μ Opioid receptor: role for the amino terminus as a determinant of ligand binding affinity

Molecular Brain Research 76 Ž2000. 64–72 www.elsevier.comrlocaterbres

Research report

m Opioid receptor: role for the amino terminus as a determinant of ligand binding affinity Kirti Chaturvedi, Mandana Shahrestanifar, Richard D. Howells ) Department of Biochemistry and Molecular Biology, UniÕersity of Medicine and Dentistry of New Jersey, New Jersey Medical School, 185 South Orange AÕenue, Newark, NJ 07103, USA Accepted 16 November 1999

Abstract The importance of the amino-terminal domain of the m opioid receptor ŽMOR. as a component of the high affinity ligand-binding pocket was evaluated. A deletion mutant lacking 64 amino acids from the amino-terminus of MOR Ž DN64. was constructed and expressed in HEK 293 cells. The affinities of bremazocine and cyclazocine were similar for the truncated and full-length MORs. Affinities of the m receptor antagonist, naloxone, and the m receptor agonist, morphine, were decreased 3.5-fold and 6-fold, respectively, for the truncated receptor relative to the wild-type MOR. Similarly, the affinities of the opioid peptide agonists, DAMGO ŽTyr-D-AlaGly-MePhe-Gly-ol., b-endorphin, and DADL ŽTyr-D-Ala-Gly-Phe-D-Leu., for the DN64 receptor were decreased from 3- to 8-fold as a result of the deletion. In contrast, the affinities of the alkaloid agonists, methadone and fentanyl, and the peptide agonists, endomorphin 1 and endomorphin 2, for the truncated receptor relative to MOR were reduced dramatically by 20- to 60-fold. MOR is glycosylated when expressed in HEK 293 cells; however, analysis of N-glycosidase F-treated membranes indicated that N-glycan chains within the amino-terminal domain of MOR do not contribute significantly to ligand affinities. These results indicate that amino acid residues within the amino-terminal domain of MOR play a crucial role in the composition of the binding pocket for a select group of agonists. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Morphine; Methadone; Endomorphin; Fentanyl; Opioid receptor

1. Introduction Opioid drugs are powerful analgesic agents that are the drugs of choice for the treatment of pain. The pharmacological effects of opioid drugs and the physiological effects of endogenous opioid peptides are initiated through the binding and activation of membrane-bound opioid receptors that are prevalent in the central and peripheral nervous systems. Opioid receptors have been classified into three different types, m, d and k, on the basis of extensive pharmacological and behavioral studies w23,35,40x. The existence of the proposed opioid receptor types has been confirmed by molecular cloning w6,9,10,18,30,45,48,54 x. The amino acid sequences of putative transmembrane spanning segments and the three intracellular loops are highly conserved among opioid receptor types, whereas the extracellular amino termini, second and third extracellular loops, and the intracellular carboxyl termini are divergent. ) Corresponding [email protected]

author.

Fax:

q1-973-972-5594;

e-m ail:

Overall, the opioid receptors exhibit approximately 60% identity in their amino acid sequences. The opioid receptor types display characteristic ligand selectivity profiles w13,37x and neuroanatomical distribution patterns w26x. The m opioid receptor ŽMOR. is of particular clinical and social importance since the more potent analgesic drugs, such as morphine, heroin, fentanyl and methadone, elicit their beneficial pharmacological effects as well as their addictive liability through activation of the m receptor w24,27,41x. Opioid receptors belong to the family of guanine nucleotide binding protein ŽG protein.-coupled receptors w42,51x. G-protein-coupled receptors share several features: they contain seven transmembrane domains, ligands approach and engage the receptor from the extracellular space, and receptor activation results in coupling to heterotrimeric G proteins on the intracellular face of membrane. The amino-termini of nearly all G-protein-coupled receptors contain consensus amino acid sequences for asparagine-linked glycosylation, Asn-X-SerrThr, where X is any amino acid w21x. The opioid receptors have consensus

0169-328Xr00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 9 . 0 0 3 3 2 - 0

K. ChaturÕedi et al.r Molecular Brain Research 76 (2000) 64–72

65

N-linked glycosylation sites in the amino-terminal domains: two in the d and k receptors, and five in the m receptor w40x. The diversity of cellular responses to G-protein-coupled receptor activation arises from differential coupling of receptors to various G proteins, which regulate the activity of distinct intracellular effector systems. Several studies have confirmed that opioid receptors interact with multiple members of the pertussis toxin-sensitive Gi and Go protein families, to regulate adenylyl cyclase, Ca2q and Kq channels w5,22,35,36x. In the course of cloning the rat MOR, Wang et al. w48x isolated a partial cDNA, that when transcribed and translated, yielded a receptor lacking the N-terminal 64 amino acids. The truncated MOR Ž DN64. was capable of binding tritiated DAMGO and naloxone, and activation of the receptor led to inhibition of adenylyl cyclase activity w43x. Our group has also reported previously that the DN64 receptor bound w3 Hxbremazocine with an affinity that was very similar to its affinity for the full-length MOR w39x. The aim of this study was to characterize further the contribution of the m receptor amino terminus toward high affinity interactions with an expanded set of peptide and alkaloid ligands. Based on this study, the amino-terminal domain of the MOR appears to be an important constituent of the high affinity binding pocket for a select group of m receptor agonists.

trifugation, and resuspended in 0.32 M sucrose, 50 mM Tris HCl, pH 7.5, for storage at y808C. Opioid receptor binding assays were conducted in duplicate or quadruplicate on membrane preparations that had been resuspended in 50 mM Tris HCl, pH 7.5, utilizing w9-3 Hxbremazocine Žspecific activity, 26.5 Cirmmol, NEN, Boston, MA. as radioligand and 10 mM cyclazocine to define nonspecific binding w17,39x. Following 1 h incubation at 228C, binding assays were terminated by filtration through Whatman GFrB filters. Filters were soaked in Ecoscint liquid scintillation solution ŽNational Diagnostics, Manville, NJ. and filter-bound radioactivity was measured using a Packard Tri-Carb 2100 TR liquid scintillation analyzer. Receptor binding data were analyzed by nonlinear regression of saturation and competition curves using Prism 2.0 software ŽGraphPad Software, San Diego, CA.. Protein concentrations were determined as described previously w39x, using bovine serum albumin as standard. The opioid peptides, DADL ŽTyr-D-Ala-Gly-Phe-D-Leu., DAMGO ŽTyr-D-AlaGly-MePhe-Gly-ol. and b-endorphin were obtained from Sigma ŽSt. Louis, MO., while endomorphin 1 ŽTyr-ProTrp-Phe-NH 2 . and endomorphin 2 ŽTyr-Pro-Phe-Phe-NH 2 . were products of Peptides International ŽLouisville, KY.. Cyclazocine, naloxone, morphine, methadone, and fentanyl were obtained from the Division of Basic Research, National Institute on Drug Abuse ŽRockville, MD..

2. Materials and methods

2.3. Deglycosylation and immunoblotting of the m receptor

2.1. Construction of the D N64 m opioid receptor DN64, which contains a deletion of 64 amino acids from the amino terminus of the wild-type rat MOR, was generated using PCR, and ligated into the pCR 3.1 mammalian expression vector ŽInvitrogen, Carlsbad, CA. as described previously w39x. In the DN64 construct, the AUG codon for Met 65 of the full-length MOR serves as the translational initiator codon for the remainder of the receptor open reading frame. 2.2. Transfection and radioligand binding assays HEK 293 cells ŽATCC CRL, 1573. were transfected with expression plasmids encoding the wild-type rat m receptor Žobtained originally from Dr. L. Yu., the FLAGtagged mouse m receptor Žobtained from Dr. M. Van Zastrow. and the DN64 truncated rat m receptor using the calcium phosphate method, or by electroporation, as described previously w38,39x. Cells stably expressing opioid receptors were selected in media containing 0.5 mgrml G418 ŽLife Technologies, Gaithersburg, MD.. Cell membrane preparations were obtained by harvesting attached cells in 50 mM Tris–HCl, pH 7.5, homogenizing the cells with a Tekmar Tissuemizer, and centrifuging the homogenate for 20 min, 48C at 39,000 = g. Membrane pellets were washed twice by homogenization and recen-

Cell membranes were resuspended in 50 mM Tris–HCl, pH 7.5, and incubated with or without N-glycosidase F Ž40 Unitsrmg membrane protein, Roche Molecular Biochemicals, Indianapolis, IN. at 378C for 3 h. Membranes were then further diluted in 50 mM Tris HCl, pH 7.5, for radioligand binding assays as described above. For immunoblotting, aliquots of treated and untreated membranes were diluted with an equal volume of lysis buffer Ž150 mM Tris–HCl, pH 7.5, 300 mM NaCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 1% Triton X-100, 10% glycerol. and incubated for 1 h on ice. Proteins were resolved using 10% SDSrPAGE and transferred to Immobilon P SQ PVDF membranes ŽMillipore, Bedford, MA.. Receptor proteins were detected by incubation with anti-FLAG M1 monoclonal antibody, followed by rabbit anti-mouse IgG conjugated with alkaline phosphatase ŽSanta Cruz Biotech, Santa Cruz, CA., and developed using CDP Star western blot chemiluminescence reagent ŽNEN.. 3. Results 3.1. Stable expression of m and D N64 opioid receptors in HEK 293 cells The DN64 receptor has a deletion of 64 amino acids at the amino terminus, and lacks all five of the asparagine-

66

K. ChaturÕedi et al.r Molecular Brain Research 76 (2000) 64–72

linked glycosylation sites at positions 9, 31, 38, 46, and 53, as well as cysteine residues present at positions 13, 22, 43 and 57 ŽFig. 1.. Based on an empirical rule for predicting the orientation of eukaryotic membrane-spanning proteins w14x, it is assumed that the truncated DN64 receptor has the same transmembrane orientation as the full-length m receptor, that is, with the shortened amino-terminus facing the exoplasmic face of the membrane and the carboxyl-terminus facing the cytosolic face. To determine the contribution of the amino-terminal domain of the m receptor to the composition of the high affinity ligand binding pocket, the full-length and DN64 m receptors were stably expressed in HEK 293 cells, and radioligand binding assays were conducted using membrane fractions prepared from the cell lines. Bremazocine, a benzomorphan ligand with high affinity for full-length and DN64 m receptors w39,50x, was used as the radioligand in competition analysis to compare the affinities of selected ligands for the two receptor types. Nonlinear regression analysis of saturation curves revealed that bremazocine has approximately equal affinity for both the m and DN64 receptors Ž K D s 0.8 and 1.6 nM, respectively, Fig. 2 and Table 1., in excellent agreement with our previously reported observations w39x. The Bmax for the DN64 cell line was 205 fmolrmg protein, which was approximately 10-fold lower than that of the cell line expressing the full-length m receptor used in these studies. The DN64 receptor can be activated by agonists, resulting in inhibition of adenylyl cyclase activity w43x and activation of MAP kinase Žunpublished observations..

Fig. 2. Saturation curve of w3 Hxbremazocine binding to the DN64 receptor. Membrane preparations from HEK293 cells expressing the DN64 truncated m receptor were used for saturation binding of w3 Hxbremazocine. Data points are averages of duplicate determinations; a representative data set from a series of three independent experiments is shown. Nonlinear regression analysis of the curves yielded an apparent K D of 1.6 nM and a Bma x of 205 fmolrmg protein.

3.2. Comparison of binding affinities of Õarious non-peptide ligands for the full-length and truncated D N64 MORs The hypothesis that the amino-terminal domain of the m receptor was dispensable for high affinity ligand binding was tested further. The affinities of various other nonpeptide ligands for the m receptor were compared with their affinities for the truncated DN64 receptor, as determined by competition analysis using w3 Hxbremazocine as radioligand. As shown in Fig. 3 and Table 1, the affinity of cyclazocine, which is structurally related to bremazocine, was very similar for both the full-length and truncated m

Fig. 1. The amino acid sequences and proposed transmembrane topology of MOR and DN64. The amino-terminus is on the extracellular side and the carboxyl-terminus is on the intracellular side of plasma membrane. Transmembrane helices 1–7 are shown from left to right. The arrow indicates the site of the DN64 amino-terminal deletion. The presumed disulfide bond between Cys140 and Cys 217 is depicted, as is the palmitoylation site on Cys 351 . Putative recognition sequences for N-linked glycosylation ŽNXŽSrT., where X is any amino acid. in the amino-terminal domain are shown schematically with stars. There is evidence suggesting that the putative a-helical character of several transmembrane domains extends beyond the plane of the plasma membrane w51x.

K. ChaturÕedi et al.r Molecular Brain Research 76 (2000) 64–72 Table 1 Effect of N-terminal truncation of the MOR on ligand affinities Ligand dissociation constants Ž K i . for the m and DN64 opioid receptors were determined by nonlinear regression of competition curves, using w3 Hxbremazocine as radioligand. Ratios Ž K i DN64rK i m . reflect the decrease in ligand affinity resulting from truncation of the 64 amino acid MOR amino-terminal domain. Values represent means"S.E.M. of three to five analyses, each conducted in duplicate. K i ŽnM. m Alkaloids Cyclazocine Bremazocine Naloxone Morphine Methadone Fentanyl Peptides DAMGO b-endorphin DADL Endomorphin 1 Endomorphin 2

DN64

Ratio

1.1"0.3 0.8"0.1 9.6"1.4 19.6"6.9 3.4"0.3 3.9"0.7

1.6"0.5 1.6"0.5 34.1"4.7 119.0"10.2 60.8"12.8 123.0"21.2

1.5 2.0 3.5 6.1 18.0 31.0

6.7"1.6 20.3"7.2 84.9"9.4 29.9"2.9 18.2"2.4

22.3"3.0 98.0"31.7 725.0"161 721.0"184 1110.0"234

3.3 4.8 8.5 24.0 61.0

receptors Ž K i’s were 1.1 and 1.6 nM, respectively.. It appears, therefore, that the amino-terminal domain of the MOR is not a major component of the high affinity binding site for benzomorphan ligands, since it can be deleted without consequence to the affinity of bremazocine and cyclazocine. The data also suggest that the aminoterminal domain is not required to maintain a properly folded conformation of the MOR.

67

The affinity of the prototypical m receptor antagonist, naloxone, was decreased 3.5-fold by removal of the m receptor amino terminus ŽTable 1.. The ability of morphine, the prototypical m receptor agonist, to displace w3 Hxbremazocine binding was affected to a greater extent by deletion of the m receptor amino terminus. The apparent dissociation constant of morphine for the DN64 receptor Ž120 nM. was 6-fold higher than the K i observed for the full-length m receptor ŽFig. 3, Table 1.. The affinities of two m receptor agonists of clinical importance, methadone and fentanyl, were particularly sensitive to the loss of the m receptor amino terminus ŽFig. 3, Table 1.. The apparent dissociation constant of methadone increased 18-fold, from 3.4 to 61 nM, when the m receptor amino terminus was deleted, while the K i of fentanyl was shifted 32-fold, from 3.9 to 123 nM. 3.3. Comparison of the binding affinities of peptide ligands for the m and truncated D N64 opioid receptors Five different opioid peptide agonists were also analyzed for their ability to bind to the DN64 receptor, and the results obtained were similar to those observed with the non-peptide ligands: the magnitude of the shift in affinity was ligand-dependent. DAMGO and b-endorphin binding were the least affected by the absence of the amino-terminal domain. Their affinities for the DN64 receptor were 3- to 5-fold lower than affinities for the wild-type m receptor ŽFig. 4, Table 1.. The shift in the K i of DADL was intermediate, being 8.5-fold higher for the truncated receptor with respect to the full-length receptor

Fig. 3. Competition analysis of non-peptide ligand binding to MOR and DN64 receptors. Membrane preparations from HEK293 cells expressing full-length and truncated DN64 receptors were used to determine the ability of cyclazocine, morphine, methadone and fentanyl to displace w3 Hxbremazocine binding. Data points are averages of duplicate determinations; a representative data set from three to five independent experiments is shown. Nonlinear regression analysis of the curves yielded IC 50 values, which were converted to apparent dissociation constants Ž K i ., using the Cheng–Prusoff equation K i s IC 50r1 q CrK D , where C is the concentration and K D is the apparent dissociation constant of w3 Hxbremazocine.

68

K. ChaturÕedi et al.r Molecular Brain Research 76 (2000) 64–72

Fig. 4. Competition analysis of peptide ligand binding to MOR and DN64 receptors. The ability of DAMGO, b-endorphin, endomorphin 1 and endomorphin 2 to displace w3 Hxbremazocine binding was determined as described in Fig. 3. Data points are averages of duplicate determinations; a representative data set from three to five independent experiments is shown.

ŽTable 1.. In contrast, the affinities of two peptide ligands, endomorphin 1 and endomorphin 2, were affected dramatically by deletion of the m receptor amino terminus. The apparent dissociation constant of endomorphin 1 for the DN64 receptor, 721 nM, was 24-fold higher than that observed for the m receptor, 30 nM. The shift in affinity of endomorphin 2 was even more drastic: a 61-fold increase

in K i was observed, from 18 nM for the m receptor to 1110 nM for the truncated receptor ŽFig. 4, Table 1.. 3.4. Hydrolysis of N-glycan chains and effect on ligand-binding affinities Based on our observations that the affinities of methadone, fentanyl, endomorphin 1 and endomorphin 2

Fig. 5. Deglycosylation of FLAG-tagged MOR with N-glycosidase F and its effect on ligand binding. ŽA. Cell membranes were incubated with or without N-glycosidase F as described in Section 2. Proteins were resolved using 10% SDSrPAGE and transferred to Immobilon P SQ PVDF membranes. Receptor proteins were detected by incubation with anti-FLAG M1 monoclonal antibody, followed by rabbit anti-mouse IgG conjugated with alkaline phosphatase, and developed using CDP Star western blot chemiluminescence reagent. Treatment of the membrane preparation with N-glycosidase F caused essentially complete conversion of the receptor to a form that migrated as a much sharper band with increased electrophoretic mobility. B. Following N-glycosidase F treatment, membranes were diluted in 50 mM Tris–HCl, pH 7.5, for competition analysis as described in Fig. 3. Hydrolysis of FLAG-tagged m receptor N-glycan chains with N-glycosidase F caused a modest less than 3-fold decrease in the affinity of methadone, from K i s 6 nM to K i s 16 nM, and had little to no effect on the binding affinities of bremazocine, fentanyl, endomorphin 1 and endomorphin 2 Ždata not shown..

K. ChaturÕedi et al.r Molecular Brain Research 76 (2000) 64–72

were decreased significantly following deletion of the m receptor amino terminus, we sought to establish whether interactions of the ligands with N-glycan chains within this domain contributed to ligand affinities. To do so, membrane preparations from cells expressing the full-length FLAG-tagged m receptor were treated with the enzyme, N-glycosidase F, which hydrolyzes all types of N-glycan chains from the asparagine side chain of proteins. The efficacy of the enzyme was assessed by Western blotting ŽFig. 5A.. Analysis of protein extracts from membranes expressing the FLAG-tagged m receptor indicated that the glycosylated receptor migrated as a broad band with an apparent molecular mass of 75 kDa. Treatment of the membrane preparation with N-glycosidase F caused essentially complete conversion of the receptor to a form that migrated as a much sharper band with increased electrophoretic mobility. The apparent molecular mass of the deglycosylated receptor was approximately 39 kDa ŽFig. 5A.. Hydrolysis of FLAG-tagged m receptor N-glycan chains with N-glycosidase F caused a modest less than 3-fold decrease in the affinity of methadone, from K i s 6 to 16 nM Žcompare Fig. 3 with Fig. 5B., and had little to no effect on the binding affinities of bremazocine, fentanyl, endomorphin 1 and endomorphin 2 Ždata not shown..

4. Discussion The major outcomes and conclusions drawn from this study are as follows. Ž1. The amino terminus of the m receptor plays a key role as a determinant of the high affinity binding site for a subset of agonists, including methadone, fentanyl, endomorphin 1, and endomorphin 2. For these ligands, there was a 20- to 60-fold decrease in affinity for the truncated DN64 receptor relative to the full-length m receptor. Ž2. MOR is glycosylated when expressed in HEK 293 cells. N-glycan chains within the amino-terminal domain do not contribute significantly to the affinity of agonists for the m receptor. Ž3. Deletion of 64 amino acids from the m receptor amino terminus had an intermediate effect on the affinities of another ligand subset. The affinities of naloxone, morphine, DAMGO, DADL, and b-endorphin were 3- to 8-fold lower for the truncated receptor as compared to their affinities for the wild-type m receptor. Ž4. The affinities of the benzomorphans, bremazocine and cyclazocine, were not affected by removal of the amino-terminal domain of the m receptor. Ž5. These data provide evidence that the determinants of the high-affinity m receptor binding pocket differ from ligand to ligand. It is important to note, however, that there must also be significant overlap in the manner in which ligands engage the receptor, since every ligand examined in this study was capable of completely displacing w3 Hxbremazocine binding to both the full-length and Nterminally truncated m receptors.

69

The cloning of opioid receptor cDNAs has facilitated detailed analysis of receptor structure and function. This study has yielded new information regarding the properties and composition of the m receptor ligand binding pocket. We have confirmed earlier observations that the amino terminus of the m receptor is dispensable for high affinity binding of some ligands w39,43x; however, this is the first report indicating that the amino terminus contributes significantly to the composition of the binding pocket for methadone, fentanyl, endomorphin 1 and endomorphin 2. The MOR engages a diverse repertoire of ligands, including small, fused-ring alkaloids such as morphine, more flexible synthetic substances such as methadone and fentanyl, endomorphin tetrapeptides, enkephalin pentapeptides, and longer peptides, such as dynorphin 1–17, and b-endorphin, a 31-amino acid peptide. Given the spectrum of physicochemical properties inherent in this diverse set of ligands, it is reasonable to assume that the MOR binding pocket can engage a variety of functional groups, some of which may be unique to a particular ligand class. It should be noted that the affinities of endomorphin 1 and 2 for the native m receptor that we observed in this study Ž K i s 29.9 and 18.2 nM, respectively, Table 1. were 83-fold and 26-fold lower, respectively, than published by Zadina et al. w55x. We tested three different batches of the endomorphins, all of which had the expected amino acid compositions and were shown to be pure by reverse-phase high performance liquid chromatography, with identical results. The reasons for the discrepancies between our observations and those of Zadina and colleagues are not known at this time, but may be due to the use of different receptor preparations and radioligands. Since the m receptor amino terminus contains five possible sites for N-linked glycosylation ŽAsn-X-SerrThr., experiments were designed to test whether loss of N-glycan chains could account for the decrease in affinity of methadone, fentanyl, endomorphin 1 and endomorphin 2 for the truncated receptor. Western blot analysis confirmed that the m receptor is a glycoprotein, as suggested previously based on avid receptor binding to immobilized wheat germ agglutinin w12x. The observation that deglycosylation of the m receptor with N-glycosidase F had minimal effects on the binding of methadone, fentanyl, endomorphin 1, and endomorphin 2, implicates particular amino acid side-chains within the amino-terminal domain, or possible disulfide links involving Cys13 , Cys 22 , Cys 43 , or Cys 57 , as relevant features that interact positively with these ligands. We have reported that bremazocine binding to the m receptor is relatively insensitive to treatment with the reducing agent, dithiothreitol w39x, and we have recently observed that methadone and endomorphin binding is also unaffected under reducing conditions Žunpublished observations.. Based on these data, it is likely that particular amino acid side chains in the amino-terminal domain of the m receptor contribute to the binding pocket for methadone, fentanyl, endomorphin 1, and endomorphin 2.

70

K. ChaturÕedi et al.r Molecular Brain Research 76 (2000) 64–72

Localization of relevant amino acids could be accomplished by analysis of a series of truncated receptors, in conjunction with site-directed mutagenesis to assess the role of individual amino-terminal amino acids in the receptor binding pocket. The involvement of extracellular domains in ligand binding has been demonstrated for other members of the G-protein-coupled receptor superfamily. Human chorionic gonadotropin binds to lutropin receptors by interaction with the large extracellular amino-terminal domain w32x, and substitution of Cys 41 in the amino-terminal region of the thyrotropin receptor leads to a loss of TSH binding to its receptor w47x. Extracellular cysteines within the aminoterminal domain and in extracellular loops are also required for peptide binding to the angiotensin receptor w53x, and the CCR5 chemokine receptor w3x. The amino terminus of the interleukin-8 receptor is also an important functional determinant of the ligand-binding domain w15x. A stretch of nine amino acids adjacent to the initiator methionine in the amino-terminal domain of the human formyl peptide receptor was found to be crucial for receptor occupancy w34x. In addition, Val 4 in the amino-terminal end of the secretin receptor has been shown by photo-affinity labeling to interact with a secretin analogue w8x. This study is supportive of a model in which contacts between the amino-terminal domain of the m receptor and functional groups on methadone, fentanyl, endomorphin 1, and endomorphin 2 contribute substantially to ligand binding affinity. It is not known at present whether those contacts have similar physicochemical properties for each of the four ligands, or whether the important interactions differ among the ligands. There is no apparent unifying feature that dictates whether the affinity of a particular ligand will be affected by the truncation: no correlation exists regarding whether the ligand is an alkaloid or peptide, selective or nonselective for the m receptor, or behaves as an agonist or antagonist. We are attempting to address this issue with the use of molecular modeling, to examine potential similarities in the structures of methadone, fentanyl, endomorphin 1, and endomorphin 2, and differences of that group from ligands in which the effect of N-terminal truncation is less severe. Many studies have focused on the composition of the opioid receptor binding pocket and the basis for receptor selectivity, by characterizing the pharmacological profiles of receptor chimeras and through the use of site-directed mutagenesis w1,2,4,7,11,16,19,20,25,28,29,31,33,38,39, 43,44,46,49,50,52x. It is clear that charged amino acids located within transmembrane domains of the m receptor are critical for ligand recognition. Substitution of Asp114 in transmembrane domain 2 decreases the affinity of agonists, while antagonist binding is unaffected w4,43x. The aspartic acid in transmembrane domain 3 ŽAsp147 . is also a constituent of the m receptor agonist binding site w1,43x. Substitution of His 297 in transmembrane domain 6 of the m receptor with Ala led to a disruption in the binding site for

all ligands tested w25,43x. In contrast, Bot et al. w4x found that the H297N mutant m receptor recognized most ligands Žwith the exception of buprenorphine. with wild-type affinities. Another His residue ŽHis 223 . in the second extracellular loop of the m receptor has been shown to be critical for high affinity binding w39x. In addition, substituting Tyr 326 in transmembrane domain 7 with Phe caused in decrease in the affinity of most ligands tested w25x. The affinity of fentanyl was particularly sensitive to the Y326F substitution, with a greater than 50-fold reduction. The present study, implicating the amino-terminal domain of the m receptor as an important constituent of the binding site for fentanyl, methadone, endomorphin 1 and endomorphin 2, adds to the current understanding of the m receptor binding pocket. These data support the concept that the determinants of the high-affinity m receptor binding pocket differ from ligand to ligand, however, some of the determinants are also conserved among ligands. A similar conclusion has been made regarding the d opioid receptor binding site, based on observations that each ligand interacted with the d receptor using a distinct combination of contacts within the receptor binding domain w2x. In contrast, it has been reported that selective agonists and antagonists bind to physically separable sites on the k opioid receptor w19x. The antagonist binding site was localized to the amino terminus, and the agonist site was localized to more distal domains. The emerging picture appears to be that there is not a single receptor binding site, but rather, that the receptor is capable of considerable plasticity regarding engagement of ligands and subsequent events leading to receptor activation.

Acknowledgements The authors thank Drs. Lei Yu and Mark von Zastrow for opioid receptor cDNAs. Technical assistance by Mr. Norihiro Chinen is gratefully acknowledged. This research was supported by grant DA 09113 from the National Institute on Drug Abuse Žto R.D.H...

References w1x K. Befort, L. Tabbara, S. Bausch, C. Chavkin, C. Evans, B. Kieffer, The conserved aspartate residue in the third putative transmembrane domain of the d-opioid receptor is not the anionic counterpart for cationic opiate binding but is a constituent of the receptor binding site, Mol. Pharmacol. 49 Ž1996. 216–223. w2x K. Befort, L. Tabbara, D. Kling, B. Maigret, B. Kieffer, Role of aromatic transmembrane residues of the d-opioid receptor in ligand recognition, J. Biol. Chem. 271 Ž1996. 10161–10168. w3x C. Blanpain, B. Lee, J. Vakili, B.J. Doranz, C. Govaerts, I. Migeotte, M. Sharron, V. Dupriez, G. Vassart, R.W. Doms, M. Parmentier, Extracellular cysteines of CCR5 are required for chemokine binding, but dispensable for HIV-1 coreceptor activity, J. Biol. Chem. 274 Ž1999. 18902–18908.

K. ChaturÕedi et al.r Molecular Brain Research 76 (2000) 64–72 w4x G. Bot, A.D. Blake, S. Li, T. Reisine, Mutagenesis of a single amino acid in the rat m-opioid receptor discriminates ligand binding, J. Neurochem. 70 Ž1998. 358–365. w5x B.D. Carter, F. Medzihradsky, Go mediates the coupling of the m opioid receptor to adenylyl cyclase in cloned neural cells and brains, Proc. Natl. Acad. Sci. U. S. A. 90 Ž1993. 4062–4066. w6x Y. Chen, A. Mestek, J. Liu, J.A. Hurley, L. Yu, Molecular cloning and functional expression of a m-opioid receptor from rat brain, Mol. Pharmacol. 44 Ž1993. 8–12. w7x P.A. Claude, D.R. Wotta, X.H. Zhang, P.L. Prather, T.M. McGinn, L.J. Erickson, H.H. Loh, P.Y. Law, Mutation of a conserved serine in TM4 of opioid receptors confers full agonistic properties to classical antagonists, Proc. Natl. Acad. Sci. U. S. A. 93 Ž1996. 5715–5719. w8x M. Dong, Y. Wang, E.M. Hadac, D.L. Pinon, E. Holicky, L.J. Miller, Identification of an interaction between residue 6 of the natural peptide ligand and a distinct residue within the amino-terminal tail of the secretin receptor, J. Biol. Chem. 274 Ž1999. 19161– 19167. w9x C.J. Evans, D.E. Keith Jr., H. Morrison, K. Magendzo, R.H. Edwards, Cloning of a delta opioid receptor by functional expression, Science 258 Ž1992. 1952–1955. w10x K. Fukuda, S. Kato, K. Mori, M. Nishi, H. Takeshima, Primary structures and expression from a cDNA of rat opioid receptor deltaand mu-subtypes, FEBS Lett. 327 Ž1993. 311–314. w11x K. Fukuda, K. Terasako, S. Kato, K. Mori, Identification of the amino acid residues involved in selective agonist binding in the first extracellular loop of the d- and m-opioid receptors, FEBS Lett. 373 Ž1995. 177–181. w12x T. Gioannini, B. Foucaud, J.M. Hiller, M.E. Hatten, E.J. Simon, Lectin binding of solubilized opiate receptors: evidence for their glycoprotein nature, Biochem. Biophys. Res. Commun. 105 Ž1982. 1128–1134. w13x A. Goldstein, A. Naidu, Multiple opioid receptors: ligand selectivity profiles and binding site signatures, Mol. Pharmacol. 36 Ž1989. 265–272. w14x E. Hartmann, T.A. Rapoport, H.F. Lodish, Predicting the orientation of eukaryotic membrane-spanning proteins, Proc. Natl. Acad. Sci. U. S. A. 86 Ž1989. 5786–5790. w15x C.A. Hebert, A. Chuntharapai, M. Smith, T. Colby, J. Kim, R. Horuk, Partial functional mapping of the human interleukin-8 type A receptor, J. Biol. Chem. 268 Ž1993. 18549–18553. w16x S.A. Hjorth, K. Thirstrup, D.K. Grandy, T.W. Schwartz, Analysis of selective binding epitopes for the k-opioid receptor antagonist norbinaltorphimine, Mol. Pharmacol. 47 Ž1995. 1089–1094. w17x R.D. Howells, T. Gioannini, J.M. Hiller, E.J. Simon, Solubilization and characterization of active opiate binding sites from mammalian brain, J. Pharmacol. Exp. Ther. 222 Ž1982. 629–634. w18x B.L. Kieffer, K. Befort, C. Gaveriaux-Ruff, C.G. Hirth, The d opioid receptor: isolation of a cDNA by expression cloning and pharmacological characterization, Proc. Natl. Acad. Sci. U. S. A. 89 Ž1992. 12048–12052. w19x H. Kong, K. Raynor, H. Yano, J. Takeda, G.I. Bell, T. Reisine, Agonists and antagonists bind to different domains of the cloned k opioid receptor, Proc. Natl. Acad. Sci. U. S. A. 91 Ž1994. 8042–8046. w20x H. Kong, K. Raynor, K. Yasuda, S.T. Moe, P.S. Portoghese, G.I. Bell, T. Reisine, A single residue, aspartic acid 95, in the d opioid receptor specifies selective high affinity agonist binding, J. Biol. Chem. 268 Ž1993. 23055–23058. w21x R. Kornfeld, S. Kornfeld, Assembly of asparagine-linked oligosaccharides, Annu. Rev. Biochem. 54 Ž1985. 631–664. w22x K.-I. Laugwitz, S. Offermanns, K. Spicher, G. Schultz, Mu and delta opioid receptors differentially couple to G protein subtypes in membranes of human neuroblastoma SH-SY5Y cells, Neuron 10 Ž1993. 233–242. w23x P.-Y. Law, H.H. Loh, Regulation of opioid receptor activities, J. Pharmacol. Exp. Ther. 289 Ž1999. 607–624.

71

w24x H.H. Loh, H.C. Liu, A. Cavalli, W. Yang, Y.F. Chen, L.N. Wei, m Opioid receptor knockout in mice: effects on ligand-induced analgesia and morphine lethality, Mol. Brain Res. 54 Ž1998. 321–326. w25x A. Mansour, L.P. Taylor, J.L. Fine, R.C. Thompson, M.T. Hoversten, H.I. Mosberg, S.J. Watson, H. Akil, Key residues defining the m-opioid receptor binding pocket: a site-directed mutagenesis study, J. Neurochem. 68 Ž1997. 344–353. w26x A. Mansour, C.A. Fox, H. Akil, S.J. Watson, Opioid receptor mRNA expression in the rat CNS: anatomical and functional implications, Trends Neurosci. 18 Ž1995. 22–29. w27x H.W.D. Matthes, R. Maldonado, F. Simonin, O. Valverde, S. Slowe, I. Kitchen, K. Befort, A. Dierich, M. Le Meur, P. Dolle, E. Tzavara, J. Hanoune, B.P. Roques, B.L. Kieffer, Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the m-opioid-receptor gene, Nature 383 Ž1996. 819–823. w28x F. Meng, M.T. Hoversten, R.C. Thompson, L. Taylor, S.J. Watson, H. Akil, A chimeric study of the molecular basis of affinity and selectivity of the k and the d opioid receptors, J. Biol. Chem. 270 Ž1995. 12730–12736. w29x F. Meng, Y. Ueda, M.T. Hoversten, R.C. Thompson, L. Taylor, S.J. Watson, H. Akil, Mapping the receptor domains critical for the binding selectivity of d-opioid receptor ligands, Eur. J. Pharmacol. 311 Ž1996. 285–292. w30x F. Meng, G.-X. Xie, R.C. Thompson, A. Mansour, A. Goldstein, S.J. Watson, H. Akil, Cloning and pharmacological characterization of a rat kappa opioid receptor, Proc. Natl. Acad. Sci. U. S. A. 90 Ž1993. 9954–9958. w31x M. Minami, T. Nakagawa, T. Seki, T. Onogi, Y. Aoki, Y. Katao, S. Katsumata, M. Satoh, A single residue, Lys108, of the d-opioid receptor prevents the m-opioid-selective ligand wD-Ala2 , N-MePhe 4 , Gly-ol 5 xenkephalin from binding to the d-opioid receptor, Mol. Pharmacol. 50 Ž1996. 1413–1422. w32x W.R. Moyle, R.K. Campbell, S.N. Rao, N.G. Ayad, M.P. Bernard, Y. Han, Y. Wang, Model of human chorionic gonadotropin and lutropin receptor interaction that explains signal transduction of the glycoprotein hormones, J. Biol. Chem. 270 Ž1995. 20020–20031. w33x M.-C. Pepin, S.Y. Yue, E. Roberts, C. Wahlestedt, P. Walker, Novel ‘‘restoration of function’’ mutagenesis strategy to identify amino acids of the d-opioid receptor involved in ligand binding, J. Biol. Chem. 272 Ž1997. 9260–9267. w34x H.D. Perez, L. Vilander, W.H. Andrews, R. Holmes, Human formyl peptide receptor ligand binding domainŽs., J. Biol. Chem. 269 Ž1994. 22485–22487. w35x E.T. Piros, T.G. Hales, C.J. Evans, Functional analysis of cloned opioid receptors in transfected cell lines, Neurochem. Res. 21 Ž1996. 1277–1285. w36x P.L. Prather, T.M. McGinn, L.J. Erickson, C.J. Evans, H.H. Loh, P.Y. Law, Ability of d-opioid receptors to interact with multiple G-proteins is independent of receptor density, J. Biol. Chem. 269 Ž1994. 21293–21302. w37x K. Raynor, H. Kong, Y. Chen, K. Yasuda, L. Yu, G.I. Bell, T. Reisine, Pharmacological characterization of the cloned k-, d-, and m-opioid receptors, Mol. Pharmacol. 45 Ž1994. 330–334. w38x M. Shahrestanifar, R.D. Howells, Sensitivity of opioid receptor binding to N-substituted maleimides and methanethiosulfonate derivatives, Neurochem. Res. 21 Ž1996. 1295–1299. w39x M. Shahrestanifar, W.W. Wang, R.D. Howells, Studies on inhibition of m and d opioid receptor binding by dithiothreitol and N-ethylmaleimide, J. Biol. Chem. 271 Ž1996. 5505–5512. w40x V.K. Singh, K. Bajpai, S. Biswas, W. Haq, M.Y. Khan, K.B. Mathur, Molecular biology of opioid receptors, Neuroimmunomodulation 4 Ž1997. 285–297. w41x I. Sora, N. Takahashi, M. Funada, H. Ujike, R.S. Revay, D.M. Donovan, L.L. Miner, G.R. Uhl, Opiate receptor knockout mice define m receptor roles in endogenous nociceptive responses and morphine-induced analgesia, Proc. Natl. Acad. Sci. U. S. A. 94 Ž1997. 1544–1549.

72

K. ChaturÕedi et al.r Molecular Brain Research 76 (2000) 64–72

w42x C.D. Strader, T.M. Fong, M.R. Tota, D. Underwood, R.A.F. Dixon, Structure and function of G-protein-coupled receptors, Annu. Rev. Biochem. 63 Ž1994. 101–132. w43x C.K. Surratt, P.S. Johnson, A. Moriwaki, B.K. Seidleck, C.J. Blaschak, J.B. Wang, G.R. Uhl, m-Opioid receptor: charged transmembrane domain amino acids are critical for agonist recognition and intrinsic activity, J. Biol. Chem. 269 Ž1994. 20548–20553. w44x K. Thirstrup, C.E. Elling, S.A. Hjorth, T.W. Schwartz, Construction of a high affinity switch in the k-opioid receptor, J. Biol. Chem. 271 Ž1996. 7875–7878. w45x R.C. Thompson, A. Mansour, H. Akil, S.J. Watson, Cloning and pharmacological characterization of a rat mu opioid receptor, Neuron 11 Ž1993. 903–913. w46x M. Valiquette, H.K. Vu, S.Y. Yue, C. Wahlestedt, P. Walker, Involvement of Trp-284, Val-296, and Val-297 of the human dopioid receptor in binding of d-selective ligands, J. Biol. Chem. 271 Ž1996. 18789–18796. w47x H.L. Wadsworth, D. Russo, Y. Nagayama, G.D. Chazenbalk, B. Rapoport, Studies on the role of amino acids 38–45 in the expression of a functional thyrotropin receptor, Mol. Endocrinol. 6 Ž1992. 394–398. w48x J.B. Wang, Y. Imai, C.M. Eppler, P. Gregor, C.E. Spivak, G.R. Uhl, m Opiate receptor: cDNA cloning and expression, Proc. Natl. Acad. Sci. U. S. A. 90 Ž1993. 10230–10234. w49x J.B. Wang, P.S. Johnson, J.M. Wu, W.F. Wang, G.R. Uhl, Human k

w50x

w51x w52x

w53x

w54x

w55x

opiate receptor second extracellular loop elevates dynorphin’s affinity for human m rk chimeras, J. Biol. Chem. 269 Ž1994. 25966– 25969. W.W. Wang, M. Shahrestanifar, J. Jin, R.D. Howells, Studies on m and d opioid receptor selectivity utilizing chimeric and site-mutagenized receptors, Proc. Natl. Acad. Sci. U. S. A. 92 Ž1995. 12436– 12440. J. Wess, Molecular basis of receptorrG-protein-coupling selectivity, Pharmacol. Ther. 80 Ž1998. 231–264. J.-C. Xue, C. Chen, J. Zhu, S.P. Kunapuli, J.K. de Riel, L. Yu, L.-Y. Liu-Chen, The third extracellular loop of the m opioid receptor is important for agonist selectivity, J. Biol. Chem. 270 Ž1995. 12977– 12979. Y. Yamano, K. Ohyama, S. Chaki, D.-F. Guo, T. Inagami, Identification of amino acid residues of rat angiotensin II receptor for ligand binding by site directed mutagenesis, Biochem. Biophys. Res. Commun. 187 Ž1992. 1426–1431. K. Yasuda, K. Raynor, H. Kong, C.D. Breder, J. Takeda, T. Reisine, G.I. Bell, Cloning and functional comparison of k and d opioid receptors from mouse brain, Proc. Natl. Acad. Sci. U. S. A. 90 Ž1993. 6736–6740. J.E. Zadina, L. Hackler, L.-J. Ge, A.J. Kastin, A potent and selective endogenous agonist for the m-opiate receptor, Nature 386 Ž1997. 499–502.