Abstract
The ligand selectivity of human (hCRF2A) and Xenopus laevis (xCRF2) forms of the corticotropin-releasing factor type 2 (CRF2) receptor differs. The purpose of this study was to identify amino acids in these two CRF2receptors conferring these differences. An amino acid triplet in the third extracellular domain (Asp262Leu263Val264 in hCRF2A or Lys264Tyr265Ile266 in xCRF2) was found to diverge between both receptors. When binding and signaling characteristics of receptor mutants hR2KYI and xR2DLV were assessed, the tri-amino acid motif replacement produced receptors with binding properties resembling the xCRF2receptor. The converse mutation created a mutant receptor with a binding pharmacology identical to the profile of the hCRF2Areceptor. This effect was most notable for xR2DLV, which possessed a binding affinity for astressin ∼15-fold greater for astressin than sauvagine. In contrast, the binding profiles of the hCRF2Areceptor and hR2KYI did not differ. These data indicate that another domain of the xCRF2 receptor mediated low-affinity binding of astressin. Two amino acids in the first extracellular domain differ in xCRF2 (Asp69Ser70) and hCRF2A (Glu66Tyr67) receptors. The hCRF2A receptor mutant (hR2DS-KYI) bound astressin with a low affinity indistinguishable from the xCRF2 receptor. Therefore, these data demonstrate that ligand selectivity differences between amphibian and human forms of the CRF2A receptor are governed by these two motifs of the extracellular domains of the xCRF2 receptor.
Corticotropin-releasing factor (CRF), a 41-amino acid peptide originally isolated from hypothalamus (Vale et al., 1981), is the main integrator of the stress response (Dunn and Berridge, 1990; Arborelius et al., 1999; Hauger and Dautzenberg, 1999). Central and peripheral effects of CRF and its structurally related analogs urocortin (UCN) (Vaughan et al., 1995;Donaldson et al., 1996), fish urotensin I (Lederis et al., 1982), and frog sauvagine (Montecucchi and Henschen, 1981) are mediated by their binding and activation of two CRF receptors (CRF1and CRF2), which belong to the class B subfamily of G protein-coupled receptors (Vale et al., 1997). CRF1 and CRF2 receptors are ∼70% homologous and couple to stimulatory GTP-binding proteins (reviewed in Dautzenberg et al., 2001a). Three biologically active splice variants (CRF2A-C) have been identified for the CRF2 receptor (see Kilpatrick et al., 1999).
Despite a high degree of sequence homology, the specificity of CRF agonist binding to CRF1 and CRF2 proteins differs to a considerable extent. The mammalian CRF1 receptor nonselectively recognizes CRF, UCN, urotensin I, and sauvagine. These four CRF peptides bind to the CRF1 receptor with similar degrees of high affinity and equipotently stimulate intracellular cAMP accumulation (Vaughan et al., 1995; Donaldson et al., 1996; Dautzenberg et al., 1997, 1999; Palchaudhuri et al., 1998). In contrast, theXenopus laevis CRF1 receptor (xCRF1) selectively binds CRF agonists in a highly selective manner whereby human CRF (hCRF), X. laevisCRF (xCRF), urotensin I, and rat UCN are recognized with a significantly higher affinity than the structurally related analogs ovine CRF (oCRF) and sauvagine (Dautzenberg et al., 1997).
The mammalian and X. laevis CRF2receptors display substrate specificities that differ from the mammalian CRF1 and the xCRF1 receptor (Donaldson et al., 1996;Dautzenberg et al., 1997, 1999; Ardati et al., 1999; Palchaudhuri et al., 1999). The CRF peptides hCRF, oCRF, and xCRF bind with significantly lower affinity than UCN, urotensin I, and sauvagine.
The identification of regions forming the binding pocket or being critical for ligand selectivity of the mammalian CRF1, xCRF1, and human CRF2A receptors has been the subject of various studies (Liaw et al., 1997a,b; Dautzenberg et al., 1998, 1999; Perrin et al., 1998; Wille et al., 1999; Assil et al., 2001). From those studies, it became evident that both receptors use amino acids that are within the extracellular (EC) domains of the receptor or at the interface between the EC domains and the transmembrane helices. The ligand-selective regions of the CRF1 and CRF2 receptor are located, however, in different regions of these two proteins. The major determinant for high-affinity ligand binding of the CRF1 receptor resides in its N-terminal EC1 domain (Perrin et al., 1998; Wille et al., 1999;Assil et al., 2001), whereas the ligand-selective domains of human CRF2A (hCRF2A) have been identified in EC2 and at the junction of EC3 and transmembrane 5 (Liaw et al., 1997a,b). Replacement of the amino acids of hCRF1 with residues at equivalent positions of hCRF2A created a mutated receptor that no longer bound hCRF with high affinity (Liaw et al., 1997a). In agreement with these findings, the agonist-selective domains of the xCRF2 receptor have been mapped to regions other than EC1, indicating that this receptor uses domains similar to its human CRF2A counterpart (Dautzenberg et al., 1999). Recently, we reported the first evidence that the binding modes of mammalian CRF2 receptors and the xCRF2 receptor differed; furthermore, depending on the radioligand used, rank order binding profiles differed (Dautzenberg et al., 2001b). When competition binding experiments were performed with astressin, a nonselective antagonist (Gulyas et al., 1995), this ligand bound to the xCRF2 receptor with an affinity more than 10-fold higher compared with all other CRF radioligands (Dautzenberg et al., 2001b). Because the ligand-selective domains mapped to the hCRF2A receptor are not well conserved in the xCRF2 receptor (Fig.1), we speculated that one or more of these regions might be responsible for the differential binding profile of the xCRF2 receptor.
In this study, we generated mutated hCRF2 and xCRF2 receptors and tested their binding profile using the radioligands 125I-sauvagine and125I-astressin. In addition, we compared the ability of astressin and anti-sauvagine-30 (aSVG) to inhibit agonist-mediated cAMP accumulation in HEK293 cells stably transfected with the human and amphibian wild-type or mutant receptors.
Experimental Procedures
Materials, Peptides, and Reagents.
All cell culture media and reagents were purchased from Invitrogen (Basel, Switzerland). The CRF peptides (purity, >95%) were obtained from Bachem Corp. (Bubendorf, Switzerland), whereas aSVG (purity, >95%) was synthesized in-house according to a previously published method (Rühmann et al., 1996; Brauns et al., 2001).
Radiochemicals.
125I-Astressin (2200 Ci/mmol) was purchased from PerkinElmer Life Sciences (Boston, MA), whereas 125I-Tyr0-sauvagine (125I-sauvagine; 2000 Ci/mmol) was purchased from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK).
Construction of Mutated Receptors.
The residues Glu66Tyr67 and Asp262Leu263Val264of the hCRF2A receptor were mutated into the corresponding amino acids of the xCRF2 receptors (Asp69Ser70 and Lys262Tyr263Ile264) to create the receptor mutants hR2DS, hR2KYI, and hR2DS-KYI. The residues Asp69Ser70 and Lys264Tyr265Ile266of the xCRF2 receptor were mutated to the amino acids occupying these positions in the hCRF2Areceptor to generate the xR2EY, xR2DLV, and xR2EY-DLV mutants. Mutagenesis was accomplished using the QuickChange kit (Stratagene, La Jolla, CA) as reported previously (Dautzenberg et al., 1998; Wille et al., 1999). The wild-type and mutant receptors were cloned into the pcDNA3 vector (Invitrogen). Sequences obtained by polymerase chain reaction were verified by DNA sequencing using an ABI 310 DNA sequencer (Applied Biosystems, Weiterstadt, Germany); the GCG software package (Accelrys, Cambridge, UK) was used for analysis.
Cell Transfections and Radioreceptor Binding Assays.
cDNAs of hCRF2A, xCRF2, and mutated receptors, all inserted into the pcDNA3 vector (2 μg each), were stably transfected into HEK293 cells with the Transfectam reagent (BioSepra, Inc., Villeneuve La Garenne, France) as reported previously (Dautzenberg et al., 2000). Two days after transfection, geneticin selection (500 μg/ml) was initiated, and clones expressing low to moderate receptor levels (500–1200 fmol/mg) were selected.
Membranes from stably transfected HEK293 cells were prepared as described previously (Dautzenberg et al., 1997; Hauger et al., 1997). Scatchard analyses using 0.1 nM 125I-astressin or125I-sauvagine were performed with 1 to 30 μg of membrane proteins in the scintillation proximity assay format as described previously (Dautzenberg et al., 2000, 2001b). Under these conditions, less than 10% of the total radioactivity was specifically bound by the various receptor constructs. The dissociation constantK d and the inhibition constantK i were calculated by the Xlfit program (IDBS, Guildford, UK). Scatchard analyses revealed a one-site model for these four CRF receptors.
cAMP Assays.
HEK 293 cells, stably expressing hCRF2A, xCRF2, or mutated receptors, were plated at 10,000 to 50,000 cells per well in 96-well dishes. Transfected cells were exposed to CRF peptides for a 10-min stimulation period at 37°C (5% CO2). During the 10-min incubation period, no desensitization of the cAMP signal was observed. However, longer CRF stimulation periods resulted in considerable desensitization of the cAMP signal, especially at agonist concentrations higher than 100 nM (sauvagine and UCN; unpublished observations). The antagonistic experiments with aSVG and astressin were conducted by simultaneous application of the respective antagonist and sauvagine followed by a 10-min incubation as described above. Measurement of intracellular cAMP levels was performed as described previously (Dautzenberg et al., 2001c). The data were analyzed by two-way analysis of variance, and significance between groups was determined by post hoc analysis using Dunnett's test.
Results
Binding Properties of hCRF2A, xCRF2, and Two Mutated CRF2 Receptors.
cDNAs were synthesized to encode the following two different mutations in the EC3 domain of the human and X. laevis CRF2 receptors: hR2KYI (amino acids Lys264Tyr265Ile266of xCRF2) and xR2DLV (residues Asp262Leu263Val264of hCRF2A). After these constructs were stably transfected into HEK293 cells, binding profiles using a radiolabeled agonist (125I-sauvagine) and an antagonist (125I-astressin) were determined. Because potential differences in the binding profiles of the mutated receptors could result from differences in G protein-coupling properties (seeKenakin, 1997), we excluded this possibility by assessing binding in the presence of increasing concentrations of GTPγS or Gpp(NH)p. Both GTP analogs potently inhibited 125I-sauvagine binding to the receptor preparations to 56 to 72% (Table1). In addition, the GTP analog inhibited125I-sauvagine binding to the CRF2 receptor preparation with similar affinity (Table 1). In agreement with its antagonist properties, binding of125I-astressin was not inhibited by Gpp(NH)p and GTPγS (data not shown). Thus, we concluded that the native and mutant CRF2 receptors exhibited the same degree of coupling to the endogenous Gs proteins.
When 125I-sauvagine was used as the competed ligand, the rank orders of CRF ligand binding differed between the hCRF2A and xCRF2 receptor. The hCRF2A receptor bound hUCN, sauvagine, and aSVG with subnanomolar and astressin with low nanomolar affinity, whereas the K i values for hCRF and oCRF were markedly higher (Table 2). In contrast to hCRF2A, the xCRF2 receptor bound hUCN with subnanomolar and sauvagine and aSVG with low nanomolar affinity (Table 2). As reported previously (Dautzenberg et al., 2001b), when125I-sauvagine was used as the competed ligand, astressin exhibited an 8-fold lower affinity at the xCRF2 compared with the hCRF2A receptor (Fig.2A, Table 2). hCRF and oCRF competed for125I-sauvagine binding to the xCRF2 receptor at high nanomolar concentrations similar to their affinities at the hCRF2Areceptor (Table 2). Importantly, mutation of the amino acid triplet (Lys264Tyr265Ile266) in the xCRF2 receptor, analogous to the sequence of its human counterpart (Asp-Leu-Val) to create the xR2DLV receptor, converted the xCRF2 binding profile to that of the hCRF2A receptor. The xR2DLV receptor exhibited the following binding: hUCN ∼ aSVG ∼ sauvagine ∼ astressin ≫ hCRF ≫ oCRF (Table 2). Surprisingly, however, mutation of the ligand-selective amino acids of the EC3 domain of the hCRF2A receptor to the corresponding sequence of its amphibian counterpart to form the hR2KYI mutant failed to replicate the full binding profile of the xCRF2 receptor when125I-sauvagine was used as the competed ligand. Although the hR2KYI mutant, similar to the xCRF2receptor, bound sauvagine and aSVG with ∼6-fold lower affinity than hUCN, the affinity for astressin was ∼17-fold higher at the hR2KYI receptor compared with the xCRF2 receptor (Fig.2A; Table 2).
When 125I-astressin was used as the radioligand instead of 125I-sauvagine, substantial binding differences were observed for the two native and the mutated receptors. For the hCRF2A receptor, significant rightward shifts of the dose-response curves were observed for all agonists except hUCN, which retained affinity <1 nM (Table3). In contrast, using125I-astressin, binding affinities of antagonists to the hCRF2A receptor were only minimally affected (Fig. 2B; Table 3). When these agonists and antagonists competed with 125I-astressin at the hR2KYI receptor, the binding profile resembled the hCRF2A profile (Fig. 2B; Table 3). Conversely, the hR2KYI mutant produced the same binding profile as the xCRF2 receptor when125I-astressin was used as the competed radioligand: hUCN ∼ astressin > sauvagine > aSVG > hCRF ≫ oCRF (Table 3). Notably, hCRF was bound with an affinity ∼2-fold higher at the hR2KYI and xCRF2receptors compared with the xR2DLV and hCRF2Areceptors when 125I-astressin instead of125I-sauvagine was the competed radioligand. However, the affinities for sauvagine and aSVG binding to the hR2KYI and xCRF2 receptors were decreased ∼6- to 7-fold when the radioligand was the antagonist astressin. The only difference between the xCRF2 and hR2KYI receptor was the binding profile of astressin, which was not affected by the radioligand in the case of the hR2KYI receptor but was 15-fold better at the xCRF2 receptor compared with its affinity to compete for 125I-sauvagine binding (Fig. 2; Tables 2 and 3).
Binding Affinities of Human and Amphibian CRF2Receptors Carrying Point Mutations in the EC1 or EC1/EC3 Domains.
Mutations in the EC3 domain did not completely reverse the differential binding profiles of the hCRF2A and xCRF2 receptor. Consequently, experiments were performed to compare binding affinities for ligands at human and amphibian CRF2 receptors carrying point mutations restricted to the EC1 domain or a combination of EC1 and EC3 mutations. Because a two-amino acid motif in the ligand-selective domain of the xCRF1 receptor differs in hCRF2A(Glu66Tyr67) and xCRF2(Asp69Ser70) receptors (Dautzenberg et al., 1998), the following four mutated receptors were constructed: hR2DS (EC1 mutation), hR2DS-KYI (EC1/EC3 mutation), xR2EY (EC1 mutation), and xR2EY-DLV (EC1/EC3 mutation). After these mutated receptors were stably transfected into HEK293 cells, their binding properties were characterized using125I-sauvagine as the radioligand.
The EC1 mutants hR2DS and xR2EY retained the pharmacology of their respective wild-type receptors (Table 4). The rank order of binding affinities for the hR2DS mutant (aSVG ∼ hUCN ∼ sauvagine > astressin > hCRF ≫ oCRF) was identical to the native hCRF2A receptor (Table 4). The xR2EY mutant displayed a binding rank order (hUCN > aSVG ∼ sauvagine > astressin ≫ hCRF > oCRF) identical to the xCRF2 receptor (Tables 2 and 4). In contrast, binding profiles of EC1/EC3 mutants hR2DS-KYI and xR2EY-DLV were shifted completely compared with profiles of the wild-type receptors (Fig. 3). The rank order for the human CRF2A receptor mutant hR2DS-KYI was indistinguishable from the rank order for the xCRF2 receptor (Table 4). Conversely, rank orders for the xCRF2 receptor mutant xR2EY-DLV and the hCRF2A receptor were identical (Table 4). Importantly, similar to binding data for the hCRF2A, xCRF2, hR2KYI, and xR2DLV (Table 3), hR2DS, hR2DS-KYI, xR2EY, and xR2EY-DLV bound astressin with equal affinity when 125I-astressin was the competed ligand (Fig. 3).
Stimulation of cAMP Accumulation in HEK293 Cells Expressing hCRF2A, xCRF2, and Mutated Receptors.
The ability of CRF agonists to stimulate cAMP accumulation was assessed in HEK293 cells stably expressing native or mutated CRF2 receptors. A similar potency rank order (sauvagine > UCN > hCRF > oCRF) was observed among the four receptors (Table 5). Because sauvagine stimulated intracellular cAMP accumulation with the greatest potency, we assessed the inhibitory effects of the nonselective CRF receptor antagonist astressin and the selective CRF2 receptor antagonist aSVG on cAMP stimulation produced by 1 nM sauvagine, which is a concentration slightly above the EC50 value observed in the four receptor lines (Table 5). When cells expressing the four different CRF2 receptor proteins were coincubated with 100 nM astressin or aSVG, the sauvagine-stimulated cAMP accumulation was significantly reduced in the four receptor lines, indicating antagonist actions (Figs. 4 and5).
Next, Schild plots were generated for the antagonist potencies of astressin and aSVG on sauvagine-stimulated cAMP accumulation in the four receptor lines. Astressin and aSVG behaved as competitive antagonists at all CRF2 receptor-expressing lines (Fig. 5; Table 6). The two antagonists differed, however, in their inhibitory potencies. Astressin markedly inhibited sauvagine-stimulated cAMP accumulation in cells expressing hCRF2A, hR2DS, hR2KYI, xR2DLV, and xR2EY-DLV receptors. When Schild plots were calculated for hR2DS-KYI, xR2EY, and xCRF2-expressing cells, 5- to 6-fold higher concentrations of astressin were required to shift the sauvagine dose-response curve to the right (Table 6). In contrast, aSVG was a more potent antagonist than astressin at the hCRF2A, hR2DS, xR2DLV, and xR2EY-DLV receptors. However, in HEK293 cells expressing hR2KYI, hR2DS-KYI, xR2EY, or xCRF2 receptors, aSVG at a 10-fold higher concentration than used in the other receptor lines was required to inhibit sauvagine-stimulated cAMP accumulation (Table 6).
Discussion
The purpose of this study was to identify the residues of the xCRF2 receptor that govern its higher degree of binding selectivity compared with its human counterpart (Dautzenberg et al., 2001). Although the mammalian and amphibian CRF1 receptor use amino acids located in the N-terminal EC1 domain for high-affinity ligand binding and substrate recognition (Dautzenberg et al., 1998; Perrin et al., 1998; Wille et al., 1999; Assil et al., 2001), the hCRF2A and xCRF2 receptor were reported to use different exofacial domains. For the hCRF2A receptor, three regions within the EC2 and EC3 domain of this receptor have been identified to be critical for selective binding of and activation by CRF agonists (Liaw et al., 1997a). In our recent study, similar regions in EC2 and EC3 seemed to confer its agonist selectivity (Dautzenberg et al., 1999).
Interestingly, the amino acid motifs that probably mediate the ligand selectivity of hCRF2A are not well conserved between the human and X. laevis receptors (Dautzenberg et al., 1997, 1999). Moreover, we have recently shown that a histidine residue reported to be located at position 185 and to play a crucial role for the binding specificity of hCRF2A (Liaw et al., 1997a) most likely represents a polymerase chain reaction artifact. Instead, our sequencing experiments identified an arginine residue (Arg185), located at position 185 of the hCRF2A cDNAs, isolated from a variety of tissues, which is conserved in the hCRF2A gene as well as other vertebrate CRF1 and CRF2 receptors (Dautzenberg et al., 2000). Furthermore, the second important domain for the ligand selectivity of the hCRF2A receptor, residues Val172Asp173His174(Liaw et al., 1997a), are almost identical to the equivalent region of the xCRF2 receptor (Ile174Asp175His176) (Dautzenberg et al., 1997).
The above findings led us to first focus our investigation on the third ligand-selective domain, residues Asp262Leu263Val264of the hCRF2A receptor (Liaw et al., 1997a). The equivalent domain of the xCRF2 receptor, residues Lys264Tyr265Ile266(Dautzenberg et al., 1997), differ strongly from its human counterpart.
Indeed, mutagenesis of these three amino acids altered the binding pharmacology of the two mutants. The xR2DLV mutant, encoding the residues of the hCRF2A receptor, displayed the same binding preferences with both radioligands,125I-sauvagine and125I-astressin, as the human receptor. For the human mutant hR2KYI, the presence of the Lys264Tyr265Ile266triplet of the xCRF2 receptor resulted in a pharmacology profile closely resembling the amphibian CRF2 receptor. The largest differences in binding-affinity profiles were observed for the agonists hCRF and sauvagine and for the antagonists aSVG and astressin. Interestingly, the hR2KYI mutant and the xCRF2 receptor bound hCRF with low affinity when 125I-sauvagine was the competed radioligand. For the xR2DLV and hCRF2A receptor, a low binding affinity for hCRF was observed in the presence of 125I-astressin, consistent with the concept that binding of an agonist is reduced in the presence of a radiolabeled antagonist (Sleight et al., 1996;Dautzenberg et al., 2001b). Furthermore, sauvagine binding in the presence of 125I-sauvagine occurred with a lower affinity to the xCRF2 and hR2KYI receptor compared with other ligands, whereas sauvagine bound with high affinity to the hCRF2A and xR2DLV receptor. However, in the presence of 125I-astressin, the agonist showed no differences in binding to the four receptors. The CRF2-selective antagonist aSVG, like sauvagine, bound with a lower affinity to the xCRF2 and hR2KYI receptor than to the hCRF2A receptor and the xR2DLV mutant. However, unlike classical antagonists, which bind independent of the agonistic or antagonistic nature of the competed radioligand (Sleight et al., 1996; Perrin et al., 1999), the affinity of aSVG was shifted to the right with all four receptors. This effect was strongest for the xCRF2 receptor and hR2KYI mutant, which showed only a moderate affinity of ∼20 nM for the antagonist. This unusual behavior for a receptor antagonist suggests that aSVG binding selectively depends on the agonistic or antagonistic nature of the competed radioligand. Nevertheless, both the nonradioactive and the iodinated form of the peptide have been shown to behave as antagonists (Rühmann et al., 1998; Higelin et al., 2001).
Notably, the mutagenesis approach within the EC3 domain did not unravel completely the unusual binding profile of astressin to the xCRF2 receptor. Although incorporation of the amino acid triplet Asp-Leu-Val into the xCRF2receptor created a mutant with binding preferences indistinguishable from that of the hCRF2A receptor, the converse result was not obtained when the corresponding human sequence was replaced by the triplet Lys-Tyr-Ile in the hR2KYI mutant. This mutant bound astressin with high affinity in the presence of125I-sauvagine and thus differed from the xCRF2 receptor, which showed a lower affinity for astressin when 125I-sauvagine was the competed radioligand (Dautzenberg et al., 2001b).
Thus, we concluded that an unidentified region of the xCRF2 receptor interacts with the amino acid triplet Lys264Tyr265Ile266to negatively regulate astressin binding. Similarly, a recent study demonstrated that the binding of astressin differed from CRF and UCN at rat CRF1 receptor (Perrin et al., 1998). Although astressin binding to the rat CRF1 receptor only requires the EC1 domain, the EC4 domain is also needed for binding of CRF and UCN (Perrin et al., 1998). Furthermore, a microheterogeneity of agonist binding to the hCRF2A receptor was also reported (Liaw et al., 1997b). Although introduction of the ligand-selective regions of the hCRF2A receptor into the sequence of the hCRF1 receptor strongly impaired CRF and sauvagine binding, UCN binding was insensitive to this mutation (Liaw et al., 1997b). These findings are not only restricted to the CRF receptor system because a similar observation was reported for another member of the class B G protein-coupled receptor subfamily, the parathyroid hormone receptor, which shows involvement of different amino acids for the binding of different parathyroid hormone variants (Lee et al., 1995).
We focused our search for the additional extracellular region that impairs binding of astressin to the xCRF2receptor on the N-terminal EC1 domain. Recently, we identified an amino acid doublet within the ligand-binding site of the mammalian CRF1 receptor (Wille et al., 1999; Assil et al., 2001) in a parallel study investigating the site for astressin binding to the xCRF1 receptor (S. Wille, J. Higelin, and F. M. Dautzenberg, manuscript in preparation). This doublet is conserved in xCRF1(Glu70Tyr71) and hCRF2A(Glu66Tyr67) but diverges in hCRF1(Ala70Phe71) and the xCRF2 receptor (Asp69Ser70). Interestingly, mutagenesis of this amino acid doublet did not alter binding of astressin to the hR2DS and xR2EY receptors. In contrast, replacement of EC1/EC3 residues of the hCRF2Areceptor with the corresponding X. laevis motifs resulted in a xCRF2 pharmacology. Likewise, replacement of EC1/EC3 residues of the xCRF2 receptor with the corresponding human motifs shifted its ligand rank order to an hCRF2A profile.
Rank orders for sauvagine-stimulated cAMP accumulation resembled rank orders obtained with binding studies where125I-sauvagine was used as the radioligand. Although astressin and aSVG were highly potent and competitive antagonists at the hCRF2A, hR2DS, xR2DLV, and xR2EY-DLV receptors, both antagonists were significantly less potent at the hR2DS-KYI, xCRF2, and xR2EY receptors. However, astressin was a more potent antagonist than aSVG in hR2KY-expressing cells in agreement with astressin possessing a higher binding affinity than aSVG at the hCRF2Areceptor. Thus, the binding properties of both antagonists correspond closely when 125I-sauvagine rather than125I-astressin is used as the radioligand. Furthermore, these data demonstrate that the binding pocket of xCRF2 for astressin differs from its binding pockets for agonists and other antagonists. Importantly, the slopes for the antagonism of aSVG and astressin of sauvagine-stimulated cAMP accumulation were ∼1. Although our aSVG data agree with the findings of a recent study (Brauns et al., 2001), the slopes for astressin antagonism differ. Because our transfected cell lines express CRF2 receptors at a level significantly lower than the CRF2 receptor expression level used byBrauns et al. (2001), our data may reflect more native cell lines endogenously expressing CRF receptors.
In conclusion, our site-directed mutagenesis experiments have identified two important regions mediating the differential binding of CRF analogs to the amphibian CRF2 receptor: a) an amino acid triplet Lys264Tyr265Ile266in the EC3 domain and b) a two-amino acid motif, Asp69Ser70, in the EC1 domain of the xCRF2 receptor. Replacement of this region by the corresponding amino acids of the hCRF2A receptor (Glu66Tyr67 and Asp262Leu263Val264) generated a mutant with a binding pharmacology indistinguishable from that of the hCRF2A receptor. The converse replacement of this region in the human CRF2Areceptor with the corresponding X. laevis sequence shifted the binding profile to that of the xCRF2receptor. Finally, antagonism of sauvagine-stimulated cAMP accumulation by astressin and aSVG followed competitive binding data using125I-sauvagine as a radioligand. Therefore, microheterogeneity within the ligand-binding pocket seems to be present in the amphibian CRF2 receptor.
Footnotes
- Received July 30, 2001.
- Accepted January 24, 2002.
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R.L.H. was supported by a Veterans Affairs Merit Review grant, the Veterans Affairs Mental Illness Research, Education and Clinical Center (MIRECC) of VISN22, and the National Institute of Mental Health (PHS MH20914-14) Mental Health Clinical Research Center.
Abbreviations
- CRF
- corticotropin-releasing factor
- aSVG
- anti-sauvagine-30
- CRF1
- CRF type 1 receptor
- CRF2
- CRF type 2 receptor
- UCN
- urocortin
- hCRF
- human CRF
- oCRF
- ovine CRF
- xCRF
- X. laevis CRF
- hUCN
- human UCN
- EC
- extracellular domain
- GTPγS
- guanosine 5′-O-(3-thio)triphosphate
- Gpp(NH)p
- guanosine 5′-(β,γ-imido)triphosphate
- The American Society for Pharmacology and Experimental Therapeutics