Vol.
172,
October
No.
BIOCHEMICAL
2, 1990
30,
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
1990
ISOLATION
OF ANTI-ENDOTHELIN
MONOCLONAL
ANTIBODIES
503-510
RECEPTOR
FOR USE IN RECEPTOR
CHARACTERIZATION Motohiro Kondoh, Hitoshi Miyazaki, Hirotoshi Watanabe, Takeshi Shibata, Masashi Yanagisawa#, Tomoh Masaki#, and Kazuo Murakami Institute of Applied Biochemistry,
Gene Experiment Center, and
#Basic Medical Sciences, University of Tsukuba, Ibaraki 305, Japan Received
August
22,
1990
ABSTRACT: Monoclonal antibodies reactive with endothelin (ET) receptors have been prepared by immunization of mice with rat lung membranes. Of four clones isolated, three clones preferentially recognized 32,000-dalton ET receptor and the other has a higher affinity for the 45,000-dalton receptor. The binding of 1251-ET-1 to detergent-solubilized ET receptors which were adsorbed to the antibodies was displaced by increasing concentrations of unlabeled ET isopeptides. These results demonstrate that the four clones specific for the receptor have the potential to be a useful tool in the characterization of ET receptors. 0 1990m2adernrc Press,hc.
Endothelin
(ET),
a novel
physiological
functions
and vascular
smooth
originally cells,
isolated
muscle from
(6,
7).
have demonstrated in
as well
several
species
(l-5).
media
the existence and
tissues
has a variety
binding
of multiple (6,
pressure
In addition
to ET-l,
aortic
isopeptides, and
8-16).
of
of blood
of porcine
that two other Ligand
peptide,
as the increase
constriction
culture
it has been found
are present
vasoactive
endothelial
ET-2
and ET-3,
pharmacological
studies
types These
of ET receptors findings
raise
0006.291W90 503
$1.50
Vol.
172,
No.
2, 1990
questions
BIOCHEMICAL
concerning
receptor
subtypes,
One
the
of
the and
powerful We with
for
the
RESEARCH
among
ET-induced
of each type
for each ET receptor
BIOPHYSICAL
relationships many
keypoints
characterization
AND
ET
isopeptides,
physiological
elucidation
of
of the receptor.
subtype
COMMUNICATIONS
are therefore
these
functions. issues
Antibodies regarded
the
is the specific
as potentially
tools. report
here
ET receptors
the isolation using
distinct
types
present
in the rat lung.
different
of ET
affinities
for
of monoclonal
antibodies
membranes
as the antigen;
rat lung
receptors
of 45,000
In result, these
two
and
monoclonal ET
receptor
32,000 antibodies subtypes
reactive
daltons
two are
exhibiting have
been
obtained.
MATERIALS
AND
METHODS
Materials: r 251-ET- 1 was obtained from Amersham International plc. (Bucks, U. K.). ET-l, -3, and sarafotoxin S6b were purchased Sp2/0-Ag 14 from the Peptide Institute, Inc. (Osaka, Japan). myeloma cell was from Flow Laboratories. Protein A-Sepharose CL-4B was from Pharmacia. IgGsorb, an IgG absorbent, was obtained from the Enzyme Center. Binding buffer: 10 mM Tris-HCl (7.4) containing 10 mM MgCl,, 0.5 mM PMSF, 2 mM EDTA, 20 uglml leupeptin, and 10 ug/ml antipain. PBS (phosphate-buffered saline): 8.1 mM Na2HP0,, 1.5 mM KH,PO, containing 137 mM NaC1 and 2.7 mM KCI. BALB/c mice were immunized with Immunization and fusion: rat lung membranes prepared as previously described (10); the was approximately 4 pmol/mg concentration of ET receptors Mice were immunized intraperitoneally at membrane protein. intervals of two weeks with a series of four injections of the The antigen was membranes (1 mg of protein for each injection). mixed with complete Freund’s adjuvant for the primary immunization, incomplete adjuvant for the next two injections, and Three days after the last injection, in PBS for the final injection. spleens were harvested and fused with Sp2/0-Ag14 myeloma cells using 50% polyethylene glycol 1500. Screening solubilized
for from
ET receptors monoclonal antibodies: rat lung membranes (1 mg protein/ml) with 504
were 0.3%
Vol.
172,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
digitonin; the protein concentration of solubilized fractions -300 pg/ml. Screening of hybridomas for anti-ET receptors carried out as follows. 1251-ET-l (-20,000 cpm/lO ~1) incubated with 100 ul of rat lung solubilized fractions, diluted
was was was I:9
with binding buffer, at 25°C for 2 hr to form ‘251-ET-l-ET receptor complexes, added to a mixture of hybridoma supernatants (100 ~1) and the IgGsorb diluted with PBS (100 ul), and rotated at 4OC overnight. The resultant mixtures were filtrated through Whatman No. 2 filters and the filters were counted in a gamma counter after washing with PBS containing 0.1% Tween 20. Filters with high counts indicate the existence of anti-ET receptor monoclonal antibodies. Positive hybridomas were subcloned at a limiting dilution at least twice. Subtype analysis: Subtypes of monoclonal antibodies produced by hybridoma cells were determined using a MOUSE TYPER kit from Bio-Rad according to the instructions provided by the manufacturer except that microtiter plates were precoated with anti-receptor antibodies purified by affinity chromatography on Protein A-Sepharose and alkaline phosphatase was used instead of horse radish peroxidase. Affinity labeling immunoprecipitation: Rat lung and solubilized ET receptors were prepared by the method of (17) except that NaCl was omitted, and the solubilized fractions were diluted with a lo-fold volume of 50 mM sodium phosphate (7.4) containing 150 mM NaCl, 5 mM MgCl,, 0.5 mM PMSF, 20 ug/ml leupeptin, and 10 ug/ml antipain, then affinity-labeled with 1251ET-l using the cross-linking reagent disuccinimidyl tartarate (DST) as described (8). The complexes of ‘251-ET-1 and the receptors were immunoprecipitated with 20 ug of each monoclonal antibody followed by the addition of 2 mg of Protein A-Sepharose. The resulting pellets were heated at 100°C for 3 min in SDS-gel sample buffer in the presence of the reducing reagent 2-mercaptoethanol and subjected to SDS-PAGE (12% acrylamide) followed by autoradiography. Competitive displacement of the solubilized receptors bound to the monoclonal antibodies: Digitonin-solubilized fractions (100 ul), diluted 1:9 with binding buffer, were incubated at 25OC for 2 hr with 50 pM *251-ET-1 in the presence or absence of increasing concentrations of unlabeled ET-l, -3, or sarafotoxin S6b (10 pM-100 nM), and immunoprecipitated with 20 yg of the antibodies. Other procedures were essentially the same as those for anti-ET receptor antibody screening except that Protein ASepharose was used instead of IgGsorb.
Vol.
172,
No.
2,
1990
BIOCHEMICAL
AND
RESULTS Four
hybridomas
receptors
AND
rat
lung
E7,
and A2,
GlO,
subclasses
of these monoclonal
and
G9,
G9,
from
designated
GlO
and
immunoprecipitation and
ET
these
clones
particular,
the
l-labeled
receptors
for the receptors.
immunoprecipitate the antibodies
and
reacted
with
4,000
by
antigen
and
The
In contrast,
and
1 shows
Fig.
of complexes digitonin
the
of 1251-ET- 1 from
rat
lung
immunoprecipitated
by
manner.
recovered
In in
that control
itself
G9
has
mouse
the IgG2,
highest did not
to the complexes, which
1.0
15
20
antibody (pg)
of 1251-ET-1 and ET receptor rat Iung membranes by various amounts of the monoclonal antibodies GlO, G9, E7. and A2. 125IET-l-labeled ET receptors were incubated with mixtures of increasing amounts of the monoclonal antibodies GlO ( o ), G9 ( l ), E7 ( X ), A2 ( n ), and control mouse IgG,, ( A) and Protein AOther Sepharose (2 mg) at 4OC overnight with constant mixing. experimental conditions are exactly the same as those for a experiment described under competitive displacement MATERIALS AND METHODS. Fig.
1.
complexes
Immunoprecipitation solubilized from
506
all
was not bound
/&;. / ? 0.5
the
value of 50% of the 1251-ET-
In addition
ET receptor
classes
A2.
receptors
revealing
the complexes.
the
a dose-dependent
a maximum
added,
ET
for the clones
of the complexes in
with
were IgG2,
0.3%
t 25 I-ET- 1 -labeled
by G9 reached
affinity
E7
with
increased
precipitate
as
respectively.
antibodies for
amount
reactive
membranes
solubilized
The
four
IgG,
COMMUNICATIf
approximately
by these antibodies
receptors
membranes.
antibodies
isolated
with
RESEARCH
DISCUSSION
producing
were
immunization
BIOPHYSICAL
Vol.
172,
No.
2,
BIOCHEMICAL
1990
to the ligand clones
affinity with
for
ET-3
in
solubilized reagent
the
DST
the
rat
in Fig.
35,000
daltons
directly precipitation
receptors was
2.
that all four
of the two different
types of ET
Two
A
-2 have
than
for
against
and
major
the
when
the
TVo ET of
using
the
resulting
on B).
The
cross-linking
the
were
The result daltons
G9,
were
immunoE7,
35,000-dalton
and
A2 band
0
L
u
t
92.5K-
Fig. 2. Immunoprecipitation of affinity-labeled ET receptors with 1251-ET-1 by monoclonal antibodies GlO, G9, E7, A2, and control mouse IgG,,. Lanes A and B indicate autoradiograms following SDS-PAGE of affinity-labeled ET receptors with 1251-ET-1 without immunoprecipitation. To detect specific bands, unlabeled ET-l (100 nM) was added in lane B. Lanes C-G reveal autoradiograms of immunoprecipitated affinity-labeled ET receptors with 1251-ET1. Other procedures are detailed under MATERIALS AND METHODS. 507
is and
receptors without
clones
receptor
cross-linked
of 48,000
SDS-PAGE
the
precipitates
affinity-labeled
immunoprecipitated
A
the
bands
type
To investigate
by autoradiography. specific
higher
the 32,000-dalton
(10).
1251-ET-1
followed
and
a slightly
immunoprecipitation
with
electrophoresed (lanes
and
antibodies
performed
appeared
preferentially
COMMUNICATIONS
indicate
interacts
lung,
to SDS-PAGE
shown
type
preferentially of
ET
subjected
ET-l
lung;
the 45,000-dalton
immunoreactivity subtypes
the existence
the rat
which
RESEARCH
for ET receptors.
We have identified in
BIOPHYSICAL
These results
(data not shown).
are specific
receptors
AND
Vol.
172,
No.
2,
although the
1990
BIOCHEMICAL
they interacted
clone
GlO
G).
Thus,
(lane
with
the
45,000-dalton estimated
bands
reacted
the two receptor
receptor
receptor; by subtracting
with
that is
RESEARCH
COMMUNICATIONS
D-F).
In contrast,
(lanes the
subtypes
suggesting
32,000-dalton
BIOPHYSICAL
both
exclusively
but are not identical, that
AND
48,000-dalton
are antigenically
they
are different
a proteolytic
the
molecular
mass of
3,000
daltons
of ‘251-ET-l
the
band.
were not observed
with
control
mouse
bands
which
migrated
a little
than
35,000-dalton
that
two
48,000-dalton detected
band
in every
produced
disappeared
disulfide
This
receptors,
(data
confirm
ET
out with
unlabeled
ET-l,
than
of
the
receptor
was
and DST
from
these two
(lane C).
bands
It should be
slower
than band
the were
to be artifacts
of IgG because IgG of -160,000
and -25,000
daltons
supported
on SDS-PAGE
specificity
displacement
receptors
rather
by cleavage
of
by the fact that
the
in the absence
of the
not shown). the
competitive
solubilized
IgG2,
the
is further
completely
reduction further
chains
to -50,000
bonds.
hand,
These two bands are thought
lane.
is reduced
the disulfide
To
faster
by heavy and light
daltons
bands
and
On the other
related
product
the size of the labeled
noted
band
adsorbed
of of
to the
concentration
antibodies
1251-ET-1
-3, and sarafotoxin
agonist
the
antibodies S6b.
for
binding were Fig.
to the carried
3 shows
(-IogM)
Fig. 3. Competitive displacement of 1251-ET-l binding to solubilized ET receptors bound to the antibody G9 with unlabeled Experimental conditions are detailed in ET isopeptides. MATERIALS AND METHODS. ET-l ( l ); ET-3 ( o ); sarafotoxin S6b (Xl.
508
ET
a
Vol.
172,
No.
2,
BIOCHEMICAL
1990
typical
displacement
pattern
against
the receptors.
AND
using
by
with
= 0.13 nM for ET-l.
nM
for
increasing
ET-3
clones
GlO,
under E7,
displacement confirm
as
all
four
the
dose
unlabeled
conditions.
exhibited (data
isolated
peptides
S6b, and 34
The other
a similar
not
three
dose-dependent
shown).
These
certainly
recognize
clones
titer
dependently
2.8 nM for sarafotoxin
also G9
COMMUNICATIONS
has the highest was
of
our experimental
pattern
that
binding
concentrations
and A2
RESEARCH
which
G9,
1251-ET-1
inhibited IC,,
BIOPHYSICAL
results ET
receptors. In summary, ET receptors the
we have isolated by immunization
immunogen.
recognized
Three
32,000-dalton
higher
affinity
should
therefore
for
the
provide
monoclonal
of mice
of the
four
antibodies
with
rat lung
clones
isolated
ET receptor.
In contrast,
45,000-dalton
receptor.
useful
tools
specific
for
membranes
as
preferentially the other
These
has a
antibodies
for the characterization
of ET
receptors. ACKNOWLEDGMENTS
We
would
like
comments
in
Foster
her help
for
supported
by
to
preparing
thank
Yasuo
monoclonal
in preparing
a Grant-in-Aid
Science and Culture
Dr.
Uchiyama
antibodies
the manuscript. from
the
and This
Ministry
of Japan, and a grant from Chichibu
of
for
helpful
Ms.
Norma
work
was
Education, Cement
Co.
REFERENCES 1)
Yanagisawa, M. Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K. and Masaki, T. (1988) Nature 332,
2)
3)
411-415.
de Nucci, G., Thomas, R., D’Orleans-Juste, P., Antunes, E., Walder. C., Warner, T. D., and Vane, J. R. (1988) Proc. Natl. Acad. Sci. USA 85, 9797-9800. Fukuda, Y., Hirata, Y., Yoshimi, H., Kojima, T., Kobayashi, Y., Yanagisawa, M., and Masaki. T. (1988) Biochem. Biophys. Res. Commun. 155, 167-172. 509
Vol.
172,
4) 5) 6)
7) 8)
9)
10)
1 1) 12)
13) 14) 1.5) 16) 17)
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Rakugi, H., Nakamura, M., Saito, H., Higaki, J., and Ogihara, T. (1988) Biochem. Biophys. Res. Commun. 155, 1244-1247. Morishita, R., Higaki, J., and Ogihara, T. (1989) Biochem. Biophys. Res. Commun. 160, 628-632. Inoue, A., Yanagisawa, M., Kimura, S., Kasuya, Y., Miyauchi, T., Goto, K. & Masaki, T. (1989) Proc. Natl. Acad. Sci. USA 86, 2863 -2867. Saida, K., Mitsui, Y. and Ishida, N. (1989) J. Biol. Chem. 264, 14613-14616. Miyazaki, H., Kondoh, M., Watanabe, H., Masuda, M., Murakami, K., Takahashi, M., Yanagisawa, M., Kimura, S., Goto, K., and Masaki, T. (1990) Eur. J. Biochem., 187, 125-129. Watanabe, H., Miyazaki, H., Kondoh, M., Masuda, Y., Kimura, S., Yanagisawa, M., Masaki, T. and Murakami, K. (1989) Biochem. Biophys. Res. Commun. 161, 1252-l 259. Masuda, M., Miyazaki, H., Kondoh, M., Watanabe, H., Yanagisawa, M., Masaki, T., and Murakami. K. (1989) FEBS Lett. 257, 208-210. Kloog, Y., Bousso-Mittler, D., Bdolah, A., and Sokolovsky, M. (1989) FEBS Lett. 253, 199-202. Sugiura, M., Snajdar, R. M., Schwartzberg, M., Badr, K. F., and Inagami, T. (1989) Biochem. Biophys. Res. Commun. 162, 1396-1401. Gomez-Sanchez, C. E., Cozza, E. N., Foecking, M. F., Chiou, S., and Ferris, M. W. (1990) Hypertension 15, 744-747. Warner, T. D., de Nucci, G., and Vane, J., R. (1989) Eur. J. Pharmacol. 159, 325-326. Emori, T., Hirata, Y., and Marumo, F. (1990) FEBS Lett. 263, 261-264. Chang, T. F. W., Ishida, N., Saida, K., Mitsui, Y., Okamoto, Y., and Nozawa, Y. (1989) FEBS Lett. 257, 351-353. Wada, K., Tabuchi, H., Ohba, R., Satoh, M., Tachibana, Y., Akiyama, N., Hiraoka, O., Asakura, A., Miyamota, C., and Furuichi, Y. (1990) Biochem. Biophys. Res. Commun. 167, 251-257.
510
172,
October
No.
BIOCHEMICAL
2, 1990
30,
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
1990
ISOLATION
OF ANTI-ENDOTHELIN
MONOCLONAL
ANTIBODIES
503-510
RECEPTOR
FOR USE IN RECEPTOR
CHARACTERIZATION Motohiro Kondoh, Hitoshi Miyazaki, Hirotoshi Watanabe, Takeshi Shibata, Masashi Yanagisawa#, Tomoh Masaki#, and Kazuo Murakami Institute of Applied Biochemistry,
Gene Experiment Center, and
#Basic Medical Sciences, University of Tsukuba, Ibaraki 305, Japan Received
August
22,
1990
ABSTRACT: Monoclonal antibodies reactive with endothelin (ET) receptors have been prepared by immunization of mice with rat lung membranes. Of four clones isolated, three clones preferentially recognized 32,000-dalton ET receptor and the other has a higher affinity for the 45,000-dalton receptor. The binding of 1251-ET-1 to detergent-solubilized ET receptors which were adsorbed to the antibodies was displaced by increasing concentrations of unlabeled ET isopeptides. These results demonstrate that the four clones specific for the receptor have the potential to be a useful tool in the characterization of ET receptors. 0 1990m2adernrc Press,hc.
Endothelin
(ET),
a novel
physiological
functions
and vascular
smooth
originally cells,
isolated
muscle from
(6,
7).
have demonstrated in
as well
several
species
(l-5).
media
the existence and
tissues
has a variety
binding
of multiple (6,
pressure
In addition
to ET-l,
aortic
isopeptides, and
8-16).
of
of blood
of porcine
that two other Ligand
peptide,
as the increase
constriction
culture
it has been found
are present
vasoactive
endothelial
ET-2
and ET-3,
pharmacological
studies
types These
of ET receptors findings
raise
0006.291W90 503
$1.50
Vol.
172,
No.
2, 1990
questions
BIOCHEMICAL
concerning
receptor
subtypes,
One
the
of
the and
powerful We with
for
the
RESEARCH
among
ET-induced
of each type
for each ET receptor
BIOPHYSICAL
relationships many
keypoints
characterization
AND
ET
isopeptides,
physiological
elucidation
of
of the receptor.
subtype
COMMUNICATIONS
are therefore
these
functions. issues
Antibodies regarded
the
is the specific
as potentially
tools. report
here
ET receptors
the isolation using
distinct
types
present
in the rat lung.
different
of ET
affinities
for
of monoclonal
antibodies
membranes
as the antigen;
rat lung
receptors
of 45,000
In result, these
two
and
monoclonal ET
receptor
32,000 antibodies subtypes
reactive
daltons
two are
exhibiting have
been
obtained.
MATERIALS
AND
METHODS
Materials: r 251-ET- 1 was obtained from Amersham International plc. (Bucks, U. K.). ET-l, -3, and sarafotoxin S6b were purchased Sp2/0-Ag 14 from the Peptide Institute, Inc. (Osaka, Japan). myeloma cell was from Flow Laboratories. Protein A-Sepharose CL-4B was from Pharmacia. IgGsorb, an IgG absorbent, was obtained from the Enzyme Center. Binding buffer: 10 mM Tris-HCl (7.4) containing 10 mM MgCl,, 0.5 mM PMSF, 2 mM EDTA, 20 uglml leupeptin, and 10 ug/ml antipain. PBS (phosphate-buffered saline): 8.1 mM Na2HP0,, 1.5 mM KH,PO, containing 137 mM NaC1 and 2.7 mM KCI. BALB/c mice were immunized with Immunization and fusion: rat lung membranes prepared as previously described (10); the was approximately 4 pmol/mg concentration of ET receptors Mice were immunized intraperitoneally at membrane protein. intervals of two weeks with a series of four injections of the The antigen was membranes (1 mg of protein for each injection). mixed with complete Freund’s adjuvant for the primary immunization, incomplete adjuvant for the next two injections, and Three days after the last injection, in PBS for the final injection. spleens were harvested and fused with Sp2/0-Ag14 myeloma cells using 50% polyethylene glycol 1500. Screening solubilized
for from
ET receptors monoclonal antibodies: rat lung membranes (1 mg protein/ml) with 504
were 0.3%
Vol.
172,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
digitonin; the protein concentration of solubilized fractions -300 pg/ml. Screening of hybridomas for anti-ET receptors carried out as follows. 1251-ET-l (-20,000 cpm/lO ~1) incubated with 100 ul of rat lung solubilized fractions, diluted
was was was I:9
with binding buffer, at 25°C for 2 hr to form ‘251-ET-l-ET receptor complexes, added to a mixture of hybridoma supernatants (100 ~1) and the IgGsorb diluted with PBS (100 ul), and rotated at 4OC overnight. The resultant mixtures were filtrated through Whatman No. 2 filters and the filters were counted in a gamma counter after washing with PBS containing 0.1% Tween 20. Filters with high counts indicate the existence of anti-ET receptor monoclonal antibodies. Positive hybridomas were subcloned at a limiting dilution at least twice. Subtype analysis: Subtypes of monoclonal antibodies produced by hybridoma cells were determined using a MOUSE TYPER kit from Bio-Rad according to the instructions provided by the manufacturer except that microtiter plates were precoated with anti-receptor antibodies purified by affinity chromatography on Protein A-Sepharose and alkaline phosphatase was used instead of horse radish peroxidase. Affinity labeling immunoprecipitation: Rat lung and solubilized ET receptors were prepared by the method of (17) except that NaCl was omitted, and the solubilized fractions were diluted with a lo-fold volume of 50 mM sodium phosphate (7.4) containing 150 mM NaCl, 5 mM MgCl,, 0.5 mM PMSF, 20 ug/ml leupeptin, and 10 ug/ml antipain, then affinity-labeled with 1251ET-l using the cross-linking reagent disuccinimidyl tartarate (DST) as described (8). The complexes of ‘251-ET-1 and the receptors were immunoprecipitated with 20 ug of each monoclonal antibody followed by the addition of 2 mg of Protein A-Sepharose. The resulting pellets were heated at 100°C for 3 min in SDS-gel sample buffer in the presence of the reducing reagent 2-mercaptoethanol and subjected to SDS-PAGE (12% acrylamide) followed by autoradiography. Competitive displacement of the solubilized receptors bound to the monoclonal antibodies: Digitonin-solubilized fractions (100 ul), diluted 1:9 with binding buffer, were incubated at 25OC for 2 hr with 50 pM *251-ET-1 in the presence or absence of increasing concentrations of unlabeled ET-l, -3, or sarafotoxin S6b (10 pM-100 nM), and immunoprecipitated with 20 yg of the antibodies. Other procedures were essentially the same as those for anti-ET receptor antibody screening except that Protein ASepharose was used instead of IgGsorb.
Vol.
172,
No.
2,
1990
BIOCHEMICAL
AND
RESULTS Four
hybridomas
receptors
AND
rat
lung
E7,
and A2,
GlO,
subclasses
of these monoclonal
and
G9,
G9,
from
designated
GlO
and
immunoprecipitation and
ET
these
clones
particular,
the
l-labeled
receptors
for the receptors.
immunoprecipitate the antibodies
and
reacted
with
4,000
by
antigen
and
The
In contrast,
and
1 shows
Fig.
of complexes digitonin
the
of 1251-ET- 1 from
rat
lung
immunoprecipitated
by
manner.
recovered
In in
that control
itself
G9
has
mouse
the IgG2,
highest did not
to the complexes, which
1.0
15
20
antibody (pg)
of 1251-ET-1 and ET receptor rat Iung membranes by various amounts of the monoclonal antibodies GlO, G9, E7. and A2. 125IET-l-labeled ET receptors were incubated with mixtures of increasing amounts of the monoclonal antibodies GlO ( o ), G9 ( l ), E7 ( X ), A2 ( n ), and control mouse IgG,, ( A) and Protein AOther Sepharose (2 mg) at 4OC overnight with constant mixing. experimental conditions are exactly the same as those for a experiment described under competitive displacement MATERIALS AND METHODS. Fig.
1.
complexes
Immunoprecipitation solubilized from
506
all
was not bound
/&;. / ? 0.5
the
value of 50% of the 1251-ET-
In addition
ET receptor
classes
A2.
receptors
revealing
the complexes.
the
a dose-dependent
a maximum
added,
ET
for the clones
of the complexes in
with
were IgG2,
0.3%
t 25 I-ET- 1 -labeled
by G9 reached
affinity
E7
with
increased
precipitate
as
respectively.
antibodies for
amount
reactive
membranes
solubilized
The
four
IgG,
COMMUNICATIf
approximately
by these antibodies
receptors
membranes.
antibodies
isolated
with
RESEARCH
DISCUSSION
producing
were
immunization
BIOPHYSICAL
Vol.
172,
No.
2,
BIOCHEMICAL
1990
to the ligand clones
affinity with
for
ET-3
in
solubilized reagent
the
DST
the
rat
in Fig.
35,000
daltons
directly precipitation
receptors was
2.
that all four
of the two different
types of ET
Two
A
-2 have
than
for
against
and
major
the
when
the
TVo ET of
using
the
resulting
on B).
The
cross-linking
the
were
The result daltons
G9,
were
immunoE7,
35,000-dalton
and
A2 band
0
L
u
t
92.5K-
Fig. 2. Immunoprecipitation of affinity-labeled ET receptors with 1251-ET-1 by monoclonal antibodies GlO, G9, E7, A2, and control mouse IgG,,. Lanes A and B indicate autoradiograms following SDS-PAGE of affinity-labeled ET receptors with 1251-ET-1 without immunoprecipitation. To detect specific bands, unlabeled ET-l (100 nM) was added in lane B. Lanes C-G reveal autoradiograms of immunoprecipitated affinity-labeled ET receptors with 1251-ET1. Other procedures are detailed under MATERIALS AND METHODS. 507
is and
receptors without
clones
receptor
cross-linked
of 48,000
SDS-PAGE
the
precipitates
affinity-labeled
immunoprecipitated
A
the
bands
type
To investigate
by autoradiography. specific
higher
the 32,000-dalton
(10).
1251-ET-1
followed
and
a slightly
immunoprecipitation
with
electrophoresed (lanes
and
antibodies
performed
appeared
preferentially
COMMUNICATIONS
indicate
interacts
lung,
to SDS-PAGE
shown
type
preferentially of
ET
subjected
ET-l
lung;
the 45,000-dalton
immunoreactivity subtypes
the existence
the rat
which
RESEARCH
for ET receptors.
We have identified in
BIOPHYSICAL
These results
(data not shown).
are specific
receptors
AND
Vol.
172,
No.
2,
although the
1990
BIOCHEMICAL
they interacted
clone
GlO
G).
Thus,
(lane
with
the
45,000-dalton estimated
bands
reacted
the two receptor
receptor
receptor; by subtracting
with
that is
RESEARCH
COMMUNICATIONS
D-F).
In contrast,
(lanes the
subtypes
suggesting
32,000-dalton
BIOPHYSICAL
both
exclusively
but are not identical, that
AND
48,000-dalton
are antigenically
they
are different
a proteolytic
the
molecular
mass of
3,000
daltons
of ‘251-ET-l
the
band.
were not observed
with
control
mouse
bands
which
migrated
a little
than
35,000-dalton
that
two
48,000-dalton detected
band
in every
produced
disappeared
disulfide
This
receptors,
(data
confirm
ET
out with
unlabeled
ET-l,
than
of
the
receptor
was
and DST
from
these two
(lane C).
bands
It should be
slower
than band
the were
to be artifacts
of IgG because IgG of -160,000
and -25,000
daltons
supported
on SDS-PAGE
specificity
displacement
receptors
rather
by cleavage
of
by the fact that
the
in the absence
of the
not shown). the
competitive
solubilized
IgG2,
the
is further
completely
reduction further
chains
to -50,000
bonds.
hand,
These two bands are thought
lane.
is reduced
the disulfide
To
faster
by heavy and light
daltons
bands
and
On the other
related
product
the size of the labeled
noted
band
adsorbed
of of
to the
concentration
antibodies
1251-ET-1
-3, and sarafotoxin
agonist
the
antibodies S6b.
for
binding were Fig.
to the carried
3 shows
(-IogM)
Fig. 3. Competitive displacement of 1251-ET-l binding to solubilized ET receptors bound to the antibody G9 with unlabeled Experimental conditions are detailed in ET isopeptides. MATERIALS AND METHODS. ET-l ( l ); ET-3 ( o ); sarafotoxin S6b (Xl.
508
ET
a
Vol.
172,
No.
2,
BIOCHEMICAL
1990
typical
displacement
pattern
against
the receptors.
AND
using
by
with
= 0.13 nM for ET-l.
nM
for
increasing
ET-3
clones
GlO,
under E7,
displacement confirm
as
all
four
the
dose
unlabeled
conditions.
exhibited (data
isolated
peptides
S6b, and 34
The other
a similar
not
three
dose-dependent
shown).
These
certainly
recognize
clones
titer
dependently
2.8 nM for sarafotoxin
also G9
COMMUNICATIONS
has the highest was
of
our experimental
pattern
that
binding
concentrations
and A2
RESEARCH
which
G9,
1251-ET-1
inhibited IC,,
BIOPHYSICAL
results ET
receptors. In summary, ET receptors the
we have isolated by immunization
immunogen.
recognized
Three
32,000-dalton
higher
affinity
should
therefore
for
the
provide
monoclonal
of mice
of the
four
antibodies
with
rat lung
clones
isolated
ET receptor.
In contrast,
45,000-dalton
receptor.
useful
tools
specific
for
membranes
as
preferentially the other
These
has a
antibodies
for the characterization
of ET
receptors. ACKNOWLEDGMENTS
We
would
like
comments
in
Foster
her help
for
supported
by
to
preparing
thank
Yasuo
monoclonal
in preparing
a Grant-in-Aid
Science and Culture
Dr.
Uchiyama
antibodies
the manuscript. from
the
and This
Ministry
of Japan, and a grant from Chichibu
of
for
helpful
Ms.
Norma
work
was
Education, Cement
Co.
REFERENCES 1)
Yanagisawa, M. Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K. and Masaki, T. (1988) Nature 332,
2)
3)
411-415.
de Nucci, G., Thomas, R., D’Orleans-Juste, P., Antunes, E., Walder. C., Warner, T. D., and Vane, J. R. (1988) Proc. Natl. Acad. Sci. USA 85, 9797-9800. Fukuda, Y., Hirata, Y., Yoshimi, H., Kojima, T., Kobayashi, Y., Yanagisawa, M., and Masaki. T. (1988) Biochem. Biophys. Res. Commun. 155, 167-172. 509
Vol.
172,
4) 5) 6)
7) 8)
9)
10)
1 1) 12)
13) 14) 1.5) 16) 17)
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Rakugi, H., Nakamura, M., Saito, H., Higaki, J., and Ogihara, T. (1988) Biochem. Biophys. Res. Commun. 155, 1244-1247. Morishita, R., Higaki, J., and Ogihara, T. (1989) Biochem. Biophys. Res. Commun. 160, 628-632. Inoue, A., Yanagisawa, M., Kimura, S., Kasuya, Y., Miyauchi, T., Goto, K. & Masaki, T. (1989) Proc. Natl. Acad. Sci. USA 86, 2863 -2867. Saida, K., Mitsui, Y. and Ishida, N. (1989) J. Biol. Chem. 264, 14613-14616. Miyazaki, H., Kondoh, M., Watanabe, H., Masuda, M., Murakami, K., Takahashi, M., Yanagisawa, M., Kimura, S., Goto, K., and Masaki, T. (1990) Eur. J. Biochem., 187, 125-129. Watanabe, H., Miyazaki, H., Kondoh, M., Masuda, Y., Kimura, S., Yanagisawa, M., Masaki, T. and Murakami, K. (1989) Biochem. Biophys. Res. Commun. 161, 1252-l 259. Masuda, M., Miyazaki, H., Kondoh, M., Watanabe, H., Yanagisawa, M., Masaki, T., and Murakami. K. (1989) FEBS Lett. 257, 208-210. Kloog, Y., Bousso-Mittler, D., Bdolah, A., and Sokolovsky, M. (1989) FEBS Lett. 253, 199-202. Sugiura, M., Snajdar, R. M., Schwartzberg, M., Badr, K. F., and Inagami, T. (1989) Biochem. Biophys. Res. Commun. 162, 1396-1401. Gomez-Sanchez, C. E., Cozza, E. N., Foecking, M. F., Chiou, S., and Ferris, M. W. (1990) Hypertension 15, 744-747. Warner, T. D., de Nucci, G., and Vane, J., R. (1989) Eur. J. Pharmacol. 159, 325-326. Emori, T., Hirata, Y., and Marumo, F. (1990) FEBS Lett. 263, 261-264. Chang, T. F. W., Ishida, N., Saida, K., Mitsui, Y., Okamoto, Y., and Nozawa, Y. (1989) FEBS Lett. 257, 351-353. Wada, K., Tabuchi, H., Ohba, R., Satoh, M., Tachibana, Y., Akiyama, N., Hiraoka, O., Asakura, A., Miyamota, C., and Furuichi, Y. (1990) Biochem. Biophys. Res. Commun. 167, 251-257.
510
