(Received for publication, September 18, 1995)
From the
We previously identified two isoforms of the mouse prostaglandin
E receptor EP3 subtype, EP3 and EP3
, with different
carboxyl-terminal tails, produced through alternative splicing and
showing different efficiency in inhibition of adenylate cyclase
(Sugimoto, Y., Negishi, M., Hayashi, Y., Namba, T., Honda, A., Watabe,
A., Hirata, M., Narumiya, S., and Ichikawa, A.(1993) J. Biol. Chem. 268, 2712-2718). To assess the role of the carboxyl-terminal
tails in the G protein coupling properties of the EP3 receptor, we
examined the G
activities of EP3
, EP3
, and the
mutant receptor, in which the carboxyl-terminal tail was truncated at
the splicing site. The EP3
receptor showed marked
agonist-independent constitutive inhibition of adenylate cyclase, while
EP3
receptor had no agonist-independent inhibition. On the other
hand, the truncated receptor showed only agonist-independent
constitutive inhibition. The constitutive activity of these receptors
on the stimulation of GTPase activity of G
was also
observed. Thus, alternative splicing produced two isoforms with
different carboxyl-terminal tails and with different constitutive
activity, and the truncation of the carboxyl-terminal tail caused full
constitutive activity.
Prostaglandin E (PGE
) (
)produces a broad range of biological actions in diverse
tissues through its binding to specific receptors on plasma
membranes(1, 2) . PGE receptors are pharmacologically
subdivided into four subtypes, EP1, EP2, EP3, and EP4, on the basis of
their responses to various agonists and
antagonists(3, 4) . Among these subtypes, the EP3
receptor has been most well characterized and has been suggested to be
involved in such PGE
actions as contraction of the
uterus(5) , inhibition of gastric acid secretion(6) ,
modulation of the neurotransmitter release(7) , lipolysis in
adipose tissue(8) , and sodium and water reabsorption in the
kidney tubulus(9, 10) . Although EP3 receptor-mediated
actions are believed to be mediated by inhibition of adenylate cyclase,
the dose-response curve and potency of PGE
vary with
tissue, implying heterogeneity of EP3
receptors(11, 12) .
We have recently cloned the
mouse EP3 receptor and demonstrated that this receptor is a G
protein-coupled rhodopsin-type receptor that engages in inhibition of
adenylate cyclase(13) . Furthermore, we identified the two
isoforms of the mouse EP3 receptor with different COOH-terminal tails,
which are produced through alternative splicing and show different
efficiency in inhibition of adenylate cyclase(14) . Therefore
the COOH-terminal tails of the EP3 receptor may play an important role
in the receptor-G protein coupling. To assess the role of the
COOH-terminal tails of the EP3 receptor in coupling to G proteins, we
constructed a mutated EP3 receptor, T-335, in which the COOH-terminal
tail was truncated at the alternative splicing site, and showed that
the truncated receptor retained identical agonist binding activity and
ability to associate with G(15) . We studied in
more detail the G protein coupling properties of EP3
, EP3
,
and T-335 receptors. We report here that the two isoforms of EP3
receptor differ in agonist-independent constitutive activity, and the
mutant receptor without the COOH-terminal tail is a fully constitutive
active receptor.
Chinese hamster ovary (CHO) cells stably expressing EP3,
EP3
(14) , or the truncated receptor, T-335(15) ,
were cultured in the
-modification of Eagle's medium lacking
ribonucleosides and deoxyribonucleosides, with 10% dialyzed fetal
bovine serum under humidified air containing 5% CO
at 37
°C.
Figure 1:
Inhibition of adenylate cyclase by
EP3, EP3
, and T-335. CHO cells expressing EP3
(
),
EP3
(
), or T-335 (
) were incubated at 37 °C for
10 min with 10 µM forskolin in the absence or presence of
the indicated concentrations of M& 28767, then cAMP contents were
determined as described under `` Experimental Procedures.''
The values are means of triplicate experiments, which varied by less
than 5%.
To assess
the constitutive activities of the receptors, we examined the effect of
PT on the G activities of these receptors. As shown in Fig. 2A, PT treatment concentration-dependently
increased the forskolin-stimulated cAMP formation in the absence of the
agonist in the EP3
receptor-expressing cells. PT treatment
attenuated the M& 28767-induced inhibition of the
forskolin-stimulated cAMP formation, the cAMP level reaching the same
value as that in the absence of the agonist. Although PT treatment
completely attenuated the agonist-induced inhibition of the
forskolin-stimulated cAMP formation in the EP3
receptor, this
treatment did not increase the forskolin-stimulated cAMP formation in
the absence of the agonist, and the treatment rather slightly
suppressed the cAMP level at higher concentrations of PT (Fig. 2B). This suppression at higher concentrations of
PT was also observed in the mock-transfected cells (data not shown). In
the T-335-expressing cells, PT treatment increased the cAMP level of
the forskolin-stimulated formation in the absence and presence of the
agonist with the same concentration-dependent curve, the level reaching
maximally the value of PT-treated EP3
- and EP3
-expressing
cells (Fig. 2C). To confirm the effect of the toxin,
membrane fractions exposed to the toxin were incubated with the
activated toxin and [
-
P]NAD. The
ADP-ribosylation of G
was decreased progressively as the
concentration of the toxin used for the pretreatment was increased
(data not shown). These findings indicate that the truncated receptor
is fully constitutively active and two isoforms differ in constitutive
activity.
Figure 2:
Effect of PT treatment on
agonist-dependent or -independent inhibition of adenylate cyclase by
EP3, EP3
, or T-335. After cells expressing EP3
(A), EP3
(B), or T-335 (C) had been
treated with the indicated concentrations of PT for 6 h, they were
incubated at 37 °C for 10 min with 10 µM forskolin in
the absence (
) or presence (
) of 1 µM M&
28767, then cAMP contents were determined as described under
``Experimental Procedures.'' The values are means of
triplicate experiments, which varied by less than
5%.
Figure 3:
GTPase
activity of EP3, EP3
, or T-335. The membrane of cells
expressing EP3
(
), EP3
(
), or T-335 (
) was
assayed for GTPase activity with the indicated concentrations of
M& 28767, as described under ``Experimental
Procedures.'' The membrane (10 µg) of cells expressing
EP3
(
), EP3
(
), or T-335 (
) was incubated
with 2 µl of G
antiserum (AS/7) for 1 h at 4
°C, after which the membrane was assayed for GTPase activity with 1
µM M& 28767. The values are means of triplicate
experiments, which varied by less than 5%.
To confirm the
constitutive activation of G in these receptors, we
examined the effect of PT on the agonist-dependent or independent
activation of G
in the membrane expressing each receptor (Fig. 4). In the EP3
receptor, PT treatment suppressed the
agonist-induced stimulation of GTPase activity without any change of
the basal activity. On the other hand, PT treatment decreased both
basal activity and agonist-induced stimulation of GTPase activity in
the EP3
receptor to the level of the basal activity in the
EP3
receptor. PT treatment also decreased the GTPase activity in
the T-335 receptor in the absence and presence of the agonist to the
level of the basal activity in the EP3
receptor.
Figure 4:
Effect of PT pretreatment on
agonist-dependent or -independent stimulation of GTPase activity by
EP3, EP3
, or T-335. After cells expressing EP3
,
EP3
, or T-335 had been treated with (&cjs2108;, &cjs2113;) or
without (
,
) 10 ng/ml PT for 6 h, the membrane of cells
expressing each receptor was assayed for GTPase activity with (
,
&cjs2113;) or without (
, &cjs2108;) 1 µM M&
28767, as described under ``Experimental Procedures.'' The
values are means ± S.E. for triplicate
experiments.
Alternative splicing in transcription from a single gene
produces related protein isoforms with distinct primary structures and
adds different functional properties to the protein. We have already
demonstrated that alternative splicing generates two EP3 receptor
isoforms with different COOH-terminal tails, which are different in the
sensitivity to agonists in the adenylate cyclase
inhibition(14) . We here revealed that the two isoforms differ
in constitutive activity; the EP3 receptor has high constitutive
activity, whereas the EP3
receptor has no constitutive activity.
The EP3
and EP3
receptors are the first example of isoforms
produced through alternative splicing, showing different constitutive
activity. Several reports have been made on endogenous receptors,
having some levels of constitutive activity(17, 18) ,
and receptors having greater constitutive activities have been
speculated to show a reduced -fold stimulation due to the high basal
activity and receptors with lower or no constitutive activities to
reflect a broader spectrum of agonist responses(19) . The EP3
receptor isoforms might, therefore, underlie the diverse dose-response
curves of PGE
and provide a variety of the receptor
responses in inhibition of adenylate cyclase. In addition to the
EP3
and EP3
receptors, which are exclusively coupled to
G
, we identified a third EP3 receptor isoform, EP3
,
with a different COOH-terminal tail, which is also produced through
alternative splicing, and we demonstrated that the EP3
receptor is
coupled to multiple G proteins, G
and G
,
suggesting that the COOH-terminal tails of EP3 receptor participate in
determination of G protein specificity(20) . The truncated EP3
receptor showed neither elevation of basal adenylate cyclase activity
nor its agonist-dependent stimulation, indicating that the receptor did
not show constitutive G
activity. (
)
Lefkowitz
and co-workers have proposed a two-state model in which receptors are
in equilibrium between the inactive conformation and the active
conformation that can associate and activate G protein, and classical
agonists increase the concentration of the latter conformation of the
receptors(19) . Our findings demonstrate that most of the
EP3 receptors have the inactive conformation in the absence of the
agonist, while about half of the EP3
receptors already have the
active conformation in the absence of the agonist, and the agonist
shifts all of the EP3
receptor and the EP3
receptor with an
inactive form from inactive conformation to fully active conformation.
The domains of the receptors, which interact with and activate G
proteins, have been studied extensively(22) . These studies
showed that synthetic peptides derived from specific regions in the
second and third intracellular loops in various receptors directly
activate G proteins. Therefore, the receptors in the inactive
conformation may prevent the activating domains from association and
activation of G proteins, and an agonist-promoted conformational change
of the receptors may release the constraint, allowing the domains to
associate with G proteins. Because the two EP3 receptor isoforms with
different constitutive activity are different only in the COOH-terminal
tail, and the truncated receptor exhibited agonist-independent
constitutive activity, the COOH-terminal tails after the alternative
splicing site may suppress activation of G by the EP3
receptor to a different extent dependent on the structure of the
COOH-terminal tail. The COOH-terminal tail of the EP3
receptor
completely suppresses the activity, while that of the EP3
receptor
partially suppresses it, and agonists release the suppression by the
COOH-terminal tail. Constitutively active receptors with mutations in
the COOH-terminal domain of the third intracellular loops have been
reported(22, 23) . Recently, truncation of the
COOH-terminal tail has been shown to cause constitutive activity in the
thyrotropin-releasing hormone receptor (24) . Thus, our finding
also supports the idea that the COOH-terminal tail is the site involved
in the constraint of the receptor in its inactive conformation in
various receptors.
In summary, we present here that two EP3 receptor
isoforms differ in constitutive activity and that the COOH-terminal
tails play an important role in the constraint of the EP3 receptor in
its inactive conformation. This study will contribute not only to the
understanding heterogeneity of PGE actions but will also
help to elucidate the molecular mechanism of G protein activation
induced by receptors.