From the
The endothelin (ET) family of peptides acts via two subtypes of
guanine nucleotide-binding regulatory protein (G protein)-coupled
receptors termed ET
Guanine nucleotide-binding regulatory protein (G
protein)
The endothelins (ETs) are a
family of potent vasoactive peptides termed endothelin-1, -2, and -3
(ET-1, -2, and -3)
(14, 15) . cDNA cloning of
cell-surface receptors for ET-1 identified two subtypes of ET receptors
(ETRs), denoted ET
Initial studies indicated that both subtypes of hETR coupled with
pertussis toxin (PTX)-insensitive G protein, possibly a member(s) of
G
In a preliminary study, we found that hET
Expression of ET
Both hET
Aramori and Nakanishi
(23) demonstrated the selective coupling of bovine ET
The data on the stimulatory effect of ET-1 on cAMP formation
indicated the roles of both ICLII and -III in the selective activation
of G
The data on the inhibitory effects of ET-1 indicated
the role of ICLIII of hETR as a determinant for the selective
activation of G
That the characteristics of
structural basis for the selective coupling are shared by hET
There was a large difference in the EC
We also
demonstrated in the present study that each receptor subtype expressed
on the same cell could work independently, i.e. for hET
Clonal cell lines
expressing each receptor construct were isolated as described under
``Materials and Methods.'' Binding parameters were determined
by saturation isotherms of
and ET
. ET-1 stimulated
cAMP formation in Chinese hamster ovary (CHO) cells stably expressing
human wild-type ET
(CHO/hET
cells) while it
inhibited cAMP formation in CHO cells expressing human wild-type
ET
(CHO/hET
cells), and pharmacological
evidence indicated that the opposite effects were due to the selective
coupling of each receptor subtype with G
s/G
i. To find out a
receptor domain(s) that determined the selective coupling, a series of
chimeric receptors between hET
and hET
was
expressed on CHO cells, and the effect of ET-1 on cAMP formation in
each cell line was tested. hET
with the replacement of
second and/or third intracellular loop (ICLII and/or -III) to the
corresponding region(s) of hET
failed to transmit the
stimulatory effect of ET-1. hET
with the replacement of
ICLIII to the corresponding region of hET
failed to
transmit the inhibitory effect of ET-1. A chimeric receptor with ICLII
of hET
and with ICLIII of hET
failed to
transmit both effects. In cells expressing chimeric receptors with
ICLII of hET
and with ICLIII of hET
, ET-1
inhibited cAMP formation while it stimulated cAMP formation when cells
were pretreated with pertussis toxin. These results indicated the roles
of ICLII and -III of hETR as a major determinant of the selective
coupling of hET
and hET
with G
s/G
i,
respectively. We also demonstrated that each receptor subtype expressed
on the same cell could work independently, i.e. for hET
to activate G
s and for hET
to activate G
i,
resulting in dose-dependent dual effects of ET-1 on cAMP formation.
(
)
-coupled receptors can be thought of as
having two binding domains, a ligand-binding domain on the
extracellular side and a G protein-binding domain on the intracellular
side. As for the G protein-binding domain, early studies on adrenergic
and muscarinic receptors using receptor chimeras, deletions, or point
mutations indicated the importance of the third intracellular loop
(ICLIII)
(1, 2, 3, 4, 5, 6, 7) .
Subsequent studies including those using peptides that mimic or inhibit
receptor-G protein interaction, however, suggested the presence of
multiple points of interaction between receptors and G proteins
(8, 9) , and now it is generally assumed that the
binding surface comprises, at least, regions from ICLII, ICLIII, and
carboxyl-terminal cytoplasmic tail; each receptor region contributes to
G protein specificity in a combinatorial fashion, enhancing or
restricting interaction with particular G proteins
(10, 11) . Recent identifications of splice variants
with differential G protein specificities of prostaglandin E receptor
(12) and of pituitary adenylyl cyclase-activating polypeptide
receptor
(13) illustrated the examples of in vivo tuning of G protein selection by receptor sequences in carboxyl
terminus and ICLIII, respectively.
and ET
, both of which belong
to a family of G protein-coupling receptors
(16, 17) .
The two subtypes can be pharmacologically distinguished by different
rank orders of affinity toward the three ET isopeptides; ET
is ET-1 selective, showing an affinity rank order of ET-1
ET-2
ET-3, whereas ET
exhibits similar affinities to
all three isopeptides
(16, 17) . In other words, ET-1 is
a nonselective agonist, and ET-3 is an ET
-selective
agonist. Human ET
and ET
(hET
and
hET
) exhibit a high polypeptide sequence identity to each
other (55% overall, 74% within the putative transmembrane helices)
(18, 19) . Of the receptor subdomains, the extracellular
amino terminus and the intracellular carboxyl terminus display the
least sequence similarities, whereas the sequences both in ICLII and
-III are relatively well conserved (16 out of 21 residues of ICLII and
21 out of 29 residues of ICLIII are identical). The significance of the
heterogeneity of either amino or carboxyl terminus in the
subtype-specific receptor functions has not yet been elucidated.
q family, to activate phospholipase C
(20) . It is,
however, now apparent that both of them belong to a subfamily of G
protein-coupled receptors with a promiscuous nature, which can activate
multiple types of G proteins. Interaction of ETR with G
s has been
suggested by studies demonstrating ET-1-induced increase in cAMP
formation in rat vascular smooth muscle cells
(21) , bovine
tracheal cells
(22) , or Chinese hamster ovary (CHO) cells
expressing rat ET
(23) . Interaction of ETR with
G
i has been suggested by studies demonstrating ET-1-induced
decrease in cAMP formation in bovine endothelial cells
(21) , C6
glioma cells
(24) , or CHO cells expressing bovine ET
(23) .
and hET
, when expressed on CHOK1 cells, transmitted
opposite effects on cAMP formation in the cells. Because
pharmacological evidence indicated that the opposite effects were due
to the selective coupling of each receptor subtype with
G
s/G
i, we set out to reveal the structural basis for it using
hET
chimeras. To circumvent the potential influence of
variable receptor densities on cell responses, we generated stable
transfectants of each receptor construct and subjected clones with
similar receptor densities to the study.
and ET
are not exclusive to each other in native
cells; some types of cells express both mRNAs for the receptor subtypes
(25) . In an attempt to obtain an experimental model that
mimicked the situation, we also generated a clonal cell line that
co-expressed hET
and hET
and studied the
signaling events activated by ET-1.
Reagents
Materials were obtained from
the following sources: HAM F12 medium, fetal calf serum, lipofectamine,
G418, and blasticidin from Life Technologies, Inc. (Tokyo); synthetic
human ET-1 and ET-3 from Peptide Institute (Osaka, Japan);
I-ET-1 (74 TBq/mmol) and cAMP radioimmunoassay assay kit
from Amersham (Buckinghamshire, United Kingdom); PTX and U-73122
(1-(6-[(17
-3-methoxyestra-1,3,5
(10) -trien-17-yl)amino]hexyl)-1H-pyrrole-2,5-dione)
from Funakoshi Co. Ltd. (Tokyo, Japan); Fura-2 acetoxymethyl ester from
Dojin Chemicals (Tokyo); BCA microprotein assay kit from Pierce. The
ET
antagonist 97-139
(27- O-3-[2-(3-carboxy-acryloylamino)-5-hydroxyphenyl]-acryloyloxy
myricerone) was kindly provided by Shionogi & Co. (Osaka, Japan),
and the ET
antagonist BQ788
( N- cis-2,6-dimethylpiperidinocarbonyl - L -
- methylleucyl - D 1 -
methoxycarbonyltryptophanyl-D-norleucine) was kindly provided
by Ciba-Geigy Japan Ltd. (Hyogo, Japan). All other chemicals were of
reagent grade and were obtained commercially.
Stable Expression of Wild-type and Chimeric hETRs in
CHO Cells
To obtain a cell line stably expressing hETR, we
used SR promoter-based mammalian expression vector pME18Sf, which
carried cDNA construct encoding wild-type or chimeric
hET
. Procedures for construction and subcloning of
wild-type and chimeric receptor cDNA were exactly the same as those
previously described
(26) . Each expression plasmid was
cotransfected with pSVneo
plasmid into CHOK1 cells by
lipofection, using lipofectamine according to the manufacturer's
instructions. Cell populations expressing neo
gene
product were selected in HAM F12, 10% fetal calf serum containing G418
(0.5 mg/ml). From these selected cell populations, clonal cell lines
were isolated by colony lifting and maintained in the same selection
medium. The density of receptors expressed in each clone was determined
by saturation isotherms of
I-ET-1 binding to the crude
membrane preparations as described below. We have isolated several
clones for each expression plasmid with various receptor densities.
Cell clones showing similar levels of receptor densities were used for
the present study (see ``Results'' for the actual binding
capacities). To obtain a cell line that expressed both hET
and hET
, wild-type hET
cDNA was
cotransfected with pSVbsr
plasmid into CHOK1 cells stably
expressing wild-type hET
, and the cells were selected for
resistance against blasticidin (10 µg/ml). The ratio of the
densities of hET
/hET
expressed on each clone
was determined by displacement study of
I-ET-1 binding as
described below.
Receptor densities were determined by
I-ET-1 Binding
Assay
I-ET-1 binding assay according to the protocols
previously described
(17) with slight modifications. In brief,
crude membrane preparations were prepared by lysis of the cells
suspended in PBS by sonication followed by centrifugation at 100,000
g for 30 min. The pellets were suspended in PBS, and
protein concentrations were determined by BCA microprotein assay kit.
The membrane preparations (0.5-1.0 µg of protein/assay) were
incubated with various concentrations (10-1000 pM) of
I-ET-1 in 0.2 ml of PBS, 0.2% bovine serum albumin. After
incubation for 60 min at 25 °C, bound ligand was separated from
free ligand by filtration through Whatman GF/C glass fiber filters
pre-immersed in the ice-cold binding medium. The filters were washed
with ice-cold PBS and counted for radioactivity in a
-counter.
Nonspecific binding was defined as the binding in the presence of 100
nM unlabeled ET-1 and was always less than 10% of the total
binding activity. For displacement study of
I-ET-1
binding, the membrane preparations were incubated with 100 pM
of
I-ET-1 and various concentrations (1
pM-1 µM) of either ET-1 or ET-3.
PTX Pretreatment of the Cells and Measurement of
Cyclic AMPFormation
Cells in 48-well plates at 50%
confluency were incubated for 16 h with or without PTX (50 ng/ml). The
cells were washed twice with PBS and then incubated at 37 °C for 10
min with 0.25 ml of PBS containing a phosphodiesterase inhibitor
3-isobutyl-1-methylxanthine (IBMX) (1 mM). IBMX alone caused
no significant change in basal cAMP formations (data not shown). ETR
antagonists, when used, were included in the preincubation medium. The
cells were then stimulated with ET-1 at 37 °C for 10 min at the
concentrations indicated. The reaction was stopped by addition of 10%
(w/v) trichloroacetic acid, and the cAMP content in the trichloroacetic
acid-soluble cell extract was measured using a radioimmunoassay kit
(Amersham).
Data Analysis
All of the experiments were
done in duplicate unless otherwise indicated. When necessary,
statistical analysis was done by analysis of variance.
Stable Expression of Wild-type and Chimeric
hETRs
By cotransfecting CHOK1 cells with pMEsf/hETR and
pSVneoconstructs and then selecting for resistance against
G418, we obtained more than three individual clonal cell lines that
stably expressed hETRs at various levels for each receptor construct.
Scatchard analysis of the data obtained from saturation isotherms with
I-ET-1 detected a single class of binding sites on each
cell clone. Cell clones showing similar levels of receptor densities
were used in the subsequent study. The K
and B
values for the receptors expressed
on each clone adopted are listed in . Functional expression
of each receptor construct was verified by the
[Ca
]
response evoked
by ET-1 according to the procedures previously described
(26) .
When loaded with Ca
-sensitive fluoroprobe Fura-2 and
stimulated by ET-1, every clone showed essentially the same pattern of
[Ca
]
transient,
i.e. 1) the response was biphasic, consisting of an initial
robust peak followed by a sustained increase, 2) maximal initial peak
increments (
1 µM) were obtained by 10 nM
ET-1, and 3) EC
values for ET-1 to give half-maximum
increments of the initial peak were
0.5 nM. Pretreatment
of the cells with PTX did not affect the
[Ca
]
response evoked
by ET-1 in any of the clones (data not shown). A phospholipase C
inhibitor, U-73122, at the concentration of 10 µM,
completely suppressed the
[Ca
]
response evoked
by ET-1 (10 nM) (data not shown).
Effects of ET-1 on cAMP Formation in CHO Cells
Expressing Wild-type hETR
In contrast to the apparently
identical [Ca]
responses, there were distinct differences in the effects of ET-1
on cAMP formation between CHO/hET
and CHO/hET
cells. First, ET-1 stimulated cAMP formation in CHO/hET
cells but not in CHO/hET
cells (Fig. 1,
left). The EC
value for the stimulatory effect of
ET-1 was 6.5 ± 0.3 nM (mean ± S.E., n = 3), and the maximum effect was an
8-fold increase
above basal formation. Second, ET-1 inhibited forskolin-induced cAMP
formation in CHO/hET
cells but not in CHO/hET
cells (Fig. 1, right). The EC
value
for the inhibitory effect of ET-1 was 68 ± 5 pM (mean
± S.E., n = 3), and the maximum effect was
50% inhibition. These differential effects of ET-1 were attributed
to a direct and selective coupling of hET
and hET
with G
s and G
i, respectively, based on the following
observations. First, PTX pretreatment of the cells caused no change in
the stimulatory effect of ET-1 on cAMP formation in CHO/hET
cells while it completely abolished the inhibitory effect of ET-1
in CHO/hET
cells (Figs. 2 and 3). Second, ET-1, when
applied together with GTP, stimulated cAMP formation in membrane
preparations from CHO/hET
cells but not in those from
CHO/hET
cells (data not shown). Third, effects of
arachidonic acid metabolites such as thromboxane A
or
prostacyclin, whose production could be stimulated by the activation of
phospholipase C/Ca
/protein kinase C signaling pathway
in some cell types, were excluded because 1) a phospholipase C
inhibitor, U-73122, at the concentration of 10 µM failed
to affect both the stimulatory effect of ET-1 in CHO/hET
cells and the inhibitory effect of ET-1 in CHO/hET
cells and 2) a cyclooxygenase inhibitor indomethacin (100
µM) also failed to affect both effects (data not shown).
Figure 1:
Effects of ET-1 on cAMP formation in
CHO cells expressing wild-type hET (
) or
hET
(
). Cells were stimulated with increasing
concentrations of ET-1 for 10 min with ( right) or without
( left) 100 µM forskolin in the presence of 1
mM IBMX. The contents of cAMP in the trichloroacetic
acid-soluble cell extracts were determined by radioimmunoassay. Values
are the means ± S.E. of three determinations each done in
duplicate.
Stimulatory Effects of ET-1 on cAMP Formation in CHO
Cells Expressing Chimeric hETR
To find out a receptor
domain(s) that determined the selective coupling of hETwith G
s, the effects of ET-1 on cAMP formation in stable
transfectants of chimeric receptors were tested (Fig. 2). In the
series of B/A chimeras, substitutions of hET
domains from
amino terminus up to third transmembrane helices (TMIII) to the
corresponding regions of hET
did not affect the coupling,
whereas further substitutions including ICLII resulted in a complete
loss of coupling. In the series of A/B chimeras, substitutions of
hET
domains from carboxyl terminus up to TMVII to the
corresponding regions of hET
did not affect the coupling,
whereas further substitutions including ICLIII resulted in an apparent
loss of coupling. The effect of ET-1 transmitted through a chimeric
receptor, A(N-IV)B(V-C), was peculiar in that, although lesser in
degree compared with the effect of ET-1 on cells expressing wild-type
hET
, ET-1 did cause a significant stimulation of cAMP
formation when cells were pretreated with PTX. The same was the case
for a chimeric receptor A(N-IV)B(V-VI)A(VII-C) in which ICLIII of
hET
was replaced with that of hET
.
Figure 2:
Stimulatory effects of ET-1 on cAMP
formation in CHO cells expressing wild-type ( wt) or chimeric
hETR and effects of PTX pretreatment. Cells, either untreated ( open bars) or pretreated ( hatched bars) with
PTX (50 ng/ml for 16 h), were stimulated with 10 nM ET-1 for
10 min in the presence of 1 mM IBMX. The contents of cAMP in
the trichloroacetic acid-soluble cell extracts were determined by
radioimmunoassay. Each bar represents the mean ± S.E.
of three determinations each done in duplicate. *, p < 0.01
(significantly different from the values of basal
formation).
Inhibitory Effects of ET-1 on cAMP Formation in CHO
Cells Expressing Chimeric hETR
To find out a receptor
domain(s) that determined the selective coupling of hETwith G
i, the effects of ET-1 on forskolin-stimulated cAMP
formation in cells expressing chimeric receptors were tested
(Fig. 3). In the series of A/B chimeras, substitutions of
hET
domains from amino-terminal head up to TMV to the
corresponding regions of hET
did not affect the coupling,
whereas further substitutions including ICLIII resulted in a complete
loss of coupling. In the series of B/A chimeras, substitutions of
hET
domains from carboxyl-terminal tail up to TMVII to the
corresponding regions of hET
did not affect the coupling,
whereas further substitutions including ICLIII resulted in a complete
loss of coupling. ET-1 inhibited forskolin-stimulated cAMP formation in
cells expressing A(N-IV)B(V-VI)A(VII-C), which carried ICLIII of
hET
and ICLI, -II, and -C termini of hET
. In
every case where the inhibitory effect of ET-1 was observed, it was
completely blocked by PTX pretreatment of the cells.
Figure 3:
Inhibitory effects of ET-1 on cAMP
formation in CHO cells expressing wild-type ( wt) or chimeric
hETR and effects of PTX pretreatment. Cells, either untreated ( open bars) or pretreated ( hatched bars) with
PTX (50 ng/ml for 16 h), were stimulated either with forskolin (100
µM) alone or with forskolin (100 µM) and ET-1
(10 nM) for 10 min in the presence of IBMX (1 mM).
The contents of cAMP in the trichloroacetic acid-soluble cell extracts
were determined by radioimmunoassay. The values were expressed as
relative to the forskolin-stimulated formation (100%). There were no
significant differences in the absolute values of forskolin-stimulated
formations in the cell clones examined. Each bar represents
the mean ± S.E. of three determinations each done in duplicate.
*, p < 0.01 (significantly different from the values from
of cAMP formation stimulated with forskolin
alone).
Effects of ET-1 on cAMP Formation in CHO Cells
Expressing a Chimeric Receptor A(N-IV)B(V-VI)A(VII-C)
A
detailed dose-response analysis of the effects of ET-1 on cAMP
formation in cells expressing A(N-IV)B(V-VI)A(VII-C) confirmed the
findings described above (Fig. 4). When cells were untreated with
PTX, ET-1 alone did not stimulate cAMP formation while it inhibited
forskolin-stimulated cAMP formation. Both the ECvalue for
the inhibitory effect of ET-1 (19 ± 2 pM, mean ±
S.E., n = 3) and the maximum effect (
50%
inhibition) were compatible with those observed for the effect of ET-1
in CHO/hET
cells. When cells were pretreated with PTX, ET-1
alone stimulated cAMP formation while it failed to inhibit
forskolin-stimulated cAMP formation. The EC
value for the
stimulatory effect of ET-1 was 5.7 ± 0.8 nM (mean
± S.E., n = 3), which was similar to that
obtained in CHO/hET
cells, and the maximum effect was a
4-fold increase, which was about half of the maximum effect of
ET-1 in CHO/hET
cells.
Figure 4:
Effects of ET-1 on cAMP formation in CHO
cells expressing a chimeric receptor A(N-IV)B(V-VI)A(VII-C). Cells were
either untreated () or pretreated (
) with PTX (50 ng/ml for
16 h). They were then stimulated with increasing concentrations of ET-1
for 10 min with ( right) or without ( left) 100
µM forskolin in the presence of 1 mM IBMX. The
contents of cAMP in the trichloroacetic acid-soluble cell extracts were
determined by radioimmunoassay. Values are the means ± S.E. of
three determinations each done in duplicate.
Effects of ET-1 on cAMP Formation in CHO Cells
Expressing Both hET
Clonal
cell lines co-expressing both hET and hET
and hET
were
isolated as described under ``Materials and Methods.''
Displacement study using
I-ET-1 and ET-3 revealed the
expression of two classes of binding sites with different affinities
for ET-3. One with the higher affinity was supposed to be
hET
, and the other with the lower affinity was supposed to
be hET
. Of the several clones obtained, one with the
approximately equal densities of each receptor subtype was adopted for
the subsequent study (Fig. 5). When the cells were not pretreated
with PTX, ET-1 caused dose-dependent dual effects on cAMP formation
(Fig. 6). One was the inhibitory effect observed at the
concentrations higher than 100 pM, and the other was the
stimulatory effect observed at the concentrations higher than 3
nM. PTX pretreatment oppositely modulated the two effects of
ET-1 (Fig. 6); the pretreatment completely abolished the
inhibitory effect of ET-1 while it enhanced the stimulatory effect.
These apparently complex effects of ET-1 reflected independent and
simultaneous activations of both receptor subtypes as revealed by
applications of selective antagonists. The ET
-specific
antagonist 97-139
(27) completely suppressed the
stimulatory actions of ET-1 on cAMP formation both in PTX-untreated and
pretreated cells (Fig. 7) while it caused no change in the
inhibitory actions of ET-1 (Fig. 8). The ET
-specific
antagonist BQ788
(28) completely suppressed the inhibitory
actions of ET-1 on cAMP formation both in PTX-untreated and pretreated
cells (Fig. 8) while it enhanced the stimulatory actions of ET-1
in PTX-untreated cells but not in PTX-pretreated cells (Fig. 7).
Figure 5:
Displacement by either ET-1 () or
ET-3 (
) of
I-ET-1 binding to membrane preparations
from CHO cells expressing both hET
and hET
.
Isolation of cells co-expressing both receptor subtypes and
displacement study on the membrane preparations with
I-ET-1 and unlabeled ET-1/ET-3 were conducted as
described under ``Materials and Methods.'' The concentration
of
I-ET-1 was 100 pM. Shown are means of
triplicate determinations obtained in a single experiment. Similar
results were obtained in two other separate
experiments.
Figure 6:
Effects of ET-1 on cAMP formation in CHO
cells expressing both hET and hET
. Cells were
either untreated (
) or pretreated (
) with PTX (50 ng/ml for
16 h). They were then stimulated with increasing concentrations of ET-1
for 10 min with ( right) or without ( left) 100
µM forskolin in the presence of 1 mM IBMX. The
contents of cAMP in the trichloroacetic acid-soluble cell extracts were
determined by radioimmunoassay. Values are the means ± S.E. of
three determinations each done in duplicate. *, p < 0.01
(significantly different from the values of basal
formation).
Figure 7:
Effects of ETR antagonists on the
stimulatory effects of ET-1 on cAMP formation in CHO cells expressing
both hET and hET
. Cells, either untreated
( left) or pretreated ( right) with PTX (50 ng/ml for
16 h), were incubated for 10 min with or without the ETR antagonist
indicated and then stimulated with 10 nM ET-1 for 10 min. The
concentrations of both 97-139 and BQ788 were 1 µM.
IBMX (1 mM) was included throughout the preincubation and
stimulation periods. The contents of cAMP in the trichloroacetic
acid-soluble cell extracts were determined by radioimmunoassay. Each
bar represents the mean ± S.E. of three determinations
each done in duplicate.
Figure 8:
Effects of ETR antagonists on the
inhibitory effects of ET-1 on cAMP formation in CHO cells expressing
both hET and hET
. Cells, either untreated
( left) or pretreated ( right) with PTX (50 ng/ml for
16 h), were incubated for 10 min with or without the ETR antagonist
indicated and then stimulated with 10 nM ET-1 and 100
µM forskolin for 10 min. The concentrations of both
97-139 and BQ788 were 1 µM. IBMX (1 mM) was
included throughout the preincubation and stimulation periods. The
contents of cAMP in the trichloroacetic acid-soluble cell extracts were
determined by radioimmunoassay. The values were expressed as relative
to the forskolin-stimulated formation (100%). There were no significant
differences in the absolute values of forskolin-stimulated formations
under the indicated conditions. Each bar represents the mean
± S.E. of three determinations each done in
duplicate.
and hET
(and also all of the
chimeric receptors examined) could couple with PTX-insensitive G
protein (possibly a member(s) of G
q family) to induce
[Ca
]
response of the
cells. The [Ca
]
responses of the various clones were indistinguishable from each
other, suggesting that any combinations of ICLII, ICLIII, and carboxyl
terminus of either hET
or hET
could confer the
full ability both to select and activate G
q. These results are
consistent with the observation on deletion mutants of hET
(29) that both 10-amino acid residues in the carboxyl-terminal
stalk region of ICLIII and 13-amino acid residues in the proximal stalk
region of carboxyl terminus were required to induce ET-1-dependent
increase in [Ca
]
.
hET
and hET
share the common 10-amino acid
residues in the carboxyl-terminal region of ICLIII and a quite similar
sequence in the proximity of carboxyl terminus; 11 out of the 13 amino
acid residues are identical.
and rat ET
with G
s and G
i, respectively,
when expressed on CHO dhfr cells. The same was the case for
hET
and hET
expressed on CHOK1 cells
(Fig. 1), suggesting that the G protein specificity was a
characteristic of ETR conserved over species differences. The present
study demonstrated the roles of ICLII and -III but not that of carboxyl
terminus or of hETR in the selective activation of G
s/G
i.
s (Fig. 2). hET
with the replacement of
ICLII and/or -III to the corresponding region(s) of hET
failed to transmit the stimulatory effect of ET-1. In cells
expressing chimeric receptors with ICLII of hET
and with
ICLIII of hET
, ET-1 stimulated cAMP formation only when
cells were pretreated with PTX. These results can be interpreted as
follows. 1) Both ICLII and III of hET
were required for
full activation of G
s; 2) of the two ICLs, ICLII was an absolute
requirement for the coupling with G
s, and ICLIII played an
ancillary role to confer the higher efficacy of coupling; and 3) in
chimeric receptors with ICLII of hET
and ICLIII of
hET
(A(N-IV)B(V-C) and A(N-IV)B(V-VI)A(VII-C)), concomitant
activation of G
i counteracted the activation of G
s. A study
on chimeric muscarinic/
-adrenergic receptors suggested a similar
requirement for the original set of ICLII and -III of
-adrenergic
receptors (
AR) to confer the full ability to activate G
s
(7) .
i (Fig. 3). All of the chimeric receptors
with ICLIII of hET
, regardless of the various combinations
with ICLII/carboxyl terminus derived either from hET
or
hET
, could transmit the effects that were compatible with
that transmitted by wild-type hET
. All the chimeric
receptors with ICLIII of hET
failed to transmit the effect.
These results suggested that ICLIII of hET
was necessary
and also sufficient to confer the full activation of G
i. Studies
on chimeras between muscarinic receptor subtypes
(4, 5, 6) also indicated the role of ICLIII of m2/m4 as a major
determinant for coupling with G
i.
and
AR to activate G
s and by hET
and
muscarinic receptors to activate G
i (as described here) points to
the notion that both the three-dimensional architecture of the surface
that binds G proteins and the mechanism that activates G proteins are
highly conserved in all G protein-coupled receptors
(11) . The
carboxyl-terminal stalk portion of ICLIII has been proposed to be the
most likely region involved in receptor/G protein coupling by
mutagenesis studies of the receptor-G protein pairs, including both
AR-G
s and m2/m4-G
i
(30) . Because hET
and hET
share the common sequence in the
carboxyl-terminal stalk region of ICLIII, the region cannot determine
the G protein specificity but is quite likely to be involved in the
activation mechanism common to G
s and G
i (and also to
G
q).
values
for the stimulation and inhibition of cAMP formation by hET
and hET
, respectively (Fig. 1), and similar
differences have been observed for the efficacies of
AR to
stimulate cAMP formation and of muscarinic receptors to inhibit it
(3) . These results suggested that, besides the structural basis
for the selective coupling, the differential efficacies to
stimulate/inhibit cAMP formation may be a common feature to all the
G
s/G
i-coupled receptors. The changes in cAMP formation,
however, are the net results of receptor-G
protein-adenylate
cyclase interactions, and detailed analyses will be required to clarify
the molecular basis for the differential efficacies.
to activate G
s and for hET
to activate G
i,
resulting in dose-dependent dual effects of ET-1 on cAMP formation
(Fig. 6). Some cell types including cardiomyocytes express mRNAs
for both receptor subtypes
(25) and possibly both receptor
proteins. An intriguing observation is that, in isolated cardiomyocytes
from guinea pig, ET-1 exerted inhibitory effect on cAMP formation to
counteract
AR stimulation, and this effect was apparently
transmitted via ET
coupled with G
i
(31, 32) . Co-expression of the two receptor subtypes,
as evidenced by the data presented, cannot be the reason for the
coupling of ET
with G
i. Further study is necessary to
verify alternative explanations such as a tissue-specific expression of
receptors with structural differences or a selective sequestration of G
proteins by the cells.
Table:
Densities and affinities of wild-type and
chimeric hETexpressed on CHO cells
I-ET-1 binding assay using
membrane preparations. Shown are the values obtained from a single
experiment on each clone. These clones were selected for use in the
present study because of the similar receptor densities as listed. N,
I-VII, and C designate the extracellular amino terminus, transmembrane
helices (TM) I-VII, and cytoplasmic carboxyl terminus, respectively,
plus the adjacent loop regions when applicable. For example, the
chimeric receptor A(N-VII)B(C) has hET
sequences of amino
terminus, TMI-VI, and a part of VII plus adjacent loop regions
(Met
to Asn
) and hET
sequences of
a part of TMVII and cytoplasmic carboxyl terminus (Ser
to
Ser
). See Ref. 26 for detailed description of each
receptor construct. Progressive substitutions from the amino-terminal
side of the subdomains of hET
to the corresponding domains
of hET
gave B/A chimeras, while those from the
carboxyl-terminal side gave A/B chimeras.
AR,
-adrenergic receptor.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.