2 Division of Nephrology, University of Florida, Gainesville, Florida 32610; 3 Department of Pharmacolgy, University of Texas Southwestern Medical School, Dallas, Texas 75235; and 1 Fourth Department of Internal Medicine, Kumamoto University, Kumamoto, Japan
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ABSTRACT |
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Endothelin (ET) receptors activate heterotrimeric G proteins
that are members of the Gi,
Gq, and
Gs families but may also activate
members of other families such as
G12/13.
G
13 has multiple complex
cellular effects that are similar to those of ET. We studied the
ability of ET receptors to activate
G
13 using an assay for G
protein
-chain activation that is based on the fact that an activated (GTP-bound)
-chain is resistant to trypsinization compared with an inactive (GDP-bound)
-chain. Nonhydrolyzable guanine nucleotides and AlMgF protected
G
13 from degradation by
trypsin. In membranes from human embryonic kidney 293 cells that
coexpress ETB receptors and
13, ET-3 and
5'-guanylylimidodiphosphate [Gpp(NH)p] increased the
protection of
13 compared with
Gpp(NH)p alone. The specificity of
ETB
receptor-
13 coupling was
documented by showing that
2
receptors and isoproterenol or ETA
receptors and ET-1 did not activate
13 and that a specific
antagonist for ETB receptors
blocked ET-3-dependent activation of
13.
G protein; cell signaling; G protein-coupled receptor
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INTRODUCTION |
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ENDOTHELIN (ET) is a peptide ligand for two homologous
G protein-coupled receptors, ETA
and ETB receptors. These receptors regulate multiple intracellular signaling processes, not all of which
have been clearly associated with an individual G protein. ET's
effects include inhibition of adenylyl cyclase, presumably through
activation of a Gi; activation
of phospholipase C, presumably through activation of members of the
Gq/11 family of
-chains; stimulation of tyrosine phosphorylation by an undetermined mechanism; and activation of the mitogen-activated protein (MAP) kinases [extracellular signal-regulated protein kinases (ERK), c-Jun
NH2-terminal kinases (JNK), and
p38] (1, 34). ET has a variety of tissue-specific biological
effects. In vascular smooth muscle, ET is a potent vasoconstrictor and
stimulus for smooth muscle growth, where it stimulates endothelial
PGI2 and NO synthesis. In
myocardium, it is a positive inotrope and stimulates myocardial
hypertrophy. In the adrenal cortex ET stimulates aldosterone secretion,
in fibroblasts it stimulates
Na+/H+
exchange (NHE1), and in epithelial cells it stimulates
Na+/H+
exchange by a different NHE isoform (NHE3) (7, 34, 42).
The actions of ET may not be explained fully by its activation of G
proteins of the i or
q families. To determine
whether some of the biological effects of ET could be due to coupling of ETB receptors to
G
13, a member of a distinct
family of G protein
-chains, we studied the ability of
ETB receptors to activate G
13 in a transient cell
expression system. G
13 is
activated by thrombin, thromboxane, thyroid-stimulating hormone (TSH),
and ANG II AT1A receptors (23, 25,
31). Like ET, thrombin, TSH, and ANG II have complex biological actions
that involve activation of multiple G protein
-chains.
G
13 is capable of transforming cells, stimulating JNK activity, activating immediate early response genes, and stimulating
Na+/H+
exchange in a variety of cell types and appears to be involved in
regulation of cell motility (32). ET and
13 share a number of effects on
cells, including stimulation of JNK and
Na+/H+
exchange activities (7-9, 12, 27, 34, 41, 42, 44). Consequently,
13 could mediate some of the
biological effects of ET.
Ligand-bound receptors activate G proteins by catalyzing conformational
changes and guanine nucleotide exchange. Many assays for specific G
protein-receptor coupling are based on the measurement of
conformational changes or guanine nucleotide exchange. Activation is
characterized by reduced affinity for subunits and receptors and
sequestration of a trypsin cleavage site that is in the approximate center of the molecule (4, 15, 26-28). Resistance of the
conformation-sensitive trypsin site to cleavage by trypsin is a
sensitive and specific assay for the active conformation. Trypsin
protection has been used to study conformational changes and receptor
activation of native, expressed, and recombinant
s and transducin (14, 16, 26,
28). We determined the trypsin cleavage pattern for activated and
inactivated purified, recombinant
G
13. We then overexpressed
13 and
ETB receptors in human embryonic
kidney 293 (HEK-293) cells and measured activation of
13 in membranes by trypsin
protection. We demonstrate that
ETB receptors activate
G
13.
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MATERIALS AND METHODS |
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Materials
HEK-293 cells were obtained from the American Type Culture Collection. Tissue culture medium and serum were obtained from GIBCO; trypsin from Worthington or Sigma; guanine nucleotides from Boehringer Mannheim; ET-1, ET-3, and BQ-788 from Calbiochem; and the chemiluminescence system from Amersham. The cDNA encoding the human ETB and ETA receptors in the expression vector pMEhETB (driven by the SRExpression and Purification of Recombinant
13
Cell Culture and Transfection
HEK-293 cells were grown in Dulbecco's modified Eagle's medium with 10% fetal calf serum and 25 mM HEPES, pH 7.5, in a 10% CO2-90% air incubator. Ten-centimeter dishes of cells were transfected with the cDNA constructs by the Ca-PO4 coprecipitation method. Each transfection contained 5 µg of DNA. One microgram of each expression construct was used (Preparation of Cell Membranes
Cells were washed with phosphate-buffered saline and scraped in 1 ml of membrane buffer containing 20 mM HEPES, pH 8, 1.5 mM MgSO4, and 1 mM EDTA. The cells were disrupted by Dounce homogenization, nuclei were removed by centrifugation at 1,000 g for 3 min, and membranes were prepared by centrifugation of the supernatant at 15,000 g for 20 min. The pellets were resuspended in membrane buffer containing 10% glycerol and frozen atTrypsin Protection Assay
Purified r13-Subunit
in cell membranes. Membranes (15 µg per reaction)
were suspended in buffer containing 0.05% polyoxyethylene 10 lauryl
ether (Lubrol), 20 mM HEPES, pH 8.0, 1.5 mM
MgSO4, and 1.5 mM EDTA. Where
indicated, GDP and 5'-guanylylimidodiphosphate [Gpp(NH)p] were present at a concentration of 50 µM; AMF
was 50 µM AlCl3, 10 mM
MgCl2, and 10 mM NaF; and ET-3 and
BQ-788 (ETB receptor antagonist)
were present at 10 nM. Reactions began with the simultaneous addition
of guanine nucleotide analogs, AMF, and ET agonists or antagonists. The
reactions were incubated at 30°C for 5 min. At that time, 2.5 µl
of TPCK trypsin (0.5 mg/ml in the same buffer as above) was added for 2 min. The reactions were stopped by removing a 10-µl aliquot to a tube
containing 2.5 µl of 5× SDS gel loading buffer and 5 mg/ml
soybean trypsin inhibitor. The digestion products were separated by
SDS-PAGE on 9% polyacrylamide gels and immunoblotting with rabbit
antisera specific for the carboxy terminus of
13 (B860) (36). The blots were
visualized with the enhanced chemiluminescence system (Amersham).
Labeling of G13 and Gq With Azidoanilido [32P]GTP and Immunoprecipitation
Membranes (20 µg) were prepared as described above and suspended in buffer containing 0.1% polyoxyethylene 10 lauryl ether (Lubrol), 50 mM HEPES, pH 7.4, 30 mM NaCl, 10 mM MgCl2, 0.1 mM EDTA, and 1.0 mM benzamidine on ice. The agonists (as indicated) were added on ice, and the tubes were warmed to 30°C for 3 min. The reactions were started with the addition of 5 µl of azidoanilido [32P]GTP (10 nM or ~5 × 106 cpm, total reaction volume 60 µl) and incubated at 30°C for 5 min. The reactions were placed on ice and exposed to ultraviolet light (254 nm) for 20 s. An equal volume of immunoprecipitation buffer (final concentrations 1% Nonidet P-40, 1% deoxycholate, 0.5% SDS, 10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin) was added to each sample. The reactions were vortexed and centrifuged for 10 min at 13,000 g. The supernatants were transferred to new tubes, and 1 µl affinity-purified antibodies (B860 for ![]() |
RESULTS |
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We established the trypsin cleavage pattern for
G13 in the active and inactive
conformations using purified, recombinant protein. We found that, like
the other G proteins studied to date (transducin and
G
s), binding of a GTP analog
or activation of
13 with GDP
and AMF protects a conformation-sensitive trypsin site that is in the
approximate center of the protein (26-28). As shown in Fig.
1, trypsinization of the 43-kDa GDP-bound
pure protein produces two transient peptides of ~41 and 39 kDa that are seen in lower concentrations of trypsin (0.2 and 1 µg/ml). These
peptides are both completely degraded in the presence of 5 µg/ml
trypsin. In contrast, in the presence of AMF, a 39-kDa peptide is
preserved even after digestion with 5 µg/ml of trypsin. This same
39-kDa peptide is also protected by GTP
S in a dose-dependent fashion
over the concentration range of 2 µM-1 mM GTP
S. Protection is not
as effective as that seen with AMF, possibly because the spontaneous
rate of guanine nucleotide exchange by purified
13 is slow, whereas activation
of
-chains by AMF does not require guanine nucleotide exchange (36).
These studies demonstrate that activation of
13 by AMF or GTP analogs
produces a 39-kDa peptide that is protected from degradation by
trypsin.
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To determine whether G13 could
be activated to produce a trypsin-resistant conformation in membranes,
we activated
13 with AMF in
membranes prepared from HEK-293 cells that had been transfected with
13. Figure
2 shows that, like the pure protein,
13 expressed in cell membranes
is degraded by trypsin in the presence of GDP alone but that a
protected band of ~39 kDa is found in the presence of AMF. In
contrast to the studies with the pure protein, the protected
39-kDa band is not stable over time.
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In membranes from HEK-293 cells that express both
13 and
ETB receptors, trypsinization in
the presence of Gpp(NH)p results in a protected 39-kDa band, and the
amount of protection is enhanced approximately twofold by the addition
of ET-3 (Fig.
3A). The
ratio of the protected 39-kDa band to the 43-kDa band was 0.33 for
Gpp(NH)p alone and 0.78 for Gpp(NH)p with ET. We used Gpp(NH)p instead of GTP because the rate of hydrolysis of GTP by
13 is rapid compared with the
time required for trypsinization in this assay. In studies not shown,
neither ET-1 nor ET-3 affected trypsin protection of G13 in membranes in the absence of
expressed receptors. To demonstrate that ligand-dependent coupling of
ETB receptors to
13 is specific for
ETB receptors, we coexpressed
2-adrenergic receptors and
13 in HEK-293 cells to
determine whether a receptor that has different biological activities
from ETB receptors could enhance trypsin protection of the 39-kDa band of
13 (Fig.
3B). Incubation of membranes
containing expressed
2-adrenergic receptors and
13 with isoproterenol did not
enhance protection of the 39-kDa band of
13 compared with similar lanes
without isoproterenol. We further explored the specificity of the
interaction of ETB receptors with
G
13 by comparing the ability of
ET-1 to increase protection of
G
13 in cells that express
either ETA or
ETB receptors and
G
13. Figure
4 shows that ET-1 (an agonist with equal
potency for ETA and
ETB receptors) increases
protection of G
13 in a
dose-dependent manner in membranes that express
ETB receptors and
G
13, but not in membranes that
express ETA receptors and
G
13 (34). In the membranes that
express the ETB receptors, the
ratio of the 39-kDa band increases from 0.68 [Gpp(NH)p
alone] to 1.19 [Gpp(NH)p with 10
7 M ET-1]. However,
in the membranes with ETA
receptors and G
13, the ratio
remains relatively constant, ranging from 1.1 to 1.3.
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We used the specific ETB receptor
antagonist BQ-788 as an additional test of specificity to confirm that
the enhanced protection of expressed
13 was due to ET-3 binding to
the ETB receptor (13). As shown in
Fig. 5, ET increases protection of the
39-kDa band of G
13 by
approximately twofold, whereas BQ-788 prevents protection of
13 by ET-3. Additionally,
BQ-788 reduces the basal level of protection in the presence of
Gpp(NH)p. These studies demonstrate that protection of
13 is a specific function of
the ETB receptor.
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To confirm that the trypsin protection assay reflects incorporation of
GTP by the G protein -chain, we documented stimulation of
incorporation of azidoanilido
[32P]GTP into
13 and
q in membranes from HEK-293
cells that expressed ETB or
ETA receptors and the
-chains.
The
-chains were identified by immunoprecipitation with specific
antisera (2, 31, 39). As shown in Fig.
6A, ET-1
stimulates incorporation of azidoanilido [32P]GTP into
13 and
q in membranes from cells that
express ETB receptors. In cells
that express ETA receptors, ET-1
stimulates incorporation of azidoanilido
[32P]GTP into
q but not
13 (Fig.
6B). As a further test of
specificity, we determined whether
ETA or
ETB receptors could activate
s. Figure
6C shows that neither
ETB nor
ETA receptors with ET-3 are able
to activate G
s. However, as a
positive control,
2 receptors and isoproterenol increase photolabeling of
G
s. These results are similar
to those obtained in Figs. 3-6.
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Figure 7 is a summary of the data presented
above and represents a comparison of receptor-dependent protection of
the 39-kDa band of 13 in cells
that express
13 and the
ETB receptor, the ETA receptor, or the
2-adrenergic receptor.
Individual experiments were analyzed by calculating the ratio of the
protected 39-kDa band to the 43-kDa band in the membranes with Gpp(NH)p
alone and comparing that ratio with the ratio in the membranes with
receptor agonist. ET treatment generally resulted in a twofold increase in this ratio. In separate experiments with different membrane preparations, the ratios varied considerably, but ET treatment still
resulted in a twofold increase in the ratio. Therefore, to compare
multiple experiments using different membrane preparations, we
calculated the multiple increase in the ratio of the protected 39-kDa
band to the 43-kDa band in the experimental membranes (ET, BQ-788 + ET,
isoproterenol) over the membranes with Gpp(NH)p alone. ET consistently
increased protection of the 39-kDa band (2.13 ± 0.15-fold,
P < 0.05, ANOVA). BQ-788 inhibited
the ET-induced protection (1.3 ± 0.1-fold for ET + BQ-788 vs. 2.13 ± 0.15-fold for ET alone, P < 0.05, ANOVA). The other comparisons were not statistically different
from control [Gpp(NH)p alone].
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DISCUSSION |
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We demonstrated that ETB receptors
activate G13 when they are
coexpressed in HEK-293 cells. The specificity of the interaction of
ETB receptors with
G
13 was demonstrated by the
facts that ETA and
2-adrenergic receptors did not
increase the protection of G13
from degradation by trypsin and that the specific
ETB antagonist BQ-788 blocked the
ability of ET-3 to protect G
13.
Similar results were obtained using an independent technique,
photolabeling of
-subunits and immunoprecipitation with specific
antisera (Fig. 6). Studies by others have shown that, depending on the
cell type, ET receptors may activate members of the
s,
i, and
q/11 families (34). Our results
demonstrate that ETB receptors can
also activate a member of a fourth family of G protein
-chains,
G
13 (37). These results
indicate that, in some tissues, the complex biological effects of ET
are due to activation of G
13
and the intracellular signaling systems it controls.
Several other receptors, those for thrombin, thromboxane, TSH, and ANG
II, have been shown to activate
G13 (23, 25, 31). We studied
the activation of
13 by
ETB receptors by coexpressing the
two proteins in HEK-293 cells. Others have used essentially the same
technique to study portions of signaling pathways including receptor-G
protein interactions and demonstrated specific interactions in this
type of system (5, 6, 14, 27, 48).
The rG13 and
G
13 expressed in HEK-293 cells
behaved similarly in that both were degraded by trypsin in the presence
of GDP alone and protected by AMF and nonhydrolyzable guanine
nucleotides. However, these proteins differed in their spontaneous
rates of guanine nucleotide exchange. This rate is slow for the
purified protein, as demonstrated by the relatively poor protection of rG
13 by GTP
S. In contrast,
the rate of spontaneous guanine nucleotide exchange
[Gpp(NH)p] was substantial in cells that expressed
ETB receptors with
13 or with
13 alone. In cells that
coexpressed the ETB receptor and
13, the rate of guanine
nucleotide exchange was further stimulated by addition of the ligand
(ET-3) for the receptor. These results suggest that even without ligand
(ET-3) or ETB receptors, guanine
nucleotide exchange is catalyzed by some factor in the membranes. We
cannot determine whether the spontaneous rate of guanine nucleotide
exchange seen in the membrane preparation was due to the presence of
other receptors that activate
13 or whether some other
protein interacts with the
-chain to facilitate guanine nucleotide exchange.
The magnitude of receptor-stimulated guanine nucleotide exchange for
receptors and G protein -chains found by other investigators in
membrane preparations is comparable to that which we found with
ETB receptors and
G
13. Offermanns et al. (31) and
Laugwitz et al. (23) measured thrombin and TSH receptor-simulated
photoaffinity labeling of G13 with
azidoanilido GTP. They found that the activated receptors increased
photolabeling of G
13 by 82 and
123%, respectively, over the basal, or spontaneous, rate of guanine
nucleotide exchange (no receptor ligand). In studies using other
receptors and
-chains, they demonstrated that agonist-stimulated
receptors stimulated
-chain labeling by 95-320% (24, 33). Our
trypsin protection studies (Fig. 7) demonstrated a 2.13 ± 0.15-fold, or ~113%, increase in the amount of active
G
13 in response to
ETB receptor stimulation. Using
proteins expressed in a baculovirus expression system with [35S]GTP
S and
immunoprecipitation, Barr et al. (2) demonstrated that stimulation of
5-HT1A receptors resulted in an
approximately twofold increase in guanine nucleotide exchange compared
with receptor alone. In all of these systems, the basal rate of guanine nucleotide exchange is substantial, so that when agonist-stimulated and
"spontaneous" guanine nucleotide exchange rates are compared, the
difference is on the order of two- to fourfold. Trypsin protection studies in which rhodopsin and transducin were coexpressed in COS cells
demonstrated a greater multiple increase in protection of transducin by
light-activated rhodopsin because the spontaneous rate of guanine
nucleotide exchange in this system is much lower than for other
receptor-G protein combinations (14).
ET acts through ETA and
ETB receptors. These receptors
have different affinities for their ligands
(ETA: ET-1 ET-2 > ET-3; ETB: ET-1 = ET-2 = ET-3) and
patterns of tissue and cell-type distribution (34). The differences
between these two receptors and the intracellular signaling pathways
they activate have not been clearly established in all tissues. Most
studies to date rely on selectivity of ET-1 or ET-3 agonists and
antagonists for the different receptors. Some of the apparent
differences may be due to cell-specific expression of downstream
signaling molecules. ETA and
ETB receptors share the ability to
increase intracellular Ca2+
through activation of a member of the
G
q/11 family of G proteins as
well as regulating Ca2+ entry by
undefined mechanisms (34). They also appear to activate phospholipase
A2 and phospholipase D, which also
may be involved in Ca2+ signaling.
ETB and
ETA receptors are capable of
inhibiting cAMP production (7, 29, 34). In one report,
ETA receptors acting through
s stimulated adenylyl cyclase
(11). ETB receptors are capable of
activating three members of the MAP kinase family, ERK, JNK, and p38
(1). In several studies, G
13
activated the JNK pathway, so ETB
receptors may act through
13 to
activate JNK in these systems (8, 41, 44).
ETB receptors stimulate NHE1
activity in fibroblasts (the
Na+/H+
exchanger isoform expressed in most cells) and NHE3 in renal epithelial
cells (7, 34, 42). These transporters are regulated by similar
extracellular signals under a number of circumstances, but the precise
intracellular mechanisms that regulate them are not fully defined (30,
40). The mechanisms may involve changes in intracellular
Ca2+ levels, protein kinase C
activity, protein kinase C-independent mechanisms, and tyrosine kinase
activity (3, 30, 34). Expression studies with two G protein -chains,
G
q and
G
13, showed that they activate
NHE1 in part through increasing mRNA and protein levels (20).
Therefore, because
q and
13 activate NHE1 as does ET
through ETB receptors,
ETB receptors may activate these
-chains. G
q activates
phospholipase C and may stimulate
Na+/H+
exchange activity by that mechanism, but the mechanisms by which G
13 acts are not defined.
Preliminary studies indicate that
ETB receptors and
G
13 stimulate an NHE1
promoter-reporter construct (Shiraishi and Miller, unpublished
observations). Consequently, G
13 is a candidate for one of
the G proteins that is activated by
ETB receptors and that regulates
Na+/H+ exchange.
G13 appears to activate a
number of downstream signaling molecules. Two recent reports show that
it activates p115, an RGS protein, that is also a guanine nucleotide
exchange factor for the small GTP binding protein Rho A (18, 22). Other
molecules may also be targets of
G
13.
G
13 contributes to regulation
of hormone-stimulated Ca2+ influx
and intercellular Ca2+ by other
mechanisms that are independent of phospholipase C, K channels, the
small GTP binding protein cdc42, and the JNK pathway (19, 20, 25, 41,
46, 47, 49). These and possibly other signaling pathways contribute to
cell transformation, induction of inducible NO synthase, and activation
of
Na+/H+
exchange (7, 10, 20, 21, 42, 43, 45).
Our studies demonstrate that ETB
receptors activate G13 in
HEK-293 cells. Our studies do not address which of the potential signaling proteins are downstream of
ETB receptors and
13 in HEK-293 cells, but they
do demonstrate that the complex biological effects of ET derive in part
through signaling by ETB receptors though an additional family of G protein
-chains.
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ACKNOWLEDGEMENTS |
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K. Kitamura and N. Shiraishi contributed equally to this work.
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FOOTNOTES |
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The authors thank Paul Sternweis and Shmuel Muallem for valuable discussions.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-41726, funds from the American Heart Association with funds from the Florida affiliate (R. T. Miller), Grants GM-15359 and GM-31954, and funds from the Robert A. Welch foundation (P. C. Sternweis), and a training grant from the National Kidney Foundation (to K. Kitamura and N. Shiraishi).
Address for reprint requests and other correspondence: R. T. Miller, Division of Nephrology, Univ. of Florida, Box 100224 JHMHC, 1600 SW Archer Rd., Gainesville, FL 32610 (E-mail: tmiller{at}nervm.nerdc.ufl.edu).
Received 10 July 1997; accepted in final form 25 January 1999.
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