From The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037
Received for publication, October 11, 2002, and in revised form, February 25, 2003
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ABSTRACT |
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The heptahelical receptors for
corticotropin-releasing factor (CRF), CRFR1 and CRFR2, display
different specificities for CRF family ligands: CRF and urocortin I
bind to CRFR1 with high affinity, whereas urocortin II and III bind to
this receptor with very low affinities. In contrast, all the urocortins
bind with high affinities, and CRF binds with lower affinity to CRFR2.
The first extracellular domain (ECD1) of CRFR1 is important for
ligand recognition. Here, we characterize a bacterially
expressed soluble protein, ECD1-CRFR2 Corticotropin-releasing factor
(CRF)1 (1) is the major
neuroregulator of the hypothalamic-pituitary-adrenal axis and, among other roles, serves to integrate the endocrine, autonomic, and behavioral responses to stress. In addition to its central nervous system actions, CRF and its related ligands also affect the
cardiovascular, reproductive, gastrointestinal, skin, and immune
systems (2). The CRF ligand family includes (frog) sauvagine, (fish)
urotensins, mammalian urocortin I (3, 4), and the 38 amino acid
peptides from the newly recognized genes urocortin II (5) and urocortin III (6), also known as stresscopin-related peptide and stresscopin (7).
The actions of CRF ligands are initiated by binding to their receptors,
whose activation results in an increase of intracellular cAMP,
hydrolysis of phosphoinositol, activation of mitogen-activated protein (MAP) kinases (8, 9), and other signaling pathways (10). In
mammals, two receptor types, CRFR1 and CRFR2, have been cloned
(11-17); orthologous receptors have also been identified in many other
species including chicken (18), fish (19), and Xenopus (20).
A third receptor, CRFR3, with a high level of sequence identity to
CRFR1, has been cloned in catfish (19). CRF receptors have been
characterized in the central nervous system and various
peripheral sites including pituitary, gastrointestinal tract,
epididymis, heart, gonad, adrenal, skin, and skeletal muscle.
The CRF receptors are 7-transmembrane domain proteins with relatively
large first extracellular domains (ECD1s). Both CRFR1 and CRFR2 exist
as multiple splice variants and belong to the type B receptor family
that includes receptors for growth hormone-releasing factor, secretin,
calcitonin, vasoactive intestinal peptide, glucagon, glucagon-like
peptide (GLP), and parathyroid hormone (2).
The ligand specificities in binding to CRFR1 and CRFR2 are markedly
different. Although both CRF and urocortin I bind with equally high
affinities to CRFR1, the affinity of CRF for CRFR2 is at least 10 times
lower than that of urocortin I. There is no high affinity interaction
of either urocortin II or urocortin III with CRFR1, whereas their
affinities for CRFR2 are in the subnanomolar range. The agonist,
sauvagine, and the peptide antagonist, astressin, have equally high
affinities for both types of receptors (5, 6, 21).
The majority of differences between the sequences of the two receptors
are found in their ECD1s. Mutagenesis studies have identified regions
of the receptors that are important for differential recognition of
agonists and small molecule antagonists, as well as for governing the
ligand selectivity of the two types of receptors (22-27). Binding data
from chimeric receptors in which the ECD1 of CRFR1 is annealed to
transmembrane domains of other receptors suggested the importance of
the ECD1 in CRF/receptor interactions (28, 29). Further, a soluble
protein corresponding to the ECD1 of CRFR1 expressed either in bacteria
or mammalian cells binds astressin and urocortin with moderately high
affinity (28, 30).
Recently, 5 amino acids have been identified in the ECD1 of the type 2 CRF receptor that appear to govern the differences in ligand
recognition of the Xenopus and human receptors (31). Further
understanding of the role of the ECD1 of CRFR2 in the binding of the
CRF ligands may be gained from an investigation of their binding to the
ECD1 isolated from the rest of the receptor.
In this study, we present biochemical, biophysical, and functional
characterization of a soluble protein corresponding to the ECD1 of
mouse (m)CRFR2 ECD1-CRFR2 Chimeric Receptors, ECD1-CRFR2 Peptide Synthesis--
Astressin,
cyclo(30-33)[D-Phe12,Nle21,38,Glu30, Lys33]rat/human
CRF (12-41), was synthesized as described (33). Detailed synthetic procedures for the other peptides have been described (33). Radioiodination of [D-Tyr0]astressin
has been reported previously (21),and radioiodination of
[Tyr0,Glu1,Nle17]sauvagine is
similar to that of [D-Tyr0]astressin.
Radioreceptor Assays--
Transfections and binding to membrane
fractions were performed as described (28). The data for the chimeric
receptors are from crude membrane fractions of COS-M6 cells transiently
expressing the receptors; the data for the wild-type receptor are from
crude membrane fractions of Chinese hamster ovary cells stably
expressing mCRFR2 Disulfide Arrangement--
The ECD1-CRFR2 Circular Dichroism Spectropolarimetry--
CD spectropolarimetry
was conducted with an Aviv Model 62DS spectropolarimeter (Aviv
Associates, Lakewood, NJ). Conditions were as follows: (a)
low ionic strength case, 0.01 M sodium phosphate, pH 7.5, 22.2 µM ECD1-CRFR2 In our previous work, we were able to express, in bacteria,
milligram quantities of a functional soluble form of the ECD1 from
CRFR1 (26). Further, we found that the amino acids corresponding to the putative signal peptide of the ECD1 were cleaved in the mature
CRFR1, expressed in mammalian cells. We assume here that amino acids
1-26 of mCRFR2 An essential aspect of the biochemical characterization of
ECD1-CRFR2, corresponding to the ECD1
of mouse CRFR2
. The Ki values for binding to
ECD1-CRFR2
are: astressin = 10.7 (5.4-21.1) nM,
urocortin I = 6.4 (4.7-8.7) nM, urocortin II = 6.9 (5.8-8.3) nM, CRF = 97 (22-430) nM,
urocortin III = sauvagine >200 nM. These affinities
are similar to those for binding to a chimeric receptor in which the
ECD1 of CRFR2
replaces the ECD of the type 1B activin receptor
(ALK4). The ECD1-CRFR2
possesses a disulfide arrangement identical
to that of the ECD1 of CRFR1, namely
Cys45-Cys70,
Cys60-Cys103, and
Cys84-Cys118. As determined by circular
dichroism, ECD1-CRFR2
undergoes conformational changes upon binding
astressin. These data reinforce the importance of the ECD1 of CRF
receptors for ligand recognition and raise the interesting possibility
that different ligands having similar affinity for the full-length
receptor may, nevertheless, have different affinities for
microdomains of the receptor.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
. We find that the relative affinities of the CRF
ligands for the soluble protein are similar to those for a chimeric
receptor in which the ECD1 of CRFR2
is anchored in the plasma
membrane by a single transmembrane domain of the type 1 activin
receptor (32). Further, we present the disulfide arrangement of the
ECD1 and show that it is identical to that of the ECD1 for CRFR1.
Finally, we show that ligand binding induces a conformational change in
the protein.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Expression in Escherichia coli--
A cDNA
encoding amino acids 27-134 of mCRFR2
was inserted into pET-32a(+)
(Novagen) with KpnI and XhoI (pET-ECD1-CRFR2
), and the integrity of the construct was confirmed by automated sequencing. The E. coli strain, Origami trxB, gor
(DE3)pLysS (Novagen) was chemically transformed according to
manufacturer's directions using pET-ECD1-CRFR2
. Bacteria
were grown to an A600 nm = 0.6-1.0 and induced
with isopropyl-1-thio-
-D-galactopyranoside (1 mM) for either 2.5 h at 37 °C or 16 h at
22 °C. The bacterial pellet was solubilized, sonicated in 10 mM Tris-HCl, pH 8, (10 ml/g of wet pellet), and centrifuged
at 100,000 × g for 30 min. The soluble ECD1-CRFR2
,
obtained as a thioredoxin fusion protein in the supernatant, was
subjected to thrombin cleavage and enriched by affinity chromatography with S-protein-agarose (Novagen). After thrombin cleavage,
ECD1-CRFR2
contains 24 additional amino acids at the N
terminus derived, in part, from the S-(epitope) tag. The sequence of
ECD1-CRFR2
is GSGMKETAAAKFERQHMDSPDLGT (mouse
CRFR2
(27-134)) (the S-tag is underlined); the additional amino
acids, GS and DLGT, are part of the thrombin cleavage site and the
KpnI cloning site, respectively.
/ALK4 and
ECD1-CRFR1/ALK4--
The chimeric receptors in
which amino acids 1-135 of mCRFR2
or amino acids 1-118 of rat
CRFR1 replace the first 122 amino acids of the type 1B activin
receptor, ECD1-CRFR2
/ALK4 or ECD1-CRFR1/ALK4, respectively, were
generated by means of PCR as detailed previously (28).
. For membrane binding, 5-10 µg of membrane
protein/50 µl of assay buffer A (20 mM Na-HEPES, pH, 7.4, 0.1% bovine serum albumin, 10% sucrose, 2 mM EGTA) were
incubated with increasing concentrations of unlabeled peptides (50 µl
of assay buffer) and [125I-D-Tyr0]astressin
(~200,000 cpm/50 µl of assay buffer A + 0.02% Triton) or
[125I-Tyr0,Glu1,Nle17]sauvagine
(~100,000 cpm/50 µl of assay buffer A) in a final volume of 200 µl. For soluble protein binding, 0.2-0.5 µg of ECD1-CRFR2
/50 µl of assay buffer B (20 mM Na-HEPES, pH, 7.6, 0.1%
bovine serum albumin) were incubated with increasing concentrations of
unlabeled peptides (50 µl of assay buffer B) and
[125I-D-Tyr0]astressin
(~200,000 cpm/50 µl of assay buffer B+ 0.02% Triton) in a final
volume of 200 µl. Incubation proceeded for 90 min at room
temperature in MAGV microtiter plates (Millipore) precoated with
0.1% polyethylenimine. The mixture was aspirated under vacuum, and the
plates were washed twice with relevant assay buffer. The counts bound
in the wells were quantified by
-counting. To obtain the
Ki values, the competitive displacement data were analyzed by a non-linear regression analysis (GraphPad Prism program (GraphPad Softwares, Inc., San Diego CA)). All assays were performed in
triplicate at least three times.
(10 µg) was
initially subjected to digestion by endoproteinase Asp-N (1 µg) in 50 mM MES at pH 6.2 for 16 h. A large fragment containing
all 6 cysteine residues was isolated after HPLC purification. This
fragment was analyzed by chemical sequence analysis and yielded three
sequences beginning with residues Glu43, Asp65,
and Glu105. The endoproteinase Asp-N fragment was further
subjected to digestion by chymotrypsin, and fragments were again
isolated after HPLC separation. The HPLC fractions were analyzed by
MALDI-MS and by chemical sequence analysis. Two chymotryptic fragments
were identified that contained cysteine residues. Fragment CT-1
exhibited an intense signal at m/z = 1655.71. Chemical sequence analysis yielded two major sequences
corresponding to that of ECD1-CRFR2
: 101-109 (containing
Cys103) and 60-64 (containing Cys60). The
calculated monoisotopic mass (MH+) for this
cystine-connected fragment is 1655.72 Da. The MALDI-MS spectrum of
fragment CT-2 showed an intense signal at
m/z = 2868.33. Chemical sequence analysis
yielded two major sequences corresponding to residues 80-88
(containing Cys84) and 116-129 (containing
Cys118). The calculated monoisotopic mass (MH+)
for the cystine-connected fragment is 2868.35 Da. These fractions were
reduced by treatment with tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and reanalyzed by MALDI-MS. In both cases, the original signal
diminished in intensity giving rise to signals corresponding to the
individual cysteine-containing peptides.
neat or 20.0 µM
ECD1-CRFR2
+ 20.0 µM astressin, temperature 20.5 ± 0.1 °C, wavelength range 185-260 nm, collection frequency of 1.0 nm/datum, integration time of 1.0 s, spectral bandwidth of 1.5 nm,
averaging 5 repetitions/spectrum, quartz cell path length of 0.5 mm;
(b) high ionic strength case, 0.15 M sodium
chloride, 0.01 M sodium phosphate, pH 7.5, 19.5 µM ECD1-CRFR2
, ± 19.5 µM astressin, all
other conditions as in (a). No postcollection smoothing was
applied to the CD data. Spectral deconvolution by the method of Bohm
et al. (34) employed CDNN version 2.1 (bioinformatik.biochemtech.uni-halle.de/cdnn) and the supplied neural
network based on the 33-member basis set. The ECD1-CRFR2
used for
the CD measurements was purified and quantified by HPLC.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
encode its signal peptide and are absent in its
mature protein. Accordingly, a cDNA encoding amino acids 27-134 of
mCRFR2
was used to transform a strain of E. coli in which
there is enhanced formation of disulfide bonds as a result of mutations
in thioredoxin and glutathione reductase. The ECD1 was obtained as a
soluble trx-fusion protein. Following thrombin cleavage, the
soluble protein, ECD1-CRFR2
, was obtained. The protein contains 132 amino acids and comprises 108 amino acids of the ECD1, 15 amino acids
encoding the S-(epitope) tag, and other amino acids from the thrombin
cleavage site and the KpnI cloning site. Analyses, by
SDS-PAGE, of the fusion protein and its thrombin cleavage
products indicate that the apparent molecular size of the fusion
protein is ~29 kDa, the apparent molecular size of the trx
tag is ~14 kDa, and the apparent molecular size of the ECD1-CRFR2
is ~15 kDa (data not shown). Mass-spectrometric analysis confirms the
size of ECD1-CRFR2
: measured m/z = 14,944, calculated [MH]+ = 14,950 Da.
is an assessment of its affinity for the CRF family members. In Fig. 1A, we show
the competitive displacements by astressin, CRF, urocortins I, II, III,
or sauvagine of
[125I-D-Tyr0]astressin bound to
ECD1-CRFR2
. For comparison, we also show the competitive
displacements by the same ligands of
[125I-D-Tyr0]astressin bound to
the wild-type mCRFR2
stably expressed in CHO cells (Fig.
1B). The affinities of the agonists CRF, urocortin I, and
urocortin II as well as of astressin for the soluble ECD1 are similar
to their affinities for the complete receptor, whereas the affinities
of urocortin III and sauvagine for ECD1-CRFR2
are much lower than
their affinities for the wild-type mCRFR2
.
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Fig. 1.
Competitive displacement by astressin
( ), rat urocortin I (
), mouse urocortin II (
), mouse urocortin
III (
), rat/human CRF (
), or sauvagine (
) of
[125I-D-Tyr0]astressin bound to
ECD1-CRFR2
(A) or to
membranes from CHO cells stably expressing mCRFR2
(B).
To investigate the role of the ECD1 of mCRFR2 in the context of a
membrane environment, we studied a chimeric receptor,
ECD1-CRFR2
/ALK4, in which the ECD1 of mCRFR2
replaced the ECD of
the type 1B activin receptor (ALK4), a single transmembrane receptor
(32). The competitive displacements of
125I-[D-Tyr0]astressin bound to
this chimera by CRF family ligands are shown in Fig.
2. Again, the relative potencies of CRF,
urocortin I, urocortin II, and astressin are similar to their potencies
for the full-length receptor, whereas both urocortin III and sauvagine have very low potencies; the affinities of the CRF family ligands for
this chimeric receptor are nearly the same as those for ECD1-CRFR2
. Consistent with the lack of displacement of labeled astressin by
nanomolar concentrations of unlabeled sauvagine is the absence of
specific binding of
[125I-Tyr0,Glu1,Nle17]sauvagine
to either the ECD1-CRFR2
or the ECD1-CRFR2
/ALK4.
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These data are summarized in the first four rows of Table I. For comparison, binding to a chimeric receptor in which the ECD1 of CRFR1 replaces the ECD of ALK4 is also given. The low affinity binding of urocortin II and urocortin III for the chimera expressing the ECD1 of CRFR1 is in accord with the low affinity binding of these ligands for the full-length type 1 CRF receptor.
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Also, for comparison, we list in Table I (in the last row) the
affinities derived from competitive displacement of labeled sauvagine
bound to mCRFR2. It can be seen that the calculated affinities of
some, but not all, of the analogs are dependent on the analog used as
the radiolabel. When determined by displacement of labeled astressin,
the Ki of sauvagine is ~5 times greater, and that
of urocortin III is ~10 times greater than their Ki values determined from displacement of labeled sauvagine.
To further characterize the soluble protein at a molecular level, we
determined the Cys-Cys connectivity pattern. The disulfide arrangement
of the ECD1-CRFR2 was determined by employing a strategy similar to
the one used previously for the ECD1-CRFR1 protein (26). The protein
was digested by proteases that cleave between cysteine residues. The
fragments were then isolated, analyzed by MALDI-MS, and subjected to
chemical sequence analysis. Cystine-linked fragments exhibit a mass in
MALDI-MS analysis corresponding to the sum of the cysteine-containing
peptides. Chemical sequence analysis yields two sequences, again
corresponding to the constituent peptides. Initial digestion with
endoproteinase Asp-N was insufficient to cleave the cystine-containing
core of the protein. Instead, three N termini were observed during
sequence analysis.
The analysis of chymotryptic fragments allowed the determination of two
of the disulfide bridges. The fragment CT-1 was found to consist of the
peptides 101-109 and 60-64 from mCRFR2. This established a
connection between Cys103 and Cys60. The other
fragment, CT-2, consisted of peptides 80-88 and 116-129 and thereby
established the connection between Cys84 and
Cys118. The remaining connection between Cys45
and Cys70 was established by sequence analysis of the
original endoproteinase Asp-N digestion product. The presence of the
three N termini at residues Glu43, Asp65, and
Glu105 in a single species can only be explained by a
connection between cysteine residues 45 and 70. A schematic
representation of the disulfide pattern is shown in Fig.
3. The overall pattern of cysteine connectivity is identical to that found in the ECD1 of CRFR1 (26) and
also in the related parathyroid hormone receptor (35).
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The behavior of ECD1-CRFR2, attending ligand interaction, was
studied by circular dichroism. Difference CD spectra were collected in
the far UV (185-260 nm) of ECD1-CRFR2
in the absence and presence of astressin. The CD spectra of ECD1-CRFR2
in low ionic strength, physiological pH aqueous solution, or more physiological ionic strength
(0.15 M sodium chloride, not shown) are equivalent and are
dominated by the random coil with additional notable contributions from
the antiparallel sheet and turn conformations (Fig.
4). Deconvolution by the method of Bohm
(34) resulted in contributions of 8.1, 16.3, 3.4, 24.8, and 46.8% for
the helix, antiparallel, parallel,
-turn, and coil
conformations, respectively. To determine the structural effects of
ligand binding, ECD1-CRFR2
was incubated with equimolar astressin,
and the spectrum of the resulting mixture was compared with the
arithmetic sum of the spectra of the components Fig. 4. The resulting
difference CD spectrum (Fig. 4, inset) clearly shows the
formation of positive ellipticity with a peak near 192 nm and a slight
increase in the magnitude of the negative ellipticity in the range
220-225 nm. Deconvolution of the spectrum of the mixture gives
conformational contributions of 11.4, 28.7, 3.4, 26.0, and 31.4% for
the helix, antiparallel, parallel,
-turn, and coil conformations,
respectively. Thus, the deconvolution and graphical data suggest that
when combined with astressin, ECD1-CRFR2
loses significant coil
contribution (46.8-31.4%) with a concomitant increase in antiparallel
sheet (16.3-28.7%) and to a much lesser extent helix (8.1-11.4%).
Because the spectra shown in Fig. 4 were collected under equimolar
conditions of protein and ligand, and because the residue ratio of
ECD1-CRFR2
to astressin is 120:32, ~80% of the signal observed in
Fig. 4 is contributed by ECD1-CRFR2
. Thus, although undoubtedly a
small fraction of the difference CD spectrum (Fig. 4, inset)
arises from changes in the astressin conformation, the major CD changes
observed must reflect changes in the conformation of ECD1-CRFR2
.
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DISCUSSION |
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The CRF ligand/receptor system consists of the related ligands CRF, (frog) sauvagine, (fish) urotensin, urocortin I, the recently discovered mammalian orthologs urocortin II and urocortin III (similar to stresscopin-related peptide and stresscopin, respectively), and the receptors, CRFR1 and CRFR2, derived from two distinct genes. Each receptor exists in at least two forms as a result of alternative splicing. The CRF receptors belong to the type B G-protein-coupled receptor family whose members are characterized by comparatively large ECD1s with 6 conserved cysteines.
Given the distinct pharmacology and physiological roles of the type 2 CRF receptor, we wished to explore the ligand binding characteristics
of a soluble form of the ECD1 for mCRFR2. By means of competitive
displacement assays using radiolabeled astressin, we find that the
soluble protein, ECD1-CRFR2
, binds astressin, urocortin I, and
urocortin II with high affinities (6-10 nM), binds CRF
with moderate affinity (~100 nM), and binds urocortin III
and sauvagine with low affinities (>200 nM). Under the
conditions of our assay, there is no binding of labeled sauvagine to
ECD1-CRFR2
, and displacement of bound, labeled astressin by
urocortin III or unlabeled sauvagine is seen only at high
concentrations. This absence of binding of sauvagine was seen also for
the ECD1 of CRFR1 (26). In summary, the relative affinities for
ECD1-CRFR2
are: astressin
urocortin I
urocortin II>CRF
urocortin III
sauvagine.
These data are supported by complementary data on a chimeric receptor,
ECD1-CRFR2/ALK4, in which the ECD1 of mCRFR2
is anchored to the
plasma membrane by means of the single transmembrane domain of a type 1 activin receptor. The relative potencies of the ligands in binding to
the chimera are similar to those for binding to the ECD1-CRFR2
.
The observation that sauvagine does not displace labeled astressin bound to the soluble ECD1 of either the type 1 or type 2 receptor suggests that other regions of the complete receptor are necessary for binding of sauvagine. These data are consistent with those showing that the affinity of sauvagine was ~500 nM and that the affinity of CRF was >1000 nM in binding to a chimeric receptor in which the ECD1 of CRFR1 replaced the corresponding ECD1 of the parathyroid hormone receptor (29). In another study, it was demonstrated that sauvagine cross-links to the second extracellular loop of CRFR1 (36).
The large decrease in affinity of urocortin III for the isolated ECD1
of CRFR2 suggests that, also for this ligand, other regions of the
receptor are required for high affinity binding. Another explanation
for these observations is that sauvagine and urocortin III require
correct coupling of the receptor to a G-protein, whereas urocortin I
and urocortin II are able to bind with high affinity to receptors in
absence of G-protein coupling. These data are consistent with the lack
of effect of GTP on binding of urocortin I (21, 37) and with the
suggestion that urocortin I may possess intrinsic antagonistic
properties (38).
The data in Table I show that the apparent affinities of urocortin III
and of sauvagine for wild-type mCRFR2 depend on the nature of the
labeled analog that is used in the radioreceptor assay. For both
peptides, when labeled astressin is used, the Ki
values are higher than those obtained by displacement of labeled
sauvagine. The affinities of urocortin I and of urocortin II do not
appear to depend on the nature of the radioligand. These observations
are similar to those in another study (38) in which a similar reduction
in the affinity of sauvagine but not in the affinity of urocortin I was
seen in competitive displacement of labeled astressin, as compared with
their affinities determined from displacement of labeled sauvagine.
The biochemical characterization revealed that the pattern of disulfide
bonds in the ECD1 of CRFR2 is the same as that determined for the
ECD1 of CRFR1 (26), which in turn is the same as that of the ECD1 of
the parathyroid hormone receptor (35). The observations of high
affinity ligand binding and of a unique disulfide pattern with no
scrambling suggest that the bacterially expressed protein assumes a
conformation close to that of the native receptor. This adds further
support to the suggestion that the 3 pairs of conserved cysteines in
the ECD1 of this subgroup of receptors are arranged in a unique manner
that may characterize the family of receptors. A similar kind of
disulfide arrangement is found in a number of small bioactive peptides
including cocaine- and amphetamine-regulated transcripts (CARTs) whose
NMR structures have been analyzed (39). Despite the variations in the
loop sizes, the basic fold is identical in all these structures. It is
possible to speculate that the ECD1 of the CRF receptors assumes a
similar folding pattern. It is interesting to note that the ECD of
receptors for the TGF-
and related ligands assumes the cardiotoxin
fold that had been associated previously with a family of small
peptides (40).
The observation that the CD spectra of ECD1-CRFR2 at pH 7.5 in both
low and physiological ionic strength media are primarily random coil
(~47% by deconvolution) but that this coil conformation is
significantly reduced by the interaction with astressin suggests that
ECD1-CRFR2
exists as a mobile structure capable of large shifts in
conformation without irreversible denaturation at room temperature.
This behavior has been documented in the interaction of astressin with
the soluble ECD1 of CRFR1 (26).
Biochemical characterizations of soluble ECD1s include those for the
G-protein-coupled receptors for follicle-stimulating hormone
(41), luteinizing hormone/human chorionic gonadotropin (42),
calcium (43), parathyroid hormone (35), GLP-1 (44), and glutamate (45).
A conformational change, as determined from CD measurements, was found
for the soluble ECD1 of the follicle-stimulating hormone receptor
following binding of follicle-stimulating hormone (41). Data obtained
from an analysis of the CD spectra of the soluble ECD1-luteinizing
hormone receptor/human chorionic gonadotropin receptor-hormone complex
showed that it was characterized by more than 25% -helices (42),
and similar data from the ECD1 of the parathyroid hormone receptor
showed ~25%
-helical and 23%
-sheet secondary structures
(35). Structural data from x-ray crystallography have been obtained,
thus far, only for ECD1 of the metabotropic glutamate receptor, which
has been characterized to 2.2-Å resolution (46).
In conclusion, we have found that a soluble protein, expressed in
bacteria, corresponding to the first extracellular domain of the
mCRFR2, binds with nanomolar affinity the CRF family members, urocortin I and urocortin II, as well as a synthetic peptide
antagonist, astressin. The agonist, CRF, binds with moderately high
affinity, whereas the agonists urocortin III and sauvagine display only low affinities for the soluble ECD1. The disulfide pattern of the ECD1
of CRFR2
is the same as that of the ECD1 of CRFR1 and suggests a
common pattern for the receptor family. Further, there is a significant
conformational change in the ECD1 following binding of astressin.
It is possible to speculate that the ligands of the CRF family have
co-evolved with the receptors in such a manner that the contribution to
high affinity ligand binding of the ECD1, relative to that of other
receptor sites, is specific for each ligand. For example, the ECD1 is a
major target of high affinity interaction for CRF, urocortin I, and
urocortin II, whereas interactions with other domains of the receptor
appear to be important for high affinity binding of urocortin III and
sauvagine. A model describing the activation of CRF receptors was
suggested by our previous study of the sempiternal signaling of a
chimeric receptor in which the ECD1 of CRFR1 was replaced by the first
16 amino acids of CRF. Taken together, our data suggest that the ECD1
of the CRF receptors interacts initially with the C-terminal half of
ligands such as CRF, urocortin I, and urocortin II, which are then
positioned in the correct proximity to present the N-terminal portion
of the ligand to the remainder of the receptor for subsequent
activation. It should now be possible to refine these models with data
from molecular structure determinations using soluble proteins such as
the ECD1 of the CRF receptors.
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ACKNOWLEDGEMENTS |
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We thank Dr. C. Perrin for fruitful discussions and A. Fischer, R. Kaiser, W. Low, M. Park, K. Pham, L. Shamel, and J. Vaughan for technical assistance.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant NIDDK 26741, the Foundation for Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: The Clayton Foundation
Laboratories for Peptide Biology, The Salk Institute, 10010 North
Torrey Pines Rd., La Jolla, CA 92037; Tel.: 858-453-4100; Fax:
858-558-8763; E-mail: perrin@salk.edu.
§ The Dr. Frederik Paulsen Chair in Neurosciences Professor.
¶ A Senior Foundation for Research Investigator.
Published, JBC Papers in Press, February 28, 2003, DOI 10.1074/jbc.M210476200
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ABBREVIATIONS |
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The abbreviations used are: CRF, corticotropin-releasing factor; CRFR, CRF receptor; ECD, extracellular domain; m, mouse; MES, 4-morpholineethanesulfonic acid; HPLC, high pressure liquid chromatography; MALDI-MS, matrix-assisted laser desorption/ionization-time of flight-mass spectrometry.
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