terminal extracellular region and TM I of the -receptor with the corresponding regions of the µ-receptor (BA1) did not produce remarkable change in the affinity to morphine, and further replacement from ICL I to ECL II increased the affinity only slightly (BA2 and BA3). However, the affinity to morphine was increased 10-fold by further replacement of the region spanning TM V-VII (BA4) to be comparable with that of the wild-type µ-receptor. Similarly, the most dramatic change in the affinity to codeine, another opioid alkaloid, was found between AB3 and AB4, and between BA3 and BA4, in the AB and BA series chimeras, respectively (Table 1; Fig. 7). These data together suggest that the region spanning TM V-VII of the µ-receptor contains the major determinant for the selective high-affinity binding of µ-selective opioid alkaloids.

The results obtained for the C and D series chimeras were in agreement with this conclusion (Table 1; Fig. 6, C and D). The chimeric receptor C3, in which the region spanning TM V-VII of the -receptor is replaced with the corresponding sequence of the µ-receptor, showed the binding affinity to morphine only 2-fold lower than that of the wild-type µ-receptor. In contrast, the chimeric receptor D3, which is the mirror image of C3, exhibited a low binding affinity nearly identical with that of the wild-type -receptor. Furthermore, C5, D2, and D4, chimeric receptors possessing the region spanning TM V-VII derived from the µ-receptor, showed affinities to morphine similar as or slightly lower than that of the wild-type µ-receptor, whereas the chimeric receptors C1, C4, and D5, which contain the region spanning TM V-VII derived from the -receptor, demonstrated low affinity to morphine comparable with that of the wild-type -receptor.


DISCUSSION

In this investigation, we attempted to delineate the structural domains involved in determining the ligand binding selectivity of the opioid receptor by analysis of chimeric receptors between the rat - and µ-opioid receptors. The high-affinity binding of DPDPE, a -selective enkephalin-related peptide, was shown to be determined mainly by the region spanning TM V-VII of the -receptor and also partly by the region spanning from the carboxyl-terminal half of TM III to ECL II and the carboxyl-terminal cytoplasmic region. In contrast, the region spanning from ICL I to the amino-terminal half of TM III of the µ-receptor was demonstrated to contain the major determinant for selectivity to DAGO and DAMO, µ-selective enkephalin-related peptides. Furthermore, the major determinant for selectivity to morphine and codeine, µ-selective opioid alkaloids, was shown to exist in the region spanning TM V-VII of the µ-receptor. These results indicate that the determinants for binding of the - and the µ-selective enkephalin-related peptides exist in distinct domains and that high-affinity binding of the µ-selective enkephalin-related peptide and the µ-selective opioid alkaloid is determined by distinct domains.

The amino-terminal extracellular region of the opioid receptors differs considerably in amino acid sequence and seems likely to contribute directly or indirectly to ligand selectivity. However, our findings indicate that the amino-terminal extracellular region contributes little to selective binding of agonists. Accordingly, this region may be necessary for functions other than ligand selectivity, such as organizing proper conformation of the receptor molecule with oligosaccharide side chains and efficient expression on the cell surface in the proper transmembrane orientation. The carboxyl-terminal cytoplasmic region is also divergent in amino acid sequence between the - and µ-receptors. Our results showed that this portion of the receptor molecule contributes partly to high-affinity DPDPE binding of the -receptor. Since it is unlikely that the cytoplasmic domain contributes directly to the formation of the agonist binding site, the carboxyl-terminal cytoplasmic region may indirectly affect the affinity to DPDPE by influencing the conformation of the major determinant for DPDPE binding, which was demonstrated to exist in the region spanning TM V-VII. The amino acid sequence of the region spanning from ICL I to the amino-terminal half of TM III, which was shown to possess the major determinant for the µ-selective enkephalin-related peptides, is well conserved between the - and µ-receptors; amino acid residues are identical at 48 of 56 positions in this region of these receptors (Fig. 1). Seven of eight non-identical residues in this region are clustered around ECL I. There are three charge-modifying amino acid substitutions between the - and µ-receptors in ECL I: that is, Lys, Glu, and Glu in the -receptor corresponding to Gln, Gly, and Thr in the µ-receptor, respectively. The difference in distribution of charged amino acid residues might result in difference in binding stability between the peptide and the receptor molecule. The region spanning TM V-VII was shown to be necessary for high-affinity binding of DPDPE and the µ-selective alkaloids. It is conceivable that ECL III in this region contributes to the selectivity to DPDPE and opioid alkaloids, because the amino acid sequence of ECL III is more divergent between the - and the µ-receptor than those of ECL I and ECL II. However, it is also possible that TM V and TM VI contribute to determination of the selectivity, since contribution of amino acid residues in the transmembrane segments to ligand selectivity has been suggested in other peptide receptor systems(21, 22) .

Enkephalin-related peptides, including DPDPE, DAGO, and DAMO, are characterized by the amino-terminal sequence Tyr-X-Gly. Accordingly, it is likely that the amino-terminal sequence of these peptides interact with an equivalent domain of the opioid receptor molecule. However, our results suggest that high-affinity binding of the - and the µ-selective enkephalin-related peptides is based on interaction with different regions of the opioid receptor molecule. It is possible that the opioid peptides interact with the opioid receptor at a binding site composed of at least two subsites: one subsite binds with the amino-terminal consensus sequence of enkephalin-related peptides and is constituted from an equivalent domain of each opioid receptor type; and the other subsite is involved in determining ligand selectivity and constituted from different regions of each receptor type. According to the ``message-address'' concept(23) , these two subsites conceivably correspond to the recognition site for the message component of the opioid peptide and that for the address component, respectively.

Our results indicate that the major binding determinant for opioid alkaloids resides in a region of the µ-receptor different from that for enkephalin-related peptides. This finding suggests that the mechanisms for µ selectivity of the opioid alkaloid and the enkephalin-related peptide are different. It was suggested that the tyramine moiety in the opioid alkaloid and the amino-terminal tyrosine residue in opioid peptides, including enkephalin, play a similar role in the interaction with the opioid receptor(24) . Therefore, it is conceivable that the binding site of the µ-receptor for the opioid alkaloid and that for the enkephalin-related peptide overlap with each other, but are not completely identical. Recently, it was reported that different epitopes underlie affinity for peptide agonists and non-peptide antagonists in the cholecystokinin-B/gastrin receptor and the neurokinin-1 receptor(25, 26) . However, there has been no report that affinities for peptide and non-peptide agonists are determined by distinct regions of the G-protein-coupled receptor.

It was reported that Asp in TM II of the -receptor, which is conserved in the -, µ-, and kappa-receptors, is involved in binding of -selective agonists, including DPDPE, but not in binding of nonselective ligands(27) . However, we could not identify TM II of the -receptor, the amino acid sequence of which is completely identical with that of the µ-receptor, as a determinant for DPDPE binding, because the roles of conserved amino acid residues cannot be analyzed by the approach using chimeric receptors. The possibility that conserved amino acid residues also contribute to ligand selectivity would further increase the complexity of the mechanism for the ligand selectivity of the opioid receptor.

Studies on several G-protein-coupled receptors for peptides have identified ligand binding epitopes in the receptor molecules. It has been reported that the region spanning TM IV-VI of the endothelin B receptor constitute the binding determinant for the endothelin B-selective agonists(28) . The region spanning from TM II to ECL II has been reported to mainly determine agonist selectivity of the tachykinin receptor(29) . Furthermore, in glycohormone receptors such as lutropin/choriogonadotropin receptor and the thyrotropin receptor, the amino-terminal extracellular domains were shown to be responsible for high-affinity binding of the hormones(30, 31) . These observations, together with our findings, suggest that the mode of interaction between agonists and the receptor is different for each receptor system.

In summary, our results indicate that the determinants for binding of the - and the µ-selective enkephalin-related peptides exist in distinct regions of the opioid receptor molecules. Furthermore, the region specifying the µ-selective alkaloid binding was shown to be different from the determinant for binding of the µ-selective enkephalin-related peptides. Further studies will be necessary to identify specific amino acid residues that interact directly with agonist molecules. However, the results obtained in this investigation would provide insights into the mechanism for the ligand selectivity of the opioid receptor and facilitate development of highly selective opioid analgesics acting on the opioid receptor, which will have considerable therapeutic potentials.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Dept. of Anesthesia, Kyoto University Hospital, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-01, Japan. Tel.: 81-75-751-3441; Fax: 81-75-752-3259.

(^1)
The abbreviations used are: TM, transmembrane segment; ICL, intracellular loop; ECL, extracellular loop; EKC, ethylketocyclazocine; DPDPE, [D-Pen^2,D-Pen^5]enkephalin; DAGO, [D-Ala^2,MePhe^4,Gly-ol^5]enkephalin; DAMO, [D-Ala^2,MePhe^4,Met-ol^5]enkephalin.


ACKNOWLEDGEMENTS

We thank Keiko Komatsu for technical assistance.


REFERENCES

  1. Simon, E. J. (1986) Ann. N. Y. Acad. Sci. 463, 31-45 [Medline] [Order article via Infotrieve]
  2. Simonds, W. F. (1988) Endocr. Rev. 9, 200-212 [Abstract]
  3. Kieffer, B. L., Befort, K., Gaveriaux-Ruff, C., and Hirth, C. G. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 12048-12052 [Abstract]
  4. Evans, C. J., Keith, D. E., Jr., Morrison, H., Magendzo, K., and Edwards, R. H. (1992) Science 258, 1952-1955 [Medline] [Order article via Infotrieve]
  5. Yasuda, K., Raynor, K., Kong, H., Breder, C. D., Takeda, J., Reisine, T., and Bell, G. I. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6736-6740 [Abstract]
  6. Chen, Y., Mestek, A., Liu, J., Hurley, J. A., and Yu, L. (1993) Mol. Pharmacol. 44, 8-12 [Abstract]
  7. Fukuda, K., Kato, S., Mori, K., Nishi, M., and Takeshima, H. (1993) FEBS Lett. 327, 311-314 [CrossRef][Medline] [Order article via Infotrieve]
  8. Minami, M., Toya, T., Katao, Y., Maekawa, K., Nakamura, S., Onogi, T., Kaneko, S., and Satoh, M. (1993) FEBS Lett. 329, 291-295 [CrossRef][Medline] [Order article via Infotrieve]
  9. Nishi, M., Takeshima, H., Fukuda, K., Kato, S., and Mori, K. (1993) FEBS Lett. 330, 77-80 [CrossRef][Medline] [Order article via Infotrieve]
  10. Bzdega, T., Chin, H., Kim, H., Jung, H. H., Kozak, C. A., and Klee, W. A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9305-9309 [Abstract]
  11. Wang, J. B., Imai, Y., Eppler, C. M., Gregor, P., Spivak, C. E., and Uhl, G. R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10230-10234 [Abstract]
  12. Thompson, R. C., Mansour, A., Akil, H., and Watson, S. J. (1993) Neuron 11, 903-913 [Medline] [Order article via Infotrieve]
  13. Meng, F., Xie, G.-X., Thompson, R. C., Mansour, A., Goldstein, A., Watson, S. J., and Akil, H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9954-9958 [Abstract]
  14. Chen, Y., Mestek, A., Liu, J., and Yu, L. (1993) Biochem. J. 295, 625-628 [Medline] [Order article via Infotrieve]
  15. Li, S., Zhu, J., Chen, C., Chen, Y.-W., Deriel, J. K., Ashby, B., and Liu-Chen, L.-Y. (1993) Biochem. J. 295, 629-633 [Medline] [Order article via Infotrieve]
  16. Dohlman, H. G., Thorner, J., Caron, M. G., and Lefkowitz, R. J. (1991) Annu. Rev. Biochem. 60, 653-688 [CrossRef][Medline] [Order article via Infotrieve]
  17. Mishina, M., Kurosaki, T., Tobimatsu, T., Morimoto, Y., Noda, M., Yamamoto, T., Terao, M., Lindstrom, J., Takahashi, T., Kuno, M., and Numa, S. (1984) Nature 307, 604-608 [Medline] [Order article via Infotrieve]
  18. Gorman, C. (1985) in DNA Cloning (Glover, D. M. ed.) Vol. II, pp. 143-190, IRL, Oxford
  19. Cheng, Y.-C., and Prusoff, W. H. (1973) Biochem. Pharmacol. 22, 3099-3108 [CrossRef][Medline] [Order article via Infotrieve]
  20. Roemer, D., Buescher, H. H., Hill, R. C., Pless, J., Bauer, W., Cardinaux, F., Closse, A., Hauser, D., and Huguenin, R. (1977) Nature 268, 547-549 [Medline] [Order article via Infotrieve]
  21. Fong, T. M., Huang, R.-R. C., and Strader, C. D. (1992) J. Biol. Chem. 267, 25664-25667 [Abstract/Free Full Text]
  22. Krystek, S. R., Jr., Patel, P. S., Rose, P. M., Fisher, S. M., Kienzle, B. K., Lach, D. A., Liu, E. C.-K., Lynch, J. S., Novotny, J., and Webb, M. L. (1994) J. Biol. Chem. 269, 12383-12386 [Abstract/Free Full Text]
  23. Portoghese, P. S. (1989) Trends Pharmacol. Sci. 10, 230-235 [CrossRef][Medline] [Order article via Infotrieve]
  24. Schiller, P. W. (1984) in Peptides (Udenfriend, S., and Meienhofer, J., eds) Vol. 6, pp. 219-268, Academic Press, New York
  25. Beinborn, M., Lee, Y.-M., McBride, E. W., Quinn, S. M., and Kopin, A. S. (1993) Nature 362, 348-350 [CrossRef][Medline] [Order article via Infotrieve]
  26. Gether, U., Johansen, T. E., Snider, R. M., Lowe, J. A., III, Nakanishi, S., and Schwartz, T. W. (1993) Nature 362, 345-348 [CrossRef][Medline] [Order article via Infotrieve]
  27. Kong, H., Raynor, K., Yasuda, K., Moe, S. T., Portoghese, P. S., Bell, G. I., and Reisine, T. (1993) J. Biol. Chem. 268, 23055-23058 [Abstract/Free Full Text]
  28. Sakamoto, A., Yanagisawa, M., Sawamura, T., Enoki, T., Ohtani, T., Sakurai, T., Nakao, K., Toyo-oka, T., and Masaki, T. (1993) J. Biol. Chem. 268, 8547-8553 [Abstract/Free Full Text]
  29. Yokota, Y., Akazawa, C., Ohkubo, H., and Nakanishi, S. (1992) EMBO J. 11, 3585-3591 [Abstract]
  30. Xie, Y.-B., Wang, H., and Segaloff, D. L. (1990) J. Biol. Chem. 265, 21411-21414 [Abstract/Free Full Text]
  31. Nagayama, Y., Wadsworth, H. L., Chazenbalk, G. D., Russo, D., Seto, P., and Rapoport, B. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 902-905 [Abstract]

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