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Analgesia Research Laboratory (ARL)

Laboratory: Room 419
Phone: 918.561.8498

ARL Mission
The mission of the ARL is to investigate the mechanisms of opioid analgesia starting with the initial binding of opioid drugs to the opioid receptor and ending with the resulting analgesia measured on behavioral tests. We are keen to discover the intimate molecular details of the action of opioid analgesic drugs at their neuronal opioid receptors. In particular, there are three main types of opioid receptors expressed in mammalian CNS tissue. It is not sure which type of opioid receptor (mu, delta, or kappa) is most important for producing analgesia in humans. We approach this question of opioid receptor function using an earlier-evolved vertebrate model, by gaining an evolutionary perspective to mechanisms of opioid analgesia. Our work to date has led to the unireceptor hypothesis of opioid action, whereby opioid receptor molecules in amphibians may be primordial to the resultant m . d , and k opioid receptors in higher mammals. The functional evolution of opioid receptors is investigated by behavioral pharmacology studies, by radioligand binding studies, and by molecular pharmacology studies in our laboratory. The following bookmarks provide further information adapted from a recent NIH grant submission.

Introduction
Opioid drugs produce antinociception in rodent models and analgesia in humans by action at m , d , or k opioid receptors. All three types of opioid receptors are highly homologous products of three distinct genes in rodents and humans. 1 Behavioral pharmacology experiments in rodents and other mammals confirm type-selective opioid antinociception after using highly-selective opioid antagonists for m , d , or k opioid receptors. These results were in turn confirmed by administration of m , d , or k -specific antisense oligonucleotides leading to "knock-down" of selected opioid receptors and loss of antinociception produced by the respective opioid agonist in rodents. 2, 3. However, three separate types of opioid receptors do not appear to be mediating the antinociceptive effects of m , d , or k opioids in amphibians.

Described below, preliminary data from our lab suggests to us that selective m , d , and k opioid agonists produce their antinociceptive effects in the grass frog, Rana pipiens, at a single type of opioid receptor. We call this hypothesized promiscuous opioid receptor, the "unireceptor". Furthermore, it follows that the opioid unireceptor may be a generic opioid receptor, shown by us to mediate antinociception, which has structural motifs with considerable binding affinity for m , d , or k opioids. Background given below details the unireceptor hypothesis in terms of the molecular evolution of opioid receptors and m , d , and k selectivity in mammals. The "negative determinant" theory of receptor evolution derived from peptide hormone receptor research is introduced and applied to our developing hypotheses. A key mystery in understanding the mechanisms of opioid antinociception in amphibians is highlighted, and an original and tantalizing solution is proposed. The research plan described in the present proposal will give us the data to refute or accept our current grand scheme hypothesis of the mechanisms of opioid antinociception in amphibians. This is significant as our research efforts are the sole arena for investigating opioid mechanisms underlying antinociception in earlier-evolved vertebrates.

Specific Aims
One of the major benefits from the integration of molecular biology into pharmacology is the opportunity to correlate the amino acid sequence of a receptor protein with its pharmacological selectivity. We recently characterized opioid binding sites and cloned mu, delta, and kappa opioid-like receptor cDNA from amphibian CNS tissue. Using a comparative bioinformatics approach, the analysis of all available sets of vertebrate opioid receptors revealed original hypotheses correlating receptor primary structure and ligand selectivity (see Preliminary Data). The studies proposed here are crucial extensions of the ongoing projects soon ending. Significantly, we will obtain the sequences of all four opioid-like receptors in novel vertebrate species to create an original dataset of opioid receptor sequences. Additionally, the cloned receptors from various species will be expressed in CHO cells and ligand type-selectivity determined. The expected results will provide a correlation of pharmacological selectivity and receptor protein structure for all four members (MOR, DOR, KOR, and ORL) of the opioid receptor family with the added dimension of vertebrate evolution.

  • A1. Characterize the selectivity of nociceptin in behavioral and binding studies in amphibians.
    1. Determine the effects of intraspinally administered nociceptin and analogs and pharmacological selectivity using opioid and nociceptin antagonists on the acetic acid test in the amphibian, Rana pipiens.
    2. Characterize the affinity (KD) and density (Bmax) of [3H]-nociceptin binding sites in Rana pipiens spinal cord tissue and determine the apparent affinity (Ki) of unlabelled nociceptin and other nociceptin agents and selective opioids in competition binding against the nociceptin radioligand.

Based on preliminary data, we hypothesize that nociceptin will show a dose-dependent analgesic effect blocked by selective nociceptin antagonists but not opioid antagonists. We hypothesize that [3H]-nociceptin will bind to a single, high affinity site in amphibian spinal cord tissue with moderate receptor density. Furthermore, we hypothesize that [3H]-nociceptin will be displaced by nociceptin receptor ligands and selective opioid receptor ligands with less selectivity than observed in mammalian studies.

  • A2. Clone and sequence MOR, DOR, KOR, and ORL-like receptors expressed in novel vertebrate species.
    1. Clone and sequence the MOR, DOR, KOR, and ORL-like receptor proteins expressed in the amphibian brain tissue.
    2. Clone and sequence opioid-like receptor proteins expressed in lamprey, chicken, and alligator brain tissue.
    3. Confirm cDNA sequence identity of novel opioid-like receptors by homology to existing opioid-like receptors.

Based on preliminary data and partial opioid-like receptor cDNA obtained by other investigators, we hypothesize that PCR-based cloning techniques will yield MOR, DOR, KOR, and ORL-like receptor cDNA from brain tissue of all vertebrates examined. We also hypothesize that type assignment will be possible.

  • A3. Characterize novel MOR, DOR, KOR, and ORL-like receptors expressed in CHO cells.
    1. Transfect CHO cells with novel opioid-like receptor cDNA.
    2. Characterize the type-selectivity of each expressed receptor protein by using a panel of selective radioligands and unlabelled competitors.

Based on preliminary data and the literature, we hypothesize that four opioid family receptor proteins will be functionally expressed in CHO cells. We also hypothesize that the type-selectivity of receptors from earlier-evolved vertebrates will be less stringent than that observed in later-evolved species.

  • A4. Perform comparative bioinformatic analysis on complete vertebrate opioid receptor dataset.
    1. Determine the phylogeny of the family of opioid-like receptor proteins in vertebrates.
    2. Examine novel vertebrate OpR sequences for evidence of less divergence in earlier-evolved vertebrates.
    3. Analyze receptor domains and individual amino acid sites to discover the determinants of type-selectivity.

Based on preliminary data we hypothesize that vertebrate OpRs will give a lineage of ancestry from ORL as most basal, to KOR, DOR, and MOR as most recent. Additionally, we hypothesize that the four opioid-like receptors will be significantly more similar to each other in earlier-evolved vertebrates than in later-evolved vertebrates. We also hypothesize that conservation of specific extracellular loop domains and individual sites on vertebrate opioid receptors will be correlated with particular types of receptors.

Background and Significance
The overall goal of this 4-year project is to obtain a comprehensive dataset consisting of the nucleotide and amino acid sequences of novel vertebrate opioid receptor proteins and the opioid type-selectivity of novel cloned receptors expressed in cell lines. There are four opioid family receptor proteins identified in mammals: mu (MOR), delta (DOR), kappa (KOR), and nociceptin (ORL) receptors.1 We recently cloned and sequenced the amphibian MOR, DOR, KOR, and ORL receptor proteins (see Preliminary Data for access nos.). Adding the opioid receptor sequences from an amphibian to the existing vertebrate opioid receptor (OpR) sequences produced a dataset of nucleotide and amino acid sequences from 5 species in 3 vertebrate classes (Pisces, Amphibia, and Mammalia). Phylogenetic analysis of this novel dataset demonstrated that KOR receptor proteins were ancestral to the DOR or MOR proteins. Vertebrate ORL receptors were basal to the branch leading to all three types of opioid receptors. We found data to generate the hypothesis that vertebrate opioid receptors are more similar and less type-selective in earlier-evolved species, with the corollary that receptors are more selective in later-evolved vertebrates. Novel phylogenetic analysis restricted to particular receptor domains linked specific extracellular loop (EL) domains to type of opioid receptor. Particular amino acids within these domains were identified that may be crucial factors in the determination of opioid type-selectivity (see § C).

Given that the results of this comparative bioinformatics approach to analyzing vertebrate opioid receptors correlated with experimental findings, there is a strong mandate to pursue the same strategy in the present proposal with a more complete dataset. The comparative bioinformatic analysis proposed here is the first method to determine type-selectivity of a GPCR using natural receptor sequences expressed in the brain of diverse and representative vertebrate species. To state simply, we have discovered a way to let evolution tell us what parts of a receptor must be unchanged across the vertebrate phyla to retain the identity of a particular receptor type. To strengthen this novel evolutionary approach, we need opioid-like receptor sequence data from at least one member of all six classes of vertebrates.

The complete vertebrate OpR sequence dataset will provide the means to gain a unique insight of the molecular evolution of vertebrate opioid-like receptors, with an emphasis on ligand-selectivity and determination of type.

Direct tangible results of the project will be:

  1. lineage of vertebrate opioid receptor types;
  2. determination of the evolution of type-selectivity among vertebrates; and
  3. resolution of the determinants of type-selectivity at the receptor domain and amino acid levels for MOR, DOR, KOR, and ORL receptors.

Correlative results will be:

  1. the integration of bioinformatics and behavioral, binding, and molecular pharmacology for amphibian and mammalian opioid-like receptors;
  2. fidelity of the relationship between primary sequence divergence and type-selectivity for vertebrate opioid receptors; and
  3. the establishment of a novel, evolutionarily based method for the bioinformatic analysis of GPCR type-selectivity.

The overall significance is a more detailed understanding of mu, delta, kappa, and ORL receptor primary sequence as it relates to pharmacological selectivity. The proposed merger of pharmacology and bioinformatics will bring a novel understanding of vertebrate opioid receptor evolution and the determinants of opioid type-selectivity. By extension, the expected results and bioinformatic analysis have implications for many types of GPCRs. Practical and theoretical applications are significant. A detailed molecular understanding of ligand selectivity of the opioid receptor gene family products has important implications for the design of new opioid analgesic and dependence medications, the development of new opioid receptor bioassays, and for the eventual gene therapy treatment of pain, and opioid tolerance and dependence.

Additional background and significance for each specific aim follows.

  • B1. Characterize the selectivity of nociceptin in behavioral and binding studies in amphibians

As introduced in the next section, there are six classes of vertebrates and at present, only three classes represented in the vertebrate opioid receptor sequence dataset. As the overall goal is to characterize the functional evolution of vertebrate opioid receptors by vertical integration of molecular, binding, and behavioral data, it is important to have at least one non-mammalian species for which this hierarchal dataset is attainable. This first aim of nociceptin behavioral and homogenate binding studies in amphibians is important as this completes the integrated suite of similar data characterizing the behavioral and binding of mu, delta, and kappa opioid selective agents in Rana pipiens.2-13 Background on the studies of nociceptin pharmacology in mammals follows:

Prepronociceptin is a precursor polypeptide cleaved to at least two biologically active peptides; nociceptin (a.k.a. orphanin FQ) and nocistatin. Nociceptin/OFQ (hereafter, nociceptin) is a 17-amino acid neuropeptide implicated in the modulation of nociception and opioid-mediated effects. Two groups in 1995 isolated nociceptin from mammalian brain extracts.14;15 The prepronociceptin polypeptide that yields nociceptin is closely related to the family of endogenous opioid prepropeptides; preproopiomelanocortin, preproenkephalin and preprodynorphin. Prepronociceptin has similar gene structure (e.g. intron/exon boundaries, conserved CYS residues and dibasic cleavage sites) to opioid precursors, especially preprodynorphin.16-18 Phylogenetic analysis suggests a common ancestor for prepronociceptin and opioid precursor polypeptides, a finding made clear by the hybrid opioid- and nociceptin-like prepronociceptin precursor isolated from the early-evolved sturgeon fish.19 Prepronociceptin mRNA and nociceptin immunoreactivity is localized throughout the CNS of mammals within neuronal pathways that function in the processing of pain.20 Especially high densities of both markers are found in the dorsal horn of the mammalian spinal cord.16;21-23 There is not yet a study of nociceptin in frogs.

The initial observation of hyperalgesia following intracerebroventricular (i.c.v.) administration of nociceptin in mice was striking enough to name the peptide ‘nociceptin’.14 This hyperalgesic effect was confirmed by other investigators24;25 but nociceptin given by spinal administration showed the opposite effect of analgesia.26;27 Whereas the simple explanation that the differences were due to the route of administration was an early interpretation, further studies added many caveats to these findings. There are now numerous studies that show hyperalgesic, analgesic, or no effect of nociceptin following either route of administration.24;26;28-32 Additionally, even though nociceptin does not have an appreciable affinity for any opioid receptor and most opioids have no appreciable affinity for the ORL receptor (see below), some studies have found reversal of nociceptin effects by opioid antagonists28;30;33-35 whereas other have not.25;27;36;37 Factors cited to account for the discrepancy of nociceptin effects include species (rat or mouse), route of administration (supraspinal or spinal), algesiometric tests used (acute or chronic pain stimulus), endogenous opioid tone (stressful or non-stressful tests) strain of mouse or nociceptin dose.20;32 Much of the discordant behavioral data above may be explained by the findings from of the cellular and in vivo effects of nociceptin and opioid ligands in the rat nucleus raphe magnus (NRM).38 The NRM contains two types of opioid-sensitive cells that influence the descending pathway output. Nociceptin was shown to inhibit the cell type that is disinhibited by morphine or endogenous opioid released under stress, thus exhibiting opioid antagonism or hyperalgesia. In the absence of exogenous or endogenous opioids, nociceptin inhibits both cells types and results in analgesia. More recently, two papers strengthened the case for spinal antinociceptive effects of nociception by anatomically and functionally linking nociceptin to the inhibition of substance P.39;40

Radioligand binding studies using [3H]-nociceptin in mammalian brain tissue homogenates demonstrate a single, high-affinity site with KD ranging from 0.05 to 5.0 nM and Bmax from 143 to 254 fmol/mg protein.41;42 In rat spinal cord, [3H]-nociceptin bound with affinities ranging from 0.1 – 1.9 nM with a density of about 440 fmol/mg protein using in vitro quantitative autoradiography.43;44 In the mouse spinal cord, [3H]-nociceptin bound with an affinity of 0.031 nM and a density of 47.8 fmol/mg protein.45 Surprisingly, there is a single report of [3H]-nociceptin1-13-NH2 (an nociceptin analog) binding in amphibians using brain tissue homogenates from the European water frog, Rana esculenta.46 These results show that the nociceptin analog binds to a single, high-affinity site on brain tissue homogenates with an affinity of 0.55 nM and a density of 180 fmol/mg protein. However, given the availability of [3H]-nociceptin itself and for comparison to mammalian studies as well as our own data showing analgesic activity of nociceptin following spinal administration of nociceptin in Rana pipiens, we propose an enlarged and systematic study of [3H]-nociceptin binding of spinal cord membranes as part of our first aim. Shown below in preliminary data, we obtained an initial saturation curve using the [3H]-nociceptin radioligand available commercially.

In summary, the amphibian model is the only established non-mammalian model for the behavioral assessment of opioid analgesia (antinociception). This will allow a unique opportunity to correlate the relationship of opioid-like receptor primary sequences all the way to the pharmacological selectivity observed in vivo for at least one earlier-evolved vertebrate for comparison to the same data from later-evolved mammalian species. For other non-mammalian vertebrates species used in this project or elsewhere, there is no established method for the behavioral assessment of opioid antinociception, nor can one be easily envisioned (e.g. thrashing lamprey tail-flick test?).

  • B2. Clone and sequence MOR, DOR, KOR, and ORL-like receptors expressed in novel vertebrate species.

The evolution of the six vertebrate classes is shown in Fig. 1. The most primitive vertebrate class is Agnatha (jawless fishes) which survives today as various species of lampreys and hagfish. Cartilaginous fish and bony fish make up the two main divisions of Class Pisces, frogs, toads, and salamanders represent Class Amphibia, and alligators, snakes and turtles are members of Class Reptilia. Class Aves are birds, and humans and other warm-blooded, teat-suckling, hairy creatures are members of the Class Mammalia.

Figure 1

Fig. 1. Evolution of the classes of vertebrate species. Used from Biology (Digital), 6th Edition, by Campbell and Reece, Pearson Educational.

At present, there are only five vertebrate species from three classes of vertebrates with all four opioid-like receptor proteins cloned and sequences deposited in GenBank: zebrafish, frog, rat, mouse, and human (see Table 1, next page, for species and access numbers). The sequences in this table were derived from brain tissue mRNA and in many cases verified by additional submissions. Online availability of whole genome databases from new vertebrate species are announced on a regular basis, however these sequences are derived from shotgun or low-pass methods using nuclear DNA. Given the possibility of pseudogenes or alternative exon splicing in genomic sequences,47 clearly the best sequence database for examining the functional evolution of opioid receptor proteins is one derived from nervous system mRNA.

Table 1. Existing vertebrate species with cloned cDNA sequences for MOR, DOR, KOR, and ORL receptors and proposed representative vertebrates to be used. Access numbers are given for the NCBI nucleotide database (see Methods). Bolded sequences were recently cloned in P.I.’s lab; sequences noted TBD (to be deposited) will be cloned and sequenced as part of the proposed studies.

Table 1

There was an initial study partially cloning opioid-like receptor proteins in five classes of vertebrates from Evans and colleagues.48 They used degenerate primers targeting a short, conserved region between IL1 (intracellular loop 1) and TM3 (transmembrane region 3) to yield 162 bp (54 a.a.) partial clones from genomic DNA (full OpR-like sequences are -1200 bp and -400 a.a.). Multiple opioid-like receptor partial clones were detected in human, bovine, rat, mouse, chicken, frog, shark, bass, and hagfish. A species from Class Reptilia was not used. The hagfish is a member of Class Agnatha and a close relative of the lamprey (see Fig. 1). They did not attempt to extract mRNA or sequence the complete cDNA and given the limited sequence were not able to assign opioid receptor family type based on homology to existing OpR cDNA in all cases. A second paper focusing on mu opioid-like receptors republishes their mu-like partial clones (162 bp) from the above vertebrates and presented further, but not complete, cDNA sequence (TM-TM6) from hagfish.48 Both papers state that OpR-like cDNA was not obtained from any of the invertebrate species examined. Importantly, the same degenerate primers used in these above studies were also successful in cloning the full-length cDNA of all four members of the opioid-like receptor family in amphibians (see §C, below). There has been little systematic examination of non-mammalian opioid-like receptors in the ensuing years except results from our research group and that of Rodriguez and colleagues using the zebrafish.49-53

Preliminary data based on comparative bioinformatic analyses of the available 20 sequences (Table 1), supports novel hypotheses on the lineage of opioid receptor types, the selection pressure for increased pharmacological selectivity in vertebrate evolution, and the receptor domains and individual amino acids that may determine opioid type-selectivity (see §C, below). This preliminary data was obtained with sequence representation from only three classes of vertebrates. The complete dataset of cDNA sequences of opioid-like receptor proteins expressed in representative species from all six classes of vertebrates is needed to test and extend these important hypotheses.

  • B3. Characterize novel MOR, DOR, KOR, and ORL-like receptors in transfected cell lines.

After obtaining the sequence of novel OpR cDNA from the aim above, the pharmacological selectivity of these novel receptors will be tested. The most common way this is accomplished is by cell culture transfection for expression of novel OpRs proteins. For in vitro characterization of mammalian OpRs, numerous papers used the Chinese hamster ovary (CHO) cell line or other cells as the substrate for receptor protein production. The seminal paper by Riesine and colleagues characterized the selectivity of human MOR, DOR, and KOR expressed in CHO cells.54 Expressed opioid receptors were also used to determine the pharmacological selectivity by radioligand saturation and binding studies, agonist activation of GTP binding, and coupling to signal transduction pathways.55-62 There are more limited reports of nociceptin selectivity acting on its ORL cognate protein expressed in cell lines.63-67 Cell lines transfected with ORL cDNA display a high-affinity for nociceptin binding, and nociceptin stimulates the binding of GTPγS and the inhibition of cAMP formation.41 With the exception of lofentanil and buprenorphine, most opioids do not have appreciable affinity for the ORL receptor.64;65;67 The development of selective ORL antagonists68-71 has led to further studies demonstrating the selectivity of nociceptin at ORL receptors. ORL selectively binds nociceptin and activates a pertussis-sensitive Gαi/o signal transduction pathway leading to inhibition of cAMP formation, decrease in calcium conductance and increase in potassium channel conductance.72;73 Thus, functionally, ORL is also quite homologous to opioid receptors by identical signal transduction pathways.74 From this data and much more, a well-established and quantitative record exists of selective opioid binding affinity, competitive displacement, and selectivity ratios for all four mammalian members of the opioid-like receptor family.

In contrast, data from non-mammalian OpRs expressed in cell lines are scant. There was an initial study of the white suckerfish that reported expression of a MOR-like protein but selectivity studies were not fully completed.75 As mentioned above, Rodriguez and colleagues cloned and sequenced four members of the opioid family expressed in zebrafish but only a single OpR was transfected, expressed and characterized.50;76 Our work has begun on transfection and expression of rpOpRs (see § C) and the present project will provide the resources, brains and hands to complete these ongoing studies and characterize the new vertebrate opioid receptors cloned in the laboratory. One of the hypotheses that arose from the preliminary data was that opioid receptors are less-selective in earlier-evolved vertebrates. This important aspect of the molecular evolution of vertebrate opioid receptors is directly testable by this aim. The addition of selectivity data from expression of novel opioid receptors from species widely dispersed across vertebrate phylogeny will provide the power and the numbers to confirm or deny this hypothesis.

The same expression system as proposed for radioligand binding characterization of novel OpRs in CHO cells is also amenable to studies of post-ligand binding events. Studies could be designed to characterize the relative potency of agonist GTP-binding and second messenger/downstream effects such as intracellular cAMP or Ca+2 and K+ conductances. However, to obtain the data crucial to the project, emphasis is placed on experiments to determine the selectivity of ligand binding and assays of signal transduction pathways will only be performed to assess that the novel OpR cDNA is a functional receptor when expressed (see § D).

  • B4. Perform comparative bioinformatic analysis on complete vertebrate opioid receptor dataset.

The four members of the opioid receptor gene family are mapped to four different chromosomes (1, 6, 8, and 20) in the human genome.77 The opioid and ORL receptor gene loci are located in paralogous regions of each chromosome.77 Paralogs are members of a gene family that indicate gene duplication. If so, having four opioid-like receptor genes mapped to paralogous regions of four different chromosomes supports the hypothesis that the whole genome underwent duplication twice in early vertebrate evolution.78-80 The general schematic above reflects how four members of the opioid receptor gene family might arise given twice-occurring whole genome duplication (ovoids represent chromosomes and the black bars are gene loci). While further consideration of vertebrate genome duplication is beyond the scope of this proposal, it does highlight the importance of investigating all four member of the opioid receptor family for understanding the functional evolution of vertebrate opioid receptors. There are a few studies using bioinformatics to examine GPCRs, however, these were for the purpose of classification77;81 or only analyzed sequences from Class Mammalia.82;83 The analyses carried out in this project are novel and the first to be applied to a family of GPCRs with a complete representative vertebrate dataset.

Figure 2Fig. 2. Schematic of the proposed gene duplications that led to the four members of the opioid receptor gene family. Straight arrows represent a round of whole genome duplication, which is thought to have happened twice before the evolution of vertebrates. See text for further details.

Receptor domains and individual amino acid sites that determine type-selectivity.

Although there are three types of opioid receptors that mediate the effects of opioid analgesics, at present it is not known what regions of the receptor protein determine opioid type-selectivity and what specific amino acids in those regions are crucial to type-selectivity. Like all GPCRs, the opioid receptor protein takes a serpentine configuration in the plasma membrane. With the N-terminal outside and the C-terminal inside the cell, the seven transmembrane-spanning helices form three extracellular and three intracellular loops.84;85 The N-terminal does not appear to be critical for opioid ligand binding or opioid selectivity but the extracellular loop domains (EL1-3) may function as selectivity filters (see below). Transmembrane (TM1-7) and intracellular domains (IL1-3) have the most conserved residues among opioid receptors. The extracellular domains; N-TERM, EL1, EL2, and EL3, are the most variable and divergent sequences in receptor families and show about 40% mismatch between MOR, DOR and KOR proteins in mammalian opioid receptors.84;85

Figure 3Fig. 3. Visual of the hypothesis of opioid type-selectivity by receptor domains.

After cloning amphibian opioid receptors and performing comparative bioinformatics as part of our current project, we found evidence that pharmacological selectivity may be determined by specific domains or regions of the opioid receptor protein. Among all vertebrate MOR, the 1st extracellular loop is most conserved, for KOR the 2nd extracellular loop and for DOR the 3rd extracellular loop is most conserved (see Fig. 3, on the left). This led to our second hypothesis that specific opioid receptor domains determine type-selectivity. This hypothesis is supported by some, but not all, studies using receptor chimeras or mutants. Site-directed mutagenesis was used to show that certain residues in the first extracellular loop (EL1) of the mammalian MOR are crucial for mu opioid selectivity, the second extracellular loop (EL2) of KOR is crucial for kappa opioid selectivity, and the third extracellular loop (EL3) of DOR most important for delta opioid selectivity.86-98 Significantly, some of the amino acids identified as important for MOR type-selectivity in preliminary bioinformatics analysis also play a crucial role in MOR type-selectivity as discovered in mutagenized receptor studies (see § C4). An important difference is that our hypothesis was generated from a novel comparative analysis of extant vertebrate opioid receptors using bioinformatics. That these two methods (mutagenesis and bioinformatics) showed a congruent result strengthened our resolve to use a bioinformatics approach to investigate opioid receptor type-selectivity in the present proposal. The addition of the novel vertebrate OpR-like receptor cDNA to the existing database of vertebrate OpR-like receptor sequences allows for further novel bioinformatics as we previewed in a recent publication (see Appendix for reprint).13

In summary, the overall significance of this project is to gain a fundamental understanding of opioid receptor selectivity by using a comparative, evolutionarily based approach merging pharmacology and molecular biology techniques and analyzed with the modern tools of bioinformatics. We have obtained convincing preliminary data with a partial vertebrate opioid receptor dataset as presented in the next section. It is crucial that a complete vertebrate opioid receptor dataset is obtained to extend and test these original findings.

Preliminary Studies
Preliminary data is presented for the 4 specific aims. These data confirm the feasibility of the proposed studies and demonstrates that our small research group has the equipment, resources and expertise to carry out the proposed project.

  • C1. Characterize the selectivity of nociceptin in behavioral and binding studies in amphibians
  • C1a. Behavioral effects of intraspinal nociceptin and nociceptin antagonists in amphibian

Figure 4 at left shows the results of preliminary experiments demonstrating an antinociceptive effect of intraspinal nociceptin in frogs and pharmacological selectivity at the ORL receptor. The top panel shows that fentanyl was about 10 times more potent than either nociceptin agonist on the acetic acid test following spinal administration. Higher doses of the nociceptin agonists will be administered before calculation of ED50 values and relative potency. The middle panel demonstrated that the antinociceptive effect of intraspinal (i.s.) nociceptin and the nociceptin analog, nociceptin1-13-NH2,65 was blocked by co-administration of [Nphe1]-nociceptin1-13-NH2, a selective ORL antagonist.69;99 An equi-effective dose of i.s. fentanyl was not blocked by the nociceptin antagonist but was blocked by systemic naltrexone pretreatment (bottom panel). However, systemic naltrexone pretreatment did not block the analgesic effect of nociceptin.

Figure 4 Fig. 4. Top: Log dose-response curves of the antinociceptive effects of fentanyl; nociceptin and nociceptin amide after i.s. administration in amphibians. Middle: Nociceptin, but not fentanyl, antinociception blocked by nociceptin antagonist. Treatment groups were: Saline (5 μl), nociceptin antagonist alone (30 nmol), nociceptin plus nociceptin antagonist (30 nmol each), fentanyl (15 nmol) and fentanyl (15 nmol) plus N/OFQ antagonist (30 nmol). Bottom: Fentanyl, but not nociceptin, antinociception blocked by naltrexone. Groups: Saline (5 μl), nociceptin amide (100 nmol), naltrexone pre (100 nmol/g, s.c. 60 min before) plus nociceptin amide (100 nmol), saline pre plus fentanyl (15 nmol), and naltrexone pre plus fentanyl (15 nmol). N=6-8 animals per treatment group or dose. Significant differences determined by ANOVA and post-hoc Newman-Keuls test. Methods for administration and antinociceptive testing are described in § D.

The finding that intraspinal nociceptin in amphibians produces antinociceptive effects on the acetic acid test suggests that this behavioral assay is useful for determining the pharmacological selectivity of the ORL receptors in non-mammalian species.

  • C1. Characterize the selectivity of nociceptin in behavioral and binding studies in amphibians (cont.)
  • C1b. Binding of [3H]-nociceptin to amphibian spinal cord membranes

Pilot experiments using commercially available [3H]-nociceptin were performed to establish assays conditions for radioligand binding using amphibian CNS tissue. Recently, using a Millipore Multiscreen 96-well format (see Methods for details) a saturation curve of [3H]-nociceptin was obtained using frog spinal cord homogenates (see Fig. 5, right). Additional studies to complete this aim are the completion of saturation curves (with higher concentrations) to obtain an N of three in triplicates and [3H]-nociceptin displacement studies with cold nociceptin and opioid ligands to determine apparent affinity (Ki) of selective agents. The expected results will contribute pharmacological selectivity data of the amphibian ORL-like binding site.

Figure 5Fig. 5. Pilot saturation curve for [3H-Leu14]-nociceptin. Non-linear regression analysis gave the best fit to a single site with a KD of 6.06 nM and Bmax of 940 fmol/mg protein. Methods used are described in § D.

  • C2. Clone and sequence MOR, DOR, KOR, and ORL-like receptors expressed in novel vertebrate species.
  • C2a. Cloning and sequencing of rpMOR, rpDOR, rpKOR, and rpORL.

We recently cloned and sequenced all four opioid-like receptor proteins expressed in the nervous system of the amphibian, Rana pipiens. This was done using the methodology We recently cloned, sequenced, and deposited in GenBank the three orthologs of mammalian opioid receptors expressed in brain tissue of Rana pipiens; rpMOR (AF530571), rpDOR (AF530572), and rpKOR (AF530573). A fourth opioid-like receptor, rpORL (AY434690) was more recently sequenced and was homologous to existing ORL proteins (data not shown). Table 2 shows the percent identity (same residue) and percent similarity (residue of same class) between rpOpRs and species in the existing dataset. The cDNA of rpMOR, rpDOR, and rpKOR yielded the receptor protein sequences used for the multiple alignment shown on the next page.

Table 2Table 2. Homology of amphibian mu, delta, and kappa opioid-like receptors versus existing vertebrate opioid-like receptors from species with complete dataset available. Percent identity and similarity determined by pairwise BLAST. a

Fig. 6. CLUSTALW multiple alignment of rpMOR, rpDOR, and rpKOR with existing vertebrate OpRs. The 7 transmembrane regions are boxed. Notation above the blocks shows the extent of extracellular loop sequences used for domain specific phylogenetic analysis. Filled squares under each block show the identity at that site between ALL, MOR vs DOR, MOR vs. KOR, and DOR vs. KOR, resp. Colons (:) indicate the same class of amino acid substitution between above groups. Sequences from Table 1; h=human, r=rat, m=mouse, rp =Rana pipiens, dr=Danio rerio. See Methods for details of the CLUSTALW multiple alignment.

  • C3. Characterize novel MOR, DOR, KOR, and ORL-like receptors in transfected cell lines.
  • C3a. Transfection and expression of rpMOR in CHO-rpMOR and GH3-rpMOR clones.

With the assistance of our colleague and relatively close neighbor, Dr. Paul Prather at University of Arkansas Medical Sciences, Little Rock, AR, we began transfection studies with the first amphibian clone, rpMOR. Initial data is presented below in figure 7. These data from Paul’s lab indicate that rpMOR is indeed expressed in cells, able to bind opioid ligands, and is functionally coupled to the intracellular effector, adenylyl cyclase. We also did a single transfection and expression of rpORL in our laboratory with the assistance of Dr. Greg Sawyer in our department who provided the CHO cells (see letter in Appendix) but have not yet finished these studies (data not shown). The resources requested for the present project will enable local completion of this aim.

Figure 7 left

Fig. 7

  • C3b. [35S]GTPγS binding assays in amphibian brain tissue

The GTP-binding assay will be done using membranes of CHO cells transiently transfected with OpR cDNA (see § D). We have performed this assay using amphibian brain membranes. With the generous support of our Hungarian colleagues, Mr. Brasel from our lab spent two weeks in the laboratory of Drs. Anna Borsodi and Sándor Benyhe (Institute for Biochemical and Biological Research Center, Hungarian Academy of Sciences, Szeged) to learn how to do [35S]GTPγS binding assays on frog brain membrane homogenates. As shown in Figure 8 below, the extended enkephalin sequence, MERF, produced a max 2-fold increase in the binding of [35S]GTPγS which was blocked by EKC (which has antagonist, or even inverse agonist effects in amphibians). For the present project, CHO cell membranes embedded with novel OpR will be tested for agonist-stimulation of GTP-binding. This will ensure that the novel OpR-like protein is a functional GPCR.

Figure 8 left

Fig. 8

  • C3c. Saturation binding and competition of [3H]-naloxone in cell membrane from CHO-hMOR.

To gain experience in methods used for characterizing expressed OpRs in cell lines, we purchased a small amount of CHO-hMOR membranes from a commercial supplier (Receptor Biology, Inc., Beltsville, MD). As shown in Fig. 9, [3H]-naloxone bound to a single site in the CHO-hMOR membrane preparation and in competition studies was displaced most potently by mu-selective ligands.

Figure 9 left

Fig. 9 left

 

 

 

 

 

 

 

Figure 9 right

Fig. 9 right

  • C4. Perform comparative bioinformatic analysis on complete vertebrate opioid receptor dataset.
  • C4a. The phylogenetic analysis of mu, delta, and kappa opioid–like receptors in Rana pipiens.

As presented in above, we recently cloned, sequenced, and deposited in GenBank the four orthologs of mammalian opioid-like receptors proteins expressed in the brain tissue of Rana pipiens; rpMOR, rpDOR, rpKOR, and rpORL. Fig. 10 (next page) shows the results of the phylogenetic analysis of the current dataset of MOR, DOR, KOR and ORL proteins. For this analysis, we have used the original dataset of 5 vertebrates, 4 OpR-like receptor sequences (total n=20) and used as an outgroup to root the opioid-like receptors the corresponding vertebrate rhodopsin sequences for human, rat, mouse, Rana pipiens, and Danio rerio (access nos. U49742, Z46957, BC013125, S49004, and NM_131084, resp.). The opioid receptor family is classified as belonging to the rhodospsin-like GPCRs superfamily and the rhodopsin protein is considered the base model for these GPCR proteins.77;100;101 The vertebrate groups matched traditional character–based cladistics and the relationship is evident from both NT and a.a. sequences is such that the order of type evolution is ORL, KOR, and DOR, with MOR the most recent or least divergent of the family members (Fig. 10).

  • C4b. Comparison of mu, delta, and kappa opioid–like receptors within species.

As pharmacological selectivity is correlated to similarity of amino acid sequences at the GPCR family level (e.g. opioid receptors vs. muscarinic receptors) it is reasonable to assume that the type-selectivity within family members (e.g. MOR, DOR, KOR and ORL) is also correlated with percent identity or similarity. Pair-wise BLAST analysis (see Methods) showed that opioid receptors from earlier-evolved species are more similar to each other than opioid receptors expressed in mammals (see Table 3, next page). This finding suggests less pharmacological selectivity of these early vertebrate opioid-like receptors than those found in mammalian species. Experimental data supports this analytic finding (see Background). This led to our first hypothesis that opioid receptors in earlier-evolved vertebrates are less type-selective than those found in humans. The hypothesis that opioid receptors in earlier-evolved vertebrate are less selective is supported by behavioral and binding studies in amphibians10;102 and by binding studies on zebrafish opioid-like receptors expressed in CHO cells.50;76

Figure 10   Figure 10

Fig. 10. Phylogenetic analysis of the vertebrate opioid family of receptors. Left panel: nucleotide sequences; Right panel: amino acid sequences. For both, the neighbor-joining algorithm was used as implemented in MEGA3 (see Methods). Including our data of the sequences opioid-like receptor proteins expressed in Rana pipiens the most complete dataset at present consists of MOR, DOR, KOR, and ORL sequences for five vertebrates (h, human; r, rat; m, mouse; rp, Rana pipiens; and dr, Danio rerio). Genbank access nos. are given in Table 1, above. The corresponding vertebrate rhodopsin sequences (RHO) were used as the outgroup to provide a rooted tree for the four opioid-like receptors. Numbers at the node provide the bootstrap value at that node (2000 iterations) and the scale bar is the proportional difference (e.g. 0.05 = 5 % divergence in sequence) for given unit of branch length.

Table 3

Table 3. Homology of MOR, DOR, and KOR receptors within different vertebrate species. Percent identity and similarity determined by BLAST-P.a Table 3. Homology of MOR, DOR, and KOR receptors within different vertebrate species. Percent identity and similarity determined by BLAST-P.a

  • C4. Perform comparative bioinformatic analysis on complete vertebrate opioid receptor dataset (cont.).
  • C4c. Phylogenetic analysis of opioid receptor extracellular loop domains.

After cloning amphibian opioid receptors and performing comparative bioinformatics as part of our current OCAST, we found evidence that pharmacological selectivity may be determined by specific domains or regions of the opioid receptor protein. Among all vertebrate MOR, the 1st extracellular loop is most conserved, for KOR the 2nd extracellular loop and for DOR the 3rd extracellular loop is most conserved (see Fig. 11, below). This led to our second hypothesis that specific opioid receptor domains determine type-selectivity. This hypothesis is supported by some, but not all, studies using receptor chimeras or mutants. Site-directed mutagenesis was used to show that certain residues in the first extracellular loop (EL1) of the mammalian MOR are crucial for mu opioid selectivity, the second extracellular loop (EL2) of KOR is crucial for kappa opioid selectivity, and the third extracellular loop (EL3) of DOR most important for delta opioid selectivity.86-98 An important difference is that our hypothesis was generated from a novel comparative analysis of extant vertebrate opioid receptors using bioinformatics. That these two methods (mutagenesis and bioinformatics) showed a congruent result strengthened our resolve to use a bioinformatics approach to investigate opioid receptor type-selectivity in the present proposal.

Fig.11
Figure 11     Figure 11     Figure 11

  • C4d. Analysis of individual amino acid site rate-shfts for MOR and ORL proteins.

The phylogenetic analysis using EL domains of vertebrate OpRs is suggestive of domain-specific determinants of opioid type selectivity. If confirmed by additional vertebrate opioid receptor sequences sought in this project, this approach still does not determine which amino acids within the domain may be critical for type-selectivity. It is known that individual sites (residues) in a protein can exhibit different rates of replacement, either faster or slower, than the average rate in a gene family, due to functional constraints.103 Differences in site-specific rates (also called evolutionary rate shifts) are especially prevalent in paralogs, i.e. genes thought to arise from gene duplication events.104-107 As noted above, the family of opioid receptor proteins show strong evidence of gene duplication. The results of a likelihood ratio test108 for functional divergence among the 5 vertebrates, 4 OpRs dataset for MOR and ORL proteins is shown below (Fig. 12).

The analysis process consisted of a multiple alignment of the entire OpR dataset, removing gaps, and submitting paired groups for input. The 5 vertebrate, RHO sequences were used in the alignment and as an outgroup to anchor the test groups as previously used in the phylogenetic analysis. The partial output given in the figure shows a number of sites (the red full columns) that have significantly increased site-specific rate shifts compared to the average replacement rate. Sites displaying slower than average rates of replacement are portrayed in solid blue columns. These sites are not informative as to the difference in shifts between MOR and ORL that may underlie functional divergence but do highlight amino acids that may be important for maintaining general GPCR characteristics (note the distribution of solid blue bars in TM and IL1 regions which are highly-conserved among GPCR of the rhodopsin superfamily). More importantly for investigating type-selectivity is the sites where difference in the rate shift are significant between the test groups. These sites are marked by an asterisk in the figure. Of special interest are the two sites in EL1 (TI at 139-140 in hMOR). These amino acids show a more rapid rate of evolution or greater divergence here in the MOR group and conservation of a more basal state in the ORL. These two amino acids are therefore great candidates for site-directed mutagenesis studies of MOR proteins as they may be crucial for MOR type-selectivity. Amazingly, such studies replacing these exact two amino acids, both alone and together, were found for both rMOR and hMOR and reduced the selectivity of the receptor.109-111

Figure 12

Fig. 12. Bioinformatic analysis of individual amino acid sites that have significantly greater (red) or lesser (blue) substitution rates compared to the overall rate variation of the protein (see methods). Group analysis compared vertebrate MOR and ORL receptors with corresponding vertebrate RHO sequences as the outgroup. For rate variations that are significant between groups, different colors are indicated per group, denoted by the asterisk (*) above the blocks. Of particular interest are the two sites in EL1 that have increased rate shifts in the MOR group (TI at sites 139-140) yet are comparatively decreased in ORL. See text for further comments.

In summary, preliminary data of the comparative bioinformatic analysis highlights the promise of our novel approach. In three important areas, the hypotheses generated from the preliminary data of this aim found some experimental support: 1) there is less opioid type-selectivity in earlier-evolved vertebrates, 2) that particular receptor domains are conserved and determinate type-selectivity, and 3) that individual amino residues are crucial factors in the determination of type-selectivity. Other hypotheses generated such as gene duplication of opioid receptor family members and the lineage of opioid receptor types remain to be tested.

Breaking ground in the field growing between pharmacology and genomics is of the utmost significance for the future of opioid pharmacology. The design of better opioid analgesics and dependence medications depends on an improved understanding of the molecular recognition of an opioid ligand to its receptor. The day may arrive soon when opioid receptor gene therapy is tested and a synthetic opioid receptor might be tailored for particular type-selectivity. This project provides fundamental data that is a precursor to those possibilities. For example, in chronic pain patients, an opioid receptor might be employed with less type-selectivity to be available to a wide range of analgesic agents. In an opioid addict, opioid receptor gene therapy might use an engineered receptor that is highly tuned to endogenous opioid peptides in an attempt to mitigate withdrawal or act as an anti-craving therapy. The field of opioid pharmacology, the first among the GPCRs in so many aspects, should embrace this new field of pharmacology and bioinformatics and lead the way for other GPCRs. In some small way, the present proposal contributes to this ideology and suggests an original and practical plan on how to get there from here.

Research Designs and Methods
The specific methodologies used in this proposal include behavioral assays, radioligand binding, molecular pharmacology techniques, and a novel method of comparative bioinformatics. The research team consists of a full-time Research Assistant (senior-level technician, Mr. Chris Brasel), a Ph.D. level graduate student, and the P.I. and is determined to make this project a reality. We also have the assistance of Dr. Sawyer in our department and Dr. Prather, in Little Rock, for advice and encouragement. The timeline for the project is illustrated below and methods for each specific aim follow.

Timeline

  • D1. Characterize the selectivity of nociceptin in behavioral and binding studies in amphibians
  • D1a. Behavioral studies

Animals: Male or female Northern grass frogs, Rana pipiens, will be used for all experiments. Animals with a snout-vent length of 5-7 cm (about 25-35 g body weight) are purchased from commercial distributors (Sullivans, Nashville, TN). Upon arrival, frogs are kept in stainless steel group holding aquaria, provided with flowing water and fed crickets three to four times weekly (also supplied by wholesale distributors). Two days before the start of an experiment, frogs will be randomly assigned to individual plastic cages with soft mesh lids, for acclimatization to laboratory conditions.

The acetic acid test: The acetic acid test (AAT) to determine the nociceptive threshold (NT) in frogs consists of eleven concentrations of acetic acid serially diluted from glacial acetic acid. The concentrations are given a code number from 0 to 10 with the lowest code number equal to the lowest concentration of acetic acid.112;113 Nociceptive testing is done by placing, with a Pasteur pipette, a single drop of acid on the dorsal surface of the frog's thigh. Testing begins with the lowest concentration and proceeds with increasing concentrations until the NT is reached. The NT is defined as the code number of the lowest concentration of acid that causes the frog to vigorously wipe the treated leg. The nociceptive response of the animal is directly dependent on the pH of the acid solution applied as the noxious stimulus.113 To prevent tissue damage, the acetic acid is immediately wiped off with a gentle stream of distilled water once the animal responds or after four seconds. If the animal fails to respond, testing continues on the opposite hindlimb. An animal that fails to respond to the highest concentration (#10) is assigned the cut-off of 11.

Intraspinal (i.s.) administration of nociceptin and other agents. Agents and sources to be administered by the intraspinal route are listed in Table 4. The technique for intraspinal administration in frogs was developed by the P.I. over 20 years ago and is akin to the Hylden and Wilcox technique of intraspinal injections in mice.114 For behavioral studies, drugs are mixed in saline to give nmol/μl solutions of the free base. Opioid agonists and antagonists are administered by intraspinal (i.s.) injection into the lumbar region of the spinal cord with a microsyringe fitted with a 26-gauge needle.2 Injections are made percutaneously via the articulation between the seventh and eighth vertebrae. All injections, including co-run saline vehicle controls, are given in a volume of 5 µl/animal. Doses for selective antagonist studies will be chosen based on previous work in the amphibian.7 The nociceptin antagonists are administered concurrently with the agonist using doses determined in pilot experiments. The selective opioid antagonists will also be used to assess amphibian nociceptin receptor selectivity. Non-targeted drug effects are assessed by testing the animals for hindlimb withdrawal, corneal reflexes and their ability to right themselves. Animals displaying any untoward effects either from the spinal injection or high dose effects will be noted and eliminated from the experiment.

Data collection and analysis: For agonist effects, the NT will be determined in animals before the administration of the agonist dose (baseline NT) and at 30, 60, 120 and 180 min after administration. Raw data collected as individual animal's NT will be entered onto a spreadsheet and maximum percent effect (MPE) calculated by the formula below:

MPE

M.P.E. data will be plotted for treatment groups as the time course after administration, and the maximum M.P.E. value over that time course will be pooled from individual animals at the same treatment dose for construction of dose-response curves. Pharmacological software (Pharmacological Calculation Systems) will be used to calculate the median effective dose (ED50) and 95% confidence interval, the relative potency for each of the tested agents, the parallelism of dose-response curves, and for statistical testing of the significant differences between treatment groups.

Table 4. Treatment groups designed to test the pharmacological selectivity of nociceptin activity.

Potential Problems and Solutions: The acetic acid test in amphibians may not detect alterations in nociceptive thresholds produced by intraspinal nociceptin or the nociceptin amide analog. This is unlikely as this behavioral assay was able to detect analgesic effects of intraspinal nociceptin and the nociceptin amide analog in preliminary studies.

  • D1b. Radioligand binding studies.

Radioligand binding studies using amphibian CNS tissue homogenates were published with [3H]-diprenorphine,115 [3H]-naloxone,11;102 and the selective opioid agonists, [3H]-DAMGO, [3H]-DPDPE, and [3H]-U6959312 and the methodology is also described therein. For the binding of [3H]-nociceptin to spinal cord tissue, membrane fractions are obtained by decapitation and rapid excision of the spinal cord by the expulsion method. This is done with a saline filled syringe inserted into the caudal end of the cut vertebral canal. Spinal cord tissue is used fresh and obtained the day of the experiment. Spinal cord tissue is homogenized in approximately 100 volumes/weight of 50 mM Tris HCl with sodium EDTA, pH 7.4. Pellets are obtained by centrifugation of the homogenate at 400 rpm at 4°C for 15 min to remove cell debris followed by 14,500 rpm at 4°C for 15 min. The resulting pellet is suspended in 5 ml of 50 mM Tris HCl with 5 mM MgCl2, at pH 7.4 (working buffer) and rehomogenized for immediate use in the binding assay. Protein content is determined according to the Bradford method116 using bovine serum albumin (BSA) as the standard (BioRad, CA).

Binding assays. Radioligand binding is performed by incubating 200 ml of tissue (spinal cord; 300 mg to 400 mg of protein) with [3H]-nociceptin (NEN, spec. activity 80 Ci/nmol) in a volume of 25 µl and 25 µl of buffer with unlabeled nociceptin (at 10 μM, to determine non-specific binding) or without (for total binding). The tubes are incubated at room temperature for 30 minutes to attain binding equilibrium (determined in pilot experiments). The reaction is terminated by rapid filtration under vacuum using a 96-well Multiscreen system (Millipore) followed by a vigorous but brief washing (3 x 2 ml, approximately 15 seconds) with ice-cold buffer onto the plate well filters. Plates are allowed to dry overnight and filters are punched out and collected into vials and filled with 2.0 ml of Scintiverse scintillation fluid (Fisher, Pittsburgh, PA). Radioactivity trapped in the filters is counted using a Beckman LS1801 scintillation counter (45% efficiency) with specific binding defined as the difference between nonspecific binding and total binding.

Saturation and competition binding assays. For saturation binding, increasing concentrations of the [3H]-nociceptin (0.01 nM - 20 nM) is used to determine receptor density (Bmax) and affinity (KD) in spinal cord membranes. Data is entered into a software program for calculations of these values (see next). Competition analyses to obtain the apparent affinity (Ki) for unlabeled nociceptin and other ORL and opioid ligands (Table 2) is done by co-incubating the radioligand with various concentrations of cold competitor, as we fully described elsewhere.11;12;102;115

Statistical Analysis. Minimum runs per plot is N=3 done in triplicate. Saturation data is analyzed by the iterative nonlinear least-squares curve-fitting program, PRISM (v 4.0, San Diego, CA) to determine KD (affinity) and Bmax (density) values. Nonlinear regression analysis is used to fit the data to equations that minimize the sum of the squares of the distances of the data points to the curve in order to obtain binding parameters.117 Data are first fit to the rectangular hyperbolae (binding isotherm) function followed by linear transformation (Bound/Free versus Bound). Data transformation is performed to determine linearity, which may be suggestive of binding to a single non-interactive site. Hill co-efficients are obtained using the PRISM program. However, both KD and Bmax values are obtained from analysis of the rectangular hyperbola. Best fit models (1 or more sites) are determined by the F-test which is based on the statistical F-ratio test which compares the weighted residual sum of squares. All comparisons are considered significant at P < 0.05.

Table 5 on the next page lists the radioligand and cold (unlabeled) competitors to be used in saturation and competitive binding assays using amphibian spinal cord tissue:

Table 5

Potential Problems and Solutions:  Putative rpORL receptors may not be expressed or exist in great enough density to detect in amphibian spinal cord tissue. This is unlikely, as we have shown that nociceptin produces antinociceptive effects after intraspinal administration that is blocked by a selective nociceptin antagonist. Furthermore, preliminary binding studies using [3H]-nociceptin demonstrate a saturable binding site in frog spinal cord. Importantly, a putative cDNA for a rpORL-like receptor protein was recently cloned (see Preliminary Data).

  • D2. Clone and sequence MOR, DOR, KOR, and ORL-like receptors expressed in diverse vertebrate species.
  • D2a. Clone and sequence novel OpR-like proteins.

The following methods were used to clone and sequence the cDNA for rpMOR, rpDOR, rpKOR and rpORL receptor proteins and identical methodology will be used for the cloning of cDNAs of novel OpRs. Total RNA is extracted using Trizol (Invitrogen) from approx 2g of brain tissue according to protocol provided with the reagent. Flash-frozen chicken brain tissue has already been obtained from Dr. Ken Sufka, University of Mississippi, Oxford, MS. Alligator brain tissue will be obtained with the generous assistance of Dr. Dennis Paul, LSU Medical Center, New Orleans, LA, who has a cousin that owns an alligator farm. Live sea lampreys will be sent by Mr. Michael Twohey, of the U.S. Fish and Wildlife Service, Sea Lamprey Control Program, Marquette, MI. Upon arrival, lampreys will be decapitated and brain tissue removed by dissection. mRNA is isolated from total RNA from all brain tissue using the Fast Track 2.0 mRNA Isolation Kit (Invitrogen). Using degenerate primers (Table 6, below) that were successful in partial cloning (162 bp out of -1100) of OpR cDNA in a number of vertebrates including lamprey (see Background), PCR is done with the Advantage 2 Polymerase enzyme (Clontech) using a MJ Research PTC-200 Peltier Thermal Cycler. Parameters for the PCR are: initial denature 94° C/1 min, cycle 35 times: 94° C/30 sec and anneal/extend at 68° C/30 sec, and final extension 68° C/3 min. PCR products are electrophoresed on a 1.5% agarose/ethidium bromide gel and the predicted products identified by standard bp ladders. PCR products are cloned into the pCR4-TOPO vector using the TOPO-TA Cloning Kit (Invitrogen). TOP10 E. coli. (Invitrogen) are transformed with the ligation and plated overnight at 37° C in shaker/incubator. The next day, overnight cultures of LB/Amp (100μg/ml) are inoculated with picked colonies. Plasmids are purified using the QIAprep Spin Mini Prep Kit (Qiagen) and sequenced by the OSU-Core Sequencing Facility (at OSU main campus in nearby Stillwater, OK) using a Perkin-Elmer ABI sequencer.

Table 6. Degenerate primers used to clone novel OpR cDNAs from vertebrate brain tissue.

Table 6

Based on the 162 bp OpR cDNA fragment obtained, gene-specific primers are designed for 5’ and 3’ RACE (rapid amplification of cDNA ends)118 using the SMART RACE cDNA Amplification Kit (Clontech). First-strand cDNA synthesis and subsequent RACE reactions is performed until 5’ and 3’ ends are amplified. Another set of primers is designed from the 5’ and 3’ untranslated (UTR, flanking) regions to obtain a full-length cDNA clone using High-Fidelity 2 Polymerase (Clontech). The predicted -1.2kb product is observed on a 1.0% agarose/EtBr gel and cloned into the pCR4-TOPO vector as before. Clones will be submitted for sequencing as above and contigs assembled using SeqMan (DNAstar software). A minimum of eight, separate full-length PCRs will be performed to verify sequence. Nucleotide and deduced protein sequences of novel OpRs will be deposited in the National Center for Biotechnology Information (NCBI) databank (GenBank) maintained by NIH (http://www.ncbi.nlm.nih.gov).

Possible problems and solutions. The degenerate oligonucleotide primers may not isolate OpRs in novel vertebrate species. It is unlikely that novel OpRs sequences will not be obtained given that they have been successful in Evans hands for hagfish (a close relative of the lamprey), chicken, and a number of fish species. They also were effective in our isolation of amphibian OpRs. However, if this occurs, new primers will be designed based on the partial sequences of the 162 bp clone isolated previously from each of these vertebrates in Evans’ lab (see Background above).

  • D4b. Identification of the receptor type of novel OpRs sequences.

Bioinformatics will be performed on the novel OpRs by BLAST analyses119 using the NCBI server and will result in a comparison of percent identity and similarity of the nucleotide sequence and predicted amino acid sequences to all banked vertebrate OpRs. The identification of all novel OpR-like receptor sequences will be additionally tested by using the GPCR Sub-Family Classifier.120 Banked sequences that are partial or alternative splice variants will not be included in bioinformatic analyses.

Possible problems and solutions. The novel ancestral OpR sequences may not clearly differentiate into one of the four types of opioid-like receptor proteins by homology testing. This is unlikely as partial clones were identifiable as particular types in hagfish and zebrafish, even without the full sequence obtained in the present project (see Background). Furthermore, phylogenetic analysis (Fig. 10) shows that even at the root of all opioid-like receptor proteins, there is discrimination of each branch leading to a particular group of family members.

  • D3. Characterize novel MOR, DOR, KOR, and ORL-like receptors in transfected cell lines.
  • D3a. Transfection and expression of novel OpRs in cell lines.

Transient transfection of CHO cells. Chinese hamster ovary cells (CHO cells, American Tissue Bank, generously supplied by Dr. Greg Sawyer, see letter in Appendix) are plated at a concentration of 2 X 105 of CHO cells in 500µl F12 growth media/10% fetal bovine serum without antibiotics per each well (24 well format). The next day, plasmid DNA from above is diluted from above in a sufficient volume of Opti-MEM media for the transfection (0.8µg of DNA and 50µl of media per well) and incubated at RT for 5 minutes. Lipofectamine is diluted in a sufficient volume of Opti-MEM media for the transfection (2µl of lipofectamine and 50µl of media per well). Tubes are mixed and incubated at RT for 5 minutes. Then the DNA and the lipofectamine are mixed and incubated at RT for 20 minutes. 100µl of the DNA-lipofectamine complex are added to each well and mixed gently by rocking the plate. CHO-OpR cells are incubated at 37º C in a CO2 incubator for 24-48 hours before use below.

Possible problems and solutions. The novel opioid receptor proteins will not be expressed efficiently in mammalian CHO cells. This is not likely as we have already had some success in transfecting/expressing and characterizing rpMOR (shown above, § C), and rpDOR, rpORL (not shown ) in CHO and GH3 cells. Additionally, a number of mutagenized and chimeric opioid receptor proteins were expressed in CHO and other cells (see § B above) which suggests that this expression system is relatively permissive for receptor xenoproteins. We have the local assistance of Dr. Greg Sawyer for general cell culture and transfection advice and the expert assistance of Dr. Paul L. Prather, our collaborator and colleague (see his letter in Appendix). Dr. Prather is a recognized expert in the characterization of opioid receptor proteins in mammalian cell lines with over 20 publications in this area (Medline search).

  • D3b. Assess the expression and functional coupling of transfected OpRs

[3H]-Naloxone will be used as the test radioligand to ascertain sufficient expression levels of the MOR, DOR, KOR receptor proteins. It is a general opioid antagonist and binds with good affinity to hMOR, hDOR, and hKOR expressed in CHO cells.54 As it does not bind with appreciable affinity to ORL receptors, [3H]-nociceptin will be used to determine expression of putative ORL-like receptor proteins. Functional assays of agonist-stimulated GTP-binding will be done after initial expression experiments to determine coupling to signal transduction pathways. Clones will be selected for further studies by their ability to bind [3H]-naloxone and agonist stimulation inhibition of GTP-binding.

Whole-cell binding assay. Opioid radioligands are diluted from stock on the day of the binding assay. Radioligands to be used to determine KD and Bmax are listed below in Table 7. With peptide radioligands, preparations are made fresh from lyophilized powder to avoid freeze/thawing. A 10X concentration is conveniently used for radioligands so that 50μl can be used in a 500μl total assay volume. Growth media is aspirated from cells and 400μl of binding buffer is added to each well for nonspecific binding (NSB) samples and 450μl to each well for total binding (TB) samples. 50μl of radioligand is added to each both NSB and TB wells at appropriate concentrations ranging from 10-10 to 10-3. 50μl of cold ligand is added to NSB cells for nonspecific binding. Samples are run in triplicate. The binding reaction is incubated at 37˚C for 1 hour. Incubation times and conditions may vary depending on the results of pilot experiments. Following incubation period, buffer and ligand solutions are aspirated from each well and cells are washed 3 times with 1ml of 1X ice-cold PBS per well. Cells are lysed by adding 500μl of 0.25M NaOH to each well. Cells are incubated at room temperature for 30 minutes. Lysing of cells is neutralized by the addition of 70μl of 2.5M HCl to each well. Scintillation vials are prepared by adding 5ml of ScintiVerse (Fisher, Pittsburgh, PA) to each vial. 600μl of lysate from each well is added to the scintillation vials and counted for 5 min each in a Beckman LS1801 scintillation counter (45% efficiency) with specific binding defined as the difference between nonspecific binding and total binding. For saturation binding, 7-9 concentrations of the radioligand will be used as described above to determine receptor density (Bmax) and affinity (KD) in spinal cord membranes. Data is entered into a software program for calculations of these values (see above). Competition analyses to obtain the apparent affinity (Ki) for unlabeled opioid and nociceptin ligands (see Table 7) is done by co-incubating the radioligand using a single concentration with various concentrations of cold competitor, as we fully described elsewhere.11;12;102;115

[35S]GTPγS Binding. Receptor mediated [35S]GTPγS binding is performed as previously described for opioid stimulated GTP binding103;121-123 with slight modifications. [35S]GTPγS is available from Amersham Pharmacia Biotech (Piscataway, NJ) with high specific activity (1000-1400 Ci/mmol; t1/2 = 90 days). After 24-48h post-transfection, cells are detached by incubation with phosphate-buffered saline containing 1 mM EDTA for 5 min and centrifuged at 1000 rpm for 10 min. The cell pellets are extensively washed three times with 50 volumes of phosphate-buffered saline and stored at -80°C until use. Membranes are prepared as follows. Extensively washed, frozen cell pellets are thawed on ice and resuspended in ice-cold homogenization buffer, pH 7.4, composed of 50 mM HEPES, 1 mM MgCl2, and 1 mM EGTA. Cells are then homogenized with 10 strokes of a glass Dounce homogenizer (Wheaton, Philadelphia, PA) and centrifuged at 40,000g for 10 min at 4°C. Pellets are resuspended in homogenization buffer, homogenized, and centrifuged again as described. This procedure is repeated twice more. The final pellets are resuspended in 50 mM Tris-HCl buffer, pH 7.4, and aliquots are frozen at 80°C until use. Protein concentration is determined using bovine serum albumin as a standard.116 CHO cell membranes with OpRs (50 µg of protein) are incubated with [35S]GTPγS (0.1 nM) in a binding buffer (20 mM HEPES, pH 7.4, 10 mM MgCl2, 100 mM KCl, and 10 µM GDP) in a volume of 250 µl. Nonspecific binding is defined by the inclusion of 10 µM cold (unlabelled) GTPγS. For basal levels of GTP binding, nothing is added to this final volume. Morphine, DPDPE, U-69593, or nociceptin (depending on the type of putative OpR) are added to the incubation at a concentration of 1 µM to assess the type-selectivity of G-protein activation by OpRs. Naltrexone, a non-selective opioid antagonist, at 10 µM is added to an additional set of reactions to assess antagonism of the agonist-mediated effects for MOR, DOR, and KOR-like proteins and the nociceptin antagonist used for ORL-like proteins. The reaction is incubated for 1 h at 30°C. The reaction is terminated by rapid filtration under vacuum using a 96-well Multiscreen system (Millipore) followed by a vigorous but brief washing (3 x 2 ml, approximately 15 seconds) with ice-cold buffer onto the plate well filters. Plates are allowed to dry overnight and filters are punched out and collected into vials and filled with 2.0 ml of Scintiverse scintillation fluid (Fisher, Pittsburgh, PA). Radioactivity trapped in the filters is counted using a Beckman LS1801 scintillation counter (45% efficiency) with specific [35S]GTPγS binding defined as the difference between nonspecific binding (with 10 µM cold GTPγS) and total binding in the presence of opioid agonists.

Possible problems and solutions. The novel OpRs may not bind the radioligand and expression would be missed. This is unlikely as naloxone binding sites have been detected in all vertebrate nervous system where assayed and even quite a few invertebrate tissue (see review124). The use of the above type-selective agonists to assess GTP-binding may be too selective for novel OpR. If putative OpRs exhibit radioligand binding but do not increase GTP-binding with the selective agonists listed above, other type-selective agonists will be tried.

  • D3c. Determine the type-selectivity using of novel OpRs

Once the transfection and expression parts of this aim are finished, each clone will be subjected to a systemtic analysis of type-selectivity by employing a panel of selective opioid and nociceptin ligands (Table 7).

Table 7. Radioligand and cold competitors to be used for characterization of novel OpRs in CHO cells.

Table 7

  • D4. Perform comparative bioinformatic analysis on complete vertebrate opioid receptorataset.
  • D4a. The phylogenetic analysis of mu, delta, and kappa opioid–like receptors in Rana pipiens.

For construction of the phylogenetic trees (dendrograms) and pair-wise differences among opioid receptor domains and full amino acid sequences, the software MEGA3 (v. 3.0) will be used.125 The neighbor-joining method (NJ) is the algorithm used to determine the topology of the dendrograms. The NJ method has a high degree of accuracy126 and was used for previous studies examining receptor phylogeny.81-83 The reliability of branching points (nodes) is determined by the bootstrap method. The number of bootstrap repetitions will be 1000. The bootstrap value is noted above the internal branch points on the dendrograms, converted to percent. Thus, a bootstrap value of 95 denotes that 95% of all the trees generated (i.e. 950 out of 1000) placed the branch at that node. The branch length represents the proportion of different amino acids from each node and the value is denoted below the branch (scale bar given on dendrogram). Branch lengths are generated by pair-wise distance using the MEGA3 software package. Both NT and amino acid characters will be analyzed.

Multiple alignments will be made using the CLUSTAL-W program127 as implemented on the EBI website. (See Table 8, below, for listing of bioinformatics programs and their availability). Default programs settings will be used to obtain a complete multiple alignment of the total vertebrate dataset (N= 32 OpR-like proteins, Table 1). Using the alignment editor program bundled with MEGA3, gapped columns will be removed and the multiple alignment file saved and opened in the phylogeny subprogram of MEGA3. The neighbor-joining algorithm will be used for generating the topology of vertebrate opioid receptors. Other algorithms were used with the preliminary dataset and showed no difference in the results). For rooted trees, the rhodopsin protein will be used as an outgroup to the dataset of opioid-like receptor proteins. The output of the phylogenetic analysis will be analyzed for the lineage of opioid receptor types.

  • D4b. Comparison of MOR, DOR, KOR, and ORL-like proteins within species.

Initially, a series of 2-way BLAST-P analyses will be made for each comparison of opioid receptor family proteins in each species (MOR vs. DOR, MOR vs. KOR, MOR vs. ORL, DOR vs. KOR, DOR vs. ORL, and KOR vs. ORL). These values will be obtained for both percent identity and percent similarity as done for preliminary data in Table 3, above. A more sophisticated approach will be to use a distance matrix available in MEGA3 to directly calculate the mean distance of among all four types of OpRs for each species. This analysis will support of refute the hypothesis that OpRs expressed in earlier-evolved vertebrates are less divergent and therefore less selective.

  • D4c. Phylogenetic analysis of opioid receptor extracellular loop domains.

The extracellular, intracellular and transmembrane domains are initially assigned as previously suggested,128 however, past experience with opioid receptors demonstrates that precise boundaries between receptor domains are not definitively established.13 Guided by the results of the multiple sequence analysis, precise amino acid subsequences are determined by extending borders based on variability until clear conservation sequence resumes (see domains for opioid receptors in Fig. 6). This method works due to the greater variability of the EL domains compared to the TM domains in vertebrate opioid receptors. Extracellular loops of the putative OpRs will be analyzed as in the preliminary data shown in Fig 11 to determine if particular domains are conserved in particular types of vertebrate opioid receptors. Phylogenetic analysis will proceed as described above using the alignment editor and phylogeny programs bundled with in MEGA3.

  • D4d. Analysis of individual amino acid site rate-shfts for MOR and ORL proteins.

The evolutionary rate-shift program using a likelihood ratio test will be used to determine if particular sites along the full-length of the receptor sequence show significantly different rates of replacement during evolution.108 In this test, groups of a gene family can be tested by a pair-wise procedure, with or without rooting by an outgroup. All permutation of this analysis on the complete dataset of vertebrate opioid-like receptor proteins will be done to identify key amino acids that may be determinant of type-selectivity.

Table 8. Bioinformatic resources to be used in the proposed project.

Table 8

Possible problems and solutions. The world-wide-web is disrupted by ever increasing denial of service attacks, viruses, worms, Trojan horses, spam, and incessant pop-ups blocking access to crucial online bioinformatics tools. This is unlikely to occur; although there is no doubt that the virtual computing space of the Internet is becoming increasingly polluted with commercial and other nefarious interests. Additionally, many of the programs listed above are available as local executable files downloaded to the users machine.

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