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

Development of a selective photoactivatable antagonist for
corticotropin-releasing factor receptor, type 2 (CRF
2
)
Ines Bonk
1
and Andreas Ru¨ hmann
2
1
WITA Proteomics AG, Teltow/Berlin, Germany;
2
Institute for Molecular Biosciences, The University of Queensland, St Lucia,
Australia
A novel photoactivatable analog of antisauvagine-30 (aSvg-
30), a specific antagonist for corticotropin-releasing factor
(CRF) receptor, type 2 (CRF
2
), 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 [
125
I-Tyr0]oCRF or

[
125
I-Tyr0]Svg from membrane homogenates of human
embryonic kidney (HEK) 293 cells stably transfected with
cDNA coding for rat CRF receptor, type 1 (rCRF
1
)or
mouse CRF receptor, type 2b (mCRF
2b
). Furthermore, the
ability of ATB-[His12]Svg
(12)40)
to inhibit oCRF- or Svg-
stimulated cAMP production of transfected HEK 293 cells
expressing either rCRF
1
(HEK-rCRF
1
cells) or mCRF
2b
(HEK-mCRF
2b
cells) was determined. Unlike astressin and
photo astressin, ATB-[His12]Svg
(12)40)
showed high select-
ive binding to mCRF
2b
(K
i

¼ 3.1 ± 0.2 n
M
) but not the
rCRF
1
receptor (K
i
¼ 142.5 ± 22.3 n
M
) and decreased
Svg-stimulated cAMP activity in mCRF
2b
-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 mCRF
2b
receptor. The specificity of the photoactivatable
125
I-labeled
CRF
2b
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.
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].
CRF mediates its action through two distinct G
protein-coupled receptors: CRF receptor types 1 (CRF
1
)
and 2 (CRF
2
). While CRF
1
has been found at high
level in cortical and cerebellar structures of the brain and
pituitary, CRF
2
expression is generally confined to
subcortical structures. This distribution of CRF
2
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 CRF
1

and CRF
2
has
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
CRF
1
receptor [5]. In this view, several CRF
1
-specific
nonpeptide antagonists are currently being investigated in
clinical phase II studies as potential drugs for anxiety-
related diseases [6].
The 40-amino-acid peptide urocortin (Ucn) a naturally
occurring CRF analog was proposed to be the endogenous
ligand for the CRF
2
receptor [7]. However, Ucn also
exhibited high affinity binding to CRF
1
[7], and fibers that
express Ucn did not correlate with targets in the brain
bearing CRF
2
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 CRF
2

receptors have been
identified. The functional role of these peptides remains to
be elucidated.
We recently designed, synthesized and characterized for
the first time a CRF
2
-selective antagonist [12]. This
compound named antisauvagine-30 (aSvg-30) showed high
selectivity binding to CRF
2a
and CRF
2b
but not to the
CRF
1
receptor or the CRF binding protein [12–14]. Results
obtained from pharmacological studies have confirmed that
aSvg-30 acts as a competitive antagonist at CRF
2
receptors
[15]. Consequently aSvg-30 has helped to elucidate the role
of CRF
2
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:
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)
Eur. J. Biochem. 269, 5288–5294 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03246.x
[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 CRF
1
and CRF
2
.
Several CRF receptor 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].
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 CRF
2
-selective
antagonist based on the amino-acid sequence of aSvg-30,
which carries the 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
CRF
1
or CRF
2
receptor.
MATERIALS AND METHODS
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].
Synthesis and purification of peptides
The CRF peptides (Fig. 1) were synthesized, purified, and
characterized as described [12,21,22].
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
Pd°[PPh
3
]
4
in HOAc/N-methylaniline/dichloromethane
(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 bypreparative RP-HPLC
on a Vydac C
18
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, R
t
¼ 25.9 min; Svg: ESI MS calcd
4600.4, observed 4599.4, R
t
¼ 25.4 min; astressin: ESI MS
calculated 3565.1, observed 3563.1, R
t
¼ 24.8 min;
ATB-[Ala32stressin: ESI MS calculated 3562.1, ob-
served 3561.1, R
t
¼ 30.2 min; aSvg-30: ESI MS calculated
3652.2, observed 3650.3, R
t
¼ 21.6 min, ATB-

[His12]Svg
(12)40)
: ESI MS calculated 3716.3, observed
3715.4, R
t
¼ 26.6 min).
Fig. 1. Comparison of the amino-acid sequence
of (A) oCRF, (B) Svg, (C) astressin, (D) ATB-
[
125
I-labeled His13,Ala32]astressin, and (E)
ATB-[
125
I-labeled His12] Svg
(12-40)
.
Ó FEBS 2002 Photoactivatable CRF
2
receptor antagonist (Eur. J. Biochem. 269) 5289
Iodination to the photoactivatable aSvg-30 analog
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 (
125
I-ATB-[His12]Svg
(12)40)

:R
t
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.
Photolysis of ATB-[His12]Svg
(12)40)
, and its
radioactively labeled analog
125
I-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).
Crude membrane preparation
HEK 293 cells, permanently transfected with cDNA coding
for rCRF
1
(HEK-rCRF
1
cells), and mCRF
2b
(HEK-
mCRF
2b
cells) were maintained and subjected to membrane

preparations as described [12,35].
Binding assays with CRF peptides
Binding of the CRF analogs to the rCRF
1
and mCRF
2b
receptor was performed essentially as described previously
[12].Briefly,5lL of membrane suspension (25 lgof
protein from HEK-rCRF
1
cells; 50 lg of protein from
HEK-mCRF
2b
cells) was added to a plate containing CRF
peptides (0–1 l
M
) and 50 000 c.p.m. of either [
125
I-
Tyr0]oCRF (specific activity 81.4 TBqÆmmol
)1
, 68.25 p
M
,
DuPont NEN, Boston) for the analysis of rCRF
1
or [
125
I-
Tyr0]Svg (specific activity 81.4 TBqÆmmol

)1
, 68.25 p
M
,
DuPont NEN, Boston) for the analysis of mCRF
2b
in
100 lL incubation buffer (50 m
M
Tris/Cl, 5 m
M
MgCl
2
,
2m
M
EGTA, 100 000 kallikrein inhibitor units per litre of
Trasylol (Bayer, Leverkusen), 1 m
M
dithiothreitol,
1mgÆmL
)1
BSA, pH 7.4). After incubation (60 min,
23 °C), membrane suspension was aspirated through the
plate, followed by two washes with assay buffer (0.2 mL,
23 °C). Radioactivity of the punched filters was measured
with a 1470 WIZARD automatic gamma counter (Bert-
hold, Hannover). Specific binding of [
125
I-Tyr0]oCRF or

[
125
I-Tyr0]Svg to membranes of transfected cells was
calculated by subtraction of unspecific binding found in
the presence of 1 l
M
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.
cAMP stimulation
HEK-rCRF
1
cells or HEK-mCRF
2b
cells were incubated
with different CRF agonists in the presence or absence of
1 l
M
or 10 n
M
antagonist, or CRF antagonist (1 l
M

) alone.
After removal of the medium, cells were lyzed with aqueous
6% trichloroacetic acid (5 min, 100 °C) [21,22]. The cell
lysates were stored at )70 °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.
Photoaffinity labeling experiments with
125
I-labeled
ATB-[His12]Svg
(12)40)
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 °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).
RESULTS

Synthesis of ATB-[His12]Svg
(12)40)
and its radioactively
labeled analog
4-(1-Azi-2,2,2-trifluoroethyl)benzoic acid was successfully
linked to [His12]Svg
(12)40)
(Fig. 1). Subsequent radiolabe-
ling with
125
I gave ATB-[
125
I-His12]Svg
(12)40)
with a specific
activity of 74 TBqÆmmol
)1
.
Binding and cAMP assay
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
rCRF
1
or mCRF
2b
were used [21,36]. Scatchard analysis
indicated high-affinity binding of oCRF (K
i
¼ 0.6 ±

0.1 n
M
) and Svg (K
i
¼ 0.7±0.1n
M
)toCRF
1
but showed
significant difference in binding to the CRF
2
receptor
(Fig. 2). The photoactivatable astressin analog (compound
3) showed similar high affinity binding to CRF
1
(K
i
¼ 5.3 ± 1.3 n
M
)andCRF
2
(K
i
¼ 2.6 ± 1.1 n
M
)when
compared with astressin (CRF
1
: K
i

¼ 5.7 ± 1.6 n
M
;CRF
2
:
K
i
¼ 4.0 ± 2.3 n
M
). In contrast the photoactivatable aSvg-
30 analog (compound 1) exhibited high-affinity binding to
CRF
2
(K
i
¼ 3.1 ± 0.2 n
M
) but low affinity to CRF
1
(K
i
¼ 142.5 ± 22.3 n
M
) similar when compared with
aSvg-30 (CRF
1
: K
i
¼ 153.6 ± 33.5 n
M

;CRF
2
:
K
i
¼ 1.4 ± 0.4 n
M
). (Fig. 2, Table 1). The 46-fold preferred
binding of compound 1 to CRF
2
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 CRF
2
but low affinity to CRF
1
receptor, respectively (Table 1). The one amino acid-
truncated tyrosine-12 substituted aSvg-30 analog exhibited
very low affinity binding to either receptor.
Application of oCRF and Svg to HEK-rCRF
1
cells
stimulated the accumulation of intracellular cAMP in a
dose dependent manner with EC
50
values of
0.41 ± 0.08 n
M
and 0.19 ± 0.05 n

M
, respectively. The
potency of the two peptide agonists to enhance cAMP in
5290 I. Bonk and A. Ru
¨
hmann (Eur. J. Biochem. 269) Ó FEBS 2002
HEK-mCRF
2b
cells was significantly different (oCRF:
EC
50
¼ 11.79 ± 1.96 n
M
; Svg: EC
50
¼ 0.23 ± 0.05 n
M
)
(not shown). Ovine CRF-stimulated cAMP production in
HEK-rCRF
1
cells could be inhibited by CRF antagonists in
the following rank order: astressin (compound 4), ATB-
[Ala
32
]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-
mCRF
2b
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

125
I-labeled compound 1 and membranes of
HEK-mCRF
2b
cells (Fig. 3A). No photo cross-link was
observed in analogous experiments with membranes of
HEK-rCRF
1
cells (Fig. 3B). Binding of
125
I-labeled com-
pound 1 to the CRF
2
receptor could be efficiently inhibited
by addition of increasing concentrations of antisauvagine-
30 but not 10 l
M
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).
DISCUSSION
After the successful characterization of a photoactivatable
radioactively labeled CRF agonist and antagonists at CRF
1
receptors, we were now interested in the development of a
photoactivatable CRF
2
-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.
To this end we examined the binding affinity of photo
astressintoCRF
1
and CRF
2
receptors. Surprisingly, photo
Table 1. Binding constants of different CRF agonists and antagonists displacing [
125
I-Tyr0]oCRF from recombinant rCRF
1
or [
125
I-Tyr0]Svg from
recombinant mCRF
2b
.
Compound Peptide [
125
I-Tyr0]Svg K
i(mCRF
2b
)
(n
M
)[
125
I-Tyr0]oCRF K
i(rCRF1)

(n
M
) K
i(rCRF1)
/ K
i(mCRF
2b
)
1 ATB-[His12] Svg
(12)40)
3.1 ± 0.2 142.5 ± 22.3 45.97
2[
D
-Phe11, His12]Svg
(12)40)
f
1.4 ± 0.4 153.6 ± 33.5 109.71
3 ATB-[Ala32]astressin
b,c,g
2.6 ± 1.1 5.3 ± 1.3 2.04
4 Astressin
a
4.0 ± 2.3 5.7 ± 1.6 1.42
5 Svg 4.5 ± 0.4 0.7 ± 0.1 0.15
6[
D
-Phe11]Svg
(11–40)
3.5 ± 0.2 237.3 ± 27.7
c

67.80
7 [Tyr11,His12]Svg
(11)40)
4.9 ± 1.8 220.9 ± 89.1
d,e
45.08
8 [Tyr11]Svg
(11)40)
20.8 ± 1.6 >1000 >48.08
9 oCRF 162.4 ± 23.8
a
0.6 ± 0.1 0.00
10 [Tyr12]Svg
(12)40)
428.5 ± 118.9
b
>1000 >2.33
Statistically significant differences between the K
i(mCRF
2b
)
values of the peptides.
a
, P < 0.001 vs. 5;
b
, P < 0.0001 vs. 1–9. Statistically
significant differences between the K
i(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].
Fig. 2. Displacement of [
125
I-Tyr0]oCRF (A) or [
125
I-Tyr0]Svg (B)
bound to membrane homogenates of HEK 293 cells stably transfected
with cDNA coding for rat CRF receptor, type 1 (rCRF
1
)(A),ormouse
CRF receptor, type 2b (mCRF
2b
)(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), [
D
-Phe11]Svg
(11)40)
(compound 6, ·), [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).
Ó FEBS 2002 Photoactivatable CRF
2
receptor antagonist (Eur. J. Biochem. 269) 5291
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 CRF
2
receptors. Furthermore the potency of
photo astressin to increase or inhibit sauvagine-stimulated
second messenger production in CRF
2
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 CRF
2

when compared with
antisauvagine-30. However, this difference was not statisti-
cally significant.
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 CRF
2
expressing cells. Both compounds exhibited significantly
lower potency to suppress agonist-stimulated cAMP pro-
duction in CRF
1
cells when compared with astressin or its
photoactivatable analog thus indicating the specificity of
the novel photo ligand. It is noteworthy that the
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
CRF
2
receptors. A combination of an aromatic with a
heteroaromatic ring at the N-terminus increases the
binding affinity of antisauvagine-30 analogs to CRF
2

but
also CRF
1
receptors.
At high concentration similar to Svg
(11)40)
[12] but unlike
antisauvagine-30 the photoactivatable antisauvagine-30
analog exhibited significantly higher intrinsic activity in
CRF
1
cells when compared with control without peptide.
From experiments with chimeric receptors of CRF
1
and rat
growth hormone releasing factor it was concluded that the
first extracellular domain of the CRF
1
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 CRF
1
receptors [41] it was concluded that an initial
contact point is formed between the N and C terminus of
the CRF
1
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 G
s
-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
CRF
1
receptor may discriminate between the size and
charge of the N terminal dipeptide fragment of antisauv-
agine-30 analogs.
However, the photoactivatable antisauvagine-30 analog
did not cross-link to membrane homogenates of cells
permanently transfected with the gene coding for CRF
1
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 CRF
2
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
CRF
2
receptors.
In summary, we have designed, synthesized and charac-
terized for the first time a high-affinity photoaffinity probe
Table 2. Relative potency of CRF antagonists. The relative potency determined by the effect of 10 n
M
(mCRF
2b
)or1l
M
(rCRF
1
) CRF antagonist
on the cAMP production stimulated by 1 n
M
Svg (mCRF
2b
)or1n
M
oCRF (rCRF
1
).
Compound Peptide
HEK-mCRF
2b

cells HEK-rCRF
1
cells
cAMP prod.
Antag./Svg
k
Rel potency
Antag
l
cAMP prod.
Antag./oCRF
k
Rel potency
Antag
l
1 ATB-[His12]Svg
(12)40)
0.007 ± 0.002 0.48 ± 0.04 0.15 ± 0.01
j
0.58 ± 0.04
2[
D
-Phe11,His12]Svg
(11)40)
0.004 ± 0.001 0.42 ± 0.02 0.04 ± 0.01 0.71 ± 0.02
3 ATB-[Ala32]astressin
b,c
0.010 ± 0.001 0.30 ± 0.05 0.10 ± 0.01
j
0.11 ± 0.03

g,h
4 Astressin
a
0.004 ± 0.001 0.57 ± 0.04
c
0.10 ± 0.02
j
0.10 ± 0.02
i
5 Svg 1.00 1.00 – –
6[
D
-Phe11]Svg
(11)40)
0.005 ± 0.001 0.71 ± 0.04
a,b
0.07 ± 0.01
j
0.76 ± 0.14
7 [Tyr11, His12]Svg
(11)40)
0.008 ± 0.002 0.37 ± 0.05 0.03 ± 0.01 0.79 ± 0.07
8 [Tyr11]Svg
(11)40)
0.007 ± 0.002 0.67 ± 0.04
d,e
0.09 ± 0.01 0.82 ± 0.06
9 oCRF – – 1.00 1.00
10 [Tyr12]Svg
(12)40)

0.006 ± 0.002 0.80 ± 0.04
f
0.06 ± 0.03 0.90 ± 0.02
Control without peptide – 0.004 ± 0.001 – 0.01 ± 0.003 –
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 n
M
(mCRF
2b
)or1l
M
(rCRF
1
) CRF antagonist on
the cAMP production stimulated by 1 n
M
Svg (mCRF
2b
)or1n
M
oCRF (rCRF
1
).
5292 I. Bonk and A. Ru
¨
hmann (Eur. J. Biochem. 269) Ó FEBS 2002
specific for CRF receptor type 2b. Due to the similarity of
the pharmacological profile of mammalian CRF
2a
and
CRF
2b
, the new ligand, which we propose to name photo
antisauvagine, should serve as a useful tool to detect CRF

2
binding sites and elucidate its functional role in the brain
and peripheral organs.
ACKNOWLEDGEMENTS
We are grateful to Dr Frank M. Dautzenberg and Dr Andreas K. E.
Ko
¨
pke for providing the HEK-rCRF
1
cellsandDrChijenR.Linand
Dr Michael G. Rosenfeld for providing the HEK-mCRF
2b
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.
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