Type 1 angiotensin II receptor-associated protein ARAP1 binds and recycles the receptor to the plasma membrane

BBRC Biochemical and Biophysical Research Communications 310 (2003) 1254–1265 www.elsevier.com/locate/ybbrc

Type 1 angiotensin II receptor-associated protein ARAP1 binds and recycles the receptor to the plasma membraneq Deng-Fu Guo,a,* Isabelle Chenier,a Valerie Tardif,a Sergei N. Orlov,a and Tadashi Inagamib a

Research Centre, H^ otel-Dieu du CHUM, Department of Medicine, Universit
e de Montr
eal, Montr
eal, Qu
e., Canada H2W 1T8 b Department of Biochemistry, Vanderbilt University, School of Medicine, Nashville, TN 37232-0146, USA Received 12 September 2003

Abstract The carboxyl terminus of the type 1 angiotensin II receptor (AT1 ) plays an important role in receptor phosphorylation, desensitization, and internalization. The yeast two-hybrid system was employed to isolate proteins associated with the carboxyl terminal region of the AT1A receptor. In the present study, we report the isolation of a novel protein, ARAP1, which promotes recycling of AT1A to the plasma membrane in HEK-293 cells. ARAP1 cDNA encodes a 493-amino-acid protein and its mRNA is ubiquitously expressed in rat tissues. A complex of ARAP1 and AT1A was observed by immunoprecipitation and Western blotting in HEK-293 cells. In the presence of ARAP1, recycled AT1A showed a significant Ca2þ release response to a second stimulation by Ang II 30 min after the first treatment. Immunocytochemical analysis revealed co-localization of recycled AT1A and ARAP1 in the plasma membrane 45 min after the initial exposure to Ang II. Taken together, these results indicate a role for ARAP1 in the recycling of the AT1 receptor to the plasma membrane with presumable concomitant recovery of receptor signal functions. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Angiotensin; Receptor recycling; Hypertension; Gene expression

Agonist stimulation of G protein-coupled heptahelical receptors causes a dramatic reorganization of their intracellular distribution. Activation of such receptors triggers receptor endocytosis and, since these receptors recycle back to the plasma membrane continuously, a new steady state is reached between the internal compartments and the plasma membrane, where a significant proportion of receptors are located internally. Although the recycling of receptors back to the plasma membrane is a notable event, the molecular mechanisms of this process are not well understood. Several studies have demonstrated that heterotrimeric G proteins, G protein-coupled receptor kinases, tyrosine kinases, and arrestins interact directly with G protein-coupled heptahelical receptors [1–5]. Molecular domain studies have shown that the third q The rat ARAP1 cDNA sequence has been deposited with GenBank under Accession No. AF159049. * Corresponding author. Fax: +514-412-7204. E-mail address: [email protected] (D.-F. Guo).

0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.09.154

intracellular loop of G protein-coupled heptahelical receptors plays a key role in receptor coupling to heterotrimeric G proteins [6–9]. However, several reports have established that various physiological consequences of G protein-coupled heptahelical receptor stimulation do not seem to be mediated by heterotrimeric G protein activation. Concurrently, novel techniques for detecting protein–protein interactions, such as the yeast two-hybrid system, phage display, and fusion protein overlays, have revealed associations of G protein-coupled heptahelical receptors with a variety of intracellular partners other than heterotrimeric G proteins [10–12]. Angiotensin II (Ang II) elicits a wide range of physiological responses in a variety of cell types. This octapeptide has an important role in the regulation of the cardiovascular and renal systems via diverse mechanisms [13], which include vasoconstriction, vascular smooth muscle proliferation, and electrolyte transport in the distal tubules [14]. On the basis of its pharmacological, functional, and structural features, Ang II

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receptors have been divided into two isoforms: AT1 and AT2 [15–20]. Both isoforms of Ang II receptors have a heptahelical structure and bind Ang II with a high affinity. Recently, the carboxyl terminal region of the AT1 receptor has been reported to be directly associated with several downstream effectors [21–24]. Mutagenesis studies have also shown that this region contains discrete amino acid sequences which are essential for receptor phosphorylation [25,26], desensitization [27–30], and internalization [29,31,32]. These observations raise the possibility that the carboxyl terminal region of AT1 receptors may interact with additional intracellular proteins that may play an important role in receptor trafficking such as recycling. To investigate this possibility, we searched for proteins associated with the carboxyl terminal region of the AT1A isoform of the rat AT1 receptor with the yeast two-hybrid system. We report here the isolation of a novel rat protein called ARAP1 (type 1 angiotensin II receptor associated protein 1) which specifically binds the carboxyl terminal region of the AT1A receptor between residues 319 and 359. This region is crucial for their interaction. Functional studies suggest that ARAP1 recycles the AT1A receptor to the plasma membrane in transfected HEK-293 cells. Materials and methods Materials. Ang II and anti-Flag monoclonal antibody (M2) were purchased from Sigma. Polyclonal and monoclonal antibodies against Myc-epitope were obtained from Santa Cruz. Culture media and fetal bovine serum were purchased from Gibco. Secondary goat anti-mouse IgG antibody (C3Y) was obtained from Jackson ImmunoResearch Laboratories. Yeast two-hybrid screening. The yeast two-hybrid system was carried out essentially as described previously [33]. Briefly, the bait plasmid was pBTM116 expressing the carboxyl terminal 64 amino acid residues (295–359) of rat AT1A receptor in-frame with the lexA cDNA binding domain (pBTM116-AT1A ). The L40 yeast strain was transformed with pBTM116-AT1A , and subsequently with a pVP16 10.5day mouse embryo cDNA library. Plasmids. For the receptor binding assay experiments, rat AT1A , AT1A -T318, ARAP1, and human AT2 cDNAs were subcloned into the pcDNA3 vector, and human b2-adrenoceptor cDNA was subcloned into pRC/CMV vector. For immunoprecipitation experiments, the rat AT1A receptor and rat ARAP1 were Flag and Myc epitope-tagged, respectively, at the amino-terminus in the mammalian expression vector pcDNA3. The amino-terminus of the pcDNA3-Flag-AT1A plasmid contained the prolactin signal sequence (MDSKGSSLLCQG VVS) followed by the Flag-epitope sequence (DYKDDDD) and then by the AT1A receptor sequence beginning with Ala [34]. To facilitate immunoprecipitation, the N-glycosylation site Asn4 of AT1A was changed to Asp. The amino-terminus of the pcDNA3-Myc-ARAP1 plasmid contained MGREFEQKLISEEDLL followed by the Arg of the ARAP1 sequence. For confocal microscopy, ARAP1 was expressed as enhanced green fluorescence protein (GFP) fusion protein with pEFP2 vector. The epitopes were integrated into the cDNAs encoding rat AT1A and ARAP1 by oligonucleotide-directed mutagenesis with PCR methods, and sequences of the recombinant DNAs were confirmed by dideoxy sequencing using the chain termination method.

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Cells. HEK-293 and NIH 3T3 cells were grown in MEM with 10% fetal calf serum (FCS) at 37 °C under 5% CO2 . COS-7 cells were grown in high glucose DMEM and Sprague–Dawley rat vascular smooth muscle cells (VSMCs) were grown in low glucose DMEM containing 10% FCS, respectively. Northern blot analysis. Total RNA was extracted from different rat tissues by a modified version of the guanidinium thiocyanate procedure [35]. Poly(A)þ mRNAs were then extracted with an Oligotex mRNA kit. Two micrograms of poly(A)þ mRNA was resolved by electrophoresis in a 1.2% agarose gel containing 1.8% formaldehyde, transferred to a Hybond-N membrane, fixed, and hybridized with a 32 P-labeled probe. Full-length rat ARAP1 was used as a probe. After exposition, the membrane was stripped and rehybridized with the GAPDH probe. Receptor binding assay. HEK-293 cells were seeded in 6- or 12-well dishes the day before the transfections. DNAs were transfected by a modified calcium phosphate method. Forty-eight hours after the transfection, the cells were treated with 100 nM Ang II or 1 lM isoproterenol for indicated durations. They were then washed once with acidic buffer (10 mM NaCl, 50 mM glycine, pH 3.0) on ice for 3 min and washed twice with PBS. Receptor expression was measured by saturation binding of 125 I-Sar-Ile-Ang II for Ang II receptors and 125 ICYP for b2-adrenoceptors. Recycling kinetic studies. HEK-293 cells were seeded in 12-well dishes a day before transfection. Cells were transiently transfected with pcDNA3 + AT1A or pcDNA3-ARAP1 + AT1A , respectively. Forty eight hours after transfection, the cells were pretreated with 10 nM Ang II for 15 min at 37 °C. After an acidic wash (pH 3.0), the cells were incubated in fresh medium for different time periods at 37 °C to allow recycling of the receptor. Cells were finally incubated with 125 I-Sar-IleAng II for 3 h at 4 °C. Interaction of ARAP1 with AT1A . Transfected HEK-293 cells in 10cm dishes were stimulated with 100 nM of Ang II for indicated durations at 37 °C. After three washes with cold PBS, the cells were lysed in lysis buffer (50 mM Tris–HCl, pH 7.4, 100 mM NaCl, 20 mM NaF, 10 mM Na pyrophosphate, 5 mM EDTA, 1% Triton X-100, 10 mg/ml leupeptin, 1 mM PMSF, 10 mg/ml pepstatin, and 1 mM orthovanadate). Five micrograms of antibody was added to 600 lg of cell lysate. After incubation for 2 h at 4 °C, immunocomplexes were collected by the addition of protein A–conjugated Sepharose beads. After three washes with lysis buffer, bound proteins were eluted with SDS sample buffer at 65 °C for 15 min, subjected to SDS–polyacrylamide gel (10%), electrophoresis, and then transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk in TBST buffer (50 mM Tris–HCl, pH. 7.4, 150 mM NaCl, and 0.2% Tween 20) and incubated with primary antibody in TBST buffer for 1 h at room temperature. The membranes were then washed with TBST buffer without skim milk, followed by incubation with protein A-HRP in 5% skim milk in TSBT buffer for 1 h. Next, the membranes were washed with TBST buffer, incubated with ECL working solution for 1 min, and exposed to an X-ray film. The antibodies used were rabbit polyclonal IgG against Myc-epitope tag, mouse monoclonal IgG1, M2 to Flag-epitope tag for Western blotting or mouse monoclonal IgG1, 9E10 to Myc epitope tag and M2 for immunoprecipitation. Measurement of Ca2þ . Intracellular Ca2þ concentration was determined using the Ca2þ -sensitive dye fluo-3 [36]. Fluorescence was measured in buffer A containing 1.2 mM EGTA ([Ca2þ ] less than 1 lM) with a FluoroMax Spectrofluorometer (SPEX FluoroMax spectrofluorometer) with excitation wavelengths of 483 and 523 nm. Free intracellular Ca2þ was measured as a function of Ca2þ ¼ Kd ðF
Fmin ÞðFmax
F Þ
1 , where Fmax and Fmin are maximal and minimal values of F measured in the presence of 0.5% Triton X-100 and 3 mM CaCl2 or 10 mM EGTA (pH 8.9), and Kd is the dissociation constant of the Ca2þ -Fluo-3 complex (864 nm at 37 °C). Confocal microscopy analysis. One day before the experiments, HEK-293 cells were passaged on glass coverslips and transfected transiently with 2 lg of Flag epitope-tagged AT1A and 2 lg of GFP-

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ARAP1. Two days after transfection, the cells were treated with 100 nM Ang II for indicated periods, fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.2% Tween, and then stained with 10 lg/ ml of monoclonal Flag-epitope M2 antibody for 60 min. Subsequently, secondary antibodies (goat anti-mouse CY3, Jackson ImmunoResearch Laboratores) were diluted 1:200 and applied in blocking buffer for 1 h at room temperature. The cells were visualized with a Zeiss 410 laser scanning microscope through 1.63  1.4 N.A. objective. Images were processed by Adobe Photoshop software.

residues of rat ARAP1 (residues 126–217) was isolated. Clone A11 was subsequently retested in the yeast twohybrid assay with the truncated AT1A receptor (residues 295–317). Although the entire AT1A carboxyl terminus domain (residues 295–359) interacted with mouse clone A11, the truncated fragment AT1A -T318, consisting of residues 295–318, failed to do so (Fig. 1C). The result indicates that the region between residues 319–359 of AT1A is required for interaction with ARAP1 in the yeast two-hybrid system.

Results Isolation of rat ARAP1 cDNA Yeast two-hybrid screening To identify proteins that recognize the carboxyl terminal region of the AT1A receptor, a yeast two-hybrid screen was performed in a system which included the AT1A (residues 295–359) fused to the lexA DNA-binding domain (Fig. 1A) and a mouse embryo library [33] fused to the VP16 transactivation domain. One positive clone, A11 (Fig. 1B), which contained 92 amino acid

A full length 2138 bp rat ARAP1 cDNA clone was subsequently isolated from a kgt11 rat VSMC cDNA library using mouse clone A11 as a probe. ARAP1 had a putative ATG start codon, which started at adenine 389, preceded by a consensus Kozak sequence, AGGACCATG [37], and an open reading frame of 1479 bp encoding a hydrophilic protein of 493 amino acid residues with a calculated molecular mass of 57,156 Da

Fig. 1. Interacting molecules detected in the yeast two-hybrid assay. (A) Depiction of the organization of the seven transmembrane-spanning domains, three intracellular and extracellular loops in the AT1A receptor, and the carboxyl terminal region employed as bait in the yeast two-hybrid assay. (B) Depiction of ARAP1 structural organization and the region encompassed by clone A11 obtained as prey in the yeast two-hybrid system. PEP12-like domain (residues 95–202), PRS: proline-rich sequence (residues 212–228), and the fibrinogen-like domain (residues 272–487). (C) Interaction of ARAP1 with the wild type and truncated mutant AT1A receptor in the yeast two-hybrid system.

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(Fig. 2A). The mobility of the in vitro translation product was in agreement with the molecular mass predicted for ARAP1 (data not shown). A 10 bp poly(A) tail and a polyadenylation signal AAUAAA sequence were identified at the 30 -untranslated terminus of the cDNA. The ARAP1 protein contains a proline-rich region in the middle of the protein, which may interact with the Src-homology 3 (SH3) domain-containing proteins [38]. Amino acid sequence database search revealed a region of 20% identity over a 108 amino acid residue stretch between ARAP1 (residues 95–202) and yeast PEP12 without any gaps (Fig. 2B). The yeast PEP12 protein is localized in the pre-vacuolar endosome and its activity is required for transporting proteins from the Golgi to the vacuoles [39,40]. This suggests that ARAP1 may function in the sorting of AT1 in rat cells. A region of 43% identity over 216 amino acid residues

A

aactgaggctgctgctgtcggcctggg gatggaccccaagccctgagtggtgctgttggacccaggacctgcaaggagcacgcacta aggcagctacggaccacgctgtgagggagagcaggttgggagcagccccagtgacaccag agccagcctcatccctaagagcttctaagagcatagactgctgccagctgaggccagtaa ggcagggctgctcggcggccagtccagcctaagactcgggacctctcctggaggccacgg ccaggctgtcccgctgatggcaccgggaagcatgtgaagaccctgctccagggccaagca ggagagaagaggtttcaagagacctcattcataaagaccaaggagcacactgcaaggacc ATGAGGCCACTGTGTATGACTTACTGGTGGCTTGGACTGCTGGCCACCGTGGGAGCTGTT M R P L C M T Y W W L G L L A T V G A V ACAGGCCCAGAGGCTGATGTTGAGGGCGCAGAGGATGGTTCGCAGAGAGAGTACATTTAC T G P E A D V E G A E D G S Q R E Y I Y CTCAACAGGTACAAGAGGGCAGGTGAGTCCCCAGACAAGTGCACCTACACTTTCATTGTG L N R Y K R A G E S P D K C T Y T F I V CCCCAGCAGCGGGTCACAGGTGCCATTTGTGTCAACTCCAAAGAGCCCGAGGTGCACCTG P Q Q R V T G A I C V N S K E P E V H L GAGAACCGTGTGCACAAGCAGGAGCTGGAGCTGCTCAACAATGAGCTGCTTAAGCAGAAG E N R V H K Q E L E L L N N E L L K Q K CGGCAGATCGAGACGCTGCAGCAGCTGGTAGAGGTAGATGGCGGCATCGTGAGCGAGGTG R Q I E T L Q Q L V E V D G G I V S E V AAGCTCGTGCGCAAGGAGAGCCGCAACATGAACTCTCGGGTCACACAGCTGTACATGCAG K L V R K E S R N M N S R V T Q L Y M Q CTTCTACACGAGATCATTCGCAAGCGAGACAATGCGCTGGAGCTTTCCCAGCTGGAGAAC L L H E I I R K R D N A L E L S Q L E N AGGATCCTGAACCAGACAGCTGACATGCTGCAGCTGGTGAGCAAGTACAAGGACCTGGAG R I L N Q T A D M L Q L V S K Y K D L E CACAAGTTCCAGCACCTGGATATGCTGGCACACAACCAATCAGAGGTCATTGCCCAGCTT H K F Q H L D M L A H N Q S E V I A Q L GAAGAGCACTGCCAACGTGTACCTGCAGCCAGGCCTGTGCCCCAGCCACCCCCAGCCACG E E H C Q R V P A A R P V P Q P P P A T CCACCTCGGGTCTACCAGCCACCAACCTACAACCGCATCATCAACCAGATCTCCACTAAT P P R V Y Q P P T Y N R I I N Q I S T N GAGATCCAGAGTGACCAGAATCTGAAGGTGCTGCCACCCTCCCTGCCCACCATGCCTGCC E I Q S D Q N L K V L P P S L P T M P A CTTACCAGTCTCCCATCTTCCACTGATAAGCCATCAGGTCCATGGAGAGATTGTCTACAG L T S L P S S T D K P S G P W R D C L Q GCCCTGGAGGATGGTCACAGCACCAGCTCCATCTACCTGGTGAAGCCGGAGAATACCAAC A L E D G H S T S S I Y L V K P E N T N CGCCTTATGCAGGTGTGGTGCGACCAGAGACATGACCCTGGGGGTTGGACTGTCATCCAG R L M Q V W C D Q R H D P G G W T V I Q AGACGCCTGGATGGCTCTGTCAACTTCTTCAGGAACTGGGAGACCTATAAGCAAGGGTTT R R L D G S V N F F R N W E T Y K Q G F GGGAACATCGACGGCGAATACTGGCTGGGCCTGGAGAACATCTACTGGCTGACGAACCAA G N I D G E Y W L G L E N I Y W L T N Q GGCAACTACAAATTGCTTGTAACCATGGAGGACTGGTCTGGCCGCAAAGTCTTTGCAGAG G N Y K L L V T M E D W S G R K V F A E TATGCTAGCTTCCGACTGGAGCCAGAAAGCGAGTACTATAAGCTGCGGCTGGGGCGTTAT Y A S F R L E P E S E Y Y K L R L G R Y CACGGCAACGCAGGCGACTCCTTTACCTGGCACAACGGCAAACAGTTCACCACCCTGGAC H G N A G D S F T W H N G K Q F T T L D AGGGACCATGATGTCTACACAGGAAACTGTGCCCACTATCAGAAGGGAGGATGGTGGTAC R D H D V Y T G N C A H Y Q K G G W W Y AATGCCTGTGCTCACTCCAACCTCAATGGGGTCTGGTACCGTGGGGGCCATTACCGGAGC N A C A H S N L N G V W Y R G G H Y R S CGATACCAGGATGGGGTCTACTGGGCTGAGTTCCGAGGAGGATCTTACTCACTCAAGAAG R Y Q D G V Y W A E F R G G S Y S L K K GTGGTGATGATGATTCGGCCCAACCCCAACACTTTCCATTAAgctctctctgcctggcca V V M M I R P N P N T F H * cttacggcattgccagaagccatcccaactgtgcgacgtcagcacagctcttcactggcc cacctcaggctgggaggacagagtgctggactctgctctccaagtggctgtcagatgatg gagatgaaagggcttctctgcccctcctgccttcttttacacccagccatccctgtattc caggacaggacagaactgcaatcttccaatcagttaagtcttaataaaaatttcaactgc caaaaaaaaaa

-360 -300 -240 -180 -120 -60 60 20 120 40 180 60 240 80 300 100 360 120 420 140 480 160 540 180 600 200 660 220 720 240 780 260 840 280 900 300 960 320 1020 340 1080 360 1140 380 1200 400 1260 420 1320 440 1380 460 1440 480 1500 493 1560 1620 1680 1740

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between ARAP1 (residues 272–487) and fibrinogen was also identified by database searches with three gaps of 23 residues (Fig. 2C). The predicted ARAP1 amino acid sequence was used to search available EST databases by means of the BLAST program. ARAP1 had high homology to two mouse EST clones (Accession Nos. AI151857 and AA638040) and one human EST clone (Accession No. N47466). Tissue and cell distribution of rat ARAP1 Northern blot analysis showed that ARAP1 mRNA is expressed in the heart, brain, lung, liver, kidney, and testis, but is particularly abundant in the heart (Fig. 3A). ARAP1 mRNA was also detected in cultured rat VSMC, human HEK-293 cells, the monkey kidney cell line Cos-7, and the mouse fibroblast cell line NIH 3T3

B

ELLKQKRQIETLQQLVEVDGGIVSEVKLVRKESRNMNSRVTQLYMQLLHEIIRK ARAP1 ELFEINGQISTLQQFTATLKSFIDRGDVSAKVVERINKRSVAKIEEIGGLIKKI PEP12 Consensus EL-----QI-TLQQ----------------K-----N-R-----------I--RDNALELSQLENRILNQTADMLQLVSKYKDLEHSFQEFQGIQPQFTQVMKQVNER NTSVKKMDAIEEASLDKTLQIIAREKLVRDVSYKFQHLDMLAHNQSEVIAQLEEH ----------E---L--T-----------D----FQ-----------V--Q--E-

C

ARAP1 SGPWRDCLQALEDGHS--TSSIYLVKPENTNRLMQVWCDQRHDPGGWIVIQRR Fibrinogen SRPVRDCDDVLQTHPSGTQSGIFNIKLPGSSKIFSVYCDQETSLGGWLLIQQR Consensus S-P-RDC---L----S---S-I---K---------V-CDQ----GGW--IQ-R LDGSVNFFRNWETYKQGFGNI----DGEYWLGLENIYWLTNQGNYKLLVTMEDWSG MDGSLNFNRTWQDYKRGFGSLNDEGEGEFWLGNDYLHLLTQRGSV-LRVELEDWAG -DGS-NF-R-W--YK-GFG-------GE-WLG------LT--G---L-V--EDW-G RKVFAEYASFRLEPESEYYKLRLGRYHGNAGDS-----------FTWHNGKQFTTL NEAYAEY-HFRVGSEAEGYALQVSSYEGTAGDALIEGSVEEGAEYTSHNNMQFSTF ----AEY--FR---E-E-Y-L----Y-G-AGD-------------T-HN--QF-TDRDHDVYTGNCAHYQKGGWWYNACAHSNLNGVWYRGGHYRSR------YQDGVYWA DRDADQWEENCAEVYGGGWWYNNCQAANLNGIYYPGGSYDPRNNSPYEIENGVVWV DRD-D----NCA----GGWWYN-C---NLNG--Y-GG-Y--R---------GV-WEFRGGSYSLKKVVMMIRP SFRGADYSLRAVRMKIRP -FRG--YSL--V-M-IRP

Fig. 2. Sequence of rat ARAP1. (A) cDNA sequence and predicted amino acid sequence of rat ARAP1. (B) Comparison of ARAP1 with the yeast PEP12 sequence. (C) Comparison of the ARAP1 sequence with fibrinogen.

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Fig. 3. ARAP1 mRNA expressed in different rat tissues and different cell types. (A) Tissue distribution of ARAP1 mRNA in the rat. Br, brain; He, heart; Ki, kidney; Li, liver; Lu, lung; and Te, testis. (B) Expression of ARAP1 mRNA in different cell lines.

(Fig. 3B). Only a single ARAP1 transcript was found in the rat, whereas two ARAP1 transcripts were detected in mice, monkeys, and humans, presumably due to alternative splicing in the 30 -untranslated region. The level of ARAP1 mRNA was not affected by addition of 100 nM Ang II for 60 min to cultured rat VSMC (Fig. 3B), suggesting that Ang II does not affect the transcriptional regulation of ARAP1 in a short time period. Interaction between rat ARAP1 and the AT1 receptor in HEK-293 cells To examine whether the interaction of AT1A and ARAP1 occurs in HEK-293 cells, Flag epitope-tagged AT1A receptors were created. As shown in Fig. 4A, two forms of the receptors, glycosylated and nonglycosylated, were detected by Western blot analysis after immunoprecipitation with the monoclonal anti-Flag M2 antibody. The glycosylated form of the wild-type AT1A and the truncated mutant AT1A -T318 migrated at 70 and 66 kDa, respectively, in a 10% SDS–PAGE, while the non-glycosylated forms migrated at 40 and 36 kDa, respectively. On the other hand, Myc epitope-tagged rat ARAP1 had only a single band detected at the expected position of 57 kDa (data not shown). Since the sizes of the non-glycosylated form of the receptor and rat ARAP1 were 40 and 57 kDa, respectively, the membranes were cut at the 50 kDa position into two parts in order to reach maximal detection by Western blotting analysis in the following co-immunoprecipitation studies.

To examine whether ARAP1 forms a complex with AT1A and the truncated mutant AT1A -T318, co-immunoprecipitation experiments were performed with transiently transfected HEK-293 cells. HEK-293 cells expressing the Flag epitope-tagged wild type AT1A , or the truncated mutant AT1A -T318 and Myc epitopetagged ARAP1, were incubated for 30 min in the presence of 100 nM Ang II. Myc epitope-tagged ARAP1 was immunoprecipitated and co-immunoprecipitated Flag epitope-tagged receptors were detected by Western blotting with anti-Flag antibody (Fig. 4B). Only wild type AT1A could be detected in the Myc epitope-tagged ARAP1 immunoprecipitation complex, indicating that the carboxyl terminal region between residues 319–359 of the receptor is important for association with rat ARAP1. These data are consistent with those obtained with the yeast two-hybrid system where ARAP1 did not interact with the truncated mutant AT1A -T318 receptor. To determine whether Ang II promotes the association between the receptor and rat ARAP1, co-immunoprecipitation experiments were performed with transiently transfected HEK-293 cells. HEK-293 cells expressing Flag epitope-tagged AT1A receptors and Myc epitope-tagged ARAP1 were incubated for the indicated durations with 100 nM Ang II. The receptors were immunoprecipitated with M2 antibody, and immunocomplexes were separated and transferred onto nylon membranes. Myc epitope-tagged ARAP1 could be detected in the absence of agonist, but its level was increased after Ang II treatment (Fig. 4C). Maximal ARAP1 and AT1A association occurred within 30– 45 min of exposure to Ang II. These results indicate that Ang II stimulates the association between rat ARAP1 and the receptors. ARAP1 promotes recycling of the AT1A receptor to the plasma membrane Upon exposure of the cells to Ang II, AT1 undergoes rapid internalization. The sequestration of receptors into the endosome is in dynamic equilibrium with receptor recycling to the plasma membrane. Receptor internalization was monitored by measuring surface receptor number by radioligand binding assay in which receptor number was decreased by 50–90%. To determine whether rat ARAP1 regulates AT1 receptor expression, the radioligand binding assay was performed in HEK-293 cells co-transfected transiently with the vector or rat ARAP1 with rat AT1A (Fig. 5A). Without transfected rat ARAP1, AT1A was rapidly internalized and reached a minimal level (40% of the original) at 30 min following exposure of the cells to 100 nM Ang II. No further decrease of receptor number on the cell surface was detected. Strikingly, a marked increase in cell surface receptor number was observed in cells transfected with ARAP1 at 30 min following exposure of the cells to

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Fig. 4. Association of Myc epitope-tagged ARAP1 with Flag epitope-tagged AT1A , but not with the truncated mutant AT1A -T318 receptor in transiently transfected HEK-293 cells. (A) Immunoprecipitation of Flag epitope-tagged AT1A and truncated mutant AT1A -T318 receptors in transiently transfected HEK-293 cells. (B) Interaction of Myc epitope-tagged ARAP1 with Flag epitope-tagged wild type AT1A , but not with the truncated mutant AT1A -T318 receptor. (C) Time course of interaction between Myc epitope-tagged ARAP1 and Flag epitope-tagged AT1A receptors.

100 nM Ang II although AT1 receptor internalization was not affected as determined at 15 min of treatment with 100 nM Ang II. Under these conditions, no further increase of cell surface receptor number was detected, suggesting that a new equilibrium had been reached after 30 min exposure of the cells to Ang II. To determine whether rat ARAP1 promotes recycling of AT1A , HEK-293 cells co-transfected with vector or rat ARAP1 with rat AT1A were pretreated with 10 nM Ang II for 15 min. After an acid wash, cells were left in a fresh medium for different time periods at 37 °C to permit recovery of the receptor binding activity. 125 ISar-Ile-Ang II binding was then performed for 3 h at 4 °C. The results show that the receptor was rapidly recycled with t1=2 around 7 min in the ARAP1 overexpressed cells, compared with t1=2 around 20 min in the control cells (Fig. 5B). Overexpression of rat ARAP1 increased the membrane AT1 receptor number by 1.6fold after stimulation for 30 min, whereas no significant rise of the receptor number was seen in the cells prior to addition of the peptide agonist (Fig. 5C). These observations clearly indicate that rat ARAP1 promotes the recycling process of AT1A receptor in HEK-293 cells. To examine whether rat ARAP1 can recycle carboxyl terminus-truncated AT1A (AT1A -T318) to the plasma

membrane, the mutant receptor was co-transfected with either the vector alone or ARAP1 in HEK-293 cells, and receptor expression was measured by radioligand binding assay (Fig. 5D). Compared with wild type AT1A , internalization of the truncated AT1A -T318 receptor was markedly attenuated to only 20% sequestration in 30 min. No effect on receptor internalization was observed in cells overexpressing rat ARAP1 and AT1A T318 receptors, suggesting that the region between residues 319–359 of AT1A may be important for interaction with ARAP1. These data are consistent with the results of the yeast two-hybrid analysis discussed above. Co-expression of rat ARAP1 with human AT2 in HEK-293 cells had no effect on AT2 receptor expression (Fig. 5E). This finding was expected because AT2 is not internalized by the agonist peptide. In view of reports that the carboxyl terminal region of the b2-adrenoceptor plays an important role in receptor internalization, we examined the possible involvement of ARAP1 in the regulation of b2-adrenoceptor expression (Fig. 5F). b2adrenoceptors were rapidly internalized; after 30 min of isoproterenol stimulation, 70% of receptors were internalized. However, ARAP1 overexpression did not affect b2-adrenoceptor expression.

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Fig. 5. Requirement of ARAP1 for the recycling of AT1 receptor to the plasma membrane. (A) Kinetics of internalization of the AT1A receptor in the presence or absence of ARAP1. (B) Kinetics study of the AT1A receptor in the presence or absence of ARAP1. (C) Effect of ARAP1 on AT1A receptor expression. (D) Effect of ARAP1 on truncated mutant AT1A -T318 receptor expression. (E) Effect of ARAP1 on AT2 receptor expression. (F) Effect of ARAP1 on b2-adrenoceptor expression. All data are mean values of at least three separate experiments except for two experiments on b2-adrenoceptor expression. Similar results were also obtained in Cos-7 cells.

Recycled receptors are resensitized and responded to a second stimulation by Ang II A characteristic of the AT1 receptor is its ability to activate the Ca2þ /inositol triphosphate signal transduction pathway [19,20]. To examine whether recycled AT1A is capable of responding to a second exposure to Ang II, HEK-293 cells stably expressing AT1A were transiently transfected with rat ARAP1. After a 48 h transfection, the cells were loaded with Fluo-3 and changes in intracellular Ca2þ were measured (Fig. 6A). Addition of 100 nM Ang II to the cells evoked a rapid,

transient increase of Ca2þ which peaked at 15 s (I1Ang II ). The increment of Ca2þ induced by Ang II varied over a range of 180–220 nM and did not differ significantly in HEK-293 cells transfected with either the vector or ARAP1 (data not shown). The second challenge with 1 lM Ang II after 30 min of exposure to 100 nM agonist led to a very small increment of Ca2þ in HEK-293 cells without ARAP1 transfection (I2Ang II < 10 nM). In contrast, in cells with exogenously transfected ARAP1, the second 1 lM Ang II challenge increased Ca2þ by 40– 50 nM (Fig. 6B). To exclude a possible effect of intracellular Ca2þ depletion during 30 min exposure to

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Fig. 6. Resensitization of recycled AT1A receptors in the plasma membrane. (A) Kinetics of Ca2þ modulation in HEK-293 cells transfected with AT1A and vector alone as control. (B) Ca2þ modulation in second addition of Ang II in HEK-293 cells transfected with AT1A and ARAP1. Fluo-3-loaded cells were exposed to Ang II (100 nM) for 30 min, followed by a second treatment with Ang II (1 lM). Five min later, 10 lM ATP was added. Representative tracings are shown for control vector alone (A) and ARAP1 transfected cells (B) and similar results were obtained from three independent experiments. (C) Relative increase of Ca2þ induced by second addition of Ang II (1 lM, IAng II ) in HEK-293 cells transfected with the vector or ARAP1. Ca2þ increment induced by ATP (10 lM, IATP ) was taken as 100%. IAng II /IATP ratio is expressed as mean + SE of three experiments.

100 nM Ang II in Ca2þ free medium, the purinergic receptor response to 10 lM ATP after 5 min of a second Ang II treatment was compared between ARAP1 and vector-transfected HEK-293 cells. Both showed similar ATP-induced increments of intracellular Ca2þ (IATP ¼ 50–70 nM; Figs. 6A and B), indicating that the ARAP1 effect was not due to prevention of Ca2þ depletion. Fig. 6C shows that the I2Ang II /IATP ratio was about 5-fold higher in ARAP1 transfected cells compared to cells transfected with vector. ARAP1 is co-localized with the AT1A receptor in the plasma membrane 45-min after stimulation with Ang II The co-immunoprecipitation of ARAP1 and AT1 from cell homogenates suggests co-localization of the two proteins in the form of a complex in intact cells. Such a possibility was investigated by immunocytochemical analysis with confocal microscopy. In HEK293 cells transfected with Flag epitope-tagged AT1A , and treated with 100 nM Ang II for 0, 15, and 45 min, AT1A was located in the plasma membrane at 0 min (Fig. 7A), but internalized into intracellular vesicles at 15 min

(Fig. 7B), and remained largely internalized at 45 min (Fig. 7C). In the cells co-transfected with Flag epitopetagged AT1A and GFP-ARAP1 fusion protein and treated with 100 nM Ang II for 0, 15, and 45 min, AT1A was located at the plasma membrane at time 0 (Fig. 7D), internalized into the endosome at 15 min (Fig. 7E), but was recycled back to the plasma membrane at 45 min (Fig. 7F). ARAP1 was located in the cytosol-like vesicles and trans Golgi network at 0 min (Fig. 7G), remained internal at 15 min (Fig. 7H), and translocated into the plasma membrane at 45 min (Fig. 7I). AT1A was colocalized in the plasma membrane with rat ARAP1 45 min after stimulation with Ang II (Fig. 7L).

Discussion In the present study, we isolated a novel gene, ARAP1, which interacts with the carboxyl terminal region of the AT1A receptor (residues 318–359), and we demonstrated that ARAP1 promotes the recycling of the AT1A receptor back to the plasma membrane. Agonist stimulation of G protein-coupled heptahelical receptors

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Fig. 7. Imaging of the recycling of AT1A receptor by ARAP1. HEK-293 cells were transfected with GFP-ARAP1 (D–L) or not (A–C) and processed for immunocytochemical analysis of the distribution of Flag epitope-tagged AT1A receptor as described in Materials and methods. Each panel shows a single confocal section, green color indicates GFP-ARAP1, red color is the AT1A receptor. Note that the receptor is localized at the plasma membrane (A,D) in unstimulated HEK-293 cells. At 15 min after addition of Ang II most of the receptor is in the cytosol in either absence (B) or presence (E) of GFP-ARAP1. However, whereas in the absence of GFP-ARAP1 the receptor is largely in the cytosol at 45 min after Ang II stimulation, the receptor in cells expressing ARAP1 was mostly in the cytoplasmic membrane at this time point. A similar distribution is observed for GFP-ARAP1 (G–I). At 45 min following Ang II stimulation, the two proteins show partial co-localization as indicated by yellow color. Bar ¼ 10 lm.

causes dramatic reorganization of their intracellular distribution. Activation of AT1 receptors triggers receptor internalization and, since the receptors recycle back to the plasma membrane continuously, a new steady state is reached where a significant proportion of them are located internally [41]. However, the molecular mechanisms of the receptor recycling back to the plasma membrane are not well understood. The novel gene ARAP1 may provide a key element involved in the receptor recycling pathway and may help us to understand the molecular mechanisms of the receptor recycling event. In addition to ARAP1, a recently discovered novel 18 kDa protein, ATRAP, has been shown to directly associate with the carboxyl terminal region of the AT1 receptor [42]. This novel protein, ATRAP, interacts with the carboxyl terminal region between residues 339 and 359 of the AT1A receptor. Although we did not narrow the detailed region of AT1A receptor which interacts with ARAP1, the residues between 319 and 359 are important for their interaction. Phospholipase C is markedly inhibited in response to Ang II stimulation in the cells overexpressing ATRAP, but AT1 receptor internalization and recycling pathways are not affected. In the present study, we showed that ARAP1 overexpression increased the receptor number back to the plasma membrane after cell exposure to Ang II for 30 min. Resensitization of recycled AT1A receptors was

confirmed by drastic increase of intracellular Ca2þ concentration triggered by the second addition of Ang II in cells transfected with ARAP1, compared to untransfected cells. Although ARAP1 and ATRAP are both AT1 receptor-associated proteins and interact with similar regions of the receptor, they have different functions in receptor regulation. By mutagenesis analysis, the carboxyl terminal region of the AT1 receptor has been shown to contain discrete amino acid motifs that are required for receptor phosphorylation [25,26,43], desensitization [29,30], and internalization [29,31,32]. Taken together, these findings suggest that the carboxyl terminal region of G protein-coupled heptahelical receptors may be important for their trafficking and recycling. The radioligand binding assays revealed that ARAP1 seems to promote the recycling of the AT1 receptor back to the plasma membrane, but not other G protein-coupled heptahelical receptors tested in the present study, such as the AT2 and the b2-adrenoceptors. ARAP1 did not heighten the internalization rate of the AT1 receptor upon exposure of the cells to Ang II, but increased receptor number in the plasma membrane 30-min after stimulation with Ang II. This finding suggests that ARAP1 is involved in the recycling pathways of the AT1 receptor to the plasma membrane rather than in the internalization process. We observed that addition of

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Myc epitope-tag or GFP protein to ARAP1 did not affect the ARAP1 as measured by receptor ligandbinding assays (data not shown) and immunocytochemical analysis (Fig. 7). Several proteins that associate with the carboxyl terminal region of G protein-coupled heptahelical receptors regulate receptor-mediated signaling. Recent studies have implicated protein kinase C and G proteincoupled kinases in heterologous and homologous desensitization of the AT1 receptor, respectively [30,44]. ARAP1 mRNA was widely expressed in all tissues tested and in commonly used cell lines. Dominant-negative or knock-out approaches would be helpful to further elucidate its function in downstream AT1 receptor-dependent signaling. Further studies should address how ARAP1 regulates AT1 receptor-mediated signaling such as receptor phosphorylation, Ang II-induced activation of Erk1/Erk2 cascades, and hypertrophic effects. ARAP1 seems to belong to a family of proteins which share a common sequence motif of the fibrinogen-like domain. This family of proteins includes angiopoietin [45], ficolin [46], tenascin [47], and the Drosophila melanogaster gene SCABROUS (sca) [48]. These proteins share a C-terminal fibrinogen-like domain, and differ in their N-terminal regions, which often contain coiled coils [49]. In the sca gene, the apparent coiled coils are required for dimerization and they are linked to the Cterminal fibrinogen-like domain by a short proline-rich sequence [38]. Expression of the sca N-terminal region could rescue the sca mutant phenotype as full-length sca cDNA and this region includes a putative receptor-interacting site [48], indicating that the N-terminal region of ARAP1 may also be important for its activity. Angiopoietin-1 is a secreted ligand for the TIE2 receptor tyrosine kinase, suggesting the possibility that ARAP1 may transport AT1 to the plasma membrane. In none of these proteins is the specific function of the fibrinogenlike domain known, despite its conservation and the medical importance of fibrin as a protein directly responsible for myocardial infarction and stroke [50]. The present study has revealed a novel function of this family of proteins, containing a fibrinogen-like domain, in the regulation of other G protein-coupled heptahelical receptors. PEP12, a yeast protein, has been implicated in the docking and fusion of intracellular transport vesicles [39]. The SNARE (soluble N-ethyl maleimide-sensitive factor attachment protein receptor) hypothesis provides a universal model of how nearly all intracellular membrane fusion events work [51,52]. Although only 20% of the 108 amino acid residues homologous between ARAP1 and PEP12 are identical, ARAP1 may participate in, or modulate the formation of, the v/t-SNARE (vesicle/target-SNARE) complexes. These complexes, formed of proteins found on the membranes of transport vesicles and acceptor compartments, are involved

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in the specific recognition of transport vesicles with their target membranes. ARAP1 specifically promotes the recycling of AT1 receptors back to the plasma membrane, but not AT2 and b2-adrenoceptors. It is not surprising that ARAP1 does not affect the AT2 receptor, because the latter does not undergo internalization and desensitization in response to agonist stimulation [16,17]. There are different mechanisms involved in the internalization of AT1 receptors and b2-adrenoceptors [53]. b2-adrenoceptors are internalized by a b-arrestin- and dynamin-dependent mechanism, whereas AT1 receptors are internalized by a mechanism independent of b-arrestin and dynamin. It is also highly likely that the recycling mechanisms for AT1 and b2-adrenoceptors are different. In conclusion, the present study defines a new protein that confers specificity on outward trafficking back to the plasma membrane of the G protein-coupled heptahelical receptor AT1 in its recycling after ligand-induced internalization. As it accelerates receptor traffic, it could also increase its receptor response to Ang II. The possibility cannot be excluded that accelerated receptor recycling may serve as a clearance mechanism for the agonist in blood. Given that the AT1 receptor plays a central role in the biological and physiological mechanisms of the renin-angiotensin system, ARAP1 may be significantly involved in the regulation of cardiovascular diseases.

Acknowledgments We are grateful to Drs. R.J. Lefkowitz and M. Bouvier for providing the b2-adrenoceptor cDNA, Mr. O.M. Da Silva for his critical reading of this manuscript. This work was supported by the Canadian Institute of Health Research (MT-14726), the Heart and Stroke Foundation of Canada, and the Canadian Foundation for Innovation (D.F.G.). D.F.G. was supported by a scholarship from the Heart and Stroke Foundation of Canada.

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