Introduction

Summary Introduction Materials & Methods Results Discussion References

The molecular cloning of G protein-coupled receptors has served to vastly increase our understanding of the biology of this class of proteins over the past several years. Although the receptors for most known neurotransmitters and neuropeptides have been identified, a large number of putative receptor sequences have also been cloned for which the natural ligands are still not known; thus, these proteins have been referred to as "orphan" receptors. As a first step toward elucidation of novel neurotransmitter systems, we searched for the endogenous ligand for an opioid receptor-like orphan receptor (LC132) (1). Although this protein shares a high degree of sequence homology to the cloned µ-, -, and -opioid receptors, no opioid ligands have been found that interact with receptor LC132 below micromolar concentrations (2). Recently, we (3) and others (4) isolated a novel neuropeptide from mammalian brain that activates the LC132 receptor with high potency. We termed this peptide OFQ and its receptor OFQ-R.

To further characterize this system, it is desirable to have tools that allow the pharmacological and biochemical characterization of this receptor and the determination of receptor expression in different preparations. For these purposes, we developed two peptidergic radioligands: one is a tritiated version of the native peptide ([³H]OFQ), and the other is a radioiodinated derivative, based on a biologically active OFQ peptide in which Leu14 is substituted by tyrosine (¹²⁵I-Tyr14-OFQ). We have previously shown that both Tyr14-OFQ and iodo-Tyr14-OFQ are full agonists with potencies identical to the parent peptide and that ¹²⁵I-Tyr14-OFQ binds in a saturable manner to the OFQ-R expressed in CHO cells (3, 5). However, the utility of both [³H]OFQ and ¹²⁵I-Tyr14-OFQ as radioligands depends on their ability to bind appropriately to a receptor site that exhibits properties consistent with G protein-coupled receptors, including saturability, reversibility, and modulation by environmental factors such as ions and guanine nucleotides. The results of the current study demonstrate that these radioligands bind in a reversible, saturable, and high affinity manner to OFQ-Rs heterologously expressed in HEK 293 or CHO cells. Furthermore, this interaction is characterized in terms of its modulation by monovalent and divalent cations and guanine nucleotides. Finally, we demonstrate that ¹²⁵I-Tyr14-OFQ can be used to specifically label sites in rat brain membranes that exhibit nearly identical pharmacological properties as the OFQ-R expressed in CHO cells.

	Materials and Methods

Summary Introduction Materials & Methods Results Discussion References

Radioligands. All peptides were synthesized at Research Genetics (Huntsville, AL). Iodination and purification of Tyr14-OFQ, in which the leucine residue at position 14 of native OFQ is replaced by tyrosine, was carried out at Amersham International PLC (Buckinghamshire, UK) using the chloramine-T method (5, 6). The product was purified by reverse-phase chromatography, yielding a specific activity of 2000 Ci/mmol (74 TBq/mmol). Eighty-two percent of the labeled product was monoiodinated on Tyr14. [³H]OFQ was prepared by Amersham via catalytic hydrogenation of dehydro-Leu14-OFQ, followed by reverse-phase high performance liquid chromatography purification, yielding a specific activity of 110 Ci/mmol.

Cell culture. CHO dhfr cells, which were stably transfected with the rat OFQ-R as previously described (3), were grown in  modified Eagle's medium containing 5% fetal calf serum and 500 µg/ml geneticin (G418) in a 5% CO₂ atmosphere at 37°. HEK 293 cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum in 5% CO₂ at 37°. For high level expression, the rat OFQ-R was subcloned into the eukaryotic expression vector pCEP4 (InVitrogen, San Diego, CA) and transfected into HEK 293 cells using Lipofectamine (Life Technologies, Gaithersburg, MD) according to the manufacturer's recommendations. Transfected cells were selected in the presence of 100 units/ml hygromycin (Calbiochem, San Diego, CA), and OFQ-R-expressing clones were identified by the ability of 10 nM OFQ to inhibit forskolin-stimulated cAMP accumulation as previously described (3). All cell culture reagents were obtained from Life Technologies.

Membrane preparation. CHO and HEK 293 cells were harvested by incubation in phosphate-buffered saline plus 1 mM EDTA. Cells were washed three times with phosphate-buffered saline before resuspension in buffer A (50 mM Tris·HCl, pH 7.8, 5 mM MgCl₂, 1 mM EGTA) and disruption with a tissue homogenizer (30 sec, setting 4, Pt 20, Kinematica, Kriens-Lucern, Switzerland). A total membrane fraction was obtained by centrifugation at 49,000 × g at 4°. This procedure was repeated twice, and the pellet was resuspended in buffer A. Aliquots were stored at 70°, and protein concentrations were determined using the BCA Protein Assay Reagent (Pierce, Rockford, IL) following the manufacturer's recommendations. Membranes from various rat brain regions were prepared essentially as for cultured cell membranes with the exception that the following protease inhibitors were added to buffer A: 10 µg/ml aprotinin, 0.1 mg/ml phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml pepstatin (all protease inhibitors were obtained from Boehringer-Mannheim Biochemica, Mannheim, Germany).

Binding assays. ¹²⁵I-Tyr14-OFQ binding assays were carried out in deep-well 96-well plates (Beckman Instruments, Fullerton, CA). The binding assay consisted of 18 µg (CHO cells) or 14 µg (rat brain tissues) of membrane protein and ¹²⁵I-Tyr14-OFQ in a final volume of 0.2 ml of buffer A containing 0.1% bovine serum albumin (Sigma Chemical, St. Louis, MO). Saturation analysis of ¹²⁵I-Tyr14-OFQ binding to 293/OFQ-R membranes was carried out in a final volume of 0.5 ml using 1 µg of membrane protein. [³H]OFQ competition and dissociation studies were carried out with 77 µg of membrane protein (HEK 293 cells) in a final assay volume of 0.5 ml of buffer A plus 0.1% bovine serum albumin and 0.01% bacitracin (Boehringer-Mannheim). For [³H]OFQ association and saturation studies, 6-7 µg of protein was used, and the final assay volume was increased to 2 ml to avoid binding >10% of the added [³H]OFQ ligand. For both [³H]OFQ and ¹²⁵I-Tyr14-OFQ, 50 nM unlabeled OFQ was used to define the nonspecific binding, and a 1-hr incubation time at room temperature was found to be optimal for saturation and competition assays. The assays were terminated by filtration through Whatman GF/C filters (Brandell, Gaithersburg, MD) [pretreated with 0.3% polyethylenimine (Sigma) and 0.1% bovine serum albumin (Sigma) for 1 hr] on a Brandell multichannel harvester, and the filters were washed six times with 1 ml of ice-cold 50 mM Tris·HCl, pH 7.5. In some cases, to avoid excessive nonspecific binding of ¹²⁵I-Tyr14-OFQ to filter materials, saturation assays were terminated by rapid cooling to 4°, followed by centrifugation at 14,000 × g for 15 min at 4°. After a brief rinse with buffer A and centrifugation for 5 min at 14,000 × g (4°), the membrane pellets were counted in a gamma counter. Binding parameters determined using this separation method did not differ from those determined using separation by filtration when nonspecific filter binding was not experienced. The association rates of ¹²⁵I-Tyr14-OFQ (0.1 nM) and [³H]OFQ (0.2 nM) were determined by incubating the assays for increasing lengths of time after the addition of membrane suspension. For determination of dissociation kinetics, assays containing 10 pM ¹²⁵I-Tyr14-OFQ or 0.2 nM [³H]OFQ were allowed to equilibrate for 30 min at room temperature. Then, 50 nM OFQ was added to the assay, and the incubation was continued for increasing lengths of time before termination by filtration.

	Results

Summary Introduction Materials & Methods Results Discussion References

Association and dissociation kinetics of [³H]OFQ and ¹²⁵I-Tyr14-OFQ. To identify appropriate conditions for conducting equilibrium binding studies of [³H]OFQ and ¹²⁵I-Tyr14-OFQ, the kinetics of association and dissociation were investigated using an HEK 293 cell line stably expressing the OFQ-R (293/OFQ-R). Under the conditions used for these assays, < 10% of the radioligand was bound at equilibrium, and with the assumption that these ligands interact with only one class of binding sites, the kinetics of association can be considered of pseudo-first-order. The association of both [³H]OFQ and ¹²⁵I-Tyr14-OFQ was rapid and monophasic, with a t_1/2 of 201 sec for 0.2 nM [³H]OFQ (Fig. 1A), in close agreement with the t_1/2 of 117 sec obtained with 0.1 nM ¹²⁵I-Tyr14-OFQ (data not shown). The apparent association rate constants (k_obs) were determined at four concentrations of [³H]OFQ (40-400 pM) (data not shown), and the association rate constant (k_a) was determined from the slope of a plot of k_obs versus [³H]OFQ concentration (9) (Table 1). The value for k_a calculated in this manner did not differ significantly from the k_a value calculated assuming pseudo-first-order conditions. Thus, k_a values for ¹²⁵I-Tyr14-OFQ binding to 293/OFQ-R and CHO/OFQ-R membranes were calculated using the pseudo-first-order simplification (9) (Table 1). Once bound, both ligands were found to dissociate in a biphasic manner, as shown for [³H]OFQ in Fig. 1B, and curve fitting to two exponentials resulted in a significantly better fit than fitting to one exponential. The t_1/2 values for the two components were 7.6 and 218 min in a proportion of 55% and 45%, respectively. These values compare well with the t_1/2 values of 3.4 and 80 min obtained using ¹²⁵I-Tyr14-OFQ, although the proportion of fast and slow dissociating sites (29% and 71%, respectively) differs somewhat (Table 1). High and low affinity equilibrium dissociation constants (K_dH and K_dL) could then be calculated, yielding for [³H]OFQ a K_dH value of 5.73 × 10¹² M and a K_dL value of 1.65 × 10¹⁰ M. These values agree very well with the K_dH value of 5.95 × 10¹² M and the K_dL value of 1.38 × 10 ¹⁰ M calculated for ¹²⁵I-Tyr14-OFQ (Table 1).

View larger version (35K): [in this window] [in a new window]   Fig. 1.   Kinetics of 125I-Tyr14-OFQ and [3H]OFQ binding. A and C, Association of [3H]OFQ to 293/OFQ-R membranes (A) and of 125I-Tyr14-OFQ to CHO/OFQ-R membranes (C). 293/OFQ-R membranes were incubated with 0.2 nM [3H]OFQ and CHO/OFQ-R membranes were incubated with 0.1 nM 125I-Tyr14-OFQ at room temperature for varying times before termination of the reaction by filtration as described in Materials and Methods. For both ligands, nonspecific binding was determined in the presence of 50 nM cold OFQ. Insets, pseudo-first-order representation of the data. Beq, amount radiolabeled OFQ at equilibrium. Bt, amount bound at each time point. B and D, Dissociation of [3H]OFQ from 293/OFQ-R membranes (B) and of 125I-Tyr14-OFQ from CHO/OFQ-R membranes (D). 293/OFQ-R membranes were incubated with 0.2 nM [3H]OFQ and CHO/OFQ-R membranes were incubated with 10 pM 125I-Tyr14-OFQ for 30 min at room temperature. Dissociation was initiated by the addition of 50 nM cold OFQ peptide, and the incubations were terminated by filtration at various times as described in Materials and Methods. Nonspecific binding was determined at the start of dissociation (time 0) by inclusion of 50 nM cold OFQ during the 30-min labeling incubation. Insets, pseudo-first-order representation of the dissociation experiments. All data are from representative experiments done in triplicate and were repeated at least three times with similar results.

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Saturation analysis of ¹²⁵I-Tyr14-OFQ and [³H]OFQ to the OFQ-R. Preliminary experiments were carried out to investigate the stability of OFQ radioligand binding. These experiments revealed that although binding parameters were stable from 1 through 3 hr, binding was not stable for longer periods. Incubation at elevated temperatures further decreased the stability of the binding preparation. Thus, an incubation time of 1 hr at room temperature was chosen for saturation analysis. In additional experiments, a variety of protease inhibitors were tested for their ability to enhance the stability of binding; only 0.01% bacitracin was found to have a stabilizing effect, and it was routinely included in the binding assays.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Saturation binding of [3H]OFQ to 293/OFQ-R membranes and of 125I-Tyr14-OFQ to CHO/OFQ-R membranes. A, Saturation binding of [3H]OFQ binding to 293/OFQ-R membranes. 293/OFQ-R membranes were incubated with increasing concentrations of [3H]OFQ (3- 800 pM) in a final volume of 2 ml for 1 hr at room temperature. Incubations were terminated by rapid filtration as described in Materials and Methods. Nonspecific binding was determined in the presence of 50 nM cold OFQ and was < 10% of total binding at all concentrations tested. Specific binding only is shown for the sake of clarity. Inset, Scatchard transformation of specific [3H]OFQ binding. B, Saturation binding of 125I-Tyr14-OFQ to CHO/OFQ-R membranes. CHO/OFQ-R membranes were incubated with increasing concentrations of 125I-Tyr14-OFQ (2- 900 pM) in a final volume of 200 µl for 1 hr at room temperature. Incubations were terminated by rapid filtration as described in Materials and Methods. Nonspecific binding was determined in the presence of 50 nM cold OFQ and was generally ~15% of total binding at 50 nM 125I-Tyr14-OFQ. Specific binding only is shown for the sake of clarity. Inset, Scatchard transformation of specific 125I-Tyr14-OFQ binding. Data shown are of representative experiments performed in triplicate.

Effect of monovalent and divalent cations on OFQ radioligand binding. To determine whether cations are able to modulate the binding of OFQ ligands to the OFQ-R, we investigated the effects of several different cations on [³H]OFQ binding to 293/OFQ-R membranes and compared them with the effects on ¹²⁵I-Tyr14-OFQ binding to CHO/OFQ-R membranes. The effect of the cation Mg²⁺ was first examined. Decreasing the MgCl₂ concentration to 1 mM had no significant effect on either ¹²⁵I-Tyr14-OFQ or [³H]OFQ binding, as determined by saturation analysis (data not shown). However, saturation binding carried out in 0.125 mM MgCl₂ yielded decreased binding levels that could not be appropriately analyzed (data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Effect of sodium on the binding of [3H]OFQ to 293/OFQ-R membranes. Scatchard transformation of specific [3H]OFQ saturation binding to 293/OFQ-R membranes in the absence () or presence () of 120 mM NaCl. Saturation binding assays were carried out and analyzed as described in Materials and Methods. Data are representative of four independent experiments carried out in duplicate.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Inhibition of 125I-Tyr14-OFQ binding to CHO/OFQ-R membranes by various divalent ions. Increasing concentrations of ZnCl2 (), CaCl2 (), MnCl2 (), or NaCl () were added to CHO/OFQ-R membranes prepared in buffer A containing 0.125 mM MgCl2. Incubations were carried out with 0.1 nM 125I-Tyr14-OFQ for 1 hr at room temperature before termination of the assay by filtration as described in Materials and Methods. Nonspecific binding was estimated using 50 nM cold OFQ. Specific 125I-Tyr14-OFQ binding in the absence of added ions (other than MgCl2) was ~12,600 dpm, whereas nonspecific binding was ~2200 dpm. Values represent the percentage of specific binding in the absence of added ions and are mean ± standard error of three independent experiments performed in triplicate.

Effect of guanine nucleotides on the binding of OFQ ligands. Coupling of the OFQ-R with G proteins was explored by evaluating the effect of guanine nucleotides on [³H]OFQ and ¹²⁵I-Tyr14-OFQ binding. The addition of 200 µM of the nonhydrolyzable GTP analog Gpp(NH)p to 293/OFQ-R membranes affected the binding of [³H]OFQ by shifting the proportion of high to low affinity sites from 47% high/53% low to 15% high/85% low (Fig. 5A). There was no significant change in the total binding capacity or in the K_d value of either affinity site. In CHO/OFQ-R membranes, the addition of 200 µM Gpp(NH)p reduced the affinity of ¹²⁵I-Tyr14-OFQ binding by 43%, from 57 to 134 pM, with little change in the B_max value (94-83 fmol/mg of protein) (Fig. 5B). Under these conditions, nonlinear curve fitting revealed the presence of only a single class of binding sites, as was observed in the absence of Gpp(NH)p.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Effect of Gpp(NH)p on the binding of [3H]OFQ to 293/OFQ-R membranes and of 125I-Tyr14-OFQ to CHO/OFQ-R membranes. A, Scatchard transformation of [3H]OFQ saturation binding to 293/OFQ-R membranes in the absence () or presence () of 200 µM of Gpp(NH)p. Nonlinear curve fitting confirmed the presence of two affinity states under both conditions. Solid lines, the two calculated affinity sites for the control condition. Dashed lines, the two calculated affinity sites for the +Gpp(NH)p condition. B, Scatchard transformation of 125I-Tyr14-OFQ saturation binding to CHO/OFQ-R membranes in the absence () or presence () of 200 µM of Gpp(NH)p. Nonlinear curve fitting indicated the presence of a single affinity state under both conditions. For both A and B, membranes were incubated with increasing amounts of radioligand in the presence or absence of Gpp(NH)p for 1 hr at room temperature as described in Materials and Methods. Data are representative of two or three experiments performed in triplicate.

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View larger version (32K): [in this window] [in a new window]   Fig. 6.   Effect of nucleotides on [3H]OFQ binding to 293/OFQ-R membranes. Membranes from 293/OFQ-R cells were incubated with 40 pM [3H]OFQ and increasing concentrations of the indicated nucleotides for 1 hr at room temperature before filtration as described in Materials and Methods. Data represent the percentage of specific [3H]OFQ bound in the absence of added nucleotide and are mean ± standard error of three independent experiments performed in duplicate.

¹²⁵I-Tyr14-OFQ binding in rat brain and peripheral tissue membranes. Because the mRNA for OFQ-R has been shown to be widely distributed in the brain (1, 2), we attempted to determine whether ¹²⁵I-Tyr14-OFQ could be used to detect OFQ-R binding activity in membranes prepared from different brain regions. As shown in Fig. 7, specific, saturable ¹²⁵I-Tyr14-OFQ binding could be detected in membranes prepared from cerebral cortex. Analysis of the saturation isotherm yielded a K_d value of 65.9 ± 6.8 pM (three experiments), which is in good agreement with the value observed in CHO/OFQ-R cells. Next, we evaluated the level of OFQ-R expressed in different rat brain regions. Table 3 indicates that highest expression level of OFQ-R was detected in the hypothalamus, followed by the hippocampus, thalamus, and cortex (507, 489, 249, and 226 fmol/mg of protein, respectively). Other brain regions, including the striatum, cerebellum, and midbrain, contained modest-to-low levels of OFQ-R with no detectable binding in the pituitary. Again, analysis by nonlinear curve fitting revealed only a single class of binding sites in all regions examined, with Hill coefficients close to unity. Interestingly, the apparent affinity of ¹²⁵I-Tyr14-OFQ in all brain regions was in close agreement with the K_d value for CHO/OFQ-R cells (Table 3). No specific binding of ¹²⁵I-Tyr14-OFQ was detected in peripheral tissues, including the spleen, thymus, kidney, intestine, heart, lung, and skeletal muscle.

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Saturation binding of 125I-Tyr14-OFQ binding to rat cerebral cortex membranes. Specific binding of increasing concentrations of 125I-Tyr14-OFQ (23 pM to 2 nM) to membranes from rat cerebral cortex. Nonspecific binding was defined with 50 nM OFQ as described in Materials and Methods. Total and nonspecific binding data are not shown for the sake of clarity. Inset, Scatchard transformation of specific 125I-Tyr14-OFQ binding. Data are representative of three experiments performed in triplicate.

	Discussion

Summary Introduction Materials & Methods Results Discussion References

In the present study, we characterized the interaction of two peptidergic radioligands with the receptor for a novel neuropeptide, OFQ. The results of this study revealed that both the tritiated, native OFQ ([³H]OFQ) and the radioiodinated, Tyr14-substituted analog (¹²⁵I-Tyr14-OFQ) interacted with the OFQ-R in a virtually identical manner. These radioligands met the criteria of saturability, reversibility, and low nonspecific binding that are necessary for useful radioligands. In addition, the binding of these radioligands was found to be modulated by environmental factors known to modulate agonist binding in other receptor systems.

In our first studies of [³H]OFQ and ¹²⁵I-Tyr14-OFQ binding, we examined the kinetics of association and dissociation from the OFQ-R expressed in CHO or HEK 293 cells. Both radioligands associated rapidly, in a monophasic manner, with the OFQ-R, with t_1/2 values of 201 and 158 sec for 0.2 nM [³H]OFQ and 0.1 nM ¹²⁵I-Tyr14-OFQ, respectively. The binding of both ligands was stable for 60 min at room temperature and was blocked by an excess of OFQ peptide. Dissociation of both [³H]OFQ and ¹²⁵I-Tyr14-OFQ from OFQ-R expressed in HEK 293 cells occurred in a distinctly biphasic manner. In contrast, dissociation of ¹²⁵I-Tyr14-OFQ from OFQ-R expressed in CHO cells occurred in a monophasic manner, with a relatively long t_1/2 value of 3.2 hr. Comparison of the slow components of dissociation from 293/OFQ-R with dissociation of ¹²⁵I-Tyr14-OFQ from CHO/OFQ-R suggested that both ligands recognize the same high affinity state of the receptor in both cell lines but that the low affinity state is not present in the CHO cell line. Such a result may be due to the difference in expression level of OFQ-R between the two cell lines and suggests that in the CHO cell line, the OFQ-R may be very tightly coupled to G proteins.

In accord with the clear presence of two affinity states for agonist binding to the OFQ-R in HEK 293 cells, as revealed by kinetic experiments, two affinity states were likewise observed in saturation binding experiments. The affinities for these sites, calculated by nonlinear curve fitting of the saturation isotherm, agreed well with the kinetically determined values for both the high and low affinity sites for both radioligands, indicating that under the conditions used for saturation binding, both radiolabeled OFQ ligands bind to two affinity states of the OFQ-R according to the law of mass action.

The presence of two affinity states for agonist binding is common in G protein-coupled receptors and is often taken to indicate a functional interaction with signal-transducing G proteins. That this is the case for the OFQ-R is demonstrated by the effect of the nonhydrolyzable guanine nucleotide analog Gpp(NH)p on radiolabeled OFQ binding. In the 293/OFQ-R cells, the addition of Gpp(NH)p resulted in a shift in the proportion of high to low affinity states of the OFQ-R. In the absence of guanine nucleotides, ~50% of the OFQ-R is in the high affinity state, presumably coupled to G proteins. In the presence of Gpp(NH)p, the percentage of high affinity sites is reduced to 15%, whereas the total number of binding sites remains virtually the same. This indicates that the bulk of the receptor has been shifted to the low affinity state, which is considered to be uncoupled from G proteins. In CHO/OFQ-R cells, 100% of the receptor appears to be in the high affinity, G protein-coupled state in the absence of guanine nucleotide, as demonstrated by monophasic kinetic and saturation binding analysis. The addition of Gpp(NH)p to membranes from these cells resulted in virtually complete conversion of receptor sites from high affinity to low affinity. That this effect is mediated by G proteins is indicated by the finding that only guanine, not adenine, nucleotides are able to modulate OFQ radioligand binding.

For G protein-coupled receptors, ligand binding, and agonist ligand binding in particular, is often also modulated by the presence of ions such as Na⁺ and Mg²⁺. In general, Na⁺ seems to reduce the affinity of agonist binding, whereas antagonist binding is less affected (10). In contrast, Mg²⁺ tends to promote high affinity agonist binding (Refs. 10-21 and references therein). In the case of the OFQ-R, and independent of the radioligand used, Mg²⁺ concentrations of 1-5 mM were required to demonstrate high affinity binding. Surprisingly, Mg²⁺ concentrations of < 1 mM did not give rise to a low affinity uncoupled state of the receptor as predicted but instead yielded an unusual binding mode, suggesting that alternative sites of action for Mg²⁺ may need to be considered for the OFQ-R (11).

On the other hand, specific binding of radioligands to the OFQ-R was decreased by increasing concentrations of Na⁺ with an IC₅₀ value of 20 mM. Although the site for Na⁺ modulation of ligand binding has been proposed to be a conserved aspartic acid residue in transmembrane region 2 for a wide variety of G protein-coupled receptors, including the rat OFQ-R, the precise molecular mechanism remains to be elucidated (22).

Other divalent cations were also found to modulate the binding of both OFQ radioligands. The addition of Mn²⁺ and Ca²⁺ to the binding buffer resulted in inhibition of specific binding; this is surprising considering that Mn²⁺ and Ca²⁺ have been reported to improve agonist and antagonist binding to opiate receptors (9). It is also interesting that neither of these cations is able to substitute for the Mg²⁺ required for high affinity binding of the OFQ radioligands. At the present time, we are unable to account for these discrepancies because our knowledge on the mechanism of action of the cations is limited. In contrast, the inhibition of OFQ binding by Zn²⁺ agrees well with a previous finding regarding the effect of this cation on enkephalin binding to opiate receptors (23).

Having shown that ¹²⁵I-Tyr14-OFQ is a high specific activity agonist and a valid ligand for heterologously expressed OFQ-Rs, we used it to quantify OFQ binding sites in different tissues. The highest levels of ¹²⁵I-Tyr14-OFQ binding were found in the hypothalamus and hippocampus, followed by the thalamus and cerebral cortex. Lower levels of binding were observed in the striatum, midbrain, and cerebellum. In all regions where specific binding was observed, ¹²⁵I-Tyr14-OFQ bound to a single class of saturable, high affinity sites, with K_d values that were not significantly different from the those for OFQ-R expressed in CHO cells. Interestingly, saturation binding analysis revealed that in general, ¹²⁵I-Tyr14-OFQ binding correlated very well with the abundance and distribution of mRNA for the OFQ-R previously reported by several groups (1, 2, 24).

We chose to further characterize the properties of ¹²⁵I-Tyr14-OFQ binding in brain by examining the ability of several OFQ peptide analogs to displace its binding from cortical membranes. For all peptides tested, both the affinity and rank order of inhibition of ¹²⁵I-Tyr14-OFQ binding were similar to those seen in membranes from CHO cell membranes. These results indicate that in brain membranes, ¹²⁵I-Tyr14-OFQ labels a receptor site with pharmacological properties identical to those exhibited by the OFQ-R heterologously expressed in CHO cell membranes.

The results presented here demonstrate that the radioligands ¹²⁵I-Tyr14-OFQ and [³H]OFQ are high specific activity agonists that can be used for analyzing the properties and functions of the OFQ-R in transfected cells and native tissue preparations. Both ligands behave in a nearly identical manner; however, each ligand offers distinct advantages. ¹²⁵I-Tyr14-OFQ, with its very high specific activity, will be useful for studies of the OFQ-R in native tissues in which expression levels may be relatively low. [³H]OFQ, on the other hand, has the advantage of being identical to the native peptide and thus is likely to be a preferable ligand for detailed studies of the interactions between the OFQ peptide and its receptor, as well as for pharmacological characterizations of the OFQ-R. Given their respective advantages, the use of these radioligands will promote an enhanced understanding of the biology of this novel neuropeptide system at the molecular level.