©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Chimeric Study of the Molecular Basis of Affinity and Selectivity of the and the Opioid Receptors
POTENTIAL ROLE OF EXTRACELLULAR DOMAINS (*)

Fan Meng (§) , Mary T. Hoversten , Robert C. Thompson , Larry Taylor , Stanley J. Watson , Huda Akil

From the (1) Mental Health Research Institute, University of Michigan, Ann Arbor, Michigan 48109

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

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, , 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) .

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 and types of opioid receptors as a first step in achieving an overview of the structure-function relationship of this family of receptors.


EXPERIMENTAL PROCEDURES

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) .


RESULTS

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 .

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, 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.

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.() 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.

-Selective Non-peptide Ligands

Among the four ligands considered here, U50,488, U63,640, and ICI204,488 share a similar binding profile across constructs. Chimeric receptor -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.

-Selective Peptide Ligands

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 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.

-Selective Non-peptide Ligands

All three ligands tested in this category are 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.

In general, the results lead us to the conclusion that the -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.

It is very clear from that the -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.

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 -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.

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 -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.


DISCUSSION

The experiments described above lead us to the following conclusions. (i) The selectivity of the 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.

A particularly surprising result is the observation that the 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.

If the extended prodynorphin peptides, when binding the 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.

Given the above arguments, we would suggest that the 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) .

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 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.

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.

  
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)

The values in the first data column represent the change in K 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.



FOOTNOTES

*
This work was supported by National Institute on Drug Abuse Grant RO1 DA02265 (to H. A. and S. J. W.), Markey Grant (from the Lucille P. Markey Charitable Trust) 88-46 (to H. A. and S. J. W.), the Gut Center Grant P30-AM34933 (to H. A. and S. J. W.), and the Gut Center pilot feasibility study grant (to F. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Mental Health Research Institute, University of Michigan, 205 Zina Pitcher Place, Ann Arbor, MI 48109. Tel: 313-763-3771; Fax: 313-763-4130.

The abbreviations used are: U50,488, (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzene acetamide methanesulfonate; U63,640, spiradoline-(-)-enantiomer; BNTX, (5)-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.

F. Meng and H. Akil, manuscript in preparation.

F. Meng, M. T. Hoversten, R. C. Thompson, L. Taylor, S. J. Watson, and H. Akil, manuscript in preparation.

A. Mansour, J. Fine, M. T. Hoversten, R. C. Thompson, L. Taylor, S. J. Watson, and H. Akil, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. Henry I. Mosberg of the University of Michigan for providing us the highly -selective ligand JOM13 and for valuable discussions.


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