From the 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
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ABSTRACT |
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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,
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: 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, 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 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 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 DH5 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, 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.
, and
) 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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-endorphin, melanocyte-stimulating hormone-associated peptides),
proenkephalin (end products: Met-enkephalin, Leu-enkephalin), and
prodynorphin (end products: dynorphins and
-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,
, and
opiate receptors) (4,
5).
, and
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,
, and
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.
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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
80 °C until used for mRNA isolation.
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.
,
, 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,
, and
.
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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (27K):
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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 . 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, , or
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,
, and
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
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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The predicted sturgeon F undecapeptide
(Phe137-Tyr147; Fig. 1) failed to bind either
the ORL1 or the opioid receptors to any measurable degree, and
affinity for the µ and
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.
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DISCUSSION |
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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, , or
opioid receptors. Rather, it is the ligand for the
ORL1 orphan receptor, a putative homolog of the
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 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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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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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 -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.
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ACKNOWLEDGEMENT |
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We thank Dr. Fan Meng for assistance.
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FOOTNOTES |
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* 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
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ABBREVIATIONS |
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The abbreviations used are: N, nociceptin; ORL1, opioid receptor-like 1; RACE, rapid amplification of cDNA ends; bp, base pair(s); EKC, ethylketocyclazocine.
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