Studies on inhibition of mu and delta opioid receptor binding by dithiothreitol and N-ethylmaleimide. His223 is critical for mu opioid receptor binding and inactivation by N-ethylmaleimide.

The sensitivity of m and d receptor binding to dithiothreitol and N-ethylmaleimide was examined to probe receptor structure and function. Binding to both receptor types was inhibited by dithiothreitol (IC50 values 5 250 mM), suggesting the presence of inaccessible but critical disulfide linkages. m receptor binding was inhibited with more rapid kinetics and at lower N-ethylmaleimide concentrations than d receptor binding. Ligand protection against N-ethylmaleimide inactivation suggested that alkylation was occurring within, or in the vicinity of, the receptor binding pocket. Sodium ions dramatically affected the IC50 of N-ethylmaleimide toward both receptor types in a ligand-dependentmanner. Analysis of receptor chimeras suggested that the site of N-ethylmaleimide alkylation on the m receptor was between transmembrane domains 3 and 5. Substitution of cysteines between transmembrane domains 3 and 5 and elsewhere had no effect on receptor binding or sensitivity toward N-ethylmaleimide. Serine substitution of His in the putative second extracellular loop linking transmembrane domains 4 and 5 protected against Nethylmaleimide inactivation. TheH223S substitution decreased the affinity of bremazocine 25-fold, highlighting the importance of this residue for the formation of the high affinity bremazocine binding site in the m opioid receptor.

Three major types of opioid receptor, ␦, , and , have been cloned and characterized extensively (reviewed in Refs. 1 and 2). There is approximately 60% amino acid sequence identity between the opioid receptor types. The ␦, , and opioid receptors have unique ligand specificities, anatomical distributions, and physiological functions (3). Morphine, related opioid drugs, and the endogenous opioid peptides activate signal transduction pathways by binding to opioid receptors (4), which are members of the G protein-coupled receptor family (5). G protein-coupled receptors are seven-transmembrane domain (TM) 1 proteins that mediate signal transduction across the plasma membrane. The ligands approach and engage the receptor from the extracellular side, and receptor activation results in the coupling to heterotrimeric G proteins on the intracellular face of the membrane. Opioid receptor types interact with multiple G proteins (6 -8) to regulate adenylyl cyclase, Ca 2ϩ channels, and K ϩ channels.
It has been known from early studies on the characterization of opioid receptors that specific binding is inhibited by sulfhydryl reagents, such as iodoacetamide, N-ethylmaleimide (NEM), and p-hydroxymercuribenzoate (9 -11). Preincubation with opioid ligands protected against receptor inactivation, suggesting that the sensitive sulfhydryl group was located within or near the binding site. Evidence has been obtained that analogs of Leu-enkephalin and morphine, containing activated sulfhydryl groups, form mixed disulfide linkages with opioid receptors (12,13). The covalently bound agonists caused receptor activation that persisted following extensive washing, yet was naloxone-reversible. The results suggested that the agonists became tethered to the receptor via a mixed disulfide linkage that was in the vicinity of the receptor binding site. Other studies provided evidence that NEM affected opioid agonist binding by at least two mechanisms, direct inhibition (as mentioned above) and indirect inhibition due to uncoupling of receptors from G proteins (14,15).
The goals of this study were 1) to examine the sensitivity of and ␦ receptor binding to reduction with dithiothreitol (DTT), in order to determine whether disulfide linkages were necessary for maintenance of the binding site, and 2) to characterize the sensitivity of and ␦ receptor binding to alkylation with NEM and identify the reactive groups involved. Due to the proximity of the NEM-reactive group to the ligand binding site of the receptor, knowledge of its location is essential for construction of accurate molecular models of the binding pocket.

EXPERIMENTAL PROCEDURES
Transfection and Radioligand Binding Assays-Human embryonic kidney 293 cells (ATCC CRL 1573) were transfected with and ␦ opioid receptor expression plasmids (obtained from Drs. L. Yu and C. Evans, respectively) using the calcium phosphate method, as described (27). Cells stably expressing opioid receptors were selected in media containing 0.5 mg/ml G418 (Life Technologies, Inc.). Opioid receptor binding assays (28) (29), using bovine serum albumin as the standard. Treatment of Membranes with DTT and NEM-To determine the effect of disulfide bond reduction on and ␦ receptor binding, DTT (1-100 mM) was added immediately prior to performing radioligand binding assays. The effect of NEM alkylation on and ␦ receptor binding was generally determined by preincubating membranes in the absence and presence of varying concentrations of NEM (0.5 M to 5 mM) at 37°C for 15 min, followed by the addition of 5 mM reduced glutathione to all samples to quench the reaction. Initially, membranes were washed by centrifugation in order to remove the NEM and glutathione prior to the start of the radioligand binding assays; however, the washes were subsequently found to have no effect on the outcome of the assay. Protection of and ␦ receptor binding against NEM inactivation was assessed by preincubating membranes in the absence and presence of ligands (100 nM or 1 M) for 10 min at 37°C, prior to reaction with 0.5 mM NEM for 15 min at 37°C. Samples were chilled on ice, and glutathione was added to 5 mM. Ligands were removed by centrifugation at 35,000 ϫ g for 20 min. Membranes were resuspended in 50 mM Tris-HCl, 1 mM Na 4 EDTA buffer, pH 7.4, incubated for 10 min at 37°C to promote ligand dissociation, and then centrifuged and resuspended two more times prior to initiating the radioligand binding assay.
Construction of Receptor Chimeras-Receptor chimeras were constructed using a two-step polymerase chain reaction process followed by direct subcloning into the pCR3 expression vector, as described previously (30). Receptor chimeras D2M, D3M, D5M, and the reciprocal chimeras M2D and M5D were used in these studies. Designations for chimeras indicate the origin of the amino-terminal domain on the left and the carboxyl-terminal domain on the right (M represents , and D represents ␦), separated by a number, which refers to the transmembrane helix that is the site of the junction. Thus, the chimera referred to as D2M contains the amino terminus derived from the ␦ receptor, the site of the ␦/ junction is in TM2, and the carboxyl terminus is derived from the receptor. All chimeric receptor constructs were fully sequenced to verify the location of the junction site and to ensure that no mutations were introduced during synthesis.
The ⌬N 64 amino-terminal deletion was generated in a similar manner, with the following sense strand oligonucleotide, ATGGTCACAGC-CATTACC. The ⌬N 89 amino-terminal deletion was an unexpected byproduct of the polymerase chain reaction reaction used to generate the C321S mutation.

Effect of DTT on and ␦ Opioid Receptor
Binding-The importance of disulfide bonds for the maintenance of active opioid receptor conformations was studied by comparing the effect of the disulfide reducing agent, DTT, on and ␦ opioid receptor binding. [ 3 H]Bremazocine, a ligand of the benzomorphan series with high affinity for and ␦ opioid receptors (30), was used to measure specific binding to both receptor types in the absence and presence of varying concentrations (1-100 mM) of dithiothreitol. Binding to both opioid receptor types was inhibited to approximately 60% of control levels, but only at relatively high concentrations of the disulfide reducing agent (Fig. 1). Nonlinear regression analysis of the DTT inhibition curves yielded extrapolated IC 50 values of 230 -250 mM for inhibition of both and ␦ opioid receptor binding.
In the course of determining the optimal concentration of glutathione to use to quench NEM reactions, we were surprised to observe that [ 3 H]bremazocine binding to and ␦ receptors was considerably more sensitive to incubation with reduced glutathione than with DTT. The IC 50 of glutathione was approximately 15 mM for inhibition of [ 3 H]bremazocine binding to and ␦ receptors, and the slopes of the inhibition curves were very steep (data not shown). Similar results were reported for binding to opioid, neurokinin-1, and kainic acid receptors (31). We found, however, that the inhibition of binding by glutathione was due to lowering the pH of the buffer solution, due to the acidic nature of the tripeptide. We suggest, therefore, that the results on glutathione inhibition of binding to neurokinin-1 and kainic acid receptors be interpreted with caution.
Comparison of the Effect of NEM on Wild-type and ␦ Opioid Receptor Binding and Protection by Ligand-The kinetics of NEM inactivation were significantly more rapid for receptor binding than for ␦ receptor binding (Fig. 2). Half-lives of inactivation were calculated by nonlinear regression analysis to be 8 and 56 min for and ␦ opioid receptor binding, respectively. Pseudo-first-order rate constants were 0.09 min Ϫ1 and 0.01 min Ϫ1 for inactivation of and ␦ opioid receptor binding, respectively. The inhibitory effect of NEM on receptor binding was due primarily to an 8-fold reduction in the maximum number of binding sites, with little change in the affinity of the remaining receptors for bremazocine (Table I).
The ability of agonist and antagonist ligands to protect against NEM inactivation of and ␦ opioid receptor binding was determined. Both receptor types were protected against NEM inactivation by preincubation with ligands, although protection of receptor binding was more complete (Fig. 3). All ligands tested were capable of protection, including type-selective peptide agonists (DAMGO and DSLET), alkaloid agonists (morphine and etorphine), and the antagonist, naloxone. The data indicated that the NEM-reactive group on both receptor types resided within, or in the vicinity of, the ligand binding crevice.
Sodium Ions Differentially Affect the NEM Sensitivity of Peptide and Bremazocine Binding to and ␦ Opioid Receptors-The specific binding of [ 3 H]DAMGO to the receptor was slightly more susceptible to inactivation by NEM than the specific binding of [ 3 H]bremazocine when assayed in Tris-EDTA buffer (Fig. 4). The presence of 100 mM NaCl in the buffer, however, differentially altered the sensitivity of DAMGO and bremazocine binding to NEM inactivation. The IC 50 of NEM toward inactivation of DAMGO binding to the receptor decreased significantly in the presence of 100 mM NaCl from 32 to 3 M, while the IC 50 of NEM toward inactivation of bremazocine increased from 85 to 330 M (Fig. 4).
Binding to the ␦ receptor was considerably less sensitive to NEM inactivation than was binding to the receptor. [ 3 H] DSLET and [ 3 H]bremazocine binding to the ␦ receptor were also differentially affected by inclusion of 100 mM NaCl in the buffer (Fig. 5), in a similar manner to that observed with receptor binding. The IC 50 of NEM toward inactivation of DSLET binding to the ␦ receptor decreased markedly in the presence of 100 mM NaCl from 450 to 8 M, while the IC 50 of NEM toward inactivation of bremazocine increased from 660 M to 3.6 mM.
Utilization of /␦ Receptor Chimeras to Search for the Site of NEM Alkylation on the Opioid Receptor-The receptor contains 17 cysteines (Fig. 6). A strategy utilizing /␦ receptor chimeras was devised to determine the location of the NEM alkylation site on the receptor, based on the large difference in sensitivity of [ 3 H]bremazocine binding to and ␦ receptors toward NEM inactivation. A panel of chimeric receptors was generated, utilizing junction sites shown in Fig. 6. A schematic illustration of the structures of the /␦ receptor chimeras is displayed in Fig. 7. D2M, D3M, and D5M contain ␦ receptor sequences from the amino termini to junction sites in TM2, TM3, and TM5, respectively, followed by sequences derived from the receptor from the junction site to the carboxyl termini. M2D and M5D are reciprocal chimeras, with aminoterminal domains derived from the receptor, junction sites in TM2 or TM5, respectively, followed by ␦ receptor-derived sequences to the carboxyl termini.
The ability of NEM to inactivate [ 3 H]bremazocine binding to wild-type and ␦ receptors and /␦ receptor chimeras was compared (Table II). [ 3 H]Bremazocine binding to wild-type receptors was 10 times more sensitive to NEM inactivation   when compared with binding to ␦ receptors (NEM IC 50 values were 0.16 and 1.6 mM, respectively). Binding to the D2M receptor chimera was inactivated by NEM at similar concentrations as binding to the receptor (IC 50 ϭ 0.29 mM), while the sensitivity of the D5M chimera was even greater than the wild-type ␦ receptor (IC 50 ϭ 4.3 mM). These data suggested than the NEM-reactive groups in the and ␦ receptor resided between TM2 and TM5. Data from the reciprocal M2D and M5D chimeras were consistent with this assumption; the M2D chimera behaved like the ␦ receptor, and the M5D chimera was even more sensitive than the receptor with respect to the NEM IC 50 for inactivation of [ 3 H]bremazocine binding (Table  II). Analysis of the NEM sensitivity of the D3M chimera (with a receptor-like IC 50 ϭ 0.05 mM) led to the more focused prediction that the NEM-reactive group in the receptor was in the region between the junction sites in TM3 and TM5.

Utilization of Truncated and Site-specific Receptor Mutants to Determine the Site of NEM Alkylation on the Opioid
Receptor-Based on the results regarding the NEM sensitivity of [ 3 H]bremazocine binding to /␦ receptor chimeras, cysteines in TM3, TM4, and TM5 of the receptor were individually substituted with Ser and then evaluated for their ability to bind [ 3 H]bremazocine and for their sensitivity toward NEM inhibition of binding. The Ser for Cys substitutions did not affect the affinity of the mutated receptors for bremazocine (data not shown). Furthermore, the NEM IC 50 values of the three Ser-substituted receptors (0.11-0.28 mM) were all in the same range as that of the wild-type receptor (0.16 mM, Table III). These data indicated that Cys 159 , Cys 190 , and Cys 235 were not the sites of NEM alkylation that resulted in inhibition of [ 3 H]bremazocine binding.
Based on these results, Ser was substituted for other cysteines residing outside of the region between TM3 and TM5, and the mutant receptors were tested for sensitivity to NEM. Ser substitution of either Cys 292 or Cys 321 , located in TM6 and TM7, respectively, did not affect the ability to bind [ 3 H]bremazocine or the sensitivity toward NEM inhibition of binding (Table III). Deletion of 64 amino acids from the amino-terminal domain (⌬N 64), which contains four cysteines at positions 13, 22, 43, and 57 (see Fig. 6), did not affect the affinity of the truncated receptor for [ 3 H]bremazocine (K D ϭ 1.3 nM versus 0.8 nM for the wild-type receptor). It has also been reported previously that this deletion did not affect the binding of [ 3 H]naloxone and [ 3 H]DAMGO to the receptor (32). The concentration of NEM required for inactivation of bremazocine binding to the truncated receptor was increased 3-fold relative to the wild-type receptor (Table III); however, the IC 50 was still in the submillimolar range (0.57 mM). We also tested the effect of removal of 89 amino acids from the amino terminus of the C321S mutant receptor. The deleted region of this construct, referred to as ⌬N 89, included the amino-terminal domain and most of putative TM1, including Cys 79 (Fig. 6) The affinity of [ 3 H]bremazocine for the ⌬N 89 construct decreased 20-fold. 2 The IC 50 of NEM for the ⌬N 89 receptor, however, was similar to the IC 50 of the ⌬N 64 receptor (Table III), suggesting that the cysteines in the amino-terminal domain and Cys 79 were not the relevant targets for NEM alkylation. Substitution of Cys 140 with Ser completely blocked the ability of the mutant receptor to bind [ 3 H]bremazocine (data not shown). Although this mutated receptor could not be tested for NEM sensitivity, Cys 140 is thought to be linked by a disulfide bond with Cys 217 (Fig. 6); hence, it would not be reactive with NEM.
The data suggested that none of the cysteines that were substituted with Ser or deleted were likely targets for NEM alkylation. Based on the chimeric receptor data, which indicated that the NEM-reactive group resided in the region between TM3 and TM5, the sequence of the receptor was reexamined for amino acids other than Cys that might be reactive toward NEM. It has been reported that reaction of lysozyme and ribonuclease with NEM resulted in the alkylation of ⑀-amino groups and imidazole groups (33). The receptor contains a His residue at position 223 in the putative second extracellular loop connecting TM4 and TM5 (Fig. 6). According to our alignment of the opioid receptor sequences, 3 (Table III). The H223S substitution also decreased the affinity for [ 3 H]bremazocine dramatically (Table IV). Preliminary data from competition analyses indicated that the affinity of the H223S-substituted receptor for etorphine and naloxone was also decreased Ն25-fold (data not shown). In addition to the 25-fold decrease in the affinity constant for bremazocine, the cell line that expressed the H223S mutant receptor also had a significantly lower B max than the cell line that expressed the wild-type receptor (Table IV). DISCUSSION The following observations and conclusions were made based on these studies. 1) [ 3 H]Bremazocine binding to and ␦ opioid receptors was inhibited to an equal extent by high concentrations of DTT, implying the presence of relatively inaccessible but critical disulfide linkages for both receptor types. 2) [ 3 H]Bremazocine binding to the receptor was considerably more sensitive to treatment with NEM than binding to the ␦ receptor. This finding suggested that the functional group that was the site of alkylation on the receptor was more accessible to and/or reactive with NEM than the relevant group on the ␦ receptor. 3) Ligand protection against NEM inactivation of binding to and ␦ opioid receptors was consistent with the site of alkylation being within, or in the vicinity of, the receptor binding crevice. 4) Dose-response curves of NEM inactivation of and ␦ receptor binding in the presence of sodium ions suggested that at least two reactive groups were subject to alkylation. Alkylation of and ␦ receptor sites at low concentrations of NEM resulted in inhibition of  (⌬N 64 and ⌬N 89) are indicated. The presumed disulfide bond between Cys 140 and Cys 217 , in putative extracellular loops 2 and 3, respectively, is indicated with a connecting bar. One of the two cysteines in the carboxyl-terminal tail is shown with a schematic palmitoyl group inserted into the cytoplasmic side of the plasma membrane. Junction sites used to construct /␦ receptor chimeras are indicated with boldface circles. These amino acids are encoded by contiguous homologous nucleotide sequences. Possible sites of N-linked glycosylation (NX(S/T), where X is any amino acid except P) in the amino-terminal domain are shown schematically with core oligosaccharides. of the G protein-coupled receptor superfamily, such as the cannabinoid, ␤ 1 -and ␤ 2 -adrenergic, and ␣ 1 -and ␣ 2 -adrenergic receptors, was considerably more sensitive to treatment with DTT (20,25,35,36). There is evidence for the presence of a disulfide bond between two conserved Cys residues in the first and second extracellular loops of the ␤ 2 -adrenergic receptor and rhodopsin (36 -38). The corresponding residues in the opioid receptor are Cys 140 and Cys 217 (Fig. 6). Our finding that the C140S substitution completely blocked [ 3 H]bremazocine binding to the receptor was consistent with the presence of a disulfide bond between Cys 140 and Cys 217 that is required for maintenance of an active receptor conformation. The mutagenesis data also suggested that Cys 13 , Cys 22 , Cys 43 , Cys 57 , Cys 159 , Cys 190 , Cys 235 , Cys 292 , and Cys 321 were not involved in disulfide linkages that were critical for ligand binding.
Comparison of the Effect of NEM on Wild-type and ␦ Opioid Receptor Binding and Protection by Ligand-It was obvious that radioligand binding to the receptor was more sensitive to inactivation by NEM than binding to the ␦ receptor, suggesting that the functional group that is the site of alkylation on the receptor was more exposed and/or more reactive. This finding corroborates previous studies involving selective labeling of rat brain membrane preparations that found that the rank order of receptor sensitivity to NEM was Ͼ ␦ Ͼ (39). Our analysis of saturation isotherms indicated that inhibition of bremazocine binding to the ␦ receptor by NEM resulted from a decrease in the total number of binding sites, with no appreciable effect on the affinity of the receptor for the ligand. This observation suggested that alkylated receptors totally lose the ability to bind ligand and that residual receptor binding in the presence of NEM concentrations that gave sub-maximal inhibition was due to the presence of intact, nonalkylated receptors. When high enough concentrations of NEM were used, binding to and ␦ receptors was totally abolished.
The observation that preincubation with opioid ligands protected against NEM inactivation of binding to and ␦ receptors was consistent with the site of alkylation being within, or in the vicinity of, the ligand binding crevice of both receptor types. Early studies performed before the realization that there were multiple opioid receptor types also demonstrated ligand protection against N-ethylmaleimide inactivation of receptor binding to rat brain membranes (10,11 Differential effects of sodium ions on the ability of NEM to inhibit agonist and antagonist binding have been reported previously (11,39). The pharmacological profile of bremazocine,    In the presence of sodium ions, bremazocine binding was increased slightly (data not shown), and the doseresponse curve of NEM was shifted to the right. There is pharmacological evidence that suggests that bremazocine acts as an agonist at receptors and as an antagonist at and ␦ receptors (40 -43), although it has also been reported that high concentrations of bremazocine caused inhibition of forskolinstimulated cAMP accumulation in COS cells expressing the ␦ receptor (44). Additional studies using cloned opioid receptors will be necessary to clarify this issue. The observation that [ 3 H]peptide agonist binding to and ␦ receptors was reduced to Ͻ20% of control values at NEM concentrations that had minimal effects on [ 3 H]bremazocine binding provided strong evidence for the involvement of at least two NEM-reactive groups. The shift to the left of the NEM doseresponse curve for inhibition of [ 3 H]DAMGO and [ 3 H]DSLET binding may be partially due to NEM alkylation of a GTPbinding protein (15), resulting in receptor uncoupling and a consequent decrease in agonist affinity. [ 3 H]Bremazocine binding, in contrast, is not affected by the state of receptor coupling (45). 4 Localization of the Site of N-Ethylmaleimide Alkylation on the Opioid Receptor-Analysis of the N-ethylmaleimide sensitivity of [ 3 H]bremazocine binding to a panel of /␦ receptor chimeras led to the conclusion that the reactive group on the receptor resided in the region between transmembrane domains 3 and 5. Due to the lack of dramatic effects involving substitution or deletion of cysteines, the region between transmembrane domains 3 and 5 was reexamined for other possible reactive side chains. Although NEM is widely regarded as a sulfhydryl-specific reagent, it has been reported that the imidazole group of His can also be alkylated (33,46).
The H223S substitution (in putative extracellular loop 2 connecting TM4 and TM5) resulted in pronounced effects on basal [ 3 H]bremazocine binding and NEM sensitivity of [ 3 H]bremazocine binding. The affinity of bremazocine was lowered 20-fold, and bremazocine binding was rendered insensitive toward NEM. There are at least two plausible explanations for these findings. 1) His 223 makes direct contact with bremazocine in the binding site, and NEM alkylation of His or substitution with Ser abolishes the ability of bremazocine to bind to the receptor. In this case, the H223S mutant receptor would be insensitive to NEM since the reactive group had been removed. 2) His 223 makes an important contribution to the overall active conformation of the receptor. NEM alkylation of His or substitution with Ser would be presumed to disrupt the active conformation, leading to a loss of high affinity bremazocine binding. Again, the H223S mutant receptor would be insensitive to N-ethylmaleimide since the reactive group had been removed, or alternatively, the conformational change resulting from the H223S substitution could conceivably shield other reactive groups (presumably one of the cysteines that was not subjected to deletion or mutagenesis) from interaction with NEM. Investigations are under way to distinguish between these interpretations.
Evidence for essential histidyl residues within opioid receptors has been reported previously (47). In these studies, chemical modification of opioid receptors with two different histidylspecific reagents resulted in complete inhibition of [ 3 H]etorphine binding to rat brain membranes. Further support for His being the actual site of NEM alkylation was ob-tained by studying the pH dependence of the NEM inactivation of opioid receptor binding. Childers and Jackson (48) found that the apparent pK a value of the N-ethylmaleimide-reactive groups on opioid receptors was between 5.4 and 6.0, which is much closer to the average pK a of histidine (pK a ϭ 6.5) than cysteine (pK a ϭ 8.5) (49).
The data from these studies add to the growing body of knowledge regarding the constituents of opioid receptor binding sites. Previous mutagenesis experiments have highlighted the importance of the Asp in TM2 of the and ␦ receptor for high affinity selective agonist binding (32,44). Mutation of Asp 147 in TM3 and His 297 in TM6 of the receptor inhibited both agonist and antagonist binding (32). These amino acids are also conserved in ␦ and receptors. Regarding receptor selectivity, analysis of / and ␦/ receptor chimeras revealed that the second extracellular loop of the receptor was required for high affinity binding of dynorphin-(1-17), dynorphin-(1-13), ␣-neoendorphin, and dynorphin B (50 -52). Evidence has also been provided that the binding site for antagonists in the opioid receptor differs substantially from the antagonist site of the and ␦ opioid receptors (53). The amino terminus of the opioid receptor was found to be necessary for high affinity naloxone binding and for reversal of agonistmediated inhibition of forskolin-stimulated cAMP accumulation by naloxone. In contrast, Glu 297 in the putative third extracellular loop of the receptor plays a major role in binding the -selective antagonist, norbinaltorphimine (54). Our group and others (30,52,55) have reported recently that a major binding determinant for ␦-selective peptides resides in the region spanning TM5 to TM7 of the ␦ receptor, in excellent agreement with our studies regarding the role of the Arg residues in the putative third extracellular loop (30). Our finding on the importance of the putative first extracellular loop for DAMGO binding using /␦ receptor chimeras (30) has also been recently reported independently (55,56). In contrast, Xue et al. (57) found that the third extracellular loop of the receptor was important for agonist selectivity using / receptor chimeras. This discrepancy was clarified recently with the important finding that DAMGO distinguishes between and ␦ opioid receptors at a site different from that for the distinction between and opioid receptors (58).
Until high resolution experimental data is obtained from crystallography, insights from analysis of receptor chimeras and mutagenesis will provide information on the structure and function of opioid receptors, with the aid of molecular modeling and computer simulation. Understanding of the molecular mechanisms involved in receptor activation and G protein coupling triggered by agonist engagement of the opioid receptor binding site remain as long term goals of these studies. It is anticipated that an understanding of opioid receptor structure and function will lead to the development of novel therapeutic agents.