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Three-amino acid motifs of urocortin II and III determine their CRF receptor subtype selectivity
Neuropharmacology 47 (2004) 233–242 www.elsevier.com/locate/neuropharm
Three-amino acid motifs of urocortin II and III determine their CRF receptor subtype selectivity Olaf Jahn, Hossein Tezval, Lars van Werven, Klaus Eckart, Joachim Spiess Department of Molecular Neuroendocrinology, Max Planck Institute for Experimental Medicine, Hermann-Rein-Str. 3, 37075 Goettingen, Germany Received 9 September 2003; received in revised form 17 March 2004; accepted 23 March 2004
Abstract Corticotropin-releasing factor (CRF) and the CRF-like peptide urocortin I (UcnI) exert their activity through two different CRF receptors, CRF1 and CRF2. Recently, UcnII and UcnIII have been discovered as potential endogenous agonists selective for CRF2 known to be involved in brain functions such as learning and anxiety, as well as in cardiovascular functions. A structure–affinity relationship study using chimeric peptides was designed to characterize mouse UcnII (mUcnII) and mUcnIII further and to investigate the structural basis of their receptor subtype selectivity. In the framework of this study, mUcnII (IC50 ¼ 4:4 nM) but not mUcnIII was identified as high-affinity ligand for the rat CRF binding protein. Such affinity had previously not been observed for the human version of this protein. On the basis of secondary structure predictions, it was hypothesized that the amino acid motifs Pro-Ile-Gly of mUcnII and Pro-Thr-Asn of mUcnIII decrease a-helicity and thereby impair binding to CRF1. In support of this hypothesis, binding affinity to CRF1 of the chimeric peptides [Pro11Ile12Gly13]h/rCRF, [Pro11Thr12Asn13]h/rCRF, and the corresponding rUcnI analogs was found to be decreased by three orders of magnitude, whereas binding affinity to CRF2 was much less affected. The dramatic decrease in binding affinity to CRF1 correlated with a decrease in a-helicity as indicated by the data of circular dichroism spectroscopy. # 2004 Elsevier Ltd. All rights reserved. Keywords: Corticotropin-releasing factor (CRF); Urocortin; CRF receptor; CRF binding protein; Secondary structure
1. Introduction The 41 amino acid peptide corticotropin-releasing factor (CRF) (Spiess et al., 1981), originally identified as an early chemical signal in the mammalian responses to stress stimuli (Vale et al., 1981), has been more recently recognized to be also involved in complex brain functions. CRF is a neuromodulator of learning and memory, anxiety, and food intake and is linked to the pathogenesis of various anxiety, mood, and eating disorders (Behan et al., 1996; Eckart et al., 2002). CRF and its natural analog urocortin I (UcnI) (Vaughan et al., 1995) exert their biological activity by binding to two subtypes of the G protein-coupled CRF receptor, CRF1 and CRF2, which have distinct expression profiles and different physiological functions (Eckart et al., Corresponding author. Tel.: +49-551-3899-258; fax: +49-5513899-359. E-mail address: spiess.offi[email protected] (J. Spiess).
0028-3908/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2004.03.018
2002; Perrin and Vale, 1999). Gene-deletion (Bale et al., 2000; Coste et al., 2000; Kishimoto et al., 2000; Smith et al., 1998; Timpl et al., 1998) and pharmacological (Radulovic et al., 1999) studies provided a growing body of evidence to support a partial functional antagonism of these receptor subtypes. Thus, in the central nervous system (CNS), activation of CRF1 accessible through the brain ventricle system predominantly enhances anxiety, whereas CRF2 reached through the ventricles acts anxiolytically. These differential effects of CRF may be also affected by the CRF binding protein (CRFBP) (Potter et al., 1991) which serves as a local reservoir of endogenous ligand (Behan et al., 1995; Karolyi et al., 1999). In the periphery, CRF-like peptides acting at CRF2 modulate cardiovascular functions possibly protecting against cardiac failure (Parkes et al., 2001). As in the CNS, these actions may be also affected by circulating CRFBP—at least in humans. In contrast to rodents, humans are distinguished by a
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significant level of CRFBP in their blood (Potter et al., 1991). Because of its anxiolytic potential and cardioprotective effects, CRF2 has become an interesting target of physiological and pharmacological research. Recent progress in this research was the identification of human stresscopin-related peptide and stresscopin (Hsu and Hsueh, 2001), and the corresponding murine orthologs UcnII and UcnIII (Lewis et al., 2001; Reyes et al., 2001) on the basis of predictions using genomic DNA information and subsequent cDNA cloning. These two new members of the CRF peptide family were identified as agonists selective for CRF2. On the basis of physicochemical measurements (Dathe et al., 1996; Lau et al., 1983; Pallai et al., 1983; Romier et al., 1993) and structure–activity relationship studies (Hernandez et al., 1993; Miranda et al., 1994), it has been suggested that CRF receptor ligands assume an a-helical conformation when interacting with their receptors. By the development of N-terminally truncated CRF-like peptides as peptidic antagonists (Hernandez et al., 1993; Rivier et al., 1984), it was recognized that the N-terminal part of the peptide is not only crucial for receptor activation, but may also induce and stabilize the a-helix structure as the conformation required for high-affinity binding to CRF receptors. In agreement with this observation, the affinity of the antagonists developed on the basis of the sequence of h/rCRF9-41 (Rivier et al., 1984) or h/rCRF12-41 (Hernandez et al., 1993) to both CRF receptor subtypes was found to be significantly decreased compared to that of the parent peptide h/rCRF (Perrin and Vale, 1999). Since the decrease in affinity is more profound for CRF1, it was observed that such truncated h/rCRF analogs display a clear preference for CRF2 (Perrin and Vale, 1999). This differential effect on receptor binding was found to be even more significant when the 40 amino acid CRF-like peptide sauvagine (Svg) (Montecucchi and Henschen, 1981) was N-terminally truncated. Peptides derived from Svg by N-terminal truncation lost their affinity to CRF1 almost completely, while retaining their affinity to CRF2. This finding finally led to the development of the highly selective CRF2 antagonist antisauvagine-30 (Ru¨hmann et al., 1998). In agreement with these observations, a recent study using various N-terminally truncated CRF-like peptides emphasized that the structural requirements of CRF1 for high-affinity binding of peptidic ligands are more stringent than the corresponding requirements of CRF2 (Brauns et al., 2002b). Furthermore, the affinity of N-terminally truncated CRF-like peptides, especially to CRF1, can be restored by introduction of a lactam bridge between the side chains of amino acid residues 30 and 33 of h/rCRF (Gulyas et al., 1995) or 29 and 32 of Svg (Brauns et al., 2002a). Since a lactam bridge functions as a structural constraint probably stabilizing the a-helical confor-
mation of the peptide (Gulyas et al., 1995), these findings further support the assumption that an a-helix structure is required for high-affinity binding—at least to CRF1. Taken together, there is suggestive evidence that the affinity of a peptide to CRF1 may be enhanced by its a-helicity, whereas binding to CRF2 appears to be less sensitive to a decrease in a CRF-like peptide’s a-helicity. The objective of the present study was to investigate the structural basis for the CRF2 selectivity of UcnII and UcnIII on the basis of the hypothesis that these peptides contain amino acid motifs which disturb the integrity of their a-helix structure and thereby impair binding to CRF1. To identify such motifs, we compared the amino acid sequences of the CRF2-selective agonists mUcnII and mUcnIII (Lewis et al., 2001) with the sequences of the other natural rodent agonists human/rat CRF (h/rCRF), preferably binding to CRF1, and rat UcnI (rUcnI), exhibiting no selectivity (Behan et al., 1996; Eckart et al., 2002). On the basis of sequence alignments and secondary structure predictions, candidate amino acid motifs were identified in mUcnII and mUcnIII. These motifs possibly affect a-helicity and thereby receptor selectivity of these peptides. Our hypothesis was tested by introduction of the identified motifs into h/rCRF and rUcnI, respectively. The pharmacological properties of the resulting chimeric peptides were investigated by using stably transfected cell lines producing either rat CRF1 (rCRF1), mouse CRF2b (mCRF2b) or rat CRFBP (rCRFBP). Circular dichroism (CD) spectroscopy was used to analyze the peptides’ secondary structures. Parts of this work have been presented as abstracts in preliminary form to the Society for Neuroscience (Jahn et al., 2002) and the Endocrine Society (Jahn et al., 2003).
2. Methods 2.1. Peptide synthesis Peptides were synthesized, purified, and characterized by mass spectrometry as described recently (Jahn et al., 2001). The final concentration of peptide stock solutions was determined by amino acid analysis with a Beckman high performance ion exchange analyzer system 6300. Total hydrolysis of the peptides was carried v out with 6 M HCl for 24 h at 100 C in the presence of 1 nmol norvaline as internal standard. 2.2. Binding assays Crude membrane fractions were prepared from transfected human embryonic kidney (HEK) 293 cells stably producing either rCRF1 (HEK-rCRF1 cells) or mCRF2b (HEK-mCRF2b cells) as described (Ru¨hmann
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et al., 1998). rCRFBP was produced in HEK 293 cells stably transfected with cDNA coding for rCRFBP C-terminally fused with a His-tag as described (Jahn et al., 2001). The utilization of a scintillation proximity assays (SPA) for competition binding analysis of CRF receptors (Eckart et al., 2001; Hofmann et al., 2001) and CRFBP (Eckart et al., 2001) was recently established in our laboratory. For binding analysis of CRF-like peptides to CRF receptors, wheat germ agglutinin-coated SPA beads (neuropeptide Y receptor SPA binding assay, Amersham Pharmacia Biotech) were used as described (Hofmann et al., 2001). [125ITyr0]h/rCRF and [125I-Tyr0]Svg (NEN) were employed as radiolabeled ligands for rCRF1 and mCRF2b, respectively. Binding of CRF-like peptides to His-tagged rCRFBP was carried out in nickel-chelate-coated 96 well SPA microtiter plates (Flash Plate PLUS2, NEN) as described (Eckart et al., 2001). [125I-Tyr0]h/ rCRF (NEN) was employed as radiolabeled ligand. Competition binding curves were analyzed by nonlinear regression using the Prism computer program (GraphPad Software). 2.3. Measurement of intracellular cAMP accumulation HEK 293 cells were plated into 24 well cell culture plates and stimulated as described (Eckart et al., 2001) using increasing concentrations of the peptides under investigation. Intracellular cAMP was measured with the Biotrak2 cAMP [125I] SPA system (Amersham Pharmacia Biotech) according to the manufacturer’s product manual. Dose–response curves were analyzed by non-linear regression using the Prism computer program (GraphPad Software). 2.4. Secondary structure prediction For secondary structure prediction, the amino acid sequences of the CRF-like peptides were submitted to the PredictProtein server (http://www.emblheidelberg.de/predictprotein). On the basis of the submitted sequence, an automatic PSI-BLAST multiple sequence alignment was performed against the SWISSPROT database and this alignment was used as input for the PROFsec secondary structure prediction algorithm (Rost and Sander, 1993). 2.5. CD spectroscopy The CD spectra were recorded in the range of 190– 260 nm as described recently (Brauns et al., 2002a). The CRF-like peptides were dissolved in 10 mM sodium phosphate buffer (pH 7.4; Suprapure, Merck) containing various concentrations of 2,2,2-trifluoroethanol (TFE) as a-helix structure-inducing organic co-solvent. Relatively low final peptide concentrations in the
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range of 10 lM were used because it has been shown— at least for h/rCRF—that higher concentrations lead to association which is accompanied by an increase in the a-helicity of the peptide (Dathe et al., 1996). The exact peptide concentrations were determined by amino acid analysis as described above. The CD spectra were deconvoluted by using the method of Bo¨hm et al. (1992) (CDNN).
3. Results 3.1. Design of the chimeric peptides The aligned amino acid sequences of h/rCRF, rUcnI, mUcnII, and mUcnIII were first compared in view of the conformational preferences of the single amino acid residues (Williams et al., 1987) with the objective to identify candidate amino acid motifs that possibly affect the integrity of the a-helix structure of mUcnII and mUcnIII, respectively (Fig. 1). This comparison revealed that mUcnII contains Pro and Gly in positions 8 and 10 in contrast to h/rCRF and rUcnI containing Thr and His in the corresponding positions (Fig. 1). mUcnIII like mUcnII contains Pro and Asn in positions 8 and 10 (Fig. 1). Interestingly, Pro, Gly, and Asn have high propensities to form reverse turn structures (Williams et al., 1987) and, thus, rarely occur in the interiors of a-helical structures. Therefore, it was hypothesized that the amino acid sequences Pro-Ile-Gly (PIG motif) and Pro-Thr-Asn (PTN motif) assume a-helix-breaking conformations and thereby decrease the extent of a-helix structure of mUcnII and mUcnIII. In agreement with this hypothesis, secondary structure predictions indicated that the a-helices in mUcnII and mUcnIII were shifted C-terminally by three to four residues in comparison to their location in h/rCRF and rUcnI, respectively (Fig. 1). This shift corresponded to approximately one complete a-helix turn consisting of 3.6 amino acid residues (Fig. 1). To test whether the presence of the PIG and PTN motifs in mUcnII and mUcnIII, respectively, impaired binding to CRF1, the chimeric peptides [Pro11Ile12Gly13]h/ rCRF, [Pro11Thr12Asn13]h/rCRF, [Pro10Ile11Gly12] rUcnI, and [Pro10Thr11Asn12]rUcnI were synthesized and subsequently tested for their pharmacological properties. With an inverse approach, the corresponding chimeric peptides [Thr8Phe9His10]mUcnII and [Thr8Phe9His10]mUcnIII were also included in these experiments. 3.2. Pharmacological properties of mUncnII and mUcnIII In the present study, the novel CRF-like peptides mUcnII and mUcnIII were first tested for their binding
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Fig. 1. Amino acid sequence alignment of h/rCRF, rUcnI, mUcnII, and mUcnIII. The amino acid sequences used for replacement are underlaid in gray. Above each residue, its conformational preference is indicated: a ¼ a-helix, b ¼ b-strand, t ¼ reverse turn. Below the sequences, the predicted secondary structures are presented: H, helix; E, extended (loop); dash, other (loop). The black squares represent the C-terminal amide groups.
affinity to rCRF1, mCRF2b, and rCRFBP using the assay systems established in our laboratory (Eckart et al., 2001; Hofmann et al., 2001). In agreement with the data of the original study on the characterization of these peptides (Lewis et al., 2001), mUcnII exhibited high affinity to mCRF2b (IC50 ¼ 0:25 nM), but not to rCRF1 (IC50 >350 nM) (Table 1). Similarly, mUcnIII bound with moderate affinity to mCRF2b (IC50 ¼ 14 nM), whereas specific binding of this peptide to rCRF1 was not detectable (IC50 >2000 nM) (Table 1). Thus, the high selectivity of mUcnII and mUcnIII for CRF2 was confirmed. Interestingly, mUcnII bound to rCRFBP with an affinity in the low nanomolar range (IC50 ¼ 4:4 nM), whereas mUcnIII did not exhibit any detectable affinity (IC50 >2000 nM) to this protein (Table 1). Although the affinity of mUcnII to rCRFBP
was lower by approximately one order of magnitude than the affinities of h/rCRF (IC50 ¼ 0:54 nM) and rUcnI (IC50 ¼ 0:98 nM), respectively, mUcnII was found to be a high-affinity ligand for rCRFBP (IC50 ¼ 4:4 nM). The biological potency of mUcnII and mUcnIII was evaluated by the determination of EC50 values for agonist-induced intracellular accumulation of cAMP in HEK-rCRF1 or HEK-mCRF2b cells. In agreement with the data of the original study (Lewis et al., 2001), high potencies of mUcnII (EC50 ¼ 0:43 nM) and mUcnIII (EC50 ¼ 0:83 nM) to stimulate HEK-mCRF2b cells were found (Table 1). Despite its low affinity for rCRF1 (IC50 >350 nM), mUcnII displayed a considerable potency to stimulate the cAMP production of HEK-rCRF1 cells (EC50 ¼ 19 nM) (Table 1).
Table 1 Pharmacological properties of h/rCRF, rUcnI, mUcnII, mUcnIII, and their chimeric analogs Peptide
h/rCRF rUcnI mUcnII mUcnIII [Pro11Ile12Gly13]h/rCRF [Pro11Thr12Asn13]h/rCRF [Pro10Ile11Gly12]rUcnI [Pro10Thr11Asn12]rUcnI [Thr8Phe8His10]mUcnII [Thr8Phe9His10]mUcnIII
IC50 (nM)
EC50 (nM)
rCRF1
mCRF2b
rCRFBP
rCRF1
mCRF2b
1.6 (1.3–1.9) 0.17 (0.11–0.23) 350 (320–380) >2000 >2000 >2000 340 (180–500) 550 (420–680) 22 (11–32) 1300 (750–1900)
42 (25–59) 0.86 (0.67–0.92) 0.25 (0.08–0.43) 14 (10–19) 160 (150–170) 60 (49–71) 47 (35–59) 51 (43–58) 0.26 (0.16–0.35) 4.0 (3.1–5.0)
0.54 (0.38–0.71) 0.98 (0.66–1.3) 4.4 (3.7–5.2) >2000 n.d. n.d. n.d. n.d. n.d. n.d.
0.33 (0.26–0.40) 0.89 (0.78–1.0) 19 (15–24) >2000 110 (63–160) 78 (8.0–150) 30 (20–40) 210 (130–290) 0.45 (0.20–0.70) 22 (4.7–39)
1.6 (1.0–2.2) 0.84 (0.35–1.3) 0.43 (0.28–0.57) 0.83 (0.56–1.1) 21 (13–28) 25 (15–36) 2.5 (1.8–3.2) 7.6 (5.8–9.3) 0.61 (0.43–0.79) 4.0 (1.8–6.0)
IC50 values are the mean of four independent competition binding assays. EC50 values are the mean of three independent cell stimulation experiments. 95% confidence intervals are given in parentheses. n.d., not determined.
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3.3. The effect of the PIG and PTN motifs on receptor binding The sequence Thr11Phe12His13 of h/rCRF was first replaced by the PIG motif of mUcnII. In comparison to the high affinity of the parent peptide h/rCRF to rCRF1 (IC50 ¼ 1:6 nM), the affinity of the chimeric peptide [Pro11Ile12Gly13]h/rCRF to this receptor subtype (IC50 >2000 nM) was almost completely abolished (Fig. 2A; Table 1). In contrast, its affinity to mCRF2b was only slightly decreased (IC50 ¼ 160 nM) compared to that of h/rCRF (IC50 ¼ 42 nM) (Fig. 2B; Table 1). With an inverse approach, the PIG motif of mUcnII was replaced by the corresponding sequence Thr-PheHis present in h/rCRF. In comparison to the highly selective CRF2 ligand mUcnII, the chimeric peptide [Thr8Phe9His10]mUcnII exhibited a relatively high affinity to rCRF1 (IC50 ¼ 22 nM) (Fig. 2A; Table 1), whereas the affinities of mUcnII and [Thr8Phe9 His10]mUcnII (IC50 ¼ 0:26 nM) to mCRF2b did not deviate significantly from each other (Fig. 2B; Table 1).
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For a more detailed investigation of the effects of the PIG and PTN motifs on receptor binding, the chimeric approach described above was extended to rUcnI and mUcnIII. First, the sequence Thr11Phe12His13 of h/rCRF was replaced by the PTN motif of mUcnIII. In agreement with the lack of affinity to rCRF1 observed for [Pro11Ile12Gly13]h/rCRF, the affinity of [Pro11Thr12Asn13]h/rCRF (IC50 >2000 nM) to this receptor subtype was also almost completely abolished (Table 1). In contrast, its affinity to mCRF2b was not significantly changed (IC50 ¼ 60 nM) compared to that of h/rCRF (Table 1). A similar observation—at least in view of binding to rCRF1—was made when the sequence Thr10Phe11His12 of rUcnI was replaced either by the PIG motif of mUcnII or by the PTN motif of mUcnIII. The high affinity of rUcnI to rCRF1 (IC50 ¼ 0:17 nM) was decreased by three orders of magnitude upon introduction of the PIG or PTN motifs as demonstrated by the low affinities of [Pro10Ile11Gly12]rUcnI (IC50 ¼ 340 nM) and [Pro10 Thr11Asn12]rUcnI (IC50 ¼ 550 nM) to rCRF1 (Table 1). The affinities of rUcnI, [Pro10Ile11Gly12]rUcnI, and [Pro10Thr11Asn12]rUcnI to mCRF2b were also decreased, but to a lesser extent, as indicated by the respective IC50 values of 0.86, 47, and 51 nM (Table 1). Furthermore, the PTN motif of mUcnIII was replaced by the corresponding sequence Thr-Phe-His present in rUcnI. When the highly selective CRF2 ligand mUcnIII and the chimeric peptide [Thr8Phe9His10]mUcnIII (IC50 ¼ 1300 nM) were compared, no marked increase in affinity to rCRF1 was detected as was for [Thr8Phe9 His10]mUcnII (Table 1). While mUcnII and [Thr8Phe9 His10]mUcnII were similar in view of their affinity to mCRF2b, [Thr8Phe9His10]mUcnIII (IC50 ¼ 4:0 nM) exhibited a slightly higher affinity to this receptor subtype than the parent peptide mUcnIII (Table 1). 3.4. The effect of the PIG and PTN motifs on biological potency
Fig. 2. Binding characteristics of h/rCRF, mUcnII, and their chimeric analogs. Competition curves are shown for the binding of h/rCRF, [Pro11Ile12Gly13]h/rCRF, mUcnII, and [Thr8Phe9His10] mUcnII to rCRF1 (A) or mCRF2b (B). Competition curves were normalized by total binding in the absence of competitor (B0). Data points represent pooled data from four independent experiments performed in duplicate.
The biological potency was determined by the intracellular cAMP accumulation in HEK-rCRF1 and HEK-mCRF2b cells, respectively, after stimulation with the ligand under investigation. The chimeric peptides [Pro11Ile12Gly13]h/rCRF, [Pro11Thr12Asn13]h/r CRF, [Pro10Ile11Gly12]rUcnI, [Pro10Thr11Asn12]rUcnI, [Thr8Phe9His10]mUcnII, and [Thr8Phe9His10]mUcnIII were subjected to this assay. The high potency of h/rCRF to stimulate the cAMP production of HEKrCRF1 cells (EC50 ¼ 0:33 nM) was decreased by two orders of magnitude upon introduction of the PIG and PTN motifs as demonstrated by the low potencies of [Pro11Ile12Gly13]h/rCRF (EC50 ¼ 110 nM) and [Pro11 Thr12Asn13]h/rCRF (EC50 ¼ 78 nM) (Table 1). Similarly, compared with the potency of the parent peptide rUcnI (EC50 ¼ 0:89 nM), the potencies of [Pro10
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Ile11Gly12]rUcnI (EC50 ¼ 30 nM) and [Pro1011 12 Thr Asn ]rUcnI (EC50 ¼ 210 nM) to stimulate rCRF1 were also significantly decreased (Table 1). In contrast, when these peptides were tested for their potencies to stimulate HEK-mCRF2b cells, a less profound decrease in potencies was observed for [Pro11Ile12Gly13]h/rCRF (EC50 ¼ 21 nM), [Pro11Thr12 13 10 11 Asn ]h/rCRF (EC50 ¼ 25 nM), [Pro Ile Gly12]rUcnI (EC50 ¼ 2:5 nM), and [Pro10Thr11Asn12]rUcnI (EC50 ¼ 7:6 nM) in comparison to the potencies of the parent peptides h/rCRF (EC50 ¼ 1:6 nM) and rUcnI (EC50 ¼ 0:84 nM) (Table 1). Upon introduction of the sequence Thr-Phe-His, the potencies of mUcnII and mUcnIII to stimulate HEK-rCRF1 cells were increased by two orders of magnitude as shown by the relatively low EC50 values of [Thr8Phe9His10]mUcnII (EC50 ¼ 0:45 nM) and [Thr8Phe9His10]mUcnIII (EC50 ¼ 22 nM) (Table 1). In contrast, the potencies of these peptides to stimulate HEK-mCRF2b cells were not significantly changed for [Thr8Phe9His10]mUcnII (EC50 ¼ 0:61 nM) or slightly decreased for [Thr8Phe9 His10]mUcnIII (EC50 ¼ 4:0 nM) in comparison to the parent peptides mUcnII and mUcnIII, respectively (Table 1). All peptides tested had similar agonist efficacies as indicated by similar maximal cAMP responses in the range of 100 pmol cAMP/105 HEK-rCRF1 cells and of 170 pmol cAMP/105 HEK-mCRF2b cells (data not shown). In agreement with the extremely low potency of mUcnIII to stimulate HEK-rCRF1 cells (Table 1), its agonist efficacy could not be determined on the basis of the peptide concentrations applied (up to 1 lM). 3.5. The effect of the PIG and PTN motifs on peptide secondary structure According to our hypothesis, a reduction of a-helicity of a ligand should impair binding to CRF1. Therefore, we assumed that the extremely low affinities of [Pro11Ile12Gly13]h/rCRF, [Pro11Thr12Asn13]h/rCRF, 10 11 12 [Pro Ile Gly ]rUcnI, and [Pro10Thr11Asn12]rUcnI to rCRF1 were due to a decrease in the extent of a-helix structure caused by the presence of the PIG or PTN motifs. We tested this hypothesis by CD spectroscopic analysis of the secondary structures of these peptides. The CD spectra of the peptides were recorded in the presence of various concentrations of the a-helix structure-inducing organic co-solvent TFE (0–30%) with the objective to investigate the inducibility of the a-helix structure rather than maximal a-helicity. In the presence of relatively low TFE concentrations (0–15% TFE), the extent of a-helix structure of [Pro11 Ile12Gly13]h/rCRF and [Pro11Thr12Asn13]h/rCRF was found to be decreased by approximately one to three complete a-helix turns in comparison to the parent peptide h/rCRF (Fig. 3A). When the concentration of
Fig. 3. a-Helicity derived from CD spectra. The a-helicities are shown for h/rCRF, [Pro11Ile12Gly13]h/rCRF, and [Pro11Thr12Asn13] h/rCRF (A), and rUcnI, [Pro10Ile11Gly12]rUcnI, and [Pro10Thr11 Asn12]rUcnI (B). By deconvolution of the CD spectra, the portions of a-helix structure were obtained in percent, transformed to the number of a-helical amino acid residues and displayed as a function of TFE concentration.
TFE was increased to 20% and higher, the a-helicity of h/rCRF and its chimeras reached a common plateau level represented by approximately eight to nine complete a-helix turns (Fig. 3A). A similar pattern of a-helicity was observed for rUcnI and its chimeras with the difference that more than 50% of the residues of rUcnI formed an a-helix structure already under aqueous conditions (0% TFE) (Fig. 3B). Especially with low TFE concentrations (10% TFE and below), the effect of the PIG and PTN motifs on the secondary structure of rUcnI was even more profound than observed for h/rCRF as indicated by a decrease in the extent of a-helix structure by up to four complete a-helix turns (Fig. 3B). Unfortunately, mUcnII, mUcnIII, and their chimeric and [Thr8Phe9 peptides [Thr8Phe9His10]mUcnII 10 His ]mUcnIII were found to be not sufficiently soluble so that the concentration of 10 lM peptide required for the CD measurements could not be reached. Especially with relatively low TFE concentrations (20% TFE and below) the maximal solubility of these pep-
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tides was in part less than 2 lM, a concentration that was not compatible with the methodological requirements of CD spectroscopy. This low solubility of mUcnII and mUcnIII at physiological pH was consistent with our previous experience when we tried to dissolve these peptides in artificial cerebrospinal fluid for injections into the mouse brain (Olaf Jahn, Cedomir Todorovic, and Joachim Spiess; unpublished observations).
4. Discussion The pharmacological properties of mUcnII and mUcnIII observed here strongly confirm the notion that these novel peptides are selective and potent agonists for CRF2. In agreement with the original study on the pharmacology of mUcnII and mUcnIII (Lewis et al., 2001), we found that these peptides were equipotent in view of their potency to stimulate the cAMP production of HEK-mCRF2b cells. However, the observed biological potencies of mUcnII and mUcnIII did not match their binding affinities. Whereas mUcnII exhibited a subnanomolar affinity to mCRF2b, mUcnIII was bound to mCRF2b with an affinity that was decreased in comparison to the affinity of mUcnII by a factor of 56 (Table 1). This difference in affinity may be due to the competition binding assay used. As already shown in the framework of the characterization of CRF receptors (Perrin et al., 1999; Ru¨hmann et al., 1999), one has to consider that the absolute values of the affinity constants determined by competition binding assays depend on the pharmacological properties of the radiolabeled peptide employed. Thus, the differences in the efficiencies of mUcnII and mUcnIII to displace radiolabeled Svg bound to mCRF2b may be explained by the possibility that different parts of this receptor are involved in the interaction with mUcnII and mUcnIII, respectively. This assumption is supported by a recent study showing that mUcnII, but not mUcnIII binds with high affinity to the soluble N-terminal extracellular domain of mCRF2b (Perrin et al., 2003). The differential effects on CRF receptor binding which were observed upon introduction of the sequence Thr-Phe-His into mUcnII and mUcnIII, respectively (Table 1), could be interpreted as further evidence for pharmacological differences between these peptides. Similarly, the binding of h/rCRF and rUcnI to mCRF2b was differentially affected by introduction of the PIG and PTN motifs. These motifs only led to a slight decrease in affinity to mCRF2b when introduced into h/rCRF, but significantly decreased the affinity to mCRF2b by two orders of magnitude when introduced into rUcnI (Table 1). This discrepancy may be also explained by different binding mechanisms of h/rCRF and rUcnI. For example, different requirements of
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receptor-G protein coupling for binding of ovine CRF and rUcnI to CRF receptors were proposed on the basis of the observation that binding of ovine CRF, but not rUcnI is modulated by guanyl nucleotides (Perrin et al., 1999). A clear correlation between a lack of specific binding to rCRF1 and a lack of any detectable biological potency to stimulate the cAMP production of HEKrCRF1 cells was observed for mUcnIII (Table 1). In contrast, despite its low affinity for rCRF1, mUcnII displayed a considerable potency to stimulate the cAMP production of HEK-rCRF1 cells (Table 1). In view of its efficacy, mUcnII elicited maximal cAMP responses similar to those observed with CRF1 agonists such as h/rCRF and rUcnI (data not shown). It was speculated that the unexpected high cAMP levels elicited by mUcnII were possibly typical for the HEKrCRF1 cells used and, thus, a consequence of the transfection. In support of this speculation, the relatively high potency of mUcnII to stimulate the cAMP production of HEK-rCRF1 cells could not be confirmed for Y79 human retinoblastoma cells—a cell line of neuronal origin which produces endogenous CRF1 (Olaf Jahn and Joachim Spiess, unpublished observations). Such differences between natural and recombinant receptor systems may be explained by the altered relative stoichiometry of receptors to host membrane components such as G proteins. There are numerous examples reported to show that the quantity and/or quality of agonist efficacy can change in recombinant test systems due to receptor overexpression (reviewed in Kenakin, 1997). In contrast to the original finding that neither UcnII nor UcnIII exhibit appreciable affinity to human CRFBP (Lewis et al., 2001), we identified mUcnII but not mUcnIII as a high-affinity ligand for rCRFBP. This discrepancy may be explained by species differences. Interestingly, mUcnII contains Ala, a small hydrophobic residue, in position 19 of the amino acid sequence (Fig. 1). The presence of Ala in the corresponding position of the sequence is a common feature shared by h/rCRF and rUcnI (Fig. 1), as well as by other CRF-like peptides binding with high affinity to rCRFBP (Jahn et al., 2001). In contrast, the bulky negatively charged Asp residue present in position 19 of mUcnIII (Fig. 1) may be responsible for the lack of binding of mUcnIII to rCRFBP in agreement with our single amino acid switch concept (Eckart et al., 2001). In this previous study, it was found that the corresponding residue Ala-22 of h/rCRF was responsible for this peptide’s high affinity to rCRFBP, whereas Glu located in the corresponding position of Svg and [Glu22]h/rCRF prevented high-affinity binding to rCRFBP (Eckart et al., 2001). Although still far from being completely understood, the physiological role of CRFBP in the brain may be to limit the availability of
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free ligand for CRF receptor-mediated actions (Karolyi et al., 1999). Thus, as a consequence of the relatively high affinity of mUcnII to rCRFBP, it has to be taken into account that the levels of free mUcnII may be also controlled by binding to this protein as it was proposed for CRF on the basis of the finding that 50% of the total brain CRF are bound to CRFBP in humans (Behan et al., 1995). In view of the co-localization of UcnII (Reyes et al., 2001), CRFBP (Potter et al., 1992), and CRF2 (Van Pett et al., 2000) in the rodent brain, substructures of the hypothalamus and the brain stem may be considered as possible sites for a modulation of UcnII actions by CRFBP. On the basis of secondary structure predictions, we hypothesized that the PIG and PTN motifs present in mUcnII and mUcnIII, respectively, decreased the extent of a-helicity of these peptides. Although secondary structure prediction algorithms are normally designed to predict the structural features of proteins and not of peptides, the predicted a-helical structure of h/rCRF (Fig. 1) was in agreement with data from nuclear magnetic resonance (NMR) studies (Dathe et al., 1996; Romier et al., 1993). It was therefore concluded that the prediction algorithm applied was at least suitable to estimate the helical content of the peptides under investigation. In support of the assumption that a decrease in the ligand’s a-helicity impairs binding to CRF1, replacement of the sequence Thr-Phe-His of h/rCRF and rUcnI by the PIG motif of mUcnII or the PTN motif of mUcnIII dramatically decreased the binding affinity of h/rCRF and rUcnI to CRF1 as well as their potency to stimulate the cAMP production of HEK-rCRF1 cells. This significant pharmacological effect was found to correlate with a decrease in the peptides’ a-helicity as suggested by the data obtained by secondary structure predictions and CD spectroscopy measurements of the chimeric peptides [Pro11 Ile12Gly13]h/rCRF, [Pro11Thr12Asn13]h/rCRF, [Pro10 Ile11Gly12]rUcnI, and [Pro10Thr11Asn12]rUcnI. Interestingly, the PIG motif of mUcnII and the PTN motif of mUcnIII are almost completely conserved in all of their respective orthologs known to date. Thus, the PIG motif is also present in human (Lewis et al., 2001) and rat (accession number Q91WW1) UcnII. Similarly, the PTN motif is also present in human (Hsu and Hsueh, 2001; Lewis et al., 2001), rat (accession number XP_225546), and Takifugu pufferfish (Hsu and Hsueh, 2001) UcnIII. Only in Tetraodon pufferfish UcnIII (Hsu and Hsueh, 2001), Asn of the PTN motif is replaced by Ser and thus by a residue that also has a high propensity to form reverse turn structures. It may be therefore concluded that the presence of an a-helixbreaking amino acid motif in the N-terminal part of CRF2-selective agonists is a common structural feature that impairs binding to CRF1 and thereby determines selectivity for CRF2. However, on the basis of the data
presented, it cannot be ruled out completely that functional interactions of the amino acids of the PIG and PTN motifs with the CRF receptor subtypes may also contribute to the selectivity of CRF2 agonists. Additional structural studies such as the NMR measurements recently reported for receptor-bound neurotensin (Luca et al., 2003) will be needed to elucidate the exact conformation of CRF-like peptides during interaction with their receptors.
Acknowledgements Thomas Liepold and Dr. Bodo Zimmermann are gratefully acknowledged for characterizing the peptides by amino acid analysis and mass spectrometry, respectively. We thank Dr. Astrid Sasse and Cedomir Todorovic for helpful discussions.
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Three-amino acid motifs of urocortin II and III determine their CRF receptor subtype selectivity Olaf Jahn, Hossein Tezval, Lars van Werven, Klaus Eckart, Joachim Spiess Department of Molecular Neuroendocrinology, Max Planck Institute for Experimental Medicine, Hermann-Rein-Str. 3, 37075 Goettingen, Germany Received 9 September 2003; received in revised form 17 March 2004; accepted 23 March 2004
Abstract Corticotropin-releasing factor (CRF) and the CRF-like peptide urocortin I (UcnI) exert their activity through two different CRF receptors, CRF1 and CRF2. Recently, UcnII and UcnIII have been discovered as potential endogenous agonists selective for CRF2 known to be involved in brain functions such as learning and anxiety, as well as in cardiovascular functions. A structure–affinity relationship study using chimeric peptides was designed to characterize mouse UcnII (mUcnII) and mUcnIII further and to investigate the structural basis of their receptor subtype selectivity. In the framework of this study, mUcnII (IC50 ¼ 4:4 nM) but not mUcnIII was identified as high-affinity ligand for the rat CRF binding protein. Such affinity had previously not been observed for the human version of this protein. On the basis of secondary structure predictions, it was hypothesized that the amino acid motifs Pro-Ile-Gly of mUcnII and Pro-Thr-Asn of mUcnIII decrease a-helicity and thereby impair binding to CRF1. In support of this hypothesis, binding affinity to CRF1 of the chimeric peptides [Pro11Ile12Gly13]h/rCRF, [Pro11Thr12Asn13]h/rCRF, and the corresponding rUcnI analogs was found to be decreased by three orders of magnitude, whereas binding affinity to CRF2 was much less affected. The dramatic decrease in binding affinity to CRF1 correlated with a decrease in a-helicity as indicated by the data of circular dichroism spectroscopy. # 2004 Elsevier Ltd. All rights reserved. Keywords: Corticotropin-releasing factor (CRF); Urocortin; CRF receptor; CRF binding protein; Secondary structure
1. Introduction The 41 amino acid peptide corticotropin-releasing factor (CRF) (Spiess et al., 1981), originally identified as an early chemical signal in the mammalian responses to stress stimuli (Vale et al., 1981), has been more recently recognized to be also involved in complex brain functions. CRF is a neuromodulator of learning and memory, anxiety, and food intake and is linked to the pathogenesis of various anxiety, mood, and eating disorders (Behan et al., 1996; Eckart et al., 2002). CRF and its natural analog urocortin I (UcnI) (Vaughan et al., 1995) exert their biological activity by binding to two subtypes of the G protein-coupled CRF receptor, CRF1 and CRF2, which have distinct expression profiles and different physiological functions (Eckart et al., Corresponding author. Tel.: +49-551-3899-258; fax: +49-5513899-359. E-mail address: spiess.offi[email protected] (J. Spiess).
0028-3908/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2004.03.018
2002; Perrin and Vale, 1999). Gene-deletion (Bale et al., 2000; Coste et al., 2000; Kishimoto et al., 2000; Smith et al., 1998; Timpl et al., 1998) and pharmacological (Radulovic et al., 1999) studies provided a growing body of evidence to support a partial functional antagonism of these receptor subtypes. Thus, in the central nervous system (CNS), activation of CRF1 accessible through the brain ventricle system predominantly enhances anxiety, whereas CRF2 reached through the ventricles acts anxiolytically. These differential effects of CRF may be also affected by the CRF binding protein (CRFBP) (Potter et al., 1991) which serves as a local reservoir of endogenous ligand (Behan et al., 1995; Karolyi et al., 1999). In the periphery, CRF-like peptides acting at CRF2 modulate cardiovascular functions possibly protecting against cardiac failure (Parkes et al., 2001). As in the CNS, these actions may be also affected by circulating CRFBP—at least in humans. In contrast to rodents, humans are distinguished by a
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significant level of CRFBP in their blood (Potter et al., 1991). Because of its anxiolytic potential and cardioprotective effects, CRF2 has become an interesting target of physiological and pharmacological research. Recent progress in this research was the identification of human stresscopin-related peptide and stresscopin (Hsu and Hsueh, 2001), and the corresponding murine orthologs UcnII and UcnIII (Lewis et al., 2001; Reyes et al., 2001) on the basis of predictions using genomic DNA information and subsequent cDNA cloning. These two new members of the CRF peptide family were identified as agonists selective for CRF2. On the basis of physicochemical measurements (Dathe et al., 1996; Lau et al., 1983; Pallai et al., 1983; Romier et al., 1993) and structure–activity relationship studies (Hernandez et al., 1993; Miranda et al., 1994), it has been suggested that CRF receptor ligands assume an a-helical conformation when interacting with their receptors. By the development of N-terminally truncated CRF-like peptides as peptidic antagonists (Hernandez et al., 1993; Rivier et al., 1984), it was recognized that the N-terminal part of the peptide is not only crucial for receptor activation, but may also induce and stabilize the a-helix structure as the conformation required for high-affinity binding to CRF receptors. In agreement with this observation, the affinity of the antagonists developed on the basis of the sequence of h/rCRF9-41 (Rivier et al., 1984) or h/rCRF12-41 (Hernandez et al., 1993) to both CRF receptor subtypes was found to be significantly decreased compared to that of the parent peptide h/rCRF (Perrin and Vale, 1999). Since the decrease in affinity is more profound for CRF1, it was observed that such truncated h/rCRF analogs display a clear preference for CRF2 (Perrin and Vale, 1999). This differential effect on receptor binding was found to be even more significant when the 40 amino acid CRF-like peptide sauvagine (Svg) (Montecucchi and Henschen, 1981) was N-terminally truncated. Peptides derived from Svg by N-terminal truncation lost their affinity to CRF1 almost completely, while retaining their affinity to CRF2. This finding finally led to the development of the highly selective CRF2 antagonist antisauvagine-30 (Ru¨hmann et al., 1998). In agreement with these observations, a recent study using various N-terminally truncated CRF-like peptides emphasized that the structural requirements of CRF1 for high-affinity binding of peptidic ligands are more stringent than the corresponding requirements of CRF2 (Brauns et al., 2002b). Furthermore, the affinity of N-terminally truncated CRF-like peptides, especially to CRF1, can be restored by introduction of a lactam bridge between the side chains of amino acid residues 30 and 33 of h/rCRF (Gulyas et al., 1995) or 29 and 32 of Svg (Brauns et al., 2002a). Since a lactam bridge functions as a structural constraint probably stabilizing the a-helical confor-
mation of the peptide (Gulyas et al., 1995), these findings further support the assumption that an a-helix structure is required for high-affinity binding—at least to CRF1. Taken together, there is suggestive evidence that the affinity of a peptide to CRF1 may be enhanced by its a-helicity, whereas binding to CRF2 appears to be less sensitive to a decrease in a CRF-like peptide’s a-helicity. The objective of the present study was to investigate the structural basis for the CRF2 selectivity of UcnII and UcnIII on the basis of the hypothesis that these peptides contain amino acid motifs which disturb the integrity of their a-helix structure and thereby impair binding to CRF1. To identify such motifs, we compared the amino acid sequences of the CRF2-selective agonists mUcnII and mUcnIII (Lewis et al., 2001) with the sequences of the other natural rodent agonists human/rat CRF (h/rCRF), preferably binding to CRF1, and rat UcnI (rUcnI), exhibiting no selectivity (Behan et al., 1996; Eckart et al., 2002). On the basis of sequence alignments and secondary structure predictions, candidate amino acid motifs were identified in mUcnII and mUcnIII. These motifs possibly affect a-helicity and thereby receptor selectivity of these peptides. Our hypothesis was tested by introduction of the identified motifs into h/rCRF and rUcnI, respectively. The pharmacological properties of the resulting chimeric peptides were investigated by using stably transfected cell lines producing either rat CRF1 (rCRF1), mouse CRF2b (mCRF2b) or rat CRFBP (rCRFBP). Circular dichroism (CD) spectroscopy was used to analyze the peptides’ secondary structures. Parts of this work have been presented as abstracts in preliminary form to the Society for Neuroscience (Jahn et al., 2002) and the Endocrine Society (Jahn et al., 2003).
2. Methods 2.1. Peptide synthesis Peptides were synthesized, purified, and characterized by mass spectrometry as described recently (Jahn et al., 2001). The final concentration of peptide stock solutions was determined by amino acid analysis with a Beckman high performance ion exchange analyzer system 6300. Total hydrolysis of the peptides was carried v out with 6 M HCl for 24 h at 100 C in the presence of 1 nmol norvaline as internal standard. 2.2. Binding assays Crude membrane fractions were prepared from transfected human embryonic kidney (HEK) 293 cells stably producing either rCRF1 (HEK-rCRF1 cells) or mCRF2b (HEK-mCRF2b cells) as described (Ru¨hmann
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et al., 1998). rCRFBP was produced in HEK 293 cells stably transfected with cDNA coding for rCRFBP C-terminally fused with a His-tag as described (Jahn et al., 2001). The utilization of a scintillation proximity assays (SPA) for competition binding analysis of CRF receptors (Eckart et al., 2001; Hofmann et al., 2001) and CRFBP (Eckart et al., 2001) was recently established in our laboratory. For binding analysis of CRF-like peptides to CRF receptors, wheat germ agglutinin-coated SPA beads (neuropeptide Y receptor SPA binding assay, Amersham Pharmacia Biotech) were used as described (Hofmann et al., 2001). [125ITyr0]h/rCRF and [125I-Tyr0]Svg (NEN) were employed as radiolabeled ligands for rCRF1 and mCRF2b, respectively. Binding of CRF-like peptides to His-tagged rCRFBP was carried out in nickel-chelate-coated 96 well SPA microtiter plates (Flash Plate PLUS2, NEN) as described (Eckart et al., 2001). [125I-Tyr0]h/ rCRF (NEN) was employed as radiolabeled ligand. Competition binding curves were analyzed by nonlinear regression using the Prism computer program (GraphPad Software). 2.3. Measurement of intracellular cAMP accumulation HEK 293 cells were plated into 24 well cell culture plates and stimulated as described (Eckart et al., 2001) using increasing concentrations of the peptides under investigation. Intracellular cAMP was measured with the Biotrak2 cAMP [125I] SPA system (Amersham Pharmacia Biotech) according to the manufacturer’s product manual. Dose–response curves were analyzed by non-linear regression using the Prism computer program (GraphPad Software). 2.4. Secondary structure prediction For secondary structure prediction, the amino acid sequences of the CRF-like peptides were submitted to the PredictProtein server (http://www.emblheidelberg.de/predictprotein). On the basis of the submitted sequence, an automatic PSI-BLAST multiple sequence alignment was performed against the SWISSPROT database and this alignment was used as input for the PROFsec secondary structure prediction algorithm (Rost and Sander, 1993). 2.5. CD spectroscopy The CD spectra were recorded in the range of 190– 260 nm as described recently (Brauns et al., 2002a). The CRF-like peptides were dissolved in 10 mM sodium phosphate buffer (pH 7.4; Suprapure, Merck) containing various concentrations of 2,2,2-trifluoroethanol (TFE) as a-helix structure-inducing organic co-solvent. Relatively low final peptide concentrations in the
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range of 10 lM were used because it has been shown— at least for h/rCRF—that higher concentrations lead to association which is accompanied by an increase in the a-helicity of the peptide (Dathe et al., 1996). The exact peptide concentrations were determined by amino acid analysis as described above. The CD spectra were deconvoluted by using the method of Bo¨hm et al. (1992) (CDNN).
3. Results 3.1. Design of the chimeric peptides The aligned amino acid sequences of h/rCRF, rUcnI, mUcnII, and mUcnIII were first compared in view of the conformational preferences of the single amino acid residues (Williams et al., 1987) with the objective to identify candidate amino acid motifs that possibly affect the integrity of the a-helix structure of mUcnII and mUcnIII, respectively (Fig. 1). This comparison revealed that mUcnII contains Pro and Gly in positions 8 and 10 in contrast to h/rCRF and rUcnI containing Thr and His in the corresponding positions (Fig. 1). mUcnIII like mUcnII contains Pro and Asn in positions 8 and 10 (Fig. 1). Interestingly, Pro, Gly, and Asn have high propensities to form reverse turn structures (Williams et al., 1987) and, thus, rarely occur in the interiors of a-helical structures. Therefore, it was hypothesized that the amino acid sequences Pro-Ile-Gly (PIG motif) and Pro-Thr-Asn (PTN motif) assume a-helix-breaking conformations and thereby decrease the extent of a-helix structure of mUcnII and mUcnIII. In agreement with this hypothesis, secondary structure predictions indicated that the a-helices in mUcnII and mUcnIII were shifted C-terminally by three to four residues in comparison to their location in h/rCRF and rUcnI, respectively (Fig. 1). This shift corresponded to approximately one complete a-helix turn consisting of 3.6 amino acid residues (Fig. 1). To test whether the presence of the PIG and PTN motifs in mUcnII and mUcnIII, respectively, impaired binding to CRF1, the chimeric peptides [Pro11Ile12Gly13]h/ rCRF, [Pro11Thr12Asn13]h/rCRF, [Pro10Ile11Gly12] rUcnI, and [Pro10Thr11Asn12]rUcnI were synthesized and subsequently tested for their pharmacological properties. With an inverse approach, the corresponding chimeric peptides [Thr8Phe9His10]mUcnII and [Thr8Phe9His10]mUcnIII were also included in these experiments. 3.2. Pharmacological properties of mUncnII and mUcnIII In the present study, the novel CRF-like peptides mUcnII and mUcnIII were first tested for their binding
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Fig. 1. Amino acid sequence alignment of h/rCRF, rUcnI, mUcnII, and mUcnIII. The amino acid sequences used for replacement are underlaid in gray. Above each residue, its conformational preference is indicated: a ¼ a-helix, b ¼ b-strand, t ¼ reverse turn. Below the sequences, the predicted secondary structures are presented: H, helix; E, extended (loop); dash, other (loop). The black squares represent the C-terminal amide groups.
affinity to rCRF1, mCRF2b, and rCRFBP using the assay systems established in our laboratory (Eckart et al., 2001; Hofmann et al., 2001). In agreement with the data of the original study on the characterization of these peptides (Lewis et al., 2001), mUcnII exhibited high affinity to mCRF2b (IC50 ¼ 0:25 nM), but not to rCRF1 (IC50 >350 nM) (Table 1). Similarly, mUcnIII bound with moderate affinity to mCRF2b (IC50 ¼ 14 nM), whereas specific binding of this peptide to rCRF1 was not detectable (IC50 >2000 nM) (Table 1). Thus, the high selectivity of mUcnII and mUcnIII for CRF2 was confirmed. Interestingly, mUcnII bound to rCRFBP with an affinity in the low nanomolar range (IC50 ¼ 4:4 nM), whereas mUcnIII did not exhibit any detectable affinity (IC50 >2000 nM) to this protein (Table 1). Although the affinity of mUcnII to rCRFBP
was lower by approximately one order of magnitude than the affinities of h/rCRF (IC50 ¼ 0:54 nM) and rUcnI (IC50 ¼ 0:98 nM), respectively, mUcnII was found to be a high-affinity ligand for rCRFBP (IC50 ¼ 4:4 nM). The biological potency of mUcnII and mUcnIII was evaluated by the determination of EC50 values for agonist-induced intracellular accumulation of cAMP in HEK-rCRF1 or HEK-mCRF2b cells. In agreement with the data of the original study (Lewis et al., 2001), high potencies of mUcnII (EC50 ¼ 0:43 nM) and mUcnIII (EC50 ¼ 0:83 nM) to stimulate HEK-mCRF2b cells were found (Table 1). Despite its low affinity for rCRF1 (IC50 >350 nM), mUcnII displayed a considerable potency to stimulate the cAMP production of HEK-rCRF1 cells (EC50 ¼ 19 nM) (Table 1).
Table 1 Pharmacological properties of h/rCRF, rUcnI, mUcnII, mUcnIII, and their chimeric analogs Peptide
h/rCRF rUcnI mUcnII mUcnIII [Pro11Ile12Gly13]h/rCRF [Pro11Thr12Asn13]h/rCRF [Pro10Ile11Gly12]rUcnI [Pro10Thr11Asn12]rUcnI [Thr8Phe8His10]mUcnII [Thr8Phe9His10]mUcnIII
IC50 (nM)
EC50 (nM)
rCRF1
mCRF2b
rCRFBP
rCRF1
mCRF2b
1.6 (1.3–1.9) 0.17 (0.11–0.23) 350 (320–380) >2000 >2000 >2000 340 (180–500) 550 (420–680) 22 (11–32) 1300 (750–1900)
42 (25–59) 0.86 (0.67–0.92) 0.25 (0.08–0.43) 14 (10–19) 160 (150–170) 60 (49–71) 47 (35–59) 51 (43–58) 0.26 (0.16–0.35) 4.0 (3.1–5.0)
0.54 (0.38–0.71) 0.98 (0.66–1.3) 4.4 (3.7–5.2) >2000 n.d. n.d. n.d. n.d. n.d. n.d.
0.33 (0.26–0.40) 0.89 (0.78–1.0) 19 (15–24) >2000 110 (63–160) 78 (8.0–150) 30 (20–40) 210 (130–290) 0.45 (0.20–0.70) 22 (4.7–39)
1.6 (1.0–2.2) 0.84 (0.35–1.3) 0.43 (0.28–0.57) 0.83 (0.56–1.1) 21 (13–28) 25 (15–36) 2.5 (1.8–3.2) 7.6 (5.8–9.3) 0.61 (0.43–0.79) 4.0 (1.8–6.0)
IC50 values are the mean of four independent competition binding assays. EC50 values are the mean of three independent cell stimulation experiments. 95% confidence intervals are given in parentheses. n.d., not determined.
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3.3. The effect of the PIG and PTN motifs on receptor binding The sequence Thr11Phe12His13 of h/rCRF was first replaced by the PIG motif of mUcnII. In comparison to the high affinity of the parent peptide h/rCRF to rCRF1 (IC50 ¼ 1:6 nM), the affinity of the chimeric peptide [Pro11Ile12Gly13]h/rCRF to this receptor subtype (IC50 >2000 nM) was almost completely abolished (Fig. 2A; Table 1). In contrast, its affinity to mCRF2b was only slightly decreased (IC50 ¼ 160 nM) compared to that of h/rCRF (IC50 ¼ 42 nM) (Fig. 2B; Table 1). With an inverse approach, the PIG motif of mUcnII was replaced by the corresponding sequence Thr-PheHis present in h/rCRF. In comparison to the highly selective CRF2 ligand mUcnII, the chimeric peptide [Thr8Phe9His10]mUcnII exhibited a relatively high affinity to rCRF1 (IC50 ¼ 22 nM) (Fig. 2A; Table 1), whereas the affinities of mUcnII and [Thr8Phe9 His10]mUcnII (IC50 ¼ 0:26 nM) to mCRF2b did not deviate significantly from each other (Fig. 2B; Table 1).
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For a more detailed investigation of the effects of the PIG and PTN motifs on receptor binding, the chimeric approach described above was extended to rUcnI and mUcnIII. First, the sequence Thr11Phe12His13 of h/rCRF was replaced by the PTN motif of mUcnIII. In agreement with the lack of affinity to rCRF1 observed for [Pro11Ile12Gly13]h/rCRF, the affinity of [Pro11Thr12Asn13]h/rCRF (IC50 >2000 nM) to this receptor subtype was also almost completely abolished (Table 1). In contrast, its affinity to mCRF2b was not significantly changed (IC50 ¼ 60 nM) compared to that of h/rCRF (Table 1). A similar observation—at least in view of binding to rCRF1—was made when the sequence Thr10Phe11His12 of rUcnI was replaced either by the PIG motif of mUcnII or by the PTN motif of mUcnIII. The high affinity of rUcnI to rCRF1 (IC50 ¼ 0:17 nM) was decreased by three orders of magnitude upon introduction of the PIG or PTN motifs as demonstrated by the low affinities of [Pro10Ile11Gly12]rUcnI (IC50 ¼ 340 nM) and [Pro10 Thr11Asn12]rUcnI (IC50 ¼ 550 nM) to rCRF1 (Table 1). The affinities of rUcnI, [Pro10Ile11Gly12]rUcnI, and [Pro10Thr11Asn12]rUcnI to mCRF2b were also decreased, but to a lesser extent, as indicated by the respective IC50 values of 0.86, 47, and 51 nM (Table 1). Furthermore, the PTN motif of mUcnIII was replaced by the corresponding sequence Thr-Phe-His present in rUcnI. When the highly selective CRF2 ligand mUcnIII and the chimeric peptide [Thr8Phe9His10]mUcnIII (IC50 ¼ 1300 nM) were compared, no marked increase in affinity to rCRF1 was detected as was for [Thr8Phe9 His10]mUcnII (Table 1). While mUcnII and [Thr8Phe9 His10]mUcnII were similar in view of their affinity to mCRF2b, [Thr8Phe9His10]mUcnIII (IC50 ¼ 4:0 nM) exhibited a slightly higher affinity to this receptor subtype than the parent peptide mUcnIII (Table 1). 3.4. The effect of the PIG and PTN motifs on biological potency
Fig. 2. Binding characteristics of h/rCRF, mUcnII, and their chimeric analogs. Competition curves are shown for the binding of h/rCRF, [Pro11Ile12Gly13]h/rCRF, mUcnII, and [Thr8Phe9His10] mUcnII to rCRF1 (A) or mCRF2b (B). Competition curves were normalized by total binding in the absence of competitor (B0). Data points represent pooled data from four independent experiments performed in duplicate.
The biological potency was determined by the intracellular cAMP accumulation in HEK-rCRF1 and HEK-mCRF2b cells, respectively, after stimulation with the ligand under investigation. The chimeric peptides [Pro11Ile12Gly13]h/rCRF, [Pro11Thr12Asn13]h/r CRF, [Pro10Ile11Gly12]rUcnI, [Pro10Thr11Asn12]rUcnI, [Thr8Phe9His10]mUcnII, and [Thr8Phe9His10]mUcnIII were subjected to this assay. The high potency of h/rCRF to stimulate the cAMP production of HEKrCRF1 cells (EC50 ¼ 0:33 nM) was decreased by two orders of magnitude upon introduction of the PIG and PTN motifs as demonstrated by the low potencies of [Pro11Ile12Gly13]h/rCRF (EC50 ¼ 110 nM) and [Pro11 Thr12Asn13]h/rCRF (EC50 ¼ 78 nM) (Table 1). Similarly, compared with the potency of the parent peptide rUcnI (EC50 ¼ 0:89 nM), the potencies of [Pro10
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Ile11Gly12]rUcnI (EC50 ¼ 30 nM) and [Pro1011 12 Thr Asn ]rUcnI (EC50 ¼ 210 nM) to stimulate rCRF1 were also significantly decreased (Table 1). In contrast, when these peptides were tested for their potencies to stimulate HEK-mCRF2b cells, a less profound decrease in potencies was observed for [Pro11Ile12Gly13]h/rCRF (EC50 ¼ 21 nM), [Pro11Thr12 13 10 11 Asn ]h/rCRF (EC50 ¼ 25 nM), [Pro Ile Gly12]rUcnI (EC50 ¼ 2:5 nM), and [Pro10Thr11Asn12]rUcnI (EC50 ¼ 7:6 nM) in comparison to the potencies of the parent peptides h/rCRF (EC50 ¼ 1:6 nM) and rUcnI (EC50 ¼ 0:84 nM) (Table 1). Upon introduction of the sequence Thr-Phe-His, the potencies of mUcnII and mUcnIII to stimulate HEK-rCRF1 cells were increased by two orders of magnitude as shown by the relatively low EC50 values of [Thr8Phe9His10]mUcnII (EC50 ¼ 0:45 nM) and [Thr8Phe9His10]mUcnIII (EC50 ¼ 22 nM) (Table 1). In contrast, the potencies of these peptides to stimulate HEK-mCRF2b cells were not significantly changed for [Thr8Phe9His10]mUcnII (EC50 ¼ 0:61 nM) or slightly decreased for [Thr8Phe9 His10]mUcnIII (EC50 ¼ 4:0 nM) in comparison to the parent peptides mUcnII and mUcnIII, respectively (Table 1). All peptides tested had similar agonist efficacies as indicated by similar maximal cAMP responses in the range of 100 pmol cAMP/105 HEK-rCRF1 cells and of 170 pmol cAMP/105 HEK-mCRF2b cells (data not shown). In agreement with the extremely low potency of mUcnIII to stimulate HEK-rCRF1 cells (Table 1), its agonist efficacy could not be determined on the basis of the peptide concentrations applied (up to 1 lM). 3.5. The effect of the PIG and PTN motifs on peptide secondary structure According to our hypothesis, a reduction of a-helicity of a ligand should impair binding to CRF1. Therefore, we assumed that the extremely low affinities of [Pro11Ile12Gly13]h/rCRF, [Pro11Thr12Asn13]h/rCRF, 10 11 12 [Pro Ile Gly ]rUcnI, and [Pro10Thr11Asn12]rUcnI to rCRF1 were due to a decrease in the extent of a-helix structure caused by the presence of the PIG or PTN motifs. We tested this hypothesis by CD spectroscopic analysis of the secondary structures of these peptides. The CD spectra of the peptides were recorded in the presence of various concentrations of the a-helix structure-inducing organic co-solvent TFE (0–30%) with the objective to investigate the inducibility of the a-helix structure rather than maximal a-helicity. In the presence of relatively low TFE concentrations (0–15% TFE), the extent of a-helix structure of [Pro11 Ile12Gly13]h/rCRF and [Pro11Thr12Asn13]h/rCRF was found to be decreased by approximately one to three complete a-helix turns in comparison to the parent peptide h/rCRF (Fig. 3A). When the concentration of
Fig. 3. a-Helicity derived from CD spectra. The a-helicities are shown for h/rCRF, [Pro11Ile12Gly13]h/rCRF, and [Pro11Thr12Asn13] h/rCRF (A), and rUcnI, [Pro10Ile11Gly12]rUcnI, and [Pro10Thr11 Asn12]rUcnI (B). By deconvolution of the CD spectra, the portions of a-helix structure were obtained in percent, transformed to the number of a-helical amino acid residues and displayed as a function of TFE concentration.
TFE was increased to 20% and higher, the a-helicity of h/rCRF and its chimeras reached a common plateau level represented by approximately eight to nine complete a-helix turns (Fig. 3A). A similar pattern of a-helicity was observed for rUcnI and its chimeras with the difference that more than 50% of the residues of rUcnI formed an a-helix structure already under aqueous conditions (0% TFE) (Fig. 3B). Especially with low TFE concentrations (10% TFE and below), the effect of the PIG and PTN motifs on the secondary structure of rUcnI was even more profound than observed for h/rCRF as indicated by a decrease in the extent of a-helix structure by up to four complete a-helix turns (Fig. 3B). Unfortunately, mUcnII, mUcnIII, and their chimeric and [Thr8Phe9 peptides [Thr8Phe9His10]mUcnII 10 His ]mUcnIII were found to be not sufficiently soluble so that the concentration of 10 lM peptide required for the CD measurements could not be reached. Especially with relatively low TFE concentrations (20% TFE and below) the maximal solubility of these pep-
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tides was in part less than 2 lM, a concentration that was not compatible with the methodological requirements of CD spectroscopy. This low solubility of mUcnII and mUcnIII at physiological pH was consistent with our previous experience when we tried to dissolve these peptides in artificial cerebrospinal fluid for injections into the mouse brain (Olaf Jahn, Cedomir Todorovic, and Joachim Spiess; unpublished observations).
4. Discussion The pharmacological properties of mUcnII and mUcnIII observed here strongly confirm the notion that these novel peptides are selective and potent agonists for CRF2. In agreement with the original study on the pharmacology of mUcnII and mUcnIII (Lewis et al., 2001), we found that these peptides were equipotent in view of their potency to stimulate the cAMP production of HEK-mCRF2b cells. However, the observed biological potencies of mUcnII and mUcnIII did not match their binding affinities. Whereas mUcnII exhibited a subnanomolar affinity to mCRF2b, mUcnIII was bound to mCRF2b with an affinity that was decreased in comparison to the affinity of mUcnII by a factor of 56 (Table 1). This difference in affinity may be due to the competition binding assay used. As already shown in the framework of the characterization of CRF receptors (Perrin et al., 1999; Ru¨hmann et al., 1999), one has to consider that the absolute values of the affinity constants determined by competition binding assays depend on the pharmacological properties of the radiolabeled peptide employed. Thus, the differences in the efficiencies of mUcnII and mUcnIII to displace radiolabeled Svg bound to mCRF2b may be explained by the possibility that different parts of this receptor are involved in the interaction with mUcnII and mUcnIII, respectively. This assumption is supported by a recent study showing that mUcnII, but not mUcnIII binds with high affinity to the soluble N-terminal extracellular domain of mCRF2b (Perrin et al., 2003). The differential effects on CRF receptor binding which were observed upon introduction of the sequence Thr-Phe-His into mUcnII and mUcnIII, respectively (Table 1), could be interpreted as further evidence for pharmacological differences between these peptides. Similarly, the binding of h/rCRF and rUcnI to mCRF2b was differentially affected by introduction of the PIG and PTN motifs. These motifs only led to a slight decrease in affinity to mCRF2b when introduced into h/rCRF, but significantly decreased the affinity to mCRF2b by two orders of magnitude when introduced into rUcnI (Table 1). This discrepancy may be also explained by different binding mechanisms of h/rCRF and rUcnI. For example, different requirements of
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receptor-G protein coupling for binding of ovine CRF and rUcnI to CRF receptors were proposed on the basis of the observation that binding of ovine CRF, but not rUcnI is modulated by guanyl nucleotides (Perrin et al., 1999). A clear correlation between a lack of specific binding to rCRF1 and a lack of any detectable biological potency to stimulate the cAMP production of HEKrCRF1 cells was observed for mUcnIII (Table 1). In contrast, despite its low affinity for rCRF1, mUcnII displayed a considerable potency to stimulate the cAMP production of HEK-rCRF1 cells (Table 1). In view of its efficacy, mUcnII elicited maximal cAMP responses similar to those observed with CRF1 agonists such as h/rCRF and rUcnI (data not shown). It was speculated that the unexpected high cAMP levels elicited by mUcnII were possibly typical for the HEKrCRF1 cells used and, thus, a consequence of the transfection. In support of this speculation, the relatively high potency of mUcnII to stimulate the cAMP production of HEK-rCRF1 cells could not be confirmed for Y79 human retinoblastoma cells—a cell line of neuronal origin which produces endogenous CRF1 (Olaf Jahn and Joachim Spiess, unpublished observations). Such differences between natural and recombinant receptor systems may be explained by the altered relative stoichiometry of receptors to host membrane components such as G proteins. There are numerous examples reported to show that the quantity and/or quality of agonist efficacy can change in recombinant test systems due to receptor overexpression (reviewed in Kenakin, 1997). In contrast to the original finding that neither UcnII nor UcnIII exhibit appreciable affinity to human CRFBP (Lewis et al., 2001), we identified mUcnII but not mUcnIII as a high-affinity ligand for rCRFBP. This discrepancy may be explained by species differences. Interestingly, mUcnII contains Ala, a small hydrophobic residue, in position 19 of the amino acid sequence (Fig. 1). The presence of Ala in the corresponding position of the sequence is a common feature shared by h/rCRF and rUcnI (Fig. 1), as well as by other CRF-like peptides binding with high affinity to rCRFBP (Jahn et al., 2001). In contrast, the bulky negatively charged Asp residue present in position 19 of mUcnIII (Fig. 1) may be responsible for the lack of binding of mUcnIII to rCRFBP in agreement with our single amino acid switch concept (Eckart et al., 2001). In this previous study, it was found that the corresponding residue Ala-22 of h/rCRF was responsible for this peptide’s high affinity to rCRFBP, whereas Glu located in the corresponding position of Svg and [Glu22]h/rCRF prevented high-affinity binding to rCRFBP (Eckart et al., 2001). Although still far from being completely understood, the physiological role of CRFBP in the brain may be to limit the availability of
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free ligand for CRF receptor-mediated actions (Karolyi et al., 1999). Thus, as a consequence of the relatively high affinity of mUcnII to rCRFBP, it has to be taken into account that the levels of free mUcnII may be also controlled by binding to this protein as it was proposed for CRF on the basis of the finding that 50% of the total brain CRF are bound to CRFBP in humans (Behan et al., 1995). In view of the co-localization of UcnII (Reyes et al., 2001), CRFBP (Potter et al., 1992), and CRF2 (Van Pett et al., 2000) in the rodent brain, substructures of the hypothalamus and the brain stem may be considered as possible sites for a modulation of UcnII actions by CRFBP. On the basis of secondary structure predictions, we hypothesized that the PIG and PTN motifs present in mUcnII and mUcnIII, respectively, decreased the extent of a-helicity of these peptides. Although secondary structure prediction algorithms are normally designed to predict the structural features of proteins and not of peptides, the predicted a-helical structure of h/rCRF (Fig. 1) was in agreement with data from nuclear magnetic resonance (NMR) studies (Dathe et al., 1996; Romier et al., 1993). It was therefore concluded that the prediction algorithm applied was at least suitable to estimate the helical content of the peptides under investigation. In support of the assumption that a decrease in the ligand’s a-helicity impairs binding to CRF1, replacement of the sequence Thr-Phe-His of h/rCRF and rUcnI by the PIG motif of mUcnII or the PTN motif of mUcnIII dramatically decreased the binding affinity of h/rCRF and rUcnI to CRF1 as well as their potency to stimulate the cAMP production of HEK-rCRF1 cells. This significant pharmacological effect was found to correlate with a decrease in the peptides’ a-helicity as suggested by the data obtained by secondary structure predictions and CD spectroscopy measurements of the chimeric peptides [Pro11 Ile12Gly13]h/rCRF, [Pro11Thr12Asn13]h/rCRF, [Pro10 Ile11Gly12]rUcnI, and [Pro10Thr11Asn12]rUcnI. Interestingly, the PIG motif of mUcnII and the PTN motif of mUcnIII are almost completely conserved in all of their respective orthologs known to date. Thus, the PIG motif is also present in human (Lewis et al., 2001) and rat (accession number Q91WW1) UcnII. Similarly, the PTN motif is also present in human (Hsu and Hsueh, 2001; Lewis et al., 2001), rat (accession number XP_225546), and Takifugu pufferfish (Hsu and Hsueh, 2001) UcnIII. Only in Tetraodon pufferfish UcnIII (Hsu and Hsueh, 2001), Asn of the PTN motif is replaced by Ser and thus by a residue that also has a high propensity to form reverse turn structures. It may be therefore concluded that the presence of an a-helixbreaking amino acid motif in the N-terminal part of CRF2-selective agonists is a common structural feature that impairs binding to CRF1 and thereby determines selectivity for CRF2. However, on the basis of the data
presented, it cannot be ruled out completely that functional interactions of the amino acids of the PIG and PTN motifs with the CRF receptor subtypes may also contribute to the selectivity of CRF2 agonists. Additional structural studies such as the NMR measurements recently reported for receptor-bound neurotensin (Luca et al., 2003) will be needed to elucidate the exact conformation of CRF-like peptides during interaction with their receptors.
Acknowledgements Thomas Liepold and Dr. Bodo Zimmermann are gratefully acknowledged for characterizing the peptides by amino acid analysis and mass spectrometry, respectively. We thank Dr. Astrid Sasse and Cedomir Todorovic for helpful discussions.
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