Introduction

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Corticotropin-releasing factor (CRF) is the principle mediator of the hypothalamic-pituitary-adrenal axis in the body's response to stress (Vale et al., 1981; Rivier and Vale, 1983). This 41 amino-acid peptide binds to and activates the CRF₁ receptor (Chen et al., 1993), which belongs to family B of the G protein-coupled receptor (GPCR) superfamily. The CRF₁ receptor is activated by peptides related in amino acid sequence to CRF, including urocortin I (UCN I) and the amphibian peptide sauvagine (Dautzenberg and Hauger, 2002). Physiological studies have strongly implicated alteration of the CRF system in anxiety and depression (Holsboer, 1999; Gilligan et al., 2000; Grigoriadis et al., 2001). Based on these studies CRF₁ receptor antagonism has been proposed as a potential treatment for these conditions. Many nonpeptide antagonists of the CRF₁ receptor have been described, such as CP 154,526 (Chen et al., 1997), SC241 (Gilligan et al., 2000), NBI 27914 (Chen et al., 1996), antalarmin (Webster et al., 1996), DMP-696 (He et al., 2000), and R121919 (Grigoriadis et al., 2000). These compounds are CRF₁ receptor-selective, block CRF₁ receptor signaling in vitro, and demonstrate in vivo efficacy for reducing stress-related modulators and behaviors in animal models of neuropsychiatric disorders (Holsboer, 1999; Gilligan et al., 2000; Grigoriadis et al., 2001).

Mechanisms of peptide-ligand interaction with CRF receptors have been extensively investigated (Perrin and Vale, 1999; Grigoriadis et al., 2001). The extreme C terminus of CRF is required for high-affinity binding (Vale et al., 1981), whereas the N-terminal region of CRF is required for receptor activation (Rivier et al., 1984; Nielsen et al., 2000). These findings have been used to develop a high-affinity peptide antagonist, astressin [cyclo(30-33)[D-Phe¹²,Nle^21,38,Glu³⁰,Lys³³]CRF(12-41) (Miranda et al., 1994)]. CRF receptors are predicted to consist of a large extracellular N-terminal domain (N-domain), connected to the juxtamembrane region consisting of the transmembrane domains and intervening loops (J-domain) (Perrin and Vale, 1999; Grigoriadis et al., 2001). The N-domain is a determinant of high-affinity peptide ligand binding (Liaw et al., 1997b; Dautzenberg et al., 1998; Perrin et al., 1998; Wille et al., 1999; Assil et al., 2001; Hofmann et al., 2001; Perrin et al., 2001). Regions and residues in the J-domain are involved in receptor activation by peptide ligands (Sydow et al., 1999; Nielsen et al., 2000; Assil et al., 2001) and contribute to ligand binding affinity (Liaw et al., 1997a,b; Perrin et al., 1998; Sydow et al., 1999). Collectively, these results suggest that the N-terminal portion of the ligand binds the J-domain of the receptor (for activation), and the C-terminal ligand region binds the receptor's N-domain (for high-affinity binding).

In contrast to peptide ligands, little is known regarding the receptor interactions of nonpeptide ligands for the CRF₁ receptor. Receptor mutation has suggested that NBI 27914 binds to a site at least partially distinct from the peptide ligand binding regions (Liaw et al., 1997a). SC241 modulates peptide ligand dissociation and reduces E_max in adenylyl cyclase assays (Zaczek et al., 1997). Thus, some qualitative evidence suggests that nonpeptide ligands may act allosterically to inhibit peptide ligand binding to the CRF₁ receptor. (Allosterism is defined here as the ability of ligand binding to one site to influence the binding of ligand to a second, at least partially distinct site on the receptor.) However, little or no quantitative data exist to support this hypothesis.

In this study, we have comprehensively evaluated the functional mechanism by which nonpeptide ligands antagonize peptide ligand binding to the CRF₁ receptor. We have applied a quantitative model to ligand binding data to test the hypothesis that nonpeptide antagonists inhibit peptide ligand binding to the CRF₁ receptor via an allosteric mechanism. Moreover, the extent to which receptor-G protein interaction affects the nonpeptide antagonist mechanism is unknown. (The pharmacological behavior of GPCR ligands is frequently dependent upon the conformational state of the receptor; Kenakin, 2002). Here, the effect of receptor-G protein interaction has been investigated and shown to profoundly affect the inhibitory activity of the compounds. Finally, antagonist mechanisms have previously only been assessed indirectly using unlabeled compounds. In this study the use of [³H]NBI 35965 enabled direct measurement of the antagonist's receptor binding kinetics and allowed us to validate the proposed allosteric mode of action of the compound.

	Materials and Methods

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Materials. The peptides rat/human CRF, rat UCN I, sauvagine, astressin, and [Tyr⁰]astressin were synthesized by solid phase methodology on a Beckman Coulter 990 peptide synthesizer (Fullerton, CA) using t-Boc-protected amino acids. The assembled peptide was deprotected with hydrogen fluoride. The crude peptide product was purified by preparative HPLC, and the purity of the final product was assessed by analytical HPLC and mass spectrometric analysis using an ion-spray source. The peptides were dissolved in 10 mM acetic acid/0.1% bovine serum albumin (BSA) at a concentration of 1 mM and stored in 10- to 20-µl aliquots at 80°C. Aliquots were used once and any remaining solution discarded. ¹²⁵I-[Tyr⁰]sauvagine and ¹²⁵I-[Tyr⁰]ovine CRF were obtained from PerkinElmer Life Sciences (Boston, MA) (specific activity of 2200 Ci/mmol). ¹²⁵I-[Tyr⁰]astressin was synthesized using the chloramine T method and purified by HPLC (specific activity 2200 Ci/mol). [³H]NBI 35965 was custom synthesized by American Radiolabeled Chemicals (St. Louis, MO) (specific activity 25 Ci/mmol). Low-binding 96-well plates (no. 3605) were from Corning (Palo Alto, CA). G418 (geneticin), Dulbecco's phosphate-buffered saline (DPBS), and cell culture supplies were from Invitrogen (Carlsbad, CA). Fetal bovine serum was from Hyclone Laboratories (Logan, UT).

Cell Culture. Ltk cells stably transfected with the human CRF₁ receptor (Grigoriadis et al., 1994) (termed L-CRF₁) were grown in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 50 IU/ml penicillin, 50 µg/ml streptomycin, and 200 µg/ml G418.

Isolation of Cell Membranes. L-CRF₁ cells were grown in 500-cm² tissue culture plates until confluent. The medium was removed and the cell monolayer washed once with 50 ml of DPBS per plate. Cells were then dislodged by scraping in 50 ml of DPBS per plate. Cells were collected in 250-ml centrifuge tubes and then pelleted by centrifugation at 800g for 10 min at 4°C in a Beckman Coulter GS-6R centrifuge. The cell pellet was then resuspended in assay buffer [DPBS (1.5 mM KH₂PO₄, 8.1 mM Na₂HPO₄, 2.7 mM KCl, and 138 mM NaCl) supplemented with 10 mM MgCl₂, 2 mM ethylene glycol-bis[-aminoethyl]-N,N,N',N'-tetraacetic acid, pH 7.4, with NaOH], using 3 ml of buffer/500-cm² plate of cells. Cell lysis was then performed using a pressure cell, applying N₂ at a pressure of 900 psi for 30 min at 4°C. Unbroken cells and larger debris were removed by centrifugation at 1200g for 10 min at 4°C in a Sorvall RC 5C centrifuge (SM24 rotor). The cell membrane supernatant was then centrifuged at 45,000g (Sorvall RC 5C centrifuge, SM24 rotor) and the resulting membrane pellet homogenized in assay buffer using a Biospec Products (Bartlesville, OK) model 985-370 tissue homogenizer on setting 5 for 30 s on ice. Membrane protein concentration was determined using the Coomassie method (Pierce Chemical, Rockford, IL), using BSA as the standard. Membranes were stored at 80°C before use.

Radioligand Binding Assays. Equilibrium binding of unlabeled ligands was measured in duplicate by inhibition of radioligand binding (¹²⁵I-sauvagine, ¹²⁵I-CRF, ¹²⁵I-astressin, or [³H]NBI 35965) to L-CRF₁ cell membranes. Buffer (30 µl), 20 µl of unlabeled ligand, 50 µl of radioligand, and 100 µl of L-CRF₁ cell membranes were sequentially added to low protein-binding 96-well plates (no. 3605; Corning). In some assays guanosine 5'-O-(3-thiotriphosphate) (GTPS, 30 µM final concentration) was included, added in the 30 µl of buffer, to measure ligand binding to the G protein-uncoupled state of the receptor. In some assays GTPS and NBI 35965 were included, added sequentially in volumes of 10 and 20 µl, respectively. The concentration of radioligand used was approximately 90 pM or 2 nM for ¹²⁵I-sauvagine, 200 pM for ¹²⁵I-sauvagine in the presence of GTPS, 90 pM for ¹²⁵I-CRF, 60 pM for ¹²⁵I-astressin, and 2.5 nM for [³H]NBI 35965. The amount of membrane used per well was 2 to 5 µg for the peptide radioligands and 10 µg for [³H]NBI 35965. Dilution series of unlabeled ligands were prepared in low protein-binding 96-well plates. The assay mixture was incubated for 2 h at 21°C, a time period long enough to allow radioligand binding to closely approach its equilibrium binding asymptote (determined from radioligand association experiments; t_1/2 determined from the observed association rate constant of 21, 5, and 15 min for ¹²⁵I-sauvagine, ¹²⁵I-astressin, and [³H]NBI 35965, respectively). Bound and free radioligand were then separated by rapid filtration, using UniFilter GF/C filters (PerkinElmer Life Sciences) on a UniFilter-96 vacuum manifold (PerkinElmer Life Sciences). GF/C filters were pretreated for 20 to 40 min with 0.1% polyethylenimine in DPBS and then pretreated, immediately before harvesting, by filtration with 0.2 ml/well 1% BSA/0.01% Triton X-100 in DPBS. The filter was washed four times with 0.2 ml/well 0.01% Triton X-100 in DPBS and then dried under electric fans for 40 min to 1 h. After addition of scin-tillation fluid (40 µl/filter disc, Microscint 20; PerkinElmer Life Sciences), scintillation counts were measured in a Topcount NXT. The cpm resulting from emission of beta particles from ³H and Auger electrons from ¹²⁵I were converted to dpm, using the predetermined counting efficiency of 30%. In all assays total radioligand bound to the filter (total binding) was less than 20% of the total amount of radioligand added (6-15% for ¹²⁵I-sauvagine, 2-3% for ¹²⁵I-sauvagine with 30 µM GTPS present, 14-19% for ¹²⁵I-astressin, and 9-15% for [³H]NBI 35965). Nonspecific binding was determined as the measured value in the presence of an excess of the unlabeled analog of the radioligand (320 nM for peptide radioligands and 1 µM for NBI 35965). Nonspecific binding, as a percentage of total radioligand added, was 0.7 to 1.0% for ¹²⁵I-sauvagine, 0.5 to 0.9% for ¹²⁵I-sauvagine with 30 µM GTPS present, 2 to 4% for ¹²⁵I-astressin, and 2 to 4% for [³H]NBI 35965. The total binding: nonspecific binding ratio was 6 to 17 for ¹²⁵I-sauvagine, 3 to 4 for ¹²⁵I-sauvagine with 30 µM GTPS present, 5 to 11 for ¹²⁵I-astressin, and 3 to 6 for [³H]NBI 35965. The amount of radioactivity recovered after the 2-h incubation was measured by withdrawing all the assay solution from the well and counting it. The amount recovered was >95% for ¹²⁵I-sauvagine and ¹²⁵I-astressin, and >85% for [³H]NBI 35965, indicating minimal depletion of the radioligand concentration by nonspecific binding to the plate surface. The amount of radioactivity recovered was not affected by the presence of a high concentration (1 µM) of NBI 35965 or sauvagine. The total amount of radioligand added was measured by using a PerkinElmer Life Sciences Cobra II gamma counter for ¹²⁵I-labeled peptides (78% efficiency) and by using a PerkinElmer Life Sciences 1600TR liquid scintillation counter for [³H]NBI 35965 (55% efficiency).

Data Analysis. Inhibition of radioligand binding was fitted to one-affinity state or two-affinity state competition models, and the best fit determined using a partial F-test, using GraphPad Prism 3.0 (GraphPad Software Inc., San Diego, CA). K_i was calculated using the method of Cheng and Prusoff (1973). Radioligand saturation data were fitted to one- and two-site saturation equations using Prism 3.0, and the best fit determined using a partial F-test. (In all cases, the one-site model provided the best fit to the data (p > 0.05).) Radioligand dissociation data were analyzed using the following monoexponential and biexponential decay functions, and the best fit determined using a partial F-test, using Prism 3.0:


where [RL] is total radioligand bound at time t, [RL_t = 0] is total radioligand bound at 0 min, k₁ the dissociation rate constant, and NSB is nonspecific radioligand bound. In the biexponential equation, P_(fast) is the percentage of [RL_t = 0] that dissociates at the faster of the two rates, k_1(fast) is the dissociation rate constant of the faster dissociating component, and k_1(slow) is the dissociation rate constant of the slower dissociating component. In these analyses [RL_t = 0] and NSB were held constant. [RL_t = 0] was determined from linear regression of the time course of total binding measured as a control in the dissociation phase of the assay, as the extrapolated value at 0 min. NSB was determined from the same analysis of the time course of nonspecific binding.

	Results

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The mechanism of receptor regulation by nonpeptide antagonists was investigated by measuring ligand binding to the CRF₁ receptor. In this study, we evaluated the binding mechanism at the different conformational states of the CRF₁ receptor in Ltk cell membranes. Ligand binding to the CRF₁ receptor is regulated by receptor-G protein interaction, an almost universal characteristic of GPCRs. The uncoupled receptor state (R) binds agonists with lower affinity and can be measured using the antagonist ¹²⁵I-astressin with 30 µM GTPS present. The receptor bound to G protein (RG) occupies a state with high affinity for agonists and can be measured using the agonist radioligand ¹²⁵I-sauvagine. A third, minor state of the CRF₁ receptor was identified (named here as R_O), which is insensitive to GTPS but which binds agonist with high affinity.^{1 125}I-Sauvagine saturation experiments indicated that the R_O state of the receptor was present in the absence of GTPS. Neither the B_max nor K_d of ¹²⁵I-sauvagine for the R_O state was affected 30 µM GTPS, whereas ¹²⁵I-sauvagine binding to RG was rendered undetectable by GTPS.¹ Ligand binding to this third state can be measured using ¹²⁵I-sauvagine with 30 µM GTPS present. We measured the effect of nonpeptide antagonists on peptide radioligand binding to these three states of the CRF₁ receptor and also measured the effect of peptide ligands on [³H]NBI 35965 binding to the R state.

Modulation of Equilibrium Peptide Antagonist Binding to the R State of the CRF₁ Receptor by Nonpeptide Antagonists. We first examined the regulation of the R state of the CRF₁ receptor. Initially, the effect of nonpeptide antagonists on radiolabeled antagonist binding was evaluated, by measuring the effect of nonpeptide antagonists on equilibrium ¹²⁵I-astressin binding to L-CRF₁ membranes in the presence of 30 µM GTPS. ¹²⁵I-Astressin binding was not affected by any concentration of GTPS tested (31.6 pM-100 µM), and the K_i value of unlabeled astressin was not significantly different for the R and RG states.¹ Antalarmin, NBI 27914, NBI 35965, and DMP-696 failed to completely inhibit specific ¹²⁵I-astressin binding to the R state (Fig. 1A). At saturating concentrations (defined as the lower plateau of the inhibition curve), the compounds inhibited 20 to 32% of ¹²⁵I-astressin binding (Fig. 1A; Table 1). All three antagonists displayed high affinity for inhibiting ¹²⁵I-astressin binding (1.1-8.6 nM; Table 1). The partial inhibition of radioligand binding is suggestive of an allosteric mode of inhibition: Binding of ¹²⁵I-astressin to receptor saturated with nonpeptide antagonist is consistent with at least partially distinct binding sites for the two ligands (at the R state). In the Appendix, the ¹²⁵I-astressin inhibition data are analyzed using a quantitative model of allosteric modulation, the allosteric ternary complex model (Stockton et al., 1983; Ehlert, 1988; Lazareno and Birdsall, 1995). The fitted parameter estimates are presented in Table 1.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Modulation of 125I-astressin binding to the R state of the CRF1 receptor by NBI 35965. Binding of 125I-astressin to L-CRF1 cell membranes was measured as described under Materials and Methods, in the presence of 30 µM GTPS. A, inhibition of equilibrium 125I-astressin binding by antalarmin, NBI 27914, NBI 35965, and DMP-696. The curves are the best fits to the allosteric ternary complex model (eq. 1; Appendix). The mean of the fitted values to eq. 1 are in Table 1. Data were normalized as the percentage of specific binding in the absence of nonpeptide antagonist, with nonspecific binding defined as binding in the presence of 320 nM astressin. Data points are the mean ± range of duplicate determinations. Data are from representative experiments that were performed three or four times with similar results. B, modulation of 125I-astressin dissociation by NBI 35965. Dissociation of 125I-astressin from L-CRF1 cell membranes, in the presence of 30 µM GTPS, was measured as described under Materials and Methods, in the absence or presence of a range of concentrations of NBI 35965. The curves are fits to a biexponential decay function, which provided a significantly better fit than a monoexponential function in all cases (p < 0.05). In the absence of NBI 35965, the mean fitted dissociation parameters were P(fast) = 79 ± 12%, k1(fast) = 0.060 ± 0.030 min1, k1(slow) = 0.0066 ± 0.0021 min1. Data points are mean ± range of duplicate measurements. Data are from a representative experiment that was performed three times with similar results. D, effect of NBI 35965 on the half-time (t1/2) of 125I-astressin dissociation. The curve is the best fit to a four-parameter logistic equation. Data are the mean ± S.E.M. of values from three experiments.

View this table: [in this window] [in a new window]   TABLE 1 Binding of nonpeptide antagonists to R, RG, and RO states of the CRF1 receptor in L-CRF1 cell membranes Displacement of radioligand binding to L-CRF1 cell membranes was measured as described under Materials and Methods. Inhibition of 125I-sauvagine binding provides a measure of ligand affinity for the RG state of the CRF1 receptor. Ligand affinity for the R state was measured by displacement of 125I-astressin or [3H]NBI 35965 binding in the presence of 30 µM GTPS. A third state of the receptor was identified in L-CRF1 cell membranes, which bound agonists with high affinity in a GTPS-insensitive manner (RO). Ligand affinity for RO was measured by displacement of 125I-sauvagine binding with 30 µM GTPS present. pKi values were obtained by fitting the displacement data to a single affinity-state competition model, followed by conversion of IC50 to Ki (Cheng and Prusoff, 1973). [In all cases, a two affinity-state model did not significantly improve the goodness of fit (p > 0.05)]. The Kd value used in the Ki calculation was 43 pM and 1.4 nM for 125I-sauvagine at RG and RO, respectively, 0.56 nM for [3H]NBI 35965, and 70 pM for 125I-astressin.

Modulation of Peptide Antagonist Dissociation from the R State of the CRF₁ Receptor by Nonpeptide Antagonist. Modulation of peptide antagonist binding to R was investigated further in ¹²⁵I-astressin dissociation experiments. NBI 35965 accelerated dissociation of ¹²⁵I-astressin from L-CRF₁ membranes (with 30 µM GTPS present) in a concentration-dependent, saturating manner, consistent with allosteric modulation of ¹²⁵I-astressin binding (Fig. 1B). NBI 35965 reduced the t_1/2 for ¹²⁵I-astressin dissociation with a pEC₅₀ value of 7.89 ± 0.33 (EC₅₀ = 13 nM; Fig. 1C), lower than the compound's potency for displacing equilibrium ¹²⁵I-astressin binding to R (pK_i = 8.87, K_i = 1.4 nM; Table 1). The compound produced a maximal reduction of t_1/2 of 1.5 ± 0.1-fold (Fig. 1C). ¹²⁵I-Astressin dissociation was biphasic in the absence and presence of NBI 35965 (Fig. 1, legend). The mechanism underlying biphasic dissociation is unknown. The observation might be due to multiple points of contact between ¹²⁵I-astressin and the receptor.

Modulation of Equilibrium Peptide Agonist Binding to the R State of the CRF₁ Receptor by Nonpeptide Antagonists. The measurable binding of ¹²⁵I-astressin in the presence of nonpeptide antagonists enabled us to examine modulation of unlabeled agonist binding to the R state. (Agonist binding to R was too weak to measure directly using agonist radioligands; Fig. 2.) Modulation of agonist binding was measured by inhibition of ¹²⁵I-astressin binding by peptide agonist with GTPS present, in the absence and presence of a range of concentrations of NBI 35965 (Fig. 2). NBI 35965 produced a rightward shift of the sauvagine, CRF, and UCN I inhibition curve (Fig. 2), indicating inhibition of agonist binding to the R state. However, incremental increases of NBI 35965 did not produce incremental increases of agonist IC₅₀, as predicted by a competitive interaction between the two ligands. Rather, the extent of increase of agonist IC₅₀ seemed to approach a limiting value (Fig. 2). In consequence, the fold-shift of agonist IC₅₀ at 100 nM NBI 35965 (4.0 ± 0.8, 6.5 ± 2.2 and 7.1 ± 0.8 for sauvagine, CRF and UCN I, respectively) was much less than the fold-increase of agonist affinity predicted by competitive inhibition (57-fold, calculated using the Cheng-Prusoff equation; Cheng and Prusoff, 1973), assuming the affinity of NBI 35965 for the R state is 1.8 nM (Table 1). These observations are consistent with an allosteric interaction between NBI 35965 and agonist ligands at the R state: The limited increase of agonist IC₅₀ suggests that binding of NBI 35965 only partially reduces the affinity of agonist binding to the receptor. In the Appendix, the allosteric ternary complex model has been used to quantify the allosteric effect, and the fitted parameters are provided in Table 2.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Effect of NBI 35965 on peptide agonist binding to the R state of the CRF1 receptor. The effect of NBI 35965 on binding of unlabeled peptide agonists to the R state was evaluated by measuring inhibition of 125I-astressin binding by agonist in the presence of 30 µM GTPS, in the absence and presence of NBI 35965. The unlabeled agonist peptides tested were sauvagine (A), CRF (B), and UCN I (C). Data were fitted to a model of allosteric modulation of peptide agonist binding by NBI 35965 (eq. 3). In this model, agonist and 125I-astressin bind to a common site and NBI 35965 binds to a distinct site from which it allosterically regulates peptide agonist or 125I-astressin binding. The curves are the best-fit curves to the data. Data were normalized as the percentage of specific binding in the absence of peptide agonist, with nonspecific binding defined as binding in the presence of 320 nM astressin. Note that NBI 35965 reduced the percentage specific binding in the absence of agonist, similar to the inhibition of 125I-astressin by NBI 35965 in Fig. 1A. NBI 35965 did not detectably affect nonspecific binding (data not shown). Data points are the mean ± range of duplicate determinations. Data are from a representative experiment performed three times with similar results.

View this table: [in this window] [in a new window]   TABLE 2 Estimates of the NBI 35965 affinity and cooperativity for allosteric modulation of peptide ligand binding to the R state of the CRF1 receptor The fitted parameters are those for the R state of the CRF1 receptor in L-CRF1 cell membranes. (30 µM GTPS was included in the assays.) To determine the cooperativity () between the binding of NBI 35965 and astressin, data for inhibition of 125I-astressin binding (Fig. 1A) were fitted to the allosteric ternary complex model (eq. 1, see Appendix). For the peptide agonists (sauvagine, CRF, and UCN I) KN and  were estimated by fitting the data of Fig. 2 to the allosteric ternary complex model (eq. 3). Data are the mean ± S.E.M (n = 3). In all analyses the affinity constant of 125I-astressin was fixed at the value measured in saturation experiments (1.43 × 1010 M1).

Measurement of [³H]NBI 35965 Binding to the CRF₁ Receptor. In the experiments mentioned above, binding of nonpeptide antagonists has been measured indirectly, by measuring effects of the unlabeled compound on peptide radioligand binding. Although these experiments provide an estimate of the compounds' affinity for the CRF₁ receptor, they do not provide estimates of other important parameters of binding, such as B_max and association and dissociation rate constants. In addition, radiolabeled nonpeptide antagonist binding would enable measurement of the effects of peptide ligands on nonpeptide binding, to further investigate the mechanism of action of the compounds. We therefore directly measured binding of [³H]NBI 35965 to the CRF₁ receptor.

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View larger version (21K): [in this window] [in a new window]   Fig. 3.   Binding of [3H]NBI 35965 to the CRF1 receptor. A, [3H]NBI 35965 saturation of L-CRF1 cell membranes. The dependence of specific radioligand binding on the free radioligand concentration was fitted to a single affinity-state binding isotherm. [A two affinity-state model did not significantly improve the fit (p > 0.05).] Data are from a representative experiment, performed five times with similar results. The pKd value for [3H]NBI 35965 from the experiment shown was 9.39 (Kd of 0.41 nM). B, association time course of [3H]NBI 35965 binding to L-CRF1 cell membranes. The total concentration of [3H]NBI 35965 added was 2.4 nM. The observed association rate constant was 0.0681 min1. The calculated association rate constant from this experiment was 2.5 × 107 M1 min1 (assuming a dissociation rate constant of 0.0087 min1; see text). Nonspecific binding was measured by including 1 µM NBI 35965, total binding measured in the absence of NBI 35965, and specific binding calculated by subtracting the former from the latter. Data are from a representative experiment performed four times with similar results. C, dissociation time course of [3H]NBI 35965 binding to L-CRF1 cell membranes. "Dissociation" binding was measured by adding NBI 35965 (1 µM final concentration) after equilibration of [3H]NBI 35965 and the CRF1 receptor. "Total" binding was measured by adding the same volume of buffer after equilibration. "Nonspecific" binding measured by adding NBI 35965 (1 µM) before equilibration. Data are from a representative experiment performed five times with similar results. The curves are fits to monoexponential association and dissociation equations. The straight lines are linear regression fits. Data points are the mean ± range of duplicate measurements.

View larger version (29K): [in this window] [in a new window]   Fig. 4.   Inhibition of [3H]NBI 35965 binding to the R state of the CRF1 receptor by unlabeled ligands. Inhibition of [3H]NBI 35965 binding to L-CRF1 cell membranes was measured in the presence of 30 µM GTPS, by nonpeptide antagonists (A) and by peptide ligands (B). In addition, the effect of a range of concentrations of GTPS on [3H]NBI 35965 binding is also shown (A). The curves for nonpeptide antagonists are fits to a single-affinity state inhibition model. [A two-site model did not significantly improve the goodness of fit (p > 0.05).] The curves for CRF and UCN I are the best fit to the allosteric ternary complex model (eq. 2; see Appendix). Data were normalized as the percentage of specific binding in the absence of unlabeled ligand, with nonspecific binding defined as binding in the presence of 1 µM NBI 35965. Data points are the mean ± range of duplicate determinations. Data are from representative experiments, performed three to seven times with similar results.

Inhibition of [³H]NBI 35965 Binding to the R State of the CRF₁ Receptor by Nonpeptide Antagonists. The ability to directly label the nonpeptide antagonist binding site using [³H]NBI 35965 enabled us to test the hypothesis that antalarmin, NBI 27914, NBI 35965, and DMP-696 bind a common site on the CRF₁ receptor. All the ligands fully inhibited [³H]NBI 35965 binding to the R state (Fig. 4A; Table 1; measured in the presence of 30 µM GTPS), consistent with either competitive or strong allosteric inhibition. NBI 27914 and DMP-696 at 1 µM concentration did not affect the dissociation rate of [³H]NBI 35965 (Fig. 5D), arguing against allosteric inhibition. These findings suggest that the nonpeptide antagonists bind the same site on the CRF₁ receptor.

View larger version (37K): [in this window] [in a new window]   Fig. 5.   Modulation of [3H]NBI 35965 dissociation from the R state of the CRF1 receptor by peptide ligands. [3H]NBI 35965 dissociation from L-CRF1 cell membranes was measured in the presence of 30 µM GTPS, in the presence of sauvagine (A), CRF (B), and UCN I (C). The curves are the best fit to a monoexponential dissociation equation. (In all cases, the biexponential equation did not improve the goodness of fit.) Data points are the mean ± range of duplicate determinations. Data are from representative experiments performed twice with similar results. The peptide diluent buffer (10 mM acetic acid/0.1% BSA, 25 µl in assay volume of 200 µl) did not affect the dissociation rate constant (0.0069 ± 0.0017 min1 versus the control value of 0.0086 ± 0.0020 min1). D, effect of peptide and nonpeptide ligands on the dissociation rate constant of NBI 35965 (k1). The curves are the best fit to a four-parameter logistic equation. Data are the mean ± range of values from two experiments.

Modulation of Equilibrium [³H]NBI 35965 Binding to the R State of the CRF₁ Receptor by Peptide Ligands. The findings mentioned above suggest NBI 35965 allosterically regulates peptide agonist and antagonist binding to the R state of the CRF₁ receptor. We examined the reciprocal effect of peptide ligands on NBI 35965 binding to the R state using [³H]NBI 35965.

View this table: [in this window] [in a new window]   TABLE 3 Estimates of peptide ligand affinity, cooperativity, and maximal inhibition of radioligand binding, for allosteric modulation of [3H]NBI 35965 binding to the R state of the CRF1 receptor The binding parameters are for the R state of the CRF1 receptor in L-CRF1 cell membranes (Fig. 4B, 30 µM GTPgS was included in the assays). The maximal extent of [3H]NBI 35965 inhibition produced by peptide ligand was calculated as the percentage of specific binding inhibited by 10 µM sauvagine, 10 µM CRF, 3.2 µM UCN I, and 3.2 µM astressin. Data for inhibition of [3H]NBI 35965 binding by CRF or UCN I (Fig. 4B) were fitted to the allosteric ternary complex model (eq. 2; Appendix) to estimate the affinity of peptide ligand for the free receptor (not occupied by [3H]NBI 35965, KL) and to determine the cooperativity () between the binding of peptide ligand and [3H]NBI 35965. (The data for sauvagine and astressin could not be reliably fitted using eq. 2, because of the weak maximal inhibition by the ligands.) In this analysis the affinity constant for [3H]NBI 35965 was fixed at the value measured in saturation experiments (1.80 × 109 M1).

Modulation of [³H]NBI 35965 Dissociation from the R State of the CRF₁ Receptor by Peptide Ligands. Allosteric regulation of [³H]NBI 35965 binding to the R state was further tested by measuring dissociation of the radioligand in the presence of GTPS. The agonists sauvagine, CRF, and UCN I accelerated dissociation of [³H]NBI 35965 in a concentration-dependent and saturating manner (Fig. 5, A-C), consistent with allosteric modulation of [³H]NBI 35965 binding. The effect was quantified by measuring the concentration dependence of the ligands for increasing the dissociation rate constant (k₁) of [³H]NBI 35965 (Fig. 5D). (Dissociation of [³H]NBI 35965 was monophasic in the absence and presence of peptide ligands.) The pEC₅₀ value for sauvagine, CRF, and UCN I was 6.24 ± 0.04, 6.38 ± 0.01, and 7.33 ± 0.02 respectively, with a corresponding maximal increase of the dissociation rate of 3.6 ± 0.5-, 5.3 ± 0.1-, and 7.0 ± 0.1-fold (n = 2). A saturating concentration of astressin (3.2 µM) did not significantly affect the dissociation rate of [³H]NBI 35965 (Fig. 5D). This finding is in contrast to the modulation of ¹²⁵I-astressin dissociation by NBI 35965 (Fig. 1). The reason for this difference is not presently clear.

Modulation of Equilibrium Peptide Agonist Binding to the RG State of the CRF₁ Receptor by Nonpeptide Antagonists. Modulation of agonist binding to RG was first evaluated in equilibrium binding assays, by measuring inhibition of ¹²⁵I-sauvagine binding to L-CRF₁ cell membranes in the absence of GTPS. In these assays it was not possible to detect the RG state as a homogeneous population of binding sites, owing to the detection of the R_O state by ¹²⁵I-sauvagine.¹ However, we were able to maximize the occupancy of RG relative to R_O by using a low concentration of the radioligand (90 pM), because ¹²⁵I-sauvagine binds with higher affinity to RG (43 pM) than to R_O (1.4 nM). Under these conditions the RG state represented 93% of the receptor-specific ¹²⁵I-sauvagine binding (calculated from the dissociation constants above and B_max values of 1.4 and 1.2 pmol/mg for RG and R_O states, using a two independent affinity-state model.¹

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Inhibition of 125I-sauvagine binding to the RG state of the CRF1 receptor by nonpeptide antagonists. A, inhibition of binding of low 125I-sauvagine concentrations (79-94 pM) by antalarmin, NBI 27914, and DMP-696, measured as described under Materials and Methods. The curves are fits to a single-affinity state inhibition model. [A two-site model did not significantly improve the goodness of fit (p > 0.05).] B, inhibition of a low (92 pM) and high (1.3 nM) concentration of 125I-sauvagine binding by NBI 35965. The curves are fits to a single-affinity state inhibition model. [A two-site model did not significantly improve the goodness of fit (p > 0.05).] Data for the high 125I-sauvagine concentration were also fitted to the allosteric ternary complex model (eq. 1), for which the fitted curve is superimposable on the fit to the single-affinity state inhibition model. The mean of the fitted value to eq. 1 were as follows:  = 0.0056 ± 0.0012 and pKN = 9.15 ± 0.06. These fitted mean parameters for the high concentration of 125I-sauvagine were used to simulate a 125I-sauvagine versus NBI 35965 inhibition curve for the low concentration of 125I-sauvagine (dashed line). Data were normalized as the percentage of specific binding in the absence of antagonist, with nonspecific binding defined as binding in the presence of 320 nM sauvagine. Data points are the mean ± range of duplicate determinations. Data are from representative experiments that were performed three or four times with similar results.

Modulation of Peptide Agonist Dissociation from the RG State of the CRF₁ Receptor by Nonpeptide Antagonist. Deviation from competitive inhibition of peptide binding to RG by NBI 35965 was tested further in radiolabeled agonist dissociation experiments. NBI 35965 slowed dissociation of ¹²⁵I-sauvagine and ¹²⁵I-CRF from L-CRF₁ cell membranes in a concentration-dependent and saturating manner (Fig. 7, A and B). The slowing of radiolabeled agonist dissociation by NBI 35965 was in marked contrast to the effect of GTPS, which accelerated dissociation of ¹²⁵I-sauvagine and ¹²⁵I-CRF (Fig. 7, A and B).

View larger version (32K): [in this window] [in a new window]   Fig. 7.   Modulation of radiolabeled agonist dissociation from the RG state of the CRF1 receptor by NBI 35965. Dissociation of 125I-sauvagine (A) and 125I-CRF (B) from L-CRF1 cell membranes was measured as described under Materials and Methods, in the absence of antagonist or in the presence of a range of concentrations of NBI 35965. The curves are fits to a biexponential decay function, which provided a significantly better fit than a monoexponential function in all cases (p < 0.05). In the absence of NBI 35965, the mean fitted parameters for 125I-sauvagine dissociation were P(fast) = 63 ± 9%, k1(fast) = 0.20 ± 0.09 min1, and k1(slow) = 0.0098 ± 0.0041 min1 and for 125I-CRF were P(fast) = 38 ± 3%, k1(fast) = 0.24 ± 0.07 min1, and k1(slow) = 0.0089 ± 0.00051 min1. Data points are mean ± range of duplicate measurements. Data are from representative experiments that were performed three times with similar results. C, effect of NBI 35965 on the half-time (t1/2) of 125I-sauvagine and 125I-CRF dissociation. The curves are the best fit to a four-parameter logistic equation. Data are the mean ± S.E.M. of values from three experiments.

Modulation of Equilibrium Peptide Agonist Binding to the R_O State of the CRF₁ Receptor by Nonpeptide Antagonists. A minor fraction of the CRF₁ receptor population in L-CRF₁ cell membranes (16%) exists in a conformation that binds agonists with high affinity, but which is insensitive to GTPS (termed R_O).¹ The pharmacological profile of nonpeptide antagonist activity at this state was measured by inhibition of ¹²⁵I-sauvagine binding to L-CRF₁ cell membranes in the presence of 30 µM GTPS. In this assay, antalarmin NBI 27914, NBI 35965, and DMP-696 fully inhibited binding of a low concentration of ¹²⁵I-sauvagine (150-240 pM), displaying high affinity for this effect (Fig. 8; Table 1).

View larger version (24K): [in this window] [in a new window]   Fig. 8.   Inhibition of 125I-sauvagine binding to the RO state of the CRF1 receptor by nonpeptide antagonists. Inhibition of 125I-sauvagine binding to L-CRF1 cell membranes was measured in the presence of 30 µM GTPS, as described under Materials and Methods. Under these conditions the radioligand binds an agonist high-affinity state of the CRF1 receptor, which is insensitive to GTPS (RO). The curves are fits to a single-affinity state inhibition model. [A two-site model did not significantly improve the goodness of fit (p > 0.05).] Data were normalized as the percentage of specific binding in the absence of antagonist, with nonspecific binding defined as binding in the presence of 320 nM sauvagine. Data points are the mean ± range of duplicate determinations. Data are from representative experiments that were performed three times with similar results.

Comparison of Nonpeptide Antagonist Affinity for R, RG, and R_O States of the CRF₁ Receptor. The nonpeptide antagonist affinity for these three states of the CRF₁ receptor was compared using the K_i value for inhibition of [³H]NBI 35965 binding in the presence of GTPS, ¹²⁵I-sauvagine binding, and ¹²⁵I-sauvagine in the presence of GTPS, respectively. None of the antagonists appreciably discriminated between these states: the largest difference of affinity was only 3.3-fold (between RG and R_O for NBI 35965; Table 1). The nonpeptide antagonist affinity for R, RG, and R_O was not significantly different for antalarmin, NBI 27914, and DMP 696 (p = 0.10, 0.09, and 0.12, respectively; single-factor ANOVA). The affinity values were significantly different for NBI 35965 (p = 0.0057; single-factor ANOVA): the affinity for R_O (4.6 nM) was significantly different from the affinity for R (1.8 nM; p < 0.01) and RG (1.4 nM; p < 0.01, post hoc analysis using the Newman-Keuls test).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Appendix References

Numerous nonpeptide antagonists have been developed for the CRF1 receptor, as potential therapies for CRF-associated disorders such as anxiety and depression (Holsboer, 1999; Gilligan et al., 2000; Grigoriadis et al., 2001). However, little is known regarding their functional mechanism of action at the receptor level. The aim of this study was to quantitatively evaluate the mechanism of action of four nonpeptide antagonists: antalarmin, NBI 27914, NBI 35965, and DMP-696. In addition, we compared the effects of these molecules at the G protein-coupled (RG) and uncoupled (R) states of the CRF₁ receptor in Ltk cell membranes. The principle findings are as follows: 1) At the R state, nonpeptide antagonists only partially inhibited peptide ligand binding and accelerated ¹²⁵I-astressin dissociation. 2) Reciprocally, peptide agonists only partially inhibited [³H]NBI 35965 binding to the R state and accelerated [³H]NBI 35965 dissociation. 3) Antalarmin, NBI 27914, NBI 35965, and DMP-696 likely bind a common site on the receptor and modulate peptide ligand binding in a quantitatively similar manner. 4) Nonpeptide antagonists bind with similar affinity to the R and RG state. 5) At the RG state nonpeptide antagonists strongly inhibited peptide agonist binding (in marked contrast to their behavior at the R state), explaining their antagonist effect. 6) At the RG state deviations from simple competitive inhibition were detected. As described below, findings 1 and 2 for the R state support an allosteric mechanism by which nonpeptide antagonist and peptide ligand inhibit each other's binding. Findings 5 and 6 for the RG state are consistent with either strong allosteric inhibition or competitive inhibition at one of the peptide agonist binding sites.

At the R state of the CRF₁ receptor, four observations were consistent with an allosteric mechanism for nonpeptide antagonism, in which peptide and nonpeptide ligands bind to at least partially distinct sites (Appendix; Stockton et al., 1983; Ehlert, 1988; Lazareno and Birdsall, 1995): 1) Saturating concentrations of nonpeptide antagonists only partially inhibited equilibrium ¹²⁵I-astressin binding and only partially reduced peptide agonist binding affinity. This suggests that peptide ligands can bind the receptor saturated with nonpeptide antagonist, consistent with at least partial spatial independence of their binding sites. 2) Reciprocally, saturating concentrations of peptide agonists only partially inhibited equilibrium [³H]NBI 35965 binding, suggesting that nonpeptide antagonist can bind the receptor saturated with peptide ligand. 3) Nonpeptide antagonist (NBI 35965) accelerated dissociation of ¹²⁵I-astressin, consistent with nonpeptide antagonist binding the receptor-¹²⁵I-astressin complex. (We were unable to measure the effect of NBI 35965 on peptide agonist dissociation from R, because binding of peptide agonist radioligands to R could not be detected.) 4) Peptide agonists accelerated [³H]NBI 35965 dissociation, suggesting peptide agonist binding to the receptor-[³H]NBI 35965 complex.

Other potential models were considered to explain these four findings for the R state. In the first model, nonpeptide antagonist binds to only a subpopulation of the receptor population bound by ¹²⁵I-astressin. This model could explain partial inhibition of ¹²⁵I-astressin binding by nonpeptide antagonists. However, a number of findings argue against this model. First, the model can only explain partial ¹²⁵I-astressin inhibition if the receptor subpopulation that can bind nonpeptide antagonist is independent of the subpopulation that cannot (i.e., the populations do not interconvert). Under these conditions, NBI 35965 could not affect dissociation of ¹²⁵I-astressin. Furthermore, the B_max value of [³H]NBI 35965 (6.0 pmol/mg) was similar to that for ¹²⁵I-astressin (7.7 pmol/mg), arguing against NBI 35965 selectively binding to a minor fraction of the receptor population. Finally, nonpeptide antagonists bound with similar affinity to the known different states of the receptor in L-CRF₁ cell membranes (R, RG, and R_O; Table 1). In the second potential model, two binding regions of the peptide ligand bind to two corresponding, spatially independent sites on the receptor (site 1 and site 2). This model is consistent with the known peptide binding mechanism (Perrin and Vale, 1999; Grigoriadis et al., 2001). In this model nonpeptide antagonist competitively inhibits peptide binding to the site 1, without affecting peptide binding to site 2. Examination of this model using simulated data indicates that it allows for partial inhibition of peptide binding by nonpeptide antagonist, partial inhibition of [³H]NBI 35965 binding by peptide ligand (provided that the peptide affinity for the site 1 is weak compared with site 2), and modulation of peptide ligand dissociation.¹ However, the model does not allow modulation of [³H]NBI 35965 dissociation by peptide ligand. Therefore, of the models considered, only allosteric modulation fully accounts for the data obtained for the R state of the CRF₁ receptor.

For other GPCRs, allosteric modulation is consistent with a theoretical model, the allosteric ternary complex model (Stockton et al., 1983; Ehlert, 1988; Lazareno and Birdsall, 1995; Trankle et al., 1999; Leppik and Birdsall, 2000). In this model, the behavior of the allosteric ligand (e.g., NBI 35965) is defined by its affinity for the receptor and by the cooperativity between binding of allosteric and orthosteric ligand (e.g., CRF). Data for the R state of the CRF₁ receptor were fitted to the allosteric ternary complex model to quantify the allosteric effect. The analysis indicated negative cooperativity between NBI 35965 and peptide agonist binding. The negative cooperativity was weak; the greatest effect of NBI 35965 was on UCN I binding ( = 0.11, indicating that NBI 35965 binding reduces the affinity of UCN I by only 9-fold). Equilibrium binding and radioligand dissociation data are in good agreement with the model (Appendix), indicating that allosteric modulation is sufficient to account for the data for the R state. In particular, the data are fully consistent with the reciprocity of the allosteric effect, that the cooperativity of NBI 35965 on peptide agonist binding is equal to the cooperativity of peptide agonist on [³H]NBI 35965 binding (Tables 2 and 3; Appendix). This reciprocal relationship has been demonstrated for gallamine and N-methylscopolamine at the M₂ muscarinic acetylcholine receptor (Trankle et al., 1999).

At the RG state, the effect of the nonpeptide antagonists on peptide agonist binding differed markedly from the R state. Nonpeptide antagonists antalarmin, NBI 27914, NBI 35965, and DMP-696 strongly inhibited agonist binding to RG, in contrast to their weak inhibition of binding to R. This finding demonstrates, for the first time, that the inhibitory action of a family B GPCR antagonist is dependent upon the conformational state of the receptor. The strong inhibition of peptide agonist binding to RG explains the antagonist properties of the compounds, because this state of the receptor is coupled, via subsequent G protein activation, to intracellular signaling pathways. At the RG state, deviations from competitive behavior were observed: NBI 35965 slowed radiolabeled agonist dissociation and incompletely inhibited ¹²⁵I-sauvagine binding at high radioligand concentrations. These observations can be explained by strong allosteric inhibition by the nonpeptide antagonist (Appendix) or by a model that assumes competitive inhibition at one of two peptide agonist-binding sites (see above). We could not distinguish these two models because it was not possible to unambiguously define [³H]NBI 35965 binding to the RG state, to determine whether peptide ligands affect [³H]NBI 35965 dissociation from RG (a necessary experiment to discriminate the models for the R state; see above).

In this study, we have evaluated the functional mechanism of nonpeptide antagonism of the CRF₁ receptor. The molecular mechanism underlying the effects requires further investigation. In our view, the data in this study are consistent with three plausible molecular mechanisms (Fig. 9). These mechanisms assume that peptide binds to the N- and J-domains (Perrin and Vale, 1999; Grigoriadis et al., 2001), that nonpeptide antagonist binds only the J-domain (Liaw et al., 1997a; Nielsen et al., 2000), and that an allosteric interaction is at least partially involved in the inhibition of peptide binding by nonpeptide antagonist (see above). In mechanism 1, nonpeptide antagonist binds to a site distinct from the peptide-binding site in the J-domain, and allosterically inhibits peptide binding to the J-domain (Fig. 9A). In mechanism 2, nonpeptide antagonist binding to the J-domain allosterically inhibits peptide binding to the N-domain (Fig. 9B). In mechanism 3, an extension of mechanism 2, nonpeptide antagonist binds to the same site in the J-domain as the peptide, competitively inhibiting peptide binding to the J-domain, whereas allosterically inhibiting peptide binding to the N-domain (Fig. 9C). Molecular biological approaches will be required to distinguish these models. The currently limited data are consistent with mechanism 1: mutation of His 199 (in transmembrane 3) to Val and Met276 (in transmembrane 5) to Ile increased the K_i value of NBI 27914 for the CRF₁ receptor (40- and 200-fold, respectively), without affecting the binding affinity of CRF (Liaw et al., 1997a).

View larger version (18K): [in this window] [in a new window]   Fig. 9.   Potential molecular models of nonpeptide antagonist action at the CRF1 receptor. These models of nonpeptide ligand and peptide interaction with the receptor are consistent with the data in the present study. Peptide ligand is assumed to consist of two binding regions, which interact with two corresponding binding domains of the receptor (the N- and the J-domain). Nonpeptide antagonist (NPA) is assumed to bind only the J-domain. The dashed line indicates an allosteric interaction between spatially distinct binding sites. A, nonpeptide antagonist allosterically inhibits peptide binding to the J-domain. B, nonpeptide antagonist allosterically inhibits peptide binding to the N-domain. C, nonpeptide antagonist competitively inhibits peptide binding to the J-domain and allosterically inhibits peptide binding to the N-domain. An allosteric component was included in each mechanism because allosteric inhibition was demonstrated for the R state. Note that the figure is a schematic representation and so may not accurately reflect the precise location of the receptor's binding sites.

In summary, for the first time we have quantitatively evaluated the inhibitory mechanism of nonpeptide antagonists for the CRF₁ receptor. The allosteric ternary complex model was necessary and sufficient to account for the data for the R state. The compounds are weak allosteric inhibitors of peptide binding to the R state. In contrast, at the RG state nonpeptide antagonists strongly inhibited peptide agonist binding, demonstrating a previously unknown effect of R-G coupling on nonpeptide antagonist activity. The strong inhibitory activity at RG could be explained by either strong allosteric inhibition or competitive inhibition at one of the two peptide-binding sites. Strong inhibition of peptide binding to RG explains the antagonist activity of the compounds. These findings will be relevant to the further study and discovery of nonpeptide antagonists for the CRF₁ receptor, and potentially for other family B GPCRs.