(Received for publication, November 19, 1996, and in revised form, January 24, 1997)
From the Astra Research Centre Montreal, Montreal, Québec H4S 1Z9, Canada
A novel "restoration of function" mutagenesis
strategy was developed to identify amino acid sequence combinations
necessary to restore the ability to bind -selective ligands to an
inactive
/µ receptor chimera in which 10 amino acids of the third
extracellular loop of the
receptor were replaced by the
corresponding amino acids from the µ receptor (
/µ291-300). This
chimera binds a nonselective opioid ligand but is devoid of affinity
for
-selective ligands. A library of mutants was generated in which
some of the 10 amino acids of the µ sequence of
/µ291-300 were
randomly reverted to the corresponding
amino acid. Using a ligand
binding assay, we screened this library to select mutants with high
affinity for
-selective ligands. Sequence analysis of these
revertants revealed that a leucine at position 300, a hydrophobic
region (amino acids 295-300), and an arginine at position 291 of the human
-opioid receptor were present in all revertants. Single and
double point mutations were then introduced in
/µ291-300 to
evaluate the contribution of the leucine 300 and arginine 291 residues
for the binding of
-selective ligands. An increased affinity for
-selective ligands was observed when the tryptophan 300 (µ residue) of
/µ291-300 was reverted to a leucine (
residue). Further site-directed mutagenesis experiments suggested that the presence of a tryptophan at position 300 may block the access of
-selective ligands to their docking site.
The opioid receptors are widely distributed throughout the central
nervous system and mediate the diverse effects of endogenous opioid
peptides and opiate drugs (1). Pharmacological studies have defined at
least three classes of opioid receptors, named , µ, and
, that
differ in their affinity for ligands and in their distribution in the
nervous system (1-6).
The antinociception mediated by supraspinal opioid receptors is thought
to act via the µ-opioid receptor subtype (7-10). The numerous side
effects accompanying opioid treatment, including respiratory depression
and addiction (11), are generally thought to be mediated by the
stimulation of µ receptors. However, there is growing evidence
suggesting that selective stimulation of the receptor may also
mediate antinociception (12-19). The strongest indication of an
involvement of
-opioid receptors in supraspinal antinociception
follows from studies with selective antagonists (14-16). These studies
demonstrated that the antinociception produced by
intracerebroventricular injection of morphine and
[D-Ala2,MePhe4,Gly-ol5]enkephalin
(µ agonists) was antagonized by
-funaltrexamine and naloxonazine
(µ antagonists) but not by ICI-174864 (
antagonists). Conversely,
the antinociception produced by intracerebroventricular injection of
DPDPE1 (
agonist) was antagonized by
ICI-174864 but not by
-funaltrexamine and naloxonazine. Moreover,
studies have shown that an antisense oligodeoxynucleotide to the cloned
-opioid receptor given intrathecally lowers
but not µ or
spinal (20) and central (21) analgesia. These studies confirm, at the
molecular level, traditional pharmacological studies implying distinct
receptor mechanisms for
, µ, and
analgesia. The development of
selective and potent
-opioid agonists therefore presents the
potential for the discovery of novel analgesic agents with reduced
accompanying side effects.
The recent cloning of the genes encoding the opioid receptors
showed that they are members of the seven transmembrane G
protein-coupled receptor family (22-28). There is about 60% amino
acid identity among the sequences of the three subtypes µ, , and
. The highest sequence homology between the three opioid receptor
subtypes resides in the transmembrane domains and the intracellular
loops. Lower sequence homology is seen in the N and C termini,
transmembrane domain 4, and extracellular loops 2 and 3. It is likely
that some of these divergent regions contain elements responsible for
the discrimination among these receptors by the subtype-selective opioid ligands. The construction of chimeric receptors is a powerful approach to investigate the structural basis for the subtype
specificity of G protein-coupled receptor (29-34). Previous studies
from our group (35) and others (36-40) have demonstrated, using
chimeras, the importance of the third extracellular loop of the
-opioid receptor for
ligand selectivity.
Most mutagenesis experiments designed to analyze the structure and
function of G protein-coupled receptor involve a strategy based on the
loss of function. Thus, even in well controlled studies, the
interpretation of these experiments is often difficult since a loss of
function may result from various causes. A mutant receptor may lack
affinity for a ligand because a critical residue of the binding pocket
has been hit but also because the mutated receptor is unable to traffic
efficiently to the cell surface or because the mutation induces protein
misfolding, allosteric changes, or gross structural defect.
Furthermore, the direct determination of G protein-coupled receptor
three-dimensional structure is hampered by technical difficulties
limiting their overexpression and purification in quantities that would
permit crystallographic studies. Today, only relatively low resolution
structural information has been obtained for the bacteriorhodopsin and
bovine rhodopsin from two-dimensional cryo-electromicroscopic
experiments (41, 42). For these reasons, we have designed a mutagenesis
strategy based on the restoration of a lost function to identify amino
acid sequence combinations critical to confer -selective ligand
binding to an opioid receptor.
pcDNA3-hDOR
consists of a 1.2-kilobase cDNA EcoRI-XhoI
fragment of the -opioid receptor (23) sub-cloned at the
EcoRI-XhoI site of pcDNA3 (Invitrogen). Using
unique site elimination (USE) mutagenesis (43), pcDNA3-hDOR was
mutated to pcDNA3-
/µ291-300 by replacing 10 amino acids
(positions 291-300) of the third extracellular loop of the
receptor with the corresponding amino acids from the µ receptor. The
selection primer was designed to mutate the unique PvuI site
of pcDNA3-hDOR to a EcoRV site (5
-GCT CCT TCG GTC CTC
GAT ATC TTG TCA GAA GTA AGT TGG C-3
) and primer
/µ291-300 (5
-GTC TGG ACG CTG GTG GAC ATC GAC CCA GAA ACT ACG TTC CAG ACT GTT TCT
TGG CAC CTG TGC ATC GCG CTG GGT TAC-3
) was used to produce the
pcDNA3-
/µ291-300 chimera. The library of mutants of the
/µ291-300 chimera was produced by USE mutagenesis (43) using
pcDNA3-
/µ291-300 as the parental vector. A degenerated
primer,
/µ291-300.degenerated (5
-GTC TGG ACG CTG GTG GAC ATC GAC
C c/g A g/c a/g A a/g c/a T a/c CG TT c/g c/g a/t G a/g c/t T G t/c T
t/g CT T g/t G CAC CTG TGC ATC GCG CTG GGT TAC-3
) was designed to
randomly and independently revert amino acids of the µ sequence to
the original
sequence (16,384 possible combinations). Each
degenerated position contained an equal ratio of the nucleotide from
the µ or the
sequence. This degenerated primer was used with a
selection primer EcoRV to PvuI (5
-GCT CCT TCG
GTC CTC CGA TCG TTG TCA GAA GTA AGT TGG C-3
) to perform a mutagenesis
reaction on pcDNA3-
/µ291-300. This synthesis mixture was
transformed into Escherichia coli DH5
cells, and pools of
clones were randomly selected. Plasmid DNA from each pool was isolated
using QIAprep 8 plasmid kit (Qiagen, Chatsworth, CA) and used for
transfection into HEK 293s cells.
Single and double point mutations
were introduced into /µ291-300 chimera using Clontech
site-directed mutagenesis kit which is based on the method of Deng and
Nickoloff (43). In a first mutant, we introduced a single mutation that
converts tryptophan residue 300 to leucine residue (
/µ291-300
(W300L)). Another mutant of the chimera was constructed into which
proline residue 291 was converted to arginine residue (
/µ291-300
(P291R)). A double mutation was also introduced into the
/µ291-300 chimera that converts proline 291 to arginine and
tryptophan 300 to leucine (
/µ291-300 (P291R/W300L)). Sequences of
the primers were 5
-CTG GTG GAC ATC GAC CGA GAA ACT ACG TTC CAG-3
,
5
-TTC CAG ACT GTT TCT TTG CAC CTG TGC ATC GCG-3
, and 5
-CTG GTG GAC
ATC GAC CGA GAA ACT ACG TTC CAG ACT GTT TCT TTG CAC CTG TGC ATC GCG-3
to obtain P291R, W300L, and P291R/W300L mutations, respectively.
Human embryonic
kidney 293s cells (obtained from Michael Matthew, Cold Spring Harbor)
were grown in Dulbecco's modified Eagle's medium supplemented with
10% fetal calf serum. Cells were transiently transfected according to
the procedure of Chen and Okayama (44). Transfections were performed
using 15 µg of expression vectors and 1 × 106 cells
per 25-cm2 flasks. Binding of -selective ligands
(SNC-121 and DPDPE) to the transfected cells was monitored 48 h
after transfection.
Pools containing 50 clones each were screened for the
presence of revertant mutants with affinity for -selective ligands using a radioactive ligand binding assay. Cells transfected with pools
of the library were assayed 48 h post-transfection for the binding
of
-selective ligands [3H]DPDPE (peptide agonist) and
[3H]SNC-121 (non-peptide agonist) (45). Glycerol stocks
of E. coli transformants corresponding to positive pools
were partitioned into smaller pools of 10 clones using a row/column
strategy.
One hundred colonies from each positive pool were inoculated on a Petri dish using 10 rows × 10 columns pattern. After overnight incubation, the 10 colonies from each row and each column were pooled into 5 ml of Luria-Bertani (LB) broth and incubated overnight at 37 °C. DNA from each pool was prepared as described and transfected into 293s cells for ligand binding analysis. Colonies at the intersection of a positive column and a positive row were selected for sequencing and further pharmacological characterization.
The sequence of the revertants was determined by dideoxy nucleotide
chain termination method using T7-DNA polymerase (Pharmacia Biotech
Inc.) and -35S-dATP (DuPont NEN).
For the receptor binding study,
HEK 293s cells expressing pools of mutant receptors were harvested
48 h after transfection and resuspended in 1.5 ml of membrane
buffer (50 mM Tris-HCl, pH 7.4, 320 mM
sucrose). Cells were then frozen/thawed, and an aliquot was used for
the radioligand binding assay. Cells (50 µl) were incubated in a
final volume of 150 µl of binding buffer (50 mM Tris-HCl,
pH 7.4, 3 mM MgCl2, 0.1% bovine serum albumin) with [3H]bremazocine (6.9 nM, specific
activity 30 Ci/mmol) (DuPont NEN) (46), 3H-labeled
[D-Pen2,D-Pen5]enkephalin
([3H]DPDPE) (6 nM, specific activity 31.4 Ci/mmol) (DuPont NEN), or [3H]SNC-121 (tritiated version
of SNC-80) (6 nM, specific activity 26.3 Ci/mmol) (from
John Partilla, NIDA) for 90 min at room temperature. For saturation
experiments, duplicates contained 0.3-15 nM
[3H]bremazocine, [3H]DPDPE, or
[3H]SNC-121. Nonspecific binding was determined using 10 µM naloxone. For competition experiments, duplicate
contained 2.5 nM [3H]bremazocine and 1 pM to 10 µM DPDPE or SNC-80. Reactions were terminated by filtration on polyethyleneimine-precoated GF/B Whatman filters. The filters were washed three times with ice-cold buffer (50 mM Tris-HCl, pH 7.4, 3 mM MgCl2)
and dried. Bound radioactivity was counted using a scintillation
counter. Specific binding was determined as the difference between
binding in the absence or presence of an excess of unlabeled naloxone
(10 µM). Curve fitting and analysis of the binding data
were performed using the GraphPad Prism program version 1.03 (1994).
The three-dimensional model of
human -opioid receptor was constructed following a general procedure
to build G protein-coupled receptors. There are three steps in this
procedure. First, we identified the transmembrane helical domains from
sequence alignments of the opioid receptor subfamily. Using the
identified sequences, we built the initial helices bundle and then
searched for the maximum interactions among these seven helices using a
mixed molecular dynamics and conformational search procedure with the
restraints from the projection density maps of rhodopsin (41). Finally we added the extracellular loops obtained from the Protein Data base
based on the sequence homology analysis. The sequence of human
-opioid receptor (47) (Genbank P41143[GenBank]) was first submitted to the
TMAP procedure of EMBL (48) to search for the opioid receptor family
and to identify the transmembrane helical regions. The assumption that
the arginine and lysine residues are most likely at the end of helices
(49) was further used to adjust the helical regions. The initial helix
building, the sequence homology, and the final structure refinement
were performed using Quanta/CHARMM (Biosym/MSI). The mixed molecular
dynamics and conformational search procedure was developed
in-house.
Fig. 1 illustrates the
mutagenesis strategy we have designed to identify residues critical for
the binding of -selective ligands. This strategy relied on the
restoration of a function rather than the loss of a function. A
chimeric receptor unable to bind the
-selective ligands was used as
template in a mutagenesis reaction. The amino acid sequence over the µ region (Fig. 1) was mutated using a degenerated oligonucleotide.
The resulting library of mutants was then separated in pools of 50 clones. The plasmid DNA from each pool of clones were isolated and
transfected into HEK 293s cells. The presence of a receptor mutant
within a pool was detected if the transfected cells expressed a binding
site for the selective ligand. The pool was then gradually split to isolate the clone responsible for the
-selective ligand binding activity. The DNA sequences of revertant clones were determined and
compared to identify common structural features.
Construction of the
Evidence from different groups has suggested that the
third extracellular loop of -opioid receptors is involved in the
binding of selective ligands (35-40, 50). A chimeric
-opioid
receptor in which 10 amino acids of the third extracellular loop (amino acids 291 to 300) were replaced by the corresponding amino acids from
the µ receptor was constructed. HEK 293s cells were transfected with
the plasmid DNA coding for the chimera, and radioligand binding assays
using the nonselective [3H]bremazocine or
-selective
ligands [3H]SNC-121 and [3H]DPDPE were
performed 48 h post-transfection. The radioligand binding
properties of
/µ291-300 chimera are shown in Fig.
2A and summarized in Table I.
This chimera binds the nonselective opioid ligand bremazocine with the
same affinity as the wild-type receptor (Fig. 2A and Table
I). However, it does not bind
-selective ligands such as
[3H]DPDPE or [3H]SNC-121 (Fig.
2A and Table I).
|
We then randomly and independently substituted µ residues of this
chimera with the corresponding residues. To this end, we used a
degenerated primer (
/µ291-300.deg) to mutate this 10-amino acid
region. The primer was designed to allow each residue of this stretch
to be either of the µ or the
sequence. Owing to the design of the
primer, some positions could also code for non-µ and non-
residues
(see Fig. 2B). Using the pcDNA3-
/µ291-300 plasmid
as a template and
/µ291-300.degenerated as the mutagenic primer,
we performed a mutagenic synthesis theoretically generating all the
possible combinations of µ,
, or non-µ, non-
residues over
the 10 amino acids of the third extracellular loop of
/µ291-300 (Fig. 2B). This represents 16,384 possible combinations.
To evaluate the frequency of amino acid substitution over the targeted
10-amino acid stretch, 50 clones of the mutant receptor library were
randomly selected and subjected to DNA sequencing. Sequence analysis of
the clones showed that 90% of these clones were mutated and contained
48.3% amino acid substitution at a position where a combination of two
residues was possible and a percentage of substitution increased to
76% at a position where a combination of four different residues was
possible (Fig. 3). These results indicate that amino
acid substitution occurred randomly and without any preference for one
or the other sequence.
Screening of the Library
Preliminary experiments were performed to determine the size of the pools where a single revertant would be detected using radioligand binding assays. We transfected HEK 293s cells with different dilutions of the wild-type hDOR expression vector corresponding to pools of 1-10,000 clones that would contain a single colony encoding a wild-type hDOR receptor. In this experiment, we observed that statistically significant specific binding can be detected using pools of 500 clones (one of which being hDOR) using [3H]SNC-121 (6 nM) or [3H]DPDPE (6 nM) as radioligands (data not shown). After performing a series of preliminary experiments to determine the ideal pool size, we decided to screen pools of 50 clones.
DNA from pools of 50 clones each were then transfected into HEK 293s
cells according to the procedure of Chen and Okayama (44). Radioligand
binding assays using -selective ligands were performed 48 h
after transfection. Forty pools of 50 clones were transfected into HEK
293s cells and screened independently with [3H]DPDPE or
[3H]SNC-121 in the presence or absence of 10 µM naloxone. From this first screen, 6 positive pools
(pool 2, 16, 18, 29, 32, and 37) were selected since they displayed
significant affinity for [3H]DPDPE and
[3H]SNC-121. These
-selective binding pools were
subdivided into smaller pools using the row/column strategy described
under "Experimental Procedures." Each pool contained one revertant
except pools 16 and 32 from which two positive clones were obtained,
yielding a total of eight revertants (Table I).
The
plasmid DNAs encoding the eight revertant receptors were transfected
into HEK 293s cells, and the transfected cells were analyzed 48 h
post-transfection. Saturation binding experiments using
[3H]SNC-121, [3H]DPDPE, and
[3H]bremazocine were performed to determine the
respective Kd values for each of the revertants.
Saturation binding experiments with [3H]bremazocine were
used to evaluate binding site levels. The examination of
Bmax values (Table I) indicates that mutations
under study do not drastically modify receptor expression in HEK 293s
cells. Expression levels of most of the mutants are not significantly modified. Bmax values of 29.4, 37.5, 32.55, 16.9.9 and hDOR(R291P) mutants are slightly decreased, but radioligand
binding remains nevertheless easily detectable. All the identified
revertants display affinity for the -selective ligand
([3H]SNC-121 and [3H]DPDPE) and
nonselective ligand ([3H]bremazocine) similar to the
wild-type
-opioid receptor. Interestingly, all the revertants
regained the ability to bind both [3H]SNC-121 and
[3H]DPDPE. No statistical differences were noted between
the Kd values measured for the different
revertants.
The sequences of
the revertant mutants were determined and are shown in Fig.
4. Analysis of the sequence of these revertants revealed
that an arginine at position 291 and a leucine at position 300 (from
the sequence) were present in all the revertants (Fig. 4). Amino
acids from either the
, µ, or non-µ/non-
sequence were found
at positions 292-294 suggesting that these residues are not critical
for the binding of
-selective ligand (Fig. 4). Moreover, all
revertants had acquired a stretch of hydrophobic residues at positions
295-300 (valine, alanine, and leucine), a characteristic of the
receptor in this region (Figs. 4 and 5). The hydrophobic
residues found at positions 295-300 were either from the
sequence,
the µ sequence, or from non-
or non-µ sequence.
Ligand Binding Properties of Single and Double Point Mutants of
To evaluate the independent or
simultaneous contribution of the two amino acids leucine 300 and
arginine 291 to the binding of -selective ligands, single and double
point mutations were generated in
/µ291-300 chimera. Using this
chimera as the template, tryptophan 300 was reverted to a leucine
residue (
/µ291-300 (W300L)), proline 291 was reverted to an
arginine residue (
/µ291-300 (P291R)), or both mutations were
introduced simultaneously (
/µ291-300 (P291R/W300L)). As
determined by saturation binding experiments, all these mutant receptors bind [3H]bremazocine with the same affinity as
the wild-type receptor and their Bmax values
show that these mutations do not considerably alter the expression
level. Competition assays using [3H]bremazocine as tracer
and the
-selective ligands DPDPE or SNC-80 as competitors were
performed, and the Ki values are listed in
Table II.
|
We observed that reversion of tryptophan 300 (µ residue) to a leucine
( residue) (
/µ291-300 (W300L)) increases the affinity for both
DPDPE and SNC-80 (Table II), although it remains 15 times lower than
wild-type hDOR.
Double reversion to the sequence of residues located at positions
291 and 300 (
/µ291-300 (P291R/W300L)) did not produce a further
increase in affinity toward
-selective ligands.
Similarly, reversion of proline 291 (µ residue) to an arginine ( residue) (
/µ291-300 (P291R)) did not increase affinity for
-selective ligands DPDPE and SNC-80 as compared with the
/µ291-300 chimera.
Three-dimensional computer modeling was used to gain
some insight into the orientation of the residues that are present in all of the revertant mutants (Fig. 6). The
three-dimensional model of the human -opioid receptor was
constructed following a general procedure for G protein-coupled
receptors that has been described under "Experimental Procedures."
In this model, the seventh transmembrane domain starts at valine 296 which is 5 residues ahead of leucine 300, and these 2 residues are
separated by a hydrophobic region. According to our model, the arginine
localized at position 291 (Arg-291) (shown in yellow in Fig.
6) points toward the outside of the receptor suggesting that arginine
291 does not interact directly with ligand. The leucine localized at
position 300 (Leu-300) (shown in yellow in Fig. 6) faces the
inner side of the binding pocket and could directly interact with the
-selective ligand SNC-121 which is represented in red in
this figure. The hydrophobic region from amino acids 295-300,
represented in green in Fig. 6, is localized at the top of
the seventh transmembrane domain.
In this paper we describe the design and use of a "restoration
of function" mutagenesis strategy to identify residues of the human
-opioid receptor involved in the binding of subtype-selective ligands. Leucine 300 has been identified as a critical residue, and we
proposed that residues at this particular position in other opioid
receptor subtypes may play a role of exclusion of
-selective ligands.
First, we generated a chimeric receptor (/µ291-300) in which 10 amino acids of the third extracellular loop of the human
-opioid
receptor were replaced by the corresponding amino acid sequence of the
µ-opioid receptor. This protein binds nonselective opioid ligands but
is devoid of affinity for
-selective ligands. Our results are in
agreement with results from previous studies using
/
or
/µ
chimeric receptors that have shown that
-selective ligands interact
mainly with the region containing the sixth transmembrane domain and
the third extracellular loop of the
-opioid receptor (35-40, 50).
In this study, we have delimited this region to 10 amino acids that are
located between arginine residue at position 291 and leucine residue at
position 300.
Using this chimeric construct as the template, we generated a library
containing theoretically 16,384 mutants in which combinations of amino
acids of the third loop were reverted to the corresponding sequence. Next, we used radioactive
-specific ligands to select from
this receptor library mutants that had regained the ability to bind
-selective ligands with high affinity. Using this novel strategy, we
showed that a leucine at position 300, a hydrophobic region (amino
acids 295-300), and an arginine at position 291 of the human
-opioid receptor were present in all revertants suggesting a
possible role of these residues in the binding of
-selective
ligand.
The binding characteristics of the /µ291-300 chimera demonstrates
that replacing amino acids 291-300 of the
-opioid receptor by the
corresponding amino acids of the µ receptor abolishes
-selective binding while preserving nonselective opioid ligand binding. This suggests that the overall structure of the chimera is preserved and
that
amino acids 291-300 contribute to
selectivity either by
making specific contacts with the
ligand or by inducing
conformational change in the receptor that would favor migration of the
ligand to a binding pocket located more deeply in the receptor.
Another hypothesis developed by Metzger and Ferguson (51) could be
applied in this model. They suggest that opioid ligands would bind to their receptors into a pocket formed by the transmembrane helices and
this pocket would be common to all opioid receptor subtypes. Selectivity would be conferred by the extracellular loops that would
act as a gate to allow the passage of certain ligands while excluding
others. In our model, µ residues located at positions 291-300 of the
chimera could inhibit the passage of
-selective ligand to the
transmembrane binding pocket.
Comparison of amino acid sequences of the selected revertants allows a
number of observations to be made. All revertants have substituted
tryptophan 300 and proline 291 (µ sequence) from /µ291-300 chimera with a leucine and an arginine (
sequence), respectively, suggesting that these positions might play a role in determining
specificity. To more precisely define the contribution of leucine 300 and/or arginine 291 to the restoration of
-selective ligand binding,
these residues were mutated singly or in combination in
/µ291-300
chimera.
/µ291-300 is devoid of any detectable affinity for
ligands.
The reversion of tryptophan 300 (µ residue) to a leucine (
residue) in the construct (
/µ291-300 (W300L)) partially restores the affinity for both
-selective ligands DPDPE
(Ki = 72 nM) and SNC-80
(Ki = 102 nM) (Table II). Nevertheless, these Ki values remain 15 times higher than observed for the wild-type hDOR. The presence of a leucine at position 300 is
not an absolute requirement for
-selective ligand binding, since a
mutant of the
-opioid receptor in which this leucine residue is
substituted for an alanine binds SNC-80 with wild-type affinity.2 It appears that the absence of
tryptophan at position 300 is more important than the presence of a
leucine. It is conceivable that the presence of a bulky tryptophan
residue at position 300 blocks the access of
ligands to their
docking site. Our three-dimensional model of the receptor (Fig. 6)
suggests that the leucine at position 300 points toward the inside of
the binding pocket. Therefore its replacement by a tryptophan would
obstruct the access to the central pore of the receptor where the
ligand docking site is likely located. These observations are in
agreement with a recently proposed hypothesis (51) suggesting that the
selectivity within the opioid receptor family may be imparted through a
mechanism of exclusion, rather than specific pharmacore recognition
within the extracellular loops.
Single reversion of proline 291 (µ residue) to arginine ( residue)
is not sufficient to restore the binding of
-selective ligand
(
/µ291-300 (P291R)) (Table II). The tryptophan residue at
position 300 is still present in this construction and may inhibit
binding of
-selective ligands. However when mutations reverting
tryptophan 300 to a leucine and proline 291 to an arginine are
introduced simultaneously in
/µ291-300 chimera, there is no
increase in binding affinity as compared with single reversion of
tryptophan 300 to leucine (
/µ291-300 (W300L)). This result indicates that in this sequence context, arginine 291 does not improve
-selective binding. Therefore, the possible involvement of arginine
291 in the binding of
-selective ligands remains unclear and has to
be elucidated.
A mutant of hDOR with an alanine residue at position 291 instead of an
arginine binds -selective ligand DPDPE and SNC-80 with wild-type
affinity suggesting that arginine 291 (
residue) is not critical for
the binding of
-selective ligand (35). However, the adjacent residue
at position 292 is also an arginine which may compensate for the
substitution at position 291. This interpretation is supported by the
observation of Wang and co-workers (50) that has shown that a double
mutation of arginine 291 and 292 abolishes the ability to bind DSLET (a
-selective ligand) while retaining nonselective ligand binding
properties.
We observed that residues 295-300 were hydrophobic in all the selected
revertants. This result is supported by the work of Valiquette et
al. (35) where they have shown that the valine 296 and the valine
297 residues of the hDOR are involved in the binding of -selective
ligand. The present study suggests that it is the overall hydrophobic
character of this region rather than its specific primary amino acid
sequence that is important for
-selective binding since the primary
sequence of most revertants is divergent from the
receptor
sequence.
Amino acids 292-294 do not seem critical for the binding of
-selective ligands. Indeed, binding of [3H]DPDPE and
[3H]SNC-121 is observed with mutants bearing
-, µ-,
or non-
or non-µ amino acids at positions 292, 293, and 294, suggesting that strict residue identity is not required at these
positions for
-selective binding.
The restoration of function strategy we have used presents some
advantages over the traditional "loss of function" strategy. In the
traditional mutagenesis strategy, the residues that are identified as
critical are those causing a loss of binding function when mutated. In
interpreting the results from such experiments, one needs to explain
the reasons for the loss of function which could be due to the
substitution of a residue essential for ligand binding, to a low level
of expression of the mutant, a decrease in the stability of the mutant
receptor, or inefficient traffic to the cell surface. By using careful
controls, one can eliminate possible explanations, but there often
remain some possibilities for misinterpretation. Unlike the traditional
mutagenesis strategy, which analyzes the contribution of a single amino
acid, the restoration of function strategy permits the identification
of multiple combinations of residues thus allowing a specific function
to be restored. Moreover, this positive approach allows us to identify
nonessential positions or positions that can tolerate various
substitutions. We thus have observed in this study that residues at
positions 292-294 were not critical for the binding of -selective
ligand since µ,
, or non-µ/non-
residues with different
physicochemical properties were found at these positions. Finally, this
positive approach can identify specific physicochemical properties
(like hydrophobic characteristics) of a region or area required for the
function. This situation is observed when residues are not reverted to a specific sequence but to residues sharing similar physicochemical characteristics.
In this study, we have developed a novel and efficient method for the analysis of the structure-function of the opioid receptors or possibly any G protein-coupled receptor. This method is based on a positive approach that allows identification of positions within the receptors that are essential, deleterious, or neutral for the interaction with different ligands.
We are grateful to Dr. Brigitte Kieffer for
providing us with the human receptor cDNA. We also thank Dr.
Manon Valiquette for critical review of the manuscript and Huy K. Vu
for technical assistance.