Vol. March
183,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
16, 1992
CHARACTERIZATION TO THE
OF A POLYCLONAL ANGIOTENSIN II TYPE-l
781-788
ANTI-PEPTIDE ANTIBODY (AT,) RECEPTOR
Blanka Zelezna, Elaine M. Richards, Wei Tang, Di Lu, Cohn Stunners, and Mohan K. Raizada Department of Physiology, College of Medicine University of Florida, Gainesville, FL 32610
Received
January
31,
1992
A polyclonal antibody has been prepared against a synthetic peptide corresponding to amino acids 14-23 of the angiotensin II type-l (AT,) receptor. The antibody is of high titer and mono-specific. Western blot analysis of membranes from rat liver, kidney, and adrenal gland showed that the antibody specifically recognizes a protein band of MW 70,000 whose amounts are highest in the liver, followed by kidney and adrenals. In addition, a relatively less prominent band of MW 95,000 was also detected. The relative distribution of this protein correlates well with the values obtained for [“HI-DuP753 binding and AT, receptor mRNA. Q1992Rcademlc PreSS,1°C.
The renin angiotensin system (RAS) is vitally important in the control of fluid homeostasis, and blood pressure. In the periphery, the RAS modifies blood pressure by influencing smooth muscles, renal absorption of water and sodium, and the secretion of aldosterone. Centrally, the RAS influences blood pressure by regulating the release of ACTH and vasopressin, sympathetic outflow, and dipsogenic responses (for review see 1). While many of the physiological effects of this system are well studied in normal and pathological conditions, such as hypertension (2), the cellular and molecular mechanisms of these effects are not clearly understood. This is, in part, due to the fact that very little has been known about the regulation of angiotensin II (Ang II) receptors, and the mechanism by which Ang II exerts its actions. Conventional approaches to study the receptor, such as photo-affinity and chemical crosslinking have yielded limited information (3-7). Studies with cells in culture from both brain and peripheral target tissues have been informative, limited (2, 8-11).
but
Recently, two significant advances have been made in elucidating Ang II receptor function. 1) The development of nonpeptide Ang II receptor antagonists leading to pharmacological classification into type-l (AT,)
and type-2 (AT?) receptors
(12). Specific distribution of these receptor subtypes in various tissues has been reported (13-15). 2) The AT, receptor DNA has been cloned, and its cDNA anti protein
sequence reported (16,17).
These are important
breakthroughs towards
Vol.
183, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
understanding the cellular and molecular mechanisms of Ang II receptor regulation, especially as many of the physiological actions of Ang II including blood pressure (18), and dipsogenic (19) responses are mediated via AT, receptors.
In the present
study we have synthesized a 10 amino acid peptide corresponding to amino acids 1423 of the AT, receptor. This peptide was used to produce a polyclonal antiserum to AT, receptors, and our observations suggest that the antibody specifically recognizes AT,
receptors from various peripheral tissues.
METHODS AT, Recentor Per>tide Svnthesis and Antibodv Production: A ten amino acid peptide was synthesized corresponding to amino acids 14-23 of the published AT, receptor sequence (16). This peptide was synthesized using the multiple antigemc protein procedure of Tam (20), with the help of a model 430A peptide synthesizer. The peptide (0.5 mg) was emulsified in complete Freund’s adjuvant and injected into New Zealand white rabbits at 20 sites on the back subcutaneously. Rabbits were boosted with the same amount of the peptide and bled 7-10 days after the boost. Measurement of r3H1-DuP753 Binding: The nonpeptide Ang II receptor antagonist, DuP753, is specific for AT, receptors (12). [“HI-DuP753 has been used to quantitate AT, receptor binding in various peripheral tissues. Liver, kidney, and adrenal glands were removed and immediately homogenized in 10 volumes of Tris buffer (0.15 M NaCI, 5 mM EDTA, 0.1 mM PMSF, 50 mM Tris pH 7.4). The homogenate was centrifuged, the pellets were homogenized in Tris buffer, and suspended to a protein concentration of 2 mg/ml. Membranes (200 pg protein) were incubated with [3H]-DuP753 (5 nM) containing varying concentrations (1 nM 1 PM) of unlabelled DuP753 and 1 mg/ml bacitracin and 0.25 pg/ml leupeptin in a final volume of 400 ~1. After incubation for 30 min at room temperature, 1 ml ice-cold Tris buffer was added to the reaction mixtures and bound radioactivity was separated by centrifugation. The pellet was washed once with ice-cold Tris buffer. dissolved in 0.1 N NaOH and radioactivity counted. Scatchard analysis of the data was conducted with the use of the “Ligand” program. Western Blots: Liver. kidney, and adrenals from male Sprague Dawley rats (250400 g) were collected and immediately homogenized in PBS containing protease inhibitors (30 pg PMSF, 300 pg EDTA, and 0.5 pg leupeptin/ml). Homogenates were centrifuged at 20,OOOxg for 10 min, and pellets resuspended in PBS with protease inhibitors. Membranes were rinsed twice by centrifugation and finally suspended in 10 volumes of lysis buffer (0.5% SDS, 1.57~ D?T. 20% glycerol, and 0.05 mM Tris-HCl pH 6.8). Samples were incubated for 5 min in a boiling water bath, spun at 1OOOxgfor 10 min and 400 fig protein was electrophoresed in 4%/7.S% SDS-PAGE by the method of Laemmli (21). Separated proteins were electroblotted overnight onto a nitrocellulose membrane. After blocking and washing (22), the membranes were incubated for 4 hours at room temperature in wash buffer containing a 1:lOOO dilution of anti-AT, receptor peptide antiserum, and 0.4% bovine serum albumin (BSA) (22). The excess primary antibody was removed by washing. The membranes were treated with [““I-labeled protein A (100,000 DPM/ml) in 0.4% BSA for 5 hours at 4°C. Membranes were washed thoroughly, dried and subjected to autoradiography for 48 hours at -70°C as described previously (22). The singlePreparation of Single-Stranded r3’Pl-Labeled AT, Recentor Probe: stranded [“PI-labeled anti-sense AT, receptor probe was prepared as follows: Two oligonucleotide primers were syntheszzed, one complementary to bases 894-916 of the AT, receptor sequence, and another identical to bases 195-218 (16). These primers were used to synthesize 721bp of double-stranded cDNA for the AT, receptor by PCR. Five pg of total RNA prepared from rat liver was mixed with 50 PM of each dNTP, 20 units of RNAsin, 200 units of MoMuLV reverse transcriptase, and 10 782
Vol.
183,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
pmole of complementary primers in lx polymerase chain reaction (PCR) buffer. After incubation for 15 min at room temperature, the mixture was incubated at 4pC for 1 hour, heated at 95°C for 5 min followed by quick chilling on ice. Fifty pmole of each primers, 5 units of Taq polymerase were added and the volume brought to 100 ~1 with lx PCR buffer. After running for 40 cycles at 95°C for 2 min, 55°C for 1.5 min, 72°C for 1 min, and 72°C for 10 min, the mixture was extracted by chloroform and DNAse-free (10 fig) pancreatic RNAse was added, followed by incubation at room temperature for 1 hour, PCR products (10 ~1) were digested by EcoRI DNA restriction enzyme and PCR products were run on a 1.2% agarose gel in lx TAE buffer at 40 volts for 3 hours. A band of the expected 721 bases in the undigested sample, and a band of the expected 360 bases in the digested sample were found. This 721bp cDNA probe was identical to that reported previously (16). Further purification of the double-stranded PCR products was carried out by the LMP gel procedure (23). One ng of double-stranded PCR product was mixed with 50 PM dTTP, 50 PM dTTP, 50 PM dATP, 300 pmole of complementary primer, 5 units of Taq polymernse, and 150 DCi of v2P]-dCTP in a final volume of 100 ~1 lx PCR buffer. The solution was heated at 95°C for 10 min and cooled to room temperature slowly. After running for 60 cycles of 95°C for 3 min, 55°C for 3 min, and 7pC for 3 min, per cycle, DNA was purified by Push Column (Stratagene) and radioactivity was counted.
RESULTS A peptide was
synthesized
Figure
consisting
and used to generate
1 shows
a slot-blot
immunoreactivity 1A).
A
was
dilution
immunoreaction eliminated adrenal
of
was
a protein
band
in
MW did
the
of the antiserum.
with
not
exceed
and subjected specific
liver
>
of the
all
Figure
>
from
degradation
of
product serum
However,
the
70,000
adrenals.
failed to consistently occasionally
band,
recognize
a high-MW 783
band
An
additional
any protein of
-200,000
of this band
were variable A
membranes.
its
and
to determine
The density
This protein since
completely
that the antibody
70,000 band.
was observed.
in
This
kidney
analysis
concentrations
of the MW
MW
blot
2A reveals
to 95,000 MW was also seen in the adrenal to 53,000 MW
peptide
rat liver,
of 70,000.
whose
increase
experiments.
0.5 mM
to Western
receptor.
of 1:4000 - 1:lOOO (Fig.
further
with
to a MW
kidney
density
a band corresponding
Preimmune
for
proteins.
band corresponding
l/10
dilution
Membranes
95,000 was also seen in liver membranes
corresponding
2B).
chosen
to the AT,
A dose-dependent
an antiserum
B and C).
prepared
order;
antiserum
since preabsorption
(Fig.
recognized
recognized
a polyclonal
1:lOOO was
specific,
glands were
if the antiserum
analysis
observed
the reaction
was
of the sequence of amino acids 14-23 of the AT, receptor
faint
was
on Western was
but band
Additionally,
appeared
amount
of
seen
to be a variable. blot (Fig. in liver
Vol.
183, No.
2, 1992
BIOCHEMICAL
AND
A
BIOPHYSICAL
pmoles
pmoles
RESEARCH
B
COMMUNICATIONS
C
200 50 10 2 1:1000
1:2000
1:4000
1
2
1
2
FIGURE 1. Characterization of Antiserum to The AT, ReceDtor Peotide bv SlotBlot Analvsis: (A) Indicated amounts of AT, receptor peptide were applied onto the membranes in TBS buffer and incubated with three dilutions of the antiserum followed by incubation with [“‘I]-protein A, essentially as described in “Methods”, and elsewhere (22). (B and C) The antiserum (1:lOOO dilution) was preincubated in the absence (B) or presence (C) of AT, receptor peptide for 48 hours at 4°C in the presence of 0.4% BSA. The control and preabsorbed immune sera were then used to determine immunoreactivity as described under A. Lanes 1 and 2 represent buffer control and the peptide, respectively.
membranes with
both
preimmune
and immune sera.
Figure
2C shows that
preabsorption of the antiserum with 0.5 mM peptide eliminated the 70,000, the 95,000, and the 53,000 MW bands. These observations indicate that the major band recognized in peripheral tissues by the anti-AT,
receptor peptide antibody was a 70
KDa protein. Quantitation
of [3H]-DuP753 binding and AT, receptor mRNA was done to
determine if the amounts of the 70,000 and 95,000 MW measured on the Western blot were comparable with the levels of AT, receptor binding and its mRNA in these
95 KD
70 K.
UI 1
2
3
1
2
3
a
b
FIGURE 2. Western-Blot Analvsis of Membranes from Kidnev. Adrenals. and Liver: Rat kidney, adrenal, and liver (1, 2, and 3 respectively) membranes (A and B) were prepared and electrophoresed, and subjected to Western blotting, essentially as Serum. described under “Methods.” (A) I mmune serum and (B) Preimmune (C) Effect of preabsorbed immune serum: Liver membranes were electrophoresed and proteins transferred. The blot was incubated with preabsorbed immune serum (prepared essentially as described in the legend to Figure 1) (a), or immune serum (lanes b) as primary antibody followed by [““I]-protein A, as described in the “Methods.” 784
Vol.
183,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
20
15
. m v 5
.
. . V
kidney
> and
adrenals.
DISCUSSION In
this
polyclonal
study
we
antibody
receptor,
to a peptide
and suggest that the antibody
The peptide,
polyclonal since
antibody
completely
to react with
peptides
shown).
synthesized
The antibody
peptide
shows
lowest
in
-95,000
the
a high titer
MW
adrenals. neither
was
preimmune
strongly
nor preabsorbed
suggest this:
95,000 MW
band
photoaffinity
labeling
consistent
with
molecular
weight
recognized
that the native AT,
AT,
mRNA
amount (iii)
The
sera detected
and B,,
receptor
antiserum receptor
(ii) failed
(data not
recognition
of the
detected
sequencing
of the peptide
these protein
(3-7).
The
MWs
by the antibody,
using the FASTA 786
the size of our of Ang
II and
receptor
are
the predicted to be -40,000.
(ii) The levels of
are in a similar i.e., liver
band
of these bands
is highly glycosylated. receptors
arguments
protein
In addition,
suggest the AT,
since
This raises
The following
crosslinking
of
in the
specific,
bands.
higher MW around
band
extent
appear to be highly
by the antibody.
AT,
prominent
and to a lesser
by covalent
receptor
of radioactivity
less
of a 60,000 - 78,000 MW
sequence
for
a band of -70,000
highest in the liver and
the AT, receptor.
II receptors
This indicates receptor
in liver
band of a relatively
from the cDNA
of the AT,
a significantly
has been demonstrated
the proteins
and (iii)
were
(i) The predominance
of Ang
the AT,
immunoreaction,
Its concentrations
represents
and a second, less prominent
any
recognizes
of these proteins
that this protein
for
that the antibody
addition,
recognit!on
14-23 of the AT,
and a dose-dependent
also seen predominantly
Antibody
the possibility
In
show
a
of 1:16,000.
and adrenals.
adrenals.
specific
from other regions
blot analysis showed
in liver, kidney,
to
of
receptors.
the immunoreaction,
can be seen to a dilution Western
failed
characterization
acids
for AT,
to be highly
serum
neutralized
to amino
is specific
seems
(i) preimmune
preabsorption
MW
corresponding
and
> kidney
and TFASTA
order
as the
> adrenals. programs
of
Vol.
183,
Pearson
No.
2, 1992
and Lipman
Research
Foundation’s
conducted. This indicated
The
BIOCHEMICAL
(24) and comparison database
only homology
that the peptide
AND
BIOPHYSICAL
for homology
RESEARCH
with
and the Genbank
Genetic
of the peptide
found was
sequence
is highly specific
COMMUNICATIONS
the National Sequence with
Biomedical
database
the AT,
for the AT,
were
receptor.
receptor.
ACKNOWLEDGMENTS We wish to thank Dr. William Dunn of the Department of Anatomy and Cell Biology for his comments and constant input. Blanka Zelezna is a Visiting Fellow from the Institute of Molecular Genetics, Czechoslovak Academy of Sciences, Prague, Czechoslovakia. The research was supported by NIH grant HL33610. We also wish to acknowledge the help and expertise of the Protein Chemistry and Computer Cores of the University of Florida Interdisciplinary Center for Biotechnology Research for Peptide Synthesis and Sequence Analysis.
REFERENCES 1.
2. ;’ 5: ‘7: 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Phillips, M.I., Richards, E.M. and Van Eckelen A. (1985) In Central and Peripheral Mechanism of Cardiovascular Regulation (A. Mapro, W. Osswald, D. Reis and P. Vanhoutte, Eds.), Vol. 109 pp. 385-441. Plenum Press, New York, NY. Sumners, C., Myers, L.M., Kalberg, C. and Raizada, M.K. (1990) Prog. Neurobiol. 34, 355-385. Capponi, A.M. and Catt, K.J. (19S8) J. Biol. Chem. 255, 120Sl-12086. Guillemett, G. and Esher, E. (1983) Biochemistry 23, 5591-5596. Carson, M.C., Harper, C.M., Baukal, A.J., Aguilera, G. and Catt, K.J. (1957) Mol. Endocrinol. l., 147-1.53. Paglin, S. and Jamteson, J.D. (1982) Proc. Natl. Acad. Sci. USA 79, 3739-3743. Siemens, I.R., Adler, H.J., Mah, S.J. and Fluharty, S.J. (1991) Mol. Pharmacol. 40, 717-726. Olson, J.A., Shiverick, K.T., Ogilvie, S., Buhi, W.C. and Raizada, M.K. (1991) Proc. Natl. Acad. Sci. USA 88, 1928-1932. Rydzewski, B., Zelezna, B., Tang, W., Sumners. C. and Raizada, M.K. (1002, In Press) Endocrinology. Catt, K.J., Carson, M.C. and Hausdorff, W.P. (1957) J. Steroid Biochem. 27, 915-927. Schelling, P.. Fischer, H. and Ganten, D. (1991) J. Hypertension 9. 3-15. Timmermans, P.B.M.W.M., Wong, P.C., Chiu, A.T. and Herhlin, W.F. (1992) TIPS Reviews 12, 5.5-61. Sumners, C., Tang, W., Zelezna, B. and Raizada. M.K. (1901) Proc. Natl. Acad. Sci. USA 88, 7567-7571. Millan, M.A., Jacobowitz, D.M., Aguilera, G. and Catt, K.J. (1991) Proc. Natl. Acad. Sci. USA 88, 11440-11444. Gasparo, M. de, Whitebread, S., Mele, M., Motani, A.S., Whitcombe, P.J., Ramjoue, H-P and Kamber, B. (1990) J. Cardiovasc. Pharmacol. 16, SZl-S3.5. Murphy, T.J., Alexander, R.W., Griendling, K.K., Runge, MS. and Beinestein, K.E. (1991) Nature 351, 233-236. Sasaki, K., Yamano, Y., Bardhan, S., Iwai, N., Murrv, J.J., Hasegawa, M., Matsuda, Y. and Inagami, T. (1991) Nature 351, 230-233. Koepke, J.P., Bovy, P.R., McMahon, E.G., Olins, CM., Reitz, D.B., Salles, K.S., Schun, J.R., Trapani, A.J. and Blame, E.D. (1990) Hypertension 15, 841-847. Wong, P.C., Hart, S.D., Zaspel, A.M., Chiu, A.T., Ardecky, R.J., Smith, R.D. and Timmermans, P.B.M.W.M. (1990) J. Pharmacol. Exp. Ther. 255, 584-592.
Vol.
183,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Tam, J.P. (1988) PNAS (USA) 85, 5409-5413. 227 Laemmli, U.K. (1970) Nature (Lond.) 227, 680-683. 22: Olson, J.A., Shiverick, K.T., Ogilvie, S., Buhi, W.C. and Raizada, 23. 24.
M.K. (1991) Endocrinology 129, 1066-1074. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning, A Laboratory Manual. Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor. Pearson, W.R. and Lipman, D.J. (1988) Proc. Natl. Acad. Sci. USA 85, 24442448.
788
183,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
16, 1992
CHARACTERIZATION TO THE
OF A POLYCLONAL ANGIOTENSIN II TYPE-l
781-788
ANTI-PEPTIDE ANTIBODY (AT,) RECEPTOR
Blanka Zelezna, Elaine M. Richards, Wei Tang, Di Lu, Cohn Stunners, and Mohan K. Raizada Department of Physiology, College of Medicine University of Florida, Gainesville, FL 32610
Received
January
31,
1992
A polyclonal antibody has been prepared against a synthetic peptide corresponding to amino acids 14-23 of the angiotensin II type-l (AT,) receptor. The antibody is of high titer and mono-specific. Western blot analysis of membranes from rat liver, kidney, and adrenal gland showed that the antibody specifically recognizes a protein band of MW 70,000 whose amounts are highest in the liver, followed by kidney and adrenals. In addition, a relatively less prominent band of MW 95,000 was also detected. The relative distribution of this protein correlates well with the values obtained for [“HI-DuP753 binding and AT, receptor mRNA. Q1992Rcademlc PreSS,1°C.
The renin angiotensin system (RAS) is vitally important in the control of fluid homeostasis, and blood pressure. In the periphery, the RAS modifies blood pressure by influencing smooth muscles, renal absorption of water and sodium, and the secretion of aldosterone. Centrally, the RAS influences blood pressure by regulating the release of ACTH and vasopressin, sympathetic outflow, and dipsogenic responses (for review see 1). While many of the physiological effects of this system are well studied in normal and pathological conditions, such as hypertension (2), the cellular and molecular mechanisms of these effects are not clearly understood. This is, in part, due to the fact that very little has been known about the regulation of angiotensin II (Ang II) receptors, and the mechanism by which Ang II exerts its actions. Conventional approaches to study the receptor, such as photo-affinity and chemical crosslinking have yielded limited information (3-7). Studies with cells in culture from both brain and peripheral target tissues have been informative, limited (2, 8-11).
but
Recently, two significant advances have been made in elucidating Ang II receptor function. 1) The development of nonpeptide Ang II receptor antagonists leading to pharmacological classification into type-l (AT,)
and type-2 (AT?) receptors
(12). Specific distribution of these receptor subtypes in various tissues has been reported (13-15). 2) The AT, receptor DNA has been cloned, and its cDNA anti protein
sequence reported (16,17).
These are important
breakthroughs towards
Vol.
183, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
understanding the cellular and molecular mechanisms of Ang II receptor regulation, especially as many of the physiological actions of Ang II including blood pressure (18), and dipsogenic (19) responses are mediated via AT, receptors.
In the present
study we have synthesized a 10 amino acid peptide corresponding to amino acids 1423 of the AT, receptor. This peptide was used to produce a polyclonal antiserum to AT, receptors, and our observations suggest that the antibody specifically recognizes AT,
receptors from various peripheral tissues.
METHODS AT, Recentor Per>tide Svnthesis and Antibodv Production: A ten amino acid peptide was synthesized corresponding to amino acids 14-23 of the published AT, receptor sequence (16). This peptide was synthesized using the multiple antigemc protein procedure of Tam (20), with the help of a model 430A peptide synthesizer. The peptide (0.5 mg) was emulsified in complete Freund’s adjuvant and injected into New Zealand white rabbits at 20 sites on the back subcutaneously. Rabbits were boosted with the same amount of the peptide and bled 7-10 days after the boost. Measurement of r3H1-DuP753 Binding: The nonpeptide Ang II receptor antagonist, DuP753, is specific for AT, receptors (12). [“HI-DuP753 has been used to quantitate AT, receptor binding in various peripheral tissues. Liver, kidney, and adrenal glands were removed and immediately homogenized in 10 volumes of Tris buffer (0.15 M NaCI, 5 mM EDTA, 0.1 mM PMSF, 50 mM Tris pH 7.4). The homogenate was centrifuged, the pellets were homogenized in Tris buffer, and suspended to a protein concentration of 2 mg/ml. Membranes (200 pg protein) were incubated with [3H]-DuP753 (5 nM) containing varying concentrations (1 nM 1 PM) of unlabelled DuP753 and 1 mg/ml bacitracin and 0.25 pg/ml leupeptin in a final volume of 400 ~1. After incubation for 30 min at room temperature, 1 ml ice-cold Tris buffer was added to the reaction mixtures and bound radioactivity was separated by centrifugation. The pellet was washed once with ice-cold Tris buffer. dissolved in 0.1 N NaOH and radioactivity counted. Scatchard analysis of the data was conducted with the use of the “Ligand” program. Western Blots: Liver. kidney, and adrenals from male Sprague Dawley rats (250400 g) were collected and immediately homogenized in PBS containing protease inhibitors (30 pg PMSF, 300 pg EDTA, and 0.5 pg leupeptin/ml). Homogenates were centrifuged at 20,OOOxg for 10 min, and pellets resuspended in PBS with protease inhibitors. Membranes were rinsed twice by centrifugation and finally suspended in 10 volumes of lysis buffer (0.5% SDS, 1.57~ D?T. 20% glycerol, and 0.05 mM Tris-HCl pH 6.8). Samples were incubated for 5 min in a boiling water bath, spun at 1OOOxgfor 10 min and 400 fig protein was electrophoresed in 4%/7.S% SDS-PAGE by the method of Laemmli (21). Separated proteins were electroblotted overnight onto a nitrocellulose membrane. After blocking and washing (22), the membranes were incubated for 4 hours at room temperature in wash buffer containing a 1:lOOO dilution of anti-AT, receptor peptide antiserum, and 0.4% bovine serum albumin (BSA) (22). The excess primary antibody was removed by washing. The membranes were treated with [““I-labeled protein A (100,000 DPM/ml) in 0.4% BSA for 5 hours at 4°C. Membranes were washed thoroughly, dried and subjected to autoradiography for 48 hours at -70°C as described previously (22). The singlePreparation of Single-Stranded r3’Pl-Labeled AT, Recentor Probe: stranded [“PI-labeled anti-sense AT, receptor probe was prepared as follows: Two oligonucleotide primers were syntheszzed, one complementary to bases 894-916 of the AT, receptor sequence, and another identical to bases 195-218 (16). These primers were used to synthesize 721bp of double-stranded cDNA for the AT, receptor by PCR. Five pg of total RNA prepared from rat liver was mixed with 50 PM of each dNTP, 20 units of RNAsin, 200 units of MoMuLV reverse transcriptase, and 10 782
Vol.
183,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
pmole of complementary primers in lx polymerase chain reaction (PCR) buffer. After incubation for 15 min at room temperature, the mixture was incubated at 4pC for 1 hour, heated at 95°C for 5 min followed by quick chilling on ice. Fifty pmole of each primers, 5 units of Taq polymerase were added and the volume brought to 100 ~1 with lx PCR buffer. After running for 40 cycles at 95°C for 2 min, 55°C for 1.5 min, 72°C for 1 min, and 72°C for 10 min, the mixture was extracted by chloroform and DNAse-free (10 fig) pancreatic RNAse was added, followed by incubation at room temperature for 1 hour, PCR products (10 ~1) were digested by EcoRI DNA restriction enzyme and PCR products were run on a 1.2% agarose gel in lx TAE buffer at 40 volts for 3 hours. A band of the expected 721 bases in the undigested sample, and a band of the expected 360 bases in the digested sample were found. This 721bp cDNA probe was identical to that reported previously (16). Further purification of the double-stranded PCR products was carried out by the LMP gel procedure (23). One ng of double-stranded PCR product was mixed with 50 PM dTTP, 50 PM dTTP, 50 PM dATP, 300 pmole of complementary primer, 5 units of Taq polymernse, and 150 DCi of v2P]-dCTP in a final volume of 100 ~1 lx PCR buffer. The solution was heated at 95°C for 10 min and cooled to room temperature slowly. After running for 60 cycles of 95°C for 3 min, 55°C for 3 min, and 7pC for 3 min, per cycle, DNA was purified by Push Column (Stratagene) and radioactivity was counted.
RESULTS A peptide was
synthesized
Figure
consisting
and used to generate
1 shows
a slot-blot
immunoreactivity 1A).
A
was
dilution
immunoreaction eliminated adrenal
of
was
a protein
band
in
MW did
the
of the antiserum.
with
not
exceed
and subjected specific
liver
>
of the
all
Figure
>
from
degradation
of
product serum
However,
the
70,000
adrenals.
failed to consistently occasionally
band,
recognize
a high-MW 783
band
An
additional
any protein of
-200,000
of this band
were variable A
membranes.
its
and
to determine
The density
This protein since
completely
that the antibody
70,000 band.
was observed.
in
This
kidney
analysis
concentrations
of the MW
MW
blot
2A reveals
to 95,000 MW was also seen in the adrenal to 53,000 MW
peptide
rat liver,
of 70,000.
whose
increase
experiments.
0.5 mM
to Western
receptor.
of 1:4000 - 1:lOOO (Fig.
further
with
to a MW
kidney
density
a band corresponding
Preimmune
for
proteins.
band corresponding
l/10
dilution
Membranes
95,000 was also seen in liver membranes
corresponding
2B).
chosen
to the AT,
A dose-dependent
an antiserum
B and C).
prepared
order;
antiserum
since preabsorption
(Fig.
recognized
recognized
a polyclonal
1:lOOO was
specific,
glands were
if the antiserum
analysis
observed
the reaction
was
of the sequence of amino acids 14-23 of the AT, receptor
faint
was
on Western was
but band
Additionally,
appeared
amount
of
seen
to be a variable. blot (Fig. in liver
Vol.
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2, 1992
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A
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pmoles
pmoles
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B
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C
200 50 10 2 1:1000
1:2000
1:4000
1
2
1
2
FIGURE 1. Characterization of Antiserum to The AT, ReceDtor Peotide bv SlotBlot Analvsis: (A) Indicated amounts of AT, receptor peptide were applied onto the membranes in TBS buffer and incubated with three dilutions of the antiserum followed by incubation with [“‘I]-protein A, essentially as described in “Methods”, and elsewhere (22). (B and C) The antiserum (1:lOOO dilution) was preincubated in the absence (B) or presence (C) of AT, receptor peptide for 48 hours at 4°C in the presence of 0.4% BSA. The control and preabsorbed immune sera were then used to determine immunoreactivity as described under A. Lanes 1 and 2 represent buffer control and the peptide, respectively.
membranes with
both
preimmune
and immune sera.
Figure
2C shows that
preabsorption of the antiserum with 0.5 mM peptide eliminated the 70,000, the 95,000, and the 53,000 MW bands. These observations indicate that the major band recognized in peripheral tissues by the anti-AT,
receptor peptide antibody was a 70
KDa protein. Quantitation
of [3H]-DuP753 binding and AT, receptor mRNA was done to
determine if the amounts of the 70,000 and 95,000 MW measured on the Western blot were comparable with the levels of AT, receptor binding and its mRNA in these
95 KD
70 K.
UI 1
2
3
1
2
3
a
b
FIGURE 2. Western-Blot Analvsis of Membranes from Kidnev. Adrenals. and Liver: Rat kidney, adrenal, and liver (1, 2, and 3 respectively) membranes (A and B) were prepared and electrophoresed, and subjected to Western blotting, essentially as Serum. described under “Methods.” (A) I mmune serum and (B) Preimmune (C) Effect of preabsorbed immune serum: Liver membranes were electrophoresed and proteins transferred. The blot was incubated with preabsorbed immune serum (prepared essentially as described in the legend to Figure 1) (a), or immune serum (lanes b) as primary antibody followed by [““I]-protein A, as described in the “Methods.” 784
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20
15
. m v 5
.
. . V
kidney
> and
adrenals.
DISCUSSION In
this
polyclonal
study
we
antibody
receptor,
to a peptide
and suggest that the antibody
The peptide,
polyclonal since
antibody
completely
to react with
peptides
shown).
synthesized
The antibody
peptide
shows
lowest
in
-95,000
the
a high titer
MW
adrenals. neither
was
preimmune
strongly
nor preabsorbed
suggest this:
95,000 MW
band
photoaffinity
labeling
consistent
with
molecular
weight
recognized
that the native AT,
AT,
mRNA
amount (iii)
The
sera detected
and B,,
receptor
antiserum receptor
(ii) failed
(data not
recognition
of the
detected
sequencing
of the peptide
these protein
(3-7).
The
MWs
by the antibody,
using the FASTA 786
the size of our of Ang
II and
receptor
are
the predicted to be -40,000.
(ii) The levels of
are in a similar i.e., liver
band
of these bands
is highly glycosylated. receptors
arguments
protein
In addition,
suggest the AT,
since
This raises
The following
crosslinking
of
in the
specific,
bands.
higher MW around
band
extent
appear to be highly
by the antibody.
AT,
prominent
and to a lesser
by covalent
receptor
of radioactivity
less
of a 60,000 - 78,000 MW
sequence
for
a band of -70,000
highest in the liver and
the AT, receptor.
II receptors
This indicates receptor
in liver
band of a relatively
from the cDNA
of the AT,
a significantly
has been demonstrated
the proteins
and (iii)
were
(i) The predominance
of Ang
the AT,
immunoreaction,
Its concentrations
represents
and a second, less prominent
any
recognizes
of these proteins
that this protein
for
that the antibody
addition,
recognit!on
14-23 of the AT,
and a dose-dependent
also seen predominantly
Antibody
the possibility
In
show
a
of 1:16,000.
and adrenals.
adrenals.
specific
from other regions
blot analysis showed
in liver, kidney,
to
of
receptors.
the immunoreaction,
can be seen to a dilution Western
failed
characterization
acids
for AT,
to be highly
serum
neutralized
to amino
is specific
seems
(i) preimmune
preabsorption
MW
corresponding
and
> kidney
and TFASTA
order
as the
> adrenals. programs
of
Vol.
183,
Pearson
No.
2, 1992
and Lipman
Research
Foundation’s
conducted. This indicated
The
BIOCHEMICAL
(24) and comparison database
only homology
that the peptide
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for homology
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and the Genbank
Genetic
of the peptide
found was
sequence
is highly specific
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Biomedical
database
the AT,
for the AT,
were
receptor.
receptor.
ACKNOWLEDGMENTS We wish to thank Dr. William Dunn of the Department of Anatomy and Cell Biology for his comments and constant input. Blanka Zelezna is a Visiting Fellow from the Institute of Molecular Genetics, Czechoslovak Academy of Sciences, Prague, Czechoslovakia. The research was supported by NIH grant HL33610. We also wish to acknowledge the help and expertise of the Protein Chemistry and Computer Cores of the University of Florida Interdisciplinary Center for Biotechnology Research for Peptide Synthesis and Sequence Analysis.
REFERENCES 1.
2. ;’ 5: ‘7: 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Phillips, M.I., Richards, E.M. and Van Eckelen A. (1985) In Central and Peripheral Mechanism of Cardiovascular Regulation (A. Mapro, W. Osswald, D. Reis and P. Vanhoutte, Eds.), Vol. 109 pp. 385-441. Plenum Press, New York, NY. Sumners, C., Myers, L.M., Kalberg, C. and Raizada, M.K. (1990) Prog. Neurobiol. 34, 355-385. Capponi, A.M. and Catt, K.J. (19S8) J. Biol. Chem. 255, 120Sl-12086. Guillemett, G. and Esher, E. (1983) Biochemistry 23, 5591-5596. Carson, M.C., Harper, C.M., Baukal, A.J., Aguilera, G. and Catt, K.J. (1957) Mol. Endocrinol. l., 147-1.53. Paglin, S. and Jamteson, J.D. (1982) Proc. Natl. Acad. Sci. USA 79, 3739-3743. Siemens, I.R., Adler, H.J., Mah, S.J. and Fluharty, S.J. (1991) Mol. Pharmacol. 40, 717-726. Olson, J.A., Shiverick, K.T., Ogilvie, S., Buhi, W.C. and Raizada, M.K. (1991) Proc. Natl. Acad. Sci. USA 88, 1928-1932. Rydzewski, B., Zelezna, B., Tang, W., Sumners. C. and Raizada, M.K. (1002, In Press) Endocrinology. Catt, K.J., Carson, M.C. and Hausdorff, W.P. (1957) J. Steroid Biochem. 27, 915-927. Schelling, P.. Fischer, H. and Ganten, D. (1991) J. Hypertension 9. 3-15. Timmermans, P.B.M.W.M., Wong, P.C., Chiu, A.T. and Herhlin, W.F. (1992) TIPS Reviews 12, 5.5-61. Sumners, C., Tang, W., Zelezna, B. and Raizada. M.K. (1901) Proc. Natl. Acad. Sci. USA 88, 7567-7571. Millan, M.A., Jacobowitz, D.M., Aguilera, G. and Catt, K.J. (1991) Proc. Natl. Acad. Sci. USA 88, 11440-11444. Gasparo, M. de, Whitebread, S., Mele, M., Motani, A.S., Whitcombe, P.J., Ramjoue, H-P and Kamber, B. (1990) J. Cardiovasc. Pharmacol. 16, SZl-S3.5. Murphy, T.J., Alexander, R.W., Griendling, K.K., Runge, MS. and Beinestein, K.E. (1991) Nature 351, 233-236. Sasaki, K., Yamano, Y., Bardhan, S., Iwai, N., Murrv, J.J., Hasegawa, M., Matsuda, Y. and Inagami, T. (1991) Nature 351, 230-233. Koepke, J.P., Bovy, P.R., McMahon, E.G., Olins, CM., Reitz, D.B., Salles, K.S., Schun, J.R., Trapani, A.J. and Blame, E.D. (1990) Hypertension 15, 841-847. Wong, P.C., Hart, S.D., Zaspel, A.M., Chiu, A.T., Ardecky, R.J., Smith, R.D. and Timmermans, P.B.M.W.M. (1990) J. Pharmacol. Exp. Ther. 255, 584-592.
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Tam, J.P. (1988) PNAS (USA) 85, 5409-5413. 227 Laemmli, U.K. (1970) Nature (Lond.) 227, 680-683. 22: Olson, J.A., Shiverick, K.T., Ogilvie, S., Buhi, W.C. and Raizada, 23. 24.
M.K. (1991) Endocrinology 129, 1066-1074. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning, A Laboratory Manual. Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor. Pearson, W.R. and Lipman, D.J. (1988) Proc. Natl. Acad. Sci. USA 85, 24442448.
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