From the
Within the large family of G-protein-coupled receptors, a
picture is emerging which contrasts the binding of small ligands and
the binding of peptides to the seven-helix configuration of the
proteins. Because of its unique richness in both peptide and
non-peptide ligands, the opioid receptor family offers several
advantages for achieving a better understanding of similarities and
differences in ligand/receptor interactions across different classes of
agonists and antagonists. Since multiple, naturally occurring, ligands
interact with the multiple receptors with varying degrees of
selectivity, this family is also an excellent model for examining the
structural basis of selectivity. Thus, the molecular basis of binding
affinity and selectivity of the
It is well established that there are at least three major
opioid receptor types in the brain and periphery. These receptors are
referred to as µ,
While the above summary associates individual receptors primarily
with one or another of the three opioid precursors, proenkephalin,
prodynorphin, and proopiomelanocortin, there is in fact no clear
one-to-one segregation between a precursor family and a receptor type.
Rather, a given endogenous ligand can interact to varying extents with
multiple receptors, and a given receptor can recognize multiple ligands
deriving from any of the three opioid precursors. The endogenous
ligands share a common critical core sequence, Tyr-Gly-Gly-Phe-Met (or
-Leu), with varying C-terminal extensions which impart their unique
selectivity profiles. The structural basis of this combination of
affinity and selectivity profiles remains to be understood.
The
elucidation of the structural basis of ligand binding for different
subtypes of opioid receptors will not only enable us to better
understand the physiological function of different opioid peptides but
may also help us develop highly selective opioid drugs with minimal
side effects. The structure-function relationship of opioid receptors
has been investigated with the synthesis of a large number of ligands
based on the structure of opioid peptides and of natural opioid
alkaloids. However, this line of research, while being very fruitful in
generating ligands with high affinity and selectivity, cannot directly
address issues of receptor structure and how it relates to function. A
second approach relied on chemical modification of the opioid receptor
itself. Since it is very difficult to introduce specific modifications
to the sites that actually bind opioid ligand, the data generated are
usually circumstantial and provide only a limited view of receptor
structure. As a result, the structural basis of opioid receptor binding
to their ligands remains largely unknown.
Current knowledge of the
structure-function relationship of the seven-helix G-protein-coupled
receptor family is mainly derived from the pioneering work of
Lefkowitz, Strader, Dixon, and their co-workers on the cloned
adrenergic receptors, using site-directed mutagenesis and the
construction of receptor
chimeras
(19, 20, 21, 22) . Analysis of
the binding of small transmitters indicate that they mainly interact
with the amino acid residues in the middle of the transmembrane
domains, which can be viewed as a binding pocket for the small
molecules. Studies on a small number of receptors for large peptide
ligands have shown that the extracellular domains may play a more
important role for these ligands (23-25). Indeed some
investigators have proposed an exclusively extracellular site of
interaction for the peptide ligands
(26) . The cloning of the
three major types of opioid receptors enables us for the first time to
precisely manipulate the structure of these proteins, thus greatly
facilitating the study of their structure-function relationship. It is
now possible to determine which parts of an opioid receptor is
primarily responsible for the binding of subtype-selective ligands and
to ascertain whether all opioid receptors bind the common opioid core
Tyr-Gly-Gly-Phe-Met (or -Leu) in the same way. We have chosen the
construction of chimeras between the
To best answer the above questions
regarding domains of relevance to binding affinity and selectivity, we
shall look at these data from two perspectives. The first approach is
to analyze the binding properties of a given class of ligands across
the four chimeras and the two wild type receptors. From the binding
affinity of different categories of ligands toward various receptors,
we should be able to learn about the different structural requirements
of these ligands. Additionally, we may observe differences between
peptides and alkaloids, selective ligands and nonselective ligands,
A note regarding interpretations of the
results may be in order at this juncture. Meaningful conclusions from
chimeric constructs can be derived only if these constructs express
functional proteins free of substantial conformational distortions. We
would predict that (i) if there is no significant conformation
deterioration in the chimeric receptor and (ii) if the parent wild type
receptors bind a nonselective ligand through similar mechanisms, then
the chimeric receptor should retain good binding affinity to a
nonselective ligand. If there is a significant loss in affinity for a
nonselective ligand, either there is global conformational
deterioration in the chimeric receptor or the parent wild type
receptors bind the ligand in question in very different
ways.
In general, the results lead us to
the conclusion that the
It is
very clear from that the
It should be pointed out that for peptide
ligands, the combinations between these two large domains of
the receptors (N/M and C) maybe important. When the affinity ratios for
the same pair of segments are available from two different pairs of
receptors, they are quite different from each other for all the peptide
ligands. All of the pairs showed between an 8-fold (for E2078) and a
29-fold (for
According to the linear model, the
product of the data in the first and second data columns should be
equal to the data in the third column. For most of the small ligands
and all the peptide ligands, the product of affinity change values from
N and M segments matches well with the affinity change value of NM
segment if we consider two to three fold difference in affinity change
value as being within the error range. This suggests that, while
interactions between N/M and C were important for the peptides (see
above), the interactions between different N and M segments do not
contribute significantly to the binding of most ligands. One noticeable
exception is naltrindole. When
The experiments described above lead us to the following
conclusions. (i) The selectivity of the
A
particularly surprising result is the observation that the
If the
extended prodynorphin peptides, when binding the
Given the above arguments, we would suggest that the
While a
great deal more work remains to be done, these studies already suggest
a richer view of this subfamily of G-protein-coupled receptors. The
It should be emphasized that any results from chimeric studies may
not reflect the true structure-function relationship of the wild type
receptors, since the interactions between different segments may
contribute significantly to the binding of a given ligand. It is
essential that chimeric experiments are designed and analyzed in such a
way that one can learn whether or not the receptors are behaving
linearly, or whether there is evidence that a domain behaves
differently as a function of the protein environment in which it is
placed. Extra caution should be exercised when the receptors do not
behave linearly. In all cases, results from this class of studies
should be complemented with extensive additional experimental data and
analysis, including smaller modifications and point mutations.
Nevertheless, these studies offer a valuable starting point for
addressing the question of the structural bases of ligand binding and
ligand selectivity.
The values in the first data column represent the change
in K
We thank Dr. Henry I. Mosberg of the University of
Michigan for providing us the highly
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and the
opioid receptors
was investigated by the construction of four
/
chimeric
receptors. The pharmacological profiles of these chimeras as well as
those of the wild type
and
receptors were determined by
their binding with several different categories of opioid ligands. A
linear model was used to deduce the relative contribution of each
corresponding pairs of
-
receptor segments to the binding of
a given ligand. The results show that the
and
receptors
bind the same opioid core differently and achieve their selectivity
through different mechanisms. In addition, the interaction of a peptide
ligand with a receptor appears to be different from that of a small
ligand. Furthermore, these results point to a particularly important
role of the second extracellular loop and the top half of transmembrane
domain 4 in the binding of prodynorphin products. Together, the results
suggest that these peptide receptors can be bound and activated via
multiple binding pockets as a function of their own topography and the
nature of the interacting ligand.
, and
and have distinct
pharmacological profiles, anatomical distributions, and
functions
(1, 2, 3, 4) . The µ
receptors bind morphine-like drugs as well as several endogenous opioid
peptides. They are thought to mediate the opiate phenomena classically
associated with morphine, including analgesia, opiate dependence,
cardiovascular and respiratory functions, and several neuroendocrine
effects. The
receptors bind enkephalin-like peptides and many
other endogenous opioid peptides with high affinity. They are thought
to mediate analgesia, gastrointestinal motility, as well as a number of
hormonal functions. The
receptors exhibit a high affinity for the
products of opioid precursor prodynorphin. In addition, the
receptors interact with several pharmacological agents, including
benzomorphans, such as bremazocine, and arylacetamides, such as
U50,488
(
)
and U69,593
(5) . The
receptors mediate a spectrum of unique and distinctive functions,
including the modulation of drinking, water balance, food intake, gut
motility, temperature control, and various endocrine
functions
(6, 7, 8, 9) . The binding
profile of the
receptor is relatively unique among the opioid
receptors, while those of the µ and
receptors are more
similar to each other. Based on functional studies, opioid receptors
had been classified as members of the G-protein-coupled receptor
family, and this has been supported by their recent
cloning
(10, 11, 12, 13, 14, 15, 16, 17, 18) .
and
types of opioid
receptors as a first step in achieving an overview of the
structure-function relationship of this family of receptors.
Construction of the Chimeric Receptors
The cDNAs
encoding the rat and
receptor were cloned in our
laboratory
(18) . The rat
receptor sequence is not
published but the coding region is identical to the sequence reported
by Fukuda et al.(17) . Two conserved native restriction
sites Afl3 and Bgl2 present in both the
and
cDNA were
utilized to produce four
/
chimeric receptors. The Afl3 site
lies in the middle of TM3, and the Bgl2 site is at the end of
extracellular loop 2 (see Fig. 1). The structure of the chimeric
cDNAs was verified by extensive restriction enzyme mappings to ensure
that the arrangement of different segments is correct and to ascertain
that there are no incorrect bases at the ligation sites. The chimeric
cDNAs as well as the wild type cDNAs were subcloned into a pCMV-neo
expression vector courtesy of Dr. Mike Uhler (27).
Figure 1:
Structure
of the chimeric receptor constructs.
Expression of the Receptor and Binding
Assay
Twenty-five micrograms of plasmid DNA were transfected
into each 100-mm dish of COS-1 cells using the method of Chen and
Okayama (28). Receptor binding of the membrane preparation of the
transfected cells was performed according to Goldstein and
Naidu
(29) . About 1.5 nM of
[H]EKC (24.8 Ci/mmol, DuPont NEN) was used to
label the receptors. All assays were conducted in 50 mM Tris
buffer (pH 7.4) at room temperature. For each competing ligand, nine
different concentrations were used and all the receptors were assayed
in the same batch in duplicates. The affinity of the labeling ligand
EKC toward these receptors was determined by two independent binding
assays. The average value of two assays was used to calculate the
affinity of other ligands competing with EKC. Receptor binding results
were analyzed with the LIGAND program
(30) .
Structure of the Chimeric Receptors
Four chimeric receptors were constructed by using two
conserved restriction sites in the and
opioid receptors.
The structure of these constructs are shown in Fig. 1. The
small circles represent the net negative charges in the
extracellular domains of a receptor. In the following discussion,
``N-terminal segment'' refers to the region from the
initiator methionine residue to the Afl3 site in the middle of
TM3 in both
and
receptors; ``middle segment''
refers to the region between the Afl3 site and the
Bgl2 site, which is at the end of extracellular loop 2;
``C-terminal segment'' designates the region beyond the
Bgl2. For simplicity, the N-terminal segment from the
receptor will be referred to as
-N, that from the
receptor
as
-N, etc.
Pharmacological Profiles of the Chimeric Receptors
The binding profiles of the chimeric and wild type receptors
are summarized in .
ligands and
ligands, and possibly agonists and antagonists
as they interact with various proteins. We may also determine whether
ligands in the same category, i.e. ligands that act similarly
on the wild type receptors, achieve their behavior through the same
mechanism. The complementary approach involves the analysis of the
behavior of each component of the receptor across various ligands. This
should allow us to describe the role of individual segments in the
binding of different ligands and should provide us with a more
quantitative idea about the importance of each segment in the binding
of a particular ligand.
(
)
One would also suppose that if a given
chimeric receptor has nanomolar affinity for at least one ligand, the
protein has good global conformation.
Binding Profile of Different Ligands across Chimeras
Nonselective Alkaloids
We chose the nonselective
opioid alkaloids EKC, bremazocine, Mr2034, naloxone, and naltrexone as
control ligands because the binding mechanisms of nonselective small
ligands to the and
receptors may be more similar to each
other than to those of nonselective large peptide ligands. All four
chimeric receptors in exhibited high affinity binding to
bremazocine, Mr2034, and naltrexone. Three of the chimeric receptors
also bound EKC and naloxone very well. Chimeric receptor
-NM/
-C showed lower affinity for EKC and naloxone than both
wild type
and
receptors but in both cases it is a less than
3-fold change. Since the same receptor binds bremazocine and naltrexone
very well, this phenomenon is more likely caused by the fact that the
wild type
and
receptors bind these two ligands in somewhat
different ways than by a slight conformation change in the chimeric
receptor. In general, the binding affinities of the four nonselective
opioid alkaloids to the four chimeric receptors suggest that the
chimeric receptors have a good overall conformation capable of
supporting high affinity binding to these alkaloids.
Prodynorphin Peptide Ligands
The binding profiles
of the prodynorphin peptides DynA(1-13), -neoendorphin,
DynB, and
-selective DynA analog E2078 are very different from
those of the nonselective alkaloids. Chimeric receptor
-NC/
-M
shows the largest contrast between these two classes of ligands, with
very high affinity for the nonselective alkaloids but with lowest
affinity among all the chimeric receptors toward the nonselective
prodynorphin ligands. Chimeric receptor
-NM/
-C also exhibited
a large loss in its affinity toward these ligands. But its reciprocal
construct,
-NM/
-C showed an affinity toward prodynorphin
ligands that is equal to or better than that of either of the wild type
receptors. The affinity of chimeric receptor
-N/
-MC toward
these ligands is significantly lower than that of the wild type
receptors but is much better than that of chimeras
-NC/
-M and
-NM/
-C. From the binding profile of prodynorphin ligands, it
appears that
-M plays the most important role in their binding to
the chimeras. The
-C segment is also very important for binding
but not as critical as
-M. If the situation in the wild type
receptors is similar to that in the chimeric receptor, it would appear
that, in the
receptor, the middle segment plays a more important
role in the binding of prodynorphin peptides than either its N- or
C-terminal segments. By contrast, the C-terminal segment of the
receptor is more important for binding than its N and M segments.
Among the four
ligands considered here, U50,488, U63,640, and ICI204,488 share a
similar binding profile across constructs. Chimeric receptor
-Selective Non-peptide Ligands
-NM/
-C and the
receptor have very high affinity for
these ligands, while the reciprocal construct
-NM/
-C and the
receptor have very low affinity for these ligands. This binding
pattern suggests that the N and M segments together are largely
responsible for the selective binding of these ligands to the
receptor. From their affinity to chimeric receptor
-NC/
-M, it
appears that U50,488 binding is less dependent than U63,640 and
ICI204,488 on the
-M segment. The
-selective ligand nBNI has
a very different binding profile from the other
-selective
ligands. Among the chimeric receptors, it showed the highest affinity
for
-NM/
-C, to which other non-peptide
-selective
ligands exhibited the lowest affinity. For the remaining three chimeric
receptors, it displayed an affinity similar to its affinity for the
receptor. It is somewhat difficult to explain the binding of this
ligand by the simple addition of individual segments, but it appears
that the presence of the
-N and
-M segments is not as
important for nBNI as it is for the binding of other
-selective
ligands. It should be noted that nBNI is a
-selective antagonist
and all the other ligands in this category are
agonists. However,
the differences in binding profiles across chimeras may be due to the
difference in structure of the ligands, arylacetamides versus an alkaloid dimer, rather than to the difference in their function
as agonists and antagonist.
There are seven
ligands in this group. Five of them, Met-ENK, Leu-ENK, DSLET, DPDPE,
and JOM13, displayed a similar binding pattern across the tested
receptors. These are very highly selective -Selective Peptide Ligands
ligands with very low
affinity for the
receptor. None of them bound to chimeric
receptor
-NC/
-M and
-NM/
-C with good affinity,
while all of them exhibited moderate affinity toward chimeric receptors
-N/
-MC and
-NM/
-C. Obviously, the presence of the
-C segment is necessary for the binding of these ligands. With the
exception of Leu-ENK, usually the more
segments are included in a
construct, the higher the affinity of this construct for these ligands.
Notably, the binding properties of deltorphin II deviate significantly
from those of the other
ligands discussed here. The
K
ratio for deltorphin II between
chimeric receptor
-N/
-MC and
-NM/
-C is 1:255, while
the ratios for the other
-selective ligand ranges from 1:0.6 to
1:3. This means that unlike other
-selective ligands, the presence
of the
-M segment is very important for the binding of deltorphin
II. Another
-selective peptide ligand that has a unique binding
profile is ICI174,864. It has very high affinity for the
receptor
but its affinity to all other receptors is very low. It appears that
high affinity ICI174,864 binding strongly requires the simultaneous
presence of both
-N and
-C segments. The replacement of
either segment with a corresponding part of
receptor greatly
decreases its binding.
All three
ligands tested in this category are -Selective Non-peptide Ligands
antagonists with
morphine-like structures. They exhibited a similar pharmacological
profile among themselves. All of them have relatively low affinity for
chimeric receptor
-NM/
-C and very high affinity for its
reciprocal construct
-NM/
-C. Their affinity for
-N/
-MC is better than that for
-NC/
-M. In general,
they exhibit a smaller range than is seen with the
-selective
peptides; but this is likely related to the fact that their selectivity
between the wild type receptors is more limited. As such, they
represent an intermediate case between the
-selective peptides and
the nonselective small ligands.
-M segment is more important for the
binding of
-selective ligands, whereas the
-C segment is more
important for the binding of
-selective ligands. This working
hypothesis will be further examined below.
Relative Contribution of Individual Receptor
Segments in Binding
While the data in yield a great deal of
information about the structural requirements of different ligands,
they do not reveal the contribution of individual segments to binding
in a straightforward way. To best answer the above questions regarding
domains of relevance to binding affinity and selectivity, we shall
compare the contributions of individual segments directly.
is a dissection of the differences in their contribution
to ligand binding, expressed as -fold change in affinity when a
segment is used to replace its corresponding
segment. In this
table we obtained a ratio of the K
values
seen with related pairs of receptors, as we compared either two
chimeras, or a chimera and the wild-type; thus, these affinity ratios
are used as indicators of the magnitude of the influence of a given
segment of a receptor on the overall affinity of this receptor for a
given ligand. A linear model is implicit as a starting point in the
calculation. In other words, the total affinity change in binding is
seen as the results of the combined contributions from the individual
segments, without regard for the possible contribution of the unique
combinations of segments. As a result, in the context of the linear
model, the possible contributions to binding resulting from the
interactions among different segments, including i.e. helix-helix interactions and helix-extracellular loop
interactions, are neglected. Obviously, a linear model may not be a
good approximation for all circumstances. However, this analysis allows
us to begin to address this issue. If a given pair of segments placed
in the context of different pairs of receptors yields very different
affinity values as a function of context, this would indicate the
inadequacy of the linear model and would show that the combination of segments contributes significantly to the binding
affinity.
Similarly, if altering a large domain (e.g. N and M segments together) brings about affinity changes
significantly different from the calculated combined effects of
altering individual components of this domain (e.g. the
product of the effects of M and N replaced individually), then the
behavior of the concerned chimeric receptors is no longer linear. By
applying such an analysis systematically, we can begin to determine
when the receptors behave linearly in our experiments. Thus, a ratio of
affinity constants calculated for a corresponding pair of segments
(e.g. the
M domain and its corresponding
M domain)
is a good indication of the difference in their contribution to the
binding of a particular ligand. Furthermore, we are also able to learn
when the receptors deviate significantly from the linear model, which
would warrant particular caution in the interpretation of the changes
in affinity values. Under a nonlinear situation, an affinity ratio may
reflect a substantial contribution from the interaction between
different segments. Thus, different sets of chimeric receptors may
yield very different affinity ratios for the same pair of segments.
Contribution of the N/M Segments in Comparison with the
Contribution of the C Segment
We shall first examine the results
in the third and fourth columns in . These data are based
on the comparison of two reciprocal constructs -NM/
-C and
-NM/
-C and the wild type
and
receptors. The
values listed in the table are the geometric average of two pairs of
receptors whenever possible and should be a good reflection of the
relative contribution of the N and M segment together in comparison
with the contribution of the C segment to the binding of the
various classes of ligands when these receptors behavior linearly. As
discussed later, the receptors deviated significantly from the linear
model in the binding of some ligands (mainly peptides, see
). Under such circumstances, although affinity change
values are still used to estimate the importance of a pair of concerned
segments, we must remember that these values may contain substantial
contributions from the interactions between NM and C segments.
-NM segment is more
favorable to the binding of both
-selective and nonselective
ligands than the
-NM segment. By contrast,
-C segment
frequently exhibits a similar or lower contribution to the binding of
nonselective and
-selective ligands (except nBNI) when it is
compared to the
-C segment. When
-selective ligands are
considered, the
-C segment contributes much more to binding than
does the corresponding segment in the
receptor. Therefore, the
-selective ligands gain their selectivity predominantly through
the more favorable interactions with the first two-thirds of the
receptor. The
-selective ligands achieve their selectivity mainly
through the C-terminal segment of the
receptor although the
-NM segment also contributes in many cases. For ligands which have
similar affinities toward the
and
receptors (i.e. nonselective or partially selective), their mechanisms of
achieving high affinity binding may be very different. Many such
ligands have favorable interactions with the first two-thirds of the
receptor but their interactions with the C-terminal segment of
the
receptor are often less favorable than their interactions
with the C-terminal segment of the
receptor. Thus, it is the
balance of the interactions with the NM and C segments rather
than the identical interactions with the two parts that make the
affinity of these ligands toward the
and
receptors very
close to each other.
-neoendorphin) difference in the contribution to
binding of a given segment depending on its combination with other
segments. This strongly suggests that the specific combination of
segments significantly determines the binding of peptide ligands. In
the present case, since the product of the affinity change values for
the two reciprocal constructs is less than that for the wild type
and
receptors, the native combinations must be better for the
binding of peptide ligands than the chimeric combinations
-NM/
-C and
-NM/
-C.
By contrast, for the
non-peptide ligands, with the exception of ICI204,448, discrepancies in
relative contributions from different pairs of receptor are much
smaller than those observed for the peptide ligands. These
discrepancies are all less than 3-fold for a given segment as it is
placed in different milieus. The discrepancy for ICI204,448 in relative
contribution is 12-fold, which means that the specific combination of
segments also contributes significantly to its binding. Taken together,
these data suggest that the contribution arising from the specific
combination of these large receptor domains (N/M and C) is much more
important for the binding of peptide ligands than for the binding of
most small molecules. Therefore the interactions between NM segment and
C segments contribute significantly to the binding of peptide ligands
while they do not significantly influence the binding of small ligands.
The data also suggest that a significant loss of binding of the peptide
ligands in the chimeric receptor resulting from specific combinations
of segments should not be treated as a global conformational
deterioration, since a global conformation deterioration would have led
to a loss in affinity for the small ligands as well.
The Relative Contributions of the N versus M
Domains
Further dissection of the contributions from the N and M
segments is possible with the comparison of chimera -N/
-MC
versus the
receptor (i.e. the effect of
substituting the
-N segment with
-N) and chimera
-NC/
-M versus the
receptor (i.e. the
effect of substituting the
-M segment with
-M). Results are
summarized in data columns 1 and 2 of . For some ligands,
the contribution of the NM domain is relatively evenly distributed
across N and M segments. But for other ligands, particularly peptides,
there is a striking differentiation in the role of the two segments,
with one favorable to binding and the other detrimental to binding.
Apart from DPDPE, the
-N segment is much more favorable to the
binding of peptide ligands than
-N segment, regardless of whether
the ligand is
or
selective. The
-N segment is usually
more beneficial to the binding of small molecule ligands but very
detrimental to the binding of peptide ligands. In contrast, the
-M
segment contributes much more than the
-M segment to the binding
of peptide ligands. When the binding of small molecule ligands is
considered, the
-M and
-M segment exhibit a mixed pattern
with relatively smaller difference in contributions compared with their
effect on peptide ligands.
-N and
-M segments are
considered individually they are slightly detrimental to the binding of
naltrindole as compared to the corresponding segments in the
receptor. But
-NM binds natrindole slightly better than
-NM.
The other exception occurs in the binding of nBNI, where the
contribution of
-NM is less than the product of
-N and
-M when these segments are compared with corresponding
segments.
ligands is mainly
achieved through the favorable interactions with the structural
elements in the first two-third of the
receptor (TM1-4 and
EC2). The
ligands achieve their selectivity mainly through the
favorable interactions with the C-terminal domain of the
receptor
(TM5-7) although its N-terminal segment also appears to be
important. (ii) The
and the
opioid receptors may bind the
opioid core Tyr-Gly-Gly-Phe-Met (or -Leu) in different ways. The
receptor has a strong binding pocket for the opioid core which is
mainly located in its C-terminal region and to a lesser extent in the
N-terminal region. The
opioid receptor utilizes its middle domain
which comprises TM4 and the highly negatively charged second
extracellular domain to achieve its high affinity binding with the
prodynorphin peptides. (iii) The interaction of a small ligand with the
receptor is very different from that of a peptide ligand. The binding
of a peptide ligand is more dependent on the combination of specific
receptor segments than is the binding of a small ligand.
and
receptors rely on different domains for their binding of the
endogenous ligands. Given the very high degree of sequence identity
between these receptors, especially in the transmembrane and
intracellular domains, and given that the opioid core is perfectly
conserved across the mammalian endogenous opioids, it was reasonable to
predict that the opioid receptors would all bind this common core in a
consistent manner and that the C-terminal extensions of the peptides
would interact with the unique extracellular domains of the receptors,
thereby achieving their selectivity. This view would be consistent with
the message/address conceptualization of the interaction between
families of peptide ligands and their receptors
(31) . While
still possible, this hypothesis has become less viable, given the
present results. Possibly the best illustration of this point comes
from looking at the binding of Leu- or Met-enkephalin, i.e. the opioid core alone, across wild types and chimeras. It is clear
that these peptides have excellent selectivity against the
receptor and that their negative interactions with that molecule are
largely due to the C-terminal third of the receptor. Indeed, when
TM5-7 of
are replaced by TM5-7 of
(i.e..
NM/
C), the binding of enkephalins to this
chimeric receptor is dramatically improved (e.g. from a
K
greater than 10,000 nM to a
K
of 52 nM for Leu-ENK). Thus,
one would have to conclude that some elements in the C-terminal region
of the
receptor are particularly unfavorable for the binding of
the core, and that the binding of the extended endogenous ligands has
to occur, in spite of this mismatch, and therefore probably
relies on particularly favorable interactions elsewhere in the
molecule. This would lead us to suggest that the opioid core may
interact quite differently with the different opioid receptors, in
spite of its conservation and their high level of homology. Put
differently, while a message/address view of the opioid ligand/receptor
interactions may still be reasonable when considering the interaction
of a series of peptide with one receptor (e.g. the
binding of enkephalins and dynorphins to the
receptor), it would
not fully describe the results when one considers the binding of a set
of ligands across the
and
receptors.
receptor, do not
utilize the same region used by the enkephalins to bind the
receptor, then how do they achieve their high affinity binding to
? One of the most interesting possibilities concerns the role of
the positive charges on the prodynorphin peptides in their selectivity
toward the
receptor
(32, 33, 34) . After
the cloning of the
receptor, we noted that a salient feature of
this receptor is the presence of several net negative charges in its
extracellular domains. We proposed that these extracellular domain
negative charges may play a key role in the selective binding of
DynA(1-17), which contains several positive charges in its
C-terminal tail
(18) . However, at that time, we were unable to
predict which extracellular domain is primarily responsible for the
binding of the prodynorphin products. The binding profile of the
prodynorphin products to the chimeric receptors strongly indicates that
the middle segment of the
receptor is critical for the binding.
In addition, although Leu-ENK shares the first five amino acid residues
with DynA(1-13), DynB, and
-neoendorphin, the middle segment
of the
receptor strongly favors the binding of the peptides with
a positively charged tail. Because the major difference between the
and
receptor in this region resides in the second half
of TM4 and the adjoining extracellular loop2 (EC2), we predict
that it is this stretch (top of TM4 and all of EC2) which is primarily
responsible for the unique binding of the prodynorphin products, and
suggest that the negative charges in Extracellular Loop2 play at least
a partial role in the binding of these peptides. Our preliminary
results using site directed mutagenesis of 3 of the negatively charged
residues in EC2 lend some support to this view, although the magnitude
of the observed effect is much smaller than what is seen with the
domain swapping.
(
)
Recent reports by Wang et
al.(37) and Xue et al.(38) based on the
study of
/µ chimeras also support the importance of the second
extracellular loop of the
receptor in the binding of prodynorphin
peptides.
receptor may feature a better pocket for the opioid core, while the
binding of the
receptor may be more dependent on the interactions
of its middle domain with the C-terminal extensions of the prodynorphin
peptides including their positive charges. This nicely explains the
fact that when the C-terminal tail of the DynA(1-17) is gradually
deleted, the peptide gradually loses its selectivity for the
receptor and finally becomes
-selective
(32) .
and
receptors appear capable of binding their ligands in a
variety of ways, depending on the selectivity and chemical structure of
ligands. Furthermore, they may bind the same category of ligands (like
peptides) through different mechanisms. How do the present observations
fit with the view that small ligands may interact within the channel
formed by the transmembrane domains, whereas large peptides may
interact primarily with the extracellular loops
(26) ? Evidence
from the work of others shows that at least in the case of the
and µ receptors, residues in the TM domains such as the Aspartate
in TM3 are important for the binding of agonist peptides, suggesting
that at least the smaller peptides do enter the channel and interact
with the TM domains
(35, 36) . Unpublished work from our
laboratory shows that DynA interacts with some deep residues in the
µ receptor.
(
)
Whether the prodynorphin
peptides do indeed interact with the deeper residues in the
receptor or whether they might interact with the more superficial
aspects of TM4 and with the EC2 remains to be determined. It should be
recalled, however, that while the positive charges of prodynorphin
products determine their specificity, their interactions with the
opioid receptors are still presumed to be dependent on the presence of
the N-terminal tyrosine. The site of interaction of this critical
tyrosine with the
receptor is of great interest, as it would help
answer some of the questions regarding depth of penetration and would
sharpen the contrast between the opioid core and the extended peptide.
Table:
Pharmacological profiles of the chimeric and
wild type opioid receptors (apparent K, nM)
Table:
Contribution of corresponding and
segments to ligand binding (expressed as ratios of affinity
constants)
values due to the difference between
-N and
-N. They are calculated as ratios of the
K
values of the
receptor and
chimera
-N/
-MC. The values in the second data column
represent the difference between
-M and
-M. They are
calculated from the K
values of the
chimera
-NC/
-M and
receptor. The values in the third
data column represent the difference between
-NM and
-NM when
N and M segments are considered as one piece. They are the geometric
average of values from chimera
-NM/
-C versus the
receptor and the
receptor versus chimera
-NM/
-C. The values in the fourth data column represent the
difference between
-C and
-C. They are the geometric average
of the data from chimera
-NM/
-C versus the
receptor and the
receptor versus chimera
-NM/
-C. A value that is less than 1 suggests that the
relevant
domain is detrimental to the binding of a ligand as
compared to its corresponding
domain. An affinity change value
that is greater than 1 indicates that the
domain is beneficial to
the binding of a ligand as compared to its corresponding
domain.
)-17-(cyclopropylmethyl)-4,5-epoxy-3,14-dihydroxy-7-(phenylmethylene)-morphinan-6-one
hydrochloride; DPDPE, cyclic
[D-penicillamine
,
D-penicillamine
] enkephalin; DSLET,
[D-Ser
, Leu
]enkephalin-Thr;
Dyn, dynorphin; EKC, ethylketocyclazocine; E2078,
[N-methyl-Tyr
,N-
-methyl-Arg
,
D-Leu
] dynorphin A-(1-8) ethylamide;
ICI174,864, N,N-diallyl-Tyr-Aib-Aib-Phe-Leu;
ICI204,448,
R,S-[3-[1-[[(3,4-dichlorophenyl)acetyl]-methylamino]-2-(1-pyrrolidinyl)ethyl]phenoxy]-acetic
acid; JOM13, Tyr-c[D-Cys-Phe-D-Pen]OH;
Mr2034,
(-)-(1R,5R,9R,2`S)-5,9-dimethyl-2`-hydroxy-2-tetrahydrofurfuryl-6,7-benzomorphan-D-tartrate;
nBNI, nor-binaltrophine HCl; NTB, naltriben methanesulfonate; TM,
transmembrane domain.
-selective ligand JOM13 and
for valuable discussions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.