Cloning and Characterization of ATRAP, a Novel Protein That
Interacts with the Angiotensin II Type 1 Receptor*
Laurent
Daviet
,
Jukka Y. A.
Lehtonen,
Kouichi
Tamura,
Daniel P.
Griese,
Masatsugu
Horiuchi, and
Victor J.
Dzau§
From the Department of Medicine, Harvard Medical School, Brigham
and Women's Hospital, Boston, Massachusetts 02115
 |
ABSTRACT |
The carboxyl-terminal cytoplasmic domain of the
angiotensin II type 1 (AT1) receptor has recently
been shown to interact with several classes of cytoplasmic proteins
that regulate different aspects of AT1 receptor physiology.
Employing yeast two-hybrid screening of a mouse kidney cDNA library
with the carboxyl-terminal cytoplasmic domain of the murine
AT1a receptor as a bait, we have isolated a novel protein
with a predicted molecular mass of 18 kDa, which we have named ATRAP
(for AT1 receptor-associated protein). ATRAP interacts
specifically with the carboxyl-terminal domain of the AT1a
receptor but not with those of angiotensin II type 2 (AT2),
m3 muscarinic acetylcholine, bradykinin B2,
endothelin B, and
2-adrenergic receptors. The mRNA
of ATRAP was abundantly expressed in kidney, heart, and testis but was
poorly expressed in lung, liver, spleen, and brain. The
ATRAP-AT1a receptor association was confirmed by affinity
chromatography, by specific co-immunoprecipitation of the two proteins,
and by fluorescence microscopy, showing co-localization of these
proteins in intact cells. Overexpression of ATRAP in COS-7 cells caused
a marked inhibition of AT1a receptor-mediated activation of
phospholipase C without affecting m3 receptor-mediated activation. In conclusion, we have isolated a novel protein that interacts specifically with the carboxyl-terminal cytoplasmic domain of
the AT1a receptor and affects AT1a receptor signaling.
 |
INTRODUCTION |
G protein-coupled receptors
(GPCRs)1 interact with
different classes of intracellular proteins, including heterotrimeric G proteins, kinases, and arrestins (1-3). Although the intracellular third loop of a number of GPCRs is a key structural determinant of
coupling of the receptor to heterotrimeric G proteins (4-9), recent
studies have highlighted the functional importance of the carboxyl-terminal cytoplasmic domain in receptor signaling and desensitization (10-16).
Angiotensin II (AngII) is a key regulator of the cardiovascular system.
AngII exerts its biological effects through two major subtypes of high
affinity GPCRs designated AT1 and AT2
receptors. Recently, the carboxyl-terminal cytoplasmic domain of the
AT1 receptor has been reported to directly associate with
several downstream effectors (12-15). By means of mutational analysis, this domain has also been shown to contain discrete amino acid sequences that are required for receptor desensitization (17, 18) and
internalization (17, 19, 20). As for many GPCRs, the carboxyl-terminal
cytoplasmic domain of the AT1 receptor presumably interacts
with G protein-coupled receptor kinases and arrestins, causing
functional desensitization of the receptor (18, 21).
These observations raise the possibility that the carboxyl-terminal
cytoplasmic domain of the AT1 receptor interacts with additional cellular proteins that may play an important role in the
efficacy and/or specificity of receptor-G protein coupling. We have
investigated this possibility by searching for novel protein interactions with the carboxyl-terminal cytoplasmic domain of the
murine AT1a receptor. Using interaction cloning as well as biochemical and immunocytochemical techniques, we report here the
identification of a novel protein that specifically interacts with the
AT1 receptor tail. Functional studies suggest that this protein interaction may play a role in the regulation of
receptor-mediated signaling.
 |
EXPERIMENTAL PROCEDURES |
Plasmids--
The carboxyl-terminal cytoplasmic domain of the
murine AT1a receptor (AT1a C-ter; amino acids
297 to 359) was polymerase chain reaction-amplified and fused to the
Gal4 binding domain in the yeast shuttle vector pBD-Gal4 (Stratagene).
In a similar manner, 3 carboxyl-terminal deletions of the
AT1a receptor tail (AT1a C-ter
349,
339,
and
329) were generated by polymerase chain reaction with use of
reverse primers containing stop codons at the desired locations, and
the deleted cDNA were subcloned into pBD-Gal4. The
carboxyl-terminal cytoplasmic domains of human AT2 (amino
acids 314 to 363), human m3 muscarinic acetylcholine (amino acids 548 to 590), human bradykinin B2 (amino acids 299 to
364), endothelin B (amino acids 390 to 442), and
2-adrenergic (amino acids 328 to 413) receptors were
polymerase chain reaction-amplified and subcloned into pBD-Gal4. All
the constructs were verified by DNA sequencing using the Sanger dideoxy
termination method adapted to the Applied Biosystems model 373S
Automated DNA Sequencer.
Two-hybrid Screen--
A cDNA library from mouse kidney
poly(A)+ RNA was constructed in fusion with the Gal4
activation domain in pAD-Gal4 (Stratagene) with a cDNA synthesis
kit from Stratagene using XhoI-(dT)18 primer and
EcoRI adaptors. The yeast reporter strain YRG-2 (Stratagene) containing 2 Gal4-inducible reporter genes (HIS3 and LacZ) was sequentially co-transformed with the AT1a C-ter hybrid
expression plasmid and the mouse kidney cDNA library as described
previously (22). Double transformants were plated on yeast drop-out
media lacking tryptophan, leucine, and histidine. The transformants were grown for 3 days, and His+ colonies were then patched
on selective media, replica-plated on nitrocellulose filters, and
tested for
-galactosidase activity (23). Positive clones were
rescued and tested for specificity by retransformation into strain
YRG-2 with either AT1a C-ter or the extraneous targets
human lamin C and murine p53 (Stratagene). The cDNA inserts from
positive clones were then sequenced.
Northern Blot Analysis--
The Northern blot was purchased from
CLONTECH and hybridized with
-32P-ATRAP and
-actin cDNAs according to the
manufacturer's recommendations.
Maltose Binding Protein (MBP) Fusion Protein Affinity
Chromatography--
The cytoplasmic AT1a and
AT2 receptor tails were amplified by polymerase chain
reaction and cloned into a pMal-c2 prokaryotic expression vector (New
England Biolabs). MBP fusion polypeptides were expressed in
Escherichia coli DH5
and purified according to the
manufacturer's instructions. The MBP fusion protein load of individual
amylose resins was normalized by densitometric scanning of SDS-PAGE
gels stained with Coomassie Blue. For affinity chromatography, a
50-µl volume of the MBP fusion protein-loaded resins (50% (v/v) suspensions) was preblocked in binding buffer (50 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 1 µg of
aprotinin/ml) with bovine serum albumin (10 mg/ml) for 1 h at
4 °C. The MBP fusion protein resins were then incubated with a
lysate (100 µg of protein) of COS-7 cells transiently transfected
with an HA epitope-tagged version of ATRAP (HA-ATRAP) in binding buffer
for 16 h at 4 °C. Resins were washed four times with binding
buffer and eluted with SDS-PAGE sample buffer. Samples were then
subjected to SDS-PAGE, transferred to nitrocellulose membrane
(Hybond-ECL, Amersham Pharmacia Biotech), and probed with anti-HA
monoclonal antibody 12CA5 (Boehringer Mannheim). Epitope-tagged ATRAP
was detected with peroxidase-conjugated sheep anti-mouse secondary
antibody (Amersham Pharmacia Biotech) and enzyme-linked
chemiluminescence (ECL, Amersham Pharmacia Biotech).
Co-immunoprecipitation--
The NH2-terminal HA
epitope-tagged ATRAP in pcDNA3 was transiently co-transfected with
a FLAG-tagged murine AT1a receptor (24) in COS-7 cells
according to the LipofectAMINE protocol (Life Technologies, Inc.). The
ratio of AT1a and ATRAP DNA was 1:3. Cells were harvested
48 h after transfection, and membrane fractions prepared from the
transfected cells (25) were solubilized in 50 mM Tris-HCl
(pH 7.5), 140 mM NaCl, 1 mM CaCl2,
1 mM phenylmethylsulfonyl fluoride, and 1 µg of
aprotinin/ml (buffer A) in the presence of 1% CHAPS. The mixture was
gently agitated for 30 min at 4 °C and thereafter centrifuged at
13,000 × g for 20 min. Cleared supernatants (100 µg
of protein) were diluted 1:10 in buffer A and incubated for 16 h
at 4 °C with M1 monoclonal antibody recognizing the FLAG epitope
(Eastman Kodak Co.) and protein A/G PLUS-agarose beads (Santa Cruz
Biotechnology). The beads were then washed in buffer A, and the samples
were subjected to SDS-PAGE, transferred to nitrocellulose membrane, and
probed with anti-HA monoclonal antibody 12CA5. HA-ATRAP was detected
with peroxidase-conjugated sheep anti-mouse antibody and ECL.
Immunofluorescence Microscopy--
COS-7 cells were seeded in
glass coverslips and co-transfected with NH2-terminal
HA-tagged ATRAP and FLAG epitope-tagged AT1a using the
method described above. The cells were then fixed and permeabilized
with ice-cold methanol for 5 min. HA-ATRAP was detected with either the
monoclonal mouse antibody 12CA5 and a fluorescein isothiocyanate-conjugated rat anti-mouse IgG2b monoclonal
antibody (Pharmingen) or an HA-specific rabbit antiserum (Babco) and
CY3-goat anti-rabbit antibody (Zymed Laboratories
Inc.). AT1a receptor was detected with the monoclonal
mouse antibody M1 and a fluorescein isothiocyanate-conjugated rat
anti-mouse IgG2b monoclonal antibody.
-Galactosidase Assay--
The yeast reporter strain SFY526
was co-transformed with the deleted versions of the AT1a
C-ter and the ATRAP hybrid expression plasmid. The amounts of
-galactosidase from three independent transformants grown in liquid
selective media were measured in a chlorophenol
red-
-D-galactopyranoside-based assay (26).
Inositol Phosphate Determination--
COS-7 cells were
transiently co-transfected with NH2-terminal HA
epitope-tagged ATRAP and FLAG epitope-tagged AT1a receptor or human m3 muscarinic acetylcholine receptor using the
LipofectAMINE reagent. The ratio of receptor and ATRAP DNA was 1:3.
Transfected cells plated in 12-well plates (2 × 105
cells/well) were labeled overnight with
myo-[3H]inositol (5 µCi/ml; NEN Life Science
Products) in serum-free Dulbecco's modified Eagle's medium. After
1 h of stimulation with increasing concentrations of AngII or
carbachol in the presence of 10 mM LiCl, inositol phosphate
was extracted and separated on Dowex AG1-X8 columns (Bio-Rad). Total
inositol phosphate was eluted with 2 M ammonium formate,
0.1 M formic acid.
fos-Luciferase Assay--
Chinese hamster ovary (CHO) K1 cells
were co-transfected with the FLAG-tagged murine AT1a
receptor expression vector (24) and pSV2-Neo with the
LipofectAMINE reagent (using a 30:1 DNA ratio). Stably transfected
cells were selected in G418 (800 µg/ml; Life Technologies) for 3 weeks, and the cells expressing high levels of AT1a
receptors were sorted by fluorescence-activated cell sorting after
immunolabeling with the anti-FLAG M1 monoclonal antibody. The
immunoselected, stably transfected CHO AT1a cells were
maintained in G418 and used for up to four passages. 3.5 × 105 CHO AT1a were seeded in 6-well plates and
transiently co-transfected with pcDNA3/HA-ATRAP,
fos-luciferase reporter gene (p2FTL) and
-galactosidase
reporter gene (pCMV
; CLONTECH) by lipofection using the LipofectAMINE reagent. The fos-luciferase reporter
gene consists of two copies of the c-fos 5'-regulated
enhancer element (
357 to
276), the herpes simplex virus thymidine
kinase gene promoter (
200 to +70), and luciferase gene (4). The ratio of HA-ATRAP, fos-luciferase, and
-gal DNA was 3:1:1.
Forty-eight h after transfection, transfected cells were incubated in
serum-free medium (Ham's F12; Life Technologies) for 16 h.
Quiescent cells were then treated with 100 nM AngII for
3.5 h, washed with phosphate-buffered saline, and lysed for 10 min
with 250 µl of lysis buffer (luciferase assay system; Promega) at
4 °C. 10 µl of cell extract was mixed with 100 µl of luciferase
reagent, and the light produced was measured for 10 s using a
LUMAT LB 9507 luminometer (EG & G Berthold). Results were normalized to
the
-gal activity using a
-galactosidase enzyme assay system (Promega).
Radioligand Binding Assay--
For AT1a
receptor-transfected COS-7 and CHO K1 cells, ligand binding assays were
performed using membrane preparations as described elsewhere (25). For
m3 receptor-transfected COS-7 cells, binding assays using
N-[3H]methylscopolamine (NEN Life Science
Products) were carried out with membrane homogenates as described
previously (28). Nonspecific binding was measured in the presence of 1 µM atropine.
Statistics--
For the inositol phosphate and
fos-luciferase assay, results are expressed as mean ±S.E.
Statistical significance was assessed by t test.
 |
RESULTS AND DISCUSSION |
The yeast two-hybrid system was used to identify candidate
proteins that interact with the carboxyl-terminal cytoplasmic tail of
the mouse AT1a receptor. Screening of 1.5 × 106 transformants from a mouse kidney primary cDNA
library resulted in the isolation of three independent clones that
interacted specifically with the AT1a carboxyl-terminal
tail. No interaction with another major subtype of AngII receptor
(AT2) was observed. Sequence analysis revealed that the
three library plasmids contained different lengths of the same
cDNA. All inserts contained an open reading frame, and inspection
of the sequence of the longest cDNA revealed a potential initiator
ATG that matched well the consensus sequence for translational
initiation (29). This clone spans an open reading frame of 483 base
pairs encoding a predicted protein of 17.8 kDa (Fig.
1). We named this protein ATRAP for
AT1 receptor associated
protein. The failure of 5'-rapid amplification of cDNA ends to lead to the isolation of longer cDNA suggested that the clone isolated from the two-hybrid screen represents the full-length gene. Moreover, the mobility of an in vitro translation
product was in agreement with the molecular mass predicted for ATRAP by sequence analysis (data not shown). The ATRAP-predicted amino acid
sequence was used to search available data bases by means of the BLAST
program network server. ATRAP does not show homology with known
proteins. However, it is similar to at least three mouse EST clones
(accession numbers AA718794, AA840135, and W57121) and is homologous to
a number of human and rat EST clones. ATRAP has one potential
N-glycosylation site, one potential phosphorylation site for
protein kinase C, and one potential phosphorylation site for casein
kinase II (Fig. 1). This protein also contained several extensive
hydrophobic domains in its NH2-terminal portion.

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Fig. 1.
cDNA and predicted amino acid sequence of
ATRAP. The putative N-glycosylation and protein kinase
C phosphorylation sites are underlined and boxed,
respectively. The nucleotide sequence has been deposited in the
GenBankTM under the accession number AF102548.
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Northern blot analysis of messenger RNA from various mouse tissues,
with full-length ATRAP cDNA as a probe, revealed two transcripts of
1.2 and 0.8 kilobases; this result further suggested that the cDNA
clone represents the full-length gene. ATRAP was expressed at a
relatively high level in kidney, testis, and heart but at lower levels
in lung, liver, spleen, and brain (Fig.
2). Using reverse
transcription-polymerase chain reaction, we also detected ATRAP
transcripts in mouse aortic tissue and vascular smooth muscle cells
(data not shown).

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Fig. 2.
Northern blot analysis of
poly(A)+ RNAs from various mouse tissues. The blot
(purchased from CLONTECH) was hybridized with ATRAP
cDNA (top) and -actin cDNA (bottom)
according to the manufacturer's recommendations; sk.
muscle, skeletal muscle.
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To biochemically confirm the association between ATRAP and
AT1a C-ter, in vitro interactions were examined
in studies using AT1a and AT2 receptor
cytoplasmic tails fused to MBP. When added to detergent-solubilized
extracts of ATRAP-transfected COS-7 cells, ATRAP was recovered with the
recombinant MBP-AT1a C-ter but not with MBP-AT2
C-ter or the MBP alone (Fig.
3A). These results further suggested that ATRAP associates specifically with the AT1
receptor.

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Fig. 3.
Specific binding of ATRAP to the
AT1a receptor. A, in vitro
binding of recombinantly expressed ATRAP to AT1a and
AT2 receptor tails expressed as MBP fusion proteins.
Affinity chromatography of detergent-solubilized extracts of
HA-ATRAP-transfected COS-7 cells on immobilized fusion proteins was
performed as described under "Experimental Procedures."
B, co-immunoprecipitation of ATRAP with the AT1a
receptor. COS-7 cells were transiently co-transfected with
NH2-terminally HA epitope-tagged ATRAP and FLAG-tagged
AT1a receptor. Membrane fractions of the cells were
prepared, and receptor complexes were immunoprecipitated as described
under "Experimental Procedures." Whole-cell extracts
(lane1, Extr.) or immunoprecipitates (lanes
2-4, IP) were subjected to SDS-PAGE, blotted, and
probed for the presence of the HA epitope.
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The binding of ATRAP to full-length AT1a receptors in
vivo was confirmed by co-immunoprecipitation from transfected
COS-7 cells. The AT1a receptor was tagged at the
amino-terminal extracellular domain with a FLAG epitope to facilitate
specific immunoprecipitation of receptors (24). For the immunodetection
of ATRAP, the protein was HA-tagged at the amino terminus, and a
polypeptide of the expected size was observed by immunoblotting in
transfected cells (Fig. 3B, 1st lane). ATRAP was
co-immunoprecipitated specifically from cell membrane lysates in
association with the AT1a receptor (Fig. 3B, 4th
lane). ATRAP was not detected in control immunoprecipitates, including those prepared from cells expressing ATRAP without
FLAG-tagged receptors (Fig. 3B, 2nd lane); this result
confirmed the specificity of this protein association in
vivo. We did not observe a significant difference in the amount of
ATRAP co-immunoprecipitated with the AT1 receptor before or
after AngII stimulation (data not shown).
The subcellular localization of epitope-tagged ATRAP was examined in
transfected COS-7 cells by fluorescence microscopy. Using optical
sectioning of antibody-labeled cells by confocal microscopy, ATRAP was
visualized in a diffuse cytoplasmic distribution, with a more intensive
staining near the cell periphery (Fig.
4A). Immunoblotting of
extensively washed membrane fractions prepared from transfected cells
confirmed that a significant fraction of ATRAP was membrane-associated (data not shown). The association of ATRAP with AT1a
receptors was further examined by immunofluorescence co-localization
experiments. COS-7 cells were co-transfected with HA-tagged ATRAP and
FLAG-tagged AT1a receptors and were co-stained with
anti-FLAG (Fig. 4B, green) and anti-HA
(panel C, red) antibodies. Superposition of these images showed considerable co-localization (panel D,
yellow) of the two proteins at the periphery of the cells
and in intracellular compartments.

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Fig. 4.
Immunocytochemical co-localization of ATRAP
and AT1a receptor. COS-7 cells were transiently
co-transfected with HA-tagged ATRAP protein and FLAG-tagged
AT1a receptor. Immunostaining and fluorescence microscopy
were carried out as described under "Experimental Procedures."
A, in addition to its cytoplasmic distribution, ATRAP
localized to the plasma membrane. B-D, co-transfected and
immunostained cells were imaged by dual-color confocal microscopy.
AT1a receptor (green channel, B) and ATRAP
(red channel, C) co-localized to the plasma membrane and in
intracellular compartments as shown in yellow in the
two-color merged image (D).
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Several proteins that associate with the carboxyl-terminal cytoplasmic
domain of GPCRs regulate receptor-mediated signaling. Mutational
analysis of the AT1 receptor tail has demonstrated that it
contains discrete amino acid sequences that are important for receptor
desensitization (17, 18) and internalization (17, 19, 20). Recent
studies have implicated protein kinase C and G protein-coupled receptor
kinases in the heterologous and homologous desensitization of the
AT1a receptor, respectively (18, 21). Three protein kinase
C phosphorylation sites and a sequence that has partial homology to the
consensus G protein-coupled receptor kinases phosphorylation motif
(amino acids 343 to 348) are present within the carboxyl-terminal
cytoplasmic domain of the AT1a receptor (30). To gain
insight into the functional significance of the ATRAP-AT1a
receptor association, we first localized the binding site for ATRAP
within the AT1a receptor tail. By generating serial
deletions in the receptor tail, we found that the ATRAP-binding site
localized within the last 20 carboxyl-terminal amino acids (339 to 359)
of the receptor (Table I). It is
interesting that this sequence comprises two of the three protein
kinase C sites and the unique potential G protein-coupled receptor
kinases phosphorylation motif found in the AT1a cytoplasmic domain (30). Moreover, a recent functional characterization of
truncated AT1a receptors lacking varying lengths of the
cytoplasmic tail demonstrated that the region encompassing residues 328 to 348 plays an important role in the desensitization of the
AT1a receptor (18). To examine the possibility that ATRAP
affects receptor desensitization, the effect of ATRAP overexpression on agonist-dependent activation of phospholipase C (PLC) was
examined in AT1a receptor-transfected cells. As shown in
Fig. 5, overexpression of ATRAP markedly
inhibited the PLC response over a wide range of agonist concentrations.
PLC activation was maximally inhibited by an average of 35%
(10
8 M Ang II) in cells co-transfected with
ATRAP and AT1a receptors when compared with cells
co-transfected with AT1a receptors and the control plasmid.
Radioligand binding assays performed with the same populations of
transfectants used in the PLC assay indicated that ATRAP overexpression
did not significantly affect the affinity or the number of
AT1a receptors (Table II).
The magnitude of the inhibitory effect of ATRAP overexpression may be
influenced by the expression of endogenous ATRAP in COS-7 cells, as
detected by Northern blot analysis (data not shown). Furthermore, the
effect of ATRAP on receptor signaling may depend on its association
with other cellular partners that may be present in limiting amounts relative to overexpressed ATRAP. To assess whether ATRAP associates with other GPCRs, we examined its interaction with the
carboxyl-terminal cytoplasmic domains of several Gq-coupled
receptors in the yeast two-hybrid system. ATRAP did not interact with
the carboxyl-terminal cytoplasmic tails of the m3
muscarinic, bradykinin B2, or endothelin B receptors, nor
did it associate with the Gs-coupled
2-adrenergic receptor (data not shown). Accordingly, no
effect of ATRAP overexpression was observed on m3
receptor-mediated PLC activation over a wide range of agonist
concentrations (10
9 to 10
5 M
carbachol) or on basal PLC activity. Moreover, ATRAP did not affect PLC
(
and
isoforms) expression level as determined by immunoblot
analysis (data not shown). Taken together, these observations are
consistent with the hypothesis that ATRAP specifically inhibits signaling by interacting directly with AT1a receptors
rather than by affecting receptor expression or downstream signaling
components. Moreover, the specificity of this inhibition is consistent
with the specificity of ATRAP association with the receptor tail
in vitro. The observation that AngII binding did not affect
the ATRAP-AT1 receptor interaction would suggest that ATRAP
function may be regulated by other means such as post-transcriptional
modifications (phosphorylation/dephosphorylation) or alternatively by
its association with others cellular proteins in response to
AT1 receptor stimulation.
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Table I
Yeast two-hybrid interaction of ATRAP to deletion mutants of the
AT1a receptor tail
Yeast reporter strain YSF526 was co-transformed with the indicated Gal4
hybrid vectors. The levels of -galactosidase expression were
quantified as described under "Experimental Procedures."
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Fig. 5.
Effect of ATRAP overexpression on
agonist-dependent activation of phospholipase C in
transfected COS-7 cells. Total inositol phosphate (IP) production
was measured in COS-7 cells co-transfected either with AT1a
receptor and the control vector ( ) or with AT1a receptor
and ATRAP ( ) in the presence of increasing amounts of Ang II.
Results are expressed as the ratio of stimulated to unstimulated cells
normalized to maximal binding capacity (Bmax).
Values are means ±S.E. of 4 independent experiments performed in
duplicate. * p < 0.05.
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Table II
Binding parameters of 125I-[sarcosine1,
Ile8]AngII in transiently co-transfected COS-7 cells
COS-7 cells were co-transfected either with AT1a receptor and a
control vector (pcDNA3) or with AT1a receptor and ATRAP.
The dissociation constant and the number of binding sites
(Bmax) for 125I-[sarcosine1,
Ile8]AngII were determined by Scatchard analysis as described
under "Experimental Procedures." Data represent the mean ± S.E. from three separate experiments.
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To assess whether ATRAP may influence a more downstream AT1
receptor-dependent signaling event, we examined the effect
of ATRAP overexpression on AngII-induced c-fos gene
expression. CHO AT1a cells (Kd = 0.3 ± 0.05 nM; Bmax = 4.2 ± 1.2 pmol/mg of membrane protein) were transiently co-transfected
with a fos-luciferase reporter gene together with ATRAP or a
control vector. The fos-luciferase reporter construct
contains the serum response element of the c-fos promoter
(4), which has been shown to be sufficient for AngII-induced activation
of the c-fos promoter (27). AngII-induced c-fos
expression was determined by measuring the increase in
fos-luciferase activity in lysates of co-transfected cells
after AngII treatment. ATRAP overexpression did not significantly
affect AngII-dependent increase in c-fos
expression when compared with cells transfected with the control
plasmid (data not shown). AngII-induced activation of the serum
response element of the c-fos promoter has been proposed to
involve protein kinase C and ERK1/2 (extracellular signal-regulated kinase) stimulation (30). In contrast to the proximal effector PLC,
induction of c-fos expression is a downstream signaling
event that requires the activation of a cascade of effectors.
Therefore, it is conceivable that the attenuated PLC response observed
in cells overexpressing ATRAP is insufficient to affect the downstream effectors involved in the induction of the c-fos reporter
gene expression.
In conclusion, we have identified a novel, membrane-localized protein
that interacts specifically with the carboxyl-terminal cytoplasmic
domain of the AT1a receptor and blunts
agonist-dependent PLC activation. It is conceivable that
ATRAP attenuates receptor-mediated signaling by regulating a known
mechanism of receptor desensitization such as phosphorylation. Because
of the wide expression of ATRAP in commonly used cell lines,
dominant-negative or knock-out approaches would be helpful to further
elucidate its function in AT1 receptor signaling. Based on
our present results, ATRAP appears to function as a negative regulator
of AT1 receptor-mediated signaling. Given that the
AT1 receptor is a key mediator in the biologic mechanisms of the renin-angiotensin system, ATRAP may play a significant role in
the regulation of cardiovascular physiology.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Nathalie Blin for advice on the
m3 receptor ligand binding assay, Dr. Eugene Chen for the
mouse kidney cDNA library, Dr. Jaime Escobedo for the
fos-luciferase reporter vector (p2FTL), Dr. Lutz Hein for
the FLAG-AT1a receptor construct, Dr. Peter Marks for
advice and assistance on the confocal microscopy study, Dr. Ulrike
Mende for the m3 muscarinic acetylcholine receptor construct, Dr. Anne Pizard for the bradykinin B2 receptor
construct, and Dr. Masashi Yanagisawa for the endothelin B receptor
construct. We are grateful to Dr. Richard Pratt, Dr. Stefano Marullo,
and Dr. Clara Nahmias for valuable discussions.
 |
FOOTNOTES |
*
This study was supported by National Institute of Health
Grants HL46631, HL35252, HL35610, HL48638, HL07708, and HL58616.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF102548.
Recipient of a postdoctoral fellowship from the American Heart
Association, Massachusetts affiliate, and of an international research
fellowship from INSERM, France.
§
Recipient of National Institutes of Health Merit Award HL35610. To
whom correspondence should be addressed: Dept. of Medicine, Brigham and
Women's Hospital, 75 Francis St., Boston, MA 02115. Tel.:
617-732-6340; Fax: 617-732-6439.
 |
ABBREVIATIONS |
The abbreviations used are:
GPCR, G
protein-coupled receptor;
AT1, angiotensin II type 1 receptor;
AT2, angiotensin II type 2 receptor;
AngII, angiotensin II;
C-ter, carboxyl-terminal cytoplasmic domain;
ATRAP, AT1 receptor-associated protein;
MBP, maltose-binding
protein;
PLC, phospholipase C;
m3, human m3
muscarinic acetylcholine receptor;
HA, hemagglutinin;
PAGE, polyacrylamide gel electrophoresis;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
CHO, Chinese hamster ovary.
 |
REFERENCES |
-
Hein, L.,
and Kobilka, B. K.
(1995)
Neuropharmacology
34,
357-366[CrossRef][Medline]
[Order article via Infotrieve]
-
Kobilka, B.
(1992)
Annu. Rev. Neurosci.
15,
87-114[CrossRef][Medline]
[Order article via Infotrieve]
-
Sterne-Marr, R.,
and Benovic, J. L.
(1995)
Vitam. Horm.
51,
193-234[Medline]
[Order article via Infotrieve]
-
Wang, C.,
Jayadev, S.,
and Escobedo, J. A.
(1995)
J. Biol. Chem.
270,
16677-16682[Abstract/Free Full Text]
-
Hausdorff, W. P.,
Hnatowich, M.,
O'Dowd, B. F.,
Caron, M. G.,
and Lefkowitch, R. J.
(1990)
J. Biol. Chem.
265,
1388-1393[Abstract/Free Full Text]
-
Eason, M. G.,
and Ligget, S. B.
(1996)
J. Biol. Chem.
271,
12826-12832[Abstract/Free Full Text]
-
Tseng, M.-J.,
Coon, S.,
Stuenkel, E.,
Struk, V.,
and Logsdon, C. D.
(1995)
J. Biol. Chem.
270,
17884-17891[Abstract/Free Full Text]
-
Guiramand, J.,
Montmayeur, J.-P.,
Cearline, J.,
Bhatia, M.,
and Borrelli, E.
(1995)
J. Biol. Chem.
270,
7354-7358[Abstract/Free Full Text]
-
Conchon, S.,
Barrault, M.-B.,
Miserey, S.,
Corvol, P.,
and Clauser, E.
(1997)
J. Biol. Chem.
272,
25566-25572[Abstract/Free Full Text]
-
Klein, U.,
Ramirez, T. M.,
Kobilka, B. K.,
and von Zastrow, M.
(1997)
J. Biol. Chem.
272,
19099-19102[Abstract/Free Full Text]
-
Hall, A. R.,
Premont, R. T.,
Chow, C.-W.,
Blitzer, J. T.,
Pitcher, J. A.,
Claing, A.,
Stoffel, R. H.,
Barak, L. S.,
Shenolikar, S.,
Weinman, E. J.,
Grinstein, S.,
and Lefkowitz, R. J.
(1998)
Nature
392,
626-630[CrossRef][Medline]
[Order article via Infotrieve]
-
Ali, M. S.,
Sayeski, P. P.,
Dirksen, L. B.,
Hayzer, D. J.,
Marrero, M. B.,
and Bernstein, K. E.
(1997)
J. Biol. Chem.
272,
23382-23388[Abstract/Free Full Text]
-
Venema, R. C.,
Ju, H.,
Venema, V. J.,
Schieffer, B.,
Harp, J. B.,
Ling, B. N.,
Eaton, D. C.,
and Marrero, M. B.
(1998)
J. Biol. Chem.
273,
7703-7708[Abstract/Free Full Text]
-
Ju, H.,
Venema, V. J.,
Marrero, M. B.,
and Venema, R. C.
(1998)
J. Biol. Chem.
273,
24025-24029[Abstract/Free Full Text]
-
Sano, T.,
Ohyama, K.,
Yamano, Y.,
Nakagomi, Y.,
Nakazama, S.,
Kikyo, M.,
Shirai, H.,
Blank, J. S.,
Exton, J. H.,
and Inagami, T.
(1997)
J. Biol. Chem.
272,
23631-23636[Abstract/Free Full Text]
-
Premont, R. T.,
Inglese, J.,
and Lefkowitz, R. J.
(1995)
FASEB J.
9,
175-182[Abstract/Free Full Text]
-
Conchon, S.,
Peltier, N.,
Corvol, P.,
and Clauser, E.
(1998)
Am. J. Physiol.
274,
E336-E345[Abstract/Free Full Text]
-
Tang, H.,
Guo, D. F.,
Porter, J. P.,
Wanaka, Y.,
and Inagami, T.
(1998)
Circ. Res.
82,
523-531[Abstract/Free Full Text]
-
Thomas, W. G.,
Thekkumkara, T. J.,
Motel, T. J.,
and Baker, K. M.
(1995)
J. Biol. Chem.
270,
207-213[Abstract/Free Full Text]
-
Hunyady, L.,
Bor, M.,
Balla, T.,
and Catt, K. J.
(1994)
J. Biol. Chem.
269,
31378-31382[Abstract/Free Full Text]
-
Oppermann, M.,
Freedman, N. J.,
Alexander, R. W.,
and Lefkowitz, R. J.
(1996)
J. Biol. Chem.
271,
13266-13272[Abstract/Free Full Text]
-
Benichou, S.,
Bomsel, M.,
Bodéus, M.,
Durand, H.,
Douté, M.,
Letourneur, F.,
Camonis, J.,
and Benarous, R.
(1994)
J. Biol. Chem.
269,
30073-30076[Abstract/Free Full Text]
-
Breeden, L.,
and Nasmyth, K.
(1985)
Cold Spring Harbor Sympt. Quant. Biol.
50,
643-650[Medline]
[Order article via Infotrieve]
-
Hein, L.,
Meinel, L.,
Pratt, R. E.,
Dzau, V. J.,
and Kobilka, B. K.
(1997)
Mol. Endocrinol.
11,
1266-1277[Abstract/Free Full Text]
-
Mukoyama, M.,
Nakajima, M.,
Horiuchi, M.,
Sasmura, H.,
Pratt, R. E.,
and Dzau, V. J.
(1993)
J. Biol. Chem.
268,
24539-24542[Abstract/Free Full Text]
-
Iwabuchi, K.,
Li, B.,
Bartel, P.,
and Fields, S.
(1993)
Oncogene
8,
1693-1696[Medline]
[Order article via Infotrieve]
-
Sadoshima, J.,
and Izumo, S.
(1993)
Circ. Res.
73,
424-438[Abstract]
-
Blin, N.,
Yun, J.,
and Wess, J.
(1995)
J. Biol. Chem.
270,
17741-17748[Abstract/Free Full Text]
-
Kozak, M.
(1986)
Cell
44,
283-292[Medline]
[Order article via Infotrieve]
-
Berk, B. C.,
and Corson, M. A.
(1997)
Circ. Res.
80,
607-616[Abstract/Free Full Text]
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