Localization of Ligand-Binding Domains of Human Corticotropin-Releasing Factor Receptor: A Chimeric Receptor Approach
Chen W. Liaw,
Dimitri E. Grigoriadis,
Timothy W. Lovenberg,
Errol B. De Souza and
Richard A. Maki
Departments of Molecular Neurobiology and Neuroscience,
Neurocrine Biosciences, Inc., San Diego, California 92121
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ABSTRACT
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Two CRF receptors, CRFR1
and CRFR2, have recently been cloned and
characterized. CRFR1 shares 70% sequence
identity with CRFR2, yet has much higher
affinity for rat/human CRF (r/hCRF) than CRFR2.
As a first step toward understanding the interactions between rat/human
CRF and its receptor, the regions that are involved in receptor-ligand
binding and/or receptor activation were determined by using chimeric
receptor constructs of the two human CRFR subtypes,
CRFR1 and CRFR2,
followed by generating point mutations of the receptor. The
EC50 values in stimulation of intracellular
cAMP of the chimeric and mutant receptors for the peptide ligand were
determined using a cAMP-dependent reporter system. Three regions of the
receptor were found to be important for optimal binding of r/hCRF
and/or receptor activation. The first region was mapped to the junction
of the third extracellular domain and the fifth transmembrane domain;
substitution of three amino acids of CRFR1 in
this region (Val266,
Tyr267, and Thr268) by
the corresponding CRFR2 amino acids
(Asp266, Leu267, and
Val268) increased the
EC50 value by approximately 10-fold. The other
two regions were localized to the second extracellular domain of the
CRFR1 involving amino acids 175178 and
His189 residue. Substitutions in these two
regions each increased the EC50 value for
r/hCRF by approximately 7- to 8-fold only in the presence of the amino
acid 266268 mutation involving the first region, suggesting that
their roles in peptide ligand binding might be secondary.
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INTRODUCTION
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CRF is a 41-amino acid (aa) peptide (1) that functions as a
neurohormone to integrate the electrophysiological, autonomic, and
behavioral responses to stress (2, 3, 4). Two CRFR subtypes,
CRFR1 and CRFR2, have recently been cloned
and characterized (5, 6, 7, 8, 9, 10, 11, 12, 13). For the CRFR2, two alternatively
spliced forms (CRFR2
and CRFR2ß) with
different 5'-coding sequences have been identified (9). Both receptors
belong to the superfamily of G protein-coupled receptors (GPCR)
characterized by the presence of seven transmembrane domains.
For GPCRs that bind small molecule ligands such as catecholamines and
acetylcholine, extensive mutagenesis studies have localized the
ligand-binding sites to the transmembrane domains (14, 15, 16, 17). Since some
of the peptide ligands are considerably larger, it is conceivable that
the ligand-binding pocket of their receptors might extend beyond the
transmembrane regions. Indeed, it has been shown that both the
extracellular segments as well as the transmembrane domains of the
neurokinin receptors are required for the high affinity binding of
substance P and neurokinins (18, 19, 20).
Human CRFR1 and CRFR2
are 70% identical in
aa sequence. The 30% difference in primary sequence translates into
more than 2 orders of magnitude difference in EC50 value in
stimulating intracellular cAMP for the peptide ligand rat/human CRF
(r/hCRF), i.e. the EC50 values are
0.16 and
60 nM for CRFR1 and CRFR2
,
respectively. Those different aa residues between CRFR1 and
CRFR2
that cause such a shift in EC50 for
r/hCRF presumably are important for the binding of r/hCRF and/or
receptor activation. As there is a fairly good correlation between
shift in EC50 value and change in binding affinity for
CRFR1 and CRFR2
, the change in
EC50 value is more likely to reflect a change in binding
affinity than in receptor activation. Thus, as a first step toward
localizing the regions of CRFR involved in r/hCRF binding, we
constructed a series of chimeric receptors with various regions of the
CRFR1 sequence replaced by the corresponding
CRFR2
sequence. By determining the EC50
values of these chimeras for r/hCRF, we found three regions, one in the
second extracellular domain (EC2), one at the junction of EC2 and
transmembrane domain 3 (TM3), and one at the junction of EC3 and TM5,
that caused shift in EC50 value and were likely to be
important for the high affinity binding of r/hCRF.
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RESULTS
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To localize the regions of the CRFR involved in binding of r/hCRF,
four chimeric receptors, R1334R2,
R1243R2, R1228R2, and
R1166R2, with increasing portions of
CRFR1 C-terminal sequences replaced by corresponding
CRFR2 sequences, were constructed (Fig. 1
).
As shown in Fig. 2
and Table 1
,
R1334R2 and R1166R2 had
similar EC50 values for r/hCRF to CRFR1
(
0.16 nM) and CRFR2
(
60
nM), respectively, whereas R1243R2
and R1228R2 had an intermediate
EC50 value of
1.5 nM. These results
suggested that the sequence differences between CRFR1 and
CRFR2
for the 166 N-terminal aa, the region between aa
228243, and the C-terminal region starting at aa 334, did not
significantly change the affinity of r/hCRF binding. On the other hand,
there were two regions that influenced r/hCRF binding. A 40-fold
increase in the EC50 value (1.5 to 63.8 nM) was
observed when aa 166228 of CRFR1 were replaced by the
corresponding region of CRFR2
. The other region found to
be important was between aa 243334, which resulted in an increase in
the EC50 value of approximately 1 order of magnitude (0.10
to 1.6 nM) when replaced by the corresponding region of
CRFR2
.

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Figure 1. Schematic Representation with Sequence Alignment of
Human CRFR1 and CRFR2
The conserved aa are shown in solid circles. The
divergent aa are shown in open symbols, with the aa of
CRFR1 shown on the left and those of
CRFR2 shown on the right. The aa residues
that have been mutated are shown in squares, and the
junctions of chimeric receptors are indicated by arrows.
The numbers indicate the aa positions of those residues flanking the TM
domains. All numberings are based on the sequence of
CRFR1.
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Figure 2. Stimulation of Intracellular cAMP of Wild Type
CRFR1, CRFR2 , and Four Chimeric Receptors by
r/hCRF
VIP2.0Zc cells transiently transfected with various receptors were
incubated with different concentrations of r/hCRF for 7 h, and
cAMP-induced ß-galactosidase activity was measured. Each data
point is the average of triplicate determinations. Each
curve is representative of at least four independent
experiments.
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To further map the region(s) involved in r/hCRF binding between aa
243334, two chimeric receptors, R1266R2 and
R1228R2268R1, were constructed. In
the latter chimera, aa 229268 of the CRFR1 sequence were
replaced by the corresponding CRFR2
sequence. An
intermediate EC50 value of R1266R2
(0.25 nM) between R1243R2 (1.6
nM) and R1334R2 (0.1
nM; Fig. 3
) suggested that both regions, aa
244266 and aa 267334 were involved in r/hCRF binding. On the other
hand, R1228R2268R1 had an
EC50 value of 1.4 nM, approximately the same as
that of R1243R2, suggesting that it was the
region containing the CRFR2
sequence shared by the two
chimeric receptors, i.e. the region from aa 244268, that
was responsible for the
10-fold increase in the EC50
value over CRFR1. Point and double mutations were then
introduced into each of the six aa that are different between
CRFR1 and CRFR2
within aa 244268. As shown
in Table 2
, substitution of three aa at the junction of
the third EC domain and the fifth TM domain, Val266,
Tyr267, and Thr268, each caused
a 2- to
3-fold increase in the EC50 value when replaced by the
corresponding CRFR2
aa (see Table 2
, D266, D266L267, and
D266V268). Triple mutations with all three aa replaced by corresponding
aa of CRFR2
(D266L267V268 in Table 2
) had an
EC50 value of 1.5 nM, similar to that of
R1243R2.

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Figure 3. Stimulation of Intracellular cAMP of Four Chimeric
CRFR1/CRFR2 by r/hCRF
Each curve is representative of at least four
independent experiments. The calculated EC50 values
(mean ± SEM) for R1334R2,
R1266R2,
R1228R2268R1, and
R1243R2, are 0.10 ± 0.02, 0.25 ±
0.04, 1.4 ± 0.2, and 1.6 ± 0.4 nM,
respectively.
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Four additional chimeric receptors, R1174R2,
R1178R2, R1188R2, and
R1191R2, were constructed to localize the
region(s) involved in r/hCRF binding between aa 166228. As shown in
Table 3
, the EC50 value of
R1191R2 (1.3 nM) was similar to
that of R1228R2 (1.5 nM),
suggesting that none of the aa changes between CRFR1 and
CRFR2
within the aa 192228 region caused a significant
change in the EC50 value. On the other hand, both
R1178R2 and R1188R2 had
an EC50 value (
9.7 nM)
7-fold higher than
that of R1191R2, yet significantly lower than
that of either R1174R2 or
R1166R2. These data suggested that the aa
differences between CRFR1 and CRFR2
within
the region aa 178188 did not change the binding affinity for r/hCRF,
while two regions, one between aa 174178 and another between aa
188191 were important for r/hCRF binding.
Point mutations of each of the three aa at aa 189, 190, and 191 to the
corresponding CRFR2
aa showed that only mutation of the
Arg residue at aa 189 to His (H189) had a slight effect on the
EC50 value (see H189, C190, and I191 in Table 4
). This shift in the EC50 value was much
less than what was observed between R1188R2 and
R1191R2. As the difference between H189 mutant
receptor and R1188R2 chimeric receptor is that
the R1188R2 chimera contains the CRFR2 sequence
C-terminal to the mutation site, it is possible that some C-terminal
CRFR2 sequence might be required for the full expression of this H189
mutant phenotype. One likely candidate for such a C-terminal sequence
is aa 266268, which was shown above to be important for r/hCRF
binding. To address this, a point mutation at aa 189 was combined with
the D266L267V268 triple mutation; the resulting mutant receptor H189DLV
had an EC50 value of 12.3 nM (Table 4
) and an
8-fold increase over that of D266L267V268 (Table 2
) and was comparable
to that of R1188R2 (Table 3
).
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Table 4. EC50 of r/hCRF Stimulation of
Intracellular cAMP for CRFR1 Mutants with Mutations in EC2
and at Junction of EC2 and TM3
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Unexpectedly, although the R1174R2 chimera
contains more CRFR1 sequences than the
R1166R2 chimera and wild type
CRFR2, it had a higher EC50 value than either
R1166R2 or CRFR2 (see
Discussion). The
20-fold shift of EC50 values
between R1174R2 and
R1178R2 (Table 3
) indicated that aa 175178
was important for r/hCRF binding. However, when this region was
replaced by corresponding CRFR2
sequence
(R1174R2178R1), there was no change
in the EC50 value (Table 4
). Similar to the H189 mutation,
when mutation of aa 175178 was combined with the D266L267V268 triple
mutation (R1174R2178DLVR1 in Table 4
), the resulting mutant receptor had an EC50 value of
11 nM, 7-fold higher than that of the D266L267V268
mutant.
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DISCUSSION
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Chimeric receptors have been used extensively as an approach to
map the regions that are critical for agonist/antagonist binding of
GPCR (21, 22, 23, 24). The present study was designed to use such an approach
to localize some of the regions of the CRFR involved in r/hCRF binding.
This was followed by generating point mutations to more specifically
map the sites within the receptor that are important for peptide ligand
binding. The assay we used to monitor the affinities of various
chimeric receptors for r/hCRF is a colorimetric assay that measures the
induction of intracellular cAMP. As there is a fairly good correlation
between the change in the EC50 of stimulation of
intracellular cAMP and the shift in binding affinity for the two CRFR
subtypes, as previously described (25), and the two CRFR have identical
aa sequence in the third intracellular loop, which has been shown to be
a critical determinant of receptor activation, the changes in
EC50 of various chimeric receptors observed in the current
study are more likely to correspond to changes in binding affinity than
to those in functional activation. However, the possibility that the
regions mapped in the current study that result in changes in
EC50 may be involved in receptor activation cannot be
excluded.
As CRFR1 has more than 2 orders of magnitude higher
affinity for r/hCRF than CRFR2, it was expected that as
more CRFR1 sequences were systematically replaced by the
corresponding CRFR2 sequences, there would be a gradual
decrease in the affinity for r/hCRF. This was generally the case,
except in one instance where R1174R2 had a
lower affinity for r/hCRF than R1166R2 or
CRFR2. One possible explanation for this is that aa
166174 are incompatible with some other region(s) of the receptor
molecule in this particular chimeric environment, and the resulting
conformational change directly or indirectly affects the binding
affinity of the peptide ligand r/hCRF.
By monitoring the EC50 values of various CRF chimeric and
mutant receptors, three regions that affected the binding affinity of
r/hCRF have been localized. Two of the regions were within the second
EC domain, i.e. aa 175178 and His189 at the
junction of EC2 and TM3, whereas the third region was at the junction
of EC3 and TM5 involving three aa residues, Val266,
Tyr267, and Thr268. A mutant CRFR1
with aa 266268 replaced by the corresponding CRFR2
sequence had a 10-fold lower affinity for r/hCRF than the wild type
CRFR1 and could completely account for the shift in the
EC50 value of the R1228R2 chimera.
Whether all three aa are directly interacting with r/hCRF peptide, or
some residues are playing a more indirect role, such as maintaining the
local conformation for ligand binding, is not known.
Although substitution of the aa 266268 sequence to the corresponding
CRFR2
sequence could completely account for the 10-fold
shift of the EC50 value of chimeric receptor
R1228R2, neither substitution of aa 175178
nor mutation of His189 to the corresponding
CRFR2
sequence lowered the affinity for r/hCRF to the
same degree as predicted from chimeric receptors involving the two
regions. That is, although R1178R2 had 20- and
6-fold higher affinities for r/hCRF than
R1174R2 and R1166R2,
respectively, the R1174R2178R1
mutant had essentially the same affinity for r/hCRF as wild type
CRF1. Mutation of Arg189 to His increased the
EC50 value by less than 2-fold, whereas comparison of
EC50 values of R1188R2 and
R1191R2 would predict approximately a 7-fold
decrease in affinity. As the difference between the mutant and chimeric
receptors is that chimeric receptors have more CRFR2
sequence C-terminal to the mutation sites, the two EC2 mutants were
each combined with the downstream mutation involving aa 266268. In
both instances, the resulting mutants
R1174R2178DLVR1 and H189DLV showed
a 7- to 8-fold increase in EC50 value compared with the
D266L267V268 mutant. One possible explanation for such results is that
although aa 266268 play a primary role in securing the binding of
r/hCRF peptide, the roles of aa 174178 and His189 are
secondary, such that only when the interaction between peptide ligand
r/hCRF and aa 266268 of the receptor is weakened (as in the case of
D266L267V268 mutant) do these two regions in EC2 play a significant
role in binding r/hCRF.
Recently, a new member of the CRF peptide family, urocortin, was
cloned from rats (26) and humans (25). With 45% sequence identity to
CRF, urocortin shows only limited selectivity for CRFR1vs. CRFR2 (3- to 4-fold difference in
affinity) (25), and thus, the shift in the EC50 value was
also expected to be much smaller in magnitude for the chimeric
receptor. Indeed, the three regions mapped in the current study each
appeared to contribute about a 1.5- to 2-fold shift in EC50
value in response to urocortin (data not shown).
Finally, it is important to note that when the chimeric receptor
approach was used to map the regions that are important for ligand
binding, as in the current study, only those regions that are different
between the two receptor subtypes used to construct the chimeras were
examined. It is conceivable that some of those conserved aa between
CRFR1 and CRFR2 are critical for the binding of
r/hCRF, and the importance of those regions would be revealed only when
a more extensive study involving chimeric receptors constructed from
CRFR and a more distant GPCR family member is performed.
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MATERIALS AND METHODS
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All chimeric and mutant receptors were constructed from human
CRFR1 and CRFR2
by using either naturally
occurring restriction enzyme sites or sites generated by PCR. Sequences
derived from PCR and synthetic oligonucleotides were confirmed by DNA
sequencing.
All receptors were transiently expressed in LVIP2.0Zc cells, a cell
line containing a cAMP-responsive ß-galactosidase reporter gene (27),
and the EC50 values of r/hCRF in increasing the levels of
intracellular cAMP for various receptors were determined as previously
described (28). Briefly, 24 h after transfection, the cells were
plated in 96-well plates and, 1 day later, incubated with various
concentrations of r/hCRF. The intracellular cAMP levels induced by CRF
were then indirectly determined by assaying the ß-galactosidase
activity as previously described (28).
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ACKNOWLEDGMENTS
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We thank Drs. Michael Brownstein and Monika König for
providing the LVIP2.0Zc cell line, and Dr. Wylie Vale for the human
CRFR1 receptor complementary DNA clone. We also thank Dr.
Nick Ling for synthesizing r/hCRF, and Guy Barry for technical
assistance.
This work was supported in part by SBIR grant from NINDS (R43 NS34203).
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FOOTNOTES
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Address requests for reprints to: Chen W. Liaw, Ph.D., 3050 Science Park Road, San Diego, California 92121.
Received for publication February 6, 1997.
Revision received March 13, 1997.
Accepted for publication March 14, 1997.
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