(Received for publication, September 27, 1995; and in revised form, December 20, 1995)
From the
The sensitivity of µ and receptor binding to
dithiothreitol and N-ethylmaleimide was examined to probe
receptor structure and function. Binding to both receptor types was
inhibited by dithiothreitol (IC
values = 250
mM), suggesting the presence of inaccessible but critical
disulfide linkages. µ receptor binding was inhibited with more
rapid kinetics and at lower N-ethylmaleimide concentrations
than
receptor binding. Ligand protection against N-ethylmaleimide inactivation suggested that alkylation was
occurring within, or in the vicinity of, the receptor binding pocket.
Sodium ions dramatically affected the IC
of N-ethylmaleimide toward both receptor types in a
ligand-dependent manner. Analysis of receptor chimeras suggested that
the site of N-ethylmaleimide alkylation on the µ receptor
was between transmembrane domains 3 and 5. Substitution of cysteines
between transmembrane domains 3 and 5 and elsewhere had no effect on
receptor binding or sensitivity toward N-ethylmaleimide.
Serine substitution of His
in the putative second
extracellular loop linking transmembrane domains 4 and 5 protected
against N-ethylmaleimide inactivation. The H223S substitution
decreased the affinity of bremazocine 25-fold, highlighting the
importance of this residue for the formation of the high affinity
bremazocine binding site in the µ opioid receptor.
Three major types of opioid receptor, ,
, and µ,
have been cloned and characterized extensively (reviewed in (1) and (2) ). There is approximately 60% amino acid
sequence identity between the opioid receptor types. The
,
,
and µ opioid receptors have unique ligand specificities, anatomical
distributions, and physiological functions(3) . Morphine,
related opioid drugs, and the endogenous opioid peptides activate
signal transduction pathways by binding to opioid
receptors(4) , which are members of the G protein-coupled
receptor family(5) . G protein-coupled receptors are
seven-transmembrane domain (TM) (
)proteins that mediate
signal transduction across the plasma membrane. The ligands approach
and engage the receptor from the extracellular side, and receptor
activation results in the coupling to heterotrimeric G proteins on the
intracellular face of the membrane. Opioid receptor types interact with
multiple G proteins (6, 7, 8) to regulate
adenylyl cyclase, Ca
channels, and K
channels.
It has been known from early studies on the characterization of opioid receptors that specific binding is inhibited by sulfhydryl reagents, such as iodoacetamide, N-ethylmaleimide (NEM), and p-hydroxymercuribenzoate(9, 10, 11) . Preincubation with opioid ligands protected against receptor inactivation, suggesting that the sensitive sulfhydryl group was located within or near the binding site. Evidence has been obtained that analogs of Leu-enkephalin and morphine, containing activated sulfhydryl groups, form mixed disulfide linkages with opioid receptors(12, 13) . The covalently bound agonists caused receptor activation that persisted following extensive washing, yet was naloxone-reversible. The results suggested that the agonists became tethered to the receptor via a mixed disulfide linkage that was in the vicinity of the receptor binding site. Other studies provided evidence that NEM affected opioid agonist binding by at least two mechanisms, direct inhibition (as mentioned above) and indirect inhibition due to uncoupling of receptors from G proteins(14, 15) .
Several other, but not all, G
protein-coupled receptors are also sensitive to sulfhydryl reagents.
Susceptible receptors include the thyrotropin-releasing
hormone(16) , D1 and D2 dopamine(17, 18) ,
substance P(19) , 1 and
2
adrenoreceptor(20) , platelet-activating factor(21) ,
leukotriene B
(22) , vasopressin(23) ,
follicle-stimulating hormone(24) , and cannabinoid
receptors(25) . Recently, a cysteine in TM3 of the D2 dopamine
receptor has been identified that reacts with sulfhydryl reagents and
results in inhibition of binding(26) .
The goals of this
study were 1) to examine the sensitivity of µ and receptor
binding to reduction with dithiothreitol (DTT), in order to determine
whether disulfide linkages were necessary for maintenance of the
binding site, and 2) to characterize the sensitivity of µ and
receptor binding to alkylation with NEM and identify the reactive
groups involved. Due to the proximity of the NEM-reactive group to the
ligand binding site of the receptor, knowledge of its location is
essential for construction of accurate molecular models of the binding
pocket.
The N 64 amino-terminal deletion was
generated in a similar manner, with the following sense strand
oligonucleotide, ATGGTCACAGCCATTACC. The
N 89 amino-terminal
deletion was an unexpected byproduct of the polymerase chain reaction
reaction used to generate the C321S mutation.
Figure 1:
Effect of DTT on µ and opioid
receptor binding. DTT (1-100 mM) was added to membrane
preparations from cells stably expressing either µ or
receptors immediately prior to conducting
[
H]bremazocine (2 nM) binding assays.
Specific binding to both receptor types was inhibited slightly at high
concentrations of dithiothreitol. Each curve represents the
average of two independent experiments.
In the course of determining the optimal
concentration of glutathione to use to quench NEM reactions, we were
surprised to observe that [H]bremazocine binding
to µ and
receptors was considerably more sensitive
to incubation with reduced glutathione than with DTT. The IC
of glutathione was approximately 15 mM for inhibition of
[
H]bremazocine binding to µ and
receptors, and the slopes of the inhibition curves were very steep
(data not shown). Similar results were reported for binding to µ
opioid, neurokinin-1, and kainic acid receptors(31) . We found,
however, that the inhibition of binding by glutathione was due to
lowering the pH of the buffer solution, due to the acidic nature of the
tripeptide. We suggest, therefore, that the results on glutathione
inhibition of binding to neurokinin-1 and kainic acid receptors be
interpreted with caution.
Figure 2:
Kinetics of NEM inactivation of µ and
opioid receptor binding. Membrane preparations from cells stably
expressing either µ or
receptors were incubated at 37 °C
in the absence and presence of 0.5 mM NEM. All samples were
quenched with 5 mM glutathione at the indicated times and then
assayed for specific binding of 2 nM [
H]bremazocine. Data points are the means
± S.E. from three or four independent
experiments.
The ability of agonist and antagonist ligands to
protect against NEM inactivation of µ and opioid receptor
binding was determined. Both receptor types were protected against NEM
inactivation by preincubation with ligands, although protection of
µ receptor binding was more complete (Fig. 3). All ligands
tested were capable of protection, including type-selective peptide
agonists (DAMGO and DSLET), alkaloid agonists (morphine and etorphine),
and the antagonist, naloxone. The data indicated that the NEM-reactive
group on both receptor types resided within, or in the vicinity of, the
ligand binding crevice.
Figure 3:
Protection against NEM inactivation of
[H]bremazocine binding to µ and
opioid
receptors by opioid ligands. A, protection of µ opioid
receptor binding. B, protection of
opioid receptor
binding. Membrane preparations from cells stably expressing either
µ or
receptors were preincubated at 37 °C for 10 min in
the absence and presence of opioid ligands (either 100 nM or 1
µM) and then incubated either for 15 min in the absence or
presence of 0.5 mM NEM (for µ opioid receptor binding) or
for 30 min in the absence or presence of 2.5 mM NEM (for
opioid receptor binding). Glutathione (5 mM) was added to all
samples, and then ligands were removed by centrifugation at 35,000
g for 20 min. Membranes were resuspended in 50 mM Tris-HCl, 1 mM Na
EDTA buffer, pH 7.4,
incubated for 10 min at 37 °C to promote ligand dissociation, and
then rewashed twice by centrifugation prior to initiating the
radioligand binding assay using 2 nM [
H]bremazocine. Values are the means
± S.E. of four independent
experiments.
Figure 4:
NEM inactivation of
[H]DAMGO and [
H]bremazocine
binding to the µ opioid receptor in the presence and absence of
NaCl. Membrane preparations from cells stably expressing µ opioid
receptors were incubated for 15 min at 37 °C with various
concentrations of NEM in Tris-EDTA buffer with or without 100 mM NaCl and then assayed for specific binding of
[
H]DAMGO (4 nM) or
[
H]bremazocine (2 nM). The presence of
NaCl caused the NEM inactivation curve of
[
H]DAMGO binding to shift to the left and the NEM inactivation curve of
[
H]bremazocine binding to shift to the right. The curves are from one data set that is
representative of four independent
experiments.
Binding to the
receptor was considerably less sensitive to NEM inactivation than was
binding to the µ receptor. [
H] DSLET and
[
H]bremazocine binding to the
receptor were
also differentially affected by inclusion of 100 mM NaCl in
the buffer (Fig. 5), in a similar manner to that observed with
µ receptor binding. The IC
of NEM toward inactivation
of DSLET binding to the
receptor decreased markedly in
the presence of 100 mM NaCl from 450 to 8 µM,
while the IC
of NEM toward inactivation of bremazocine increased from 660 µM to 3.6 mM.
Figure 5:
NEM inactivation of
[H]DSLET and [
H]bremazocine
binding to the
opioid receptor in the presence and absence of
NaCl. Membrane preparations from cells stably expressing
opioid
receptors were incubated for 15 min at 37 °C with various
concentrations of NEM in Tris-EDTA buffer with or without 100 mM NaCl and then assayed for specific binding of
[
H]DSLET (4 nM) or
[
H]bremazocine (2 nM). The presence of
NaCl caused the NEM inactivation curve of
[
H]DSLET binding to shift to the left and the NEM inactivation curve of
[
H]bremazocine binding to shift to the right. The curves are from one data set that is
representative of four independent
experiments.
Figure 6:
The amino acid sequence and proposed
transmembrane topology of the rat µ opioid receptor. The amino
terminus is on the extracellular side and the carboxyl terminus is on
the intracellular side of the plasma membrane. Transmembrane helices
1-7 are shown from left to right. Cys residues
and His are shaded. Cysteines that were
substituted with serine and His
are numbered.
Sites of amino-terminal deletions (
N 64 and
N 89) are
indicated. The presumed disulfide bond between Cys
and
Cys
, in putative extracellular loops 2 and 3,
respectively, is indicated with a connecting bar. One of the
two cysteines in the carboxyl-terminal tail is shown with a schematic
palmitoyl group inserted into the cytoplasmic side of the plasma
membrane. Junction sites used to construct µ/
receptor
chimeras are indicated with boldface circles. These amino
acids are encoded by contiguous homologous nucleotide sequences.
Possible sites of N-linked glycosylation (NX(S/T),
where X is any amino acid except P) in the amino-terminal
domain are shown schematically with core
oligosaccharides.
Figure 7:
Schematic illustration of the structures
of µ/ receptor chimeras used in these studies along with the
wild-type receptors. DOR, wild-type
opioid receptor; MOR, wild-type µ opioid receptor. Designations for
chimeras indicate the origin of the amino-terminal domain on the left and the carboxyl-terminal domain on the right (µ = M,
= D), separated by a number which refers to the transmembrane helix that is the site of the
junction.
opioid receptor sequences are shown in white;
µ opioid receptor sequences are shaded; junction sites are
depicted as black boxes.
The ability of NEM to inactivate
[H]bremazocine binding to wild-type µ and
receptors and µ/
receptor chimeras was compared (Table 2). [
H]Bremazocine binding to
wild-type µ receptors was 10 times more sensitive to NEM
inactivation when compared with binding to
receptors (NEM
IC
values were 0.16 and 1.6 mM, respectively).
Binding to the D2M receptor chimera was inactivated by NEM at similar
concentrations as binding to the µ receptor (IC
= 0.29 mM), while the sensitivity of the D5M
chimera was even greater than the wild-type
receptor (IC
= 4.3 mM). These data suggested than the
NEM-reactive groups in the µ and
receptor resided between TM2
and TM5. Data from the reciprocal M2D and M5D chimeras were consistent
with this assumption; the M2D chimera behaved like the
receptor,
and the M5D chimera was even more sensitive than the µ receptor
with respect to the NEM IC
for inactivation of
[
H]bremazocine binding (Table 2). Analysis
of the NEM sensitivity of the D3M chimera (with a µ receptor-like
IC
= 0.05 mM) led to the more focused
prediction that the NEM-reactive group in the µ receptor was in the
region between the junction sites in TM3 and TM5.
Based on these
results, Ser was substituted for other cysteines residing outside of
the region between TM3 and TM5, and the mutant receptors were tested
for sensitivity to NEM. Ser substitution of either Cys or
Cys
, located in TM6 and TM7, respectively, did not affect
the ability to bind [
H]bremazocine or the
sensitivity toward NEM inhibition of binding (Table 3). Deletion
of 64 amino acids from the amino-terminal domain (
N 64), which
contains four cysteines at positions 13, 22, 43, and 57 (see Fig. 6), did not affect the affinity of the truncated receptor
for [
H]bremazocine (K
= 1.3 nMversus 0.8 nM for the
wild-type µ receptor). It has also been reported previously that
this deletion did not affect the binding of
[
H]naloxone and [
H]DAMGO to
the µ receptor(32) . The concentration of NEM required for
inactivation of bremazocine binding to the truncated receptor was
increased 3-fold relative to the wild-type µ receptor (Table 3); however, the IC
was still in the
submillimolar range (0.57 mM). We also tested the effect of
removal of 89 amino acids from the amino terminus of the C321S mutant
receptor. The deleted region of this construct, referred to as
N
89, included the amino-terminal domain and most of putative TM1,
including Cys
(Fig. 6) The affinity of
[
H]bremazocine for the
N 89 construct
decreased 20-fold. (
)The IC
of NEM for the
N 89 receptor, however, was similar to the IC
of the
N 64 receptor (Table 3), suggesting that the cysteines in
the amino-terminal domain and Cys
were not the relevant
targets for NEM alkylation. Substitution of Cys
with Ser
completely blocked the ability of the mutant receptor to bind
[
H]bremazocine (data not shown). Although this
mutated receptor could not be tested for NEM sensitivity, Cys
is thought to be linked by a disulfide bond with Cys
(Fig. 6); hence, it would not be reactive with NEM.
The
data suggested that none of the cysteines that were substituted with
Ser or deleted were likely targets for NEM alkylation. Based on the
chimeric receptor data, which indicated that the NEM-reactive group
resided in the region between TM3 and TM5, the sequence of the µ
receptor was reexamined for amino acids other than Cys that might be
reactive toward NEM. It has been reported that reaction of lysozyme and
ribonuclease with NEM resulted in the alkylation of -amino groups
and imidazole groups(33) . The µ receptor contains a His
residue at position 223 in the putative second extracellular loop
connecting TM4 and TM5 (Fig. 6). According to our alignment of
the opioid receptor sequences, (
)the corresponding amino
acids in the
and
receptor are Ser
and
Asp
, respectively. Substitution of His
with
Ser in the µ receptor completely abolished the ability of NEM to
inhibit [
H]bremazocine binding, even at
concentrations 10-fold higher than the IC
for inhibition
of binding to the wild-type µ receptor (Table 3). The H223S
substitution also decreased the affinity for
[
H]bremazocine dramatically (Table 4).
Preliminary data from competition analyses indicated that the affinity
of the H223S-substituted receptor for etorphine and naloxone was also
decreased
25-fold (data not shown). In addition to the 25-fold
decrease in the affinity constant for bremazocine, the cell line that
expressed the H223S mutant receptor also had a significantly lower B
than the cell line that expressed the
wild-type µ receptor (Table 4).
The following observations and conclusions were made based on
these studies. 1) [H]Bremazocine binding to µ
and
opioid receptors was inhibited to an equal extent by high
concentrations of DTT, implying the presence of relatively inaccessible
but critical disulfide linkages for both receptor types. 2)
[
H]Bremazocine binding to the µ receptor was
considerably more sensitive to treatment with NEM than binding to the
receptor. This finding suggested that the functional group that
was the site of alkylation on the µ receptor was more accessible to
and/or reactive with NEM than the relevant group on the
receptor.
3) Ligand protection against NEM inactivation of binding to µ and
opioid receptors was consistent with the site of alkylation being
within, or in the vicinity of, the receptor binding crevice. 4)
Dose-response curves of NEM inactivation of µ and
receptor
binding in the presence of sodium ions suggested that at least two
reactive groups were subject to alkylation. Alkylation of µ and
receptor sites at low concentrations of NEM resulted in
inhibition of [
H]DAMGO and
[
H]DSLET binding, respectively, with minimal
effect on [
H]bremazocine binding. Alkylation of
µ and
receptor sites at much higher concentrations of NEM
resulted in inhibition of [
H]bremazocine binding
to both receptor types. 5) Analyses of the NEM sensitivity of
[
H]bremazocine binding to µ/
opioid
receptor chimeras were consistent with a location of the reactive group
in the region between TM3 and TM5. 6) Site-specific substitution of
His
in the µ receptor abolished the inactivation of
[
H]bremazocine binding by NEM and led to a
dramatic reduction in the affinity for bremazocine. This result
suggested that either His
was the site of NEM alkylation
or the H223S substitution caused a conformational change in the
receptor that shielded the reactive group from the reagent.
The observation that preincubation with opioid ligands
protected against NEM inactivation of binding to µ and
receptors was consistent with the site of alkylation being within, or
in the vicinity of, the ligand binding crevice of both receptor types.
Early studies performed before the realization that there were multiple
opioid receptor types also demonstrated ligand protection against N-ethylmaleimide inactivation of receptor binding to rat brain
membranes(10, 11) .
Differential effects of sodium ions on the ability of NEM
to inhibit agonist and antagonist binding have been reported
previously(11, 39) . The pharmacological profile of
bremazocine, however, is not entirely clear. In our studies,
[H]bremazocine binding to µ and
receptors had the characteristics of antagonist binding. In the
presence of sodium ions, bremazocine binding was increased slightly
(data not shown), and the dose-response curve of NEM was shifted to the
right. There is pharmacological evidence that suggests that bremazocine
acts as an agonist at
receptors and as an antagonist at µ and
receptors(40, 41, 42, 43) ,
although it has also been reported that high concentrations of
bremazocine caused inhibition of forskolin-stimulated cAMP accumulation
in COS cells expressing the
receptor(44) . Additional
studies using cloned opioid receptors will be necessary to clarify this
issue.
The observation that [H]peptide agonist
binding to µ and
receptors was reduced to <20% of control
values at NEM concentrations that had minimal effects on
[
H]bremazocine binding provided strong evidence
for the involvement of at least two NEM-reactive groups. The shift to
the left of the NEM dose-response curve for inhibition of
[
H]DAMGO and [
H]DSLET
binding may be partially due to NEM alkylation of a GTP-binding
protein(15) , resulting in receptor uncoupling and a consequent
decrease in agonist affinity. [
H]Bremazocine
binding, in contrast, is not affected by the state of receptor coupling (45) . (
)
The H223S substitution (in
putative extracellular loop 2 connecting TM4 and TM5) resulted in
pronounced effects on basal [H]bremazocine
binding and NEM sensitivity of [
H]bremazocine
binding. The affinity of bremazocine was lowered 20-fold, and
bremazocine binding was rendered insensitive toward NEM. There are at
least two plausible explanations for these findings. 1) His
makes direct contact with bremazocine in the binding site, and
NEM alkylation of His or substitution with Ser abolishes the ability of
bremazocine to bind to the µ receptor. In this case, the H223S
mutant receptor would be insensitive to NEM since the reactive group
had been removed. 2) His
makes an important contribution
to the overall active conformation of the µ receptor. NEM
alkylation of His or substitution with Ser would be presumed to disrupt
the active conformation, leading to a loss of high affinity bremazocine
binding. Again, the H223S mutant receptor would be insensitive to N-ethylmaleimide since the reactive group had been removed, or
alternatively, the conformational change resulting from the H223S
substitution could conceivably shield other reactive groups (presumably
one of the cysteines that was not subjected to deletion or mutagenesis)
from interaction with NEM. Investigations are under way to distinguish
between these interpretations.
Evidence for essential histidyl
residues within opioid receptors has been reported
previously(47) . In these studies, chemical modification of
opioid receptors with two different histidyl-specific reagents resulted
in complete inhibition of [H]etorphine binding to
rat brain membranes. Further support for His being the actual site of
NEM alkylation was obtained by studying the pH dependence of the NEM
inactivation of opioid receptor binding. Childers and Jackson (48) found that the apparent pK
value of
the N-ethylmaleimide-reactive groups on opioid receptors was
between 5.4 and 6.0, which is much closer to the average
pK
of histidine (pK
=
6.5) than cysteine (pK
= 8.5)(49) .
The data from these studies add to the growing body of knowledge
regarding the constituents of opioid receptor binding sites. Previous
mutagenesis experiments have highlighted the importance of the Asp in
TM2 of the µ and receptor for high affinity selective agonist
binding(32, 44) . Mutation of Asp
in TM3
and His
in TM6 of the µ receptor inhibited both
agonist and antagonist binding(32) . These amino acids are also
conserved in
and
receptors. Regarding receptor selectivity,
analysis of µ/
and
/
receptor chimeras revealed that
the second extracellular loop of the
receptor was required for
high affinity binding of dynorphin-(1-17),
dynorphin-(1-13),
-neoendorphin, and dynorphin
B(50, 51, 52) . Evidence has also been
provided that the binding site for antagonists in the
opioid
receptor differs substantially from the antagonist site of the µ
and
opioid receptors(53) . The amino terminus of the
opioid receptor was found to be necessary for high affinity
naloxone binding and for reversal of
agonist-mediated inhibition
of forskolin-stimulated cAMP accumulation by naloxone. In contrast,
Glu
in the putative third extracellular loop of the
receptor plays a major role in binding the
-selective antagonist,
norbinaltorphimine(54) . Our group and
others(30, 52, 55) have reported recently
that a major binding determinant for
-selective peptides resides
in the region spanning TM5 to TM7 of the
receptor, in excellent
agreement with our studies regarding the role of the Arg residues in
the putative third extracellular loop(30) . Our finding on the
importance of the putative first extracellular loop for DAMGO binding
using µ/
receptor chimeras (30) has also been recently
reported independently(55, 56) . In contrast, Xue et al.(57) found that the third extracellular loop of
the µ receptor was important for agonist selectivity using
µ/
receptor chimeras. This discrepancy was clarified recently
with the important finding that DAMGO distinguishes between µ and
opioid receptors at a site different from that for the
distinction between µ and
opioid receptors(58) .
Until high resolution experimental data is obtained from crystallography, insights from analysis of receptor chimeras and mutagenesis will provide information on the structure and function of opioid receptors, with the aid of molecular modeling and computer simulation. Understanding of the molecular mechanisms involved in receptor activation and G protein coupling triggered by agonist engagement of the opioid receptor binding site remain as long term goals of these studies. It is anticipated that an understanding of opioid receptor structure and function will lead to the development of novel therapeutic agents.