Cloning and Characterization of ATRAP, a Novel Protein That Interacts with the Angiotensin II Type 1 Receptor*

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 AT1areceptor but not with those of angiotensin II type 2 (AT2), m3 muscarinic acetylcholine, bradykinin B2, endothelin B, and β2-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.

G protein-coupled receptors (GPCRs) 1 interact with different classes of intracellular proteins, including heterotrimeric G proteins, kinases, and arrestins (1)(2)(3). Although the intracellular third loop of a number of GPCRs is a key structural deter-minant 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 AT 1 and AT 2 receptors. Recently, the carboxyl-terminal cytoplasmic domain of the AT 1 receptor has been reported to directly associate with several downstream effectors (12)(13)(14)(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 AT 1 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 carboxylterminal cytoplasmic domain of the AT 1 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 AT 1a 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 AT 1 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 AT 1a receptor (AT 1a 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 AT 1a receptor tail (AT 1a 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 AT 2 (amino acids 314 to 363), human m 3 muscarinic acetylcholine (amino acids 548 to 590), human bradykinin B 2 (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) ϩ 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 AT 1a C-ter hybrid expression plasmid and the mouse kidney cDNA library as described previously (22). Double transformants were plated on yeast drop-out * 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. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Northern Blot Analysis-The Northern blot was purchased from CLONTECH and hybridized with ␣-32 P-ATRAP and ␤-actin cDNAs according to the manufacturer's recommendations.
Maltose Binding Protein (MBP) Fusion Protein Affinity Chromatography-The cytoplasmic AT 1a and AT 2 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-l 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 g 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 g 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 NH 2 -terminal HA epitope-tagged ATRAP in pcDNA3 was transiently co-transfected with a FLAG-tagged murine AT 1a receptor (24) in COS-7 cells according to the Lipo-fectAMINE protocol (Life Technologies, Inc.). The ratio of AT 1a 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 CaCl 2 , 1 mM phenylmethylsulfonyl fluoride, and 1 g 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 ϫ g for 20 min. Cleared supernatants (100 g 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 NH 2 -terminal HA-tagged ATRAP and FLAG epitope-tagged AT 1a 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 IgG 2b monoclonal antibody (Pharmingen) or an HA-specific rabbit antiserum (Babco) and CY3-goat anti-rabbit antibody (Zymed Laboratories Inc.). AT 1a receptor was detected with the monoclonal mouse antibody M1 and a fluorescein isothiocyanate-conjugated rat anti-mouse IgG 2b monoclonal antibody.
␤-Galactosidase Assay-The yeast reporter strain SFY526 was cotransformed with the deleted versions of the AT 1a 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 cotransfected with NH 2 -terminal HA epitope-tagged ATRAP and FLAG epitope-tagged AT 1a receptor or human m 3 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 ϫ 10 5 cells/well) were labeled overnight with myo-[ 3 H]inositol (5 Ci/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 AT 1a receptor expression vector (24) and pSV 2 -Neo with the LipofectAMINE reagent (using a 30:1 DNA ratio). Stably transfected cells were selected in G418 (800 g/ml; Life Technologies) for 3 weeks, and the cells expressing high levels of AT 1a receptors were sorted by fluorescence-activated cell sorting after immunolabeling with the anti-FLAG M1 monoclonal antibody. The immunoselected, stably transfected CHO AT 1a cells were maintained in G418 and used for up to four passages. 3.5 ϫ 10 5 CHO AT 1a 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 5Ј-regulated enhancer element (Ϫ357 to Ϫ276), the herpes simplex virus thymidine kinase gene promoter (Ϫ200 to ϩ70), 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 l of lysis buffer (luciferase assay system; Promega) at 4°C. 10 l of cell extract was mixed with 100 l 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 AT 1a receptor-transfected COS-7 and CHO K1 cells, ligand binding assays were performed using membrane preparations as described elsewhere (25). For m 3 receptor-transfected COS-7 cells, binding assays using N-[ 3 H]methylscopolamine (NEN Life Science Products) were carried out with membrane homogenates as described previously (28). Nonspecific binding was measured in the presence of 1 M atropine.
Statistics-For the inositol phosphate and fos-luciferase assay, results are expressed as mean ϮS.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 AT 1a receptor. Screening of 1.5 ϫ 10 6 transformants from a mouse kidney primary cDNA library resulted in the isolation of three independent clones that interacted specifically with the AT 1a carboxyl-terminal tail. No interaction with another major subtype of AngII receptor (AT 2 ) 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 AT 1 receptor associated protein. The failure of 5Ј-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 ATRAPpredicted 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 NH 2 -terminal portion.
Northern blot analysis of messenger RNA from various mouse tissues, with full-length ATRAP cDNA as a probe, re-vealed two transcripts of 1.2 and 0.8 kilobases; this result further suggested that the cDNA clone represents the fulllength 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).
To biochemically confirm the association between ATRAP and AT 1a C-ter, in vitro interactions were examined in studies using AT 1a and AT 2 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-AT 1a C-ter but not with MBP-AT 2 C-ter or the MBP alone (Fig. 3A). These results further suggested that ATRAP associates specifically with the AT 1 receptor.
The binding of ATRAP to full-length AT 1a receptors in vivo was confirmed by co-immunoprecipitation from transfected COS-7 cells. The AT 1a 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 AT 1a 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 AT 1 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 AT 1a receptors was further examined by immunofluorescence co-localization experiments. COS-7 cells were co-transfected with HAtagged ATRAP and FLAG-tagged AT 1a 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.
Several proteins that associate with the carboxyl-terminal cytoplasmic domain of GPCRs regulate receptor-mediated signaling. Mutational analysis of the AT 1 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 AT 1a receptor, respectively (18,21). Three protein kinase C phosphorylation sites and a sequence that has partial homology to the consen-sus G protein-coupled receptor kinases phosphorylation motif (amino acids 343 to 348) are present within the carboxyl-terminal cytoplasmic domain of the AT 1a receptor (30). To gain insight into the functional significance of the ATRAP-AT 1a receptor association, we first localized the binding site for ATRAP within the AT 1a 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 AT 1a cytoplasmic domain (30). Moreover, a recent functional characterization of truncated AT 1a 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 AT 1a 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 AT 1a 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 Ϫ8 M Ang II) in cells co-transfected with ATRAP and AT 1a receptors when compared with cells co-transfected with AT 1a receptors   4. Immunocytochemical co-localization of ATRAP and AT 1a receptor. COS-7 cells were transiently co-transfected with HAtagged ATRAP protein and FLAG-tagged AT 1a 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. AT 1a 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). 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 AT 1a 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 G q -coupled receptors in the yeast two-hybrid system. ATRAP did not interact with the carboxyl-terminal cytoplasmic tails of the m 3 muscarinic, bradykinin B 2 , or endothelin B receptors, nor did it associate with the G s -coupled ␤ 2 -adrenergic receptor (data not shown). Accordingly, no effect of ATRAP overexpression was observed on m 3 receptor-mediated PLC activation over a wide range of agonist concentrations (10 Ϫ9 to 10 Ϫ5 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 AT 1a 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-AT 1 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 AT 1 receptor stimulation.
To assess whether ATRAP may influence a more downstream AT 1 receptor-dependent signaling event, we examined the effect of ATRAP overexpression on AngII-induced c-fos gene expression. CHO AT 1a cells (K d ϭ 0.3 Ϯ 0.05 nM; B max ϭ 4.2 Ϯ 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-termi-nal cytoplasmic domain of the AT 1a receptor and blunts agonistdependent 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 AT 1 receptor signaling. Based on our present results, ATRAP appears to function as a negative regulator of AT 1 receptor-mediated signaling. Given that the AT 1 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.