Introduction

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After prolonged or repeated activation, most G protein-coupled receptors (GPCRs) show reduced responsiveness to agonists. Three distinct processes have been demonstrated that occur over different time scales: desensitization (seconds to hours), internalization (minutes to hours), and down-regulation (hours to days) (for reviews, see Ferguson et al., 1998; Krupnick and Benovic, 1998; Lefkowitz et al., 1998; Tsao and von Zastrow, 2000). Stimulation of GPCRs by agonists, in addition to activating downstream effectors, enhances phosphorylation of the activated receptors by GPCR kinases (GRKs), mostly in the C-terminal domain and/or the third intracellular loop. Receptor phosphorylation facilitates binding of arrestins, leading to uncoupling of the GPCRs from G proteins and hence reduced responsiveness to cognate agonists. Arrestins, in turn, bind clathrin and other adaptor proteins, resulting in movement of the receptors into clathrin-coated vesicles or uncoated vesicles and then into endocytic vesicles and endosomes, where they are unavailable for signal transduction. Even more prolonged agonist exposure causes down-regulation, which involves proteolytic degradation of the receptor in lysosomes and proteasomes (Li et al., 2000; Chaturvedi et al., 2001) or at plasma membranes (Kojro and Fahrenholz, 1995) and leads to a reduction in the receptor number.

Opiates and opioids act on opioid receptors to produce effects. After the cloning of the -opioid receptor, the µ- and -opioid receptors were cloned (for review, see Knapp et al., 1995). Opioid receptors belong to the rhodopsin subfamily of the GPCR family. Activation of -opioid receptors produces many effects, including analgesia (von Voigtlander et al., 1983; Dykstra et al., 1987), dysphoria (Pfeiffer et al., 1986; Dykstra et al., 1987), water diuresis (von Voigtlander et al., 1983; Dykstra et al., 1987), hypothermia (Adler and Geller, 1993), and modulation of immune responses (Taub et al., 1991). -Opioid receptors are coupled via pertussis toxin-sensitive G proteins to affect a variety of effectors, which include adenylate cyclase, potassium channels, and calcium channels and the p42/p44 mitogen-activated protein kinase pathway (for review, see Law et al., 2000b). Chronic use of -opioid agonists causes tolerance (Murray and Cowan, 1988; Bhargava et al., 1989) that can be partially accounted for at the receptor level (von Voigtlander et al., 1983; Bhargava et al., 1989; Morris and Herz, 1989; Joseph and Bidlack, 1995).

Opioid receptors have been shown to undergo desensitization, internalization, and down-regulation (for review, see Law et al., 2000b). We previously observed that after exposure to ()U50,488H, the human -opoid receptor (hkor) expressed in CHO cells underwent phosphorylation, desensitization, internalization, and down-regulation (Zhu et al., 1998; Li et al., 1999, 2000, 2001b). In contrast, the rkor stably expressed in CHO cells did not undergo phosphorylation, desensitization, internalization, and down-regulation when activated by ()U50,488H (Li et al., 1999, 2000, 2001b; Jordan et al., 2000). The differences between rkor and hkor receptors in CHO cells provided a unique opportunity to delineate the structural determinants in the receptors underlying ()U50,488H-induced regulation of the -receptor. The amino acid sequences of the hkor and the rkor are ~95% identical (Li et al., 1993; Zhu et al., 1995). We generated chimeric and mutant receptors of the hkor and the rkor and investigated whether the chimeras and mutants were internalized and down-regulated by ()U50,488H treatment to delineate the structural basis for the species differences and explore the relationship among the regulatory processes.

	Materials and Methods

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Materials. [³H]Diprenorphine (58 Ci/mmol) was purchased from PerkinElmer Life Sciences (Boston, MA). ()U50,488H was provided by Upjohn (Kalamazoo, MI). Naloxone was a gift from DuPont Merck Pharmaceutical Co. (Wilmington, DE). Diprenorphine was provided by the National Institute on Drug Abuse (Bethesda, MD). M₁ anti-FLAG mouse monoclonal antibody was purchased from Sigma-Aldrich (St. Louis, MO). Goat anti-mouse IgG (H+L) conjugated with Alexa-Fluor 488 was purchased from Molecular Probes (Eugene, OR). Normal goat serum (NGS) was purchased from Organon Teknika (West Chester, PA). Geneticin was purchased from Mediatech (Herndon, VA). Enzymes and chemicals used in molecular biology and mutagenesis experiments were purchased from Invitrogen (Carlsbad, CA), Promega (Madison, WI), Roche Applied Science (Indianapolis, IN), and QIAGEN (Valencia, CA).

Generation of FLAG-Tagged Wild-Type, Chimeric, and Mutant Receptors. The human and rat -opioid receptor cDNAs used are those we cloned (Li et al., 1993; Zhu et al., 1995). An ~130-base pair fragment containing a signal peptide and the FLAG-tag sequence was excised with HindIII and NcoI from a construct of FLAG-tagged ₂-adrenergic receptor in pcDNA3, with FLAG-tagged 5' to the initiation codon (Guan et al., 1992). The cDNA clones of FLAG-tagged hkor (FLAG-hkor), FLAG-rkor, FLAG-tagged hkor1-338/rkor339-380 (FLAG-h/rkor), FLAG-tagged rkor1-338/hkor339-380 (FLAG-r/hkor), S358N mutant of the FLAG-hkor (FLAG-hkorS358N), and N358S mutant of the FLAG-rkor (FLAG-rkorN358S) were generated by ligating the fragment with each -receptor construct at 5' to the initiation codon and cloned into the mammalian expression vector pcDNA3 (Li et al., 2001b).

Establishment of CHO Cell Lines and Cell Culture. Clonal CHO cell lines stably expressing the hkor, rkor, FLAG-hkor, FLAG-rkor, FLAG-r/hkor, FLAG-h/rkor, FLAG-hkorS358N, and FLAG-rkorN358S receptors were established previously (Li et al., 2001b). Cells were cultured in 100-mm culture dishes in Dulbecco's modified Eagle's medium/F-12 HAM supplemented with 10% fetal calf serum, 0.2 mg/ml geneticin, 100 units/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere consisting of 5% CO₂ and 95% air at 37°C.

Pretreatment with the -Agonist ()U50,488H. At ~90% confluence, cells were treated without (control) or with the -opioid agonist ()U50,488H (1 µM) in the medium for 30 min for internalization experiments or for 4 h for down-regulation experiments. Cells were washed four times with cold Krebs-Ringer-HEPES buffer solution (110 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 25 mM glucose, 55 mM sucrose, and 10 mM HEPES, pH 7.4) on ice to remove ()U50,488H.

Internalization of the -Receptor after Agonist Exposure. Intracellular receptors were assessed as we described previously (Li et al., 1999). CHO-hkor cells cultured in 24-well plates were incubated with ()U50,488H at 37°C and washed. Binding was performed on intact CHO-hkor cells with [³H]diprenorphine in Krebs-Ringer-HEPES buffer solution. Total receptor levels were assessed by binding with 2 nM [³H]diprenorphine in the presence or absence of 1 µM diprenorphine, whereas surface receptors were measured by binding with 2 nM [³H]diprenorphine in the presence or absence of 1 or 3 µM dynorphin A(1-17). Binding was performed at room temperature for 60 min. We found that maximal inhibition of [³H]diprenorphine binding to the FLAG-hkor in intact cells was reached at 3 µM naloxone or 0.3 µM diprenorphine and nonspecific binding defined by 10 µM naloxone or 1 µM diprenorphine was ~15%. In addition, dynorphin A caused maximal inhibition of [³H]diprenorphine binding to the FLAG-hkor in intact CHO cells at 1 µM. We found previously that Na⁺ had no or only a small effect on agonist binding affinity for the human -opioid receptor (Zhu et al., 1997). Thus, 1 or 3 µM was used to define nonspecific binding for surface receptor binding. Diprenorphine, a hydrophobic ligand, can bind to both cell surface and intracellular receptors, whereas dynorphin A(1-17), a hydrophilic ligand, binds only to the cell surface receptors. Thus, the difference between total receptor binding and cell surface receptor binding represents binding to the intracellular receptor pool. An increase in intracellular [³H]diprenorphine binding over the basal level after agonist exposure provides a quantitative measure of internalized receptors.

Membrane Preparation. Membranes were prepared according to Zhu et al. (1997) with some modifications. Briefly, the CHO cells were pelleted, frozen at 80°C for at least 30 min, thawed in cold lysis buffer (5 mM Tris-HCl, 5 mM EDTA, 5 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, 10 mM sodium fluouride, and 10 mM tetrasodium pyrophosphate, pH 7.4), and vortexed. Cell suspension was passed through a 1-ml 29-gauge 3/8 syringe needle five times and centrifuged. Pellets were resuspended in 50 mM Tris-HCl buffer/2.5 mM EDTA, pH 7.4, passed through the syringe needle, and centrifuged at 100,000g for 30 min, and the processes were repeated. Membranes were suspended in 50 mM Tris-HCl buffer/1 mM EGTA, pH 7.4, and protein concentration was determined by the bicinchoninic acid method of Smith et al. (1985).

Saturation Binding of [³H]Diprenorphine. Saturation binding of [³³H]diprenorphine to the wild-type, chimeric, and mutant -opioid receptors was performed with at least eight concentrations of [³³H]diprenorphine (ranging from 16 pM to 4 nM), and K_d and B_max values were determined. Binding was carried out in 50 mM Tris-HCl buffer containing 1 mM EGTA, pH 7.4, at room temperature for 1 h in duplicate in a final volume of 1 ml with ~10 to 20 µg of membrane protein. Naloxone (10 µM) was used to define nonspecific binding. Binding data were analyzed with the EBDA program (McPherson, 1983).

Immunofluorescence Staining. CHO cells stably transfected with a FLAG-tagged wild type, chimera, or mutant of the -opioid receptors were cultured in 100-mm dishes, transferred into slide chambers (Lab-Tek II; Lab-Tek, Naperville, IL), and cultured overnight. Cells were treated with 1 µM ()U50,488H or left untreated for 30 min at 37°C, washed three times with ice-cold 10 mM phosphate-buffered saline (PBS) (Na₂HPO₄ 8.1 mM, NaH₂PO₄ 1.9 mM, NaCl 154 mM, CaCl₂ 1 mM), fixed with 4% paraformaldehyde in PBS for 10 min at room temperature, and washed three times with PBS to remove the fixative. Subsequently, cells were permeabilized using 0.05% Triton X-100 for 10 min at room temperature and incubated with 4% NGS at room temperature for 10 min to block nonspecific binding. Cells were incubated with anti-FLAG mouse M₁ antibody (4 µg/ml; Sigma-Aldrich) in PBS containing 4% NGS and 0.05% Triton X-100 at 37°C for 30 min, rinsed three times with PBS containing 0.05% Triton X-100 at room temperature, and incubated with goat anti-mouse IgG (H+L) conjugated with Alexa-Fluor 488 (2 to 4 µg/ml; Molecular Probes) in PBS containing 4% NGS and 0.05% Triton X-100 at room temperature for 30 min. After three washes with PBS containing 0.05% Triton X-100 at room temperature, cells were mounted with Slow-Fade mounting medium (Sigma-Aldrich), and coverslips were sealed with nail polish. Two controls were used: anti-FLAG mouse M₁ antibody (4 µg/ml) pretreated with an excessive amount of the FLAG peptide (100 µg/ml) before incubation and omission of the anti-FLAG mouse M₁ antibody from the procedures. Cells were examined under a fluorescence microscope (ELIPSE TE300; Nikon, Tokyo, Japan) equipped with a 60× numerical aperture 1.4 objective and fluorescein filter sets or with a confocal fluorescence microscope (model IX70; Olympus, Tokyo, Japan) equipped with a 60× numerical aperture 1.4 objective (Carl Zeiss, Thornwood, NY).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Effect of ()U50,488H on Internalization and Down-Regulation of the hkor, rkor, FLAG-hkor, and FLAG-rkor. FLAG-tagged wild-type and mutant hkor and rkor were used in the study along with untagged hkor and rkor to allow detection of FLAG-tagged receptor by immunochemical method using anti-FLAG antibodies and to facilitate correlation with phosphorylation and desensitization studies performed previously (Li et al., 2001b). The FLAG-hkor and the FLAG-rkor had similar binding affinities for [³H]diprenorphine as the hkor and the rkor, and ()U50,488H displayed similar potencies for the FLAG-hkor and the FLAG-rkor in enhancing [³⁵S]GTPS binding as for the untagged receptors (Li et al., 2001b). Pretreatment of the hkor, rkor, FLAG-hkor, and FLAG-rkor stably expressed in CHO cells with 1 µM ()U50,488H for 30 min induced significant internalization of both the hkor and the FLAG-hkor, but the rkor and the FLAG-rkor underwent no internalization (Fig. 1A).

View larger version (29K): [in this window] [in a new window]   Fig. 1.   ()U50,488H-induced internalization and down-regulation of -opioid receptors: differences between human and rat receptors. CHO cells stably transfected with hkor, rkor, FLAG-hkor, or FLAG-rkor were pretreated without (basal) or with 1 µM ()U50,488H at 37°C for 30 min (A) or 4 h (B) followed by washing to remove the agonist. A, internalized receptors were determined by [3H]diprenorphine binding and presented as percentage of the total receptor with the basal value (~15% of total receptors) subtracted. Each value represents mean ± S.E.M. of seven (the hkor and the FLAG-hkor) and four (the rkor and the FLAG-rkor) independent experiments in duplicate. B, membranes were prepared, saturation [3H]diprenorphine binding was performed, and Kd and Bmax values were determined. Kd values were not changed by the treatment. Receptor numbers were expressed as percentage of the untreated control. Each value represents mean ± S.E.M. of three independent experiments in duplicate. **, p < 0.01; *, p < 0.05, compared with the untreated control by one-tail unpaired Student's t test. ##, p < 0.01; #, p < 0.05, compared with the hkor (for the rkor) or the FLAG-hkor (for the FLAG-rkor) by one-tail unpaired Student's t test.

View this table: [in this window] [in a new window]   TABLE 1 Effect of pretreatment with ()U50,488H on [3H]diprenorphine binding to the wild-type, chimeric, and mutant  receptors CHO cells stably transfected with a wild-type, chimeric, or mutant receptor were pretreated without (control) or with 1 µM ()U50,488H at 37°C for 4 h followed by washing to remove the agonist. Membranes were prepared, saturation [3H]diprenorphine binding was performed, and Kd and Bmax values were determined. Each value represents mean ± S.E.M. of at least three independent experiments in duplicate.

Effects of Monensin on Intracellular Pools of the rkor. To determine whether the apparent lack of ()U50,488H-induced internalization of the rkor was due to rapid resurfacing of the internalized receptor, we examined the effect of monensin treatment on the intracellular pool of receptors. Monensin, a sodium ionophore that prevents acidification of intracellular vesicles and blocks the recycling of endocytosed receptors (Pippig et al., 1995) did not affect the fraction of the rkor that is intracellular, both with and without ()U50,488H treatment (Fig. 2). Thus, the lack of ()U50,488H-induced internalization of the rkor is not the result of rapid recycling of internalized receptor.

View larger version (35K): [in this window] [in a new window]   Fig. 2.   Effect of monensin on intracellular pool of the rkor (as percentage of total receptors) without or with ()U50,488H treatment. CHO-FLAG-rkor cells were pretreated without or with 50 µM monensin for 1.5 h and treated without or with 1 µM ()U50,488H for the last 30 min. Intracellular receptors were determined as described under Materials and Methods. Each value represents the mean ± S.E.M. of three independent experiments in duplicate.

Role of the C-Terminal Domain of the -Opioid Receptor in ()U50,488H-Induced Internalization and Down-Regulation. For many GPCRs, the intracellular regions, particularly the third intracellular loops and the C-terminal domains, play important roles in internalization and down-regulation (Cvejic et al., 1996; Chu et al., 1997; Afify et al., 1998). The amino acid sequences of intracellular regions of the rkor and the hkor are highly homologous with only some differences in the C-terminal domain (Fig. 3). To understand the structural basis of the differences in ()U50,488H-induced internalization and down-regulation between the hkor and the rkor, we constructed two FLAG-tagged chimeric receptors, FLAG-h/rkor [FLAG-hkor(1-338)/rkor(339-380)] and FLAG-r/hkor [FLAG-rkor(1-338)/hkor(339-380)], in which the C-terminal domains were exchanged. The chimeras exhibited similar binding affinities for [³H]diprenorphine as the wild types (Table 1), and ()U50,488H displayed similar potency in stimulating [³⁵S]GTPS binding mediated by the wild types and the chimeras (Li et al., 2001b). Unlike the rkor or the FLAG-rkor, the FLAG-r/hkor underwent ()U50,488H-promoted internalization and down-regulation (Fig. 4; Table 1). In addition, in contrast to the hkor and the FLAG-hkor, the FLAG-h/rkor pretreated with ()U50,488H did not exhibit significant internalization and down-regulation (Fig. 4; Table 1). These results demonstrate that the C-terminal domain plays a crucial role in the observed species differences.

View larger version (82K): [in this window] [in a new window]   Fig. 3.   Amino acid sequence comparison between the hkor (Zhu et al., 1995) and the rkor (Li et al., 1993), points of exchange for generation of chimeric receptors and amino acid residues mutated in the hkor-S358N and rkor-N358S mutants (Li et al., 2001b). Amino acid residue numbers are indicated on both sides. Seven putative transmembrane domains (TMs) are shaded. Chimeras FLAG-r/hkor (rkor1-338/hkor339-380) and FLAG-h/rkor (hkor1-338/rkor339-380) were generated by an exchange of the C-terminal domain fragments 338 to 380 as indicated by an arrow. Amino acid residues 358 are indicated by *. Ser358 in the hkor was mutated to Asn and Asn358 in the rkor was substituted with Ser.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   ()U50,488H-induced internalization and down-regulation of chimeric -opioid receptors. CHO cells stably transfected with the FLAG-h/rkor or the FLAG-r/hkor were pretreated without (control) or with 1 µM ()U50,488H at 37°C for 30 min (A) or 4 h (B) followed by washing to remove the agonist. A, internalized receptors were determined by [3H]diprenorphine binding and presented as percentage of the total receptor. Each value represents mean ± S.E.M. of six (for the FLAG-r/hkor) and eight (for the FLAG-h/rkor) independent experiments in duplicate. B, membranes were prepared and saturation [3H]diprenorphine binding was performed and Kd and Bmax values were determined. Kd value was not changed by the treatment. Receptor numbers were expressed as percentage of the untreated control. Each value represents mean ± S.E.M. of four independent experiments in duplicate. **, p < 0.01; *, p < 0.05, compared with the untreated control by one-tail unpaired Student's t test. ##, p < 0.01; #, p < 0.05, compared with the FLAG-hkor (for the FLAG-h/rkor) or the FLAG-rkor (for the FLAG-r/hkor) by one-tail unpaired Student's t test.

Role of the Residues 358 in the C-Terminal Domains of hkor and rkor in ()U50,488H-Induced Internalization and Down-Regulation. There are only seven residues that are different in the C-terminal domains of the hkor and the rkor (Fig. 3). One notable difference is the locus 358, where it is Ser in the hkor, but Asn in the rkor. We generated the two mutants FLAG-hkor-S358N and FLAG-rkor-N358S to further delineate the structural basis of the observed species differences. Both mutants displayed similar binding affinities for [³H]diprenorphine as the wild types (Table 1), and ()U50,488H had similar potencies in stimulating the wild types and the two mutants to enhance [³⁵S]GTPS binding (Li et al., 2001b).

View larger version (28K): [in this window] [in a new window]   Fig. 5.   ()U50,488H-induced internalization and down-regulation of the FLAG-rkorN358S and the FLAG-hkorS358N mutants. CHO cells stably transfected with the FLAG-rkorN358S mutant or the FLAG-hkorS358N mutant were treated without (control) or with 1 µM ()U50,488H at 37°C for 30 min (A) or 4 h (B) followed by washing to remove the agonist. A, internalized receptors were determined by [3H]diprenorphine binding and presented as percentage of the total receptor. Each value represents mean ± S.E.M. of eight independent experiments in duplicate. B, membranes were prepared and saturation [3H]diprenorphine binding was performed, and Kd and Bmax values were determined. Kd value was not changed by the treatment. Receptor numbers were expressed as percentage of the untreated control. Each value represents mean ± S.E.M. of four independent experiments in duplicate. **, p < 0.01; *, p < 0.05, compared with the untreated control by one-tail unpaired Student's t test. #, p < 0.05, compared with the FLAG-hkor (for the FLAG-hkorS358N) or the FLAG-rkor (for the FLAG-rkorN358S) by one-tail unpaired Student's t test.

Detection of Receptor Internalization by Immunofluorescence Staining. Immunofluorescence staining with the M₁ anti-FLAG antibody and goat anti-mouse IgG conjugated with Alexa-Fluor 488 was performed to visualize distribution of FLAG-tagged receptors with and without ()U50,488H treatment. Although in untreated cells most of the FLAG-hkors or FLAG-rkors were on plasma membranes, ()U50,488H caused a great increase in intracellular fluorescence staining and a decrease in cell surface staining of the FLAG-hkor, but not the FLAG-rkor (Fig. 6). The intracellular fluorescence was punctate and seemed to accumulate in the perinuclear region. In addition, ()U50,488H treatment induced an increase in intracellular staining of the FLAG-r/hkor and FLAG-rkorN358S, and, to a less extent, FLAG-hkorS358N (Fig. 6). However, there was no increase in intracellular staining of the FLAG-h/rkor after ()U50,488H incubation (Fig. 6).

View larger version (29K): [in this window] [in a new window]   Fig. 6.   ()U50,488H-induced internalization of FLAG-hkor, FLAG-rkor, FLAG-h/rkor, FLAG-r/hkor, FLAG-hkorS358N, and FLAG-rkorN358S visualized by immunofluorescence microscopy. Transfected CHO cells expressing one receptor were incubated in the presence or the absence (control) of 1 µM ()-U50,488H at 37°C for 30 min and then fixed by 4% paraformaldehyde and permeabilized using 0.05% Triton X-100. Immunofluorescence staining was performed with anti-FLAG M1 mouse monoclonal antibody as primary antibody and goat anti-mouse IgG conjugated with Alexa-Fluor 488 as secondary antibody as described under Materials and Methods. The figures represent one of the three experiments performed with similar results.

	Discussion

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We have shown that after exposure to ()U50,488H, hkor and FLAG-hkor were internalized and down-regulated, but rkor and FLAG-rkor were not. The C-terminal domains contribute to the differences in internalization and down-regulation between the hkor and the rkor. The 358 locus plays an important role in differences in internalization; however, its role in down-regulation is not clear. Thus, in addition to differences in ()U50,488H-promoted phosphorylation and desensitization (Li et al., 2001b), the rkor and hkor also exhibit differences in internalization and down-regulation. To the best of our knowledge, the differential regulation between the hkor and rkor represents the first demonstration of such species difference in the regulation of GPCRs. In addition, this study provides the first evidence for the importance of Ser358 in ()U50,488H-induced internalization and down-regulation of the hkor.

Determination of Receptor Internalization by Binding and Immunofluorescence Staining. Total and cell surface receptors were determined by use of hydrophobic and hydrophilic ligands, respectively, allowing determination of intracellular receptors. Immunofluorescence staining of the receptor gives qualitative results and permits visualization of the receptor distribution. The two methods yielded similar results for internalization experiments on the wild-types, chimeras, and mutants of the -receptors.

Internalization and Down-Regulation of hkor and FLAG-hkor. The hkor and the FLAG-hkor were readily internalized by ()U50,488H pretreatment for 30 min, consistent with our previous report (Li et al., 1999). In addition, a 4-h pretreatment down-regulated the hkor and FLAG-hkor. The observation and the extent of down-regulation (~30%) are similar to our previous studies (Zhu et al., 1998; Li et al., 2000) and those of Blake et al. (1997).

Lack of Internalization and Down-Regulation of rkor and FLAG-rkor. A 30-min incubation with 1 µM ()U50,488H did not cause internalization of the rkor and the FLAG-rkor (Fig. 1). This finding is similar to our previous observation (Li et al., 1999) and those of Chu et al. (1997) and Jordan et al. (2000). The lack of internalization was not due to rapid recycling of the rkor because monensin had no effect on intracellular pools of the receptor with or without ()U50,488H treatment. In addition, incubation with ()U50,488H for 4 or 24 h did not promote down-regulation of the rkor and the FLAG-rkor (Fig. 1), which is similar to our previous report (Li et al., 2000). However, our results are different from those of Joseph and Bidlack (1995), who showed that the -opioid receptor in murine R1.1 thrymoma cells were down-regulated after incubation with 0.1 µM ()U50,488H for 24 h. Because the amino acid sequences of the mouse and rat -receptors are 99% identical overall and 100% identical in intracellular regions, this difference may be a reflection of the different cell systems.

Internalization and Down-Regulation of Chimeric and Mutant Receptors. Pretreatment of FLAG-r/hkor, but not FLAG-h/rkor, with ()U50,488H resulted in internalization and down-regulation, demonstrating that the C-terminal domains contribute to the difference between the hkor and the rkor.

Discrepancy in ()U50,488H-Promoted Down-Regulation between FLAG-r/hkor and FLAG-rkorN358S. ()U50,488H induced down-regulation of the FLAG-r/hkor, but not the FLAG-rkorN358S mutant. These results indicate that sequence differences in the C-terminal domain, besides the S versus N at the 358 locus, between the hkor and rkor also contribute to the differences in down-regulation. The dissimilarity between the sequences may lead to conformational differences of the C-terminal domain between the hkor and the rkor, which in turn lead to differential interactions of GRKs with this region.

Relationship between Internalization and Down-Regulation. After exposure to ()U50,488H, the hkor underwent internalization and down-regulation, but the rkor did not (Li et al., 1999, 2000). In contrast to ()U50,488H, etorphine did not cause internalization or down-regulation of the hkor (Li et al., 1999, 2000). In addition, expression of the dominant negative mutants arrestin-2(319-418) or dynamin I-K44A, which attenuated ()U50,488H-promoted internalization of the hkor, significantly reduced ()U50,488H-induced down-regulation of the receptor (Li et al., 1999, 2000). These findings indicate that internalization of the -opioid receptor is required for its down-regulation. Similar findings have been reported for the ₂-adrenergic receptor (Gagnon et al., 1998). This relationship seems to hold true for the FLAG-r/hkor, FLAG-h/rkor, and FLAG-hkorS358N. However, the FLAG-rkorN358S mutant undergoes ()U50,488H-induced internalization, but not down-regulation. Such dissociation between internalization and down-regulation has been demonstrated for GPCR mutants. Some mutants exhibited receptor internalization equivalent to that of the wild type, yet showed blunted receptor down-regulation in response to agonists; for example, several mutants of the ₂AR with substitutions in the third intracellular loop and C-terminal domain (Campbell et al., 1991) and Tyr459 mutants in the C-terminal domain of the m₂ mAChR (Goldman and Nathanson, 1994). Conversely, several GPCR mutants have been shown to have greatly reduced agonist-mediated receptor internalization, yet still retain the ability to undergo down-regulation; for example, the Y326A mutant of the ₂AR (Barak et al., 1994) and the -opioid receptor mutant lacking the C-terminal 15 amino acids (Cvejic et al., 1996; Trapaidze et al., 1996). These observations led to the suggestion that internalization and down-regulation may be mediated by distinct mechanisms. However, in view of our results on rkor and hkor and their mutants, it is likely that GPCR mutants may not be trafficked in the same manner as the wild-type receptors. Another likely explanation is that internalization was determined after a short period of agonist treatment, whereas down-regulation was measured after a longer treatment period. An alteration in the rate or extent of internalization may not affect the degree of down-regulation.

Relationship between ()U50,488H-Induced Phosphorylation and Internalization or Down-Regulation. The FLAG-hkor and -r/hkor, but not the FLAG-rkor and -h/rkor, underwent ()U50,488H-induced phosphorylation (Li et al., 2001b), internalization, and down-regulation. Although the FLAG-hkorS358N was not phosphorylated (Li et al., 2001b), internalized, and down-regulated, the FLAG-rkorN358S was not phosphorylated (Li et al., 2001b) or down-regulated, but was internalized. Our results on FLAG-hkor, -r/hkor, rkor, -h/rkor, and -hkorS358N are consistent with the observation of Whistler et al. (2001). These researchers have demonstrated that unphosphorylated C-terminal domain of the full-length -opioid receptor serves as a brake for receptor endocytosis and agonist-induced phosphorylation releases the brake allowing endocytosis to occur. However, findings on the FLAG-rkorN358S are not in accord with their observations. Similar results that full-length GPCR mutants were not phosphorylated, but were internalized, have been reported (Law et al., 2000a). It is likely that arrestins have sufficiently high affinity for the FLAG-rkorN358S to permit internalization of the unphosphorylated receptor.

Up-Regulation of rkor, FLAG-rkor, and FLAG-hkorS358N by ()U50,488H. It is intriguing that incubation with ()U50,488H for 4 h did not induce down-regulation of rkor, FLAG-rkor, and FLAG-hkorS358N, rather it caused a significant up-regulation of these receptors. This is probably due to stabilization of the receptor proteins. We have shown that upon incubation at 37°C in the presence of protease inhibitors, the rat µ-opioid receptor is denatured and an agonist or an antagonist can stabilize the structure (Li et al., 2001). Thus, the action of ()U50,488H on the -receptors may be a combination of causing internalization and down-regulation and stabilizing the receptor proteins. When the receptor is not down-regulated by the agonist, the stabilization effects may become evident. All cDNA constructs used in the study, which do not contain the promoter region of the -receptors, were cloned into the mammalian expression vector pcDNA3, and the expression of these receptors is driven by the constitutively active cytomegalovirus promoter. ()U50,488H treatment most likely had no effect on the expression of these receptors.

Concluding Remarks. The differences in ()U50,488H-induced regulation between the hkor and the rkor demonstrated in this study may have significant implications when extrapolating studies on regulation of -opioid receptors from rats to humans.