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CELLULAR AND MOLECULAR

Ligand-Selective Activation of µ-Opioid Receptor: Demonstrated with Deletion and Single Amino Acid Mutations of Third Intracellular Loop Domain

Department of Pharmacology, Medical School, University of Minnesota, Minneapolis, Minnesota

Received for publication December 5, 2002
Accepted March 6, 2003.

	Abstract

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The mechanism for the differential regulation of the µ-opioid receptor by agonists is investigated by identifying the receptor domains used to define the relative efficacies of three µ-opioid receptor-selective agonists: [D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin (DAMGO), morphine, and [N-MePhe³,D-Pro⁴]-morphiceptin (PL017) to inhibit forskolin-stimulated intracellular cAMP production in human embryonic kidney 293 cells. This was accomplished by systematically deleting four to five amino acids clusters within the third intracellular loop of rat µ-opioid receptor, Arg²⁵⁸ to Arg²⁸⁰, followed by Ala substitution and scanning studies of the ²⁷⁶RRITR²⁸⁰ sequence, the putative G protein-coupling motif. Deletion of the four to five amino acid clusters resulted in differential effects on the affinities of the agonists and antagonists, and also on the potencies and coupling efficiencies of the three opioid agonists. Ala scanning studies of the ²⁷⁶RRITR²⁸⁰ sequence revealed also the differences between [D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin (DAMGO), morphine, and PL017. Substitution of Arg²⁷⁶ or Ile²⁷⁸ with Ala reduced the potency of DAMGO but not that of morphine PL017. Meanwhile, mutation of Thr²⁷⁹ to Ala increased the potencies of morphine and PL017 but not that of DAMGO. The I278A mutation decreased the DAMGO coupling efficiency but increased the PL017 coupling efficiency. The R280A mutation resulted in the increase in PL017 potency and coupling efficiency without altering those of DAMGO and morphine. Thus, these mutation studies suggested that the activation of µ-opioid receptor and interaction between the critical domains such as RRITR within third intracellular loop and the G proteins are agonist-selective.

Several studies have investigated the domains of the opioid receptors that are involved in G protein interaction and activation. The receptor chimera studies with -opioid receptor and SSTR1 somatostatin receptor (Reisine et al., 1993) have defined the direct involvement of third intracellular loop in the receptor regulation of adenylyl cyclase activity. The use of peptides corresponding to the distal and proximal portions of third intracellular loop inhibited the agonist high-affinity binding and the agonist-induced GTPase activity or agonist-induced guanosine 5'-O-(3-thio)triphosphate binding to the membrane (Merkouris et al., 1996; Georgoussi et al., 1997). Mutation of the BBXXB motif within the third intracellular loop suggested that Arg²⁸⁰ participated in the DAMGO-induced µ-opioid receptor-mediated inhibition of the adenylyl cyclase activity (Wang et al., 1999). Other domains of the opioid receptors also seemed to be involved in the receptor G protein coupling. Truncation of the carboxyl tail of the µ-opioid receptor abolished the ability of DAMGO but not morphine ability to inhibit the adenylyl cyclase activity (Surratt et al., 1994). Similar truncation of the carboxyl tail of the -opioid receptor resulted in the attenuation of [D-Pen²,D-Pen⁵]-enkephalin-mediated activation of the phospholipase C (Hirst et al., 1998). Mutation of the Asp¹²⁸ and Tyr¹²⁹ within the third TM domain or the Tyr³⁰⁸ within the seventh TM domain resulted in the constitutive activation of the -opioid receptor (Befort et al., 1999; Cavalli et al., 1999). Because these residues could contribute to an intramolecular interaction that stabilizes the -opioid receptor in its inactive form, these mutations would allow the movement within the third TM domain and suggested the involvement of second intracellular loop of the opioid receptor in the G protein activation.

There is evidence that suggests agonist selectivity in the opioid receptor activation of the G proteins. Truncation of the µ- or -opioid receptors did not affect the nonpeptidic agonists inhibition of the adenylyl cyclase activity but attenuated the peptidic agonists activities (Surratt et al., 1994; Hirst et al., 1998). Such differences could be attributed to the differences in the domains involved in the receptor binding of these two groups of agonists (for review, see Law et al., 1999). The divergence in the agonist-receptor conformations also have been implicated in the observations that DAMGO but not morphine could induce rapid phosphorylation and internalization of the µ-opioid receptor (Arden et al., 1995; Zhang et al., 1996) and that cAMP-dependent protein kinase could phosphorylate in vitro the µ-opioid receptor in the presence of morphine but not DAMGO (Chakrabarti et al., 1998). These studies and others suggested the receptor domains involved in G protein activation could be agonist-dependent. Hence, in current studies, the µ-opioid receptor selective agonists DAMGO, morphine, and [N-MePhe³,D-Pro⁴]-morphiceptin (PL017) were used to activate the receptor. The differences in domains involved in the receptor activation by these agonists were examined with the deletion and single amino acid mutation of the third intracellular loop of the µ-opioid receptor.

	Materials and Methods

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Deletion and Mutation of the Third Intracellular Loop of µ-Opioid Receptor. The µ-opioid receptor was mutated by using QuickChangesite-directed mutagenesis kit (Stratagene, La Jolla, CA). The oligodeoxynucleotide primers containing at least 15 nucleotides of both 5' and 3' sequence flanking the deletion motif or the single nucleotide mutation were synthesized with the Expedite oligo synthesizer (Millipore Corporation, Bedford, MA) and were extended during temperature cycling by Pfu DNA polymerase to generate respective mutants. Each 50 µl of PCR reaction contained (final concentration) 40 ng of pCDNA3 plasmid with µ-opioid receptor, 44 nM of each primer, 2.5 mM of each deoxynucleotide, and 2.5 U of Pfu DNA polymerase. PCR conditions were as follows: denatured at 95°C for 30 s for one cycle, followed by denaturing at 95°C for 30 s, annealing at 58°C for 1 min, and extension at 68°C for 14 min for 18 cycles. The PCR products were treated with 10 U of DpnI at 37°C for 1 h to remove the template. The mutated plasmids were transformed into XL-1Blue. The presence of the mutation was identified with nucleotide sequencing using dideoxynucleotide termination method.

Establishing Human Embryonic Kidney (HEK) 293 Cells Stably Expressing the Mutant or Wild-Type µ-Opioid Receptor. HEK293 cells cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 µg/ml streptomycin, and 100 IU/ml penicillin in 10% CO₂ atmosphere at 37°C were transfected with 15 µg of the plasmids containing either mutant or wild type µ-opioid receptor using the calcium phosphate precipitation method as described previously (Chen and Okayama, 1987). The cell colonies formed after 10 to 14 days of geneticin (1 mg/ml) selection were isolated and cultured further in 17-mm culture dishes with reduced geneticin concentration (0.2 mg/ml). Clones expressing the mutant or wild type µ-opioid receptor were detected by whole cell binding using 1 nM [³H]diprenorphine in 25 mM HEPES buffer, pH 7.6, and 5 mM MgCl₂. Nonspecific binding was defined with 10 µM naloxone. After incubating at room temperature for 90 min., the binding reactions were terminated by collecting the cells onto a GF/B filter paper, and the filters were washed three times with 5 ml of 25 mM HEPES buffer, pH 7.6, at 4°C. The positive clones identified were maintained in standard growth medium supplemented with 0.2 mg/ml geneticin.

Saturation and Competition Binding Assays. Membranes from the HEK293 cells expressing the µ-opioid receptor were prepared as described previously (Law et al., 2000). Saturation or competition binding assays were carried out with 50 to 100 µg of these membranes, depending on the level of receptor expression. Saturation binding assays were carried with various concentrations of [³H]diprenorphine (0.1–10 nM) in the absence or presence of 10 µM naloxone, 10 mM MgCl₂, in 25 mM HEPES buffer, pH 7.6, for 90 min at room temperature. The membranes were collected on GF/B filters, washed, and radioactivity determined with scintillation counting using a Beckman 5000 scintillation counter. The dissociation constants, K_D, of [³H]diprenorphine were calculated using GraphPad Prism software (GraphPad Software, Inc., San Diego, CA). Competition binding assays were performed by incubating 75 to 150 µg of membrane protein with 1.0 nM [³H]diprenorphine with or without nonradioactive ligands [0.1 nM–10 µM DAMGO, morphine, PL017, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH₂ (CTOP), or naloxone], and 10 mM MgCl₂ in 25 mM HEPES buffer at pH 7.6. The K_i values of unlabeled ligand were determined by using the GraphPad Prism software. The one-site or two-site curve-fitting models were used to analyze the optimal fit of the competition binding data. Protein concentration was determined by the Lowry method.

Measurement of Intracellular cAMP Level. Intracellular cAMP levels in the HEK293 cells expressing either wild type or mutant µ-opioid receptor were measured by the radioimmunoassay as described previously (Maestri-El Kouhen et al., 2000). Inhibition of forskolin-stimulated adenylyl cyclase activity was carried out in the presence of various concentrations of agonists, DAMGO, morphine, or PL017. Briefly, cells were cultured in 96-well plates to 80 to 100% confluence. On the day of experiments, growth media were replaced with 0.5 ml of incubation buffer consisting of 0.5 mM isobutylmethylxanthine, 10 µM forskolin, and various concentrations of agonists (0.001 nM–10 µM) in Krebs-Ringer-HEPES buffer (100 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 25 mM glucose, 55 mM sucrose, and 10 mM HEPES at pH 7.4). The cells were incubated at 37°C for 15 min, and the reactions were terminated by the addition of 75 µl of 2.5 N perchloric acid. After incubating the cells in perchloric acid for 30 min at 4°C to allow complete cell lysis, the acid was neutralized by the addition of 150 µl of the mixture of 2 M KOH, 1 M Tris, and 60 mM EDTA. The supernatant was collected for cAMP measurement after 1 h at 4°C. One hundred microliters of diluted samples or standard cAMP solution (10–300 pg/µl) was added with 2 µl of acetylation mixture [2:1 (v/v), triethanolamine/acetaldehyde]. Then 100 µl of antigen buffer (0.05 M sodium acetate, pH 6.2, 0.06 M MgCl₂, and 4 nM of ¹²⁵I-cAMP-TME) and 100 µl of antibody buffer (rabbit anti-cAMP polyclonal antibodies diluted 1:100 in 0.9% bovine serum albumin in 66 mM sodium acetate, pH 6.2, and 0.5% -globulin) was added in the assay mixture. The incubation was carried out at 4°C for at least 12 h, and the cAMP antigen-antibody complexes were precipitated by the addition of 2 ml of 95% ethanol at 4°C. After incubating for 30 min at 4°C, the precipitated complexes were pelleted by centrifuging at 15,000g for 10 min. The amount of radioactivity was determined with a Beckman gamma 5500 counter, and the concentration of cAMP produced in each sample was calculated from the standard cAMP curve generated for each experiment. The EC₅₀ values of the agonists were obtained by curve fitting of the dose-response curves using the GraphPad Prism software program.

Fluorescence-Activated Cell Sorting Analysis (FACS). HEK293 cells stably expressing wild-type or mutant µ-opioid receptor were seeded in six-well plates. The following day, the cells were treated with either 1 µM DAMGO or etorphine for 24 h. Afterwards, the cells were washed with 1 to 2 ml of MEM and incubated with 1:500 monoclonal anti-HA antibody (Babco, Richmond, CA) in MEM media for 1 h at 4°C. The cells were washed with MEM to remove nonbound primary antibody. Then 1:400 fluorophore-labeled goat anti-mouse IgG (Alexa 488; Molecular Probe, Eugene, OR) was added to each well in the volume of 0.5 ml of MEM and incubated at 4°C in the dark for 2 h. Afterwards, the cells were washed with 1 ml of MEM, harvested, and fixed with 3.7% formaldehyde in phosphate-buffered saline at pH 7.4. The fixed cells were suspended in 1 ml of phosphate-buffered saline-EDTA solution for FACS analysis (FA-Calibur, Beckman Coulter, Inc., Fullerton, CA)

Materials. Pfu polymerase for the mutagenesis was supplied by Stratagene. Restriction endonucleases were obtained from Roche Diagnostics (Indianapolis, IN). Expression vector pCDNA3 were purchased from Invitrogen (San Diego, CA). QiaPrep 500 was purchased from QIAGEN (Valencia, CA). Cell culture reagents, minimum essential medium, fetal calf serum, and G418 were supplied from Invitrogen (Carlsbad, CA). Sequenase Version 2.0 DNA sequencing kit and [³H]diprenorphine were purchased from Amersham Biosciences Inc. (Piscat-away, NJ) and ¹²⁵I-labeled acetylated cAMP was purchased from Linco Research (St. Charles, MO). Polyclonal antibodies for acetylated cAMP were generated in rabbits as described previously (Law et al., 2000). Mouse monoclonal anti-HA antibody was purchased from Babco. Alexa 488 goat anti-mouse IgG was purchased from Molecular Probes. DAMGO, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH₂ (CTOP), etorphine morphine, PL017, and naloxone were supplied by the National Institute on Drug Abuse. Other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

	Results

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Sequential Deletion of Third Intracellular Loop Amino Acids on Antagonist Binding. To determine which region within the third intracellular loop of the rat µ-opioid receptor is involved in the G protein coupling and activation, a series of mutants with the consecutive deletion of the amino acids in the third intracellular loop were generated as shown in Fig. 1. The expression of receptor mutants in HEK293 cells was determined using intact cell binding assay. Four mutants (i3-2, i3-3, i3-4, i3-5) were expressed in HEK293 cells at levels 1.7 ± 0.7 to 11 ± 0.39 pmol/mg of protein (Table 1). These levels of receptor expression compared favorably with that of the wild-type receptor (9.4 ± 0.77 pmol/mg of protein). In contrast, the i3-1 deletion mutant receptor did not express in HEK293 cells at a high level. It was determined in a separate study that the deletion of the amino acids in this i3-1 mutant resulted in the intracellular retention of the mutant receptor (V. Chipatikul, L. J. Erickson-Hebrandson, H. H. Loh, and P. Y. Law, unpublished observation). Treatment of the HEK293 cells with the opioid antagonist naloxone or agonist etorphine for 48 h led to the cell surface expression of the i3-1 mutant receptor. Hence in subsequent studies with the i3-1 mutant, HEK293 cells were treated with 10 µM naloxone for 48 h, and the properties of the receptor were determined after the removal of naloxone by repeated washings. When saturation binding studies were carried out with [³H]diprenorphine, the i3-2 and i3-3 deletion mutants possessed K_D values similar to that of wild-type receptor, whereas the rest of the deletion mutants exhibited <2-fold but significant decrease in the [³H]diprenorphine affinity (Table 1). Although the i3-1 deletion mutant also exhibited similar decrease in the [³H]diprenorphine affinity, whether the decrease was due to residual unwashed naloxone used to rescue the cell surface expression of this mutant or the deletion itself cannot be distinguished.

View larger version (41K): [in this window] [in a new window]   Fig. 1. Amino acid sequence of the wild-type µ-opioid receptor and receptor mutants with deletion or alanine substitution of the third intracellular loop. Figure shows the one-letter amino acid representation for the wild type (WT) receptor, and mutants with the deleted or mutated amino acids show as dashes or italic letters, respectively. The TMs and amino acid positions are shown above the sequence.

Competition binding studies were carried out with these receptor mutants to assess the possible role of the third intracellular loop of µ-opioid receptor in G protein coupling. In the extended ternary complex or cubic ternary complex model, agonist has high-affinity for the receptor/G protein complex, or the high-affinity states (R_H) of the receptor (Kenakin, 2002). Uncoupling of receptor from G proteins resulted in the formation of the low-affinity binding states, R_L. Unlike agonist, antagonists bind to the G protein-coupled and -uncoupled forms with similar affinities. If these third intracellular deletion mutants did not form complexes with G proteins, a drastic reduction of R_H will be observed for agonists, whereas the antagonist binding should be affected minimally. When competition binding studies were carried out with the antagonists CTOP and naloxone, <3-fold reduction in affinities were observed (Table 2). There were differential alterations in the affinities between these two antagonists. The i3-4 and i3-5 deletion mutants significantly increased the CTOP affinities, whereas the i3-5 deletion significantly reduced the naloxone affinity. In contrast, i3-2 mutation reduced the naloxone affinity by 1.6-fold and did not affect the CTOP affinity. The i3-3 deletion increased the naloxone affinity but not that of CTOP (Table 2). These small but significant differential changes in the affinities of CTOP and naloxone suggested that deletion of four to five amino acids in the third intracellular loop affected the binding pockets of the µ-opioid receptor.

Effects of Third Intracellular Loop Deletion on Agonist-Induced Receptor/G Protein Interaction. When competition binding studies were carried out with the µ-opioid receptor agonists DAMGO, morphine, and PL017, multiple affinity-state binding characteristics were observed. Figure 2A summarized the binding of DAMGO to membranes isolated from HEK293 cells expressing the wild-type and deletion mutants. The binding curves reflected the differences in the percentages of these µ-opioid receptors existed in the R_H. Significant changes in the K_H values for DAMGO compared with that of the wild-type receptor were not observed with the i3-2 and i3-3 mutants, but were observed with the i3-4 mutant (Table 2). A decrease in the fraction of receptor population in R_H was observed with the i3-2 and i3-4 deletion mutants. If the R_H represented the receptor/G protein complexes, the reduction in the percentage of receptor in R_H suggested the involvement of i3-2 and i3-4 domains in the DAMGO-induced receptor-G protein interaction.

View larger version (21K): [in this window] [in a new window]   Fig. 2. DAMGO potency and receptor affinity for the wild-type and deletion mutants of the µ-opioid receptor. A, DAMGO affinity for the wild-type and mutant receptors were determined with the competition binding assays, as described under Materials and Methods. The amino acids deleted in the i3-2 to i3-4 mutants are summarized in Fig. 1. The values represent mean ± S.E.M. of two to four separate competition experiments in duplicate. B, ability of DAMGO to inhibit 10 µM forskolin-stimulated intracellular cAMP production in HEK293 cells was determined as described under Materials and Methods. Cells were treated with forskolin in the presence or absence of different concentrations of agonist, 10-11 to 10-5 M. The values represent mean ± S.E.M. of three to seven separate experiments performed in triplicate.

In comparison, domains i3-1 and i3-5 were determined to be critical for µ-opioid receptor/G protein interaction. Deletion of these domains resulted in mutant receptors that did not exhibit high-affinity binding for DAMGO (Table 2). Furthermore, the DAMGO low-affinity binding constants, K_L, in these two deletion mutants were significantly greater than that of the wild-type receptor, suggesting probable differences in the low-affinity binding sites of these mutants. The importance of these domains in G protein coupling could be demonstrated further by the substitution of the amino acids within the domains with alanine residues. When the RRITR sequence of the i3-5 domain was substituted with Ala, the 5A mutant, inability to form the high-affinity states for DAMGO binding was observed (Table 2). Interestingly, the low-affinity states for DAMGO binding was similar to that of wild-type receptor, and significantly different from that of the i3-5 mutant.

The absolute requirement of specific domains within the third intracellular loop for receptor/G protein interaction could be demonstrated further by agonist-induced down-regulation of the receptor. Chakrabarti et al. (1997) demonstrated the absolute requirement of receptor/G protein interaction for agonist-induced down-regulation of the µ-opioid receptor. Hence, HEK293 cells expressing the wild-type or the third intracellular loop deletion mutants were treated with either 1 µM DAMGO or etorphine for 48 h, and the cell surface receptor levels were determined with FACS analyses. Chronic agonist treatment of HEK293 cells expressing with i3-1 deletion mutant was not carried out because the removal of naloxone that was needed for cell surface expression of this receptor resulted in a rapid disappearance of the receptor (V. Chipatikul, L. J. Erickson-Hebrandson, H. H. Loh, and P. Y. Law, unpublished observation). When the HEK293 cells expressing the wild-type or the deletion mutants (i3-2, i3-3, i3-4) were exposed to 1 µM DAMGO for 48 h, comparable magnitude of reduction in cell surface receptor was observed (Fig. 3). After exposure to DAMGO for 48 h, 39 ± 6% of wild-type receptor remained on the cell surface, whereas 53 ± 9, 40 ± 9, and 37 ± 9% of receptor were determined on the cell surface of HEK293-expressing i3-2, i3-3, and i3-4 mutants, respectively. However, when the HEK293 cells expressing the i3-5 deletion mutant were treated with DAMGO similarly, the level of cell surface receptor was not altered by the agonist treatment (Fig. 3). Identical results were observed when the HEK293 cells expressing the wild-type and deletion mutant receptors were treated with 1 µM etorphine. Both DAMGO and etorphine were unable to down-regulate the 5A mutant receptor expressed in HEK293 cells also (data not shown). Thus, the i3-5 domain containing the RRITR sequence is absolutely needed for the µ-opioid receptor to interact with G protein.

View larger version (21K): [in this window] [in a new window]   Fig. 3. Inability of DAMGO to induce receptor down-regulation in HEK293 cells expressing the i3-5 receptor mutant. HEK293 cells expressing wild-type receptor (WT) or receptor mutants with various amino acids deleted in third intracellular loops, i3-2 to i3-5, were treated with 1 µM DAMGO for 48 h. Cell surface receptor numbers were measured by FACS analyses, as described under Materials and Methods. The amount of cell fluorescence in HEK293 cells after agonist treatment was compared with that in cells not treated. The columns represent the average and S.E.M. of two experiments carried out in triplicate.

When competition binding studies were carried out with morphine and PL017, similar to DAMGO results, µ-opioid receptor lacking the RRITR sequence did not exhibit high-affinity binding for these two agonists (Table 2). The deletion of the i3-2 domain within the third intracellular loop of µ-opioid receptor reduced the morphine high-affinity binding by 2-fold without any effect on PLO17 affinity. However, in contrast to the DAMGO competition binding studies, deletion of either i3-2 or i3-4 domains did not affect significantly the percentage of receptor in R_H in the presence of either morphine or PL017 (Table 2). Thus, these data suggested that the morphine or PL017 receptor complexes of these deletion mutants had similar affinities for the G proteins compared with that of wild-type receptors. The binding of morphine and PL017 to the deletion mutants also exhibited differences. The i3-4 deletion resulted in a significant increase in the affinity for PL017 but not for morphine (Table 2). The increase in agonist affinity after i3-4 deletion seems to be restricted to the peptide agonists DAMGO and PL017 and not observed with the alkaloid morphine.

Deletion of the Third Intracellular Loop Sequence on the Activation of µ-Opioid Receptor by Agonists. The effect of the third intracellular loop deletion on the µ-opioid agonists DAMGO, morphine, and PL017 activities were determined by measuring agonist inhibition of intracellular cAMP production. Deletion of the either i3-1 or i3-5 domains resulted in receptors having low-affinity binding for the agonists (Table 2). The inability of these receptors to interact with G proteins should reflect in the absence of agonist-dependent adenylyl cyclase inhibition. As summarized in Table 3, minimal inhibition of the forskolin-stimulated adenylyl cyclase was observed in HEK293 cells expressing the i3-1 or i3-5 deletion mutants. At the maximal concentration of DAMGO used, 10 µM, the agonist inhibited the adenylyl cyclase, respectively, by 29 and 10% in cells expressing the i3-1 and i3-5 deletion mutants. On the other hand, DAMGO inhibited the cAMP production in HEK293 expressing the wild-type and other deletion mutants, i3-2, i3-3, i3-4, in a concentration-dependent manner (Fig. 2B). Deletion of these domains did not significantly alter the DAMGO potency or maximal inhibition level in regulating the adenylyl cyclase activity (Table 3). This was surprising considering that both i3-2 and i3-4 deletions reduced the percentage of the receptor in the high-affinity R_H states (Table 2). The deletion of i3-2 or i3-3 domain did not alter the ratio of K_I/K_H, or the coupling efficiency, whereas the deletion of i3-4 domain reduced the coupling efficiency by 8-fold (Table 3). Thus, the deletion of amino acids within the third intracellular loop resulted in differential effects on DAMGO-induced µ-opioid receptor activation.

Deletion of the domains within the third intracellular loop affected the morphine's ability to inhibit adenylyl cyclase differently. Although the deletion of i3-1 and i3-5 domains also abolished morphine inhibition of adenylyl cyclase activity, the deletion of i3-4 resulted in minimal reduction in the coupling efficiency (2-fold; Table 3). On the other hand, deletion of i3-3 increased the potency of morphine without altering the maximal inhibition level or the coupling efficiency. The greatest difference between DAMGO and morphine activities was observed with the i3-2 deletion. The deletion of i3-2 domain resulted in both a decrease in the potency of morphine and the maximal level of inhibition. This is in contrast with the observation in which DAMGO activity was not affected by the i3-2 deletion (Table 3).

When the PL017 activities were measured, the overall effect of the deletion was more similar to those observed with morphine than with DAMGO. Again, deletion of the i3-1 and i3-5 resulted in the inability of PL017 to inhibit adenylyl cyclase activity. Similar to effect on DAMGO and morphine, deletion of the i3-4 did not alter the potency or maximal inhibitory level of PL017, but decreased the coupling efficiency of the ligand (Table 3). Similar to morphine, deletion of i3-2 significantly decreased the potency, the maximal inhibition level, and the coupling efficiency of PL017. In contrast to both DAMGO and morphine, deletion of i3-3 increased the potency and the coupling efficiency of PL017 by 5-fold (Table 3), signifying the maximal inhibition was achieved without full occupancy of the receptor by this peptide agonist.

Alanine Scanning of ²⁷⁶RRITR²⁸⁰ and the Effect on Agonist Affinity. With the studies on the deletion mutants, we have established that there are selective effects on agonist activation of the µ-opioid receptor. However, eliminating four to five amino acids could result in the repositioning of the domains, and hence the observed differences among the agonists. To distinguish further the differences, alanine scanning studies were carried out. The motif ²⁷⁶RRITR²⁸⁰ was chosen because the deletion, or the substitution of the amino acids within this motif with alanine resulted in a complete uncoupling of the µ-opioid receptor from G proteins (Tables 2 and 3). If an amino acid within this domain is critical to receptor G protein coupling and activation, substitution of the amino acid with alanine should result in an alteration in agonist activity. As summarized in Table 4, the K_D values of [³H]diprenorphine for all of the alanine mutants were comparable with the K_D value for the wild-type receptor. This is in contrast with the i3-5 deletion mutant that exhibited a decrease in [³H]diprenorphine binding (Table 1). The levels of the mutant receptor expressed at the cell surface of HEK293 were comparable with that of wild-type receptor. Also in contrast to the observations with the i3-5 deletion mutant, substitution of the ²⁷⁶RRITR²⁸⁰ sequence with alanine did not alter the affinities of the antagonists, CTOP, and naloxone (Table 5). Thus, the alanine substitution did not affect the overall ligand binding sites, as shown by the antagonist binding.

When competition binding experiments were carried out, DAMGO, morphine, or PL017 exhibited multiple affinities binding profiles in all the alanine mutants (Table 5). This is in direct contrast with the i3-5 deletion mutant competition binding studies. When the ²⁷⁶RRITR²⁸⁰ sequence was deleted from the third intracellular loop, only the low-affinity binding site was observed (Table 2). These data suggested that the alanine mutants, unlike the i3-5, could form high-affinity complexes with G proteins. Only the mutation of Iso²⁷⁸ to Ala caused a reduction in the percentage of receptor in the R_H. With the I278A mutation, the effect was limited to DAMGO and PL017 only, whereas the percentage of receptor in high-affinity state for morphine remained the same as wild-type receptor (Table 5). Again, the single alanine substitution within the ²⁷⁶RRITR²⁸⁰ sequence did not uncouple the receptor from the G proteins. Combination of the two-Arg mutation, Arg²⁷⁶ and Arg²⁷⁷, could not mimic the i3-5 deletion either (Table 5). The percentage of the double Arg mutant receptor in high-affinity states was comparable with that of wild type, with an increased in the PL017 affinity. Because the 5A mutant could completely uncouple the receptor from G protein (Table 2), the inability of the single alanine substitution, or double alanine substitution within the ²⁷⁶RRITR²⁸⁰ sequence to completely uncouple the receptor from G protein suggested multiple residues within this sequence were involved in receptor G protein coupling.

With the exception of R277A mutant, the K_H values of DAMGO and morphine for the receptor mutants were similar to that of wild-type receptor (Table 5). Mutation of Arg²⁷⁷ to Ala increased the K_H values of DAMGO and morphine, but not that of PL017. Again, the combination of the Arg²⁷⁶ mutation with the Arg²⁷⁷ mutation did not further decrease the agonists' affinities for the high-affinity binding sites. Interestingly, changes in the low-affinity binding sites' dissociation constants, K_L, of these agonists were observed with R277A and T279A mutants. In the R277A mutant, the affinities of morphine and PL017 for the R_L were significantly reduced by the alanine substitution. Meanwhile, in the T279A mutant, the affinities of DAMGO and PL017 were reduced by the mutation (Table 5). These alterations in the agonist affinities for R_L suggested R_L probably consisted of protein complexes other than the receptor alone.

Alanine Scanning of ²⁷⁶RRITR²⁸⁰ and the Effect on Agonist Inhibition of Adenylyl Cyclase Activity. To further examine the individual amino acids within the ²⁷⁶RRITR²⁸⁰ motif in agonist-selective G protein activation, all mutants with single or double alanine substitution mutants were evaluated for their abilities to mediate the agonist inhibition of adenylyl cyclase activity. As summarized in Table 6, the mutation of Arg²⁷⁷ to Ala greatly reduced the potencies of morphine and PL017. In this R277A mutant, DAMGO potency to inhibit the forskolin-stimulated adenylyl cyclase was unchanged, while the affinity of the agonist for R_H was reduced by 3-fold. Furthermore, the maximal level of morphine and DAMGO inhibition in HEK293 cells expressing the R277A mutant were significantly reduced, whereas that of PL017 was unaltered (Table 6). This R277A mutant increased the coupling efficiency of DAMGO and morphine on one hand, and decreased the coupling efficiency of PL017 on the other.

Mutation of other residues within the ²⁷⁶RRITR²⁸⁰ motif to alanine also resulted in differential responses in the agonist inhibition of the adenylyl cyclase activity (Table 6). Mutation of Arg²⁷⁶ to alanine reduced the DAMGO potency significantly by 3.5-fold, reduced the coupling efficiency of DAMGO and morphine, and with no effect on morphine and PL017 potencies or PL017 coupling efficiency. Combination of the Arg²⁷⁶ and Arg²⁷⁷ mutations within this ²⁷⁶RRITR²⁸⁰ motif did not reduce the DAMGO potency further, but reduced morphine and PL017 potencies (Table 6). The coupling efficiency of DAMGO was further decreased by the double Arg mutations. The same double Arg mutations resulted in the decrease of PL017 coupling efficiency also. The mutation of Iso²⁷⁸ to Ala within this motif did not significantly alter the potencies of the three agonists tested, but it reduced the coupling efficiency of the DAMGO dramatically with no alteration in the coupling efficiencies of morphine and PL017. The coupling efficiency of PL017 increased in the T279A and R280A mutants due to the increase in the potency of this agonist (Table 6). The increase in PL017 potency in the I278A mutant did not result in an increase in coupling efficiency. Thus, the individual residue within the ²⁷⁶RRITR²⁸⁰ motif exhibited differential effects on the DAMGO, morphine, and PL017 mediated inhibition of adenylyl cyclase activity.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
This study showed that the amino-terminal (²⁵⁸RSKSV²⁶², i3-1) and the carboxyl-terminal (²⁷⁶RRITR²⁸⁰, i3-5) regions of the third intracellular loop of rat µ-opioid receptor are critical regions of receptor to interact with G proteins. Absence of either motif resulted in loss of receptor to G protein interaction that stabilized the µ-opioid receptor in the high-affinity state for agonist binding. There are two explanations for the loss of high-affinity to agonist of the i3-1 and i3-5 mutants. First, deletion of five amino acids that extended from the TM5 or TM6 might disturb the structure of the receptor or arrangement of these two TM domains. Studies with several GPCRs and opioid receptor chimera studies have indicated that interaction among TM domains results in the formation of ligand binding sites (Fukuda et al., 1995; Wess, 1998). However, the deletion of i3-1 and i3-5 domains simultaneously disturbed the binding of DAMGO, morphine, and PL017, which has been demonstrated to have overlapping but different binding domains within the receptor (Law and Loh, 1999). Furthermore, the substitution of Ala residues with the ²⁷⁶RRITR²⁸⁰ (i3-5) also resulted in the loss of high-affinity binding. Thus, the hypothesis of disturbing agonist-binding sites by deletion is not consistent with the loss of high-affinity binding for both peptide and nonpeptide agonists seen in mutants i3-1 and i3-5.

Alternatively, the i3-1 and i3-5 domains might be the binding sites for G proteins. These two mutants displayed only the low-affinity binding state for agonist binding, while retaining the G protein-independent high-affinity binding for the antagonists (Table 2). This supposition is supported by the mutation of the region within the human PAF receptor corresponding to the ²⁷⁶RRITR²⁸⁰ motif of the µ-opioid receptor also resulted in the low-affinity binding of agonist that was not guanosine 5'-O-(3-thio)triphosphate-sensitive (Parent et al., 1996). The involvement of both amino and carboxyl termini of the third intracellular loop in G protein coupling also was demonstrated with the _2A-adrenergic receptor (Liggett et al., 1991). Using peptides that included the i3-1 and i3-5 sequences, Georgoussi et al. (1997) and Merkouris et al. (1996) were able to alter the high-affinity binding for opioid agonists. The impairment in the G protein and receptor interaction was demonstrated further by the down-regulation experiments (Fig. 3), in which agonist could not induce the down-regulation of i3-5 mutant. G protein and receptor interaction has been reported to be a prerequisite for agonist induced opioid receptor down-regulation (Chakrabarti et al., 1997). Thus, the interaction of the G proteins with µ-opioid receptor requires the participation of these two intracellular domains.

Results from the present study indicate that the activation of G proteins by the receptor could be dependent on the agonist used. Using the deletion mutants of the third intracellular loop, we demonstrated that DAMGO, morphine and PL017 potencies, maximal inhibition levels and the coupling efficiencies were differentially altered. Particularly, it is intriguing to note that deletion of the cluster ²⁶³RMLS²⁶⁶, i3-2, adjacent to the i3-1 domain resulted in a dramatic decrease of the PL017 activities and not in DAMGO activities. Although there was a reduction in the percentage of receptor in high affinity state, DAMGO inhibition of adenylyl cyclase was not altered in the i3-2 mutants. The high-affinity state reflects the receptor/G protein complexes (Kenakin, 2002). Thus, these data suggested that activation of the G protein as measured by adenylyl cyclase inhibition required steps in addition to the receptor/G protein complexes.

Deletion of the ²⁶⁷GSKEK²⁷¹, i3-3 domain from the third intracellular loop resulted in an increase in the potency of morphine and PL017 to inhibit adenylyl cyclase activity (Table 3). Particularly, the coupling efficiency of PL017 was increased 5-fold in this mutant, suggesting that unlike the other two agonists, PL017 could fully inhibit the adenylyl cyclase without full receptor occupancy. A possible explanation might be the loss of the Ca²⁺/calmodulin binding sites within the third loop. Wang et al. (1999, 2000) suggested a direct interaction of Ca²⁺/calmodulin with the receptor and that agonist binding resulted in a dissociation of Ca²⁺/calmodulin binding from the receptor. The binding of Ca²⁺/calmodulin to the third loop interfered with the G protein/receptor interaction. Because the binding of Ca²⁺/calmodulin to the third loop serves as a negative regulator of G protein interaction, the deletion of the ²⁶⁷GSKEK²⁷¹ domain could eliminate Ca²⁺/calmodulin binding and could increase the potency and efficacy of the agonist. It is of interest to note than if such scenario exists, it is not uniform across the agonists. In contrast to morphine and PL017, DAMGO potency or efficacy was not altered by the deletion of the i3-3 motif (Table 3). In their transactivation studies, Belcheva et al. (2001) reported the DAMGO effect was Ca²⁺/calmodulin-dependent and involved the binding of Ca²⁺/calmodulin to Lys²⁷³ of the human µ-opioid receptor. Whether the differences between these three agonists tested in the current studies are caused by the Ca²⁺/calmodulin binding remains to be demonstrated.

The role of the ²⁷²DRNL²⁷⁵, i3-4, domain in µ-opioid receptor/G protein interaction and activation is varied. Although the deletion of this domain increased the affinities of DAMGO and PL017 for the receptor and reduced the percentage of receptor in the R_H states, the potencies and maximal inhibition levels observed with these agonists were unchanged. This has resulted in the decrease in coupling efficiency. Thus, the decrease in percentage of high-affinity state is reflected in the coupling efficiency and not in the maximal inhibition level. The activation of the G proteins and subsequent inhibition of the adenylyl cyclase activity must involve the isomerization of R_H to R_H* as suggested by Kenakin (2002). The efficiency of this isomerization will determine the coupling efficiency of the drug.

The participation of the ²⁷⁶RRITR²⁸⁰, i3-5 domain in the G protein interaction has been discussed previously. The importance of the individual amino acids within this ²⁷⁶RRITR²⁸⁰ domain in G protein coupling and activation could be demonstrated by alanine scanning. In agreement with a previous study (Wang et al., 1999), none of the opioid receptor mutants with a single point mutation in the ²⁷⁶RRITR²⁸⁰ domain exhibited a loss of function (Table 6). Mutation of Arg²⁷⁷ to alanine exhibited a decrease in both the efficacy and potency of morphine, a decrease in the potency of PL017, and no change in DAMGO potency. The coupling efficiency of DAMGO in the R277A mutant was increased by 5-fold. Interestingly, the DAMGO potency and coupling efficiency were decreased with R276A and the double Arg²⁷⁶ and Arg²⁷⁷ mutants. Because there was no significant change in the binding parameters of these mutants for DAMGO, these data suggested that Arg²⁷⁶ is critical for the µ-opioid receptor activation of but not for the receptor interaction to the G proteins. Mutation of other arginine residues within the ²⁷⁶RRITR²⁸⁰ domain such as Arg²⁸⁰ did not alter the activation of the agonists DAMGO and morphine. Such data were in contrast to those reported by Wang et al. (1999). In that study, the author concluded that Arg²⁸⁰ was the crucial amino acid for G protein activation by µ-opioid receptor. The discrepancy could be the result of differences in size and hydrophobicity between the amino acids being substituted for arginine. In the studies reported by Wang et al. (1999), Arg²⁸⁰ was substituted with either leucine or methionine. The physicochemical features of the intracellular region of the receptor, including charge distribution (Higashijima et al., 1990), chemical nature of amino acid side chain interaction (Cheung et al., 1992), and volume and hydrophobicity moment (Higashijima et al., 1990), have been described as the properties governing the function of the domain in the G protein activation. Not only the amphiphatic helical structure but also orientation of the dipole of the amphipathic helix within the intact receptor could be critical for G protein activation (Cheung et al., 1992). This discrepancy points to the physical properties of the ²⁷⁶RRITR²⁸⁰ domain rather than the primary sequence as the major cause in governing the function of this motif for G protein activation.

From our current studies, the different agonists tested, DAMGO, morphine, and PL017, seem to use different surface of the third intracellular loop to activate G proteins. There are critical motifs within the third intracellular loop that by the deletion of the sequence, all three agonists' activities are impeded greatly. However, mutation of the individual amino acid within such motif, e.g., ²⁷⁶RRITR²⁸⁰, revealed differences among the three agonists tested. Mutation of I²⁷⁸ decreased the potency and coupling efficiency of DAMGO, whereas the same mutation increased the PL017 potency and coupling efficiency. Furthermore, mutation of Thr²⁷⁹ reduced the coupling efficiency of DAMGO by 4-fold, whereas this mutation increased the PL017 coupling efficiency by 4-fold (Table 6). Such differences in the agonist response suggest the relative spatial orientation of the amino acids within such domain after agonist binding in respect to other residues within the receptor is critical in determining the efficiency of the receptor to activate the G proteins. The hypothesis that binding of agonists would remove a single constraint resulting in subsequent activation of the receptor is not supported by our current data. Multiple constraints must be overcome in order for the efficient activation of the G proteins by the receptor. In view of the recent data suggesting different G proteins are coupled with the constitutive active µ-opioid receptor and the DAMGO-activated receptor (Liu et al., 2001), the removal of a single constraint thus activating the receptor constitutively would not reflect the general mechanism of agonist activation of the receptor.

	Footnotes

 
This research is supported in part by National Institutes of Health Grants DA07339 and DA11806. H.H.L. and P.Y.L. are recipients of K05 DA70544 and K05 DA00513, respectively.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

DOI: 10.1124/jpet.102.046219.

ABBREVIATIONS. GPCR, G protein-coupled receptor; PAF, platelet-activating factor; TM, transmembrane; DAMGO, [D-Ala²,MePhe⁴,Gly-ol⁵]-enkephalin; PL017, [N-MePhe³,D-Pro⁴]-morphiceptin; PCR, polymerase chain reaction; HEK, human embryonic kidney; CTOP, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-amide; FACS, fluorescence-activated cell sorting; MEM, minimal essential medium; HA, hemagglutinin.

Address correspondence to: Dr. P. Y. Law, Department of Pharmacology, Medical School, University of Minnesota, 6-120 Jackson Hall, 321 Church St. S.E., Minneapolis, MN 55455-0217. E-mail: lawxx001{at}tc.umn.edu

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