From the
Prostaglandin (PG) E receptor EP3D is coupled to both G
Hormonal actions are initiated by agonist binding to receptors.
The agonist-bound receptor is associated with and activates a G protein
to a state in which it can regulate effectors(1) . A number of
rhodopsin-type receptors can be coupled to multiple G proteins, and
then a single agonist produces a variety of hormonal actions through
modulation of multiple signal transduction systems(2) . The
ligand binding is believed to be determined by the transmembrane
domains, and multiple sites in transmembrane domains have been
suggested to contribute to the formation of a ligand binding pocket in
various receptors; several amino acid residues in the transmembrane
domains may be involved in agonist-induced receptor
activation(3) . However, the precise molecular mechanisms
through which the agonist, upon receptor binding, induces G protein
activation are still poorly understood. Especially, it is not known how
an agonist can modulate multiple G proteins through the binding to a
receptor.
Prostaglandin (PG)
There are several regions
conserved specifically among the prostanoid receptors, and the most
highly conserved regions are found in the seventh transmembrane
domain(3) . In rhodopsin, the hydrocarbon chain of retinal was
shown to be attached to Lys-296 in the seventh transmembrane
domain(14) . The positively charged arginine residue within the
seventh transmembrane domain, which is conserved in all of the
prostanoid receptors, was proposed to be the binding site of the
negatively charged
A variety of rhodopsin-type receptors have been reported to
transduce multiple signals by interacting with different G proteins
(2). The three tachykinin receptors and the thyrotropin receptor
stimulate both phospholipase C and adenylate cyclase through G
The EP3D receptor is also a
receptor coupled to multiple G proteins (13). In the present study, we
demonstrated that the arginine residue in the seventh transmembrane
domain of EP3D receptor, a putative binding site of
We examined, in turn, the contribution of the
The present study of the point mutation at the
arginine residue and actions of various EP3 agonists suggests that
charge-charge interaction of
Agonist-specific coupling of rhodopsin-type receptors to
multiple second messenger systems has been reported in the Drosophila octopamine/tyramine receptor (36) and type 1
pituitary adenylate cyclase-activating polypeptide
receptor(37) , but key amino acid residues of the receptor
structures responsible for selectivity of G protein coupling have not
yet been studied. In addition to these reports, our finding provides
support for the idea that receptors form multiple conformations, which
are differentially coupled to multiple G proteins.
In conclusion, in
this report we have shown that the arginine residue in the seventh
transmembrane domain of the EP3D receptor, a putative binding site of
The membrane (5 µg) of CT- or PT-treated cells
expressing EP3D or EP3D-R332Q receptor was incubated with 2 µl of
normal serum (NS), G
We thank A. Harazono for technical assistance and Drs.
K.-H. Thierauch of Schering, P. W. Collins of Searle, M. P. L. Caton of
Rhone-Poulenc Ltd., S. Kurozumi of Teijin Ltd., and B. M. Bain of Glaxo
Group Research Ltd. for providing sulprostone, misoprostol, M&
28767, TEI-3356 and GR 63799X, respectively.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and G
. To examine the roles of the interaction of
-carboxylic acid of PGE
and its putative binding site,
the arginine residue in the seventh transmembrane domain of EP3D, in
receptor-G protein coupling, we have mutated the arginine residue to
the noncharged glutamine. PGE
with a negatively charged
-carboxylic acid and sulprostone, an EP3 agonist with a noncharged
modified
-carboxylic acid, inhibited the forskolin-stimulated
adenylate cyclase activity via G
activation in the EP3D
receptor in the same concentration-dependent manner. In contrast, the
adenylate cyclase stimulation via G
activation by
sulprostone was much lower than that by PGE
. On the other
hand, both PGE
and sulprostone showed potent G
activity but failed to show G
activity in the mutant
receptor. EP3D receptor showed a high affinity binding for PGE
in the form coupled to either G
or G
.
Although the mutant receptor showed high affinity binding when coupled
to G
, it lost high affinity binding in the condition of
G
coupling. Furthermore, sulprostone bound to the
G
-coupled EP3D receptor with higher affinity than the
G
-coupled receptor. Among various EP3 agonists,
-carboxylic acid-unmodified agonists showed both G
and
G
activities, but the modified agonists showed only G
activity. These findings suggest that the interaction between the
-carboxylic acid of PGE
and the arginine residue of
the receptor regulates the selectivity of the G protein coupling.
(
)E
produces a broad range of biological actions in diverse tissues
through its binding to specific receptors on plasma
membranes(4, 5) . The pharmacological actions of
PGE
are diverse among tissues; PGE
causes
contraction or relaxation of vascular and nonvascular smooth muscle and
stimulates or suppresses the secretion of neurotransmitters or
hormones. The diversity of the actions of PGE
is due to the
coupling of PGE receptors to a variety of signal-transduction pathways.
PGE receptors are pharmacologically subdivided into four subtypes, EP1,
EP2, EP3 and EP4, on the basis of their responses to various agonists
and antagonists(6, 7) . We have recently revealed the
primary structures of mouse EP1, EP3, and EP4
receptors(8, 9, 10) . They belong to the G
protein-coupled rhodopsin-type receptor superfamily with seven
transmembrane domains and are coupled to the activation of the
Ca
channel, inhibition, and activation of adenylate
cyclase, respectively. In addition, we have identified three isoforms
of the mouse EP3 receptor (EP3
, -
, and -
) and four
isoforms of the bovine EP3 receptor (EP3A, -B, -C, and -D) with
different COOH-terminal tails, which are produced through alternative
splicing and differ in the specificity of coupling to G proteins.
EP3
,
and A are coupled to G
, while EP3B and C
are coupled to G
and EP3D and
are coupled to both
G
and G
(11, 12, 13) .
Among these isoforms, EP3D and -
belong to a receptor coupled to
multiple G proteins. The diversity of the cellular responses to
PGE
is in part based on the existence of functionally
different EP3 isoforms coupled to various signal transduction pathways
in addition to subtypes. Thus, functional analysis of agonist-binding
sites of PGE receptors is an urgent need for understanding diverse
physiological roles of PGE
.
-carboxyl group of prostanoid molecules by
analogy to the retinal binding site of rhodopsin(3) . Consistent
with this hypothesis, a point mutation at this arginine residue in the
seventh transmembrane domain of the human thromboxane A
receptor has been shown to result in the loss of ligand binding
activity(15) . A variety of PGE analogues with potent agonist
activity have been developed, but some EP3 agonists have the structural
features of substitution at C-1, where
-carboxylic acid is
replaced by a variety of esters or methanesulfonamido groups, its
negative charge being blocked. This suggests that the charge-charge
interaction of the
-carboxylic acid of PGE
with the
arginine residue of EP3 receptors appear not to be critical for some
agonist activities through EP3 receptors. To assess the role of the
interaction of
-carboxylic acid of PGE
with the
arginine residue of EP3 receptors in coupling to G proteins, we chose
the EP3D receptor, coupled to multiple G proteins, and mutated the
arginine in the seventh transmembrane domain to noncharged glutamine.
In the present study, we report that the arginine residue of the EP3D
receptor is not essential for G
coupling but is necessary
for G
coupling.
Materials
EP3 agonists were generous gifts from
Dr. K.-H. Thierauch of Schering (sulprostone), Dr. P. W. Collins of
Searle (misoprostol), Dr. M. P. L. Caton of Rhone-Poulenc Ltd. (M&
28767), Dr. S. Kurozumi of Teijin Ltd. (TEI-3356), and Dr. B. M. Bain
of Glaxo Group Research Ltd. (GR 63799X).
[5,6,8,11,12,14,15-H]PGE
(179
Ci/mmol), [
-
P]GTP (6,000 Ci/mmol), and the
I-labeled cAMP assay system were obtained from Amersham
Corp. PGE
was obtained from Cayman Chemical (Ann Arbor,
MI); pertussis toxin (PT) was from Seikagaku Kogyo (Tokyo, Japan),
cholera toxin (CT) was from Funakoshi Pharmaceuticals (Tokyo), and
rabbit antiserum against G
(RM/1) and that against
G
and G
(AS/7) were from DuPont
NEN. Sources of other materials are shown in the text.
Construction and Stable Expression of the Mutated
Receptor
A polymerase chain reaction-mediated mutagenesis (16) was used to convert arginine (Arg-332) of EP3D cDNA to
glutamine (Gln-332). The sequence of the mutated region in the
constructed cDNA for the mutant receptor (EP3D-R332Q) was confirmed by
sequence analysis by the dideoxynucleotide chain-termination method.
cDNA transfection was performed by lipofection, essentially as
described previously(17) . Briefly, Chinese hamster ovary (CHO)
cells deficient in dihydrofolate reductase activity
(CHO-dhfr) were transfected with the mutated cDNA
inserted into eukaryotic expression vector pdKCR-dhfr containing a
mouse dihydrofolate reductase gene as a selection marker (18).
Selection was performed using the
-modification of Eagle's
medium, lacking ribonucleosides and deoxyribonucleosides, with 10%
dialyzed fetal bovine serum (Cell Culture Laboratories). Clonal cell
lines were obtained by single-cell cloning and were screened by RNA
blotting.
Adenylate Cyclase Assay
CHO cells expressing EP3D
or EP3D-R332Q were exposed to 1 ng/ml PT or 5 µg/ml CT for 12 h.
The harvested cells were permeabilized in Hepes-NaOH (pH 7.5)
containing 0.015% digitonin, 2 mM MgCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, and 0.1 M NaCl essentially
according to the method of Lohse et al.(19) . Under
these conditions, the leakage of lactate dehydrogenase, an enzyme that
is confined to the cytosol, was complete (data not shown). After the
permeabilization, the permeabilized cells (50 µg) were incubated
for 5 min at 37 °C with 1 mM ATP, 10 µM GTP,
2 mM creatine phosphate, 1 unit of creatine phosphate kinase,
and 100 µM Ro-20-1724, and the reactions were
terminated by the addition of 5% trichloroacetic acid. The cAMP formed
was determined by radioimmunoassay with an Amersham cAMP assay system.
PGE
CHO cells
expressing EP3D or EP3D-R332Q were exposed to 1 ng/ml PT or 5 µg/ml
CT for 12 h. The harvested cells were homogenized using a
Potter-Elvehjem homogenizer in 20 mM Tris-HCl (pH 7.5),
containing 0.25 M sucrose, 10 mM MgClBinding Assay
, 1
mM EDTA, 20 µM indomethacin and 0.1 mM phenylmethylsulfonyl fluoride. After centrifugation at 800
g for 5 min, the supernatant was further centrifuged at
250,000
g for 20 min. The pellet was washed, suspended
in 20 mM Tris-HCl (pH 7.5) containing 10 mM MgCl
and 1 mM EDTA, and was used for the
[
H]PGE
binding assay. The membrane
(20 µg) was incubated with various concentrations of
[
H]PGE
at 30 °C for 1 h, and
[
H]PGE
binding to the membrane was
determined as described previously(11) . Nonspecific binding was
determined using a 1,000-fold excess of unlabeled PGE
in
the incubation mixture. The specific binding was calculated by
subtracting the nonspecific binding from the total binding.
GTPase Activity
GTPase activity was assayed by
incubating the membrane at 37 °C for 5 min in a total volume of 100
µl of 20 mM Hepes-NaOH (pH 7.5) containing 2 mM MgCl, 0.1 mM EDTA, 1 mM
dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 0.1 M NaCl, 1 mM App(NH)p, 0.2 mM ATP, and 0.1
µM [
-
P]GTP (0.5 µCi). The
reactions were initiated by the addition of the membrane (5 µg) and
stopped by the addition of 0.9 ml of ice-cold 5% Norit A and 0.1%
bovine serum albumin in 20 mM sodium phosphate (pH 7.0). The
mixtures were centrifuged (2,000
g, 5 min, 4 °C),
and the radioactivity of [
P]P
released into the supernatant (150 µl) was determined.
Nonspecific GTP hydrolysis was determined using a 1,000-fold excess of
unlabeled GTP in the incubation mixture. The specific low K
GTPase activity was calculated by
subtracting the nonspecific hydrolysis from the total hydrolysis. Under
these conditions, the time course was linear over a 5-min incubation
period.
Effects of PGE
To assess the role of the arginine
residue in the seventh transmembrane domain of the EP3D receptor in
agonist-mediated signal transduction, we constructed a mutant EP3D
receptor (EP3D-R332Q) with mutation of arginine (Arg-332) to glutamine.
CHO cells were transfected with EP3D-R332Q cDNA, and cells stably
expressing EP3D-R332Q were established. Sulprostone, a widely used PGE
analogue replaced at and Sulprostone on
G
and G
Activities of EP3D and
EP3D-R332Q Receptors
-carboxylic acid by methanesulfonamide, has
almost the same agonist potency as PGE
(20) . Thus,
we examined the effects of PGE
, an agonist with negatively
charged
-carboxylic acid, and sulprostone, an agonist with
noncharged modified
-carboxylic acid, on the adenylate cyclase
system of EP3D- or EP3D-R332Q-expressing CHO cells. The EP3D receptor
shows a biphasic, inhibitory and stimulatory response in the adenylate
cyclase system due to functional coupling to both G
and
G
(13) . To ablate receptor coupling to one or the
other G protein, the cells were treated with PT or CT. These treatments
of the cells with PT and CT completely eliminated further
ADP-ribosylation by PT and CT in the in vitro assay,
respectively (data not shown). As Fig. 1A shows,
PGE
and sulprostone concentration dependently inhibited the
forskolin-stimulated adenylate cyclase activity in CT-treated cells
expressing the EP3D receptor, the half-maximal concentrations for the
inhibition (10 nM) and levels of the maximal inhibition (75%
inhibition) being the same. PGE
and sulprostone also
inhibited adenylate cyclase activity in the EP3D-R332Q receptor
concentration-dependently, but the half-maximal concentrations for the
inhibition (100 nM) were 1 order of magnitude higher than
those in the EP3D receptor. On the other hand, the level of the maximal
inhibition in the EP3D-R332Q receptor was the same as that in the EP3D
receptor. PGE
concentration-dependently stimulated basal
adenylate cyclase activity, but sulprostone showed less potent
stimulation in PT-treated cells expressing the EP3D receptor (Fig. 1B). By contrast, neither PGE
nor
sulprostone stimulated it in the EP3D-R332Q receptor.
Figure 1:
Effects of PGE and
sulprostone on adenylate cyclase activity in CT- or PT-treated
permeabilized cells expressing EP3D or EP3D-R332Q receptor. A,
G
activity; CT-treated permeabilized cells expressing the
EP3D (
,
) or EP3D-R332Q receptor (
,
) was
assayed for adenylate cyclase activity with the indicated
concentrations of PGE
(
,
) or sulprostone
(
,
) in the presence of 3 µM forskolin, as
described under ``Experimental Procedures.'' Values are
expressed as a percentage of the control (41.5 ± 3.2
pmol/min/mg) obtained with the cells in the absence of agonist. B, G
activity; PT-treated permeabilized cells
expressing each receptor were assayed for adenylate cyclase activity
with the indicated concentrations of PGE
(
,
) or
sulprostone (
,
). The values are means of triplicate
experiments, which varied by less than 5%.
We next
examined the agonist-mediated G protein activation in the PT- or
CT-treated cell membrane expressing each receptor. Fig. 2A shows the GTPase activity of the CT-treated cell membrane, this
treatment blocking the GTPase activity of G but allowing
that of G
. PGE
and sulprostone stimulated the
GTPase activity in the CT-treated cell membrane expressing the EP3D
receptor, their concentration dependence profile being the same.
PGE
and sulprostone also stimulated it in the EP3D-R332Q
receptor concentration-dependently, but the half-maximal concentrations
for the stimulation (100 nM) were 1 order of magnitude higher
than those in the EP3D receptor. On the other hand, the level of the
maximal stimulation in the EP3D-R332Q receptor was the same as that in
the EP3D receptor. Fig. 2B shows the GTPase activity of
the PT-treated cell membrane, this treatment blocking the
receptor-mediated activation of GTPase activity of G
but
allowing that of G
. PGE
concentration-dependently stimulated the GTPase activity in the
PT-treated cell membrane expressing the EP3D receptor, but the
stimulation by sulprostone was much lower than that by PGE
.
By contrast, the agonist-induced stimulation of GTPase activity was not
observed with the EP3D-R332Q receptor. To assess the selective
determination of GTPase activity of each type of G protein, we examined
the ability of antisera against G
and G
to affect agonist-stimulated GTPase activity in the CT- and
PT-treated cell membranes, respectively. As shown in , the
antiserum against G
attenuated the
PGE
-stimulated GTPase activity in the CT-treated cell
membrane expressing the EP3D or EP3D-R332Q receptor. The antiserum
against G
attenuated the stimulation in the PT-treated
cell membrane expressing the EP3D receptor. Thus, the
PGE
-stimulated GTPase activities in CT- and PT-treated cell
membranes are ascribed to G
and G
,
respectively, two receptors are exclusively coupled to G
in
the CT-treated cell membranes, and the EP3D receptor is coupled to
G
in the PT-treated cell membrane.
Figure 2:
Effects of PGE and sulprostone
on GTPase activity in the membrane of CT- or PT-treated cells
expressing EP3D or EP3D-R332Q receptor. The membrane of CT- (A) or PT- (B) treated cells expressing the EP3D
(
,
) or EP3D-R332Q receptor (
,
) was assayed
for GTPase activity with the indicated concentrations of PGE
(
,
) or sulprostone (
,
), as described
under `` Experimental Procedures.'' The values are means of
triplicate experiments, which varied by less than
5%.
Agonist Binding to EP3D or EP3D-R332Q Receptor Coupled to
Either G
We next examined the
PGEor G
binding affinities of EP3D and EP3D-R332Q receptors in
the conditions of their selective coupling to either G
or
G
. Fig. 3shows the Scatchard analysis of PGE
binding to CT- or PT-treated cell membrane expressing the EP3D or
EP3D-R332Q receptor. EP3D receptor coupled to either G
or
G
had an apparently single high affinity binding site, the K
value (3.8 nM) of the receptor
coupled to G
being slightly lower than that (5.0
nM) of the receptor coupled to G
. On the other
hand, EP3D-R332Q receptor had also an apparently single high affinity
binding site (K
= 10.2
nM) when coupled to G
, but it showed very low
affinity (K
> 200 nM) in the
condition of coupling to G
.
Figure 3:
Scatchard
analysis of PGE binding to the membrane of CT- or
PT-treated cells expressing EP3D or EP3D-R332Q receptor. The specific
binding of [
H]PGE
(0.25-100
nM) to the membrane of CT- (
) or PT- (
) treated
cells expressing EP3D (A) or EP3D-R332Q receptor (B)
was determined, as described under ``Experimental
Procedures.'' The Scatchard plot was transformed from the values
of specific [
H]PGE
binding.
We further analyzed the
binding affinities of EP3D and EP3D-R332Q receptors, coupled to either
G or G
, for PGE
and sulprostone by
assessing the displacement of [
H]PGE
binding to the receptors. Fig. 4A shows the
binding to the EP3D receptor. PGE
inhibited the
[
H]PGE
binding to either the CT- or
PT-treated cell membrane expressing the EP3D receptor, their
half-maximal concentrations for the inhibition (10 nM) being
the same. Sulprostone also inhibited the binding to the CT-treated cell
membrane with the same half-maximal inhibition as that of
PGE
, but the displacement curve of sulprostone shifted 1
order toward the right in the PT-treated cell membrane. In contrast to
PGE
, the binding affinity of the G
-coupled
receptor for sulprostone was lower than that of the
G
-coupled receptor. Fig. 4B shows the
binding to the EP3D-R332Q receptor. Displacement curves of PGE
and sulprostone were the same in the CT-treated cell membrane. On
the other hand, we could not obtain displacement curves of PGE
and sulprostone in the PT-treated cell membrane because the
membrane did not exhibit significant specific
[
H]PGE
binding at this
[
H]PGE
concentration (4 nM)
used, as suggested by the Scatchard analysis (Fig. 3B).
Figure 4:
Displacement of
[H]PGE
binding by PGE
and
sulprostone in the membrane of CT- or PT-treated cells expressing EP3D
or EP3D-R332Q receptor. The membrane of CT- (
,
) or PT-
(
,
) treated cells expressing EP3D (A) or
EP3D-R332Q receptor (B) was incubated with 4 nM [
H]PGE
and the indicated
concentrations of PGE
(
,
) or sulprostone
(
,
). Specific [
H]PGE
binding was determined as described under ``Experimental
Procedures.'' Values are expressed as percentages of the control
(1.85 ± 0.078 pmol/mg; PT-treated EP3D; 1.70 ± 0.044
pmol/mg, CT-treated EP3D; 1.02 ± 0.037 pmol/mg, CT-treated
EP3D-R332Q). The values are means of triplicate experiments, which
varied by less than 5%.
Effects of Various EP3 Agonists on G
We examined the effects of various EP3 agonists
with a negatively charged and
G
Activities of EP3D and EP3D-R332Q
Receptors
-carboxylic acid or noncharged modified
-carboxylic acid on the adenylate cyclase system of EP3D and
EP3D-R332Q receptors. PGE
, M& 28767(21) , and
TEI-3356 (22) are EP3 agonists with negatively charged unmodified
-carboxylic acid, but instead M& 28767 and TEI-3356 are
modified at the 11-hydroxyl and 9-carbonyl groups, respectively. On the
other hand, sulprostone, misoprostol(21) , and GR 63799X (23) are EP3 agonists with a noncharged modified
-carboxylic acid; their
-carboxylic acids being substituted
by methanesulfonamide, methylester, and phenyl groups, respectively. As
shown in , agonists with a negatively charged
-carboxylic acid not only inhibited the forskolin-stimulated
adenylate cyclase activity in the CT-treated cells expressing EP3D
receptor but also stimulated the basal adenylate cyclase activity in
the PT-treated cells. By contrast, agonists with noncharged modified
-carboxylic acid strongly inhibited the forskolin-stimulated
adenylate cyclase activity, but they only very weakly stimulated the
basal adenylate cyclase activation. In the EP3D-R332Q receptor, all of
the EP3 agonists used showed only G
activity. These
findings suggest that the interaction between
-carboxylic acid of
PGE
and the arginine residue of EP3D receptor plays an
important role for the G
activation but not for the G
activation.
and G
, respectively(24, 25) . The M1
muscarinic receptor and thrombin receptor mediate both adenylate
cyclase inhibition and phospholipase C activation through G
and G
, respectively(26, 27) . The
-adrenergic receptor is simultaneously coupled to
G
and G
, signaling the stimulation and
inhibition of adenylate cyclase, respectively(28) . Concerning
ligand binding sites, several charged amino acid residues in the
transmembrane domains of
-adrenergic receptor, coupled
to both G
and G
, have been shown to be
essential for agonist binding according to site-directed mutagenesis
studies(29) . However, the residues involved in the activation
of each G protein are not understood.
-carboxylic
acid of PGE
, is essential for the PGE
-induced
adenylate cyclase stimulation but not for the inhibition (Fig. 1), and this is due to the different requirement for the
arginine residue in the receptor for PGE
-induced activation
of G
and G
(Fig. 2). In the
receptor-G
coupling, the EC
value of PGE
for the G
activation in EP3D-R332Q receptor was about
1 order of magnitude higher than that in the EP3D receptor (Fig. 2). This difference can be probably attributed to the
difference between the K
values of the
G
-coupled receptors for PGE
(Fig. 3). The
point mutation at the arginine may induce a somewhat conformational
change of the receptor structure as a whole, leading to a decrease in
the efficiency of the agonists for G
activation. However,
it is clear that the mutant receptor has the ability to fully activate
G
because the maximal levels of PGE
-induced
G
activation in the two receptors were the same. In the
receptor-G
coupling, EP3D-R332Q receptor almost completely
lost the activation of G
(Fig. 2), and this is due to
loss of high-affinity binding of the receptor for PGE
in
the condition of coupling to G
(Fig. 3B).
Thus, the arginine residue in the seventh transmembrane domain is
essential for high-affinity binding complex formation of the receptor
with G
but not for that of the receptor with G
.
-carboxylic acid
of agonists to the G protein activation. PGE
with a
negatively charged
-carboxylic acid activated both G
and G
(Fig. 2). On the other hand, sulprostone
with a noncharged modified
-carboxylic acid activated G
equally as well as did PGE
, but it did not activate
G
as well as it did PGE
(Fig. 2). This
difference was also observed in the PGE
binding
displacement by sulprostone; the G
-coupled receptor showed
lower binding affinity for sulprostone than the G
-coupled
one (Fig. 4A). PGE
has several structural
features participating in its agonist activity,
-carboxylic acid,
15-hydroxyl group, 9-carbonyl group, and cyclopentane ring(6) .
Among these features, the modification site in sulprostone is
-carboxylic acid, and other sites are not modified. Thus, the low
potency of sulprostone for G
activation may be ascribed to
the modification of
-carboxylic acid. Furthermore, this weak
effect on G
activity was also observed with other EP3
agonists with various modifications at the
-carboxylic acid (). On the other hand, EP3 agonists with an unmodified
-carboxylic acid had both potent G
and G
activity, even if they had modification at the sites other than
-carboxylic acid. These findings indicate that
-carboxylic
acid is important to the G
-receptor coupling, and this
modification significantly affects coupling. Although the agonists with
modified
-carboxylic acid had a much lower potency for G
activity than PGE
, they still showed significant
activation (). Although
-carboxylic acid is important
for determination of G
coupling, other group(s) of
PGE
structure may also contribute to this determination in
concert with
-carboxylic acid, or there might be noncharged
interaction of the modified
-carboxylic acid of the agonists with
the arginine, such as hydrogen bonding. Sulprostone is a
well-characterized PGE analogue widely used as a very valuable and
potent EP3 agonist(6) . Our finding of preferential activation
of G
by sulprostone suggests that a single receptor has a
functionally different pharmacological profile depending on the ligand
structure. PGE
at lower concentrations was previously
demonstrated to inhibit vasopressin-induced cAMP accumulation but at
high concentrations to stimulate cAMP accumulation in the cortical
collecting duct, resulting in dual regulation of water reabsorption
(30). In contrast to PGE
, sulprostone prevented the
vasopressin-induced cAMP accumulation but failed to stimulate cAMP
accumulation(30) . Considering selective expression of EP3
receptors, including EP3D-type isoform, among various PGE receptor
subtypes in the cortical collecting duct(31, 32) , the
stimulatory activity of PGE
would be mediated by EP3D, and
this idea is consistent with the weak effect of sulprostone to activate
G
.
-carboxylic acid of PGE
and the arginine residue of the receptor is required for the
receptor-G
coupling but not for the receptor-G
coupling, and this interaction is correlated with G protein
selectivity. Functional analysis of the agonist-binding site of the M3
muscarinic receptor suggested that Thr-234 and Tyr-509, which are
located at fifth and sixth transmembrane domains, respectively, were
the binding sites of acetylcholine and also played a role in
agonist-induced receptor activation(33) . These transmembrane
domains are connected by the third intracellular loop, a region known
to be critical for G protein recognition and activation by the M3
muscarinic receptor(34) . These two binding residues have been
proposed to be involved in triggering the agonist-induced
conformational change in the fifth and sixth transmembrane domains
required for the functional activation of the third intracellular loop.
In EP3 receptors, we demonstrated that EP3 receptor isoforms with
different COOH-terminal tails, which are produced through alternative
splicing, are coupled to different G proteins and showed that the
COOH-terminal tail determines the selectivity of G protein coupling
(12, 13). The seventh transmembrane domain is directly connected with
the COOH-terminal tail. Thus, a conformational change of the
COOH-terminal tail may regulate the selectivity of G protein coupling,
and the EP3D receptor may be able to form two types of conformation,
which can selectively be associated with either G
or
G
, depending on the interaction of
-carboxylic acid
and the arginine residue. Concerning coupling of receptors with G
proteins, Lefkowitz and co-workers have proposed a two-state model in
which receptors are in equilibrium between the inactive conformation
and a spontaneously active conformation that can couple to G protein,
and classic agonists increase the concentration of the latter
conformation of the receptors(35) . Although our present study
suggests that the EP3D receptor has two active conformations, coupling
different G proteins, it is not known whether the selective coupling of
the receptor to a particular G protein is a consequence of the
interaction of the ligand with the receptor or whether the G protein
coupling of the receptor influences its affinity for a particular
ligand.
-carboxylic acid of PGE
, contributes to the
selectivity of G protein coupling, and
-carboxylic acid of
PGE
plays an important role in the G protein coupling
selection, perhaps through interaction with the arginine residue. This
study will contribute not only to understanding the diverse PGE
actions, but it will also help to elucidate the molecular
mechanism of receptor-mediated multiple G protein activation.
Table: Functional attenuation of G or
G
coupling by G
or G
antiserum
antiserum (AS/7), or G
antiserum (RM/1) for 1 h at 4 °C, after which the membrane
was assayed for GTPase activity with or without 10 µM PGE
, as described under ``Experimental
Procedures.'' Values are means ± S.E. for triplicate
experiments.
Table: 0p4in
Noncharged modified
-carboxylic
acid.
,
-imidodiphosphate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.