(Received for publication, February 14, 1997, and in revised form, April 14, 1997)
From the Department of Pharmacology, Kyoto University Faculty of Medicine, Yoshida, Sakyo-ku, Kyoto 606-01, Japan
To identify domains conferring ligand binding
specificity to prostanoid receptors, we constructed a series of
chimeric receptors by successively replacing the regions from the
carboxyl-terminal tail of mouse prostacyclin (prostaglandin I (PGI))
receptor (mIP) with the corresponding regions of the mouse PGD receptor
(mDP). The mIP receptor expressed in COS 7 cells bound
[3H]iloprost, a PGI2 analog, and
[3H]PGE1 with Kd values
of 13 and 27 nM, respectively. This receptor did not bind
[3H]PGD2, [3H]PGE2,
and [3H]PGF2. The mDP receptor bound only
[3H]PGD2 with a Kd value
of 43 nM. The chimeric IPN-VII/DPC receptor with replacement of the carboxyl tail of the mIP receptor with
that of the mDP receptor showed 12-16-fold higher affinities for
[3H]iloprost and [3H]PGE1 than
the mIP receptor. The region extending from the sixth transmembrane
domain to the carboxyl terminus of the mIP receptor was next replaced
with the corresponding region of the mDP receptor. This chimeric
IPN-V/DPVI-C receptor acquired the ability to
bind [3H]PGD2 and
[3H]PGE2 without decreasing the affinities of
the mIP receptor to [3H]iloprost and
[3H]PGE1. These binding characteristics did
not change when the fourth and fifth transmembrane domains of the mIP
receptor were further replaced with the corresponding regions of the
mDP receptor. However, when the first extracellular to second
intracellular loop of the mIP receptor containing the third
transmembrane domain was further replaced with those of the mDP
receptor, the affinities for [3H]PGE1,
[3H]PGE2, and [3H]iloprost were
markedly decreased, whereas that for [3H]PGD2
was increased by about 2-fold. [3H]PGF2
showed no affinity for the mIP, mDP, and all the chimeric receptors.
These results suggest that the sixth to seventh transmembrane domain of
the mIP receptor confers the specificity of this receptor to bind
selectively to PGE1 and not to PGE2 and that
the third transmembrane domain of the mDP receptor confers the
selective binding of PGD2 to this receptor.
Prostaglandins (PGs)1 contain prostanoic acid as a central structural element. PGs have two structural features in the prostanoic acid framework. First, they have functional groups on the cyclopentane ring, which classifies them into four types, D, E, F, and I. Second, they are classified into three series, 1, 2, and 3, by the number of double bonds in the side chains. Additionally, another cyclooxygenase product, thromboxane A2 has an oxane ring instead of the cyclopentane ring. These prostanoids act on eight types and subtypes of the receptors. They are the PGD receptor (DP), the EP1, EP2, EP3, and EP4 subtypes of the PGE receptor, the PGF receptor (FP), the PGI receptor (IP), and the thromboxane A2 receptor (TP) (1-4). These receptors can recognize the structural differences of prostanoid molecules. The binding affinities of these receptors to prostanoid molecules are determined primarily by the cyclopentane ring structures of ligands. For example, the DP receptor shows the highest affinities to PGD2 and PGD1, but affinities to other prostanoids are at least 2 orders of magnitude less. One exception is the IP receptor, which shows the affinity to PGE1 almost comparable to PGI analogs such as iloprost. This receptor, however, can bind PGE2 with much lower affinity, suggesting that the IP receptor can discriminate a difference in the side chains.
We have cloned cDNAs for all of these types and subtypes of the mouse prostanoid receptors (5-13). These studies revealed that the prostanoid receptors belong to the G protein-coupled rhodopsin type receptor superfamily. They have several regions conserved specifically among them. These conserved regions may participate in the construction of binding domains for structures common to prostanoid molecules, whereas the other regions may confer specificity for ligand binding. For example, the arginine in the seventh transmembrane domain, which is conserved in all of the prostanoid receptors, was proposed to be the binding site for the carboxyl group of prostanoid molecules (5, 14, 15). In fact, Funk et al. (16) have shown that a point mutation at this arginine residue in the human TP receptor results in loss of ligand binding activity. However, structural domains of the prostanoid receptors conferring specificity for ligand binding are as yet unknown.
Chimeric receptors have been used to determine the regions involved in
various functions of the receptors. For example, this approach was used
to determine the regions involved in selective agonist and antagonist
binding in adrenergic receptors (17, 18). Chimeric receptors were also
used to identify the binding site of non-peptide antagonists to the
neurokinin receptors (19-22) and to the angiotensin receptors (23) and
the G protein activation sites of the muscarinic and
1-adrenergic receptors (24). These results show that
this approach has been useful in locating functional domains of various
receptors.
To identify the domains conferring the ligand binding specificity to
the prostanoid receptors, we have constructed chimeric receptors from
the mIP and mDP receptors in this study. This strategy is based on the
high homology of their amino acid sequences as well as common signal
transduction. The prostanoid receptors can be functionally grouped into
three categories: the relaxant receptors, the contractile receptors,
and the inhibitory receptor (14). The relaxant receptors, consisting of
the IP, DP, EP2, and EP4 receptors, mediate
increases in cAMP and induce smooth muscle relaxation. The contractile
receptors, consisting of the TP, FP, and EP1 receptors,
mediate calcium mobilization and induce smooth muscle contraction. The
EP3 receptor is an inhibitory receptor that mediates
decreases in cAMP and inhibits several biological processes such as
neurotransmission, gastric acid secretion, and water reabsorption.
Sequence homology among these functionally related receptors is higher
than that among the three separate groups (25). The amino acid
sequences of the mIP and mDP receptors, which belong to the same
relaxant receptor group, show 58% identity in the transmembrane
domains (Fig. 1), and both couple to the same G protein,
Gs. Chimeric mIP/DP receptors were expressed in the COS 7 cells, and
their ligand binding properties were examined.
PGD2, PGE1,
PGE2, and PGF2 were generous gifts from Ono
Pharmaceuticals Co. Ltd. (Osaka, Japan). PGD1 was obtained
from Cayman Chemical Co. (Ann Arbor, MI).
[5,6,8,9,12,14,15-3H]PGD2 (115 Ci/mmol),
[5,6-3H]PGE1 (52 Ci/mmol), [5,
6,8,11,12,14,15-3H]PGE2 (171 Ci/mmol), and
[5,6,8,9,11,12,14,15-3H]PGF2
(179 Ci/mmol)
were obtained from DuPont NEN. Iloprost and [3H]iloprost
(15.3 Ci/mmol) were obtained from Amersham International plc, United
Kingdom.
The mIP and mDP
cDNAs were first subcloned into pCMX expression vector (26). The
BalI-EcoRV fragment of CP302, a cDNA of mIP
(11), and the Asp718-BamHI fragment of PGc9, a
cDNA of mDP (12), were subcloned into the EcoRV sites
and the Asp718 and BamHI sites of pCMX,
respectively. Six types of mIP/DP chimeric receptors were then
constructed (Fig. 2A). Six restriction sites of the mIP receptor cDNA (Asp718, PstI,
PvuII, SphI, BspHI,
BamHI), four restriction sites of the mDP receptor
cDNA (BspHI, BspEI, PstI,
BamHI), and newly introduced restriction sites
(BamHI, SpeI, HaeII) were used to
construct these chimeric receptor cDNAs so that they have no
insertion or deletion in the amino acid sequences (Fig. 2B).
The new restriction sites were introduced by PCR using oligonucleotides
designed for each site (Table I). pCMX-mIP or -mDP (5 ng) was used as a template for amplification by PCR in a reaction
mixture containing 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton
X-100, 10% dimethyl sulfoxide, 0.25 mM dNTPs, 1 unit of
Pfu polymerase (Stratagene), and 20 pmol of each primer in a
total volume of 20 µl. After a denaturation step at 94 °C for 3 min, 20 cycles of amplification step (94 °C for 1 min, 50 °C for
1 min, 72 °C for 3 min) were carried out and followed by a final
elongation step of 3 min at 72 °C. PCR products were
electrophoresed, excised, purified using DEAE membrane (Whatman International Ltd., Maidstone, U. K.), inserted into pCMX, and sequenced by the dideoxy chain termination method.
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BspHI sites at equivalent positions in the mIP and mDP receptor cDNAs (Fig. 2B) were utilized to construct this chimeric receptor. Fragment D-2 was excised from pCMX-mDP by digesting with BspHI and BamHI, and fragment I-2 was excised from pCMX-mDP by digesting with SphI and BspHI. Both excised fragments were ligated into the SphI and BamHI sites of pCMX-mIP.
The Chimeric IPN-VII/DPC ReceptorFragments I-1 and D-1 were amplified by PCR with the primer pairs shown in Table I to have a BamHI site (Fig. 2B). Fragment I-1 was digested with SphI and BamHI, and fragment D-1 was digested with BamHI and BspEI. Both digested fragments were ligated into the SphI and BspEI sites of pCMX-IPN-V/DPVI-C.
The Chimeric IPN-IV/DPV-C ReceptorFragments I-3 and D-3 were amplified by PCR with primer pairs shown in Table I to have an SpeI site (Fig. 2B). Fragment I-3 was digested with SphI and SpeI, and fragment D-3 was digested with SpeI and BspEI. Both digested fragments were ligated into the SphI and BspEI sites of pCMX-IPN-V/DPVI-C.
The Chimeric IPN-III/DPIV-C ReceptorFragments I-4 and D-4 were amplified by PCR with the primer pairs shown in Table I. In the D-4 fragment, the PvuII site was introduced (Fig. 2B). Fragment I-4 was digested with PstI and PvuII, and fragment D-4 was digested with PvuII and PstI. Both digested fragments were ligated into the PstI sites of pCMX-IPN-V/DPVI-C.
The Chimeric IPN-II/DPIII-C ReceptorFragment D-5 was amplified by PCR with the primer pairs shown in Table I. In the D-5 fragment, the PstI site was introduced (Fig. 2B). Fragment D-5 was digested with PstI and ligated into the PstI sites of pCMX-IPN-V/DPVI-C.
The Chimeric IPN-I/DPII-C ReceptorFragments I-6 and D-6 were amplified by PCR with the primer pairs shown in Table I to have HaeII (Fig. 2B). Fragment I-6 was digested with Asp718 and HaeII, and fragment D-6 was digested with HaeII and BspEI. Both digested fragments were ligated into the Asp718 and BspEI sites of pCMX-IPN-V/DPVI-C.
Ligand Binding StudiesFor transient expression of each
prostanoid receptor, COS 7 cells cultured in 15-cm dishes were
transfected with 20 µg of plasmid DNA by the lipofection method (27).
After culture for 60 h, the cells were harvested, and crude
membranes were prepared as described (11). Briefly, harvested COS 7 cells were homogenized using a Potter-Elvehjem homogenizer in a
solution containing 25 mM Tris-HCl (pH 7.5), 250 mM sucrose, 10 mM MgCl2, 1 mM EDTA, and 0.1 mM phenylmethylsulfonyl
fluoride. The homogenate was centrifuged at 800 × g
for 1 min. The supernatant was collected and centrifuged at
100,000 × g for 1 h. The pellet was suspended in
20 mM MES (pH 6.0) containing 10 mM
MgCl2 and 1 mM EDTA (the suspension buffer),
and used as crude membranes. Binding assays were performed essentially
as described previously (11). For Scatchard analysis, 50 µg of crude
membranes was incubated in the suspension buffer with various
concentrations of [3H]iloprost,
[3H]PGE1, [3H]PGE2,
[3H]PGD2, or
[3H]PGF2 in a total volume of 200 µl at
4 °C for 2 h. In competition experiments, the crude membranes
were incubated with 20 nM
[3H]PGE1 or 60 nM
[3H]PGD2 in the presence of various
concentrations of PGD1 or PGD2. The incubation
was terminated by the addition of 2 ml of the ice-cold suspension
buffer, and the mixture was rapidly filtered through GF/C filters
(Whatman). The filter was then washed with 5 ml of the ice-cold
suspension buffer three times. The radioactivity on the filter was
measured in 5 ml of Clear-Sol scintillation mixture (Nakalai Tesque,
Kyoto, Japan). Nonspecific binding was determined in the presence of a
1,000-fold excess of unlabeled ligands in the incubation mixture.
Ki values were calculated from IC50
values of radioligand binding as described previously (20).
The mIP, mDP, and six chimeric receptors were expressed in COS 7 cells, and crude membranes were prepared for binding studies. Crude
membranes were incubated with various concentrations of each of
[3H]iloprost, [3H]PGE1,
[3H]PGE2, [3H]PGD2,
and [3H]PGF2 (Fig. 3).
Saturation kinetics of these binding was obtained and subjected to
Scatchard analysis. Representative analyses are shown in Fig.
4, and the results of several analyses are summarized in
Table II. As shown in Fig. 4, A and
a, the mIP receptor showed saturation binding to
[3H]iloprost and [3H]PGE1, and
Scatchard analysis revealed respective Kd values of
13 ± 2 and 27 ± 5 nM (Table II). Binding was
observed also with [3H]PGD2 and
[3H]PGE2, but their affinities were too low
to be analyzed by the Scatchard analysis. This ligand binding
specificity of the mIP receptor is consistent with previous reports on
the cloned mIP receptor (11) and on native IP receptor in various cells
(28, 29). On the other hand, the mDP receptor showed a high affinity binding only to [3H]PGD2 with a
Kd value of 43 ± 6 nM (Fig. 4,
H and h, and Table II). This is also consistent
with previous reports on the cloned mouse and human DP receptors (12,
30) and on native human DP receptor (31). We then examined the binding
properties of the chimeric receptors. The carboxyl tail of the mIP
receptor was first replaced with that of the mDP receptor. This
chimeric IPN-VII/DPC receptor showed a
12-16-fold increase in binding affinity to [3H]iloprost
and [3H]PGE1 without an appreciable increase
in the binding of [3H]PGE2 and
[3H]PGD2 (Fig. 4, B and
b, and Table II). The sixth to seventh transmembrane domain
was then further replaced. The resultant chimeric
IPN-V/DPVI-C receptor acquired the ability to
bind [3H]PGD2 and
[3H]PGE2 with Kd values of
69 ± 16 and 40 ± 6 nM, respectively. They bound
[3H]iloprost and [3H]PGE1 with
affinities comparable to those of the mIP receptor with
Kd values of 11 ± 1 and 17 ± 8 nM, respectively (Fig. 4, C and c,
and Table II). Similar ligand binding properties were shown by the
chimeric IPN-IV/DPV-C and
IPN-III/DPIV-C receptors, which have further
substitution of the fifth and fourth transmembrane domains (Fig. 4,
D and d, E and e, and Table
II); they bound both [3H]PGE2 and
[3H]PGD2 in addition to
[3H]iloprost and [3H]PGE1 with
affinities similar to those of the chimeric
IPN-V/DPVI-C receptor. In contrast, the binding
of [3H]iloprost, [3H]PGE1, and
[3H]PGE2 was almost abolished when the first
extracellular to second intracellular loop of the mIP receptor was
replaced with that of the mDP receptor (Fig. 4, F and
f). This chimeric IPN-II/DPIII-C receptor, on the other hand, showed about a 2-fold increase in the
binding affinity for [3H]PGD2. The
Kd value of 35 ± 3 nM was close to
the value of the mDP receptor (Table II). Similar ligand binding
specificity was exhibited by the chimeric
IPN-I/DPII-C receptor (Fig. 4, G and
g, and Table II).
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The above results indicate that the mIP receptor may accommodate the
cyclopentane ring structure of PGD and that it exerts its ligand
binding specificity mainly by discriminating the structural difference
in the -side chain. To examine this hypothesis, PGD1 binding was analyzed by competition binding studies on the mIP, mDP,
and chimeric IPN-V/DPVI-C receptors using
[3H]PGE1 or
[3H]PGD2 as a radioligand (Fig.
5). PGD1 effectively displaced
[3H]PGD2 binding to the mDP receptor with a
Ki value of 990 nM. PGD1
also displaced [3H]PGE1 binding to the
chimeric IPN-V/DPVI-C receptor with the Ki values of 2.5 µM. On the other
hand, PGD1 as well as PGD2 could not displace
[3H]PGE1 binding to the mIP receptor at up to
a 10 µM concentration.
The present study used chimeric receptors and examined domains of
the prostanoid receptors conferring the ligand binding specificity of
each receptor. The receptors we used are the mIP, mDP, and chimeric
mIP/DP receptors. As shown under "Results," the mIP receptor shows
high affinity binding only to [3H]iloprost and
[3H]PGE1, and the binding of
[3H]PGD2, [3H]PGE2,
and [3H]PGF2 is negligible. These binding
properties indicate that the mIP receptor exerts its ligand binding
specificity in two ways. One is the recognition of the configuration of
the side chains. PGI has a unique configuration of the
-side chain
because of the presence of an additional ring attached to the
cyclopentane ring. It is believed that PGE1 without a
double bond in the
-side chain can mimic this configuration of the
PGI molecule and bind to the IP receptor, but this is not achieved by
PGE2. The other is the recognition of the cyclopentane ring
structure. It appears that this receptor can accommodate the
cyclopentane rings of I and E types of PG, but not D and F. However,
the specificity of this receptor for the cyclopentane ring structure
appears less strict than those of other prostanoid receptors including
the mDP receptor. As shown, the mDP receptor can bind only
[3H]PGD2 with high affinity, and the binding
affinities for E, F, and I types of PG are much lower. This indicates
that the mDP receptor has strict recognition of the cyclopentane ring
structure. Therefore, questions addressed in this study were which
region(s) of the IP receptor discriminate PGE1 and
PGE2, which region(s) of the IP receptor and how strictly
they accommodate the cyclopentane ring, and which region(s) of the DP
receptor determines the specific recognition of the cyclopentane ring
of D type.
We examined these questions by successively replacing the regions of
the mIP receptor with those of the mDP receptor from the carboxyl
terminus. Replacement of the region extending from the sixth
transmembrane to the carboxyl terminus of the mIP receptor resulted in
loss of ligand binding specificity of the mIP receptor mentioned above.
This chimeric IPN-V/DPVI-C receptor bound
[3H]PGD2 and
[3H]PGE2 as well as
[3H]iloprost and [3H]PGE1. The
fact that this chimera binds PGE2, whereas neither mIP nor
mDP binds this prostanoid, suggests that the domains recognizing the
ring structure and the side chain configuration of prostanoid molecules
are located in different regions of the prostanoid receptors. Because
such a change was not observed in the chimeric
IPN-VII/DPC receptor, these results suggest
that the sixth to seventh transmembrane domain is responsible for
recognition of the side chain configuration; this region of mDP appears
to accommodate both 1 and 2 series of the prostanoid molecules, whereas
that of mIP appears more strict, discriminating a structural difference
in the -side chain between PGE1 and PGE2. A
more detailed analysis is required to locate an exact domain conferring
this selectivity. The above results also suggest that the binding
pocket of the mIP receptor for the cyclopentane ring of prostanoid
molecules is localized in another region and can accommodate the
cyclopentane rings of not only I and E but also D type, although we
cannot exclude the possibility that the sixth to seventh transmembrane
domain of the mDP receptor has contributed to accommodate the
cyclopentane ring of D type in this chimeric receptor. Interestingly,
the affinities for [3H]PGE1 and
[3H]iloprost were not changed by further replacement of
the fourth and fifth transmembrane domains, suggesting that the binding
domain of the cyclopentane ring in the mIP receptor localizes in a
region containing the first to third transmembrane domain. We have
examined if the binding specificity of the mIP receptor is determined
solely by recognition of the side chain structure by analyzing the
binding of PGD1 to the mIP. As shown in Fig. 5, no
appreciable binding of PGD1 was observed in the mIP
receptor, suggesting that if the mIP receptor can accommodate the
cyclopentane ring of D type, the relative configuration between the
cyclopentane ring and the side chains is also important in determining
the ligand binding affinity. On the other hand, PGD1 bound
to the chimeric IPN-V/DPVI-C receptor,
suggesting that the sixth to seventh transmembrane domain of the mIP
receptor is also responsible for determining this binding specificity.
Moreover, the facts that the affinity of PGD1 for the
chimeric IPN-V/DPVI-C receptor was 1 order of
magnitude lower than that of PGD2 and that this rank of
binding is identical to that observed in the mDP receptor may indicate
that the sixth to seventh transmembrane domain of the mDP receptor is
responsible for determining these affinities.
A region determining the specificity of the mDP receptor was suggested
by further replacement of the first extracellular to second
intracellular loop of the mIP receptor with the corresponding region of
the mDP receptor. This replacement resulted in loss of the binding of
iloprost and E type of PGs but increased the binding affinity for
PGD2. These observations indicate first that this region of
the mIP receptor is indispensable for iloprost, PGE1, and
PGE2 binding, and second and more importantly that this region of mDP receptor may be responsible for the ligand binding selectivity of this receptor. Then, which domain in this region is
responsible for this selectivity? The ligand binding pocket in most
rhodopsin-type receptors for small molecules is formed by transmembrane
domains. If this is also the case for the prostanoid receptors, we can
assume that the third transmembrane domain is responsible for the above
selectivity. Surprisingly, the third transmembrane domain has only four
amino acids different between the mIP and mDP receptors (Fig. 1). If
this domain is responsible, it would be intriguing to examine if any of
these four amino acids has an important influence on the recognition of
the cyclopentane ring. The functional groups at the 9- and 11-positions
of the cyclopentane ring of PG molecules are either oxo or hydroxy
groups. One hypothesis is that these groups are involved in formation of hydrogen bonds to some amino acids of the prostanoid receptors. The
vicinal hydroxyl groups of the catechol ring are shown to be involved
in formation of hydrogen bonds to Ser204 and
Ser207 of the 2-adrenergic receptor
(32-34). These issues may be tested by construction of more
detailed chimeric receptors in this region.
This investigation has also revealed that the affinities for iloprost and PGE1 of the mIP receptor are increased 12- and 16-fold by replacement of its carboxyl tail with that of the mDP receptor. There have been several reports concerning the effects of carboxyl tails of the rhodopsin-type receptors. We observed that alternative splicing of the EP3 and TP receptor in the carboxyl tail affects the specificity and efficacy of G-protein coupling (35, 36) as well as the sensitivity to agonist-induced desensitization (37). However, none of them showed a change in ligand binding properties. Whether a difference in the carboxyl tail increases the ligand binding affinity remains to be tested because our IPN-VII/DPC receptor also contains the replacement of several residues of the seventh transmembrane domain (Fig. 1).
In summary, the present study has identified the domains of the mIP and mDP receptors which confer ligand binding specificities to each receptor. Continued application of molecular biology including introduction of a point mutation to the identified regions will provide a more detailed understanding of the molecular basis of ligand recognition by the prostanoid receptors and will help design more specific therapeutic agents.
We are grateful to T. Murata, H. Sawaragi, and T. Matsuoka of our department and H. Wise of the Chinese University, Hong Kong, for helpful discussions and comments, and to K. Okuyama for secretarial assistance.