Download A consensus sequence in the endothelin

Am J Physiol Renal Physiol 288: F732–F739, 2005.
First published December 14, 2004; doi:10.1152/ajprenal.00300.2004.
A consensus sequence in the endothelin-B receptor second intracellular loop
is required for NHE3 activation by endothelin-1
Kamel Laghmani,1 Aiji Sakamoto,2 Masashi Yanagisawa,3 Patricia A. Preisig,1,* and Robert J. Alpern1,*
1
Department of Internal Medicine and the 3Howard Hughes Medical Institute, University
of Texas Southwestern Medical Center, Dallas, Texas; and 2Division of Biotechnology
and Department of Bioscience, National Cardiovascular Center Research Institute, Osaka, Japan
Submitted 11 August 2004; accepted in final form 2 December 2004
opossum kidney cells; endothelin-A/endothelin-B chimeras; sodium/
hydrogen antiporter activity
THE ENDOTHELINS (ET) are a family of three 21-amino acid
peptides, ET-1, ET-2, and ET-3. They are involved in a variety
of physiological responses, such as vascular smooth muscle
cell contraction and dilation, development, and regulation of
renal function (17, 35). The biological effects of the ETs are
mediated by two receptor subtypes, ETA and ETB (3, 13, 32,
34). ET-1 and ET-2 bind the ETA receptor with high affinities,
whereas ET-3 binds with low affinity. The three isopeptides
bind the ETB receptor with similar high affinities. Both receptors have seven hydrophobic transmembrane domains, characteristic of G protein-coupled receptors. Human ETA and ETB
receptors exhibit significant amino acid sequence identity (55%
overall, 74% within the putative transmembrane helices) (33,
36). Of the receptor subdomains, the extracellular NH2 terminus and the intracellular COOH-terminal tail display the least
sequence similarities, whereas the sequences in intracellular
loops 2 and 3 are relatively similar. Despite their similarities,
studies have demonstrated distinct roles for these intracellular
* P. A. Preisig and R. J. Alpern contributed equally to these studies.
Address for reprint requests and other correspondence: P. Preisig, Univ. of
Texas Southwestern Medical Center, Rm. H5.122, 5323 Harry Hines Blvd.,
Dallas, TX 75390-8856 (E-mail: [email protected]).
F732
loops in the human receptors. Human ETA and ETB selectively
couple to G␣s and G␣i, respectively, with the second and third
intracellular loops of human ETA involved in G␣s activation
and the third intracellular loop of human ETB involved in G␣i
activation (22).
ET-1 increases the activity of the proximal tubule apical
membrane Na⫹/H⫹ antiporter, which mediates the majority of
proximal tubular NaCl and NaHCO3 absorption (10, 12, 29,
30). This proximal tubule apical membrane Na⫹/H⫹ activity is
mediated by NHE3 (1, 4). OKP cells, an opossum kidney cell
line with many proximal tubule characteristics, express NHE3
(2, 23, 24). Using stable transfection, we established OKP cell
lines expressing ETA and ETB receptors and showed that in
cells expressing the ETB, but not the ETA receptor, ET-1
increases NHE3 activity (7). In addition, NHE3 activation by
ET-1 was inhibited by ETB receptor blockers, but not by ETA
receptor blockers. Finally, in proximal tubules harvested from
ETB receptor knockout mice, ET-1 fails to increase NHE3
activity, whereas stimulation is seen in tubules from wild-type
mice (20). These findings agree with immunohistochemistry
and binding studies showing that the ETB receptor is the
predominant ET receptor on the renal proximal tubule (9, 42).
However, the mechanism responsible for the receptor specificity of NHE3 regulation remains unclear. We previously
showed that ET-1 causes similar patterns of protein tyrosine
phosphorylation, adenylyl cyclase inhibition, and increases in
cell [Ca2⫹] in ETA- and ETB-expressing OKP cells, implying
that an additional signaling pathway must mediate the receptor
specificity of NHE3 regulation (7, 8, 28).
To address this, in the current study human ETA/ETB receptor chimeras and site-directed mutagenesis were used to identify the ET receptor domain(s) involved in ET-1 regulation of
NHE3 activity in OKP cells. We found that ETA receptor
activation inhibits NHE3 activity, an effect for which the
COOH-terminal tail is necessary and sufficient. Activation of
NHE3 by ET-1 requires the COOH-terminal domain and the
second intracellular loop of the ETB receptor. Site-directed
mutagenesis within the ETB second intracellular loop identified
residues critical for stimulation of NHE3 activity by ET-1.
These residues define a consensus sequence, KXXXPKXXXV,
required for ET-1 stimulation of NHE3.
METHODS
Materials. All chemicals were obtained from Sigma (St. Louis,
MO) unless otherwise noted as follows. Penicillin and streptomycin
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0363-6127/05 $8.00 Copyright © 2005 the American Physiological Society
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Laghmani, Kamel, Aiji Sakamoto, Masashi Yanagisawa, Patricia A. Preisig, and Robert J. Alpern. A consensus sequence in the
endothelin-B receptor second intracellular loop is required for NHE3
activation by endothelin-1. Am J Physiol Renal Physiol 288: F732–
F739, 2005. First published December 14, 2004; doi:10.1152/ajprenal.
00300.2004.—Endothelin-1 (ET-1) increases the activity of Na⫹/H⫹
exchanger 3 (NHE3), the major proximal tubule apical membrane
Na⫹/H⫹ antiporter. This effect is seen in opossum kidney (OKP) cells
expressing the endothelin-B (ETB) and not in cells expressing the
endothelin-A (ETA) receptor. However, ET-1 causes similar patterns
of protein tyrosine phosphorylation, adenylyl cyclase inhibition, and
increases in cell [Ca2⫹] in ETA- and ETB-expressing OKP cells,
implying that an additional mechanism is required for NHE3 stimulation by the ETB receptor. The present studies used ETA and ETB
receptor chimeras and site-directed mutagenesis to identify the ET
receptor domains that mediate ET-1 regulation of NHE3 activity. We
found that binding of ET-1 to the ETA receptor inhibits NHE3
activity, an effect for which the COOH-terminal tail is necessary and
sufficient. ET-1 stimulation of NHE3 activity requires the COOHterminal tail and the second intracellular loop of the ETB receptor.
Within the second intracellular loop, a consensus sequence was
identified, KXXXVPKXXXV, that is required for ET-1 stimulation of
NHE3 activity. This sequence suggests binding of a homodimeric
protein that mediates NHE3 stimulation.
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NHE3 ACTIVATION BY ENDOTHELIN-1
tation-containing DNA using high-fidelity PfuTurbo DNA polymerase. Dpn1, an endonuclease (target sequence: 5⬘-Gm6ATC-3⬘) specific for methylated and hemimethylated DNA, was used to digest the
methylated, nonmutated parental DNA template. The circular mutated
DNA was transformed into XL1-Blue supercompetent cells for nick
repair and the DNA isolated by miniprep.
Binding experiments. To measure ET-1 binding, cells were grown
to confluence in 12-well plates, rendered quiescent, and incubated in
medium containing 0.3% BSA and 20 pM 125I-ET-1, ⫾10⫺6 M cold
ET-1 for 1 h at 37°C. After the binding solution was removed, the
cells were washed twice with cold PBS, harvested in 0.5 ml of 1 N
NaOH, placed in a scintillation cocktail, and bound ET-1 was counted
in a Beckman LS3801 model scintillation counter (Fullerton, CA), as
described (7). Nonspecific binding was defined as binding that occurred in the presence of excess (10⫺6 M) cold ET-1 and was
subtracted from total binding (assayed in the absence of cold ET-1) to
obtain specific binding. Results are expressed as a percentage of
specific binding in cells transfected with the wild-type ETB receptor.
Statistics. Data are reported as means ⫾ SE. Statistical significance
was determined using a paired Student’s t-test (NHE3 activity studies)
or ANOVA (binding studies) and set at P ⬍ 0.05.
RESULTS
We previously showed in stably transfected cells that the
ET-1-induced increase in NHE3 activity is mediated by ETB
and not ETA receptors. The first step of this study was to
compare the effect of ET-1 on NHE3 activity in wild-type OKP
cells and OKP cells transiently transfected with the ETB or
ETA receptor cDNA. In wild-type untransfected cells, 10⫺8 M
ET-1 applied for 30 min caused a consistent small, but statistically insignificant, increase (⫹15%) in NHE3 activity (Fig.
1). When the ETB receptor was expressed, 10⫺8 M ET-1
significantly increased NHE3 activity by 38%, in agreement
with our previous study (7). In contrast, in OKP cells expressing the ETA receptor, 10⫺8 M ET-1 significantly inhibited
NHE3 activity (⫺15%).
Table 1. Sequences of primers
Mutation Name
Direction
Mutant 1
I836V
Mutant 2
K837Q
Mutant 3
V841I
Mutant 4
K843L
Mutant 5 (Also X4)
W844V
Mutant 6
V847I
X1
G838V
X2
I839V
X3
G840A
X5
T845A
X6
A846V
Delete
G838
Sense
Reverse
Sense
Reverse
Sense
Reverse
Sense
Reverse
Sense
Reverse
Sense
Reverse
Sense
Reverse
Sense
Reverse
Sense
Reverse
Sense
Reverse
Sense
Reverse
Sense
Reverse
Sense
Reverse
P842A
Sequence
gtt
cat
gtt
cat
gga
ctg
gga
caa
gga
caa
ggg
ccc
gga
gct
gga
gct
gta
ctg
ggg
cca
ggg
cca
gct
gct
gga
caa
gct
ttt
gct
ttt
gta
ctg
att
aac
att
aac
gtt
aaa
gta
gtc
gta
gtc
gaa
ctg
gtt
att
gtt
aat
tct
gtc
att
ttt
AJP-Renal Physiol • VOL
tct
gga
tct
gga
gaa
tcc
ggg
aat
ggg
aat
cca
tca
gaa
cat
gaa
cat
tta
tcc
cca
caa
cca
caa
tgg
cat
ggg
cta
tgg
acc
tgg
acc
tta
att
gtt
ttc
gtt
ttc
aaa
aaa
tta
ttt
tta
ttt
aag
att
aaa
aac
aaa
aac
agt
ttt
gtt
ctg
agt aga gtt aaa gga att ggg gtt cca aaa tg
cca att cct tta act cta ctc caa gaa gca ac
agt aga att caa gga att ggg gtt cca aaa tg
cca att cct tga att cta ctc caa gaa gca ac
aag ga att g gga ttc caa aat gga cag cag
ttg gaa tcc caa ttc ctt taa ttc tac tcc
cca aaa gtg aca gca gta gaa att gtt ttg
tac tgc tgt cac ttt tgg aac ccc aat tcc
cca aaa gtg aca gca gta gaa att gtt ttg
tac tgc tgt cac ttt tgg aac ccc aat tcc
tgg aca gca ata gaa att gtt ttg att tgg g
caa ttt cta ttg ctg tcc att ttg gaa ccc c
aa gta a ttg ggg ttc caa aat gga cag c
gga acc cca att act tta att cta ctc c
aag ga gtt g ggg ttc caa aat gga cag c
gga acc cca act cct tta att cta ctc c
gaa tt gcg g ttc caa aat gga cag cag
ttg caa ccg caa ttc ctt taa ttc tac
tgg gca gca gta gaa att gtt ttg att tgg
aat tcc tac tgc tgc cca ttt tgg aac ccc
tgg aca gta gta gaa att gtt ttg att tgg
aat ttc tac tac tgt cca ttt tgg aac ccc
aga att aaa ⌬ att ggg gtt cca aaa tgg aca gc
gga acc cca att tta att cta ctc caa gaa gc
gca aaa tgg aca gca gta gaa att g
ctg tcc att ttg caa ccc caa ttc c
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were from Whittaker MA Bioproducts (Walkersville, MD);
BCECF-AM was from Molecular Probes (Eugene, OR); ET-1 was
from Peptides International (Louisville, KY); and 125I-ET-1 was from
Amersham (Arlington Heights, IL).
Cell culture and transfections. OKP cells were grown in DMEM
supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and
100 ␮g/ml streptomycin at 37°C in a CO2 incubator. To overexpress
human ETA and ETB receptors, cells were transiently transfected with
wild-type, chimera, or mutant receptors using the pME18S expression
vector driven by a constitutively active SR␣ promoter (18). OKP cells
were plated on coverslips in 35-mm plastic dishes, grown to 80%
confluence, transfected with 1.1 ␮g DNA using Lipofectamine Plus
according to the manufacturer’s instructions, and grown to 100%
confluence. Confluent cells were rendered quiescent by the removal of
serum for 24 h before study. Cells on control and experimental
coverslips were derived from the same flask and passage and were
studied on the same day. ETA/ETB receptor chimeric cDNAs were
generated as described (33).
Na⫹/H⫹ antiporter activity. Cell pH was measured in a temperature-controlled spectrofluorometer (SLM 8000C, Rochester, NY) as
the ratio of BCECF fluorescence with 500- and 450-nm excitation
(emission wavelength 530 nm), as previously described (7, 8, 27, 28).
To measure Na⫹/H⫹ antiporter activity, cells were acidified with
nigericin and the initial rate of Na-dependent intracellular alkalinization was measured, as previously described (7, 8, 28). In all studies,
control and experimental cells were from the same passage and were
assayed on the same day. Exposure to 10⫺8 M ET-1 was begun at the
time of BCECF loading (30 min before study). Control cells were
exposed to the vehicle (0.1% acetic acid).
Site-directed mutagenesis. Point mutations were generated using a
Stratagene QuikChange Site-Directed Mutagenesis Kit (La Jolla, CA),
as per kit protocol. Two primers complementary to each other were
designed to contain the desired mutation (Table 1). The plasmid
containing the wild-type receptor was grown up in a dam⫹ Escherichia coli strain, resulting in the plasmid DNA methylation. Thermal
cycling was performed as per kit instructions (12 cycles of 95°C ⫻
30 s, 55°C ⫻ 1 min, and 68°C ⫻ 1 min/kb plasmid length) to denature
the template, anneal the mutagenic primers, and synthesize the mu-
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NHE3 ACTIVATION BY ENDOTHELIN-1
To identify the domain(s) of the ETB and ETA receptors
involved in ET-1 regulation of NHE3 activity, we transfected
cells with chimeric ETA and ETB receptor cDNAs. Figure 2
shows the restriction sites used to generate the chimeric
cDNAs. Nomenclature will indicate the specific domains derived from each receptor. For example, chimera A(N-III)B(IV-C)
designates a chimeric receptor consisting of the ETA sequence
from the NH2-terminal extracellular tail to the third transmembrane domain followed by the ETB sequence from the second
intracellular loop through the COOH-terminal tail. To generate
this chimera wild-type ETA and ETB receptor, cDNA was cut
with BssHI (Fig. 2) (33). OKP cells were transiently transfected with each chimeric cDNA and NHE3 activity was
measured following exposure to 10⫺8 M ET-1 or vehicle.
Role of the COOH-terminal domain. Experiments shown in
Fig. 3 examined the role of the COOH-terminal domain.
Again, activation of the ETA receptor led to inhibition of
Fig. 2. Schematic diagram showing the location of the restriction sites used to
generate the chimeric cDNAs. The number of circles is based on the human
ETA sequence. Dotted circles are sequence gaps placed in the sequence to align
the ETA and ETB sequences. F Represent amino acid residues that are identical
between the aligned human ETA and ETB sequences; E represent nonidentical
residues. Positions of the restriction sites used to construct the chimeric
receptors are shown by arrows along with the name of the restriction enzymes.
TMD, transmembrane domain.
AJP-Renal Physiol • VOL
Fig. 3. Role of the COOH-terminal tail. Cells were grown, treated, and
assayed as in Fig. 1. A(N-VII)B(C) and B(N-VII)A(C) were generated and the
COOH-terminal tail was removed in construct B(N-VII) using the restriction
enzyme EcoRI (Fig. 2). Blue, ETB receptor sequence. Red, ETA receptor
sequence. ETA: n ⫽ 3; A(N-VII)B(C): n ⫽ 7; B(N-VII)A(C): n ⫽ 7; B(N-VII):
n ⫽ 4. *P ⬍ 0.01. **P ⫽ 0.057.
NHE3 activity (Fig. 3). Substitution of the ETA COOH-terminal tail with the ETB COOH-terminal tail [A(N-VII)B(C)]
prevented this effect, whereas substitution of the ETB COOHterminal tail with the ETA COOH-terminal tail [B(N-VII)A(C)]
mediated inhibition (⫺26%, P ⬍ 0.01). Thus the presence of
the COOH-terminal domain of the ETA receptor is necessary
and sufficient to mediate inhibition of NHE3 activity by ET-1.
By contrast, the above results using the chimera A(NVII)B(C) suggest that the ETB COOH-terminal domain is not
sufficient to mediate stimulation of NHE3 activity by ET-1.
The failure of B(N-VII)A(C) to mediate ET-1-induced stimulation of NHE3 could indicate a requirement for the COOH
terminus or could be due to ETA-induced inhibition mediated
by the ETA COOH terminus. To determine whether the ETB
COOH-terminal tail is required for ET-1 stimulation of NHE3,
we examined a truncated ETB receptor in which the COOHterminal tail was removed (18). As shown in Fig. 3, with
removal of the COOH-terminal tail ET-1 was without effect on
NHE3 activity. Taken together, these results suggest that the
ETB COOH-terminal tail is necessary but not sufficient to
mediate stimulation of NHE3 activity by ET-1.
Role of the ETB receptor intracellular loops. To identify
other key domains of the ETB receptor required for ET-1
stimulation of NHE3 activity, we used a series of chimeras in
which domains of the ETB receptor were replaced with the
corresponding region of the ETA receptor. As shown in Fig. 4,
when the ETB sequence extended from the COOH terminus to
the third intracellular loop, [A(N-V)B(VI-C) and A(N-IV)B(VC)], ET-1 had no effect on NHE3 activity. By contrast, when
the second intracellular loop of the ETB receptor was included
[A(N-III)B(IV-C)], ET-1 stimulation of NHE3 activity was
restored (⫹35%). These results suggest that in addition to the
COOH-terminal tail, a specific region including transmem-
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Fig. 1. Endothelin (ET)A and ETB receptor regulation of Na⫹/H⫹ exchanger 3
(NHE3) activity. Opossum kidney (OKP) cells were grown, transfected, and
exposed to 10⫺8 M ET-1 or vehicle, and NHE3 activity was assayed as
described in METHODS. Left: untransfected cells, n ⫽ 8. Middle: ETB receptor
sequence, n ⫽ 18. Right: ETA receptor sequence, n ⫽ 10. *P ⬍ 0.01.
NHE3 ACTIVATION BY ENDOTHELIN-1
brane helix III and/or the second intracellular loop of the ETB
receptor is required for ET-1 stimulation of NHE3 activity.
To confirm this, an additional series of chimeras were used
(Fig. 5). When the sequence extending from transmembrane
helices III to VI of the ETB receptor was replaced with the
corresponding ETA sequence [B(N-III)A(IV-VI)B(VII-C)],
ET-1 was without effect on NHE3 activity. When only the
region between transmembrane helices V and VI of the ETB
receptor was replaced with the corresponding ETA sequence
[B(N-IV)A(V-VI)B(VII-C)], ET-1 stimulation of NHE3 activity was restored (⫹36%). Together, these data confirm that the
domain containing the second intracellular loop and transmembrane helix IV of the ETB receptor is critical in mediating
ET-1-induced NHE3 activation.
To further confirm this conclusion, we used a chimera in
which both the second intracellular loop and COOH-terminal
tail were derived from the ETB receptor and all other intracellular sequences were from the ETA receptor [A(N-III)B(IVV)A(VI-VII)B(C)]. As shown in Fig. 5, in cells expressing this
chimera ET-1 increased NHE3 activity by 35%, confirming
that the second intracellular loop and COOH-terminal tail of
ETB are both required and sufficient for ET-1 stimulation of
NHE3 activity.
Amino acid residues in the ETB receptor second intracellular loop required for ET-1 stimulation of NHE3 activity. The
above studies all confirmed a key role for the domain including
the second intracellular loop and transmembrane helix IV in
NHE3 stimulation. We reasoned that the key domain was most
likely intracellular and further pursued the second intracellular
loop. The second intracellular loop is highly conserved with
only six amino acids that differ between the ETA and ETB
receptors (Fig. 6). The specific role of each of these amino
acids was analyzed by site-directed mutagenesis converting
each of the six ETB amino acids individually to their corresponding ETA receptor amino acid (Fig. 6 and Table 1). As
AJP-Renal Physiol • VOL
Fig. 5. Critical TMDs of the ETB receptor. Cells were grown, treated, and
assayed as in Fig. 1. B(N-III)A(IV-VI)B(VII-C), B(N-IV)A(V-VI)B(VII-C),
and A(N-III)B(IV-V)A(VI-VII)B(C) were generated by using restriction enzymes BssHI/ClaI, NcoI/ClaI, and BssHI/BglII/EcoRI, respectively (Fig. 2).
Blue, ETB receptor sequence. Red, ETA receptor sequence. ETB: n ⫽ 10;
B(N-III)A(IV-VI)B(VII-C): n ⫽ 7; B(N-IV)A(V-VI)B(VII-C): n ⫽ 6; A(NIII)B(IV-V)A(VI-VII)B(C): n ⫽ 5. *P ⬍ 0.01.
shown in Fig. 7, in cells expressing wild-type ETB receptor,
ET-1 increased NHE3 activity by 46% (P ⬍ 0.001). Individual
mutation of ETB residues I836 and W844 to the corresponding
ETA residues V835 and V843, respectively, did not prevent this
effect (ET-1 increased NHE3 activity 44 and 52%, respectively, both P ⬍ 0.001). By contrast, when ETB receptor
residues K837, V841, K843, and V847 were individually replaced
with the corresponding ETA receptor residues (Q836, I840, L842,
and I846, respectively), the ETB receptor lost its ability to
mediate ET-1 stimulation of NHE3 activity (Fig. 7). Thus ETB
Fig. 6. Schematic diagram of the ETA and ETB receptor second intracellular
loop sequences. The diagram of the receptor is as shown in Fig. 2. The dashed
lines below the TMD indicate the region shown in the gray box. Amino acid
residues that are the same in the ETA and ETB receptor are in black. Those that
are different between the receptors are noted in italic.
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Fig. 4. Role of ETB receptor intracellular loops. Cells were grown, treated, and
assayed as in Fig. 1. A(N-V)B(VI-C), A(N-IV)B(V-C), and A(N-III)B(IV-C)
were generated using restriction enzymes BglII, NcoI, and BssHI, respectively
(Fig. 2). Blue, ETB receptor sequence. Red, ETA receptor sequence. ETB: n ⫽
16; A(N-V)B(VI-C): n ⫽ 4; A(N-IV)B(V-C): n ⫽ 4; A(N-III)B(IV-C): n ⫽ 8.
*P ⬍ 0.01.
F735
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NHE3 ACTIVATION BY ENDOTHELIN-1
ETB receptor and those expressing key truncated, chimeric, or
mutated receptors.
DISCUSSION
receptor residues K837, V841, K843, and V847 play a critical role
in ET-1 stimulation of NHE3 activity, whereas I836 and W844
are not required.
The two pairs of K and V residues (K837 and V841; K843 and
847
V ) that were found to be required for ET-1 stimulation of
NHE3 activity are each separated by three amino acids. We
next tested the role of the six amino acid residues (G838, I839,
G840, W844, T845, and A846) lying between the two “K-V” pairs
(Fig. 6 and Table 1). W844 is one of the 6 amino acid residues
that differs between the ETB and ETA receptors. Mutation of
this residue (W844V) had already been shown to have no effect
on the ability of ET-1 to stimulate NHE3 activity (Fig. 7). The
other five potential X residues were replaced with either a
valine or alanine residue. As shown in Fig. 8, individually
mutating these residues had no effect on the ability of ET-1 to
stimulate NHE3 activity (% stimulation varied between 27 and
52%, P ⬍ 0.05 in all 6 experiments). However, removal of one
of the X residues, G838, prevented the ET-1 effect (Fig. 9).
Thus, while the specific amino acids separating the K and V
residues are not critical, the number of amino acids is important.
The two KXXXV sequences in the second intracellular loop
are separated by a proline that is conserved in ETB and ETA
receptors. Mutation of this proline residue (P842) to alanine also
prevented ET-1 from stimulating NHE3 activity (Fig. 9).
Taken together, these data suggest the presence of a consensus
sequence (KXXXVPKXXXV) in the ETB receptor second
intracellular loop that mediates ET-1 stimulation of NHE3
activity.
ET-1 binding to receptors. To address the possibility that the
varying effect of ET-1 on NHE3 activity in the above studies
was a consequence of a difference in receptor expression
and/or failure of ET-1 to bind to a mutated receptor, ligand
binding studies were performed in wild-type OKP cells and
OKP cells expressing intact, and key truncated, chimeric, or
mutated receptors in which ET-1 did not stimulate NHE3
activity (Table 2). As shown, specific ET-1 binding was not
statistically different between cells expressing the wild-type
AJP-Renal Physiol • VOL
Fig. 8. Role of conserved residues in the proposed consensus sequence. Cells
were grown, treated, and assayed as in Fig. 1. Five amino acid residues lying
between the 2 K-V pairs were individually mutated. Mutations were made as
indicated in Table 1. ETB: n ⫽ 8; G838V: n ⫽ 12; I839V: n ⫽ 8; G840A: n ⫽
7; T845A: n ⫽ 8; A846V: n ⫽ 13. *P ⬍ 0.02.
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Fig. 7. Role of amino acid residues in the second intracellular loop that differ
between the ETA and ETB receptors. Cells were grown, treated, and assayed as
in Fig. 1. Mutations were made as indicated in Table 1. ETB: n ⫽ 21; I836V:
n ⫽ 7; K837Q: n ⫽ 9; V841I: n ⫽ 9; K843L: n ⫽ 10; W844V: n ⫽ 11; V847I:
n ⫽ 8. *P ⬍ 0.01.
The ETs are a family of three conserved 21 amino acid
peptides that bind to and mediate their effects through two G
protein-coupled receptor subtypes, ETA and ETB (3, 13, 32).
Despite significant amino acid homology, the receptors can be
pharmacologically distinguished by their affinity for the three
ET isopeptides and their distinct physiological roles (22, 26,
36). The ETB receptor mediates release of relaxing factors
causing vasodilation and is involved in inflammatory pain,
whereas the ETA receptor mediates vasoconstriction, stimulates mitogenesis and matrix formation, is antiapoptotic, and is
involved in acute or neuropathic pain (26). Typically, the ETB
receptor activates G␣i, whereas the ETA receptor activates
G␣s, but this G protein coupling is not fixed in all cells (22,
36). In the kidney, while both ETA and ETB are present in renal
vessels and on tubular epithelial cells, ETB expression predominates in tubular epithelial cells (17, 31, 37).
We previously showed that ET-1 stimulates NHE3 activity
in cultured OKP cells, an effect that is mediated through the
ETB and not the ETA receptor (7, 19). ET-1/ETB stimulation of
NHE3 activity involves NHE3 phosphorylation and exocytic
insertion of NHE3 into the apical membrane (27, 28). As
shown in Fig. 1, ET-1/ETA signaling not only fails to stimulate
NHE3 activity but inhibits the transporter. The physiological
significance of NHE3 inhibition is unclear as ET-1 stimulates
the proximal tubule Na⫹/H⫹ antiporter, an effect mediated by
the ETB receptor (19, 20).
Because of these differing effects on NHE3, we initially
sought to determine signaling pathways that were differentially
activated by the ETB and ETA receptors in OKP cells. Using
inhibitors, we found that NHE3 activation by the ETB receptor
was mediated by increases in cell Ca2⫹ and activation of
Ca2⫹-calmodulin kinase and tyrosine kinases. On ET-1 addition, both receptors were found to increase cell Ca2⫹ with
patterns that were indistinguishable. In addition, activation of
NHE3 ACTIVATION BY ENDOTHELIN-1
both receptors resulted in tyrosine phosphorylation of proteins
with a pattern consistent with focal adhesion protein phosphorylation. Finally, although activation of ETA and ETB receptors
can have opposing effects on adenylyl cyclase in many cells, in
OKP cells both receptors inhibited adenylyl cyclase (7, 28).
These findings led us to hypothesize that an additional signaling pathway must be required for ETB receptor activation of
NHE3, perhaps a protein that binds selectively to the ETB
receptor. To address this, the present studies were designed to
define domains unique to the ETB receptor that were required
for NHE3 activation.
The ETA and ETB receptors exhibit 55% overall amino acid
homology (22, 36). Within the receptor domains, the least
homology is seen in the intracellular COOH-terminal domain
and the most homology is associated with the transmembrane
helices. In addition, intracellular loops, a likely site of protein
binding, are highly homologous. In the present studies, OKP
cells were transiently transfected with human ETA and ETB
receptor chimera as well as mutated ETB receptor cDNAs to
determine the domains and sequences responsible for ET-1/
ETB stimulation of NHE3. Our laboratory previously showed
that we achieve ⬃80% transfection efficiency with transient
transfection in OKP cells (14). In addition, we completely
inhibited a number of cellular processes in cells transiently
transfected with dominant-negative constructs, again suggesting high transfection efficiency (21, 39, 40).
The studies led to a number of conclusions. First, the
COOH-terminal tail of the ETA receptor is necessary and
sufficient for inhibition of the NHE3 activity by ET-1. Thus a
chimeric receptor that contains only the COOH-terminal tail of
ETA, with the remainder of the receptor derived from the ETB
receptor, inhibits NHE3. Similarly, an ETA receptor in which
the COOH-terminal domain has been replaced with the relevant ETB sequence loses the ability to inhibit NHE3 activity.
Second, the COOH-terminal tail of ETB is required for
stimulation of NHE3. Deletion of this sequence eliminates
regulation, and replacement with the corresponding ETA sequence leads to ET-1-induced inhibition of NHE3 activity.
However, unlike results with the ETA receptor, the COOHterminal tail is not sufficient for stimulation of NHE3. The
AJP-Renal Physiol • VOL
chimera containing the COOH-terminal ETB sequence with the
remainder of the protein derived from the ETA receptor fails to
regulate NHE3 activity. This suggests that in addition to the
COOH-terminal tail, at least one other sequence is required for
NHE3 activation by ET-1/ETB.
The role of the COOH-terminal tail of G protein-coupled
receptors has not been studied extensively. In the parathyroid
Ca2⫹-sensing receptor, individual point mutations of His880
and Phe882 in the COOH-terminal tail to alanine residues were
associated with decreased total inositol phosphate production
and retention of the receptors in the endoplasmic reticulum,
suggesting that the COOH-terminal tail is required for efficient
targeting of the receptor to the cell surface (6). In the present
studies, however, absence of the ETB COOH-terminal tail did
not decrease ET-1 binding, demonstrating that trafficking to
the plasma membrane had not been impaired.
In addition to a requirement for the COOH-terminal tail,
NHE3 activation by ET-1/ETB required a sequence within the
second intracellular loop. Comparing a series of chimeras,
results consistently demonstrated that the presence of sequences in the second intracellular loop and transmembrane
domain IV, between the BssHI and NcoI restriction sites, was
required for NHE3 activation. Mutational studies localized the
key sequences to the second intracellular loop and further
defined the specific sequence required: two essential lysine (K)
and two essential valine (V) resides that are spaced as follows:
KXXXVPKXXXV (Fig. 6). Both the spacing between the
lysine and valine residues and the proline (P) residue were
found to be essential to confer ET-1 stimulation of NHE3. The
presence of the repeat sequence (KXXXV) and the space
requirement for three residues between the K and V pairs
Table 2. ET-1 binding to receptors
Values are means ⫾ SE. Cells were grown and treated as in Fig. 1. Ligand
binding was assayed as described in METHODS. Wild-type opossum kidney
cells: n ⫽ 9; endothelin (ET)B cells: n ⫽ 6; ETA cells: n ⫽ 6; ETB minus C-tail;
n ⫽ 6; A(N-V)B(VI-C): n ⫽ 3; B(N-III)A(IV-VI)B(VII-C): n ⫽ 3; K837Q:
n ⫽ 3; V841I: n ⫽ 6; K843L: n ⫽ 6; V847I: n ⫽ 6; Delete G838: n ⫽ 3.
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Fig. 9. G838 deletion and P842A mutation. Cells were grown, treated, and
assayed as in Fig. 1. Mutations were made as indicated in Table 1. G838
deletion: n ⫽ 6; P842A: n ⫽ 9.
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NHE3 ACTIVATION BY ENDOTHELIN-1
AJP-Renal Physiol • VOL
this region matches more closely to the NH2 terminus (address
domain) of ET-1 compared with ET-3, and thus ETA binds
ET-1 with higher affinity than ET-3 (33). Subtle differences in
the ETB receptor allow the address recognition domain to
interact with a wider spectrum of ligand address domains,
making the ETB receptor more promiscuous (33). The COOHterminal portion of the ligand interacts with transmembrane
helices I, II, III, and VII of the ETA and ETB receptors to
transmit the ligand message (22). The present studies demonstrate that the amino acid sequence in the second intracellular
loop and first part of transmembrane helix IV transmit the
signal mediating ET-1 stimulation of NHE3 activity. The
combination of a required repeat sequence, required spacing
between essential amino acid residues, and a required proline
residue between the repeat sequences suggests that the protein
that binds the receptor might have symmetrical “arms” that
interact with the receptor. Such a protein could be either a
single protein or a homodimer.
In summary, the present studies identify the COOH-terminal
tail and a sequence within the second intracellular loop of the
ETB receptor that are necessary and sufficient to mediate ET-1
stimulation of NHE3 activity. The presence of a repeat sequence with specific space requirements and a required proline
residue separating the repeat sequences suggests that signal
transmission is mediated by the binding of a homodimeric
protein to the receptor.
ACKNOWLEDGMENTS
Technical assistance was provided by Kavita Mathi and Ebtesam AbdelSalam.
GRANTS
These studies were supported by National Institutes of Health Grant
DK-39298. M. Yanagisawa is an Investigator of the HHMI.
Present address of K. Laghmani: Institut National de la Santé et de la
Recherche Médicale, Institut Biomedicale des Cordeliers, Paris, France.
Present address of R. J. Alpern: Yale School of Medicine, New Haven, CT
06520.
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