The First Inner Loop of Endothelin Receptor Type B Is
Necessary for Specific Coupling to G
13*
Bo
Liu
§ and
Dianqing
Wu
¶
From the
Department of Genetics and Developmental
Biology, University of Connecticut Medical Center, Farmington,
Connecticut 06030 and the § Department of Pharmacology,
University of Rochester, Rochester, New York 14620
Received for publication, August 23, 2002, and in revised form, November 10, 2002
 |
ABSTRACT |
Endothelin (EDN) receptor type B (EDNRB)
activates serum response factor (SRF) via Gq/11 and
G12/13 G proteins. In this study, we investigated the
involvement of intracellular loop sequences of EDNRB in coupling to
these G proteins. EDNRB mutants were generated and tested for their
abilities to activate SRF in NIH3T3 cells and in the mouse embryonic
fibroblast cell line (Fq/11) lacking both G
q
and G
11. EDNRB can activate SRF in NIH3T3 cells via Gq/11, although it can only activate SRF through
G12/13 in Fq/11 cells. Mutants with mutations
in the second and third inner loops of EDNRB functioned in the same
manner in both cell lines, either able or unable to activate SRF. This
finding suggests that the second and third inner loops of EDNRB either
participate or not in coupling to both Gq/11 and
G12/13 but are not specific for either one. However, in the
first inner loop, a substitution of three Ala residues for
Met128-Arg129-Asn130
abolished the ability to activate SRF only in Fq/11 cells,
suggesting that this mutation might specifically disrupt the coupling
to G12/13 rather than to Gq/11. Further
characterization of this first inner loop mutant revealed that
exogenous expression of G
12 or G
q could
restore SRF activation, whereas the expression of G
13
did not. Therefore, we conclude that although the three intracellular
loops of EDNRB may be involved in coupling to G proteins, residues
Met128-Arg129-Asn130 in the first
intracellular loop are specifically required for activation of
G
13.
 |
INTRODUCTION |
Endothelin (EDN)1 is a
potent vasoactive peptide, which can induce a wide range of cellular
and physiological responses (1). In mammalian cells, there are at least
two EDN receptor subtypes, EDNRA and EDNRB. The EDN receptors belong to
the superfamily of rhodopsin-like G protein-coupled receptors (GPCRs)
that contain seven transmembrane domains. The extracellular and
transmembrane domains of GPCRs are involved in ligand binding, whereas
the intracellular domains are involved in G protein coupling and
subsequent effector regulation.
Both EDN receptors can couple to Gq proteins, resulting in
the activation of phospholipase C
(2). This pathway was shown to be necessary for EDN-induced activation of the ERK/MAPK signaling cascade (3). In addition, EDN receptors were also found to regulate
adenylyl cyclases through both Gs and Gi (2).
More recently, EDN receptors were demonstrated to couple to the
G12 subfamily of G proteins that consists of
G12 and G13 (4). In addition, EDNRA was shown
to induce stress fiber formation via G
12 in fibroblast
cells (5), whereas EDNRB was shown to couple to G
13 in
human HEK-293 cells (6).
Because many GPCRs can couple to more than one G
subunit,
understanding the structural basis for specificity between a receptor and its respective G proteins is an area of active investigation (7,
8). For example, the amino acid sequences involved in G
q
activation have been mapped to the third inner loops of the
1B-adrenergic receptor, the mt1 muscarinic
receptor, and the glutamate receptors (9-11). Our own work has
identified two basic amino acids in the second inner loop of the
interleukin-8 receptor that is required for G
16 coupling
(12). The structural specificity of G protein coupling has also been
demonstrated in EDNRs. Studies using various receptor chimeras
demonstrate that the second inner loop of EDNRA is involved in
G
s coupling, whereas the third inner loop of EDNRB
receptor is responsible for G
i coupling (13). In
addition, EDNRs are modified by palmitoylation at a cluster of cysteine
residues in the C-terminal tails, which also appear to play a role in G
protein coupling (14).
In this study, we investigated the molecular basis of
G
12/13 coupling to EDNRB. Taking advantage of a
previously established reporter gene assay system in NIH3T3 and
Fq/11 cells (derived from embryos in which the genes for
G
q and G
11 were disrupted) (4, 15), we
identified three residues in the first inner loop of EDNRB as required
for specific activation of G
13.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture, Transfection, and Luciferase Assay--
NIH3T3 and
Fq/11 cells were maintained in Dulbecco's modified
Eagle's medium containing 10% fetal bovine serum at 37 °C under 5% CO2. The Fq/11 cell line was established
from mice lacking both G
q and G
11 (4).
For transfection, cells (5×104 cells/well) were seeded
into 24-well plates the day before transfection. Cells were transfected
with 0.5 µg of DNA/well using LipofectAMINE Plus (Invitrogen) as
suggested by the manufacturer. Transfection was stopped after 3 h
by switching to Dulbecco's modified Eagle's medium containing 0.5%
fetal bovine serum. Cell extracts were collected 30 h later for
luciferase assays.
Luciferase assays were performed using a Constant Light luciferase
assay kit (Roche Molecular Biochemicals) as instructed. Cell lysates
were first determined for the fluorescence intensity emitted by GFP
proteins in a Wallac Victor2 Multicounter (PerkinElmer Life
Sciences) followed by luminescence measurement by using the same
counter after the addition of the luciferase substrate. Luminescence
intensities were normalized against fluorescence intensities as
described previously (4).
Phospholipase C Assay--
Cells were incubated with 10 µCi/ml
of myo-[2-3H]inositol in 0.25 ml of
inositol-free Dulbecco's modified Eagle's medium containing 10%
dialyzed fetal bovine serum at 37 °C under 5% CO2 for
24 h. The levels of inositol phosphates were determined as
described previously (17).
Ligand Binding Assay--
NIH3T3 or Fq/11 cells in
24-well plates were transfected with the cDNAs encoding the EDNRB
or its mutants. After 24 h, the cells were washed with
phosphate-buffered saline and incubated with varying amounts of
[125I]EDN1 (3000 Ci/mmol, PerkinElmer Life Sciences) in
200 µl of phosphate-buffered saline containing 1 mg/ml bovine serum
albumin for 1 h at 4 °C. After washing three times with
ice-cold phosphate-buffered saline containing bovine serum albumin, the
cells were lysed in 0.5 ml of 0.2 N NaOH and 0.1-ml
aliquots were taken for counting in a
-ray counter. The nonspecific
binding was determined by measuring the binding of
[125I]EDN1 to cells transfected with the LacZ expression
plasmid. The number of specific EDN1 binding sites
(Bmax) and dissociation constants
(Kd) were calculated using the Scatchard analysis.
Construction of EDNRB Mutants--
All EDNRB mutants were
generated by polymerase chain reaction with the high fidelity DNA
Pfu polymerase (Stratagene), and each of the mutations was
confirmed by DNA sequencing and restriction digestion. Hemagglutinin
epitope tags were incorporated at the C termini of EDNRB and its mutants.
Western Analysis--
NIH3T3 cells expressing LacZ, ENDRB, or
its mutants were lysed in the SDS-PAGE sample buffer and incubated at
37 °C for 30 min before electrophoretic separation in 10%
acrylamide gels. After electrophoresis, proteins were blotted to
nitrocellular membranes and probed with an anti-hemagglutinin antibody.
 |
RESULTS AND DISCUSSION |
Previously, we demonstrated that EDNRB could couple to
G12/13 (4). To further understand the interaction between
EDNRB and G12/13, we generated a series of receptor mutants
in which the intracellular loop regions were mutated and tested for
their ability to activate SRF in NIH3T3 and Fq/11 cells.
Because previous work has found that residues in the third inner loop
of GPCRs were frequently found to be involved in G protein coupling
(7-11, 20), we first tested receptor mutants with deletions in the third inner loop of EDNRB as illustrated in Fig.
1A for their ability to
activate SRF (Fig. 1, B and C). Our previous
studies have demonstrated that EDNRB activated SRF via coupling to both Gq/11 and G12/13 proteins in NIH3T3 cells,
whereas EDNRB activated SRF via G12 and/or G13
in Fq/11 cells (4). As shown in Fig. 1, B and
C, EDN1 could activate SRF in NIH3T3 or Fq/11
cells expressing mt1, mt2, mt4, or mt6 (Fig. 1, B and
C) but not in cells expressing mt3 or mt5. Because the
mutation in mt5, which covers the mutation in mt3, did not affect
ligand-binding characteristics (Table I), we conclude that the amino acids deleted in mt5 are probably required for coupling to both G
q/11 and G
12/13. In
addition, the sequences deleted in mt1, mt2, mt4, and mt6 do not appear
to be essential for coupling to either Gq/11 or
G12/13.

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Fig. 1.
Effects of mutations in the third inner loop
of EDNRB on G protein coupling. A, schematic
representation of the third inner loop of EDNRB and its mutants.
Designation for the EDNRB mutants and positions of the deletion
mutations are shown. Expression of EDNRB and its mutants in NIH3T3
cells were analyzed by Western blotting using an antibody to the
hemagglutinin tags carried by these recombinant proteins. B,
regulation of SRF in NIH3T3 cells. NIH3T3 cells were transfected with
0.1 µg of serum response element/L-luciferase reporter
plasmid, 0.1 µg of GFP expression construct, 0.2 µg of LacZ
(Lz), and 0.1 µg of wild type EDNRB or one of the mutants
(mt1-6). The next day, cells were lysed 6 h after the addition of
EDN1 (2 nM). GFP levels and luciferase activities were
determined. The luciferase activities presented are normalized against
the levels of GFP expression. Experiments were carried out in
duplicates and repeated at least three times. The representative
experiments are shown. The levels of luciferase activity in the absence
of ligand are ~2000 arbitrary units (AU). C,
regulation of SRF in Fq/11 cells. The
G q/11-deficient cells were transfected as in
B. The data collection and analyses were carried out as
described in B. The levels of luciferase activity in the
absence of ligand are ~2500 AU.
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Table I
Binding of [125I]EDN1 in NIH3T3 cells
The binding assay was carried out as described under "Experimental
Procedures." The nonspecific binding as measured with
lacZ-transfected cells is <15% of corresponding total
binding.
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We went on generating two additional mutants that contain deletions
spanning the short second inner loop (Fig.
2A). Both mutants failed to
activate SRF in response to EDN1 in NIH3T3 cells (Fig. 2B).
Given that neither of the mutations affected ligand binding (Table I),
it is probable that these mutations knock out coupling to multiple G
proteins. We also made a C-terminal deletion mutant with a deletion up
to residue Cys405, a myristoylation site. This C-terminal
deletion mutant showed increased responses to EDN1 in both 3T3 and
Fq/11 cells (data not shown), probably because of the
removal of desensitization signals localized at the C-terminal tail of
the receptor as found with many other GPCRs (16, 21).

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Fig. 2.
Effects of mutations in the second inner loop
of EDNRB on G protein coupling. A, schematic
representations of the second inner loop of EDNRB and its mutants.
B, SRF regulation. NIH3T3 cells were transfected and treated
as described in Fig. 1B.
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Thus far, specific mutations in the second and third inner loops of
EDNRB have functioned in a similar manner in both cell lines. This
suggests that althogh certain regions of the second and third inner
loops are essential for G protein coupling, they are not specific for
any particular G protein. Therefore, we shifted our focus to the first
inner loop. In this segment, there are two sets of triple amino acids
that contain basic or polar residues. We mutated these sites with three
Ala residues, respectively (Fig. 3A). In 3T3 cells, the
expression of mt9 and mt10 led to marked increases in the luciferase
activity in response to EDN1 (Fig. 3B). However, mt10 failed
to induce SRF activation in Fq/11 cells, whereas mt9 could
still activate SRF in response to EDN1 almost as well as the wild type
receptor. Similar results were obtained with two other doses of EDN1
(Fig. 4, A and B).
This finding suggests that the three residues
(Met128-Arg129-Asn130) in the first
inner loop of EDNRB are required for specific coupling to the
G13 and/or G12 proteins but not to the
Gq proteins. For further confirmation, exogenous
G
13, G
12, or G
q was
expressed in Fq/11 cells by transfection. Mutant mt10 was
unable to activate SRF in response to EDN1 (Fig. 4C) when
G
13 was expressed, confirming that this mutant receptor
is unable to couple to G13. Interestingly, when
G
12 was expressed, EDN1-induced SRF activation was
restored (Fig. 4C), suggesting that residues
Met128-Arg129-Asn130 are required
for specific coupling to G
13 but not to
G
12. The reason for the failure of activation of SRF in
the absence of transfected G
12 might be because of a low
or no expression of endogenous G
12 in these embryonic
cells. As anticipated, the expression of Gq also restored
the ability of mt10 to activate SRF in response to EDN1 (Fig.
4C), confirming that mt10 can couple to Gq. The
ability of mt10 to couple to the Gq proteins was also validated by the finding that EDN1 could stimulate the accumulation of
inositol phosphates in NIH3T3 cells expressing mt10 (data not shown).
Moreover, the ability of the wild type receptor to increase reporter
gene activity in the presence exogenous G
13,
G
12, or G
q in F q/11 cells
indicates that ENDRB is capable of coupling G
13 and that
the inability of mt10 to activate SRF in the presence of exogenous
G
13 is not because of insufficient expression of G
13 in the Fq/11 cells. Furthermore,
ligand-binding analyses, which were
carried out in both NIH3T3 and Fq/11 cells (Fig. 5 and
Tables I and II), did not reveal obvious
differences between mt10 and the wild type receptor in the values of
both Bmax and Kd, suggesting
that mt10 was expressed normally at the cell surfaces of both NIH3T3
and Fq/11 cells. Putting all of these results together, we
conclude that residues
Met128-Arg129-Asn130 in the first
inner loop of EDNRB are involved in specific coupling to
G
13 but not to G
12 or Gq.

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Fig. 3.
Effects of mutations in the first inner loop
of EDNRB on G protein coupling. A, schematic
representations of third inner loop mutations of two EDNRB mutants and
Western analysis of their expression. B and C,
SRF regulation in NIH3T3 cells and Fq/11 cells. NIH3T3
cells and Fq/11 cells were transfected and treated as
described in Fig. 1.
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Fig. 4.
Specific coupling of EDNRB mutant mt10 to
G13. A and B, SRF regulation.
NIH3T3 and Fq/11 cells were transfected as described in
Fig. 1. The next day, the cells were treated for 6 h with two
doses of EDN1 (0.5 and 5 nM). C and
D, SRF regulation in the presence of recombinant G
proteins. Fq/11 cells were transfected with 0.1 µg of
serum response element/L-luciferase reporter plasmid, 0.15 µg of GFP expression construct, 0.15 µg of mt10 or EDNRB, and 0.05 µg of G 13, G 12, G q, or
LacZ. The next day, luciferase activity was determined 6 h after
the addition of EDN1 (0.5 nM). At the present transfection
condition, G protein when expressed by itself showed little activation
of the reporter system in Fq/11 cells.
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Fig. 5.
Binding of [125I]EDN1 to NIH3T3
cells. NIH3T3 cells were transfected with plasmids
expressing LacZ, the wild type, or mt10 ENDRB. The binding of
[125I]END1 to the cells were carried out as described
under "Experimental Procedures." Results are shown after
subtracting the nonspecific binding, which is the binding to cells
transfected with the LacZ expression plasmid.
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Table II
Binding of [125I]EDN1 in Fq/11 cells
The binding assay was carried out as described under "Experimental
Procedures." The nonspecific binding as measured with
LacZ-transfected cells is <35% of corresponding total binding.
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The structural determinants of receptor G protein coupling have been
pursued vigorously in the past. Although no consensus sequences have
been identified, specific sequences in individual receptors have been
identified for coupling to distinct G proteins (7, 8). The coupling
elements are often found in the third inner loops of various GPCRs with
a few exceptions. For instance, the coupling of the interleukin-8
receptor to G
16 and the EDNRA receptor to
G
s was mapped to the second inner loop (13, 15). As for
EDNRB, different elements had previously been found to be required for
coupling to Gq and Gi proteins (13). In this report, we demonstrate for the first time that the first inner loop of
a GPCR is required for specific coupling to a G protein.
Although distinct sequence elements have been found for specific
coupling to different G proteins, evidence also suggests that there may
be multiple contacts made between a GPCR and a given G protein. It
appears that there might also be some common sequences that are
required for coupling to different G proteins. For example, the
sequences deleted in mt5, mt7, and mt8 appeared to be required for
coupling to both G13 and Gq proteins. However, although we have demonstrated that these mutations do not affect ligand-binding characteristics, we cannot eliminate the possibility that the mutations disrupt certain structures formed by the
intracellular loops, which rather than the sequences themselves are
required for G protein coupling. Nevertheless, the third inner loop
sequences of many GPCRs, particularly those near the sixth
transmembrane domain, appear to be critical in coupling to
Gq and Gi (7, 8). In this report, we added
G13 to that list.
Identification of mutations that can specifically knock out the
coupling of EDNRB to G13 lays the groundwork for future
investigations of specific physiological significance of the
EDNRB-G
13 signaling pathway. The importance of EDN in
the cardiovascular system has been well documented (1). Not long ago,
G13 was also found to play an important role in vasculature
regulation because mice lacking G
13 died from
vasculature malfunction (18). In addition, the inactivation of the
EDNRB gene produced a megacolon associated with spotted coat
color in mice (19). Similar phenotypes are also manifested in patients
with Hirschsprunger's disease. It would be very interesting to
determine whether the EDNR-G
13 signaling pathway is
involved in these processes. Our finding provides a potential tool for
such an investigation.
 |
ACKNOWLEDGEMENT |
We thank Peter Maye for comments on the paper.
 |
FOOTNOTES |
*
The work is supported by National Institutes of
Health grants and an American Heart Association Established
Investigator Award (to D. W.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed. E-mail:
dwu@neuron.uchc.edu.
Published, JBC Papers in Press, November 18, 2002, DOI 10.1074/jbc.M208683200
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ABBREVIATIONS |
The abbreviations used are:
EDN, endothelin;
EDNR, endothelin receptor;
Fq/11, a mouse embryonic
fibroblast cell lines derived from mice lacking G
q and
G
11;
GPCR, G protein-coupled receptor;
GFP, green
fluorescent protein;
ERK, extracellular signal-regulated kinase;
MAPK, mitogen-activated protein kinase;
SRF, serum response factor.
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