Transmembrane Helix 7 of the Endothelin B Receptor Regulates
Downstream Signaling*
Paul
Vichi
,
Alyn
Whelchel, and
James
Posada§
From the Department of Biomedical Technologies, School of Allied
Health, and the Department of Molecular Physiology and Biophysics,
College of Medicine, University of Vermont,
Burlington, Vermont 05405
 |
ABSTRACT |
Endothelin is a 21-amino acid peptide with a
striking diversity of important biological responses, including,
vasoconstriction, bronchoconstriction, and mitogenesis. Endothelin-1
binding to the endothelin B receptor (ETB), a member of the superfamily
of G-protein-coupled receptors, was associated with catalytic
activation of the extracellular-regulated kinase 2 (ERK2) and
stimulation of AP-1 transcriptional reporter activity. A panel of
single point mutations in transmembrane helix 6 (TM6), intracellular
loop 3, and transmembrane helix 7 (TM7) were developed to study the
structural requirements for ETB activation. Point mutations within
highly conserved regions of TM6 and intracellular loop 3 were without effect on agonist-stimulated ERK activation. However, mutations within
TM7 of the ETB significantly impacted ligand-stimulated downstream
signaling. For example, nine point mutations within TM7 of the ETB were
identified that prevented endothelin-stimulated ERK activation.
Interestingly, the TM7 mutants fell into two classes; several exhibited
greatly decreased AP-1 activity, relative to wild type ETB, whereas
others displayed augmented endothelin-stimulated AP-1 transcriptional
activity relative to wild type ETB. Our results suggest that TM7 of the
ETB is involved in its activation mechanism and regulates
agonist-stimulated ERK activation.
 |
INTRODUCTION |
Endothelin-1 (ET)1 is a
21-amino acid peptide that binds to a G-protein-coupled receptor
(GPCR), of which there are two subtypes: endothelin receptor A, and
endothelin receptor B (ETB). Receptor binding triggers a diverse
spectrum of physiological effects including vasoconstriction,
mitogenesis, and embryonic development (1-5).
Consistent with the complex biology of endothelin, binding of the
peptide to its receptor initiates several important cellular signaling
pathways, including increases in cytosolic free calcium, activation of
the Src cytosolic tyrosine kinase, tyrosine phosphorylation of the Shc
adapter protein, ERK, and c-Jun NH2-terminal kinase mitogen-activated protein kinase activation, protein kinase C involvement, activation of phosphatidylinositol 3-kinase, and stimulation of the epidermal growth factor receptor tyrosine kinase activity (3, 6, 7, 9-14).
The endothelin receptors are members of a superfamily of
G-protein-coupled receptors that are thought to activate downstream signaling networks through their dynamic interaction with
heterotrimeric G-proteins. Mutational studies of the endothelin
receptors have defined extracellular loop 2 as being critical for
ligand contact and binding, whereas the cytoplasmic tail has been
implicated as important for coupling to G-proteins (15-17).
The relationship between ligand binding affinity and receptor-G-protein
interaction is described by the ternary complex model of receptor
activation (18). It is widely thought that a small fraction of
receptors reside in the activated conformation and that agonist binding
stabilizes this conformation, which results in activation of the
associated G-protein. The dynamic interaction of
ligand-receptor-G-protein is thought to result in high affinity agonist
binding when the receptor is in the active, G-protein bound state.
Therefore, mutations that diminish or prevent receptor-G-protein binding in some cases are associated with decreased agonist binding. In
contrast, several constitutively active GPCR mutants have been identified that result in higher affinity ligand binding (17, 19,
20).
Although the molecular interactions that govern GPCR activation
have not been completely defined, mutagenesis studies, biophysical analysis, and cryoelectron microscopy of rhodopsin, a well
characterized GPCR, have provided a comprehensive model for GPCR
activation (21-23). Transmembrane helices 3 and 6 are thought to be
packed into close proximity in three-dimensional space, and ligand
binding results in rigid body movement of TM6 relative to TM3. This
displacement of helix 6 relative to helix 3 is hypothesized to induce a
conformational change in the loop connecting TM5 and TM6, which is
where GT is associated with the receptor (24).
The importance of TM6 has been confirmed through mutagenesis studies of
other GPCRs. For example, the
1B-adrenergic receptor can
be constitutively activated by mutation of alanine 293, located on the
intracellular side of TM6 (25), and the m5 muscarinic receptor can be
constitutively activated by mutations at the extracellular side of TM6
(20).
Taken together the data imply that TM6 is important in the activation
mechanism of the adrenergic and muscarinic GPCRs. We have therefore
examined the TM6 region of the ETB to define the importance of this
domain in ERK activation and AP-1 transcriptional control. Our results
indicate that the ETB is distinct from the muscarinic and adrenergic
receptors in this regard, because mutation of several highly conserved
amino acids within or near TM6 had little or no effect on activation of
ERK mitogen-activated protein kinase. In contrast, we have observed
that TM7 of the ETB is critical for proper signaling functions of the
receptor. Point mutations within the TM7 region of the ETB interfered
with ligand-stimulated ERK activation and had dual effects on AP-1
transcriptional activation.
 |
MATERIALS AND METHODS |
Cell Culture and Transfection
COS cells were grown in Dulbecco's modified Eagle's medium
(DMEM) containing 10% fetal bovine serum (FBS). For kinase assays cells approximately 50% confluent in 100-mm plates were transfected with the indicated plasmids (5 µl/µg DNA) using LipofectAMINE (Life
Technologies, Inc.). Cells were exposed to the LipofectAMINE/DNA mixture for 6 h in DMEM, the medium was changed to DMEM/10% FBS, and the cells were incubated for 48 h. Cells were serum starved by
changing the medium to DMEM/0.1% FBS for 16 h, and the cells were
treated with endothelin and harvested for immunoprecipitation. For
immunofluorescence COS cells were grown and transfected in 0.2-µm
glass slides. For AP-1 reporter assays the AP-1/luciferase plasmid was
co-transfected with wild type and mutant ETB plasmids and pCMV-
-gal,
which encodes
-galactosidase at a ratio of 5:1.
Plasmid Construction
ERK2 containing a 6-fold repeated Myc epitope fused in frame to
the 5' end was used as described previously (17). Wild type rat
endothelin B receptor (GenBankTM accession number X57765)
was subcloned into pCDNA3 containing a FLAG epitope fuse in frame
at the 3' end as described previously (17). Single point mutations were
made in TM6, TM7, and the third intracellular loop of the rat ETB using
the Transformer site-directed mutagenesis kit
(CLONTECH) following the manufacturer's instructions. All mutations were verified by sequencing. The
AP-1/lucierase plasmid contains the AP-1 transcriptional promoter
element cloned upstream of the luciferase gene (kindly provided by Dr.
Mercedes Rincon, University of Vermont).
Mutagenesis
Single point mutations were introduced into the wild type rat
ETB using the Transformer site-directed mutagenesis kit following the
manufacturer's instructions (CLONTECH). Briefly, a
selection primer was engineered that changed a unique SmaI
site in the pcDNA3 vector at nucleotide 2093 to a XhoI
site that was also unique. Additional primers were designed that
changed the desired amino acids in the ETB. After synthesizing the
mutant strand in vitro the plasmids were transformed into a
mutS Escherichia coli strain. Parental plasmids
were eliminated by digestion with SmaI, and the pool of
plasmids was transformed again. Colonies were picked, and the mutations
were confirmed by sequencing.
Luciferase Reporter Assay
COS cells were grown in 6-well plates in DMEM containing 10%
FBS. At 50% confluence, cells were transfected with 0.5 µg of ETB
DNA, 0.25 µg of AP-1/luciferase reporter plasmid, and 0.1 µg of
-gal using LipofectAMINE in serum-free DMEM. After 5 h the
medium was changed to growth medium, and the cells were grown for
24 h. Cells were changed to 0.1% FBS serum, incubated overnight, and then stimulated with endothelin for 6 h. The receptors were transfected into three individual culture dishes, and the values are
the means ± S.D. of the three independent measurements. Cell lysates were made, and luciferase activity was measured in a
scintillation counter according to the manufacturer's specifications
(Promega Corp., Madison, WI). Luciferase activity was normalized to
-gal activity.
Immunofluorescence
Cells were grown in 2-well Nunc chambered slides until
approximately 50% confluent and transfected with 1 µg of ETB DNA and 3 µl of LipofectAMINE, and the medium was changed to 10% FBS 5 h later. Cells were rinsed twice with phosphate-buffered saline, fixed
with 4% paraformaldehyde, and permeabilized with MeOH. Nonspecific binding was blocked with 5% normal goat serum, and the cells were incubated for 1 h at room temperature in the anti-flag antibody (M2, Kodak Corp. New Haven, CT) at 2 µg/ml. The cells were rinsed and
blocked again for 30 min in 5% normal goat serum. Cells were washed
five times over 30 min in TBS-T (Tris-buffered saline with 0.1% Tween
20), incubated for 45 min in the dark at room temperature in a
CY3-conjugated, affinity purified, goat anti-mouse secondary antibody
(1:2000, Jackson ImmunoResearch), and mounted with VECTASHIELD (Vector
laboratories Inc., Burlingame, CA) containing DAPI.
Kinase Assays
COS cells co-transfected with wild type or mutant ETBs and
MT-ERK2 were treated with endothelin (100 nM) for 10 min.
Cells were scraped into 600 µl of 1% Nonidet P-40, 10 mM
Hepes, 2 mM EDTA, 50 mM NaF, 0.1%
-mercaptoethanol, 1% aprotinin, 50 µM microcystin, 0.2 mM sodium orthovanadate. Insoluble material was removed
by centrifugation at 15,000 rpm for 10 min at 4 °C. The Myc-tagged ERK2 was immunoprecipitated using monoclonal antibody 9E10. The immunoprecipitates were washed and incubated in 20 mM
Hepes, pH 7.5, 10 mM MgCl2, 2 mM
dithiothreitol) in the presence of myelin basic protein (MBP) and
[
32P]ATP (5 µCi) for 15 min. The extent of
phosphorylation of MBP was analyzed by running the immunoprecipitates
on a 15% SDS gel and subsequent autoradiography.
Ligand Binding
Binding to Purified Membranes--
COS cells transfected with
empty vector, wild type ETB, or ETB mutants were lysed in 10 mM Hepes, pH 7.5, 2 mM MgCl2, 5 mM KCl, and the post-nuclear supernatants were centrifuged
at 100,000 × g for 1 h to isolate the membrane
fraction. COS cell membranes (10 µg) were incubated with 50 pM 125I-endothelin-1 for 3 h. The
membranes were applied to Whatman GF/B filters and washed extensively
prior to counting. Nonspecific binding was estimated by incubating the
125I-endothelin-1 in the presence of excess (1 µM) unlabeled endothelin. Nonspecific binding averaged
15-25% of specific binding. Binding of endothelin to ETB mutant
receptors is expressed relative to the binding observed in the
membranes expressing the wild type receptor.
125I-ET Binding to Whole Cells--
Cos cells were
transfected with wild type or mutant ETBs and grown to approximately
80% confluency. Whole cells were washed in binding buffer
(phosphate-buffered saline with 140 mM NaCl, 4 mM KCl, 1 mM MgCl2, 1.25 mM CaCl2, 10 mM glucose, pH 7.4)
and incubated at 4 °C with 125I-ET at either 50 or 150 pM for 3 h. Nonspecific binding was estimated by
incubation of vector transformed cells in the presence of 1 µM unlabeled ET. Following incubations, cells were washed
three times in ice-cold binding buffer and lysed in 0.1 N
NaOH. Lysates were collected, and 125I-ET content
determined by liquid scintillation
counting using a Packard B5005
counter.
 |
RESULTS |
Because direct structural information is not available for GPCRs,
models regarding the activation mechanism of this family of receptors
have relied primarily on mutagenesis and biophysical studies. Secondary
structure models have provided insight into the topology of rhodopsin,
including the packing of the transmembrane helices (21, 22). Recent
work on rhodopsin indicates that the activation mechanism involves
rigid body movement of TM6 (23). The primary sequence of the GPCR
superfamily reveals several highly conserved amino acids in this helix
that are also present in the ETB (Fig.
1). We wished to determine whether the
activation mechanism of ETB involves TM6. Therefore, we made
substitutions of several highly conserved residues in this region.

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 1.
Model of ETB topology and alignment of TM6
and TM7. A, the ETB is a member of the GPCR superfamily
that is thought to have seven membrane spanning helices joined by
alternating intracellular and extracellular loops. B, TM6 of
the ETB has several residues that are found to be highly conserved
among different classes of GPCRs. TM7 of these GPCRs does not have
highly conserved amino acid residues. The Swiss-Prot accession numbers
for the aligned sequences are: rat dopamine D1 receptor
(D1), P35406; rat muscarinic receptor (M2),
P10980; rat endothelin B receptor (ETB), P21451; and rat
1 adrenergic receptor (B1AR), P18090.
|
|
TM6 Mutants of the ETB--
Our results indicate that although
several residues within TM6 are almost invariably conserved among the
GPCR superfamily, mutation of these residues in the ETB appeared to be
without effect on ERK mitogen-activated protein kinase activation (Fig.
2). ETB mutants F331A, C334A, W335A,
P337A, S341K, and K345A were co-transfected into COS cells with
MT-ERK2. Endothelin stimulated ERK2 catalytic activation in the wild
type ETB and mutant receptors at approximately similar levels. The
basal level of ERK activity for unstimulated mutant receptors was also
unchanged relative to wild type receptor (Fig. 2B). Because
TM6 of the ETB is not sensitive to structural changes introduced by
mutagenesis, the data infer that this helix is not integrally involved
in the activation mechanism of this receptor.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 2.
Mutations in TM6 of the ETB do not impair ERK
activation. A, single point mutations were introduced
into the putative helix 6 region of the rat ETB. The wild type and
mutant receptors (1 µg) were co-transfected into COS cells with
MT-ERK2 (1 µg). The transfected cells were stimulated with endothelin
(100 nM) for 10 min prior to immunoprecipitating MT-ERK2.
An in vitro kinase assay was done using MBP as an in
vitro substrate to measure catalytic activity. The substitutions
did not impair endothelin-stimulated ERK activation. B,
basal ERK activity in cells expressing mutant ETBs. COS cells were
transfected as above with 1 µg of Myc-tagged ERK2 and either wild
type or mutant ETB containing site-specific mutations in loop 3, TM6,
or TM7. ERK activity was determined by immune complex kinase assay,
in vitro. Mutations in ETB did not significantly alter
observed basal ERK activity.
|
|
Intracellular Loop 3 Mutants of the ETB--
Several studies have
implicated the third intracellular loop of GPCRs, such as the
adrenergic and muscarinic receptors, as playing a role in downstream
effector function (20, 25). Therefore, we also examined the third loop
of the ETB by introducing amino acid substitutions into this area and
expressing the mutant receptors in COS cells with MT-ERK2. The third
loop of the ETB appeared to be relatively unaffected by mutation. ETB
mutants M299A, T323A, M306A, and D312A had essentially wild type levels
of ERK activation, whereas ETB mutants Q316A and K322A were slightly
reduced in their ability to activate ERK (Fig.
3) As with the TM6 region of the ETB,
analysis of the basal activity of ERK in cells transfected with mutant
receptors revealed levels of activity comparable with those of
unstimulated wild type receptors (Fig. 2B).

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 3.
Loop 3 of the ETB is not sensitive to
mutation. Amino acid substitutions were introduced into the
intracellular loop 3 region of the ETB and expressed in COS cells as in
Fig. 2. The mutant receptors were activated by ligand binding, and ERK
kinase activity was measured by immune complex kinase assay using MBP
as an in vitro substrate. Basal activity of Loop 3 mutants
is presented in Fig. 2B. The results indicate that the
mutant receptors stimulate ERK in response to ET to a similar degree as
do the wild type receptors (ETB-WT), suggesting that loop 3 is not sensitive to structural changes with respect to ERK
activation.
|
|
TM7 Mutants of the ETB--
Transmembrane helix 7 of the ETB
shares relatively little homology with other GPCRs, and in fact the
GPCR superfamily has little overall homology in this region (Fig. 1).
However, TM7 of the ETB was very sensitive to mutation. A panel of TM7
mutants were engineered and co-expressed in COS cells with MT-ERK2. ETB mutants M373K, N377P, S378K, I380A, P382K, L385A, V388K, and K390A were
all severely compromised in their ability to activate ERK, in
comparison with wild type (Fig. 4).
Western blotting of the COS cell lysates demonstrated that MT-ERK2 was
expressed at similar levels across the experiment, confirming that the
decrease in catalytic activity was due to decreased activation of
MT-ERK2 and not lower expression of the kinase. Although several TM7
mutant receptors revealed slightly elevated levels of basal ERK
activity relative to wild type receptor (Fig. 2B), this was
not reflected in an increase in ET-stimulated ERK activity relative to
wild type receptors. This suggests that introduced mutations did not produce catalytically active mutants.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 4.
TM7 regulates receptor-mediated activation of
ERK. Amino acid substitutions were introduced into the TM7 region
of the ETB, and the wild type (ETB-WT) and mutant receptors
were co-transfected with MT-ERK2 into COS cells. Kinase assays were
done using MBP as an in vitro substrate to measure catalytic
activity. Basal activity is presented in Fig. 2B. Several of
the point mutations significantly decreased endothelin-stimulated ERK
activation. Western blots were done to document that decreases in ERK
kinase activity reflected changes in the activation of the enzyme
rather than its expression level.
|
|
ETB Mutant Receptors Are Expressed and Properly
Localized--
Introducing nonconserved amino acid changes in the ETB
could reduce its downstream signaling capabilities as a result of a gross alteration in the topology of the receptor. Therefore, we investigated the expression levels and localization of the ETB mutant
receptors by immunofluorescence. Receptors bearing a FLAG tag on their
carboxyl terminus were expressed in COS cells and processed for
immunofluorescence using the anti-FLAG monoclonal antibody (Fig.
5). The mutant receptors were expressed
and localized in essentially the same manner as the wild type receptor.
The data indicate that the inability of the TM7 mutations to activate ERK resulted from a defect in the activation mechanism, rather than a
mutation-induced effect on expression or localization of the mutant
receptors.

View larger version (75K):
[in this window]
[in a new window]
|
Fig. 5.
Expression of wild type and mutant ETBs.
A FLAG epitope tag was engineered on to the 3' end of wild type and
mutant ETBs. The FLAG-tagged receptors were transfected into COS cells,
and immunofluorescence was done with the M2 anti-FLAG monoclonal
antibody (Kodak) and a Cy3 secondary antibody. The expression level and
pattern of localization is similar among the wild type and mutant
ETBs.
|
|
TM7 of the ETB Regulates AP-1 Transcriptional
Activation--
Ligand binding to the ETB triggers a diverse spectrum
of biological responses, depending on the target tissue in which the receptor is expressed. ETBs stimulate mitogenesis in selective cell
types, and this response is thought to involve the activation of key
transcriptional events, including AP-1 activity (14). Furthermore,
catalytically activated ERK is known to phosphorylate transcription
factors involved in AP-1 activity (26) and stimulate transcription.
Therefore, we wished to determine whether ERK activation is required
for AP-1 transcriptional activation. To this end we have examined
transcriptional activation of an AP-1 luciferase reporter construct by
wild type and mutant ETBs (8). The mutants fell into two distinct
classes based on their ability to stimulate AP-1 transcriptional
activation. Several of the mutations in TM7 of the ETB significantly
decreased endothelin-stimulated AP-1 transcriptional activation. For
example, N377P, S378K, P382K, A384K, L385A, K390A, and F392A were
compromised in their ability to stimulate AP-1 reporter activity when
exposed to ligand. Interestingly, a second class of TM7 mutant was
identified that displayed augmented endothelin-stimulated AP-1
transcriptional activity. TM7 mutants D367A, N372A, I383A, V388K, and
I380A when stimulated with ligand demonstrated augmented AP-1 activity
compared with wild type ETB (Fig. 6). The
I380A TM7 mutant had the largest increase in ligand-stimulated AP-1
activity. These mutants were consistently observed to have increased
endothelin-stimulated AP-1 activity; however, the basal AP-1 activity
was essentially the same as wild type (data not shown). The results
suggest that TM7 of the ETB is integrally involved in ligand-induced
activation of AP-1 activity because mutations were identified that both
prevented and augmented AP-1 responses.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 6.
AP-1 transcriptional activation by wild type
and mutant ETBs. COS cells were co-transfected with wild type
(WT) and mutant ETBs (1 µg), AP-1/luciferase reporter
plasmid (1 µg), and CMV- -gal (0.25 µg). The transfected cells
were serum starved for 48 h followed by endothelin treatment (100 nM) for 6 h. Lysates were made, and the luciferase
activity was measured and is expressed as arbitrary units. Each
receptor was transfected into three separate dishes, and each
bar represents three independent transfections and
measurements. The data presented are representative of five independent
experiments. The activity of the -gal reporter was measured in each
lysate, and the values were normalized to -gal activity, which
usually varied between 5 and 15% among replicates.
|
|
Ligand Binding Properties of the Mutant ETBs--
The ternary
complex model defines the dynamic interactions between
receptor-G-protein interactions and ligand binding (18). High affinity
agonist binding is thought to be dependent on efficient G-protein
interactions with the receptor. Furthermore, several constitutively
activated GPCRs are found to have increased affinity for agonist (19,
20, 25). The ternary complex model suggests that receptor mutations
that decrease or interfere with proper receptor-G-protein physical
interactions will result in altered agonist binding.
We conducted binding studies on both purified membranes and
transfected, whole cell preparations for each receptor mutant. Our
results are consistent with this model. Several of the TM7 mutants
displayed a range of 125I-endothelin binding, which was
generally lower than the wild type receptor, suggesting that the mutant
receptors were impaired in their ability to couple with G-protein
(Table I and Fig.
7). Other mutants displayed binding
closer to wild type levels but were unable to efficiently activate ERK
or stimulate AP-1 luciferase activity (N377P and S378K mutants; Figs. 6
and 7 and Table I). Inefficient receptor-G-protein interactions may
therefore account for the inability of ETB mutants to activate ERK.
Furthermore, any decreased binding was not a result of a lack of
receptor expression or localization, because the mutant ETBs were
expressed and localized in a manner similar to that of the wild type
receptor (Fig. 5).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 7.
125I-Endothelin-1
binding to COS cells. Ligand binding to whole cells expressing
wild type (WT) or mutant ETBs for the TM7 domain was
determined by analysis of bound radiolabeled ET at 4 °C. COS cells
transfected with 1 µg of wild type or mutant TM7 receptors were
exposed in duplicate to either 50 or 150 pM
125I-ET for 3 h at 4 °C. Cells were washed, and
bound ET was determined by scintillation counting. Values are presented
as the mean ± S.E. Nonspecific binding was measured on vector
transformed cells in the presence of 1 µM cold ET.
Although most receptors examined displayed reduced ERK activity, which
correlated with reductions in ET binding, several receptors maintained
ET binding similar to wild type levels.
|
|
 |
DISCUSSION |
GPCRs form an important and diverse group of cell surface receptor
proteins that have yet to be crystallized for structural studies.
Mutagenesis experiments involving the major classes of GPCRs have
provided a comprehensive picture regarding the molecular details of
receptor-ligand interactions and receptor-G-proteins interactions.
Studies with rhodopsin suggest a model of activation that involves
interaction of TM3 and TM6. In the case of rhodopsin, rigid body
movement of TM6 is thought to be induced by agonist binding and to
alter the conformation of the binding site for transducin, the
associated heterotrimeric G-protein (23).
In our experiments we have introduced a series of point mutations in
TM6, intracellular loop 3, and TM7 to define the regions of the ETB
that are involved in downstream signaling. Our results provide insight
into the activation mechanism of the ETB. The TM6 domain of the ETB has
several residues that are highly conserved across a broad spectrum of
GPCRs, and studies with rhodopsin implicate this region in the
ligand-induced activation mechanism. Therefore, we postulated that
these residues may be important in regulating the coupling of the ETB
with downstream effectors, such as ERK activation. Surprisingly,
systematic substitution of the highly conserved residues of TM6 had no
effect on activation of ERK (Fig. 2). Although these residues are
highly conserved, this area of the ETB does not appear to be sensitive
to structural changes. The results argue against TM6 as being
critically involved in ligand-induced ERK activation. The ETB appears
to be distinct from at least two other GPCRs in which TM6 is involved
in the activation mechanism. For example, in the case of the m5
muscarinic receptor, substitution of Ser465 at the
extracellular junction of TM6 resulted in activation of the receptor
(20). Substitution of Ala293 of the
1B-adrenergic receptor, which is in loop 3 close to the junction of TM6, resulted in constitutive activation of the receptor (25). Amino acid residues throughout TM6 and loop 3 of the ETB were
mutated without effect on basal or ligand-induced ERK activation. Although these regions in the muscarinic and adrenergic receptors are
involved in the activation mechanism of the receptors, this theme is
not applicable to the ETB.
The intracellular loops of GPCRs form a structure in the cytosol of the
cell that may be important in mediating protein-protein interactions
with heterotrimeric G-proteins. In fact, introducing mutations into
this area can impair downstream signaling functions (25). In the case
of the ETB, the putative intracellular loops are very small, relative
to other GPCRs. The intracellular loop 3 of the ETB is the largest and
was therefore hypothesized as most likely to be involved in coupling to
G-proteins and downstream signaling functions. We therefore
systematically introduced point mutations into the ETB loop 3 area and
evaluated the ability of the mutant receptors to activate the ERK
pathway. Our results indicate that, similar to the TM6 region, loop 3 is not sensitive to the introduction of nonconserved amino acid
substitutions. Several mutations were introduced into loop 3 of the ETB
with no effect on ligand-stimulated ERK activation.
In contrast, TM7 of the ETB proved to be very sensitive to mutation.
Nine residues in TM7 were identified that, when substituted with a
nonconserved residue, resulted in a significant decrease in
ligand-stimulated ERK activation (Fig. 4). Our results differ from the
rhodopsin model of GPCR activation to the extent that TM6 is postulated
to be critically involved in ligand-induced activation of rhodopsin,
whereas this region of the ETB does not appear to be important for ERK
activation. In contrast, TM7 of the ETB was very sensitive to mutation,
with respect to agonist-induced ERK activation. In the
three-dimensional model of helix packing of rhodopsin, helix 7 is close
to helix 6. Because helix 6 is thought to move during activation, it is
possible that mutations of helix 7 sterically interfere with the
ability of helix 6 to be displaced during ligand binding. However, if
helix 6 of the ETB were critically involved in activation, a reasonable
prediction would be that it should be sensitive to mutagenesis.
Therefore, the data suggest the possibilities that the activation
mechanism of the ETB is distinct from that of rhodopsin and that helix
7 of the ETB is involved in ligand-induced activation.
Some of the biological effects of ETB activation are thought to involve
activation of AP-1 mediated transcription (14). Because ERK is known to
phosphorylate transcription factors leading to increased AP-1 activity
(26), we examined the relationship between ETB-stimulated ERK
activation and AP-1 transcriptional activation. Seven of the residues
in TM7 that were sensitive to mutation, with respect to ERK activation,
demonstrated reduced AP-1/luciferase reporter activity, suggesting that
AP-1 activation and ERK activity are positively correlated.
Interestingly, there were five notable exceptions to this correlation.
ETB TM7 mutants D367A, N372A, I383A, V388K, and I380A consistently
demonstrated increased endothelin-stimulated AP-1 activity. The I380A
TM7 mutant was especially notable because it consistently demonstrated
augmented ligand-stimulated AP-1 activation. The basal ERK activity of
the receptor mutants was similar to wild type levels with minor
elevations in some TM7 mutants (F392A, S378K, p382K, and K390A).
However, no positive correlation could be shown between increased basal ERK activity and increased AP-1 transcription (compare Fig.
2B with Fig. 6). With the exception of D367A, the mutants
that had increased endothelin-stimulated AP-1 activity were unable to
stimulate ERK kinase activity (Figs. 4 and 6). The results suggest that ERK activation and AP-1 activation are separable, and that ERK activity
is not an absolute requirement for AP-1 activation. The mechanism
whereby AP-1 is activated may involve one or more of the several signal
transduction systems known to be activated by endothelin. Additional
studies will be required to dissect the layers of interaction between
the components of the endothelin signaling pathway.
 |
ACKNOWLEDGEMENT |
We thank Dr. Mercedes Rincon for providing plasmids.
 |
FOOTNOTES |
*
This work was supported by U. S. Public Health Service
Grants HL49570 and HL55327.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. Tel.: 802-656-3263;
Fax: 802-656-2191; E-mail: pvichi{at}zoo.uvm.edu.
§
Present address: Eli Lilly & Co., Research Labs, Lilly Corporate
Ctr., Indianapolis, IN 46285.
 |
ABBREVIATIONS |
The abbreviations used are:
ET, endothelin-1;
ERK, extracellular-regulated kinase 2;
ETB, endothelin B receptor;
GPCR, G-protein-coupled receptor;
TM, transmembrane helix;
DMEM, Dulbecco's modified Eagle's medium;
FBS, fetal bovine serum;
-gal,
-galactosidase;
MBP, myelin basic protein;
MT-ERK2, Myc-tagged
ERK2.
 |
REFERENCES |
-
Yanagisawa, M.,
Kurihara, H.,
Kimura, S.,
Tomobe, Y.,
Kobayashi, M.,
Mitsui, Y.,
Yazaki, Y.,
Goto, K.,
and Masaki, T.
(1988)
Nature
332,
411-415[Medline]
[Order article via Infotrieve]
-
Whelchel, A.,
Evans, J.,
and Posada, J.
(1997)
Am. J. Respir. Cell Mol. Biol.
16,
589-596[Abstract]
-
Lin, H. Y.,
Kaji, E. H.,
Winkel, G. K.,
Ives, H. E.,
and Lodish, H. F.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
3185-3189[Abstract]
-
Arai, H.,
Hori, S.,
Aramori, I.,
Ohkubo, H.,
and Nakanishi, S.
(1990)
Nature
348,
730-732[Medline]
[Order article via Infotrieve]
-
Sakurai, T.,
Yanagisawa, M.,
Takuwa, Y.,
Miyazaki, H.,
Kimura, S.,
Goto, K.,
and Masaki, T.
(1990)
Nature
348,
732-735[Medline]
[Order article via Infotrieve]
-
Shapiro, P. S.,
Evans, J. N.,
Davis, R. J.,
and Posada, J. A.
(1996)
J. Biol. Chem.
271,
5750-5754[Abstract/Free Full Text]
-
Bogoyevitch, M. A.,
Marshall, C. J.,
and Sugden, P. H.
(1995)
J. Biol. Chem.
270,
26303-26310[Abstract/Free Full Text]
-
Rincon, M.,
and Flavell, R. A.
(1996)
Mol. Cell. Biol.
16,
1074-1084[Abstract]
-
Simonson, M. S.,
and Herman, W. H.
(1993)
J. Biol. Chem.
268,
9347-9357[Abstract/Free Full Text]
-
Simonson, M. S.,
Wang, Y.,
and Herman, W. H.
(1996)
J. Biol. Chem.
271,
77-82[Abstract/Free Full Text]
-
Foschi, M.,
Chari, S.,
Dunn, M. J.,
and Sorokin, A.
(1997)
EMBO J.
16,
6439-6451[Abstract/Free Full Text]
-
Cazaubon, S. M.,
Ramos-Morales, F.,
Fischer, S.,
Schweighoffer, F.,
Strosberg, A. D.,
and Couraud, P-O.
(1994)
J. Biol. Chem.
269,
24805-24809[Abstract/Free Full Text]
-
Daub, H.,
Weiss, F. U.,
Wallasch, C.,
and Ullrich, A.
(1996)
Nature
379,
557-560[Medline]
[Order article via Infotrieve]
-
Simonson, M. S.,
Jones, J. M.,
and Dunn, M. J.
(1992)
J. Biol. Chem.
267,
8643-8649[Abstract/Free Full Text]
-
Hashido, K.,
Adachi, M.,
Gamou, T.,
Watanabe, T.,
Furuichi, Y.,
and Miyamoto, C.
(1993)
Cell. Mol. Biol. Res.
39,
3-12[Medline]
[Order article via Infotrieve]
-
Adachi, M.,
Hashido, K.,
Trzeciak, A.,
Watanabe, T.,
Furuichi, Y.,
and Miyamoto, C.
(1993)
J. Cardiovasc. Pharmacol.
22 Suppl. 8,
121-124
-
Aquilla, E.,
Whelchel, A.,
Knot, H. J.,
Nelson, M.,
and Posada, J.
(1996)
J. Biol. Chem.
271,
31572-31579[Abstract/Free Full Text]
-
De Lean, A.,
Stadel, J. M.,
and Lefkowitz, R. J.
(1980)
J. Biol. Chem.
255,
7108-7117[Abstract/Free Full Text]
-
Samama, P.,
Cotecchia, S.,
Costa, T.,
and Lefkowitz, R. J.
(1993)
J. Biol. Chem.
268,
4625-4636[Abstract/Free Full Text]
-
Spalding, T. A.,
Burstein, E. S.,
Wells, J. W.,
and Brann, M. R.
(1997)
Biochemistry
36,
10109-10116[CrossRef][Medline]
[Order article via Infotrieve]
-
Dratz, E. A.,
and Hargrave, P. A.
(1983)
Trends Biochem. Sci.
8,
128-131
-
Unger, V. M.,
and Schertler.
(1995)
Biophys. J.
68,
1776-1786[Abstract]
-
Farrens, D. L.,
Altenbach, C.,
Yang, K.,
Hubbell, W. L.,
and Khorana, H. G.
(1996)
Science
274,
768-770[Abstract/Free Full Text]
-
Franke, R. R.,
Koenig, B.,
Sakmar, T. P.,
Khorana, H. G.,
and Hoffman, K. P.
(1990)
Science
250,
123-125[Medline]
[Order article via Infotrieve]
-
Kjelsberg, M. A.,
Cotecchia, S.,
Ostrowski, J.,
Caron, M. G.,
and Lefkowitz, R. J.
(1992)
J. Biol. Chem.
267,
1430-1433[Abstract/Free Full Text]
-
Karin, M.
(1995)
J. Biol. Chem.
270,
16483-16486[Free Full Text]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.