©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of Irreversible Binding of -Funaltrexamine to the Cloned Rat Opioid Receptor (*)

(Received for publication, May 4, 1995)

Chongguang Chen Ji-Chun Xue Jinmin Zhu Yung-Wu Chen (1) Satya Kunapuli (2) J. Kim de Riel (3) Lei Yu (4) Lee-Yuan Liu-Chen (§)

From the  (1)Departments of Pharmacology, Microbiology and Immunology, and (2)Physiology, the (3)Fels Institute for Molecular Biology and Cancer Research, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, and the (4)Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, Indiana 46202

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Binding of -funaltrexamine (-FNA) to the cloned rat µ opioid receptor expressed in COS-1 cells or Chinese hamster ovary cells was examined. -FNA bound to the µ receptor with high affinity. Irreversible binding of [H]-FNA was defined as the binding that could not be dissociated by trichloroacetic acid. Na greatly enhanced the specific irreversible binding of [H]-FNA to the µ receptor, which was concentration- and time-dependent. Specific irreversible binding of [H]-FNA was potently inhibited by CTAP (a µ ligand), but not by ICI174,864 (a ligand) or U50,488H (a ligand). These results indicate that [H]-FNA binds irreversibly to the cloned µ opioid receptor. SDS-polyacrylamide gel electrophoresis and fluorography showed that [H]-FNA-labeled receptors migrated as one broad and diffuse band with a mass of 80 kDa in Chinese hamster ovary or COS cells and as one band with a mass of 67 kDa in the rat brain preparation. Upon removal of N-linked carbohydrates, labeled receptors became a sharper band with a mass of 40 kDa. [H]-FNA did not bind irreversibly to the cloned rat receptor. [H]-FNA binding to four chimeric µ/ receptors was examined. The region from the middle of the third intracellular loop to the C terminus of the µ receptor is necessary for irreversible binding of -FNA.


INTRODUCTION

The existence of at least three types of opioid receptors (µ, , and ) has been demonstrated(1) . The µ opioid receptors mediate many effects of opiates and opioid compounds, including, most notably, modulation of pain perception(1) . Activation of µ opioid receptors couples via pertussis toxin-sensitive G proteins to various effectors including adenylate cyclase and K and Ca channels(1) . Cloning of the µ opioid receptor has been reported recently by several laboratories(2, 3, 4, 5, 6, 7, 8) . Hydropathy analysis of deduced amino acid sequences of these clones indicates the presence of seven putative transmembrane helices (TMHs)()separated by intra- and extracellular loops, characteristics of G protein-coupled receptors.

Affinity ligands that form covalent bonds with receptors have been very useful in the elucidation of receptor structure. Specific incorporation of radiolabeled affinity ligand into the receptor followed by SDS-PAGE and fluorography or autoradiography has been used to identify the receptor, to determine the molecular mass of the receptor without purification, and to examine the nature of carbohydrate moieties (see, for example, (9) ). Because of the covalent nature of the bond between the ligand and the receptor, the site of incorporation, thus part of the binding domain, can be precisely determined by peptide mapping and/or determination of amino acid sequences of labeled fragments. For example, [H]SKF 102,229 labeled adrenergic receptor as a glycoprotein with a mass of 64 kDa(10) . By peptide mapping of [H]SKF 102,229 (antagonist)-labeled and [H]azidoclonidine (agonist)-labeled -adrenergic receptors, Matsui et al.(11) identified the fourth TMH to be the region that formed covalent bonds with these ligands and, thus, to be part of the binding domain.

-Funaltrexamine (-FNA), synthesized by Portoghese et al.(12) , was shown to have reversible agonist and irreversible µ antagonist activities (for a review, see (13) ). The high selectivity and irreversible nature of its action on µ opioid receptors makes -FNA a very useful pharmacological tool both in vivo and in vitro. Binding of -FNA to opioid receptors in tissue membrane preparations in vitro has also been characterized(14, 15, 16, 17) . -FNA binds to µ, , and receptors with IC values of 2.2, 14, and 78 nM, respectively(14) . There is a general agreement that -FNA binds reversibly, and not irreversibly, to opioid receptors. -FNA binds irreversibly to µ opioid receptors (14, 15) and at low concentrations (1-10 nM) [H]-FNA covalently labels only µ opioid binding sites with high specificity(18, 19, 20) . In addition, at concentrations 18 nM, -FNA also binds irreversibly to receptors(14) . [H]-FNA-labeled µ opioid receptors in the rat brain have broad molecular mass ranges from 60-75 kDa (median: 67 kDa)(9) , indicative of glycoprotein nature. Upon deglycosylation by N-glycanase®, labeled rat µ receptors became a sharp band of 39 kDa(9) .

Chimeric receptors of closely related receptors have been very useful in the delineation of ligand binding domains of receptors. By examining the binding characteristics of µ/ chimeric receptors, we (21) and Wang et al.(22) demonstrated that the second extracellular loop of the receptor was essential for the high affinity binding of dynorphin family peptides. In addition, we found that the TMHs 6 and 7 and the third extracellular (e3) loop of the receptor was critical for the binding of the selective antagonist norbinaltorphimine(21) . Moreover, we demonstrated that the TMHs 6 and 7 and the e3 loop of the µ receptor is important for binding of selective agonists(23) . Kong et al.(24) examined the binding characteristics of / chimeric receptors and found that the binding domain of selective antagonists was located in the N-terminal domain. Onogi et al.(25) reported that the determinant of selectivity of the µ receptor for Tyr-D-Ala-Gly-NMePhe-Gly-ol was located in the first extracellular loop, based on analysis of binding of [H]Tyr-D-Ala-Gly-NMePhe-Gly-ol to a series of chimeric µ/ opioid receptors

-FNA has been shown to bind irreversibly to both µ and µ receptors in brain membranes(15) . Recent cloning of the µ opioid receptor makes it possible to study the interaction of -FNA with one single type of µ receptors at the molecular level. In this study, we examined the binding of [H]-FNA to the cloned rat µ opioid receptor (RMOR) (2) , as compared to native receptors in the rat brain. In addition, we also determined the region in the µ receptor that conferred selectivity for covalent binding of -FNA. For this purpose, we examined binding of -FNA to chimeric µ/ receptors constructed from cloned rat µ and opioid receptors(2, 26) , since the RMOR does, yet the rat receptor (RKOR) does not, form a covalent bond with [H]-FNA.


MATERIALS AND METHODS

Transient Expression of Rat µ and Opioid Receptors and Chimeric µ/ Receptors in COS-1 Cells

Rat µ and receptors and chimeras were transfected into COS-1 cells with DEAE-dextran-chloroquine method and membranes of COS cells transfected with RMOR (COS-RMOR) were prepared for binding as described(21, 26) .

Stable Expression of RMOR in Chinese Hamster Ovary (CHO) Cells

RMOR is in the vector pRc/CMV, which contains a neomycin-resistant gene. Transfection of CHO cells with RMOR was performed with 30 µl of Lipofectin® (1 mg/ml) and 10 µg of DNA/100-mm dish according to Felgner et al.(27) . After transfection and 48-60 h in the growth medium, cells were subcultured at the ratio of 1:5 into the growth medium containing Geneticin (0.5 mg/ml) for 4 weeks for selection of cells expressing the neomycin-resistant gene. Clonal cells were grown in 24-well plates, and [H]diprenorphine binding was performed on cells adhering to wells(28) . CHO cells expressing the rat µ opioid receptor (CHO-RMOR) were maintained in medium containing Geneticin (0.2 mg/ml) and membranes prepared for binding(26) .

Rat Brain Membrane Preparations

Brain membranes of Sprague-Dawley rats were prepared as described(18) .

Inhibition of [H]Diprenorphine Binding by -FNA

Binding experiments were performed in the presence of 100 mM NaCl with [H]diprenorphine at a concentration close to its K for each receptor and 9 concentrations of -FNA at room temperature for 60 min as described previously(26) .

Labeling of µ Opioid Receptors with [H]-FNA

Membranes suspended in 50 mM TEL buffer (50 mM Tris-HCl buffer, 1 mM EGTA, 10 µM leupeptin) were incubated with [H]-FNA at specified concentrations in the presence of indicated concentrations of NaCl at 37 °C for specified periods of time. Each assay tube contained 20-50 µg of membrane proteins. Nonspecific labeling was performed in the presence of 10 µM naloxone. Selective drugs were added at various concentrations for characterization on inhibition of irreversible binding.

Assay for Irreversible Binding of [H]-FNA

Irreversible binding of [H]-FNA in labeled membranes was assayed according to our published method(9, 19) . Labeled membranes were precipitated with trichloroacetic acid, filtered over GF/B filters, and radioactivity on the filter determined.

Solubilization of Labeled Membranes and WGL Affinity Chromatography

Both solubilization and WGL affinity chromatography were carried at 4 °C according to our published procedure(9) . Solubilization was carried out with 2% Nonidet P-40 instead of 2% Triton X-100.

Treatment of WGL Column Eluate or Solubilized Membrane Preparation with Peptide-N[N-acetyl--glucosaminyl]Asparagine Amidase (N-Glycanase®)

N-Glycanase treatment was conducted as described previously(9) .

SDS-PAGE and Detection of Radioactivity in the Gel by Fluorography

SDS-PAGE and fluorography was performed as described previously (9) with C-labeled protein molecular mass standards .

Construction of Chimeric µ/ Receptors

Four chimeric µ/ receptors (III, IV, XI, and XII) constructed in the vector pcDNA3 or pBK-CMV from RMOR and RKOR were used in this study. Chimera III (amino acids 1-141/µ151-398) and chimera IV (amino acids µ1-150/142-380) were constructed by swapping the regions from the N terminus to the start of the TMH 3. Chimera XI (amino acids µ1-268/263-380) and chimera XII (amino acids 1-262/µ269-398) were generated by exchanging the regions from the middle of the third intracellular (i3) loop to the C terminus. Details of generation of these chimeras were described previously(21) .

Protein Determination

Protein contents of membranes and solubilized preparations were determined by the BCA method of Smith et al.(29) with bovine serum albumin as the standard.

Materials

[H]-FNA (15.6 Ci/mmol) was supplied by National Institute on Drug Abuse. [H]diprenorphine (35 Ci/mmol) was purchased from Amersham Corp. Naloxone and U50,488H were generously provided by DuPont Merck Pharmaceutical Co. and Upjohn, respectively. CTAP and ICI174,584 were purchased from Chiron Co. (San Diego, CA). WGL-Sepharose 6MB and DEAE-dextran were obtained from Pharmacia Biotech Inc.; peptide-N[N-acetyl--glucosaminyl]asparagine amidase (EC 3.5.1.52) (from Flavobacterium meningosepticum) (N-glycanase®) from Genzyme Co. (Boston, MA); Lipofectin® reagent and C-labeled standards from Life Technologies, Inc.


RESULTS

Inhibition of [H]Diprenorphine Binding to the µ Receptor by -FNA

-FNA inhibited 0.4 nM [H]diprenorphine binding to the µ receptor expressed in COS-1 cells with high affinity. The IC value was calculated to be 1.7 ± 0.2 nM (n = 3) after a 60-min incubation at 25 °C.

Effect of Incubation Time on Irreversible Binding of [H]-FNA

Membranes of COS-RMOR cells were incubated with 4 nM [H]-FNA in the presence of 100 mM NaCl at 37 °C for various periods of time. The nonspecific binding increased very slowly with time, whereas the specific irreversible binding increased rapidly with time and approached a plateau at 100-120 min (Fig. 1).


Figure 1: Time course of irreversible binding of 4 nM [H]-FNA to the µ receptor expressed in COS-1 cells. Membranes of COS-RMOR cells were pretreated with or without 10 µM naloxone for 20 min and then incubated at 37 °C with 4 nM [H]-FNA in the presence of 100 mM NaCl for various periods of time. Irreversible binding was then determined. Each value represents the mean of duplicate determinations. This experiment was performed four times in duplicate with similar results.



Effect of [H]-FNA Concentration on Its Irreversible Binding

Membranes of COS-RMOR cells were incubated in the presence of 100 mM NaCl at 37 °C for 75 min with various concentrations of [H]-FNA. While the nonspecific binding increased with [H]-FNA concentration, the specific irreversible binding of [H]-FNA reached a plateau at 3 nM (Fig. 4, RMOR). Untransfected COS-1 or CHO cells showed no specific irreversible binding of [H]-FNA.


Figure 4: Irreversible binding of [H]-FNA to RMOR, RKOR, and chimeric µ/ receptors (III, IV, XI, and XII). Membranes of COS-1 cells transfected with RMOR, RKOR, or chimeric µ/ receptors (III, IV, XI, or XII) were pretreated with or without 10 µM naloxone for 20 min, followed by incubation with various concentrations of [H]-FNA in the presence of 100 mM NaCl at 37 °C for 75 min. Irreversible binding was then determined. Symbols used are as follows: , total binding; , nonspecific binding; , specific binding. For each, receptor concentration was 50-60 fmol/ml/tube. This figure represents one of three to five experiments performed for each receptor with similar results. Variations between experiments were less than 10%.



Effect of NaConcentration on the Specific Irreversible Binding of [H]-FNA

NaCl greatly enhanced the specific irreversible binding of [H]-FNA to the µ opioid receptor with the maximal effect at 200 mM. When compared to the level of specific binding in the presence of 100 mM NaCl (designated as 100%), those in the presence of various NaCl concentrations were as follows: no NaCl, 35.1%; 50 mM, 81.4%; 200 mM, 113.8%; 300 mM, 110.6% (mean of two experiments).

Inhibition of Specific Irreversible Binding of [H]-FNA by µ, , and Ligands

Specific irreversible binding of [H]-FNA to RMOR expressed in COS-1 cells was potently inhibited by CTAP (a selective µ ligand), but not by U50,488H (a ligand) and ICI174,864 (a ligand). The IC of CTAP was approximately 70 nM. It should be noted that the IC value of CTAP, a reversible ligand, in inhibiting the irreversible binding of [H]-FNA does not represent its true affinity. In contrast, neither ICI174,864 nor U50,488H at 10 µM inhibited [H]-FNA irreversible binding at all.

SDS-PAGE and Fluorography of Labeled Preparations

As shown in Fig. 2, [H]-FNA specifically labeled one band with a mass of 70-89 kDa (median: 80 kDa) in WGL-purified COS-RMOR preparation, one band with mass of 70-89 kDa (median: 80 kDa) in CHO-RMOR membranes, and one band with mass of 61-78 kDa (median: 67 kDa) in the WGL-purified rat brain preparation. Naloxone inhibited the labeling of the 80-kDa protein bands. We have shown previously that in the WGL-purified rat brain preparation, there is only one labeled band of 67 kDa, of which labeling is greatly reduced by naloxone(9) . All these three bands were broad and diffuse, indicating that labeled receptors are glycoproteins. Membranes of COS-1 cells transfected with the vector pcDNA3 alone did not show any labeled protein band (not shown).


Figure 2: SDS-PAGE and fluorography of [H]-FNA-labeled µ opioid receptors in the WGL affinity-purified rat brain preparation (lane 1), WGL affinity-purified COS-RMOR preparation labeled in the absence (lane 2) and the presence (lane 3) of 10 µM naloxone and in CHO-RMOR membranes labeled in the absence (lane 4) and the presence (lane 5) of 10 µM naloxone. Amounts of proteins and radioactivities loaded onto each lane were as follows: lane1, 500 µg, 20,000 dpm; lane2, 300 µg, 15,000 dpm; lane3, 300 µg, 7,000 dpm; lane4, 600 µg, 24,000 dpm; lane5, 600 µg, 6000 dpm, respectively. Molecular mass standards were in kDa. Exposure time was 5 days. This experiment was performed five times with similar results.



Deglycosylation of Labeled µ Receptors with N-Glycanase®

After deglycosylation with N-glycanase® to remove Asn-linked carbohydrates, both the 80-kDa protein band in the CHO-RMOR membranes and the 67-kDa band in the rat brain preparation became sharp bands of 40 kDa (Fig. 3). These results indicate that the difference in the molecular mass of cloned receptors and native receptors reflects differences in the extent of glycosylation.


Figure 3: Deglycosylation by N-glycanase® of [H]-FNA-labeled µ opioid receptors in the WGL affinity-purified rat brain preparation and in CHO-RMOR membranes. The WGL affinity-purified rat brain preparation and solubilized CHO-RMOR membranes were treated with or without N-glycanase® for 6 h and subjected to SDS-PAGE and fluorography as described under ``Materials and Methods.'' Lanes1 and 2, rat WGL preparation, control and N-glycanase®-treated, respectively, 12,400 dpm and 300 µg of protein each. Lanes 3 and 4, CHO-RMOR, control and N-glycanase®-treated, respectively, 24,000 dpm and 600 µg of protein each. Exposure time was 5 days. This experiment was performed two times with similar results.



Binding of -FNA to Chimeric µ/ Receptors

We have previously shown that chimeras III, IV, XI, and XII bound [H]diprenorphine with high affinity, with K values of 0.30, 0.78, 0.49, and 0.14 nM, respectively(21) . These values are similar to K values of µ and receptors (0.35 and 0.26 nM, respectively)(21) . -FNA inhibited [H]diprenorphine binding to all four chimeras with high affinity with IC values of 1.7, 12.5, 3.0, 13.8, 5.5, and 3.5 nM for µ, , III, IV, XI, and XII respectively (Table 1).



Irreversible Binding of [H]-FNA to RMOR, RKOR, and Chimeric µ/ Receptors

Irreversible binding of a range of concentrations of [H]-FNA was carried out (Fig. 4). [H]-FNA bound irreversibly to RMOR, but not RKOR. While chimeras III showed high [H]-FNA specific irreversible binding, similar to RMOR, [H]-FNA did not bind irreversibly to chimeras IV (Fig. 4). Thus, the region from the TMH 3 to the C terminus of the µ receptor is necessary for irreversible binding of [H]-FNA. To further narrow down the region, we examined irreversible binding to chimeras XI and XII. XII, constructed by substitution of the portion from the middle of i3 loop to the C terminus of RKOR with that of RMOR, acquired high [H]-FNA specific irreversible binding, similar to RMOR (Fig. 4). On the other hand, XI, constructed by replacement of the portion from the middle of i3 loop to the C terminus of RMOR with that of RKOR, did not display any [H]-FNA specific irreversible binding (Fig. 4). These results indicate that the region from the middle of the i3 loop to the C terminus is essential for irreversible binding of [H]-FNA.

Chimeras IV and XI were expressed at lower levels in COS-1 cells than RKOR, RMOR, chimeras III and XII, in terms of fmol/mg of membrane protein(21) . In experiments shown in Fig. 4, receptor concentrations were adjusted to similar levels for all receptors (50-60 fmol/ml/tube). Therefore, the amount of protein in the assay was different among these receptors, which accounted for different levels of nonspecific binding of each receptor. Expression levels of a given receptor from different transfection experiments were remarkably similar(21) . Thus, the variations between experiments were small.


DISCUSSION

In this study, we demonstrated that the cloned rat µ opioid receptor bound -FNA with a high affinity and could be specifically and irreversibly labeled by [H]-FNA under defined conditions. [H]-FNA-labeled receptors in COS-1 or CHO cells appeared as one diffuse broad band of 80 kDa, as compared to 67 kDa of the µ receptor in the rat brain. Upon deglycosylation, labeled receptors became much sharper bands with similar M values of 40 kDa. By examining irreversible binding of [H]-FNA to four chimeric µ/ receptors, we demonstrated that the portion from the middle of the i3 loop to the C terminus of the µ receptor was important for covalent binding of [H]-FNA.

At 37 °C, the irreversible binding of [H]-FNA to the cloned µ receptor was time- and [H]-FNA concentration-dependent, reaching a plateau at 3 nM and 100-120 min. The presence of Na was essential for high level of [H]-FNA irreversible binding to the µ receptor. CTAP potently inhibited irreversible binding of [H]-FNA, whereas ICI174,584 and U50,488H did not. These results are consistent with our previous observation that [H]-FNA binds irreversibly to the µ receptor in brain membranes(9, 18, 19, 20) . It has been thought that -FNA reacts with a Cys residue in the vicinity of the binding site of µ receptors to form a covalent bond(12, 14) .

The µ receptors in brain membranes contain the complex type of carbohydrates linked to the Asn residue of the receptor(9) . The cloned rat µ receptor contains five consensus Asn-linked glycosylation sites (2) . Whether each Asn residue is glycosylated remains to be determined. The carbohydrate moiety comprises a high percentage of the µ receptor molecular mass. The exact function of the carbohydrate moiety is not known. Native µ receptors and cloned µ receptors expressed in COS-1 or CHO cells, although displaying different extents of glycosylation, exhibited very similar binding and signal transduction properties(2) . These observations indicate that carbohydrate moieties are not important in binding or signal transduction. Similar conclusions were reached by Pasternak et al.(30) and Law et al.(31) . Neuraminidase treatment did not affect opioid receptor binding (30) . Law et al.(31) found that glycosylation plays an important role in the structural maturation and insertion into plasma membranes of opioid receptors in NG108-15 cells, but not for ligand binding or activation of the second messenger system. To our knowledge, no similar study on the µ receptor has been performed.

Deglycosylated µ receptors have a molecular mass of 40 kDa, which is smaller than the molecular mass of 44 kDa predicted from the deduced amino acid sequence(2) . The reason for this discrepancy is not clear. There may be other post-translational modifications of µ opioid receptor proteins, such as phosphorylation, palmitoylation, or isoprenylation, which affect mobility of labeled receptors in SDS-PAGE. Another possibility that cannot be entirely ruled out is that a part of the receptor molecule is particularly susceptible to protease activities. This part may be removed during the process of [H]-FNA labeling (37 °C, 75 min) and/or N-glycanase® treatment (37 °C, 6 h), even though protease inhibitors were present during these processes.

Irreversible binding of [H]-FNA to chimeric µ/ receptors was examined to define the structural basis of µ selectivity of -FNA irreversible binding. All four chimeras bound [H]diprenorphine with high affinity, similar to µ and receptors(21) . Binding of [H]diprenorphine to all chimeras was completely blocked by naloxone. These results indicate that these chimeras retain opioid receptor conformation to some extent. All four chimeras bound -FNA with similar high affinities as µ and receptors, yet [H]-FNA bound irreversibly to chimeras III and XII, but not IV and XI. Thus, the inability of chimeras IV and XI and the receptors to bind -FNA irreversibly is not due to their inability to bind -FNA reversibly. These findings indicate that the region from the third i3 loop to the C terminus is necessary for covalent binding of [H]-FNA.

Two possibilities may exist. First, the site of -FNA covalent incorporation is within the region from the i3 loop to the C terminus. Amino acid sequences within the i3 loop, most of TMH 6 and TMH 7 are very similar between µ and receptors. The C-terminal domain of the µ receptor does not contribute to ligand binding(32) . Thus, the sequence in and around the e3 loop of the µ receptor appears to confer selectivity for -FNA irreversible binding. Second, chimeras III and XII assume favorable conformations for -FNA to form covalent bonds with the receptors, whereas chimeras IV and XI do not. Preservation of opioid receptor conformation of the chimeras was confirmed by high affinity binding of diprenorphine, naloxone, and -FNA. However, the possibilities cannot be excluded that there are potential local conformational changes in the binding pocket, or that there are alterations in direct interactions between the receptor and the ligand(33) . We are currently conducting peptide mapping analysis of [H]-FNA-labeled receptor to determine the region of [H]-FNA incorporation.

In conclusion, the cloned rat µ opioid receptor expressed in COS-1 or CHO cells could be specifically and covalently labeled by [H]-FNA. The labeled receptor appeared as one diffuse broad band of 80 kDa by SDS-PAGE and fluorography and upon deglycosylation, it became a sharp band of 40 kDa. The region between the middle of the i3 loop to the C terminus in the µ receptor is necessary for covalent bond formation with [H]-FNA. We will determine the region and eventually the amino acid residue that forms covalent bond with [H]-FNA. Since [H]-FNA is a rigid molecule, the information will be very useful for computerized modeling of the µ receptor.


FOOTNOTES

*
This work was supported by Grant DA 04745 from NIDA, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom reprint requests should be addressed: Dept. of Pharmacology, Temple University School of Medicine, 3420 N. Broad St., Philadelphia, PA 19140. Tel.: 215-707-4188; Fax: 215-707-7068; liuchen{at}sgi1.fels.temple.edu

The abbreviations: TMH, transmembrane helix; CHO, Chinese hamster ovary; e3 loop, the third extracellular loop; CHO-RMOR, CHO cell lines stably expressing the rat µ opioid receptor; COS-RMOR, COS-1 cells transiently expressing the rat µ opioid receptor; CTAP, cyclic D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH; -FNA, -funaltrexamine; i3 loop, the third intracellular loop; PAGE, polyacrylamide gel electrophoresis; RMOR, the rat µ opioid receptor; RKOR, the rat opioid receptor; WGL, wheat germ lectin.


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