(Received for publication, July 11, 1994; and in revised form, December 2, 1994)
From the
To elucidate which portions of the opioid receptor molecules are
involved in the ligand selectivity, we have expressed chimeric
receptors between the rat - and µ-opioid receptors from cDNAs
and analyzed their ligand binding properties. We demonstrate that the
major binding determinant for the
-selective enkephalin-related
peptide,
[D-Pen
,D-Pen
]enkephalin,
resides within the region comprising the transmembrane segments
V-VII and the intervening loop regions. On the other hand, the
region spanning from the intracellular loop I to the amino-terminal
half of the transmembrane segment III is shown to be involved in
determining high-affinity binding of the µ-selective
enkephalin-related peptides,
[D-Ala
,MePhe
,Gly-ol
]enkephalin
and
[D-Ala
,MePhe
,Met-ol
]enkephalin,
whereas the major determinant for binding of the µ-selective
alkaloids, morphine and codeine, is demonstrated to exist in the region
spanning the transmembrane segments V-VII. These results indicate
that distinct regions of the opioid receptor determine the selectivity
for the
- and the µ-selective enkephalin-related peptides and
that the binding determinant for the µ-selective alkaloids is
distinct from that for the µ-selective enkephalin-related peptides.
The opioid receptors exhibit a widespread distribution
throughout central nervous system and mediate actions of opioid
analgesics and endogenous opioid peptides(1) .
Pharmacologically, the opioid receptor has been classified into at
least three types (µ, , and
) on the basis of their
difference in apparent affinity for ligands(1, 2) .
Recently, the cDNAs encoding the µ-, - and
-opioid
receptors have been
cloned(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) .
Analysis of the deduced amino acid sequences showed that these
receptors possess seven putative transmembrane segments (TM
I-VII), (
)which is the major characteristic structural
feature of G-protein-coupled receptors(16) , and have high
amino acid sequence identities (
60%) to each other. The present
investigation has been designed to localize the region of the opioid
receptor molecule responsible for type-selective binding of opioid
agonists. For this purpose, chimeric opioid receptors with different
combinations of the rat
- and µ-receptors have been produced
from cDNAs in COS-7 cells and analyzed for their agonist binding
properties. The results obtained indicate that high-affinity binding of
the
- and the µ-selective enkephalin-related peptides is
determined by distinct domains of the opioid receptor molecule.
Furthermore, the binding determinant for the µ-selective
enkephalin-related peptides is indicated to be present in the domain
distinct from that for the µ-selective alkaloids.
Figure 1:
Proposed transmembrane topography of
the rat opioid receptor. The model is based on the amino acid sequences
of the rat - and µ-opioid receptors deduced from the cDNA
sequences (7) . The number of amino acid residues (circles) is based on the µ-receptor sequence; the
sequence gaps introduced into the
-receptor sequence to align the
two polypeptides are indicated by striped circles and the
insertions by arrows with the number of inserted amino acid
residues (aa). Closed circles indicate the amino acid
residues identical between the aligned sequences of the
- and
µ-receptors, and open circles indicate the non-identical
amino acid residues. Positions of the restriction sites used to
construct chimeric receptor cDNAs are indicated by thick lines with the names of the restriction
enzymes.
Figure 3:
Displacement of
[H]EKC binding to membrane preparations from
COS-7 cells transfected with cDNAs by DPDPE. Membrane was prepared from
COS-7 cells expressing the wild-type
- and µ-receptors and
chimeric receptors. Competitive ligand binding assay in the presence of
increasing concentrations of DPDPE was performed as described under
``Experimental Procedures.'' The chimeric receptors analyzed
include AB series (A), BA series (B), C series (C), and D series (D). Values for 100 and 0% were
determined by measurements in the absence of unlabeled ligands and in
the presence of 1 mM naloxone, respectively. Each curve shown is representative of three to six
experiments.
Figure 4:
Displacement of
[H]EKC binding to the wild-type and chimeric
receptors by DAGO. Binding affinities to DAGO were measured as
described in the legend to Fig. 3. The chimeric receptors
analyzed include AB series (A), BA series (B), C
series (C), and D series (D). Each curve shown is representative of three to five
experiments.
Figure 5:
Displacement of
[H]EKC binding to the wild-type and chimeric
receptors by DAMO. Binding affinities to DAMO were assessed as
described in the legend to Fig. 3. The chimeric receptors
analyzed are AB series (A) and BA series (B). Each curve shown is representative of three or four
experiments.
Figure 6:
Displacement of
[H]EKC binding to the wild-type and chimeric
receptors by morphine. Binding affinities to morphine were assessed as
described in the legend to Fig. 3. The chimeric receptors
analyzed include AB series (A), BA series (B), C
series (C), and D series (D). Each curve shown is representative of three to six
experiments.
Figure 7:
Displacement of
[H]EKC binding to the wild-type and chimeric
receptors by codeine. Binding affinities to codeine were measured as
described in the legend to Fig. 3. The chimeric receptors
analyzed are AB series (A) and BA series (B). Each curve shown is representative of three
experiments.
Figure 2:
Schematic representation of the structures
of the wild-type - and µ-receptors and chimeric receptors. Thin lines indicate the amino acid sequences derived from the
-receptor; thick lines indicate sequences derived from
the µ-receptor. The chimeric receptors are classified into AB, BA,
C, and D series. The compositions of the individual chimeric receptors
are as follows (the numbers in parentheses indicate
amino acid numbers(7) ; the junctional sequences common to the
- and µ-receptors are represented by amino acid numbers of the
-receptor): AB1,
(1-79) and µ(99-398); AB2,
(1-139) and µ(159-398); AB3,
(1-218)
and µ(238-398); AB4,
(1-330) and
µ(349-398); BA1, µ(1-92) and
(74-372);
BA2, µ(1-144) and
(126-372); BA3,
µ(1-232) and
(214-372); BA4, µ(1-330)
and
(313-372); C1,
(1-79), µ(99-144)
and
(126-372); C2,
(1-139), µ(159-232)
and
(214-372); C3,
(1-218), µ(238-330)
and
(313-372); C4,
(1-79), µ(99-232)
and
(214-372); C5,
(1-139), µ(159-330)
and
(313-372); D1, µ(1-92),
(74-139)
and µ(159-398); D2, µ(1-144),
(126-218)
and µ(238-398); D3, µ(1-232),
(214-330)
and µ(349-398); D4, µ(1-92),
(74-218)
and µ(238-398); D5, µ(1-144),
(126-330)
and µ(349-398).
To confirm this conclusion, we tested the C and D series
chimeras (Table 1; Fig. 3, C and D). The
affinities to DPDPE of C3, C5, D2, and D4, chimeric receptors
possessing the region spanning TM V-VII derived from the
µ-receptor, were much lower than that of the wild-type
-receptor and similar as that of the wild-type µ-receptor.
Replacement of the region spanning TM V-VII of the µ-receptor
with the corresponding sequence of the
-receptor (D3) increased
the affinity to DPDPE more than 12-fold. These results support the view
that the region spanning TM V-VII contains the major determinant
for the high-affinity binding to DPDPE of the
-receptor. However,
the K
value of D3 was still
13-fold higher
than that of the wild-type
-receptor. Further replacement of the
region spanning from the carboxyl-terminal half of TM III to ECL II
(D5) or the carboxyl-terminal cytoplasmic region (BA3) resulted in
2-4-fold increase in the affinity to DPDPE, confirming the small
contribution of these regions to the high-affinity DPDPE binding. The
affinities to DPDPE of C1 and C4 were comparable with those of BA2 and
BA3, respectively. This again indicates that the region spanning from
the carboxyl-terminal half of TM III to ECL II of the
-receptor
partly contributes to the high-affinity DPDPE binding and further
excludes contribution of the amino-terminal extracellular region and TM
I.
To test whether
this region is sufficient to define the binding determinant for DAGO,
the C and D series chimeras were examined (Table 1; Fig. 4, C and D). The chimeric receptor C1, in
which the region of the -receptor spanning from ICL I to the
amino-terminal half of TM III is replaced with that of the
µ-receptor, showed an affinity to DAGO comparable with that of the
wild-type µ-receptor, indicating that this region is sufficient to
define the binding determinant for DAGO. Consistently with this
conclusion, C4, D2, D3, and D5, chimeric receptors containing the
region spanning from ICL I to the amino-terminal half of TM III derived
from the µ-receptor, exhibited affinities to DAGO comparable with
that of the wild-type µ-receptor, whereas C3, C5, and D4, chimeric
receptors possessing this region derived from the
-receptor,
showed affinities to DAGO as low as that of the wild-type
-receptor.
The results obtained for the C
and D series chimeras were in agreement with this conclusion (Table 1; Fig. 6, C and D). The chimeric
receptor C3, in which the region spanning TM V-VII of the
-receptor is replaced with the corresponding sequence of the
µ-receptor, showed the binding affinity to morphine only
2-fold lower than that of the wild-type µ-receptor. In
contrast, the chimeric receptor D3, which is the mirror image of C3,
exhibited a low binding affinity nearly identical with that of the
wild-type
-receptor. Furthermore, C5, D2, and D4, chimeric
receptors possessing the region spanning TM V-VII derived from
the µ-receptor, showed affinities to morphine similar as or
slightly lower than that of the wild-type µ-receptor, whereas the
chimeric receptors C1, C4, and D5, which contain the region spanning TM
V-VII derived from the
-receptor, demonstrated low affinity
to morphine comparable with that of the wild-type
-receptor.
In this investigation, we attempted to delineate the
structural domains involved in determining the ligand binding
selectivity of the opioid receptor by analysis of chimeric receptors
between the rat - and µ-opioid receptors. The high-affinity
binding of DPDPE, a
-selective enkephalin-related peptide, was
shown to be determined mainly by the region spanning TM V-VII of
the
-receptor and also partly by the region spanning from the
carboxyl-terminal half of TM III to ECL II and the carboxyl-terminal
cytoplasmic region. In contrast, the region spanning from ICL I to the
amino-terminal half of TM III of the µ-receptor was demonstrated to
contain the major determinant for selectivity to DAGO and DAMO,
µ-selective enkephalin-related peptides. Furthermore, the major
determinant for selectivity to morphine and codeine, µ-selective
opioid alkaloids, was shown to exist in the region spanning TM
V-VII of the µ-receptor. These results indicate that the
determinants for binding of the
- and the µ-selective
enkephalin-related peptides exist in distinct domains and that
high-affinity binding of the µ-selective enkephalin-related peptide
and the µ-selective opioid alkaloid is determined by distinct
domains.
The amino-terminal extracellular region of the opioid
receptors differs considerably in amino acid sequence and seems likely
to contribute directly or indirectly to ligand selectivity. However,
our findings indicate that the amino-terminal extracellular region
contributes little to selective binding of agonists. Accordingly, this
region may be necessary for functions other than ligand selectivity,
such as organizing proper conformation of the receptor molecule with
oligosaccharide side chains and efficient expression on the cell
surface in the proper transmembrane orientation. The carboxyl-terminal
cytoplasmic region is also divergent in amino acid sequence between the
- and µ-receptors. Our results showed that this portion of the
receptor molecule contributes partly to high-affinity DPDPE binding of
the
-receptor. Since it is unlikely that the cytoplasmic domain
contributes directly to the formation of the agonist binding site, the
carboxyl-terminal cytoplasmic region may indirectly affect the affinity
to DPDPE by influencing the conformation of the major determinant for
DPDPE binding, which was demonstrated to exist in the region spanning
TM V-VII. The amino acid sequence of the region spanning from ICL
I to the amino-terminal half of TM III, which was shown to possess the
major determinant for the µ-selective enkephalin-related peptides,
is well conserved between the
- and µ-receptors; amino acid
residues are identical at 48 of 56 positions in this region of these
receptors (Fig. 1). Seven of eight non-identical residues in
this region are clustered around ECL I. There are three
charge-modifying amino acid substitutions between the
- and
µ-receptors in ECL I: that is, Lys
,
Glu
, and Glu
in the
-receptor
corresponding to Gln
, Gly
, and Thr
in the µ-receptor, respectively. The difference in
distribution of charged amino acid residues might result in difference
in binding stability between the peptide and the receptor molecule. The
region spanning TM V-VII was shown to be necessary for
high-affinity binding of DPDPE and the µ-selective alkaloids. It is
conceivable that ECL III in this region contributes to the selectivity
to DPDPE and opioid alkaloids, because the amino acid sequence of ECL
III is more divergent between the
- and the µ-receptor than
those of ECL I and ECL II. However, it is also possible that TM V and
TM VI contribute to determination of the selectivity, since
contribution of amino acid residues in the transmembrane segments to
ligand selectivity has been suggested in other peptide receptor
systems(21, 22) .
Enkephalin-related peptides,
including DPDPE, DAGO, and DAMO, are characterized by the
amino-terminal sequence Tyr-X-Gly. Accordingly, it is likely
that the amino-terminal sequence of these peptides interact with an
equivalent domain of the opioid receptor molecule. However, our results
suggest that high-affinity binding of the - and the
µ-selective enkephalin-related peptides is based on interaction
with different regions of the opioid receptor molecule. It is possible
that the opioid peptides interact with the opioid receptor at a binding
site composed of at least two subsites: one subsite binds with the
amino-terminal consensus sequence of enkephalin-related peptides and is
constituted from an equivalent domain of each opioid receptor type; and
the other subsite is involved in determining ligand selectivity and
constituted from different regions of each receptor type. According to
the ``message-address'' concept(23) , these two
subsites conceivably correspond to the recognition site for the message
component of the opioid peptide and that for the address component,
respectively.
Our results indicate that the major binding determinant for opioid alkaloids resides in a region of the µ-receptor different from that for enkephalin-related peptides. This finding suggests that the mechanisms for µ selectivity of the opioid alkaloid and the enkephalin-related peptide are different. It was suggested that the tyramine moiety in the opioid alkaloid and the amino-terminal tyrosine residue in opioid peptides, including enkephalin, play a similar role in the interaction with the opioid receptor(24) . Therefore, it is conceivable that the binding site of the µ-receptor for the opioid alkaloid and that for the enkephalin-related peptide overlap with each other, but are not completely identical. Recently, it was reported that different epitopes underlie affinity for peptide agonists and non-peptide antagonists in the cholecystokinin-B/gastrin receptor and the neurokinin-1 receptor(25, 26) . However, there has been no report that affinities for peptide and non-peptide agonists are determined by distinct regions of the G-protein-coupled receptor.
It was reported
that Asp in TM II of the
-receptor, which is
conserved in the
-, µ-, and
-receptors, is involved in
binding of
-selective agonists, including DPDPE, but not in
binding of nonselective ligands(27) . However, we could not
identify TM II of the
-receptor, the amino acid sequence of which
is completely identical with that of the µ-receptor, as a
determinant for DPDPE binding, because the roles of conserved amino
acid residues cannot be analyzed by the approach using chimeric
receptors. The possibility that conserved amino acid residues also
contribute to ligand selectivity would further increase the complexity
of the mechanism for the ligand selectivity of the opioid receptor.
Studies on several G-protein-coupled receptors for peptides have identified ligand binding epitopes in the receptor molecules. It has been reported that the region spanning TM IV-VI of the endothelin B receptor constitute the binding determinant for the endothelin B-selective agonists(28) . The region spanning from TM II to ECL II has been reported to mainly determine agonist selectivity of the tachykinin receptor(29) . Furthermore, in glycohormone receptors such as lutropin/choriogonadotropin receptor and the thyrotropin receptor, the amino-terminal extracellular domains were shown to be responsible for high-affinity binding of the hormones(30, 31) . These observations, together with our findings, suggest that the mode of interaction between agonists and the receptor is different for each receptor system.
In summary, our
results indicate that the determinants for binding of the - and
the µ-selective enkephalin-related peptides exist in distinct
regions of the opioid receptor molecules. Furthermore, the region
specifying the µ-selective alkaloid binding was shown to be
different from the determinant for binding of the µ-selective
enkephalin-related peptides. Further studies will be necessary to
identify specific amino acid residues that interact directly with
agonist molecules. However, the results obtained in this investigation
would provide insights into the mechanism for the ligand selectivity of
the opioid receptor and facilitate development of highly selective
opioid analgesics acting on the opioid receptor, which will have
considerable therapeutic potentials.