Localization of Agonist- and Antagonist-Binding Domains of Human Corticotropin-Releasing Factor Receptors
Chen W. Liaw,
Dimitri E. Grigoriadis,
Marge T. Lorang,
Errol B. De Souza and
Richard A. Maki
Departments of Molecular Neurobiology and Neuroscience
Neurocrine Biosciences, Inc. San Diego, California 92121
 |
ABSTRACT
|
---|
The CRF receptors, CRFR1
and CRFR2, are members of the G protein-coupled
receptor superfamily. Despite their considerable sequence similarity,
CRFR1 and CRFR2 have
quite different affinities for the peptide ligand rat/human CRF.
Previous studies using chimeric receptors between human
CRFR1 and CRFR2 have
identified three potentially important regions in the second and third
extracellular domains of CRF receptor for the binding of rat/human CRF.
The present report further demonstrates that these same three regions
also affect the binding of urocortin and sauvagine, two other members
of the CRF peptide family, albeit to different extents. We also show
that a fourth region in the third extracellular domain, Asp254, has
been identified to be important for sauvagine but not CRF or urocortin
binding. Thus, the three peptide ligands not only interact with a
different set of regions on CRFR1 and
CRFR2 but also differentially interact with
some of the same regions. These data could, at least in part, account
for the much higher affinity of CRFR2 for
urocortin and sauvagine compared with rat/human CRF. We have also
identified two amino acid residues, His199 in the third transmembrane
domain and Met276 in the fifth transmembrane domain, that are important
for binding the non-peptide high-affinity CRFR1
antagonist NBI 27914. Mutations of His199 and Met276 to the
corresponding amino acids in CRFR2 each
decreased the binding affinity of NBI 27914 for
CRFR1 by 40- and 200-fold, respectively. This
suggests that the transmembrane regions are critically important in
forming the binding pocket for the non-peptide antagonist.
 |
INTRODUCTION
|
---|
CRF is the principal regulator that integrates the bodys
endocrine, autonomic, and behavioral responses to stress (1, 2, 3). The
CRF peptide family consists of CRF itself (4), fish urotensin I
(5), frog sauvagine (6), and the recently characterized mammalian
urocortin (7, 8). CRF mediates its actions by binding to high-affinity
membrane receptors that are coupled to Gs and transduces the signal
through an increase in the intracellular level of cAMP. Two CRF
receptors (CRFR1 and CRFR2) with approximately
70% sequence identity and distinct tissue distribution pattern and
pharmacology have been cloned and characterized (9, 10, 11, 12, 13, 14, 15, 16, 17). Radioligand
binding and second messenger studies show that CRFR1 has
comparable high affinity for CRF, urocortin, and sauvagine, while
CRFR2 binds urocortin and sauvagine with much higher
affinity than CRF (8).
By taking advantage of the large difference in the affinity of CRF for
the two CRF receptors, we previously generated and analyzed a series of
chimeric receptors between human CRFR1 and
CRFR2. In these studies, we identified three regions that
are potentially important for rat/human CRF (r/hCRF) binding (18). Here
we have further examined the roles of these regions in binding
sauvagine and urocortin and demonstrated that they have different
relative contributions in binding the three peptide ligands. We have
also identified a fourth region in the third extracellular domain,
Asp254, which is important for sauvagine binding yet has little effect
on r/hCRF or urocortin binding when mutated to Glu, the corresponding
amino acid (aa) in CRFR2.
In situ hybridization studies have shown that
CRFR1 and CRFR2 mRNA each have a distinct
distribution pattern in the brain (19), suggesting the two receptor
subtypes might have distinct functional roles. This differentiation of
functions by distinct receptor subtypes allows the development of
subtype-specific non-peptide ligands that have the therapeutic
potential to target different aspects of CRF-mediated disorders with
minimal side effects. To aid in the design of such subtype-specific
non-peptide ligands, it is important to understand the molecular
interactions between receptors and the ligands. Recently, we have
described the synthesis of a series of non-peptide antagonists for the
CRFR1 receptor (20). These antagonists are highly selective
for the CRFR1 receptor subtype and have no affinity for the
CRFR2 subtype. In the present study, we have identified two
aa within the transmembrane domains (TMs) that are important for
binding one of these antagonists termed NBI 27914 (see compound 3b in
Ref.20), suggesting that TMs are important in forming at least part of
the binding pocket for the non-peptide ligands.
 |
RESULTS AND DISCUSSION
|
---|
We have previously identified three regions in the human CRF
receptor that are potentially important for binding the peptide ligand
r/hCRF (18). One is at the junction of the third extracellular domain
and fifth TM involving three consecutive aa, Val266, Tyr267, and
Thr268; the other comprises two regions mapped to the second
extracellular domain involving aa 175178 and His189 residue (Fig. 1
). Since the other two members of the
CRF peptide family, urocortin and sauvagine, have only modest sequence
identity to r/hCRF (
45%) (7) and have much higher affinity for
CRFR2 than r/hCRF, we thought it was important to examine
whether the three regions described above were also important for the
binding of urocortin and sauvagine.

View larger version (33K):
[in this window]
[in a new window]
|
Figure 1. Schematic Model of the hCRF Receptor
Sequence alignment of CRFR1 and CRFR2 are shown
between EC2 and TM5 with the aa of CRFR1 shown on the
left and those of CRFR2 shown on the
right. The conserved aa are shown in solid
circles and divergent aa are shown in open
symbols. The regions that are important for binding all three
peptide ligands r/hCRF, urocortin, and sauvagine, are shown in
squares; the region that is important for binding
sauvagine only is shown as a diamond; the two aa that
are important for NBI 27914 binding are shown in
triangles. The numbers indicate the aa positions of
those residues flanking the postulated TMs. All numberings are based on
the sequence of CRFR1.
|
|
The two CRF receptors (CRFR1 and CRFR2) have
more than 2 orders of magnitude difference in binding affinity for
r/hCRF. Each of the three regions described above, when mutated from
CRFR1 to the corresponding CRFR2 sequences,
shifted the apparent EC50 value in stimulating
intracellular cAMP by
7- to 10-fold (18). As shown in Table 1
, each of the three regions caused a
2- to 3-fold shift in the apparent EC50 value for
urocortin when changed from the CRFR1 to the corresponding
CRFR2 sequences. These results suggest that either these
three regions play a less significant role relative to the rest of the
molecule in binding urocortin than binding r/hCRF or that these
specific CRFR1 to CRFR2 sequence changes in the
three regions are more compatible with urocortin binding than r/hCRF
binding. Overall, the relatively minimal change in EC50
value for urocortin caused by chimeric mutation in each of these three
regions is consistent with the fact that the two CRF receptors have a
more similar affinity for urocortin (
7-fold difference) than for
r/hCRF (
360-fold difference).
View this table:
[in this window]
[in a new window]
|
Table 1. EC50 of r/hCRF, Urocortin and
Sauvagine in Stimulation of Intracellular cAMP for CRFR1,
CRFR2, and the Three Chimeric Receptors that have been shown to
have decreased affinity for r/hCRF
|
|
For sauvagine, the EC50 value of CRFR2
was
6-fold higher than that of CRFR1 (Table 1
).
Unexpectedly, the D266L267V268 triple mutation alone caused an almost
50-fold shift in EC50 value for sauvagine (Table 1
). This
same triple mutation increased the EC50 value for r/hCRF by
a much smaller magnitude (
10-fold), suggesting that either
aa 266268 plays a more significant role in sauvagine binding than
r/hCRF binding, or that this particular triple mutation
(CRFR1 to CRFR2 sequence) decreased the binding
affinity for sauvagine more so than for r/hCRF.
The Arg to His mutation at aa 189, in conjunction with the D266L267V268
mutations (H189DLV), increased the EC50 value for sauvagine
by another 10-fold (Table 1
), a magnitude similar to that for r/hCRF
(
8-fold); the EC50 value of the chimeric
receptor R1174R2178DLVR1 for
sauvagine was only slightly higher (
1.5-fold) than that of
D266L267V268 mutant, a relatively small change compared with that for
r/hCRF (
7-fold). Previously, we have shown that while aa
266268 plays a primary role in securing the binding of r/hCRF, the
roles of His189 and aa 175178 appear to be secondary and become
significant only in the presence of the D266L267V268 mutations (18).
This also seems to be the case for sauvagine and urocortin binding
since both H189 and R1174R2178 R1
mutants have EC50 values for sauvagine and urocortin
comparable to those of CRFR1 (Table 1
).
The fact that the D266L267V268 triple mutation alone had significantly
higher EC50 value (lower binding affinity) for sauvagine
than CRFR2 (Table 1
), suggests that some other
CRFR1 to CRFR2 sequence change(s) is (are)
capable of rescuing or reverting part of the affinity decrease for
sauvagine caused by the D266L267V268 mutations. To localize such a
rescuer region, the EC50 value for sauvagine of the
chimeric receptor R1228R2268R1, in
which aa 229265 of CRFR1 was replaced by the
corresponding CRFR2 sequence, in conjunction with the
D266L267V268 triple mutation, was determined and found to be 12-fold
lower than that of D266L267V268 mutant (Table 2
), indicating that the rescuer is
located between aa 229 and aa 265.
View this table:
[in this window]
[in a new window]
|
Table 2. EC50 of Sauvagine and r/hCRF in
Stimulation of Intracellular cAMP for the Chimeric Receptors Containing
D266L267V268 Mutations
|
|
To further map the rescuer region, two chimeric receptors,
R1228R2238DLVR1 and
R1238R2268R1, were constructed and
assayed. The chimeric receptor
R1228R2238DLVR1, with aa 229238
of CRFR1 replaced by the corresponding CRFR2
sequences in conjunction with the D266L267V268 mutations, had very
similar EC50 value to the D266L267V268 mutant (Table 2
); on
the other hand, the R1238R2268R1
chimeric receptor, with aa 239265 replaced by the corresponding
CRFR2 sequence in conjunction with the D266L267V268
mutations, had an EC50 value of 0.3 nM
(Table 2
), significantly lower than that of the D266L267V268
mutant and similar to that of
R1228R2268R1. These results suggest
that the rescuer is located between aa 239 and aa 265, where there are
three aa differences between CRFR1 and CRFR2,
i.e. aa 254, aa 257, and aa 263 (Fig. 1
). Each of these
three aa was then individually mutated from CRFR1 to the
corresponding CRFR2 aa in conjunction with the D266L267V268
mutations. As shown in Table 2
, while Q257DLV and E263DLV still have
significantly higher EC50 values than
R1228R2268R1, E254DLV had an
EC50 value of 0.7 nM, which is 10-fold lower
than that of the D266L267V268 mutant and is comparable to the
EC50 values of
R1228R2268R1 (0.6 nM)
and R1238R2268R1 (0.3
nM).
For r/hCRF and urocortin, we did not detect the rescuing effect
described above, i.e. the EC50 values of the
D266L267V268 mutant were lower than those of CRFR2 (Table 1
) and comparable to those of the chimeric receptor
R1228R2268R1 (Table 2
). Thus, it is
not surprising that the same Asp254 to Glu254 mutation had relatively
little effect on the EC50 values of E254DLV for r/hCRF and
urocortin (compare the EC50 values of E254DLV and
D266L267V268 in Table 2
).
The Asp254 to Glu254 mutation increased the binding affinity of the
D266L267V268 mutant for sauvagine by 10-fold, implicating the
importance of aa 254 in sauvagine binding. This was further confirmed
by analyzing a mutant receptor with a CRFR2 backbone
D254R2, in which the Glu residue of CRFR2
(corresponding to CRFR1 aa 254) was mutated to Asp (for
consistency in nomenclature, the numbering of aa of CRFR2
is based on the CRFR1 sequences). With a single
conservative aa substitution by eliminating one carbon from the side
chain of aa 254, D254R2 had an EC50 value of
5.8 ± 0.9 nM (n = 3), 6.5-fold higher than that
of CRFR2. It is possible that the negatively charged
carboxyl group of Glu interacts with a basic functional group on
sauvagine such that a change in the length of the side chain (Glu to
Asp mutation) changes the effective distance of interaction and thus
lowers the binding affinity.
We have recently described the synthesis of a series of
CRFR1-specific CRF receptor antagonists (20). One of these
antagonists NBI 27914 (see compound 3b in Ref.20) has a Ki
value in the low nanomolar range for CRFR1 and no affinity
for the CRFR2 subtype (20). To initially identify some of
the aa residues involved in NBI 27914 binding, we transiently
transfected VIP2.0Zc cells with CRFR1 and two chimeric
receptors R2188R1 (where the N-terminal 188 aa
of CRFR1 was replaced by corresponding CRFR2
sequences) and R1334R2 (where the C-terminal
sequence of CRFR1 after aa 334 was replaced by the
corresponding CRFR2 sequences), all of which have about the
same affinity for r/hCRF (EC50 values are 0.16,
0.26, and 0.10 nM, respectively). We then measured the
inhibition of r/hCRF-stimulated cAMP production by NBI 27914
for the three transfectants and determined that both
R2188R1 and R1334R2
appeared to have approximately the same affinity for NBI 27914 as
CRFR1 (data not shown), suggesting that some aa differences
between CRFR1 and CRFR2 within the aa 188
(beginning of TM3) to aa 334 (end of TM6) region are responsible for
the CRFR1 selectivity of NBI 27914.
To determine more precisely which aa residues are important for the
binding of NBI 27914, some CRFR1 to CRFR2 point
mutations within TM3, TM4, and TM5 (CRFR1 and
CRFR2 have identical aa sequence for TM6) were introduced
into the CRFR1 receptor. All these mutants have
approximately the same EC50 values for sauvagine in
stimulating intracellular cAMP (0.090.18 nM) as
CRFR1. The apparent affinity of NBI 27914 for these mutants
was determined by direct measurement of [125I]sauvagine
binding. None of the six mutants with a single aa substitution within
TM4, i.e. C229, L230, L232, F233, C237, and I238
significantly altered the affinity for NBI 27914 (data not shown).
However, two point mutations, one involving a His to Val mutation at aa
199 in TM3 (mutant V199), and the other a Met to Ile mutation at aa 276
in TM5 (mutant I276), reduced the affinity of NBI 27914, while having
no effect on the ability of r/hCRF to inhibit
[125I]sauvagine binding. Figure 2
demonstrates that the apparent affinity
of NBI 27914 was shifted by approximately 40- and 200-fold, in the two
mutants V199 and I276, respectively. In the stable cell line expressing
the native CRFR1 receptor subtype, the apparent affinity of
NBI 27914 in inhibiting [125I]sauvagine binding was
17 ± 1.5 nM, and this was decreased to 750 ± 82
nM and 4162 ± 140 nM in the V199 and I276
mutants, respectively (see Fig. 2
). Thus, these two residues appear to
be either directly interacting with a critical binding site on NBI
27914 or are required to maintain the local conformation of the binding
pocket for NBI 27914. These results also suggest that the binding
domain of NBI 27914 is likely to be at least partially incorporated
within the transmembrane regions. The lack of a molecular model for CRF
receptor makes it difficult to predict whether NBI 27914 is large
enough to also interact with some aa residues in the extracellular
domains. Unfortunately, all mutations in the extracellular domains
introduced thus far also affect the binding affinity for the peptide
ligands, making measurement of the relative binding affinity of NBI
27914 for these mutants difficult. Future studies using radiolabeled
non-peptide CRFR1 antagonists may help elucidate the
absolute binding requirements. It is noteworthy however, that for all
non-peptide ligands of G protein-coupled receptors whose binding
domains have been characterized so far, whether the natural ligands are
small molecules such as biogenic amines or peptides such as
neurokinins, it is the TMs of the receptor that form the major binding
sites (for review see Ref.21).

View larger version (32K):
[in this window]
[in a new window]
|
Figure 2. Competition of r/hCRF and NBI 27914 for Native
CRFR1 and I276 and V199 Mutant Receptors
Competition of r/hCRF (top panel) or NBI 27914
(bottom panel) for [125I]sauvagine binding
was performed using radioligand binding studies described. The apparent
affinity of r/hCRF (top panel) for sauvagine binding
remained unaltered with apparent inhibition constant (Ki)
values of 3.5 ± 0.6 nM, 4.0 ± 0.6
nM, and 5.6 ± 0.3 nM for the native
CRFR1 receptor subtype (squares) and the two
mutant receptors expressed transiently, the V199 mutant
(triangles) and the I276 mutant
(circles), respectively. In contrast, the apparent
affinity of NBI 27914 (bottom panel) was drastically
reduced in the mutants from 17 ± 1.5 nM in the native
receptor (squares) to 750 ± 82 nM and
4162 ± 140 nM in the V199 (triangles)
and I276 (circles) mutants, respectively. The graphs are
representative of triplicate values from three independent
determinations. In all cases, analysis of the competition data using
nonlinear least squares curve fitting indicated binding to a single
homogeneous class of binding sites.
|
|
Since NBI 27914 is much smaller in size than r/hCRF, it is
likely that the non-peptide antagonist only interacts with a subset of
those domains that are involved in peptide ligand binding. The fact
that His199 and Met 276 are important for NBI 27914, but not r/hCRF
binding, and the competitive nature of NBI 27914 antagonism (our
unpublished results) raise the intriguing possibility that there is
some overlap in the binding pockets for NBI 27914 and r/hCRF such that
binding of the two ligands becomes mutually exclusive. A similar
phenomenon has been described for the neurokinin receptor NK1, where
mutations of His197 in TM5 and His265 in TM6 reduced the affinities for
non-peptide competitive antagonists while those same mutations had no
effect on the binding of peptide ligand substance P (22, 23).
In summary, the present study demonstrates that the three regions of
CRF receptor previously identified to be important for r/hCRF binding
are also important for urocortin and sauvagine binding. However, the
contributions of these three regions appear to be different among the
three peptide ligands. This can at least partially account for the
different affinities of CRFR2 for the three ligands. In
addition, a fourth region was identified to be important for sauvagine,
but not r/hCRF or urocortin binding, suggesting that different peptide
ligands, despite their sequence similarity, may not interact with the
same set of molecules on the receptor. We have also identified two aa
residues, His 199 and Met276, that are important for binding the
non-peptide antagonist NB I27914. Both of these aa residues are located
within TMs, suggesting that TMs are important in forming the binding
pocket for the non-peptide antagonists.
Finally, although the chimeric receptor approach has been used
extensively to localize regions that are important for ligand binding,
it is important to understand the limitations of such an approach.
First, it does not address the significance of conserved aa residues.
Second, with any specific chimeric receptor, although a lack of
observed effects on ligand binding might suggest a noncritical role for
the nonconserved region(s) that has gone through the chimeric
substitution, it is also possible that the particular chimeric sequence
change is as compatible with binding the ligand as is the native
receptor. Thus, the chimeric receptor approach used in conjunction with
specific point mutations within critical regions of the protein are
required to elucidate the importance of certain regions of the receptor
in binding the ligands.
 |
MATERIALS AND METHODS
|
---|
All chimeric and mutant receptors were constructed and
transiently expressed in LVIP2.0Zc cells (24), a cell line containing a
cAMP-responsive ß-galactosidase reporter gene as described (18). The
EC50 values of peptide ligands in increasing the levels of
intracellular cAMP for various receptors were then determined by
assaying the ß-galactosidase activity as described (18, 25). To
measure inhibition of r/hCRF-stimulated intracellular cAMP by NBI
27914, VIP2.0Zc cells were transiently transfected with various
receptors and incubated with 0.6 nM r/hCRF, which
caused
7080% of maximal cAMP induction in the absence or
presence of 0.110 µM NBI 27914.
The binding affinities of NBI 27914 and r/hCRF for the expressed
CRFR1 and various transmembrane mutant receptors were
determined using [125I]-Tyr0-sauvagine
binding in the presence of varying concentrations of unlabeled ligands
as described previously (26). All drugs and reagents were made up in
assay buffer (PBS containing 10 mM MgCl2, 2
mM EGTA, and 0.15 mM bacitracin, pH 7.0, at 22
C). Eppendorf tubes received in order, 100 µl buffer (with or without
competing r/hCRF or NBI 27914), 50 µl of
[125I]-Tyr0-sauvagine (final concentration
100200 pM), and 150 µl membrane suspension for a total
assay volume of 300 µl. The assay was incubated at equilibrium for
2 h at 22 C. Reactions were terminated by centrifugation in a
Beckman microfuge for 10 min at 12,000 x g. The
resulting pellets were washed gently with 1 ml of ice-cold PBS
containing 0.01% Triton X-100 and centrifuged again for 10 min at
12,000 x g. The supernatants were aspirated and the
tubes cut just above the pellet and placed into 12 x 75-mm
polystyrene tubes and monitored for radioactivity in a Packard Cobra II
-counter at approximately 80% efficiency. Data were analyzed using
the iterative nonlinear least-squares curve-fitting program Prism
(GraphPad Inc., San Diego, CA). Competition curves were routinely fit
to single- and multiple-site models, and the fits were compared
statistically to determine whether a more complex data model was
justified.
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. Michael Brownstein and Monika König for
providing the LVIP2.0Zc cell line, and Dr. Wylie Vale for the human
CRFR1 cDNA clone. We also thank Dr. Nick Ling for
synthesizing the peptide ligands, Dr. Chen Chen for synthesizing NBI
27914, and Guy Barry for sequencing the mutant and chimeric receptor
clones.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Chen W. Liaw, Departments of Molecular Neurobiology and Neuroscience, Neurocrine Biosciences, Inc., 3050 Science Park Road, San Diego, California 92121.
This work was supported in part by SBIR Grants R43 NS34203 and R44
NS3348902.
Received for publication August 27, 1997.
Accepted for publication September 17, 1997.
 |
REFERENCES
|
---|
-
De Souza EB, Nemeroff CB 1990 Corticotropin-Releasing
Factor: Basic and Clinical Studies of a Neuropeptide. CRC Press, Boca
Raton, FL
-
Dunn AJ, Berridge CW 1990 Physiological and behavioral
responses of corticotropin-releasing factor administration: is CRF a
mediator of anxiety of stress responses? Brain Res Rev 15:71100[Medline]
-
Owens MJ, Nemeroff CB 1991 Physiology and pharmacology of
corticotropin-releasing factor. Pharmacol Rev 43:425473[Medline]
-
Vale W, Spiess J, Rivier C, Rivier J 1981 Characterization of
a 41 residue ovine hypothalamic peptide that stimulates secretion of
corticotropin and beta-endorphin. Science 213:13941397[Medline]
-
Lederis K, Letter A, McMaster D, Moore G 1982 Complete amino
acid sequence of urotensin I, a hypotensive and corticotropin-releasing
neuropeptide from Catostomus. Science 218:162164[Medline]
-
Montecucchi PC, Anastasi A, deCastiglione R, Erspamer V 1980 Isolation and amino acid composition of sauvagine. Int J Pept Protein
Res 16:191199[Medline]
-
Vaughan J, Donaldson C, Bittencourt J, Perrin MH, Lewis K,
Sutton S, Chan R, Turnbull AV, Lovejoy D, Rivier C, Rivier J, Sawchenko
PE, Vale W 1995 Urocortin, a mammalian neuropeptide related to fish
urotensin I and to corticotropin-releasing factor. Nature 378:287292[CrossRef][Medline]
-
Donaldson CJ, Sutton SW, Perrin MH, Corrigan AZ, Lewis KA,
Rivier JE, Vaughan JM, Vale WW 1996 Cloning and characterization of
human urocortin. Endocrinology 137:21672170[Abstract]
-
Chen R, Lewis KA, Perrin MH, Vale WW 1993 Expression cloning
of a human corticotropin-releasing factor receptor. Proc Natl Acad Sci
USA 90:89678971[Abstract]
-
Vita N, Laurent P, Lefort S, Chalon P, Lelias JM, Kaghad M, Le
Fur G, Caput D, Ferrara P 1993 Primary structure and functional
expression of mouse pituitary and human brain corticotropin releasing
factor receptor. FEBS Lett 335:15[CrossRef][Medline]
-
Chang C-P, Pearse II RV, OConnell S, Rosenfeld MG 1993 Identification of a seven transmembrane helix receptor for
corticotropin-releasing factor and sauvagine in mammalian brain. Neuron 11:11871195[Medline]
-
Perrin MH, Donaldson CJ, Chen R, Lewis KA, Vale WW 1993 Cloning and functional expression of a rat brain corticotropin
releasing factor (CRF) receptor. Endocrinology 133:30583061[Abstract]
-
Lovenberg TW, Liaw CW, Grigoriadis DE, Clevenger W, Chalmers
DT, De Souza EB, Oltersdorf T 1995 Cloning and characterization of a
functionally distinct corticotropin-releasing-factor receptor subtype
from rat brain. Proc Natl Acad Sci USA 92:836840[Abstract]
-
Kishimoto T, Pearse II RV, Lin CR, Rosenfeld MG 1995 A
sauvagine/corticotropin-releasing factor receptor expressed in heart
and skeletal muscle. Proc Natl Acad Sci USA 92:11081112[Abstract]
-
Perrin M, Donaldson C, Chen R, Blount A, Berggren T,
Bilezikjian L, Sawchenko P, Vale W 1995 Identification of a second
corticotropin-releasing factor receptor gene and characterization of a
cDNA expressed in heart. Proc Natl Acad Sci USA 92:29692973[Abstract]
-
Stenzel P, Kesterson R, Yeung W, Cone RD, Rittenberg MB,
Stenzel-Poore MP 1995 Identification of a novel murine receptor for
corticotropin-releasing hormone expressed in the heart. Mol Endocrinol 9:637645[Abstract]
-
Liaw CW, Lovenberg TW, Barry G, Oltersdorf T, Grigoriadis DE,
De Souza EB 1996 Cloning and characterization of the human
corticotropin-releasing factor-2 receptor complementary
deoxyribonucleic acid. Endocrinology 137:7277[Abstract]
-
Liaw CW, Grigoriadis DE, Lovenberg TW, De Souza EB, Maki RA 1997 Localization of ligand-binding domains of human
corticotropin-releasing factor receptor: a chimeric receptor approach.
Mol Endocrinol 11:980985[Abstract/Free Full Text]
-
Chalmers DT, Lovenberg TW, De Souza EB 1995 Localization of
novel corticotropin-releasing factor receptor (CRF2) mRNA
to specific sub-cortical nuclei in rat brain: comparison with
CRF1 receptor mRNA expression. J Neurosci 15:63406350[Medline]
-
Chen C, Dagnino Jr R, De Souza EB, Grigoriadis DE, Huang CQ,
Kim K-I, Liu Z, Moran T, Webb TR, Whitten JP, Xie YF, McCarthy JR 1996 Design and synthesis of a series of non-peptide high-affinity human
corticotropin-releasing factor1 receptor antagonists.
J Med Chem 39:43584360[CrossRef][Medline]
-
Strader CD, Fong TM, Tota MR, Underwood D, Dixon RAF 1994 Structure and function of G protein-coupled receptors. Annu Rev Biochem 63:101132[CrossRef][Medline]
-
Fong TM, Yu H, Cascieri MA, Underwood D, Swain CJ, Strader CD 1994 Interaction of glutamine 165 in the fourth transmembrane segment
of the human neurokinin-1 receptor with quinuclidine antagonists.
J Biol Chem 269:1495714961[Abstract/Free Full Text]
-
Cascieri MA, Fong TM, Strader CD 1995 Molecular
characterization of a common binding site for small molecules within
the transmembrane domain of G-protein coupled receptors. J
Pharmacol Toxicol Methods 33:179185[CrossRef][Medline]
-
König M, Mahan LC, Marsh JW, Fink JS, Brownstein MJ 1991 Method for identifying ligands that bind to cloned Gs- or
Gi-coupled receptors. Mol Cell Neurosci 2:331337
-
Liaw CW, Grigoriadis DE, De Souza EB, Oltersdorf T 1994 Colorimetric assay for rapid screening of corticotropin releasing
factor receptor ligands. J Mol Neurosci 5:8392[Medline]
-
Grigoriadis DE, Liu X-J, Vaughn J, Palmer SF, True CD, Vale
WW, Ling N, De Souza EB 1996 125I-Tyr0-Sauvagine: a novel high affinity
radioligand for the pharmacological and biochemical study of human
corticotropin-releasing factor2
receptors. Mol Pharmacol 50:679686[Abstract]