Báo cáo Y học: Development of a selective photoactivatable antagonist for corticotropin-releasing factor receptor, type 2 (CRF2)

doi:10.1046/j.1432-1033.2002.03246.x

Eur. J. Biochem. 269, 5288–5294 (2002) (cid:1) FEBS 2002

Development of a selective photoactivatable antagonist for corticotropin-releasing factor receptor, type 2 (CRF2)

Ines Bonk1 and Andreas Ru¨ hmann2 1WITA Proteomics AG, Teltow/Berlin, Germany; 2Institute for Molecular Biosciences, The University of Queensland, St Lucia, Australia

photo astressin, ATB-[His12]Svg(12)40) showed high select- ive binding to mCRF2b (Ki ¼ 3.1 ± 0.2 nM) but not the rCRF1 receptor (Ki ¼ 142.5 ± 22.3 nM) and decreased Svg-stimulated cAMP activity in mCRF2b-expressing cells in a similar fashion as aSvg-30. A 66-kDa protein was identified by SDS/PAGE, when the radioactively iodinated analog of ATB-[His12]Svg(12)40) was covalently linked to mCRF2b receptor. The specificity of the photoactivatable 125I-labeled CRF2b antagonist was demonstrated with SDS/PAGE by the finding that this analog could be displaced from the receptor by antisauvagine-30, but not other unrelated pep- tides such as vasoactive intestinal peptide (VIP).

Keywords: photoaffinity labeling; antisauvagine-30; corti- cotropin-releasing factor (CRF) receptor; CRF antagonist; human embryonic kidney 293 cells.

A novel photoactivatable analog of antisauvagine-30 (aSvg- 30), a specific antagonist for corticotropin-releasing factor (CRF) receptor, type 2 (CRF2), has been synthesized and characterized. The N-terminal amino-acid D-Phe in aSvg-30 [D-Phe11,His12]Svg(11)40) was replaced by a phenyldiazirine, the 4-(1-azi-2,2,2-trifluoroethyl)benzoyl (ATB) residue. The photoactivatable aSvg-30 analog ATB-[His12]Svg was tested for its ability to displace [125I-Tyr0]oCRF or [125I-Tyr0]Svg from membrane homogenates of human embryonic kidney (HEK) 293 cells stably transfected with cDNA coding for rat CRF receptor, type 1 (rCRF1) or mouse CRF receptor, type 2b (mCRF2b). Furthermore, the ability of ATB-[His12]Svg(12)40) to inhibit oCRF- or Svg- stimulated cAMP production of transfected HEK 293 cells expressing either rCRF1 (HEK-rCRF1 cells) or mCRF2b (HEK-mCRF2b cells) was determined. Unlike astressin and

been found in the brain and pituitary of rodents when compared with humans, many of the anxiety-related behavioral effects have been suggested to be governed by CRF1 receptor [5]. In this view, several CRF1-specific nonpeptide antagonists are currently being investigated in clinical phase II studies as potential drugs for anxiety- related diseases [6].

Corticotropin-releasing factor (CRF), a 41-amino-acid polypeptide is a neuroendocrine mediator that plays a key role in the regulation of adrenocorticotropic hormone and other preopiomelanocorticotropin products from the anter- ior pituitary [1]. CRF is produced in brain and peripheral organs where it is recognized as a critical neuropeptide mediator of stress-related endocrine, autonomic, immuno- logic and behavioral responses [2].

The 40-amino-acid peptide urocortin (Ucn) a naturally occurring CRF analog was proposed to be the endogenous ligand for the CRF2 receptor [7]. However, Ucn also exhibited high affinity binding to CRF1 [7], and fibers that express Ucn did not correlate with targets in the brain bearing CRF2 receptors [8]. With the recent discovery of Ucn II [9] and Ucn III [10] also known as stresscopin-related peptide or stresscopin [11], respectively, novel peptide agonists specifically binding to CRF2 receptors have been identified. The functional role of these peptides remains to be elucidated.

CRF mediates its action through two distinct G protein-coupled receptors: CRF receptor types 1 (CRF1) and 2 (CRF2). While CRF1 has been found at high level in cortical and cerebellar structures of the brain and pituitary, CRF2 expression is generally confined to subcortical structures. This distribution of CRF2 receptor in the brain is consistent with different roles of CRF and similar ligands to control food intake and stress-related behavior (reviewed in [3,4]). Although a substantially different distribution pattern for CRF1 and CRF2 has

We recently designed, synthesized and characterized for the first time a CRF2-selective antagonist [12]. This compound named antisauvagine-30 (aSvg-30) showed high selectivity binding to CRF2a and CRF2b but not to the CRF1 receptor or the CRF binding protein [12–14]. Results obtained from pharmacological studies have confirmed that aSvg-30 acts as a competitive antagonist at CRF2 receptors [15]. Consequently aSvg-30 has helped to elucidate the role of CRF2 receptors in learning and memory function [13], anxiety [16] environmental stress [17], eating disorders [18,19] and drug addiction [20].

After the successful synthesis and characterization of photoprobes based on the amino-acid sequence of oCRF

Correspondence to A. Ru¨ hmann, Institute for Molecular Biosciences, The University of Queensland, St. Lucia QLD 4072, Australia. Fax: + 61 7 3365 1990, Tel.: + 61 7 3365 1271, E-mail: a.ruhmann@imb.uq.edu.au Abbreviations: Svg, sauvagine; aSvg-30, antisauvagine-30; CRF, corticotropin-releasing factor; h/r/oCRF, human/rat/ovine CRF; astressin, {cyclo(30-33)[D-Phe12,Nle21,38, Glu30,Lys33]h/rCRF(12- 41)}; ATB, 4-(1-azi-2,2,2-trifluoroethyl)benzoyl; HEK, human embryonic kidney. (Received 2 July 2002, revised 3 September 2002, accepted 10 September 2002)

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M A T E R I A L S A N D M E T H O D S

Synthesis of 4-(1-Azi-2,2,2-trifluoroethyl)benzoic acid

4-(1-Azi-2,2,2-trifluoroethyl)benzoic acid was synthesized in an eight step synthesis as described [21,31].

[21] and astressin [22], a conformationally constrained nonselective CRF peptide antagonist [12,23], we were now interested in the development of a potent and selective photoactivatable CRF antagonist, based on the amino-acid sequence of aSvg-30 to further investigate the different structural requirements for agonist and antagonist binding to CRF1 and CRF2.

Several CRF receptor

Synthesis and purification of peptides

The CRF peptides (Fig. 1) were synthesized, purified, and characterized as described [12,21,22].

cross-links with molecular masses in the range of 58 000–75 000 have been char- acterized applying bifunctional reagents to membranes of bovine anterior pituitary membranes [24], AtT-20 mouse pituitary tumor cells [25] rat brain, and anterior pituitary [26,27].

Labeling through monofunctional photoaffinity probes is expected to provide higher yields than labeling with chemical cross-linking methods using bifunctional reagents. Addi- tionally, photoactivation is assumed to be superior over thermal activation, because highly reactive species such as carbenes and nitrenes can be selectively formed after irradiation under mild conditions. The carbenes or nitrenes formed can insert into X-H bonds and thereby attack groups that are normally inert to chemical affinity labeling [28].

the

observed

However, a prerequisite for all experiments using a photoaffinity labeling technique is that the photoactivata- ble ligand binds with high affinity to the receptor and that the receptor is not destroyed or deactivated by the light used to activate the label [28,29]. In this respect, the aryldiazirine group has proven to be highly favorable when compared with other photoactivatable moieties: it decom- poses photochemically under mild conditions [30,31]. The first successful attempt by one of us to utilize this group for photoaffinity labeling was performed with a fatty acid derivative [32]. This methodology was applied to the synthesis of the first photoactivatable CRF2-selective antagonist based on the amino-acid sequence of aSvg-30, which carries 4-(1-azi-2,2,2-trifluoroethyl)benzoyl (ATB) residue and a histidine group for specific radioactive labeling. To further elucidate the role of the aromatic and heteroaromatic N-terminal rings of antisauvagine-30 two tyrosine-11 substituted analogs and a deleted version of aSvg-30 were synthesized and tested for selective binding to CRF1 or CRF2 receptor.

For the synthesis of the cyclized CRF analogs, amino-acid derivatives Fmoc-Glu(OAl)-OH and Fmoc-Lys(Aloc)-OH (PerSeptive Biosystems GmbH, Hamburg, Germany) were used. The side-chain protected peptides were reacted with in HOAc/N-methylaniline/dichloromethane Pd(cid:2)[PPh3]4 (2 : 1 : 40, v/v/v) for three hours and then cyclized with 1-hydroxybenzotriazole/O-(benzotriazol-1-yl)-N,N,N¢,N¢/ tetramethyluronium hexafluorophosphate in dimethylform- amide and N,N-diisopropylethylamine in N-methylpyrroli- dine for 8 h. After removal of the N-terminal Fmoc group with piperidine in N-methylpyrrolidine, 4-(1-azi-2,2,2-tri- fluoroethyl)benzoic acid was linked to the N-terminus of the peptide resin with 1-hydroxybenzotriazole/O-(benzotriazol- 1-yl)-N,N,N¢,N¢/tetramethyluronium hexafluorophosphate in dimethylformamide and N,N-diisopropylethylamine in N-methylpyrrolidine in the dark. The peptides were then cleaved from the resin and purified by preparative RP-HPLC on a Vydac C18 silica gel column (0.46 · 25 cm, 5-lm particle size, 30-nm pore size) with solvents A (0.1% trifluoracetic acid in water) and B (80% MeCN in 0.1% trifluoracetic acid in water) at a flow rate of 1 mLÆmin)1. The samples were eluted with 5% B for 5 min and then with a linear gradient of 5–95% B in 30 min (oCRF: ESI MS calcd 4670.4, observed 4669.2, Rt ¼ 25.9 min; Svg: ESI MS calcd 4600.4, observed 4599.4, Rt ¼ 25.4 min; astressin: ESI MS calculated 3565.1, observed 3563.1, Rt ¼ 24.8 min; ATB-[Ala32stressin: ESI MS calculated 3562.1, ob- served 3561.1, Rt ¼ 30.2 min; aSvg-30: ESI MS calculated 3652.2, 3650.3, Rt ¼ 21.6 min, ATB- [His12]Svg(12)40): ESI MS calculated 3716.3, observed 3715.4, Rt ¼ 26.6 min).

Fig. 1. Comparison of the amino-acid sequence of (A) oCRF, (B) Svg, (C) astressin, (D) ATB- [125I-labeled His13,Ala32]astressin, and (E) ATB-[125I-labeled His12] Svg(12-40).

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Iodination to the photoactivatable aSvg-30 analog

After removal of the medium, cells were lyzed with aqueous 6% trichloroacetic acid (5 min, 100 (cid:2)C) [21,22]. The cell lysates were stored at )70 (cid:2)C until assayed with a RIA (radioimmunoassay) kit (Amersham, Little Chalfont). Data analysis was achieved with the sigmoidal dose–response curve fitting program ALLFIT. Statistical significance was determined across groups with ANOVA, and significant differences between groups were determined by post hoc comparison using the Dunn test.

ATB-[His12]Svg(12)40) was iodinated as described [33,34] and subsequently purified with RP-HPLC and solvents A and B as described above. The sample was eluted with 45% B for 5 min and then with a linear gradient of 45–95% B in 25 min (125I-ATB-[His12]Svg(12)40): Rt ¼ 18.7 (min). A Beckman 171 Radioisotope Detector equipped with a liquid scintillation flow cell (Beckman, Fullerton, CA, USA) was used to monitor radioactivity.

Photoaffinity labeling experiments with 125I-labeled ATB-[His12]Svg(12)40)

Photolysis of ATB-[His12]Svg(12)40), and its radioactively labeled analog 125I-labeled ATB-[His12]Svg(12)40)

Photolysis was performed at a wavelength of 360 nm using a UV Stratalinker (Stratagene) equipped with five 15-W lamps and monitored with a UV spectrophotometer (Beckman DU 650 spectrometer).

The photoaffinity labeling experiments with the radiolabe- led compound and samples (25 lg of protein/tube) were carried out as described [21,22]. Samples were then heated (100 (cid:2)C, 5 min) and subjected to SDS/PAGE. Autoradio- graphy was carried out on a BAS-IP NP 2040P imaging plate. Radioactivity was monitored with a Fujix BAS 2000 scanner (Raytest, Straubenhardt). Gel documentation was accomplished with the program TINA (Raytest).

Crude membrane preparation

R E S U L T S

HEK 293 cells, permanently transfected with cDNA coding for rCRF1 (HEK-rCRF1 cells), and mCRF2b (HEK- mCRF2b cells) were maintained and subjected to membrane preparations as described [12,35].

Synthesis of ATB-[His12]Svg(12)40) and its radioactively labeled analog

Binding assays with CRF peptides

4-(1-Azi-2,2,2-trifluoroethyl)benzoic acid was successfully linked to [His12]Svg(12)40) (Fig. 1). Subsequent radiolabe- ling with 125I gave ATB-[125I-His12]Svg(12)40) with a specific activity of 74 TBqÆmmol)1.

Binding and cAMP assay

(Bayer, Leverkusen),

Ki ¼ 153.6 ± 33.5 nM;

(CRF1:

Binding of the CRF analogs to the rCRF1 and mCRF2b receptor was performed essentially as described previously [12]. Briefly, 5 lL of membrane suspension (25 lg of protein from HEK-rCRF1 cells; 50 lg of protein from HEK-mCRF2b cells) was added to a plate containing CRF peptides (0–1 lM) and 50 000 c.p.m. of either [125I- Tyr0]oCRF (specific activity 81.4 TBqÆmmol)1, 68.25 pM, DuPont NEN, Boston) for the analysis of rCRF1 or [125I- Tyr0]Svg (specific activity 81.4 TBqÆmmol)1, 68.25 pM, DuPont NEN, Boston) for the analysis of mCRF2b in 100 lL incubation buffer (50 mM Tris/Cl, 5 mM MgCl2, 2 mM EGTA, 100 000 kallikrein inhibitor units per litre of 1 mM dithiothreitol, Trasylol 1 mgÆmL)1 BSA, pH 7.4). After incubation (60 min, 23 (cid:2)C), membrane suspension was aspirated through the plate, followed by two washes with assay buffer (0.2 mL, 23 (cid:2)C). Radioactivity of the punched filters was measured with a 1470 WIZARD automatic gamma counter (Bert- hold, Hannover). Specific binding of [125I-Tyr0]oCRF or [125I-Tyr0]Svg to membranes of transfected cells was calculated by subtraction of unspecific binding found in the presence of 1 lM of oCRF or Svg from total binding, respectively. Data analysis was achieved with the nonlinear curve fitting program LIGAND. Statistical analysis was performed with ANOVA, and significant differences between groups were determined by post hoc comparison using the Dunn test. Values of P < 0.01 were considered statistically different.

For the determination of the binding affinity and the biological potency of the photoactivatable CRF antagonists, HEK 293 cell lines, stably transfected with cDNA coding for rCRF1 or mCRF2b were used [21,36]. Scatchard analysis indicated high-affinity binding of oCRF (Ki ¼ 0.6 ± 0.1 nM) and Svg (Ki ¼ 0.7 ± 0.1 nM) to CRF1 but showed significant difference in binding to the CRF2 receptor (Fig. 2). The photoactivatable astressin analog (compound 3) showed similar high affinity binding to CRF1 (Ki ¼ 5.3 ± 1.3 nM) and CRF2 (Ki ¼ 2.6 ± 1.1 nM) when compared with astressin (CRF1: Ki ¼ 5.7 ± 1.6 nM; CRF2: Ki ¼ 4.0 ± 2.3 nM). In contrast the photoactivatable aSvg- 30 analog (compound 1) exhibited high-affinity binding to CRF2 (Ki ¼ 3.1 ± 0.2 nM) but low affinity to CRF1 (Ki ¼ 142.5 ± 22.3 nM) similar when compared with aSvg-30 CRF2: Ki ¼ 1.4 ± 0.4 nM). (Fig. 2, Table 1). The 46-fold preferred binding of compound 1 to CRF2 was similar when compared with the N-terminally modified tyrosine-11 substituted aSvg-30 analogs compounds 7 and 8, which showed high and medium affinity to CRF2 but low affinity to CRF1 receptor, respectively (Table 1). The one amino acid- truncated tyrosine-12 substituted aSvg-30 analog exhibited very low affinity binding to either receptor.

cAMP stimulation

values

dependent manner with EC50

Application of oCRF and Svg to HEK-rCRF1 cells stimulated the accumulation of intracellular cAMP in a dose of 0.41 ± 0.08 nM and 0.19 ± 0.05 nM, respectively. The potency of the two peptide agonists to enhance cAMP in

HEK-rCRF1 cells or HEK-mCRF2b cells were incubated with different CRF agonists in the presence or absence of 1 lM or 10 nM antagonist, or CRF antagonist (1 lM) alone.

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HEK-rCRF1 cells could be inhibited by CRF antagonists in the following rank order: astressin (compound 4), ATB- [Ala32]astressin (compound 3) >> ATB-[His12]Svg(12)40) (compound 1) > [D-Phe11,His12]Svg(11)40) (compound 2), [D-Phe11]Svg(11)40) (compound 6), [Tyr11,His12]Svg(11)40) (compound 7), [Tyr11]Svg(11)40) (compound 8) > [Tyr12] Svg(11)40) (compound 10) (Table 2). The rank order of potencies for the CRF-related peptide antagonists to suppress Svg-stimulated cAMP production in HEK- mCRF2b cells by CRF antagonists was as follows: com- pound 3 > compound 7, compound 2, compound 1 > compound 4 > compound 8, compound 6 > compound 10 (Table 2).

Photoaffinity labeling experiments

As it was found that BSA binds to CRF analogs unspeci- fically [24,25], radioactively labeled photoactivatable com- pound 1 was stored free of any carrier protein, and photoaffinity labeling experiments were performed in buffer solutions in the absence of BSA. A 66-kDa cross-link was identified with SDS/PAGE after irradiation at 360 nm of a mixture of 125I-labeled compound 1 and membranes of HEK-mCRF2b cells (Fig. 3A). No photo cross-link was observed in analogous experiments with membranes of HEK-rCRF1 cells (Fig. 3B). Binding of 125I-labeled com- pound 1 to the CRF2 receptor could be efficiently inhibited by addition of increasing concentrations of antisauvagine- 30 but not 10 lM vasoactive intestinal peptide (VIP) in agreement with the assumed specificity of this photoprobe (Fig. 3A). Furthermore no cross-link could be identified without light activation at 360 nm (not shown).

D I S C U S S I O N

[D-Phe11]Svg(11)40) (compound 6, ·),

After the successful characterization of a photoactivatable radioactively labeled CRF agonist and antagonists at CRF1 receptors, we were now interested in the development of a photoactivatable CRF2-selective antagonist based on the amino-acid sequence of antisauvagine-30 in order to further investigate the structure–activity relationship of CRF ligands to their receptors.

HEK-mCRF2b cells was significantly different (oCRF: EC50 ¼ 11.79 ± 1.96 nM; Svg: EC50 ¼ 0.23 ± 0.05 nM) (not shown). Ovine CRF-stimulated cAMP production in

To this end we examined the binding affinity of photo astressin to CRF1 and CRF2 receptors. Surprisingly, photo

Fig. 2. Displacement of [125I-Tyr0]oCRF (A) or [125I-Tyr0]Svg (B) bound to membrane homogenates of HEK 293 cells stably transfected with cDNA coding for rat CRF receptor, type 1 (rCRF1) (A), or mouse CRF receptor, type 2b (mCRF2b) (B). Displacement was by ATB- [His12]Svg(12)40) (compound 1, d), aSvg-30 (compound 2, j), ATB- [Ala32]astressin (compound 3, m), astressin (compound 4, n), Svg (compound 5, h), [Tyr11, His12]Svg(11)40) (compound 7, +), [Tyr11]Svg(11)40) (compound 8, *), oCRF (compound 9, s), and [Tyr12]Svg(12)40) (compound 10, e).

Table 1. Binding constants of different CRF agonists and antagonists displacing [125I-Tyr0]oCRF from recombinant rCRF1 or [125I-Tyr0]Svg from recombinant mCRF2b.

f

Compound Peptide [125I-Tyr0]oCRF Ki(rCRF1) (nM) [125I-Tyr0]Svg Ki(mCRF2b) (nM) Ki(rCRF1)/ Ki(mCRF2b)

142.5 ± 22.3 153.6 ± 33.5 5.3 ± 1.3 5.7 ± 1.6 0.7 ± 0.1 237.3 ± 27.7c 220.9 ± 89.1d,e >1000 0.6 ± 0.1 1 2 3 4 5 6 7 8 9 10 3.1 ± 0.2 1.4 ± 0.4 2.6 ± 1.1 4.0 ± 2.3 4.5 ± 0.4 3.5 ± 0.2 4.9 ± 1.8 20.8 ± 1.6 162.4 ± 23.8a 428.5 ± 118.9b >1000 45.97 109.71 2.04 1.42 0.15 67.80 45.08 >48.08 0.00 >2.33 ATB-[His12] Svg(12)40) [D-Phe11, His12]Svg(12)40) ATB-[Ala32]astressin b,c,g Astressina Svg [D-Phe11]Svg(11–40) [Tyr11,His12]Svg(11)40) [Tyr11]Svg(11)40) oCRF [Tyr12]Svg(12)40)

Statistically significant differences between the Ki(mCRF2b)values of the peptides. a, P < 0.001 vs. 5; b, P < 0.0001 vs. 1–9. Statistically significant differences between the Ki(rCRF1)values of the peptides. c, P < 0.01 vs. 4; d, P < 0.01 vs. 5; e, P < 0.001 vs. 3 and 4. f Antisauvagine-30 [12]; g photo astressin [22].

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Table 2. Relative potency of CRF antagonists. The relative potency determined by the effect of 10 nM (mCRF2b) or 1 lM (rCRF1) CRF antagonist on the cAMP production stimulated by 1 nM Svg (mCRF2b) or 1 nM oCRF (rCRF1).

HEK-mCRF2b cells HEK-rCRF1 cells

cAMP prod. Antag./Svgk Rel potency Antagl cAMP prod. Antag./oCRFk Rel potency Antagl Compound Peptide

1 2 3 4 5 6 7 8 9 10 Control without peptide ATB-[His12]Svg(12)40) [D-Phe11,His12]Svg(11)40) ATB-[Ala32]astressinb,c Astressina Svg [D-Phe11]Svg(11)40) [Tyr11, His12]Svg(11)40) [Tyr11]Svg(11)40) oCRF [Tyr12]Svg(12)40) – 0.007 ± 0.002 0.004 ± 0.001 0.010 ± 0.001 0.004 ± 0.001 1.00 0.005 ± 0.001 0.008 ± 0.002 0.007 ± 0.002 – 0.006 ± 0.002 0.004 ± 0.001 0.48 ± 0.04 0.42 ± 0.02 0.30 ± 0.05 0.57 ± 0.04c 1.00 0.71 ± 0.04a,b 0.37 ± 0.05 0.67 ± 0.04d,e – 0.80 ± 0.04f – 0.15 ± 0.01j 0.04 ± 0.01 0.10 ± 0.01j 0.10 ± 0.02j – 0.07 ± 0.01j 0.03 ± 0.01 0.09 ± 0.01 1.00 0.06 ± 0.03 0.01 ± 0.003 0.58 ± 0.04 0.71 ± 0.02 0.11 ± 0.03g,h 0.10 ± 0.02i – 0.76 ± 0.14 0.79 ± 0.07 0.82 ± 0.06 1.00 0.90 ± 0.02 –

astressin bound to either receptor in a similar fashion when compared with astressin thus indicating that the substitution of the N-terminal amino-acid D-Phe by the phenyldiazirine residue does not diminish the binding affinity of photo astressin to CRF2 receptors. Furthermore the potency of photo astressin to increase or inhibit sauvagine-stimulated second messenger production in CRF2 expressing cells was comparable to astressin.

Hence, we introduced the phenyldiazirine group into antisauvagine-30. In competition binding studies the photo- activatable analog showed twofold lower preference to be bound with high affinity to CRF2 when compared with antisauvagine-30. However, this difference was not statisti- cally significant.

first extracellular domain of the CRF1 receptor contains major binding determinants for astressin and urocortin [38–40]. On the basis of these data and our findings with chimeric CRF ligands and G protein-coupled and uncou- pled CRF1 receptors [41] it was concluded that an initial contact point is formed between the N and C terminus of the CRF1 receptor and its ligand, respectively. The N-terminal phenylalanine/histidine and phenyldiazirine/ histidine motif of the peptide ligand is then presented to a core within the transmembrane domain of the receptor. Subsequent change of the receptor-ligand complex may trigger Gs-protein coupling and intracellular second mes- senger production. This effect has been found to be more pronounced in receptor–ligand complexes with photoacti- vatable antisauvagine-30 or the previously described Svg(11)40) when compared with antisauvagine-30 thus indicating that the transmembrane binding pocket of the CRF1 receptor may discriminate between the size and charge of the N terminal dipeptide fragment of antisauv- agine-30 analogs.

is noteworthy that

The photoactivatable antisauvagine-30 analog was shown to be as potent as its parent peptide when stimulating cAMP accumulation alone or suppressing agonist-induced second messenger production in CRF2 expressing cells. Both compounds exhibited significantly lower potency to suppress agonist-stimulated cAMP pro- duction in CRF1 cells when compared with astressin or its photoactivatable analog thus indicating the specificity of the novel photo ligand. the It N-terminal amino acid D-Phe in antisauvagine-30 can be replaced by a phenyldiazirine, L-Tyr or D-Tyr [37] residue without diminishing the binding affinity of the ligands to CRF2 receptors. A combination of an aromatic with a heteroaromatic ring at the N-terminus increases the binding affinity of antisauvagine-30 analogs to CRF2 but also CRF1 receptors.

However, the photoactivatable antisauvagine-30 analog did not cross-link to membrane homogenates of cells permanently transfected with the gene coding for CRF1 receptor. In contrast, a photochemical cross-link to a protein with a molecular weight of 66 kDa was formed in photoaffinity labeling studies with the CRF2 receptor. The size of the cross-link was in agreement with the careful analysis of a chemical cross-link obtained from earlier studies with radioactively labeled sauvagine [12]. Formation of the receptor-ligand cross-link was inhibited in a concen- tration-dependent manner in the presence of antisauvagine- 30 but not vasoactive intestinal peptide (VIP) again indicating the specificity of the compound to be bound to CRF2 receptors.

In summary, we have designed, synthesized and charac- terized for the first time a high-affinity photoaffinity probe

At high concentration similar to Svg(11)40) [12] but unlike antisauvagine-30 the photoactivatable antisauvagine-30 analog exhibited significantly higher intrinsic activity in CRF1 cells when compared with control without peptide. From experiments with chimeric receptors of CRF1 and rat growth hormone releasing factor it was concluded that the

Statistically significant differences between the relative potencies of the peptides: a, P < 0.001 vs. 2; b, P < 0.0001 vs. 3 and 7; c, P < 0.001 vs. 3 and 10; d, P < 0.001 vs. 2; e, P < 0.0001 vs. 3 and 7; f, P < 0.0001 vs. 1, 2, 3, and 7; g, P < 0.001 vs. 1; h, P < 0.0001 vs. 2, 6, 7, 8 and 10; i, P < 0.0001 vs. 1, 2, 6, 7, 8, and 10. Statistically significant differences between the relative agonist activities of the peptides: j, P > 0.001 vs. control without peptide. k The ratio of cAMP production of transfected HEK cells stimulated by antagonist (Antag.) or Svg or oCRF served as a measure of the intrinsic activity. l The relative potency determined by the effect of 10 nM (mCRF2b) or 1 lM (rCRF1) CRF antagonist on the cAMP production stimulated by 1 nM Svg (mCRF2b) or 1 nM oCRF (rCRF1).

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R E F E R E N C E S

1. Vale, W., Spiess, J., Rivier, C. & Rivier, J. (1981) Characterization of a 41 residue ovine hypothalamic peptide that stimulates secre- tion of corticotropin and b-endorphin. Science 213, 1394–1397. 2. De Souza, E.B. & Grigoriadis, D.E. (1995) Corticotropin-releas- ing factor. Physiology, pharmacology, and role in central nervous system and immune disorders. In Psychopharmacology: the Fourth Generation of Progress (Bloom, F.E. & Kupfer, D.J., eds), pp. 505–517. Raven Press NY.

3. Dautzenberg, F.M. & Hauger, R.L. (2002) The CRF peptide family and their receptors: yet more partners discovered. Trends Pharmacol. Sci. 23, 71–77.

4. Reul, J.M. & Holsboer, F. (2002) Corticotropin-releasing factor receptors 1 and 2 in anxiety and depression. Curr. Opin. Phar- macol. 2, 23–33.

5. Hiroi, N., Wong, M.L., Licinio, J., Park, C., Young, M., Gold, P.W., Chrousos, G.P. & Bornstein, S.R. (2001) Expression of corticotropin releasing hormone receptors type I and type II mRNA in suicide victims and controls. Mol. Psychiatry. 6, 540– 546.

6. Zobel, A.W., Nickel, T., Kunzel, H.E., Ackl, N., Sonntag, A., Ising, M. & Holsboer, F. (2000) Effects of the high-affinity corti- cotropin-releasing hormone receptor 1 antagonist R121919 in major depression: the first 20 patients treated. J. Psychiatr. Res. 34, 171–181.

7. Vaughan, J., Donaldson, C., Bittencourt, J., Perrin, M.H., Lewis, K., Sutton, S., Chan, R., Turnbull, A.V., Lovejoy, D., Rivier, C., Rivier, J., Sawchenko, P.E. & Vale, W. (1995) Urocortin, a mamalian neuropeptide related to fish urotensin I and to corti- cotropin-releasing factor. Nature (London) 378, 287–292.

8. Bittencourt, J.C., Vaughan, J., Arias, C., Rissman, R.A., Vale, W.W. & Sawchenko, P.E. (1999) Urocortin expression in rat brain: evidence against a pervasive relationship of urocortin-con- taining projections with targets bearing type 2 CRF receptors. J. Comp. Neurol. 415, 285–312.

9. Reyes, T.M., Lewis, K., Perrin, M.H., Kunitake, K.S., Vaughan, J., Arias, C.A., Hogenesch, J.B., Gulyas, J., Rivier, J., Vale, W.W. & Sawchenko, P.E. (2001) Urocortin II: a member of the corti- cotropin-releasing factor (CRF) neuropeptide family that is selectively bound by type 2 CRF receptors. Proc. Natl. Acad. Sci. USA 98, 2843–2848.

Fig. 3. Photoaffinity cross-linking of radioactively labeled compound 3 to HEK 293 cell membrane homogenates. (A) Lanes 1–5 show extracts of cells stably transfected with cDNA coding for mCRF2b. 125I-labeled compound 1 was bound in the absence (lane 1) or in the presence of 100 nM (lane 2), 1 lM (lane 3), 10 lM (4) of antisauvagine-30 (aSvg-30) or 10 lM of vasoactive intestinal peptide (VIP) (lane 5). Twenty-five lg of total membrane protein was labeled with approximately 100 000 c.p.m. of 125I-labeled compound 1 (lanes 1–5) and incubated (37 (cid:2)C, 30 min). (B) Photoaffinity cross-linking of 125I-labeled compound 1 to membrane homogenates of cells stably transfected with cDNA coding for rCRF1. 125I-labeled compound 1 was bound in the absence (lane 1) or in the presence of 10 lM of antisauvagine-30 (aSvg-30) (lane 2). Molecular masses of prestained markers are indicated on the right and left.

10. Lewis, K., Li, C., Perrin, M.H., Blount, A., Kunitake, K., Don- aldson, C., Vaughan, J., Reyes, T.M., Gulyas, J., Fischer, W., Bilezikjian, L., Rivier, J., Sawchenko, P.E. & Vale, W.W. (2001) Identification of urocortin III, an additional member of the cor- ticotropin-releasing factor (CRF) family with high affinity for the CRF2 receptor. Proc. Natl. Acad. Sci. USA 98, 7570–7575. 11. Hsu, S.Y. & Hsueh, A.J. (2001) Human stresscopin and stress- copin-related peptide are selective ligands for the type 2 cortico- tropin-releasing hormone receptor. Nat. Med. 7, 605–611.

specific for CRF receptor type 2b. Due to the similarity of the pharmacological profile of mammalian CRF2a and CRF2b, the new ligand, which we propose to name photo antisauvagine, should serve as a useful tool to detect CRF2 binding sites and elucidate its functional role in the brain and peripheral organs.

12. Ru¨ hmann, A., Bonk, I., Lin, C.R., Rosenfeld, M.G. & Spiess, J. (1998) Structural requirements for peptidic antagonists of the corticotropin-releasing factor receptor (CRF): Development of CRFR2b–selective antisauvagine-30. Proc. Natl. Acad. Sci. USA 95, 15264–15269.

13. Radulovic, J., Ru¨ hmann, A., Liepold, T. & Spiess, J. (1999) Modulation of learning and anxiety by corticotropin-releasing factor (CRF) and stress: differential roles of CRF receptors 1 and 2. J. Neurosci. 19, 5016–5025.

A C K N O W L E D G E M E N T S

14. Higelin, J., Py-Lang, G., Paternoster, C., Ellis, G.J., Patel, A. & Dautzenberg, F.M. (2001) 125I-Antisauvagine-30: a novel and specific high-affinity radioligand for the characterization of corti- cotropin-releasing factor type 2 receptors. Neuropharmacology 40, 114–122.

15. Brauns, O., Liepold, T., Radulovic, J. & Spiess, J. (2001) Phar- macological and chemical properties of astressin, antisauvagine-30 We are grateful to Dr Frank M. Dautzenberg and Dr Andreas K. E. Ko¨ pke for providing the HEK-rCRF1 cells and Dr Chijen R. Lin and Dr Michael G. Rosenfeld for providing the HEK-mCRF2b cells. Thomas Liepold is acknowledged for the performance of the amino- acid analysis. Dr Klaus Eckart is acknowledged for the performance of the mass spectrometric experiments. This work was supported by the Max-Planck Society.

(cid:1) FEBS 2002

5294 I. Bonk and A. Ru¨ hmann (Eur. J. Biochem. 269)

28. Schuster, D.I., Probst, W.C., Ehrlich, G.K. & Singh, G. (1989) Photoaffinity labeling. Photochem. Photobiol. 49, 785–804. and a-helCRF: significance for behavioral experiments. Neuro- pharmacology 41, 507–516. 29. Guillory, R.J. (1989) Design,

implementation and pitfalls of photoaffinity labelling experiments in in vitro preparations. Gen- eral principles. Pharmac. Ther. 41, 1–25. 16. Takahashi, L.K., Ho, S.P., Livanov, V., Graciani, N. & Arneric, S.P. (2001) Antagonism of CRF (2) receptors produces anxiolytic behavior in animal models of anxiety. Brain Res. 902, 135–142.

30. Bayley, H. (1987) Diazirines as Photoactivatable Reagents in Biochemistry. In: Chemistry of Diazirines, Vol. 2 (Liu, M.T.H., ed.), pp. 75–99. CRC Press, Boca Raton, FL.

17. Pelleymounter, M.A., Joppa, M., Ling, N. & Foster, A.C. (2002) Pharmacological evidence supporting a role for central corticotropin-releasing factor (2) receptors in behavioral, but not endocrine, response to environmental stress. J. Pharmacol. Exp. Ther. 302, 145–152. 31. Nassal, M. (1983) 4-(1-Azi-2,2,2-trifluoroethyl) benzoic acid, a highly photolabile carbene generationg label readily fixable to biochemical agents. Liebigs Ann. Chem. 1510–1523.

18. Cullen, M.J., Ling, N., Foster, A.C. & Pelleymounter, M.A. (2001) Urocortin, corticotropin releasing factor-2 receptors and energy balance. Endocrinology 142, 992–999. 32. Ru¨ hmann, A. & Wentrup, C. (1994) Synthesis of a photo- activatable 9-IX-oleic acid for protein kinase C labeling. Tetra- hedron 50, 3785–3796.

33. Ru¨ ckert, Y., Rohde, W. & Furkert, J. (1990) Radioimmunoassay of corticotropin-releasing hormone. Exp. Clin. Endocrinol. 96, 129–137. 19. Pelleymounter, M.A., Joppa, M., Carmouche, M., Cullen, M.J., Brown, B., Murphy, B., Grigoriadis, D.E., Ling, N. & Foster, A.C. (2000) Role of corticotropin-releasing factor (CRF) receptors in the anorexic syndrome induced by CRF. J. Pharmacol. Exp. Ther. 293, 799–806.

34. Vale, W., Vaughan, J., Yamamoto, G., Bruhn, T., Douglas, C., Dalton, D., Rivier, C. & Rivier, J. (1983) Assay of corticotropin releasing factor. Meth. In Enzymol. 103, 565–577.

20. Lu, L., Liu, D., Ceng, X. & Ma, L. (2000) Differential roles of corticotropin-releasing factor receptor subtypes 1 and 2 in opiate withdrawal and in relapse to opiate dependence. Eur. J. Neurosci. 12, 4398–4404. 35. Graham, F.L., Smiley, J., Russell, W.C. & Nairn, R. (1977) Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J. General Virol. 36, 59–72.

factor 21. Ru¨ hmann, A., Ko¨ pke, A.K.E., Dautzenberg, F.M. & Spiess, J. (1996) Synthesis and characterization of a photoactivatable analog of corticotropin-releasing factor for specific receptor labeling. Proc. Natl. Acad. Sci. USA 93, 10609–10613. 36. Kishimoto, T., Pearse, R.V., I.I.Lin, C.R. & Rosenfeld, M.G. (1995) A sauvagine/corticotropin-releasing receptor expressed in heart and skeletal muscle. Proc. Natl. Acad. Sci. USA 92, 1108–1112.

22. Bonk, I. & Ru¨ hmann, A. (2000) Novel high-affinity photo- activatable antagonists of corticotropin-releasing factor (CRF): Photoaffinity labeling studies on CRF receptor, type 1 (CRFR1). Eur. J. Biochem. 267, 3017–3024.

37. Ru¨ hmann, A., Chapman, J., Higelin, J. & Dautzenberg, F.M. (2002) Design, synthesis and pharmacological characteriza- tion of new highly selective CRF2 antagonists: development of 123I-K31440 as a potential SPECT ligand. Peptides 23, 453–460.

23. Gulyas, J., Rivier, C., Perrin, M., Koerber, S.C., Sutton, S., Corrigan, A., Lahrichi, S.L., Craig, A.G., Vale, W. & Rivier, J. (1995) Potent, structurally constrained agonists and competitive antagonists of corticotropin-releasing factor. Proc. Natl. Acad. Sci. USA 92, 10575–10579. 38. Perrin, M.H., Sutton, S., Bain, D.L., Berggren, W.T. & Vale, W.W. (1998) The first extracellular domain of corticotropin releasing factor-R1 contains major binding determinants for urocortin and astressin. Endocrinology 139, 566–570.

24. Nishimura, E., Billestrup, N., Perrin, M. & Vale, W. (1987) Identification and characterization of a pituitary corticotropin- releasing factor binding protein by chemical cross-linking. J. Biol. Chem. 262, 12893–12896.

39. Perrin, M.H., Sutton, S.W., Cervini, L.A., Rivier, J.E. & Vale, W.W. (1999) Comparison of an agonist, urocortin, and an antagonist, astressin, as radioligands for characterization of cor- ticotropin-releasing factor receptors. J. Pharmacol. Exp. Ther. 288, 729–734.

40. Nielsen, S.M., Nielsen, L.Z., Hjorth, S.A., Perrin, M.H. & Vale, W.W. (2000) Constitutive activation of tethered-peptide/cortico- tropin-releasing factor receptor chimeras. Proc. Natl. Acad. Sci. USA 97, 10277–10281.

25. Rosendale, B.E., Jarrett, D.B. & Robinson, A.G. (1987) Identifi- cation of a corticotrpin-releasing factor-binding protein in the plasma membrane of AtT-20 mouse pituitary tumor cells and its regulation by dexamethasone. Endocrinology 120, 2357–2366. 26. Grigoriadis, D.E. & De Souza, E.B. (1988) The brain cortico- tropin-releasing factor (CRF) receptor is of lower apparent molecular weight than the CRF receptor in anterior pituitary. J. Biol. Chem. 263, 10927–10931. 41. Ru¨ hmann, A., Bonk, I. & Ko¨ pke, A.K.E. (1999) High-affinity binding of urocortin and astressin but not CRF to G Protein- Uncoupled CRFR1. Peptides 20, 1311–1319.

27. Grigoriadis, D.E. & De Souza, E.B. (1989) Heterogeneity between brain and pituitary corticotropin-releasing factor receptors is due todifferential glycosylation. Endocrinology 125, 1877–1888.