A single residue, aspartic acid 95, in the delta opioid receptor specifies selective high affinity agonist binding.

The enkephalins, dynorphins, and endorphins are endogenous opioids which function as neurotransmitters, neuromodulators, and hormones and are involved in the perception of pain, modulation of behavior, and regulation of autonomic and neuroendocrine function. Pharmacological studies have defined three classes of opioid receptors, designated as delta, kappa, and mu. To investigate mechanisms by which agonists and antagonists interact with the delta opioid receptor, we have substituted aspartic acid 95 in the transmembrane segment 2 of the cloned mouse delta opioid receptor with an asparagine (D95N). The D95N mutant receptor had reduced affinity for delta receptor-selective agonists such as enkephalin, [D-Pen2,D-Pen5]enkephalin and [D-Ser2,Leu5]enkephalin-Thr6 such that it did not bind these peptides even at micromolar concentrations. The binding of delta-selective non-peptide agonists was also reduced. In contrast, the delta receptor-selective antagonists, such as naltrindole, the benzofuran analog of naltrindole, and 7-benyllidenenaltrexone, bound equally well to the wild-type and mutant receptor. Similarly, non-selective opioid agonists such as bremazocine and buprenorphine, which interact with delta, kappa, and mu opioid receptors, showed no difference in binding to the wild-type and mutant delta receptor. The D95N mutant remained coupled to G proteins, and the receptor was functionally active since it mediated agonist inhibition of cAMP accumulation. These results indicate that selective agonists and antagonists bind differently to the delta receptor and show that Asp-95 contributes to high affinity delta-selective agonist binding. The identification of a key residue involved in selective agonist binding to the delta opioid receptor will facilitate the development of novel therapeutic reagents that can be used for the treatment of chronic pain and other conditions.

The enkephalins, dynorphins, and endorphins are endogenous opioids which function as neurotransmitters, neuromodulators, and hormones and are involved in the perception of pain, modulation of behavior, and regulation of autonomic and neuroendocrine function. Pharmacological studies have defined three classes of opioid receptors, designated as 6 , K, and p. To investigate mechanisms by which agonists and antagonists interact with the 6 opioid receptor, we have substituted aspartic acid 95 in the transmembrane segment 2 of the cloned mouse 6 opioid receptor with an asparagine (D95N). The D95N mutant receptor had reduced affinity for 6 receptor-selective agonists such as enkephalin, [D-Pen2,D-Pen"]enkepha1in and [~-Ser~,Leu"]enkephalin-Thre such that it did not bind these peptides even at micromolar concentrations. The binding of 6-selective non-peptide agonists was also reduced. In contrast, the 6 receptor-selective antagonists, such as naltrindole, the benzofuran analog of naltrindole, and 7-benyllidenenaltrexone, bound equally well to the wildtype and mutant receptor. Similarly, non-selective opioid agonists such as bremazocine and buprenorphine, which interact with 6, K , and p opioid receptors, showed no difference in binding to the wild-type and mutant 6 receptor. The D95N mutant remained coupled to G proteins, and the receptor was functionally active since it mediated agonist inhibition of CAMP accumulation. These results indicate that selective agonists and antagonists bind differently to the 6 receptor and show that Asp-95 contributes to high affinity 6-selective agonist binding. The identification of a key residue involved in selective agonist binding to the 6 opioid receptor will facilitate the development of novel therapeutic reagents that can be used for the treatment of chronic pain and other conditions. * This work was supported by National Institute of Mental Health grants MH45533 and MH48518 and the Howard Hughes Medical Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ The pentapeptides methionine-and leucine-enkephalin induce diverse actions in the central nervous system, including modulation of locomotor activity and analgesia (1-3). They induce their biological effects by interacting with 6 opioid receptors. The ligand binding domain of several G proteincoupled receptors is generally believed to be composed of several charged residues within a hydrophobic pocket created by transmembrane spanning regions (4-6). A conserved aspartate in the second transmembrane spanning region of the cY2-adrenergic receptor has been shown to be critical for agonist binding (6). The recently cloned mouse 6 opioid receptor (7-9) has within its structure an aspartate at residue 95 in the second transmembrane spanning region which may be associated with the ligand binding domain. To determine the role of Asp-95 in ligand binding, we converted this residue in the cloned mouse 6 receptor to an asparagine by site-directed mutagenesis and compared the ligand binding characteristics of the mutant and wild-type proteins after expression in COS-7 cells. Our findings show that this mutation alters the receptor so that it has very low affinity for &selective agonists but binds antagonists and non-selective agonists as well as the normal receptor. These findings indicate that selective agonists and antagonists bind differently to the 6 receptor and reveal that this aspartic acid residue may contribute to stabilizing enkephalin binding. Mutagenesis of the Cloned Mouse 6 Opioid Receptor-The mouse 6 opioid receptor cDNA (9) was mutated using the Altered SitesTM in vitro mutagenesis system (Promega LKB Biotechnology Inc.). To mutate aspartic acid 95 to an asparagine, the 6 receptor cDNA was subcloned into the phagemid pALTERTM, and with the helper phage R408, a single-stranded template was produced. The 21-mer oligonucleotide (GCTTTGGCTAATGCGCTGGCC) containing the desired mutation (GAT to AAT) was annealed to the single-stranded template and elongated with T4 DNA polymerase.  Med. Chem., in press. Asp-95 in 6 Opioid Receptor Specifies Selective Ag Binding DNA was used to transform the repair-minus Escherichia coli strain BMH 71-18 mut S. Transformants were selected by growth in LB plates containing 125 pg/ml ampicillin. The mutation was confirmed by DNA sequencing. The cDNA was excised from PALTER with EcoRI and SalI and subcloned into the corresponding sites in the mammalian expression vector pCMV6c (14).

Expression of the Mouse 6 Opioid Receptor cDNA in COS-7 Cells-
The mutated and wild-type cDNA were transfected into COS-7 cells by the calcium-phosphate-mediatedprocedure as previously described (9,14). For the receptor binding studies, COS-7 cells expressing the 8 receptor were harvested 72 h after transfection in 50 mM Tris-HC1 (pH 7.8) containing 1 mM EGTA, 5 mM MgC12, 10 pg/ml leupeptin, 10 pg/ml pepstatin, 200 pg/ml bacitracin, and 0.5 pg/ml aprotinin (Buffer 1) and centrifuged at 24,000 X g for 7 min at 4 "C. The pellet was homogenized in Buffer 1 using a polytron. The homogenate was centrifuged at 48,000 X g for 20 min at 4 "C and the pellet resuspended in Buffer 1 and used in the radioligand binding assay. Cell membranes (20-30 pg of protein) were incubated with the &selective agonist [3H] DPDPE (15) (2 nM) or the 6 selective antagonist [3H]naltrindole (3, 12, 13) (1 nM) in a final volume of 200 pl for 40 min at 25 "C in the presence or absence of competing agents. Nonspecific binding was defined as radioactivity remaining bound in the presence of 1 p~ naltrindole. The binding reaction was terminated by the addition of ice-cold 50 mM Tris-HC1 (pH 7.8) and rapid filtration over Whatman GF/B glass fiber filters that were pretreated with 0.5% polyethyleneimine and 0.1% bovine serum albumin. The filters were washed with 12 ml of ice-cold buffer, and the bound radioactivity was determined using a liquid scintillation counter. Data from radioligand binding studies were used to generate inhibition curves. IC, values were obtained by curve-fitting performed by the program FITCOMP.
cAMP Accumulation-CAMP accumulation in COS-7 cells expressing the 6 receptors were measured as previously described (9,14). Briefly, COS-7 cells were subcultured in 12-well culture plates. The cells were transfected 72 h prior to the cAMP experiments. Culture medium was removed from the wells and replaced with 500 pl of fresh medium containing 0.5 mM isobutylmethylxanthine. Cells were incubated for 20 min at 37 'C. Medium was removed and replaced with fresh medium containing 0.5 mM isobutylmethylxanthine, with or without 10 p~ forskolin and various opioid agonists. The cells were incubated for 30 min at 37 "C. Medium was removed and cells sonicated in the wells in 500 pl of 1 N HC1. The HCl was evaporated off in a Speed-Vac and the cAMP analyzed using a radioimmunoassay kit from Du Pont-New England Nuclear.

RESULTS
Both the wild-type and D95N mutant receptors expressed in COS-7 cells could be labeled with the &selective agonist [3H]DPDPE (15). However, the binding of [3H]DPDPE to the mutant receptor was reduced approximately 75% compared to the wild-type receptor. The binding of 2.0 nM [3H] DPDPE to the wild-type receptor was 261 fmol/mg protein and 68 fmol/mg protein for the D95N mutant. The reduced binding to the D95N mutant could be due to uncoupling of the receptor from G proteins, low levels of expression of the receptor, or an alteration in the ligand binding properties of the receptor. The mutant receptor remained coupled to G proteins since GTPyS reduced [3H]DPDPE binding to the D95N mutant (Fig. 1). Furthermore, the D95N mutant mediated the inhibition of cAMP formation induced by the 6 agonist DSLET (16) (Fig. 2), a response requiring G protein coupling. While GTP analogs reduced high affinity agonist binding to D95N, the mutant receptor was not responsive to Na+.
[3H]DPDPE binding to the wild-type 6 opioid receptor was specifically reduced by 90 mM Na+, but agonist binding to the D95N mutant was not altered, consistent with the aspartate 95 being the site of Na+ regulation of agonist binding to this receptor.
The D95N mutant receptor was expressed at higher levels than the wild-type receptor as determined by the binding of the antagonist [3H]naltrindole. Scatchard analysis of [3H] naltrindole binding (Fig. 3)  respectively. The Bmax and K d for binding to the D95N mutant was 14.0 pmol/mg protein and 0.082 nM, respectively. These results indicate that the mutant receptor was expressed at a higher density than the wild-type receptor. Furthermore, the affinity of the mutant and wild-type receptors for the 6selective antagonist naltrindole and the &and 13~-selective antagonists (12, 13) BNTX and NTB, respectively, were similar (Table I). The normal binding affinity of the D95N mutant for antagonists indicates that the substitution of Asp-95 by Asn in the second transmembrane domain did not cause a gross conformational change in the structure of the receptor. The results imply that the mutant receptor has a selective reduction in affinity for agonists. This is further indicated by the diminished potencies of the &selective agonists Met-enkephalin, DPDPE, D-Ala2-deltorphin I1 (17), and DSLET to inhibit [3H]naltrindole binding to the mutant receptor compared to the wild-type receptor (Fig. 4, Table I). The &selective agonists tested above are all peptides. To determine whether the differences in ligand binding properties of the mutant and wild-type receptors were due to the peptide nature of the agonists or their 6 receptorselective characteristics, we tested two recently developed  potent, non-peptide &selective agonists, BW373U86 (11) and SIOM,' for their interaction with the mutant 6 receptor. Both compounds inhibit forskolin-stimulated cAMP formation in cells expressing either the wild-type or D95N mutant 6 receptor (Fig. 2), indicating that they are full agonists at the 6 receptor. BW373U86 and SIOM potently inhibit [3H]naltrindole binding to the wild-type 6 receptor (Fig. 4, Table I). In contrast, they are much less potent at binding to the mutant receptor (Fig. 4, Table I). These findings indicate that the mutant 6 receptor has reduced affinity for both peptide and non-peptide &selective agonists.
To further investigate the specificity of the diminished affinity of the mutant receptor for agonists, we tested agonists that interact with all the opioid receptors for their binding to the wild-type and D95N receptors. The non-selective opioid receptor agonist bremazocine interacts with and K opiate receptors (18) and is an full agonist at 6 receptors, since it inhibits cAMP formation in COS-7 expressing the 6 receptor (Fig. 2). It binds equally well to the mutant and wild-type 6 receptors (Table I) of this non-selective agonist to the 6 receptor from &selective agonists such as Met-enkephalin, DPDPE, deltorphin, and DSLET. The alkaloid buprenorphine also interacts potently with 1.1, K, and d opioid receptors. It is a partial 1.1 opioid agonist (10) and a full agonist at the 6 receptor, since it inhibits forskolin-stimulated cAMP formation in COS cells expressing either the mutant 6 or the wild-type receptor (Fig. 2). (-)Buprenorphine binds to both the mutant and wild-type 6 opioid receptors with similar potencies (Table I). The similar potency of bremazocine and buprenorphine at binding to the wild-type and mutant 6 receptors indicates that non-selective agonists do not bind in the same manner to the cloned 6 receptor as do &selective agonists.

DISCUSSION
The most important finding of this study is that &selective agonists bind differently to the cloned 6 opiate receptor than do &selective antagonists or non-selective opioid agonists. The reduction in affinity of the mutant receptor for &selective agonists could be explained by the necessity of Asp-95 for selective agonist binding. The similar affinity of the mutant and wild-type receptors for antagonists indicate that Asp-95 is not necessary for antagonist binding. These findings indicate that there are distinct agonist and antagonist binding domains of the 6 receptor and that Asp-95 is associated with the agonist binding domain. Alternatively, there are distinct conformational requirements for agonist and antagonist binding to the same ligand binding domain of the receptor and mutation of the Asp-95 to an Asn shifts the receptor to a conformation favoring antagonist binding. This latter possibility seems unlikely since non-selective agonists bind with equal potency to the mutant and wild-type 6 receptors, indicating that the mutant receptor does not favor antagonist binding.
If the negative charge provided by Asp-95 serves as a counterion to the positive charge of the nitrogen atom in enkephalin and &selective agonist to stabilize binding, then it is likely that the Asp-95 is in close proximity to the agonist binding domain. This would allow the negative charge of the aspartic acid to be in contact with the positive charge of selective agonists bound to the receptor. However, it is unlikely that this residue is entirely responsible for selective agonist binding to the 6 receptor since it is conserved in the Asp-95 in 6 Opioid Receptor Specifies Selective Ag Binding cloned K opioid receptor (9) which has agonist specificities distinct from those of the 6 receptor. Conversion of the Asp at residue 79 in the second transmembrane spanning region of the az-adrenergic receptor to an Asn has been reported to induce a modest reduction in agonist affinity (6). A similar mutation of the somatostatin receptor subtype SSTR2 resulted in no change in agonist binding (19). The dramatic reduction in &selective agonist binding to the D95N mutant indicates that Asp-95 of the 6 opioid receptor serves a unique role in agonist binding to this receptor.
Interestingly, the conserved Asp of the 6 opioid, a-adrenergic and somatostatin receptors is necessary for Na+ regulation of agonist binding (6,19). Na+ has been proposed to affect agonist binding by directly interacting with receptors to cause G protein uncoupling, thereby converting the receptor into a low affinity state for agonists (6). This effect of sodium to diminish agonist binding was first identified for opioid receptors (20). The findings of the present study suggest that the Asp-95 is the site of action of Na+ in regulating agonist binding to the 6 opioid receptor.
Previous behavioral studies have suggested that subtypes of 6 receptors are expressed in the central nervous system (12, 13). These findings have been based on the use of the &selective antagonist BNTX and the 6z-selective antagonist NTB. Our findings that the bz receptor subtype-selective antagonist NTB has 500-fold higher potency at binding to the cloned 6 receptor than the dl receptor-selective antagonist BNTX, suggests that the cloned receptor is a 6z subtype. Similar antagonist binding specificities have also been observed for 6 receptors endogenously expressed in NG108 cells. 3 Recently, Evans et al. (7) cloned a 6 receptor from an NG108 cDNA library, with identical amino acid sequence as the one examined in the present study, further indicating that the cloned opioid receptor is a 6z subtype.
Our findings that selective agonists and antagonists bind differently to the 6 receptor may facilitate the delevopment of more selective 6 agonists that could be used for the treat-K. Raynor, G. I. Bell, and T. Reisine, unpublished findings. ment of chronic pain. Furthermore, recent studies have suggested that 6 receptor antagonists may be useful in the treatment of cocaine (21) and ethanol (22) abuse as well as in the prevention of graft rejection (23). Identification of the unique antagonist binding domain in the 6 receptor could facilitate further development of selective antagonists at the 6 receptor and its subtypes for the treatment of these disorders.