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()U50,488H [(trans)-3,4-Dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide] InducesInternalization and Down-Regulation of the Human, but not theRat, -Opioid Receptor: Structural Basis for the DifferentialRegulation

FENGQIN ZHANG, JIN LI, JIAN-GUO LI, and LEE-YUAN LIU-CHEN

Department of Pharmacology and Center for Substance Abuse Research, Temple University School of Medicine, Philadelphia, Pennsylvania

Received November 1, 2001; accepted May 10, 2002 This article is available online at http://jpet.aspetjournals.org

ABSTRACT

We showed previously that prolonged activation by ()U50,488H[(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide] led to internalization and down-regulation ofthe human opioid receptor (hkor), but not the rat opioidreceptor (rkor). Herein, we investigated structural determinants inthe receptors underlying these differences using chimeric andmutant receptor constructs epitope tagged with FLAG and stablyexpressed in Chinese hamster ovary cells (CHO). The FLAG-hkor,but not the FLAG-rkor, underwent internalization and down-regulation after exposure to ()U50,488H. Monensin did not haveany effect on the intracellular receptor pool of the FLAG-rkor orrkor with or without ( )U50,488H treatment, indicating that thelack of ( )U50,488H-induced internalization is not due to rapidresurfacing of the rkor. Two chimeric receptors, FLAG-h/rkor and

FLAG-r/hkor, were generated, in which the C-terminal domains of

the hkor and the rkor were switched. The FLAG-r/hkor displayedsignificant ( )U50,488H-induced internalization and down-regulation, whereas the FLAG-h/rkor did not, indicating that theC-terminal domain contributes to the differences between the rkorand the hkor. To further characterize, we generated two mutants,FLAG-hkorS358N and FLAG-rkorN358S in which the locus 358was exchanged. The FLAG-hkorS358N mutant displayed greatlyreduced ( )U50,488H-induced internalization and no down-regulation compared with the FLAG-hkor, indicating that Ser358 inthe hkor is critical for these processes. However, the FLAG-rkorN358S mutant was internalized, but not down-regulated,demonstrating that N358 prevents the rkor from being internal-ized, but it may not have a role in the lack of down-regulation ofthe rkor. In addition, the trafficking of the FLAG-rkorN358S mutant

seems to be more complex than the rkor and the hkor.

After prolonged or repeated activation, most G protein-coupled receptors (GPCRs) show reduced responsiveness toagonists. Three distinct processes have been demonstratedthat occur over different time scales: desensitization (secondsto hours), internalization (minutes to hours), and down-reg-ulation (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 phos-phorylation of the activated receptors by GPCR kinases(GRKs), mostly in the C-terminal domain and/or the third

intracellular loop. Receptor phosphorylation facilitates bind-ing of arrestins, leading to uncoupling of the GPCRs from Gproteins and hence reduced responsiveness to cognate ago-nists. Arrestins, in turn, bind clathrin and other adaptorproteins, resulting in movement of the receptors into clath-rin-coated vesicles or uncoated vesicles and then into endo-cytic vesicles and endosomes, where they are unavailable forsignal transduction. Even more prolonged agonist exposure

causes down-regulation, which involves proteolytic degrada-tion 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 thereceptor number.

This work was supported by National Institute of Health Grants DA-04745and DA-11263.

ABBREVIATIONS: GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; ()U50,488H, (trans)-3,4-dichloro-N-methyl-N-

[2-(1-pyrrolidiny)-cyclohexyl]benzeneacetamide methanesulfonate; hkor, human-opioid receptor; CHO, Chinese hamster ovary; rkor, rat-opioid

receptor; FLAG epitope, DYKDDDA; FLAG-hkor, FLAG-tagged human -opioid receptor; FLAG-h/rkor, FLAG-tagged chimera of human -opioid

receptor 1-338/rat -opioid receptor 339-380; FLAG-hkorS358N, S358N mutant of the FLAG-tagged human -opioid receptor; FLAG-rkor,

FLAG-tagged rat -opioid receptor; FLAG-r/hkor, FLAG-tagged chimera of rat -opioid receptor 1-338/human -opioid receptor 339-380;

FLAG-rkorN358S, N358S mutant of the FLAG-tagged rat -opioid receptor; CHO-construct, CHO cells stably transfected with the construct; NGS,

normal goat serum; PBS, phosphate-buffered saline; GTPS, guanosine-5-O-(3-thio)triphosphate.

0022-3565/02/3023-11841192$7.00THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 302, No. 3Copyright 2002 by The American Society for Pharmacology and Experimental Therapeutics 4713/1005387JPET 302:11841192, 2002 Printed in U.S.A.

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Opiates and opioids act on opioid receptors to produce

effects. After the cloning of the -opioid receptor, the - 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 desensitiza-

tion, internalization, and down-regulation (for review, see

Law et al., 2000b). We previously observed that after expo-

sure to ()U50,488H, the human -opoid receptor (hkor)expressed in CHO cells underwent phosphorylation, desen-

sitization, 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; Jor-

dan 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 under-

lying ()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 inter-nalized 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

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-Al-

drich (St. Louis, MO). Goat anti-mouse IgG (HL) 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 Media-

tech (Herndon, VA). Enzymes and chemicals used in molecular biol-

ogy and mutagenesis experiments were purchased from Invitrogen

(Carlsbad, CA), Promega (Madison, WI), Roche Applied Science (In-

dianapolis, IN), and QIAGEN (Valencia, CA).

Generation of FLAG-Tagged Wild-Type, Chimeric, and Mu-

tant 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 se-

quence was excised with HindIII andNcoI 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 -re-

ceptor 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 Dulbeccos modifiedEagles medium/F-12 HAM supplemented with 10% fetal calf serum,0.2 mg/ml geneticin, 100 units/ml penicillin, and 100 g/ml strepto-

mycin in a humidified atmosphere consisting of 5% CO2 and 95% air

at 37C.Pretreatment with the -Agonist ()U50,488H. At 90% con-

fluence, cells were treated without (control) or with the -opioid

agonist ()U50,488H (1 M) in the medium for 30 min for internal-

ization experiments or for 4 h for down-regulation experiments. Cells

were washed four times with cold Krebs-Ringer-HEPES buffer solu-

tion (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 incu-

bated with ()U50,488H at 37C and washed. Binding was per-formed 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 M diprenorphine, whereas surface receptors were measured by

binding with 2 nM [3H]diprenorphine in the presence or absence of 1

or 3 M dynorphin A(117). Binding was performed at room temper-ature for 60 min. We found that maximal inhibition of [3H]diprenor-

phine binding to the FLAG-hkor in intact cells was reached at 3 M

naloxone or 0.3M diprenorphine and nonspecific binding defined by

10 M naloxone or 1 M diprenorphine was 15%. In addition,

dynorphin A caused maximal inhibition of [3H]diprenorphine bind-

ing to the FLAG-hkor in intact CHO cells at 1 M. We found previ-ously 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 M 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(117), ahydrophilic 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 in-

ternalized receptors.

Membrane Preparation. Membranes were prepared according

to Zhu et al. (1997) with some modifications. Briefly, the CHO cells

were pelleted, frozen at 80C for at least 30 min, thawed in cold

lysis buffer (5 mM Tris-HCl, 5 mM EDTA, 5 mM EGTA, 0.1 mMphenylmethylsulfonyl fluoride, 10 M 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 bind-

ing 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 Kd and

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Bmax values were determined. Binding was carried out in 50 mM

Tris-HCl buffer containing 1 mM EGTA, pH 7.4, at room tempera-

ture for 1 h in duplicate in a final volume of 1 ml with 10 to 20 g

of membrane protein. Naloxone (10 M) was used to define nonspe-

cific 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 over-night. Cells were treated with 1 M ()U50,488H or left untreated

for 30 min at 37C, washed three times with ice-cold 10 mM phos-phate-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

g/ml; Sigma-Aldrich) in PBS containing 4% NGS and 0.05% Triton

X-100 at 37C for 30 min, rinsed three times with PBS containing0.05% Triton X-100 at room temperature, and incubated with goat

anti-mouse IgG (HL) conjugated with Alexa-Fluor 488 (2 to 4

g/ml; Molecular Probes) in PBS containing 4% NGS and 0.05%

Triton X-100 at room temperature for 30 min. After three washeswith 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 g/ml) pretreated with an exces-

sive amount of the FLAG peptide (100 g/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 aper-

ture 1.4 objective and fluorescein filter sets or with a confocal fluo-

rescence microscope (model IX70; Olympus, Tokyo, Japan) equipped

with a 60 numerical aperture 1.4 objective (Carl Zeiss, Thornwood,

NY).

Results

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 correla-

tion with phosphorylation and desensitization studies per-

formed previously (Li et al., 2001b). The FLAG-hkor and the

FLAG-rkor had similar binding affinities for [3H]diprenor-

phine 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 M

()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).

A 4-h pretreatment with ()U50,488H caused significant

down-regulation of the hkor and the FLAG-hkor, but not the

rkor and the FLAG-rkor (Fig. 1B; Table 1). Rather,

()U50,488H pretreatment caused a slight, yet significant

up-regulation of the rkor and the FLAG-rkor (Fig. 1B; Table

1). Even after 24-h pretreatment with ()U50,488H, the rkor

was not down-regulated (data not shown).

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 acid-

ification of intracellular vesicles and blocks the recycling of

Fig. 1. ()U50,488H-induced internalization and down-regulation of-opioid receptors: differences between human and rat receptors. CHOcells stably transfected with hkor, rkor, FLAG-hkor, or FLAG-rkor werepretreated without (basal) or with 1 M ()U50,488H at 37C for 30 min(A) or 4 h (B) followed by washing to remove the agonist. A, internalizedreceptors were determined by [3H]diprenorphine binding and presentedas percentage of the total receptor with the basal value (15% of totalreceptors) subtracted. Each value represents mean S.E.M. of seven (thehkor and the FLAG-hkor) and four (the rkor and the FLAG-rkor) inde-

pendent experiments in duplicate. B, membranes were prepared, satura-tion [3H]diprenorphine binding was performed, and Kd

and Bmax

valueswere determined. Kd values were not changed by the treatment. Receptornumbers were expressed as percentage of the untreated control. Each

value represents mean S.E.M. of three independent experiments induplicate. , p 0.01; , p 0.05, compared with the untreated controlby one-tail unpaired Students t test. ##, p 0.01; #, p 0.05, comparedwith the hkor (for the rkor) or the FLAG-hkor (for the FLAG-rkor) byone-tail unpaired Students t test.

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endocytosed receptors (Pippig et al., 1995) did not affect the

fraction of the rkor that is intracellular, both with and with-

out ()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.

These results indicate that there are differences in

()U50,488H-induced internalization and down-regulationof the rat and human -receptors, which is consistent with

our previous observations (Li et al., 1999, 2000). In addition,

epitope tagging with FLAG does not affect the internaliza-

tion and down-regulation characteristics of the hkor and

rkor.

Role of the C-Terminal Domain of the -Opioid Recep-

tor in ()U50,488H-Induced Internalization and Down-

Regulation. For many GPCRs, the intracellular regions, par-

ticularly 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 differ-

ences in the C-terminal domain (Fig. 3). To understand the

structural basis of the differences in ()U50,488H-induced in-

ternalization 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-termi-

nal domains were exchanged. The chimeras exhibited similar

binding affinities for [3H]diprenorphine as the wild types (Table1), and ()U50,488H displayed similar potency in stimulating

[35S]GTPS binding mediated by the wild types and the chime-

ras (Li et al., 2001b). Unlike the rkor or the FLAG-rkor, the

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 ofchimeric receptors and amino acid residues mutated in the hkor-S358Nand rkor-N358S mutants (Li et al., 2001b). Amino acid residue numbersare indicated on both sides. Seven putative transmembrane domains(TMs) are shaded. Chimeras FLAG-r/hkor (rkor1-338/hkor339-380) andFLAG-h/rkor (hkor1-338/rkor339-380) were generated by an exchange ofthe C-terminal domain fragments 338 to 380 as indicated by an arrow.

Amino acid residues 358 are indicated by . Ser358 in the hkor wasmutated to Asn and Asn358 in the rkor was substituted with Ser.

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 M ()U50,488H at 37C for 4 h followed by washingto remove the agonist. Membranes were prepared, saturation [3H]diprenorphine binding was performed, and Kd and Bmax values were determined. Each value representsmean S.E.M. of at least three independent experiments in duplicate.

ReceptorControl ()U50,488H-Treated

Kd Bmax Kd Bmax

nM pmol/mg nM pmol/mg

hkor 0.09 0.02 0.94 0.09 0.10 0.01 0.56 0.07*rkor 0.09 0.003 1.12 0.09 0.10 0.008 1.45 0.17*FLAG-hkor 0.06 0.01 1.89 0.19 0.20 0.07 1.37 0.11*FLAG-rkor 0.11 0.03 2.09 0.18 0.17 0.03 2.63 0.56*FLAG-h/rkor 0.05 0.01 1.51 0.01 0.08 0.02 1.51 0.01FLAG-r/hkor 0.12 0.04 2.97 0.42 0.16 0.05 1.94 0.24*FLAG-hkorS358N 0.08 0.02 1.64 0.40 0.15 0.02 2.44 0.61*FLAG-rkorN358S 0.14 0.01 2.21 0.15 0.19 0.02 2.43 0.23

* p 0.05 compared with the control by unpaired Student s t test.

Fig. 2. Effect of monensin on intracellular pool of the rkor (as percentageof total receptors) without or with ()U50,488H treatment. CHO-FLAG-rkor cells were pretreated without or with 50 M monensin for 1.5 h andtreated without or with 1 M ()U50,488H for the last 30 min. Intracel-lular receptors were determined as described under Materials and Meth-ods. Each value represents the mean S.E.M. of three independentexperiments in duplicate.

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FLAG-r/hkor underwent ()U50,488H-promoted internaliza-

tion and down-regulation (Fig. 4; Table 1). In addition, in con-

trast to the hkor and the FLAG-hkor, the FLAG-h/rkor pre-

treated 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.

Role of the Residues 358 in the C-Terminal Domains

of hkor and rkor in (

)U50,488H-Induced Internaliza-tion 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 spe-

cies differences. Both mutants displayed similar binding af-

finities 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).

S358N mutation in the FLAG-hkor greatly reduced

()U50,488H-induced internalization and abolished

(

)U50,488H-caused down-regulation (Fig. 5). Rather, theagonist led to a slight, yet significant, up-regulation of the

FLAG-hkorS358N receptor (Fig. 5B; Table 1) These results

indicate that the S358 of the hkor plays a key role in

()U50,488H-induced internalization and down-regulation.

In contrast, preincubation of CHO-FLAG-rkorN358S cells

with ()U50,488H caused internalization at a level compa-

rable with that of the FLAG-hkor; however, no significant

Fig. 5. ()U50,488H-induced internalization and down-regulation of the

FLAG-rkorN358S and the FLAG-hkorS358N mutants. CHO cells stablytransfected with the FLAG-rkorN358S mutant or the FLAG-hkorS358Nmutant were treated without (control) or with 1 M ()U50,488H at 37Cfor 30 min (A) or 4 h (B) followed by washing to remove the agonist. A,internalized receptors were determined by [3H]diprenorphine bindingand presented as percentage of the total receptor. Each value representsmean S.E.M. of eight independent experiments in duplicate. B, mem-branes were prepared and saturation [3H]diprenorphine binding wasperformed, and K

dand B

maxvalues were determined. K

dvalue was not

changed by the treatment. Receptor numbers were expressed as percent-age of theuntreated control. Each value represents mean S.E.M.of fourindependent experiments in duplicate. ,p 0.01; ,p 0.05, comparedwith the untreated control by one-tail unpaired Student s t test. #, p 0.05, compared with the FLAG-hkor (for the FLAG-hkorS358N) or theFLAG-rkor (for the FLAG-rkorN358S) by one-tail unpaired Student s ttest.

Fig. 4. ()U50,488H-induced internalization and down-regulation of chi-

meric -opioid receptors. CHO cells stably transfected with the FLAG-h/rkor or the FLAG-r/hkor were pretreated without (control) or with 1 M()U50,488H at 37C for 30 min (A) or 4 h (B) followed by washing toremove the agonist. A, internalized receptors were determined by [3H]di-prenorphine binding and presented as percentage of the total receptor.Each value represents mean S.E.M. of six (for the FLAG-r/hkor) andeight (for the FLAG-h/rkor) independent experiments in duplicate. B,membranes were prepared and saturation [3H]diprenorphine bindingwas performed and K

dand B

maxvalues were determined. K

dvalue was

not changed by the treatment. Receptor numbers were expressed aspercentage 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 Studentst test. ##, p 0.01; #, p 0.05, compared with the FLAG-hkor (for theFLAG-h/rkor) or the FLAG-rkor (for the FLAG-r/hkor) by one-tail un-paired Students t test.

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down-regulation was observed for this mutant (Fig. 5B; Table

1). Even after 24 h incubation with 1 M ()U50,488H (add-

ed every 4 h), the FLAG-hkorS358N mutant was not down-

regulated. Thus, N358 of the rkor is important in its inability

to undergo internalization; however, its role in lack of down-

regulation is not clear.

Detection of Receptor Internalization by Immuno-

fluorescence Staining. Immunofluorescence staining with

the M1 anti-FLAG antibody and goat anti-mouse IgG conju-gated with Alexa-Fluor 488 was performed to visualize dis-

tribution 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 fluo-

rescence staining and a decrease in cell surface staining of

the FLAG-hkor, but not the FLAG-rkor (Fig. 6). The intra-

cellular fluorescence was punctate and seemed to accumulate

in the perinuclear region. In addition, ()U50,488H treat-

ment 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).

Discussion

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 con-

tribute to the differences in internalization and down-regu-

lation 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 regulationbetween the hkor and rkor represents the first demonstra-

tion of such species difference in the regulation of GPCRs. In

addition, this study provides the first evidence for the impor-

tance of Ser358 in ()U50,488H-induced internalization and

down-regulation of the hkor.

Determination of Receptor Internalization by Bind-

ing 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 readilyinternalized by ()U50,488H pretreatment for 30 min, con-

sistent 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).

In accord with these findings is that ()U50,488H en-

hances phosphorylation of the FLAG-hkor, which is GRK-

mediated (Li et al., 2001b). We have previously shown that

expression of dominant negative mutants of GRK2 and ar-

restin-2 reduces ()U50,488H-promoted internalization and

down-regulation of the hkor (Li et al., 1999, 2000), demon-

strating the involvement of GRKs and arrestins in these

processes. In addition, ()U50,488H-induced down-regula-

tion of the hkor involves dynamin-, rab5-, and rab7-depen-

dent mechanisms, and receptors seem to be trafficked to

lysosomes and proteasomes for degradation (Li et al., 2000).

Lack of Internalization and Down-Regulation of

rkor and FLAG-rkor. A 30-min incubation with 1 M

()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-regulationof 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 M

()U50,488H for 24 h. Because the amino acid sequences of

the mouse and rat -receptors are 99% identical overall and

Fig. 6. ()U50,488H-induced internalization of FLAG-hkor, FLAG-rkor, FLAG-h/rkor, FLAG-r/hkor, FLAG-hkorS358N, and FLAG-rkorN358Svisualized by immunofluorescence microscopy. Transfected CHO cells expressing one receptor were incubated in the presence or the absence (control)of 1 M ()-U50,488H at 37C for 30 min and then fixed by 4% paraformaldehyde and permeabilized using 0.05% Triton X-100. Immunofluorescencestaining was performed with anti-FLAG M1 mouse monoclonal antibody as primary antibody and goat anti-mouse IgG conjugated with Alexa-Fluor488 as secondary antibody as described under Materials and Methods. The figures represent one of the three experiments performed with similarresults.

Internalization and Down-Regulation of -Opioid Receptors 1189

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100% identical in intracellular regions, this difference may

be a reflection of the different cell systems.

CHO cells contain endogenous GRK2, GRK3, and GRK6 (J.

Benovic, personal communication). The lack of internaliza-

tion and down-regulation of the FLAG-rkor by ()U50,488H

may be due to insufficient levels of GRKs and arrestins for

the FLAG-rkor to undergo these processes, even though the

levels seem to be sufficient for the FLAG-hkor. However, we

found that expression of GRK2, GRK3, GRK5 or GRK6 didnot enhance ()U50,488H-induced phosphorylation of the

FLAG-rkor expressed in CHO cells (Li et al., 2001b; C. Chen,

J. Li ,and L.-Y. Liu-Chen, unpublished observation). In ad-

dition, expression of GRK2 and arrestin-2 or GRK3 and ar-

restin-3 did not enable the FLAG-rkor to be desensitized

after ()U50,488H exposure (Li et al., 2001b). Thus, the lack

of internalization and down-regulation of the FLAG-rkor is

not the result of insufficient levels of nonvisual GRKs and

arrestins.

Our results that the rkor and FLAG-rkor were not inter-

nalized or down-regulated by ()U50,488H are consistent

with the reports that ()U50,488H induced little phosphor-

ylation and desensitization of the rkor stably expressed in

CHO cells (Avidor-Reiss et al., 1995; Li et al., 2001b) andslight desensitization of the rkor expressed in Xenopus oo-

cytes (Appleyard et al., 1999). However, Tallent et al. (1998)

reported that ()U50,488H pretreatment caused desensiti-

zation of mouse -opioid receptor expressed in AtT-20 cells.

The discrepancy among these results may be due to different

cell systems and functional endpoints used.

Internalization and Down-Regulation of Chimeric

and Mutant Receptors. Pretreatment of FLAG-r/hkor, but

not FLAG-h/rkor, with ()U50,488H resulted in internaliza-

tion and down-regulation, demonstrating that the C-termi-

nal domains contribute to the difference between the hkor

and the rkor.

GRKs, which are Ser/Thr kinases, have been implicated in

()U50,488H-induced phosphorylation of the FLAG-hkor (Liet al., 2001b). The C-terminal domains of both the hkor and

rkor have two Ser and two Thr residues: S356, T357, S358,

and T363 in the hkor and S356, T357, T363, and S369 in the

rkor (Fig. 2). One Ser/Thr present in the hkor, but not in the

rkor, is S358, where it is N in the rkor. Indeed, our results

showed that S358 of the hkor played critical roles in

()U50,488H-induced internalization and down-regulation.

In contrast, N358 seems to prevent the rkor from being

internalized by ()U50,488H, but did not seem to have a role

in its lack of down-regulated.

Our results that S358 of the hkor is crucial for

()U50,488H-induced internalization and down-regulation

are consistent with those of Cheng et al. (1998), who reported

that expression of arrestin-2 reduced hkor-mediated func-

tional responses and S356/T357/S358 of the hkor play an

important role in the arrestin-2 effect.

Discrepancy in ()U50,488H-Promoted Down-Regu-

lation 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 conforma-

tional 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 un-

derwent 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 dynaminI-K44A, which attenuated ()U50,488H-promoted internal-ization of the hkor, significantly reduced ()U50,488H-in-

duced 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 dem-

onstrated for GPCR mutants. Some mutants exhibited recep-

tor internalization equivalent to that of the wild type, yet

showed blunted receptor down-regulation in response to ago-nists; for example, several mutants of the 2AR with substi-

tutions in the third intracellular loop and C-terminal domain

(Campbell et al., 1991) and Tyr459 mutants in the C-termi-

nal domain of the m2 mAChR (Goldman and Nathanson,

1994). Conversely, several GPCR mutants have been shown

to have greatly reduced agonist-mediated receptor internal-

ization, yet still retain the ability to undergo down-regula-

tion; 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 inter-

nalization 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 maynot be trafficked in the same manner as the wild-type recep-

tors. Another likely explanation is that internalization was

determined after a short period of agonist treatment,

whereas down-regulation was measured after a longer treat-

ment period. An alteration in the rate or extent of internal-

ization may not affect the degree of down-regulation.

Relationship between ()U50,488H-Induced Phos-

phorylation 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. Al-

though 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 do-

main 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

1190 Zhang et al.

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sufficiently high affinity for the FLAG-rkorN358S to permit

internalization of the unphosphorylated receptor.

In addition, there seems to be a correlation between

()U50,488H-induced phosphorylation and down-regulation

for the wild-type, chimeric, and mutant -opioid receptors.

Previous studies have shown that a major point where phos-

phorylation of the receptor regulates its lysosomal/proteoso-

mal degradation is at the internalization step (Li et al., 2000,

2001b; Maestri-El Kouhen et al., 2000; Trapaidze et al., 2000;Whistler et al., 2001). However, it is noteworthy that the

FLAG-rkorN358S was not phosphorylated or down-regu-

lated, but was internalized. Whether the lack of down-regu-

lation of this mutant is related to its lack of phosphorylation

is not clear. Using truncated and substitution mutants of the

-opioid receptor, Whistler et al. (2001) showed that after

endocytosis occurs, subsequent trafficking to lysosomes did

not require receptor phosphorylation. In addition, mutation

of S356 and S363 in the -opioid receptor did not affect

agonist-induced phosphorylation, but greatly attenuated its

down-regulation (Burd et al., 1998). Thus, there may not be

a clear relationship between phosphorylation and down-reg-

ulation of GPCR mutants and mutant receptors may be traf-

ficked differently from wild-type receptors, as mentionedabove.

Up-Regulation of rkor, FLAG-rkor, and FLAG-

hkorS358N by ()U50,488H. It is intriguing that incuba-

tion with ()U50,488H for 4 h did not induce down-regula-

tion 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 37C in the presence ofprotease inhibitors, the rat -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, thestabilization effects may become evident. All cDNA con-

structs 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 re-

ceptors 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 demon-

strated in this study may have significant implications when

extrapolating studies on regulation of-opioid receptors from

rats to humans.

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Address correspondence to: Dr. Lee-Yuan Liu-Chen, Department of Phar-macology, Temple University School of Medicine, 3420 N. Broad St., Philadel-

phia, PA 19140. E-mail: lliuche@astro.temple.edu

1192 Zhang et al.