Sturgeon Orphanin, a Molecular "Fossil" That Bridges the Gap between the Opioids and Orphanin FQ/Nociceptin*

Phillip B. DanielsonDagger §, Mary T. Hoversten, Martin Fitzpatrick||, Carl Schreck**, Huda Akil, and Robert M. DoresDagger

From the Dagger  Department of Biological Sciences, University of Denver, Denver, Colorado 80210, the  Mental Health Research Institute, University of Michigan, Ann Arbor, Michigan 48109, and the || Department of Fisheries and Wildlife and the ** Oregon Cooperative Fish and Wildlife Research Unit, United States Geological Survey-Biological Resources Division at Oregon State University, Corvallis, Oregon 97331

Received for publication, December 27, 2000, and in revised form, April 2, 2001

    ABSTRACT
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
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The elucidation of the cDNA sequence for sturgeon proorphanin provides a unique window for interpreting the evolutionary history of the opioid/orphanin gene family. The molecular "fossil" status of this precursor can be seen in several ancestral sequence characteristics that point to its origin as a duplication of either a prodynorphin- or proenkephalin-like gene. The sturgeon proorphanin cDNA encodes a precursor protein of 194 residues, and the orphanin heptadecapeptide itself binds not only the opioid receptor-like 1 (ORL1) receptor but also the classical (µ, kappa , and delta ) opioid receptors with near equal affinity. Allowing for this broad receptor specificity are several amino acid substitutions at key positions in the heptadecapeptide sequence, relative to its mammalian orthologs, that have been linked by amino acid scans and site-directed mutagenic studies to the exclusion of mammalian orphanin FQ/nociceptin from classic opioid ligands (i.e. F1Y and L14W). The unique receptor binding profile of sturgeon orphanin not only provides insight into the evolutionary history of the opioid and opioid-related peptides but also provides an ideal context in which to investigate the underlying mechanisms by which novel and often divergent physiological functions arise in receptor-ligand systems.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The expansion and functional diversification of multigene families have been a recurring theme in the evolutionary success of complex eukaryotic organisms (1), and the opioid neuropeptides clearly illustrate this phenomenon (2). The classic opioid gene family comprises three neuropeptide precursors encoded on the following genes: proopiomelanocortin (end products: beta -endorphin, melanocyte-stimulating hormone-associated peptides), proenkephalin (end products: Met-enkephalin, Leu-enkephalin), and prodynorphin (end products: dynorphins and alpha -neoendorphin). Based on the organization of these genes it appears that they were derived from a common ancestral gene (3). This hypothesis is supported by the shared neuroinhibitory action of the opioid end products and the selective binding profiles of the opioid peptides to a family of G protein-coupled receptors (µ, kappa , and delta  opiate receptors) (4, 5).

What has been difficult to resolve is the pattern of evolutionary events that has given rise to the opioid gene family. This is an issue that has been further complicated by the discovery, reported simultaneously by Meunier et al. (6) and Reinscheid et al. (7), of the 17-amino acid orphanin FQ/N,1 the endogenous ligand for the opioid receptor-like 1 (ORL1) receptor, the "orphan" opiate receptor gene. The organization of the orphanin FQ/N precursor (proorphanin or pronociceptin) indicates that this gene is evolutionarily related to the opioid-coding precursor genes. Mammalian proorphanin has the same basic intron-exon structure, conserved N-terminal cysteines, and a modified opioid-like core sequence (FGGF instead of YGGF) that are all defining features of classic opioid precursors. Similarities are also seen at the cellular level where both typical opioids and orphanin FQ/N are negatively coupled to forskolin-induced adenyl cyclase activity, the inhibition of N-type Ca2+ channels, and the activation of inwardly rectifying K+ channels. Hence, it has been proposed that the precursors for orphanin FQ/N and the classic opioids arose by sequential duplication of a common ancestral gene (2) and that subsequent divergence of these sequences has proceeded in parallel with those of the cognate receptor proteins.

Although each of the classic opioids is capable of binding and activating µ, kappa , and delta  opioid receptors with varying degrees of specificity, orphanin is alone in its affinity for the ORL1 receptor and its concomitant inability to bind or elicit any physiological effect at the µ, kappa , and delta  opioid receptors (8). The apparent receptor specificity and functional isolation of orphanin FQ/N are both unexpected and evolutionarily significant in that they may help to explain how closely related sequences attain functional separation and how novel neuromodulatory systems evolve.

Studies employing systematic alterations in peptide synthesis to change one or more residues in orphanin FQ/N suggest a close evolutionary relationship with the classic opioids and provide an indication of how readily the functional isolation of orphanin FQ/N could have been achieved. By replacing the N-terminal phenylalanine, unique to orphanin FQ/N, with a tyrosine, the defining feature of opioid peptides (9, 10), produced an orphanin FQ/N analog that bound kappa  opioid receptors with high affinity and elicited a detectable agonistic effect at concentrations over 1 µM. The Tyr-orphanin FQ/N analog study suggests a mechanism to account for the receptor selectivity of the opioid peptides and orphanin FQ/N. The current study demonstrates that the Tyr-orphanin analog is a naturally occurring product encoded in the proorphanin gene of the sturgeon, Acipencer transmontanus, a representative of an old lineage of ray-finned fish. The organization of sturgeon proorphanin provides evidence for an intermediate stage in the evolution of the vertebrate proorphanin gene. Finally, the sturgeon orphanin sequence represents a transitional state in the eventual functional isolation of the orphanin heptadecapeptide from the classical opioids.

    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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Animals-- Sexually immature white sturgeon, A. transmontanus, were obtained from the Oregon State University Department of Fisheries and Wildlife and Oregon Cooperative Fish and Wildlife Research Unit (Corvallis, Oregon). Following anesthetization with MS222 and decapitation, pituitaries were extracted, flash frozen in liquid nitrogen, and stored at -80 °C until used for mRNA isolation.

mRNA Isolation and cDNA Synthesis-- Poly(A)+ RNA for cDNA synthesis was isolated from 50 mg of pituitary tissue by direct capture onto oligo(dT25)-coated paramagnetic beads (Novagen, Madison, WI) following the procedure of Jakobsen et al. (11). First-strand cDNA synthesis by Superscript II reverse transcriptase (Life Technologies, Inc.) was primed from the poly(A) tail with a synthetic oligonucleotide (anchor dT17) having the sequence 5'-GACTCGAGTCGGATCCATCGA(T)17-3'.

Rapid Amplification of cDNA Ends (RACE) Reactions-- Full-length proorphanin cDNAs of the sturgeon were cloned using a combination of 3' and 5' RACE (12). For 3' RACE, a fully degenerate primer (CORE-1024: 5'-AA(A/G)(A/C)GITA(C/T)GGIGGITT(C/T)ATG-3' where I represents deoxyinosine and parentheses contain mixed bases) targeted to the sequence encoding the highly conserved KRYGGFM motif characteristic of opioid peptides served as the forward primer. Deoxyinosine was used at sites of 4-fold degeneracy to minimize helix instability (13). Using sequence information obtained by 3' RACE, a gene-specific reverse primer (OFQ/N-R1: 5'-GCCCATAAGTTTCCTGT-3') was used to obtain the remainder of the full-length proorphanin cDNA by 5' RACE. For these reactions, a forward primer (5'Amp dC10: 5'-GAATTCGCGGCCGCTTCAGT(C)10-3') was targeted to a homopolymeric G tail that had been synthesized at the 3' end of all first-strand cDNAs using terminal deoxynucleotidyl transferase.

Isolation and Subcloning of Polymerase Chain Reaction Products-- Polymerase chain reaction-amplified cDNAs were purified on Wizard polymerase chain reaction columns (Promega, Madison, WI), ligated into pGEM-T vector, and electroporated into Escherichia coli DH5alpha cells. Transformants were screened for inserts by polymerase chain reaction using primers targeted to the SP6 and T7 RNA polymerase promoters flanking the pGEM-T polycloning site. Plasmids containing appropriate size inserts (i.e. 300-500 bp for 3' RACE; 500-600 bp for 5' RACE) were selected for sequencing.

DNA Sequencing and Analysis-- Plasmid DNA for sequencing was isolated by the cetyltrimethylammonium bromide-based method of Del Sal et al. (14), glass milk-purified, and sequenced using an automated CEQ2000 capillary array sequencer (Beckman Coulter, Inc., Fullerton, CA). A full-length, consensus cDNA sequence was constructed with data from multiple, overlapping clones. Sequences were analyzed for similarity to known genes using the BLAST algorithm (15). Distance matrices were generated from aligned amino acid sequences using PAUP software (Phylogenetic Analysis Using Parsimony, by D. Swofford, Smithsonian Institution).

Receptor Binding Assays-- A mammalian expression vector containing the cytomegalovirus immediate-early promotor, courtesy of Dr. Michael Uhler (16), was used to express receptors in COS-1 cells. COS-1 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum at 37 °C in 5% CO2. Cells were seeded at a density of l × 106 on 10-cm plates 24 h prior to transfection. Qiagen-purified plasmid DNA (20 µg) was transfected into COS-1 cells by the calcium phosphate precipitation method of Chen and Okayama (17). Transfected cells were harvested 48 h after removal of the DNA precipitates. Affinities of test ligands for µ, kappa , delta , and orphanin FQ/N were determined by competition binding assays (18). Assays were performed in 50 mM Tris-HCl (pH 7.4) with the addition of peptidase inhibitors (final concentrations: 0.02% bovine serum albumin, 0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 mM EDTA, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 1 mM iodoacetamide). In a final volume of 250 µl, membrane protein was incubated with 2.5 nM [3H]orphanin FQ/N to label ORL1 (specific activity 51 Ci/mmol; generously provided by the National Institute on Drug Abuse drug supply system) or 2.5 nM [3H]ethylketocyclazocine (EKC) (specific activity 18.1 Ci/mmol; PerkinElmer Life Sciences) to label µ, kappa , and delta . Test ligands were evaluated in duplicate at nine concentrations in steps of 1:5 dilutions. Tubes were incubated at room temperature for 60 min and then harvested by vacuum filtration over GF/B glass fiber filters, washed with 5 ml of cold Tris-HCl, and counted for tritium. Data were plotted as percent specific bound versus log concentration of competing ligand and analyzed using a one-site competition model (GraphPad Prism, version 3.0, GraphPad Software, Inc., San Diego, CA). Ki values were determined according to the Cheng-Prussoff equation (19).

    RESULTS
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Sequencing and BLAST analysis of 40 subcloned cDNAs amplified by 3' RACE using a moderately degenerate oligonucleotide primer targeted to the sequence encoding the opioid core motif, KRYGGFM, resulted in the identification of three putative proorphanin FQ/N cDNA fragments. Using the partial cDNA sequences as a template, a gene-specific reverse primer was designed, and the remaining cDNA sequence was obtained by 5' RACE. The consensus nucleotide sequence and conceptual amino acid translation of the full-length cDNA is shown in Fig. 1. The 860-bp sturgeon proorphanin FQ/N cDNA (GenBankTM accession number AF095739) includes a 5' untranslated region of 110 bp and a 3' untranslated region of 168 bp containing a canonical polyadenylation signal (AATAAA) 32 bp upstream of the poly(A) tail. The longest open reading frame (582 bp) encodes a predicted proorphanin precursor protein of 194 residues. It should also be noted that the target sequence to which the degenerate CORE-1024 bound for amplification of the original 3' RACE fragment encoded the amino acid sequence KRFGGFM rather than KRYGGFM. The amino acid difference at the third position of the target site, however, represents only a single base mismatch between the degenerate CORE-1024 primer and the native target sequence. Given the stringency of the thermal profile used, such a mismatch was not expected to significantly impair the amplification reaction.


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Fig. 1.   Full-length nucleotide and conceptual amino acid translation of sturgeon proorphanin based on a consensus sequence of overlapping cDNAs obtained by a combination of 3' and 5' RACE. Nucleotides and amino acids are numbered on the left. Potential bioactive peptides (Phe137-Tyr147; Tyr161-Tyr177) are shaded. Flanking paired basic amino acids that could serve as targets for posttranslational proteolytic cleavage are indicated by black-down-triangle . A canonical polyadenylation signal near the 3' end of the cDNA is underlined.

Although the maximum global amino acid identity in pairwise comparisons with full-length mammalian orphanin precursor sequences was only 34.6%, the orphanin-like sequence (Tyr161-Pro177) within the precursor displayed up to 65% amino acid identity to mammalian orphanin FQ/N peptides. Although the sturgeon orphanin-like sequence begins with a tyrosine residue (Tyr161), this sequence does contain 11 of 17 residues that are absolutely conserved in mammalian orphanin peptides (i.e. Gly162-Phe164, Gly166, Arg168-Lys173, Asn176). In addition, a second opioid/orphanin-like core sequence (Phe137-Phe140) was also detected. This sequence was flanked by putative pairs of basic amino acid proteolytic cleavage sites (Lys135-Arg136 and Arg148-Lys149) but lacked all other conserved residues characteristic of orphanin peptides. Finally, a possible relic opioid sequence (Tyr180-Leu184) (2) preceded by a pair of basic amino acids (Lys178-Arg179) was also detected in the precursor sequence (Fig. 1). Notably missing from the sturgeon proorphanin sequences is any region with recognizable similarity to nocistatin, a bioactive peptide encoded within mammalian orphanin FQ/N precursors that suppresses orphanin-induced allodynia (20).

The results of receptor binding assays involving three potential sturgeon proorphanin-derived peptides (i.e. sturgeon Y: Tyr161-Pro177; sturgeon YKR: Tyr161-Arg179; sturgeon F: Phe137-Tyr147, Fig. 1) and Chinese hamster ovary cells expressing mammalian ORL1, µ, kappa , or delta  receptors are shown in Table I and Fig. 2. In these assays, dynorphin A-(1-17) and EKC served as representative ligands for the classic opioid receptors, whereas mammalian orphanin FQ/N served as the control ligand for the ORL1 receptor. As expected, mammalian orphanin FQ/N bound the ORL1 receptor with high affinity (Ki = 2.1 nM). Similarly, dynorphin A-(1-17) and EKC displayed broad affinity for µ, kappa , and delta  opioid receptors (Ki for EKC: ~4.2-5.4 nM; Ki for dynorphin A-(1-17): ~3.4-11.8 nM), although dynorphin A-(1-17), as expected, showed a preference for the kappa  receptor. Binding affinities of the sturgeon Y heptadecapeptide and YKR nonadecapeptide for the ORL1 receptor were approximately ~3-25-fold less than that recorded for the cognate mammalian orphanin FQ/N peptide. However, both sturgeon orphanin peptides showed broad affinities for all three classic opioid receptors (Ki for sturgeon Y: ~16.2-38 nM; Ki for sturgeon YKR: ~11.4-38 nM). The latter results are noteworthy given that the presence of a phenylalanine residue at the N terminus of mammalian orphanin FQ/N normally excludes binding of this peptide to classic opioid receptors (8).

                              
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Table I
Competition studies of sturgeon peptides
NB, no binding; ND, not determined; Dyn-(1-17), dynorphin A-(1-17).


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Fig. 2.   Receptor binding assays of the affinity of predicted sturgeon proorphanin-derived peptides (Sturgeon F, Phe137-Tyr147; Y, Tyr161-Pro177; YKR, Tyr161-Arg179) for orphanin (ORL1) and opioid (µ, kappa , delta ) receptors. Direct comparisons of binding by sturgeon Y and YKR peptides revealed no systematic differences. For ease of visualization in the opioid binding studies, only binding data for the YKR peptide are shown. Mammalian receptors were expressed in COS-1 cells, and affinities of test ligands were determined by competitive displacement binding assays. Data are plotted as percent specific bound versus log concentration of competing ligand.

The predicted sturgeon F undecapeptide (Phe137-Tyr147; Fig. 1) failed to bind either the ORL1 or the kappa  opioid receptors to any measurable degree, and affinity for the µ and delta  opioid receptors was 2-3 orders of magnitude less than that displayed by either a model opioid receptor ligand (EKC) or an endogenous opioid (dynorphin A-(1-17)). The absence of opioid receptor binding activity is consistent with the presence of a phenylalanine at position 1, and the lack of ORL1 receptor binding activity is consistent with the absence of conserved orphanin residues in the sturgeon F undecapeptide.

    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The difference between orphanin FQ/N and the canonical opioids (enkephalins, dynorphins, and endorphins) in terms of their physiological impact is significant. The classical opioids produce analgesia, whereas orphanin FQ/N acts in an opposing manner to induce hyperalgesia and allodynia and by reversing opioid-induced analgesia (21-23). Based on work in mammalian systems, this functional dichotomy is due, in part, to a profile of receptor binding specificities that effectively prevent cross-activation of the opioid and orphanin circuits. Orphanin FQ/N does not appear to bind or act as an agonist at the µ, kappa , or delta  opioid receptors. Rather, it is the ligand for the ORL1 orphan receptor, a putative homolog of the delta  opioid receptor (8, 9). Conversely, the classic opioid peptides do not elicit any physiologic effect at the ORL1 receptor (10, 24). Thus, although the inhibitory activity of orphanin FQ/N on a postsynaptic fiber fundamentally mirrors that of typical opioid neuropeptides, its inhibition of adenylyl cyclase is naloxone-insensitive, and its effect on neuronal modulation of pain transmission appears to be broad and pleiotropic. Intracerebroventricular administration of orphanin FQ/N in rodents, for example, has been reported to produce enhanced nociception (5, 7), hyperalgesia followed by analgesia (25, 26), and a dose-dependent reversal of morphine-induced analgesia (22). A similar variety of neuromodulatory responses, ranging from no detectable effect (27, 28) to both analgesia and hyperalgesia (29), has been observed following intrathecal administration of orphanin FQ/N.

In contrast to the functional distinction between the opioid and orphanin neuropeptides, it has been postulated that the precursor proteins from which these chemical signals are derived are all members of the same multigene family and thus share a common evolutionary ancestor. Evidence for this hypothesis can be seen in the primary sequence identity among the prohormones themselves, and the apparent coevolution of their G protein-coupled receptors (8) has provided further support for a common origin by demonstrating that only four nonsynonymous substitutions in the ORL1 receptor are needed to produce a mutant receptor with affinity for both orphanin FQ/N and several dynorphin-derived peptides. Reinscheid et al. (10) demonstrated that substitution of the N-terminal phenylalanine of orphanin FQ/N by tyrosine, an opioid hallmark (30, 31), does not lessen binding to the ORL1 receptor, and the N-terminal nonapeptide of Tyr1-substituted orphanin FQ/N has significant affinity for the kappa  opioid receptor. Although these findings provide support for a common evolutionary origin, it is important to recognize that the mutant ORL1 receptor and Tyr1-substituted orphanin FQ/N analog represent hypothetical models for the proposed ancestral receptors and ligands. The sturgeon proorphanin gene, however, encodes a naturally occurring peptide that bridges the gap between the classical opioids and orphanin in terms of both its primary sequence characteristics and receptor binding profile. That such an "evolutionary intermediate" persists to the present is consistent with the hypothesis that some extant species such as the sturgeon represent evolutionary relic species (32).

Length and primary sequence data provide the first indications that sturgeon proorphanin represents an intermediate stage in the evolutionary divergence of the orphanin and opioid neuropeptide precursors. The 194-residue sturgeon proorphanin is smaller than the 254-residue Cavia porcellus prodynorphin (33), which is the smallest classical opioid precursor yet characterized, but larger than all previously reported orphanin precursors, which range from 175 to 186 residues (34, 35). Parsimonious sequence alignments reveal that among mammalian proorphanin sequences, size variants are the result of redundancy of a hexapeptide (DAEP(E/V)A in rats and DAEPGA in mice) beginning at residue 109 in rodent sequences (Fig. 3). Repetition of such short sequence motifs commonly arises through replication slippage. By contrast, the difference in size between sturgeon and mammalian proorphanin sequences is due not to expansion of this hexapeptide repeat but rather to the presence of 24 additional residues (Phe137-Arg160) immediately N-terminal to the sturgeon orphanin sequence that contains a classic opioid core sequence (YGGF) (Fig. 3). The Phe137-Arg160 extension includes a FGGF sequence motif that may be critical for understanding the evolutionary relationship between the classical opioid precursors and proorphanin.


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Fig. 3.   Alignment of the full-length amino acid sequences, with GenBankTM accession numbers in parentheses, for sturgeon (AF095739), human (U48263), bovine (AB005251), rat (U48262), and murine (D82866) proorphanin. Amino acid sequences were aligned manually using gapped BLAST-generated alignments as a guide. Positions in the sequences that are identical are shaded. The putative F peptide and orphanin sequences are labeled and demarcated by a bar over the respective sequence. Repetitive hexapeptides responsible for mammalian proorphanin size variants are boxed.

To date, the Phe137-Arg160 extension has only been detected in sturgeon proorphanin. The absence of the Phe137-Arg160 sequence in mammalian proorphanin sequences (Fig. 3) could have been the result of an unequal crossover event as diagrammed in Fig. 4. Such an evolutionary event would account for the shorter proorphanin precursor proteins found in mammals relative to sturgeon proorphanin. The eventual deletion of the Phe137-Arg160 sequence after the divergence of the ray-finned fish and sarcopterygians (lobe-finned fish and tetrapods) may indicate that this sequence was either nonessential or nonfunctional. The receptor binding data (Fig. 2) support the latter conclusion. A peptide representing a possible bioactive product from this region (Phe137-Tyr147) was synthesized but failed to bind to mammalian opioid receptors. This observation was consistent with the presence of a phenylalanine rather than a tyrosine residue at the N-terminal position of the opioid core sequence. Furthermore, aside from a four-residue orphanin core sequence, the Phe137-Tyr147 peptide lacks even a minimal orphanin binding motif and hence cannot bind or activate the mammalian ORL1 receptor. Based on mutagenic studies of orphanin/OLR1 binding in mammals, the receptor-binding domain of orphanin is associated with the first 11 N-terminal residues of orphanin sequences (10). The 11-residue "F peptide" lacks homology to the "minimal OLR1 binding domain." The demonstrated absence of receptor binding by this critical domain obviated the utility of synthesizing and testing additional C-terminally extended forms of the "F" peptide. Finally, a BLAST analysis of the Phe137-Tyr147 sequence showed no significant similarity to any known bioactive peptides. These results lend further support to the view that this region may not contain a functional neuropeptide. However, it should be noted that these conclusions are based on binding of sturgeon peptides to mammalian ORL1 receptors. It is appreciated that the Phe137-Tyr147 sequence may bind to a native receptor in the sturgeon that is unrelated to either the proposed opioid receptors or proposed ORL1 receptor in this species.


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Fig. 4.   A, proposed evolutionary mechanism by which the modern (i.e. mammal-like) proorphanin gene is hypothesized to have arisen from a sturgeon-like proorphanin ancestor. Unequal crossover between opioid/orphanin core sequences (YGGF/FGGF) yields two distinct products. In one, there is complete elimination of the proposed F peptide region with a simultaneous Tyr to Phe substitution at the N terminus of the orphanin heptadecapeptide, whereas in the other, there is duplication of the F peptide region. B, schematic representation illustrating similarities in the structural arrangement of the precursor proteins for mammalian proenkephalin, mammalian prodynorphin, and sturgeon proorphanin. Regions in the precursors where opioid core sequences or relic opioid sequences are located are shaded and striped, respectively. Highly conserved cysteine residues near the N-terminal of each precursor are indicated by the letter C. M-RGL, methionine enkephalin Arg-Gly-Leu; M-Enk, methionine enkephalin; L-Enk, leucine enkephalin; alpha -neo, alpha -neoendorphin; Dyn A, dynorphin A; Dyn B, dynorphin B; "F", sturgeon proorphanin peptide F.

In addition to producing a shorter orphanin precursor (more consistent with mammalian orthologs), the hypothesized unequal crossover event, diagrammed in Fig. 4A at the repeated pentapeptide core sequences would result in a Y161F mutation producing the canonical orphanin core sequence found in mammals. Compared with a combination of independent transition and transversion mutations, this single deletion event represents a more parsimonious mechanism for the essential step of moving from the existing Tyr161 codon (TAC) in sturgeon proorphanin to the phenylalanine codon (TTT) found at position 1 of all orphanin core sequences.

If, as has been postulated, proorphanin arose through duplication of one of the classical opioid precursor genes, one would expect to find homology not only in the sequence and structural organization of proorphanin but also in the behavior of the bioactive peptides. Perhaps the most compelling evidence that sturgeon proorphanin represents an evolutionary intermediate stage in the evolution of the opioid/orphanin gene family comes from the receptor binding profile of the orphanin-like sequence (1YGGFIGIRKSARKWNNP17) in this precursor. Several critical amino acid substitutions place sturgeon orphanin midway between a typical mammalian orphanin FQ/N and an opioid agonist like dynorphin A. As shown in Fig. 2, sturgeon orphanin binds with almost equal affinity to both the ORL1 receptor and all three classical opioid receptors. Binding to the mammalian ORL1 receptor was anticipated given the 73% amino acid identity between the ORL1 binding domains (10) of sturgeon and mammalian orphanins. Another key amino acid involved in ORL1 binding by the sturgeon orphanin sequence may be the asparagine at position 15. The presence of an aspartic acid at this position has been linked to exclusion of dynorphin A from ORL1 (10). The opioid receptor binding activity of sturgeon orphanin appears to be the result of a tyrosine residue at position 1 and a tryptophan residue at position 14, features shared in common with mammalian dynorphin A. The latter of these substitutions occurs within the domain of mammalian orphanin that has been identified as being essential for exclusion of opioid receptor activation.

However, it is noteworthy that the binding affinity of the sturgeon orphanin peptide for the mammalian ORL1 receptor was approximately 1 order of magnitude less than that of the native mammalian ligand. This difference in binding affinity may be because of the isoleucine residues at positions 5 and 7 in sturgeon orphanin. In mammalian orphanin there are threonine and alanine residues at these positions (Fig. 3). Alanine/serine and D-amino acid scan analyses indicate that substitutions at these positions adversely impact the interaction between orphanin FQ/N and the ORL1 receptor. Finally, it is recognized that opioid ligands generally function in accordance with the "message-address" concept and that binding affinity cannot, therefore, be considered synonymous with receptor activation. Although future studies will assay directly for inhibition of forskolin-stimulated cAMP activity, the demonstration of dual affinity for orphanin and opioid receptors positions sturgeon orphanin as an intermediate in the functional divergence of the opioid/orphanin gene family.

When the results of the receptor binding studies are combined with the presence of multiple opioid/orphanin-like sequences in sturgeon proorphanin, the intermediate position of the sturgeon sequence relative to mammalian proorphanin and the classical opioid precursors emerges. As observed in Fig. 1, sturgeon proorphanin has three pentapeptide opioid-like core sequences: Phe137-Met141, Tyr161-Ile165, and a relic opioid core sequence at Tyr180-Leu184 (2). By inserting a gap in sturgeon proorphanin, the three opioid core-like sequences align neatly with the octapeptide YGGFMRGL, the penultimate Met-enkephalin, and the Leu-enkephalin domains of proenkephalin (Fig. 4B). These same regions also align equally well with the alpha -neoendorphin, dynorphin A, and dynorphin B domains of prodynorphin. These features underscore the evolutionary relationship between the classical opioid precursors and proorphanin and provide clues to the sequence of duplication events that led to the formation of the opioid/orphanin gene family.

    ACKNOWLEDGEMENT

We thank Dr. Fan Meng for assistance.

    FOOTNOTES

* This project was supported by National Science Foundation Grants IBN9517171 (to R. M. D.), IBN9810516 (to R. M. D. and P. B. D.), and R01 DA 08920 (to H. A.). Funding for maintenance of sturgeon stocks was provided by the United States Department of Agriculture.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.

AF095739.

§ To whom correspondence should be addressed: Dept. of Biological Sciences, University of Denver, 2101 E. Wesley Ave., Rm. 211, Denver, CO 80210. Tel.: 303-871-3661; Fax: 303-871-3471; E-mail: pdaniels@du.edu.

Published, JBC Papers in Press, April 4, 2001, DOI 10.1074/jbc.M011741200

    ABBREVIATIONS

The abbreviations used are: N, nociceptin; ORL1, opioid receptor-like 1; RACE, rapid amplification of cDNA ends; bp, base pair(s); EKC, ethylketocyclazocine.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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