From the F. Hoffmann-La Roche AG, CNS Research, Pharma Division, CH-4070 Basel, Switzerland and the § Department of Pharmacology, College of Medicine, University of California, Irvine, California 92697
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ABSTRACT |
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Strict pharmacological selectivity in families of
structurally related ligands and receptors may result from a key
process in evolution aiming at increasing diversity in
neurotransmission. An intriguing example of such exclusive specificity
can be found in the newly discovered orphanin FQ (OFQ) system when it
is compared with the opioid system. Both OFQ and its receptor share a
high degree of sequence similarity to the opioid peptides and their corresponding receptors, respectively. However, OFQ does not activate opioid receptors, nor do the opioid peptides elicit biological activity
at the OFQ receptor. We have therefore investigated the basis for the
inherent selectivity of the primary structures of OFQ and dynorphin A,
its closest counterpart. A series of truncated and/or chimeric peptides
led to the conclusion that both peptides contain domains which
establish their pharmacological selectivity. In the OFQ molecule we
could delineate a domain that prevents its ability to activate the
-opioid receptor by apparently repelling its binding. In both
peptides the selectivity-generating domains are composed of single
residues in key positions together with short stretches of amino acids
which do not overlap. To prove this concept, we designed a universal
agonist and found it active at both the OFQ receptor and the
-opioid
receptor. Our observations suggest that a coordinated mechanism of
evolution has separated the orphanin FQ system from the opioid
system.
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INTRODUCTION |
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Orphanin FQ (OFQ),1 also called nociceptin, is an endogenous ligand for an opioid-like G protein-coupled receptor (1, 2). This heptadecapeptide shows striking structural homology to the opioid peptides, especially dynorphin A (DynA). However, OFQ does not activate opioid receptors, nor do the opioid peptides elicit any biological effect at the orphanin FQ receptor (OFQR). Despite the high degree of homology between the two ligand/receptor systems, they seem to exert opposite physiological functions. The opioids are known to produce analgesia, whereas OFQ is able to reverse opioid-mediated stress-induced analgesia as well as morphine-induced analgesia (3). Thus, OFQ seems to function as an anti-opioid peptide in certain physiological paradigms. These observations prompted us to investigate potential structural determinants in OFQ and DynA which could ensure their pharmacological selectivity.
In a structure-activity relationship study we have recently determined the individual amino acid residues as well as some overall structural requirements responsible for the biological activity of OFQ at its receptor (4). This study indicated that OFQ exhibits a structure strikingly different from that of the opioid peptides regarding receptor binding and activation. For example, the amino-terminal Phe can be replaced by Tyr without any loss of binding affinity and biological activity. In addition, although the amino-terminal half of OFQ appeared to be more important for receptor binding than the carboxyl-terminal part, the entire OFQ molecule was required for receptor activation. This is in sharp contrast to the opioid peptides whose binding to their receptors obey the "message-address" concept in which the first part of the peptide is sufficient to activate the receptors, whereas the second part, the address, differentiates the receptor sites (5). OFQ interaction with its receptor on the other hand fits the more general model of effector and cooperative domains, where the effector part confers binding to a complementary site on the receptor but is often not sufficient to exert biological activity on its own. In turn, the cooperative domain alone is not able to induce activation of the receptor but may control specificity. The latter model implies that both effector and cooperative domains are necessary for full agonism (15, 16).
Thus far only selectivity-enhancing properties of the address domains in opioid peptides have been described, because all naturally occurring opioid peptides are promiscuous ligands at all three opioid receptors, albeit with different selectivity. This observation is surprising, because no conserved structural motif can be found in the address domains of the opioid peptides. Therefore, one cannot explain on a structural level why a peptide like Tyr1-OFQ does not activate the opioid receptors. An intriguing discovery from the structure-activity studies on OFQ is that the OFQR does not discriminate against a Tyr residue in position 1 of the peptide. This raised the question as to why the opioid peptide DynA, which contains the same amino-terminal sequence as Tyr1-OFQ and is of the same length, cannot activate the OFQR and whether structures can be identified within DynA which regulate such activity. Conversely, Tyr1-OFQ was not able to activate opiate receptors although its amino-terminal tetrapeptide contains the Tyr-Gly-Gly-Phe motif which is believed to be the minimal structure required for opioid peptide activity. The aim of this study was therefore to determine the structural basis for this exclusive selectivity.
Although Tyr1-OFQ is an artificial molecule that has so far not been identified in nature, this is not a purely academic question, because at the genetic level, just a single nucleotide exchange could create a mutation leading to the generation of Tyr1-OFQ (5). As outlined before, the OFQR would not select against such a mutant ligand.
To attempt answering these questions we designed a number of chimeric
OFQ/DynA peptides and investigated their biological activities at both
the OFQ and the opioid receptors. Our results indicate that the primary
structures of both OFQ and DynA contain domains which ensure proper
selectivity of each ligand molecule for only its cognate receptor.
Interestingly, derivatives of Tyr1-OFQ truncated at the
carboxyl terminus showed considerable activity toward the opioid
receptors, whereas the intact Tyr1-OFQ has none. This
indicates that the carboxyl-terminal half of OFQ may serve as a domain
which excludes it from binding to opioid receptors. From these studies
we were able to predict the structure of a universal agonist which
activates both the -opioid receptor (KOR) and the OFQR. Finally, our
studies may provide new insights into the evolutionary events which
could have separated the opioid from the OFQ system.
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MATERIALS AND METHODS |
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Peptides and Chemicals-- OFQ and all chimeric peptides were synthesized by Research Genetics (Huntsville, AL). Opioid peptides were from Bachem (Bubendorf, Switzerland) and non-peptidergic opioid ligands were from RBI (Natick, MA). cAMP assay reagents and [3H]OFQ were obtained from Amersham Corp. [3H]Naloxone was purchased from NEN Life Science Products. Forskolin was from Calbiochem, and tissue culture media were from Life Technologies, Inc. All other chemicals were of analytical grade and obtained from Merck or Sigma.
Transfected Cell Lines and Culture Conditions--
Cloning of
the rat OFQR as well as transfection of CHO cells and the
characterization of clones expressing the receptor have been described
previously (1, 4). The cDNAs of the rat -,
-, and µ-opioid
receptors were cloned into the eukaryotic expression vector pRcRSV
(Invitrogen) and transfected into CHO cells. Stably expressing clones
(kindly provided by D. Grandy, Vollum Institute, Portland, OR) were
selected with G418 (Life Technologies, Inc.) and screened by binding
and functional assays. All cells were cultured at 5% CO2
in modified Eagle's medium containing 5% fetal calf serum and 500 µg/ml G418.
Measurement of Adenylyl Cyclase Activity in Receptor Transfected Cells-- Adenylyl cyclase assays were carried out as described previously (1). All experiments were repeated at least three times in triplicate. Dose-response curves were fitted and calculated with Kaleidagraph.
Receptor Binding and Competition Experiments--
OFQR binding
assays were done as described before using [3H]OFQ as a
radioligand (7). -Opioid binding was determined on membranes
prepared from CHO cells expressing the rat
-opioid receptor with
[3H]naloxone as a radioligand. Incubation of membranes
(40 µg of protein) with [3H]naloxone was carried out in
the dark at room temperature for 60 min in a total volume of 500 ml of
binding buffer (50 mM Tris-HCl, pH 7.8, 1 mM
EGTA, 5 mM MgCl2, 0.1% bovine serum albumin).
For competition binding experiments 1 nM
[3H]naloxone was added together with the indicated
concentrations of unlabeled peptides. Nonspecific binding was
determined in the presence of 2 µM cold naloxone. Bound
and free ligand were separated by rapid vacuum filtration through
Unifilter GF/C glass fiber filters using a Unifilter 96 harvester
(Canberra Packard). GF/C filters had been pretreated with 0.3%
polyethylenimine containing 0.1% bovine serum albumin for 1 h at
room temperature. Filters were washed six times with 1 ml of ice-cold
50 mM Tris-HCl, pH 7.5. After washing, 60 µl of
scintillation fluid (Micro Scint, Canberrra Packard) was added and the
plates were counted in a
counter. All experiments were done at
least three times in triplicate. Binding data were analyzed by
determination of IC50 values using nonlinear curve fitting
in Kaleidagraph, and Ki values were obtained
according to the method of Cheng and Prusoff (8).
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RESULTS |
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Inactivity of OFQ at the Opioid Receptors-- As shown in Fig. 1, OFQ is not able to inhibit forskolin-stimulated adenylyl cyclase activity in CHO cells stably transfected with the three different opioid receptors. On the other hand, these three receptors responded well to challenge with both peptidergic and non-peptidergic opiates, showing their proper functional expression. The EC50 values for inhibition of forskolin-stimulated cAMP accumulation by the opioid compounds at the respective receptors are summarized in Table I.
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Activity of Tyr1-OFQ at the Opioid Receptors--
As
reported previously, the Phe residue in position 1 of OFQ can be
substituted by Tyr without loss of biological activity or binding
affinity at the OFQR (4). Tyr1-OFQ contains the
amino-terminal tetrapeptide Tyr-Gly-Gly-Phe, thus rendering this part
of the molecule identical to the opioid motif contained within all
mammalian opioid peptides (for sequences of peptides, see Table I).
However, as depicted in Fig. 2,
Tyr1-OFQ cannot activate the opioid receptors using
physiologically relevant concentrations. Only at the KOR was a weak
agonistic behavior observed above concentrations of 1 µM.
Surprisingly, Tyr1-OFQ inhibited [3H]naloxone
binding at the KOR with high affinity (Ki = 3.35 ± 2.41 nM; Fig.
3A, Table
II). Therefore, we investigated whether
Tyr1-OFQ behaved as a very inefficient partial agonist. As
shown in Fig. 3, the genuine activity of DynA or U50,488 at the KOR is not inhibited by Tyr1-OFQ. Neither coadministration of 100 nM Tyr1-OFQ with increasing concentrations of
DynA or U50,488 (Fig. 3B) nor application of increasing
amounts of Tyr1-OFQ in the presence of 0.1 or 1 nM DynA (Fig. 3C) affected the inhibition of
forskolin-stimulated adenylyl cyclase by these -opioid agonists.
Also, preincubation with Tyr1-OFQ for different time
periods did not alter the response of the KOR to DynA (results not
shown). We therefore concluded that Tyr1-OFQ, despite its
high binding affinity at the KOR, does not interfere with the mechanism
of
-opioid receptor activation by peptidergic or non-peptidergic
agonists. The Tyr-Gly-Gly-Phe sequence in Tyr1-OFQ may thus
allow for the interaction with the KOR but is not sufficient to endow
the peptide molecule with an agonistic activity.
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Uncovering Opioid Activity in Tyr1-OFQ by
Carboxyl-terminal Truncation or Alteration--
To determine which
features of Tyr1-OFQ, if any, were required to elicit
biological activity at the opioid receptors, we decided to modify the
general structure of this peptide by either truncation of
carboxyl-terminal amino acids or incorporation of DynA-specific residues. Removal of the last eight amino acids of Tyr1-OFQ
produced a molecule (Tyr1-OFQ 1-9; Table I) which
completely lost activity at the OFQR but gained potency at the KOR. As
shown in Fig. 2B and Table I, Tyr1-OFQ 1-9
inhibits forskolin-stimulated cAMP accumulation in KOR-transfected CHO
cells with an EC50 of 6.89 ± 1.71 nM.
Further truncation of this structure to Tyr1-OFQ 1-7
resulted in a loss of activity at KOR (possibly due to the loss of the
charged residues), balanced by the appearance of a weak potency at the
DOR (Fig. 2A). Thus, amino acids 10-15 of
Tyr1-OFQ appear to contain a domain which prevents the
peptide from activating opioid receptors. To investigate this
hypothesis, we created a chimeric peptide (OD Y1-17; Table I) by
exchanging amino acids 10-15 of OFQ for the corresponding residues of
DynA. This chimeric peptide is an agonist at the - and
-opioid
receptors but has lost activity at the OFQR (Fig. 2 and Table I). These results indicate that residues 10-15 of Tyr1-OFQ can act
to exclude it from activation of opioid receptors. At the same time,
the homologous region of DynA is not sufficient to activate the
OFQR.
Uncovering OFQR Activity in DynA by Incorporation of Amino Acid
Residues Critical for Biological Activity of OFQ--
Previous
structure-activity relationship studies have identified amino acid
residues 1-5 together with Arg8 in the OFQ molecule as the
most critical for biological activity (4). In addition, it was shown
that the entire OFQ peptide structure was necessary for activation of
the OFQR. Because DynA shows the highest degree of homology to OFQ and
is of identical length, we chose this peptide as a model molecule for
investigating selectivity domains which could preclude activation of
the OFQR. First, we introduced an arginine residue at position 8 of
DynA, creating Dyn R6-9 (Table I). This molecule proved to be a very potent agonist, inhibiting forskolin-stimulated cAMP accumulation with an EC50 of 0.11 ± 0.02 nM in CHO
cells expressing the KOR (Fig. 4). It
also displaced [3H]naloxone binding at the KOR with a
Ki of 0.2 ± 0.19 nM. However, Dyn
R6-9 did not display biological activity at the OFQR, although a
moderate binding affinity with a Ki of 25.3 ± 21.7 nM was detected (Table II). The next peptide tested was Dyn R8, in which the sequence of amino acids 8 and 9 of DynA were
inverted (Table I). This change resulted in a reduction of potency at
the KOR but it also produced a partial agonist at the OFQR (Fig. 4).
Dyn R8 could activate both the KOR and the OFQR with EC50
values of 10.99 ± 2.93 nM and 61.1 ± 38.3 nM, respectively. The binding potency at the KOR was high,
showing a Ki of 0.09 ± 0.08 nM,
whereas Dyn R8 displaced [3H]OFQ only moderately at the
OFQR (Ki = 34.9 ± 23.8 nM; Table
II). However, it appears that Dyn R8 either has an imperfect cooperative binding domain or contains inhibitory structures preventing full agonism at the OFQR.
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Design of a Universal Agonist for the OFQ and -Opioid
Receptor--
The existence of domains which generate selectivity in
peptides of related structure can be tested by designing an agonist which would act at both related receptors. Owing to the fact that the
OFQR appears to require a 17-amino-acid peptide for biological activity, we aimed at a heptadecapeptide structure combining all the
information about selectivity domains in OFQ as well as DynA. Our
studies on Tyr1-OFQ 1-9 and OD Y1-17 had shown that amino
acids 1-9 of Tyr1-OFQ are permissive with regard to opioid
receptor activity. We therefore used this part of the molecule as an
amino-terminal building block. This was combined with a modified
carboxyl-terminal half of DynA containing two substitutions: 1) the
proline residue in position 10 of DynA was replaced with alanine, as
proline is known to induce profound changes in the secondary structure
of peptide chains; 2) the negative charge of the aspartate at position 15 in DynA was eliminated by replacement with alanine. The resulting chimeric peptide (OD R8; Table I) proved to be a full agonist at both
the OFQR and the KOR with EC50 values of 86.7 ± 26.4 nM and 659 ± 191 nM, respectively (Fig.
4). OD R8 displaced [3H]naloxone at the KOR with a
Ki of 4.37 ± 4.89 nM. A lower
affinity value was observed at the OFQR, where OD R8 displaced [3H]OFQ with a Ki of 45.7 ± 32.5 nM (Table II). Although OD R8 is not a highly potent
molecule, by being a full agonist at both receptors it verifies our
concept of selectivity-generating domains within the two parent
peptides. These domains in both OFQ and DynA might be responsible for
the observed lack of receptor cross-activation.
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DISCUSSION |
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In the present study we have sought to investigate the structural basis of pharmacological selectivity in two closely related neuropeptides, the recently discovered orphanin FQ and dynorphin A. Our results indicate the presence of specific domains in both peptides that prevent cross-activation of inappropriate receptors. To support this concept, we designed a universal agonist in which the restrictive structures have been eliminated and showed that this peptide is active at both the OFQR and the KOR.
Within the primary structure of OFQ the amino-terminal Phe is the first
determinant which excludes it from being an opioid agonist, as all the
opioid receptors stringently require a Tyr in that position (9, 10).
However, the present study shows that OFQ also contains a domain
located between amino acids 10 and 15, which excludes it from
activating the -opioid receptor. We could also identify selectivity
generating structures in DynA residing between amino acids 7 and 10 together with Asp15. Although the message domain of DynA is
recognized by the OFQR, the lack of a cooperative binding site in the
carboxyl-terminal half of the peptide prevents activation of the OFQR.
Thus, the address or cooperative domain of DynA appears not only to
enhance the peptide's affinity for the KOR but simultaneously
restricts its biological activity, excluding it from the OFQR. Such a
dual role of the address domain in DynA could not be recognized before the discovery of OFQ. We would therefore like to propose an addition to
the traditional message-address (6) to include the existence of domains
that generate selectivity between sequentially related peptides as
depicted in Fig. 5.
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Although our studies have focused mainly on DynA and the -opioid
receptor as the closest relatives to OFQ and its receptor, it is likely
that similar results would be obtained with the other opioid peptides
and their receptors. For example, the size of the enkephalins or
-endorphin together with the absence of an arginine residue in
position 8 should be sufficient to exclude them from activating the
OFQR. Conversely, the fact that Tyr1-OFQ is unable to
activate the DOR or MOR suggests that a similar mechanism of structural
restriction excludes Tyr1-OFQ from these receptors, as
shown for KOR. However, the individual amino acids responsible for the
selectivity between OFQR and DOR or MOR might be different from the
ones identified as preventing KOR activation, because
Tyr1-OFQ 1-9 is unable to activate DOR or MOR. Further
mutation experiments will be necessary to delineate these domains in
other opioid peptides and those in Tyr1-OFQ which exclude
it to interact with DOR and MOR.
The domains that generate OFQ versus DynA selectivity are non-continuous and show virtually no overlap, indicating that a multistep mechanism of evolution, rather than a single point mutation, led to this separation. This hypothesis is supported by results from a recent study using site-directed mutagenesis showing that the OFQ receptor also contains structures which specifically exclude opioid peptides from binding (11). As few as four amino acid exchanges at remote sites of the protein are sufficient to endow the OFQR with high affinity opioid peptide binding while not affecting its affinity for OFQ itself. Thus, the wild type OFQR harbors opioid peptide-excluding structures which are clearly distinct from the binding sites for its endogenous ligand. The positions of the mutations are distributed over a larger area of the OFQR, indicating that, as proposed for the peptide ligands, a number of separate mutation events must have occurred on the receptor side. It appears that coordinate evolution in both the ligands and the receptors may have led to the complete pharmacological separation of the OFQ system from the opioid system and vice versa. This idea is also consistent with the finding that OFQ can act as an antiopioid peptide (3), as such an activity necessitates a mechanism to prevent any cross-activation of the two systems.
A similar theory has been proposed for the glycoprotein hormones lutropin and follitropin and their respective receptors. Based upon their similarity in sequence it is assumed that both the ligands and receptors might have originated from common ancestors, as in the case of the OFQ and opioid systems. However, lutropin and follitropin are clearly characterized by their distinct pharmacology, reflected by the exclusive interaction with only one specific type of receptor. In an elegant study by Moyle et al. (12) it was demonstrated that distinct domains in both the glycoprotein hormones as well as their receptors restrict ligand-binding specificity. As noted for the OFQ and the opioid systems, the selectivity determinants in lutropin and follitropin and their receptors are composed of short stretches of amino acids which are non-overlapping. In the case of the glycoprotein hormone receptors this phenomenon could be explained by exon shuffling, because the individual selectivity domains of the lutropin and the follitropin receptor could each be matched to separate exons. However, a different genetic mechanism must be postulated for both the OFQR and the KOR, as the critical parts of the proteins are both contained within the third exon of the respective coding regions (13).
Finally, one may ask why such a stringent selectivity evolved. Restrictive control of ligand/receptor selectivity is a prerequisite in cases of anatomical separation between sites of release and sites of action. Such a situation obviously exists in the case of the glycoprotein hormones, where the humoral mode of ligand distribution provides an ubiquitous appearance of the ligand but simultaneously imposes a need for the development of selectivity. OFQ and the opioids, on the other hand, are believed to exert their activity at specific synaptic junctions as do other neuropeptides. Our observations of mutual exclusion in the two systems might therefore be interpreted in several ways. First, one may still consider that important functions of OFQ and the opioids might be found in the periphery which rely on humoral transport of the transmitters. Another possibility could be a colocalization of the receptors and/or the peptides at certain synapses, although recent anatomical studies seem to disprove this idea (14). But the broad distribution of the OFQR in the central nervous system indeed suggests that it might be found in close spatial proximity to opioid receptors. What this strict selectivity indicates is a need for OFQ and the opioid peptides to be clearly differentiated, which in turn suggests that OFQ may have additional functions unrelated to those it has in the modulation of nociception.
In summary, we have determined the structural basis for pharmacological
selectivity between the OFQ and the -opioid system which prevents
cross-activation of their respective receptors. A coordinated evolution
of the ligands and the receptors must have occurred to ensure proper
separation of the physiological functions of the two systems. The
incorporation of structures that prevent activation of related but
inappropriate binding sites, rather than inventing two sets of totally
new sites, might represent a general evolutionary strategy by which
nature can gain variability in signal transduction. Knowledge of these
specific motifs will also be helpful in the development of selective
drugs to a single class of receptors.
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ACKNOWLEDGEMENTS |
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We thank D. K. Grandy and J. R. Bunzow
(Vollum Institute, Portland, OR) for kindly providing cell lines
expressing the -,
-, and µ-opioid receptors, and S. Pohl for
critically reading the manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Institute for Cell Biochemistry and Clinical
Neurobiology, University of Hamburg, Martinistr. 52, D-20246 Hamburg,
Germany.
¶ To whom correspondence should be addressed: Dept. of Pharmacology, College of Medicine, 354 Med Surge II, University of California, Irvine, CA 92717. Tel.: 714-824-2522; Fax: 714-824-4855; E-mail: ocivelli{at}uci.edu.
1
The abbreviations used are: OFQ, orphanin FQ;
OFQR, orphanin FQ receptor; DOR, -opioid receptor; KOR,
-opioid
receptor; MOR, µ-opioid receptor; DynA, dynorphin A; CHO, Chinese
hamster ovary.
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REFERENCES |
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