(Received for publication, November 10, 1995)
From the
Binding of cis-(+)-3-methylfentanyl isothiocyanate
(SUPERFIT) to cloned opioid receptors stably expressed in Chinese
hamster ovary cells was characterized. SUPERFIT inhibited
[H]diprenorphine binding with much higher
affinity for the
than the µ or
receptor. Pretreatment
with SUPERFIT followed by extensive washing reduced
binding with
an IC
value of 7.1 nM, yet it did not affect
µ and
binding up to 0.1 µM. The reduction in
binding by SUPERFIT pretreatment was due to a decrease in B
with no change in K
. These results indicate that SUPERFIT
is a highly selective
irreversible ligand. We then determined the
region in the
receptor that confered binding selectivity for
SUPERFIT by examining its binding to six µ/
chimeric
receptors. SUPERFIT bound to
, µ/
1 (amino acids
µ1-94/
76-372),
/µ3
(
1-134/µ154-398), and
/µ4
(
1-187/µ207-398) receptors with high affinity but
to µ,
/µ1 (
1-75/µ95-398),
µ/
3 (µ1-153/
135-372), and µ/
4
(µ1-206/
188-372) receptors with low affinity.
Pretreatment with SUPERFIT potently inhibited
[
H]diprenorphine binding to
, µ/
1,
/µ3, and
/µ4 but affected binding to µ,
/µ1, µ/
3, and µ/
4 only at much higher
concentrations. Thus, the segment from the beginning of the first
intracellular loop to the middle of the third transmembrane helix of
the
receptor is important for selective binding of SUPERFIT.
Opiate and opioid drugs, acting on membrane-bound receptors,
have been widely used as analgesics. They, however, also produce side
effects such as respiratory depression, decreased gastrointestinal
motility, sedation, and mood changes(1) . At least three types
(µ, , and
) of opioid receptor are present in the nervous
system(1) . Many structurally diverse opiates and opioid
compounds have been synthesized aiming to minimize the side effects and
to understand the structure-function relationship. Some drugs act
nonselectively on all three types of opioid receptors, yet there are
ligands selective for each receptor.
Among the agents synthesized
are affinity ligands or irreversible ligands. An affinity ligand is
thought to bind to the receptor and then form a covalent bond on or
near the binding site, resulting in irreversible
attachment(2, 3) . Affinity ligands have been very
useful in the purification of receptors such as the opioid
receptor (4) and in the elucidation of receptor structure.
Specific incorporation of radiolabeled affinity ligand into the
receptor followed by polyacrylamide gel electrophoresis and
fluorography or autoradiography has been used to identify the receptor,
to determine molecular mass of the receptor without purification, and
to examine the nature of carbohydrate moieties (for example, see (5) and (6) ). 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, see (7) ).
SUPERFIT, ()an
isothiocyanate derivative of cis-(+)-3-methylfentanyl (Fig. 1), was synthesized by Burke et al.(8) and found to bind irreversibly to the
receptor.
Incubation of NG108-15 cell membranes with SUPERFIT (1-5
nM) followed by extensive washing reduced
binding by
60-90%. The reduction in
binding was due to a decrease in B
with no change in K
(8) . Similar preincubation of
rat brain membranes with SUPERFIT (1-5 nM) greatly
reduced
receptor binding, without affecting µ receptor
binding. The (+)-enantiomer (SUPERFIT) was 50 times more potent
than its(-)-enantiomer(9) . The high affinity and
enantioselectivity of SUPERFIT indicate that the irreversible binding
is the result of selective interaction with the
receptor.
[
H]SUPERFIT was shown to label the
receptor
in NG108-15 cells as a 58-kDa protein band in a denaturing gel (4) . [
H]SUPERFIT-labeled
receptor
was purified to homogeneity(4) .
Figure 1: Chemical structures of SUPERFIT and fentanyl.
opioid receptors have
been cloned from several
species(10, 11, 12, 13, 14, 15, 16) .
In addition, µ and
opioid receptors have been cloned (Refs.
17 and 18 and references therein). All three opioid receptors contain
seven putative transmembrane helices (TMHs), a common structural motif
of a G protein-coupled receptor superfamily. Sequence comparison among
the three types of opioid receptors shows substantial divergence in the
N- and C-terminal domains as well as extracellular loops, while
sequences within TMHs and intracellular loops are very similar. These
divergent sequences may contribute to the binding of type-selective
ligands.
Chimeric µ/,
/
, and µ/
receptors have been used to delineate the structural basis of ligand
binding selectivity of opioid receptors. For instance, by examining the
binding characteristics of µ/
or
/
chimeric
receptors, we(19) , Wang et al.(20) , and
Meng et al.(21) demonstrated that the second
extracellular loop of the
receptor was essential for the high
affinity binding of the dynorphin family peptides. For the
receptor, by using chimeric
/
receptors, Meng et al.(21) found that replacement of TMHs 5-7 of the
receptor with those of the
receptor greatly reduced binding of
-selective peptide ligands such as cyclic
[D-penicillamine
,D-penicillamine
]enkephalin
(DPDPE),
[D-Ser
,Leu
]enkephalin-Thr,
and ICI174,864, as well as nonpeptide antagonists, including
naltrindole,
(5
)-17-(cyclopropylmethyl)-4,5-epoxy-3,14-dihydroxy-7-(phenylmethylene)morphinan-6-one
(BNTX), and naltriben. In addition, by examining binding of µ/
chimeras, Fukuda et al.(22) reported that the major
determinant for selective binding of DPDPE was in TMHs 5-7.
In
this study, we characterized the binding of SUPERFIT to cloned µ,
, and
receptors(11, 23, 24) . In
addition, we determined the region in the
receptor that conferred
selectivity for SUPERFIT binding by examining its binding to chimeric
µ/
receptors constructed from cloned rat µ and mouse
opioid receptors(11, 23) .
Figure 2:
Amino acid sequence comparison of rat
µ and mouse opioid receptors and points of exchange (A-C) for generation of chimeric receptors. Chimera
/µ1 (aa
1-75/µ95-398) and chimera
µ/
1 (aa µ1-94/
76-372) were constructed by
swapping the regions from the N terminus to point A. Chimera
/µ3 (aa
1-134/µ154-398) and chimera
µ/
3 (aa µ1-153/
135-372) were generated by
exchanging the regions from the N terminus to point B. Chimera
/µ4 (aa
1-187/µ207-398) and chimera
µ/
4 (aa µ1-206/
188-372) were constructed
by exchanging the regions from the N terminus to point C. Dash indicates the same amino acid in the µ receptor as in
the
receptor. Dots represent gaps introduced for
sequence alignment. Seven putative TMHs are underlined. Amino
acid residue numbers are indicated on both sides and in the
vicinity of points A, B, and C.
For each chimera, DNA sequence of the fragment generated by polymerase chain reaction and that in the transitional region between two receptors was determined to ensure successful in-frame construction.
Phosphate-based buffer, in place of Tris buffer, was used in
all binding experiments (10 mM KHPO
, 1
mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, 1
µM leupeptin, 10 µg/ml soybean trypsin inhibitor, pH
8.0) to avoid reaction of the isothiocyanate group of SUPERFIT with
Tris. This buffer was used in previous SUPERFIT studies (4, 8) . Saturation binding of
[
H]diprenorphine to the membranes of CHO cells
stably transfected with
, µ, or
receptor was carried out (Table 1). K
values were determined to be
0.33, 0.45, and 0.26 nM for µ,
, and
receptors,
respectively. These values are similar to those determined in Tris-HCl
buffer(11, 19, 23, 24) .
Binding
of SUPERFIT to cloned µ, , and
opioid receptors was
investigated. SUPERFIT inhibited [
H]diprenorphine
(37 °C, 60 min) binding to cloned
, µ, and
opioid
receptors with IC
values of 3.4, 680, and 747 nM,
indicating that SUPERFIT had
200-fold selectivity in this assay
for
over µ or
receptors, respectively (Fig. 3;
also see Table 3). Pretreatment of the
receptor with
various concentrations of SUPERFIT (37 °C, 45 min) followed by
extensive washing reduced binding in a concentration-dependent fashion
with an IC
value of 7.1 nM, but it did not affect
µ or
binding at
0.1 µM (Fig. 4; also
see Table 3). In contrast, pretreatment with 1 µM fentanyl (Fig. 1) did not reduce
binding, as compared
with the untreated control. Reduction in the
binding by SUPERFIT
was examined further. The
receptor was treated with 5 nM SUPERFIT or 1 µM fentanyl (control) and washed three
times, and saturation experiments with
[
H]diprenorphine were performed. SUPERFIT
pretreatment decreased the B
by 40% without
changing K
compared with fentanyl pretreatment (Table 2). These results are consistent with previous
observations of Burke et al.(8) and Kim et
al. (9) that SUPERFIT was a highly selective
irreversible ligand. It thus can be used to probe the structure of
binding pocket of the
receptor.
Figure 3:
Inhibition of
[H]diprenorphine binding to cloned µ,
,
and
opioid receptors by SUPERFIT. Membranes of CHO cells stably
transfected with
, µ, or
opioid receptors were prepared,
and inhibition of 0.4 nM [
H]diprenorphine binding by SUPERFIT was
performed as described under ``Experimental Procedures.''
Each point represents mean ± S.E. of three
experiments.
Figure 4:
Effect of pretreatment with SUPERFIT on
µ, , and
opioid receptors. Membranes of CHO cells stably
transfected with
, µ, or
opioid receptors were prepared
and pretreated with or without SUPERFIT or with 1 µM fentanyl (control), and binding was performed with 0.4 nM [
H]diprenorphine as described under
``Experimental Procedures.'' Each point represents
mean ± S.E. of three experiments.
To determine the structural
basis in the receptor for selective binding of SUPERFIT, we
examined its binding to three pairs of chimeric µ/
receptors (Fig. 5, Table 3). All chimeric µ/
receptors
bound [
H]diprenorphine with high affinity with K
values ranging from 0.13 to 0.44 nM (Table 1), indicating that these chimeric receptors retain
opioid receptor conformation to some extent. B
values of µ/
chimeric receptors stably expressed in CHO
cells varied from 200 to 700 fmol/mg of protein, which were lower than
µ and
receptors (1024 and 3062 fmol/mg of protein,
respectively). In all binding experiments, we used
[
H]diprenorphine at a concentration close to the K
and kept the receptor concentration at <10%
of the K
value. Chimeras µ/
1 and
/µ3 bound SUPERFIT with high affinity, similar to the
receptor. In contrast,
/µ1, µ/
3, and µ/
4 had
low affinity, similar to the µ receptor. Chimera
/µ4 had
intermediate affinity for SUPERFIT (Fig. 5, Table 3).
Figure 5:
Inhibition of
[H]diprenorphine binding to cloned
and
µ opioid receptors and µ/
chimeras by SUPERFIT. Membranes
of CHO cells stably transfected with
, µ, or a µ/
chimeric opioid receptor were prepared, and inhibition of 0.4 nM [
H]diprenorphine binding by SUPERFIT was
performed as described under ``Experimental Procedures.''
Each point represents mean ± S.E. of three experiments.
IC
values are shown in Table 3.
In addition, µ/ chimeras were examined for their abilities
to bind SUPERFIT irreversibly. Pretreatment with SUPERFIT followed by
three washes potently inhibited [
H]diprenorphine
binding to µ/
1 and
/µ3 with IC
values of
6.9 and 2.3 nM, respectively, but did not inhibit
[
H]diprenorphine binding to
/µ1,
µ/
3, and µ/
4 (IC
values >1
µM) (Fig. 6, Table 3). The potency for
/µ4 was moderate (IC
= 53.5 nM).
These data indicate that the segment from the N-terminal portion of the
first intracellular loop to the middle of the third TMH is important
for reversible and irreversible binding of SUPERFIT to the
receptor. Since the sequences within the first intracellular loop, the
TMH 2 and TMH 3, are highly homologous between the two receptors, the
selectivity to SUPERFIT is most likely conferred by the first
extracellular loop of the
receptor.
Figure 6:
Effect of pretreatment with various
concentrations of SUPERFIT on chimeric µ/ receptors. Membranes
were pretreated with various concentrations of SUPERFIT or 1 µM fentanyl (control), and binding was performed with
[
H]diprenorphine as described under
``Experimental Procedures.'' Each point represents
mean ± S.E. of three experiments. IC
values are
shown in Table 3.
Two possibilities may
exist in terms of the irreversible binding site of SUPERFIT. First, the
site of SUPERFIT irreversible binding is within this region. It has
been thought that the isothiocyanate group of SUPERFIT reacts with a
cysteine or lysine residue to form a covalent bond. Second, chimeras
µ/1 and
/µ3 may assume favorable conformations for
SUPERFIT to form a covalent bond with the receptor, whereas chimeras
/µ1, µ/
3, and µ/
4 do not. When
[
H]SUPERFIT is available, we will be able to
differentiate these two possibilities by limited proteolysis of
[
H]SUPERFIT-labeled
receptor and
determination of the size of labeled peptide fragments. Chimeric
receptor studies, nonetheless, provide starting points for future
studies on identification of smaller fragments and, ultimately, amino
acid residues as binding epitopes.
Although SUPERFIT is a
selective irreversible ligand, its parent compound, cis-(+)-3-methylfentanyl is a selective µ
agonist(27) . Similarly, fentanyl is a selective agonist for
the µ receptor(28) , yet its isothiocyanate derivative,
FIT, is a selective irreversible ligand for the
receptor(29) . Thus, the isothiocyanate functional group
confers not only the irreversible binding characteristics but also the
selectivity of both SUPERFIT and FIT. Determination of the site
of SUPERFIT incorporation with
receptor will provide insights
into how binding pockets of µ and
receptors differ.
Kong et al.(30) reported that an aspartate residue in the
TMH 2 of the receptor was critical for high affinity binding of
selective agonists. This Asp may also be important for the binding of
SUPERFIT. However, this residue is unlikely to be the site of
irreversible binding of SUPERFIT since 1) it is conserved among most
G-coupled receptors including µ and
opioid
receptors(23, 24) ; 2) it is also important for
agonist binding of the µ opioid receptors (31) ; and 3) the
isothiocyanate group does not react with aspartate.
In this study,
within each of the three pairs of chimeras, one is the ``mirror
image'' of the other. We demonstrated that for the two pairs of
/µ1-µ/
1 and
/µ3-µ/
3, there was not
only a reduction in SUPERFIT affinity in one of the pair but also
acquisition or preservation of high affinity binding in the other,
compared with the
receptor. By use of such pairs, our conclusion
is strengthened.
Chimera /µ4 had a lower affinity for
SUPERFIT than
/µ3, although
/µ4 had more
sequence than
/µ3. This finding is in agreement with that of
Law et al., (
)who found that, in general,
/µ4 had lower affinity for
selective ligands than
/µ3. It is likely that interactions among TMHs are important
in determining the conformation of the receptor binding pocket. The TMH
4 of
receptor may not be as compatible with the TMH 5 of the
µ receptor, thus creating a chimera (
/µ4) that does not
bind SUPERFIT as well as the
/µ3 chimera, although both
chimeras bound [
H]diprenorphine, a nonselective
ligand, with high affinity.
The region from the first intracellular
loop to the TMH 3 of the receptor confers binding selectivity for
SUPERFIT. In contrast, the specific binding sites of the
receptor
for DPDPE, naltrindole, naltriben, and BNTX are located between the TMH
5 and TMH 7(21, 22) . It is likely that depending on
stereochemical features, ligands are oriented differently in the
binding pocket.
In conclusion, SUPERFIT binds specifically and
irreversibly to the cloned mouse opioid receptor. The region
between the N-terminal portion of the first intracellular loop to the
middle of the TMH 3 is necessary for selective binding of SUPERFIT. We
will determine the amino acid residue that forms a covalent bond with
SUPERFIT. Such information will be very useful for molecular modeling
of the
receptor.