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(Received for publication, May 4, 1995) From the
Binding of The existence of at least three types of opioid receptors
(µ, 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, [ 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/
Figure 1:
Time course of irreversible binding
of 4 nM [
Figure 4:
Irreversible binding of
[
Figure 2:
SDS-PAGE and fluorography of
[
Figure 3:
Deglycosylation by N-glycanase® of
[
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. In this study, we demonstrated that the cloned rat µ
opioid receptor bound At 37
°C, the irreversible binding of [ 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 Deglycosylated µ
receptors have a molecular mass of Irreversible binding of [ Two possibilities may exist.
First, the site of In conclusion, the cloned rat µ opioid receptor
expressed in COS-1 or CHO cells could be specifically and covalently
labeled by [
Volume 270,
Number 30,
Issue of July 28, pp. 17866-17870, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-Funaltrexamine to the Cloned Rat
Opioid Receptor (*)
-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.
, 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.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, 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.
Transient Expression of Rat µ and
Rat
µ and Opioid
Receptors and Chimeric µ/ Receptors in COS-1 Cells 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 [
Binding experiments were performed in
the presence of 100 mM NaCl with
[H]Diprenorphine
Binding by -FNAH]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
[
Membranes suspended
in 50 mM TEL buffer (50 mM Tris-HCl buffer, 1 mM EGTA, 10 µM leupeptin) were incubated with
[H]-FNAH]-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
[
Irreversible binding
of [H]-FNAH]-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-Glycanase treatment
was conducted as described previously(9) .
[N-acetyl--glucosaminyl]Asparagine
Amidase (N-Glycanase®)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/
Four chimeric µ/
Receptors 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.
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
[
Membranes of
COS-RMOR cells were incubated with 4 nM
[H]-FNAH]-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).
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 [
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
Concentration on Its Irreversible BindingH]-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.
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 Na
NaCl greatly enhanced
the specific irreversible binding of [Concentration on the
Specific Irreversible Binding of
[
H]
-FNAH]-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
[
Specific irreversible binding of
[H]-FNA by µ, , and
LigandsH]-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).
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.
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
We have previously shown that chimeras III, IV, XI,
and XII bound [-FNA to Chimeric µ/
ReceptorsH]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
[
Irreversible binding of a range of
concentrations of [H]-FNA to RMOR, RKOR, and Chimeric
µ/ ReceptorsH]-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.
-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.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) .
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.
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. 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.-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.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.
;
-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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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M. M. Puig and O. Pol Peripheral Effects of Opioids in a Model of Chronic Intestinal Inflammation in Mice J. Pharmacol. Exp. Ther., December 1, 1998; 287(3): 1068 - 1075. [Abstract] [Full Text]
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C. E. Spivak, C. L. Beglan, B. K. Seidleck, L. D. Hirshbein, C. J. Blaschak, G. R. Uhl, and C. K. Surratt Naloxone Activation of µ-Opioid Receptors Mutated at a Histidine Residue Lining the Opioid Binding Cavity Mol. Pharmacol., December 1, 1997; 52(6): 983 - 992. [Abstract] [Full Text]
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J. Zhu, J. Yin, P.-Y. Law, P. A. Claude, K. C. Rice, C. J. Evans, C. Chen, L. Yu, and L.-Y. Liu-Chen Irreversible Binding of cis-(+)-3-Methylfentanyl Isothiocyanate to the [IMAGE] Opioid Receptor and Determination of Its Binding Domain J. Biol. Chem., January 19, 1996; 271(3): 1430 - 1434. [Abstract] [Full Text] [PDF]
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