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• W2034781152 abstract "Angiotensin II (Ang II) plays a central role in cardiovascular physiology and disease. Ang II type I receptor (AT1) is thought to mediate most actions of Ang II. A novel AT1 receptor intracellular partner called AT1 receptor-associated protein (ATRAP) was identified, but its exact function has not been elucidated. A yeast two-hybrid screen using ATRAP as bait identified calcium-modulating cyclophilin ligand (CAML) as an ATRAP partner. Yeast two-hybrid and coimmunoprecipitation analysis demonstrated that the N-terminal hydrophilic domain of CAML (amino acids (aa) 1–189) mediates a specific interaction between ATRAP and CAML. Our analysis also showed that aa 40–82 of ATRAP contribute to this interaction. Bioluminescence resonance energy transfer and intracellular colocalization analysis by immunofluorescence in HEK293 cells verified this association within endoplasmic reticulum vesicular structures. Functionally, transcriptional reporter assays and RNA interference ATRAP experiments demonstrated that ATRAP knockdown increased nuclear factor of activated T cells (NFAT) activity. Overexpression of ATRAP decreased Ang II- or CAML-induced NFAT transcriptional activation, whereas an ATRAP-interacting domain of CAML (aa 1–189) sensitized NFAT activation in response to Ang II. These results indicate that CAML is an important signal transducer for the actions of Ang II in regulating the calcineurin-NFAT pathway and suggest that the interaction of CAML with ATRAP may mediate the Ang II actions in vascular physiology. Angiotensin II (Ang II) plays a central role in cardiovascular physiology and disease. Ang II type I receptor (AT1) is thought to mediate most actions of Ang II. A novel AT1 receptor intracellular partner called AT1 receptor-associated protein (ATRAP) was identified, but its exact function has not been elucidated. A yeast two-hybrid screen using ATRAP as bait identified calcium-modulating cyclophilin ligand (CAML) as an ATRAP partner. Yeast two-hybrid and coimmunoprecipitation analysis demonstrated that the N-terminal hydrophilic domain of CAML (amino acids (aa) 1–189) mediates a specific interaction between ATRAP and CAML. Our analysis also showed that aa 40–82 of ATRAP contribute to this interaction. Bioluminescence resonance energy transfer and intracellular colocalization analysis by immunofluorescence in HEK293 cells verified this association within endoplasmic reticulum vesicular structures. Functionally, transcriptional reporter assays and RNA interference ATRAP experiments demonstrated that ATRAP knockdown increased nuclear factor of activated T cells (NFAT) activity. Overexpression of ATRAP decreased Ang II- or CAML-induced NFAT transcriptional activation, whereas an ATRAP-interacting domain of CAML (aa 1–189) sensitized NFAT activation in response to Ang II. These results indicate that CAML is an important signal transducer for the actions of Ang II in regulating the calcineurin-NFAT pathway and suggest that the interaction of CAML with ATRAP may mediate the Ang II actions in vascular physiology. The octapeptide pressor hormone angiotensin II (Ang II), 1The abbreviations used are: Ang II, angiotensin II; AT1, Ang II type 1 receptor; ATRAP, AT1 receptor-associated protein; GFP, green fluorescent protein; RFP, red fluorescent protein; HA, hemagglutinin; NFAT, nuclear factor of activated T cells; CAML, calcium-modulating cyclophilin ligand; aa, amino acids; BRET, bioluminescence resonance energy transfer; siRNA, small interference RNA; PBS, phosphate-buffered saline; bis-tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; X-α-gal, 5-bromo-4-chloro-3-indoyil-α-d-galactopyranoside.1The abbreviations used are: Ang II, angiotensin II; AT1, Ang II type 1 receptor; ATRAP, AT1 receptor-associated protein; GFP, green fluorescent protein; RFP, red fluorescent protein; HA, hemagglutinin; NFAT, nuclear factor of activated T cells; CAML, calcium-modulating cyclophilin ligand; aa, amino acids; BRET, bioluminescence resonance energy transfer; siRNA, small interference RNA; PBS, phosphate-buffered saline; bis-tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; X-α-gal, 5-bromo-4-chloro-3-indoyil-α-d-galactopyranoside. a key player in the renin-angiotensin system, mediates vasoconstriction and regulates salt and fluid homeostasis, contributing to the etiology of hypertension. In addition, Ang II is involved in the pathophysiology of atherosclerosis and cardiac hypertrophy and failure (1.Timmermans P.B. Wong P.C. Chiu A.T. Herblin W.F. Benfield P. Carini D.J. Lee R.J. Wexler R.R. Saye J.A. Smith R.D. Pharmacol. Rev. 1993; 45: 205-251PubMed Google Scholar, 2.Goodfriend T.L. Elliott M.E. Catt K.J. N. Engl. J. Med. 1996; 334: 1649-1654Crossref PubMed Google Scholar, 3.Sadoshima J. Izumo S. Circ. Res. 1993; 73: 413-423Crossref PubMed Scopus (1291) Google Scholar). Two distinct subtypes of Ang II receptors, type 1 (AT1) and type 2 (AT2), have been characterized (4.Sasaki K. Yamano Y. Bardhan S. Iwai N. Murray J.J. Hasegawa M. Matsuda Y. Inagami T. Nature. 1991; 351: 230-233Crossref PubMed Scopus (774) Google Scholar, 5.Murphy T.J. Alexander R.W. Griendling K.K. Runge M.S. Bernstein K.E. Nature. 1991; 351: 233-236Crossref PubMed Scopus (1167) Google Scholar, 6.Mukoyama M. Nakajima M. Horiuchi M. Sasamura H. Pratt R.E. Dzau V.J. J. Biol. Chem. 1993; 268: 24539-24542Abstract Full Text PDF PubMed Google Scholar, 7.Kambayashi Y. Bardhan S. Takahashi K. Tsuzuki S. Inui H. Hamakubo T. Inagami T. J. Biol. Chem. 1993; 268: 24543-24546Abstract Full Text PDF PubMed Google Scholar). Binding of Ang II to the G-protein-coupled receptor AT1, which is thought to mediate most of the biological responses of Ang II, leads to rapid activation of heterotrimeric G proteins (Gαq/11, Gα12/13, and Gβγ) that subsequently activate phospholipase Cβ and phospholipase Cγ to generate inositol-1,4,5-triphosphate and diacylglycerol, which in turn cause intracellular calcium mobilization and activation of protein kinase C, respectively (3.Sadoshima J. Izumo S. Circ. Res. 1993; 73: 413-423Crossref PubMed Scopus (1291) Google Scholar, 8.Ushio-Fukai M. Griendling K.K. Akers M. Lyons P.R. Alexander R.W. J. Biol. Chem. 1998; 273: 19772-19777Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 9.Hall R.A. Lefkowitz R.J. Circ. Res. 2002; 91: 672-680Crossref PubMed Scopus (182) Google Scholar). Ang II has also been shown to stimulate phospholipase A2, phospholipase D, tyrosine kinases, Janus kinase (JAK), and mitogen-activated kinases (8.Ushio-Fukai M. Griendling K.K. Akers M. Lyons P.R. Alexander R.W. J. Biol. Chem. 1998; 273: 19772-19777Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 10.Molloy C.J. Taylor D.S. Weber H. J. Biol. Chem. 1993; 268: 7338-7345Abstract Full Text PDF PubMed Google Scholar, 11.Schorb W. Peeler T.C. Madigan N.N. Conrad K.M. Baker K.M. J. Biol. Chem. 1994; 269: 19626-19632Abstract Full Text PDF PubMed Google Scholar, 12.Marrero M.B. Paxton W.G. Duff J.L. Berk B.C. Bernstein K.E. J. Biol. Chem. 1994; 269: 10935-10939Abstract Full Text PDF PubMed Google Scholar, 13.Sayeski P.P. Ali M.S. Frank S.J. Bernstein K.E. J. Biol. Chem. 2001; 276: 10556-10563Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). It appears that the diversity of Ang II actions is regulated at the level of AT1 receptor, where different adaptor proteins may preferentially recruit second messenger pathways in different types of cells to execute the distinct functions of Ang II in the target cells. Therefore understanding the AT1 receptor-associated proteins is a key step to understanding the molecular basis of Ang II signaling. A novel protein, AT1 receptor-associated protein (ATRAP), has been identified in our laboratory and shown to be an intracellular partner of the AT1 receptor, interacting physically with the receptor both in vitro and in vivo (14.Lopez-Ilasaca M. Liu X. Tamura K. Dzau V.J. Mol. Biol. Cell. 2003; 14: 5038-5050Crossref PubMed Scopus (91) Google Scholar, 15.Daviet L. Lehtonen J.Y. Tamura K. Griese D.P. Horiuchi M. Dzau V.J. J. Biol. Chem. 1999; 274: 17058-17062Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Functionally, ATRAP is capable of reducing the generation of inositol-1,4,5-triphosphate in an agonist-dependent manner and of enhancing AT1 internalization (14.Lopez-Ilasaca M. Liu X. Tamura K. Dzau V.J. Mol. Biol. Cell. 2003; 14: 5038-5050Crossref PubMed Scopus (91) Google Scholar, 15.Daviet L. Lehtonen J.Y. Tamura K. Griese D.P. Horiuchi M. Dzau V.J. J. Biol. Chem. 1999; 274: 17058-17062Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 16.Cui T. Nakagami H. Iwai M. Takeda Y. Shiuchi T. Tamura K. Daviet L. Horiuchi M. Biochem. Biophys. Res. Commun. 2000; 279: 938-941Crossref PubMed Scopus (71) Google Scholar). In this study, we analyzed the function of ATRAP in cell signaling via identification of ATRAP-interacting partners. We identified calcium-modulating cyclophilin ligand (CAML) as an ATRAP partner and present evidence that this interaction contributes to the ATRAP-mediated AT1 receptor signaling pathway. Reagents and Plasmids—ATRAP full-length cDNA or deletions were cloned into vector pGBKT7 as described (14.Lopez-Ilasaca M. Liu X. Tamura K. Dzau V.J. Mol. Biol. Cell. 2003; 14: 5038-5050Crossref PubMed Scopus (91) Google Scholar). Mouse CAML was cloned from a mouse heart cDNA library (Clontech) by standard PCR with a forward primer containing an NdeI site: 5′-cgg gaa tcc cat atg gag ccg gtg cct gcg gcc ac-3′ and a reverse primer containing a BamH1 site: 5′-gcg cgg atc ctc agg gta ctt cag gac ccc agt aat c-3′. The full-length CAML cDNA was cloned into pGADT7 vector at NdeI and BamH1 cloning sites. Full-length CAML (aa 1–296), CAML N terminus (aa 1–189), and CAML C terminus (aa 189–296) were cloned into pGFP-N2 (BioSignal Packard, Montreal, Canada) expression vector with BglII and KpnI. PCR primers were: CAML1–189, 5′-aaa gag atc ttt aat acg act c-3′ and 5′-gcg cgg tac caa ata ttc gaa aag atg caa ac-3′; CAML189–296, 5′-gga aga tct acc atg ggg ttt cga ttg gtg ggg tgc-3′ and 5′-gcg cgg tac cgg gta ctt cag gac ccc ag-3′; CAML1–296, 5′-aaa gag atc ttt aat acg act c-3′, and 5′-gcg cgg tac cgg gta att cag gac ccc a-3′. Full-length CAML also was cloned into pRluc expression vector (BioSignal Packard, Montreal, Canada) with luciferase fused to the N- or C-terminal ends. All the vectors were sequenced, and the inserts were confirmed to be in the correct reading frame. Yeast Two-hybrid Screen—A pretransformed cDNA library from mouse testis constructed in fusion with the GAL4 activation domain was purchased from Clontech. The yeast reporter strain AH109 (Clontech) containing three GAL4-inducible reporter genes (His, Ade2, and LacZ) was cotransformed with ATRAP fused to Gal4 DNA binding domain cloned into the two-hybrid expression vector pGBKT7. Double transformants were plated on yeast SD drop-out medium lacking tryptophan, leucine, adenine, and histidine (QDO). The transformants were grown, and clones were then patched on selective medium. The yeast α-galactosidase activity, expressed from the MEL1 gene in response to Gal4 activation, was determined in plates containing X-α-gal (2 mg/ml) as a chromogenic substrate. The cDNA inserts from positive clones were then sequenced. Bioluminescence Resonance Energy Transfer (BRET) Assays— ATRAP and CAML were cloned in-frame into the expression vectors pRluc and pGFP2 (Biosignal, PerkinElmer Life Sciences). Their identity was verified by DNA sequencing. For experiments on the interaction of ATRAP with CAML, HEK293 cells were transiently transfected with Lipofectamine 2000 (Invitrogen) at a ratio of 3:1 of green fluorescent protein (GFP):luciferase constructs. 48 h after transfection, HEK293 cells were detached with phosphate-buffered saline (PBS)/EDTA and washed twice with PBS. Approximately 50,000 cells/well were distributed in a 96-well microplate (White Optiplate, PerkinElmer Life Sciences). The DeepBlue coelenterazine substrate (PerkinElmer Life Sciences) was added at a final concentration of 5 μm, and readings were collected with a Victor microplate reader (PerkinElmer Life Sciences) that permits detection of signals using filters at 410 and 515 nm wavelengths. Fluorescence Microscopy—HEK293 cells were seeded in glass coverslips and cotransfected with DNA constructs expressing CAML· GFP or ATRAP·RFP. 48 h after transfection, the cells were fixed and permeabilized with 4% paraformaldehyde for 5 min and washed twice with PBS. The cells were observed under microscopy, and all images were acquired and processed with a scientific grade, cooled, charge-coupled camera in the Nikon 80i microscopy system (Thromwood, NY) and Image processing software (Media Cybernetics, Silver Spring, MD), respectively. Immunoprecipitation—2 × 106 of HEK293 cells were plated on 10-cm diameter dishes, and 5 μg of DNA was transfected with 15 μl of Lipofectamine 2000. 48 h later, the cells were lysed in buffer developed by Von Bulow and Bram (17.von Bulow G.U. Bram R.J. Science. 1997; 278: 138-141Crossref PubMed Scopus (310) Google Scholar), which contains 1% dodecyl maltoside, 20 mm HEPES (pH 7.4), 150 mm NaCl, 10% glycerol, 2 mm MgSO4, 1 mm CaCl2, and 1 mm phenylmethylsulfonyl fluoride. The lysate was clarified by centrifugation for 15 min at 4 °C. HA-tagged CAML and associated proteins were immunoprecipitated with anti-HA mouse monoclonal antibody clone 12CA5 (Roche Applied Science) conjugated to protein G-Sepharose beads (Amersham Biosciences) and subjected to protein immunoblotting. The blot was probed with polyclonal antibody to ATRAP followed by chemiluminescence detection. Parallel protein immunoblots of each sample were performed to confirm the expected expression of CAML constructs in the cells. Cell Culture and NFAT-Luciferase Assays—HEK293 cells plated in 24-well plates with Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum were transfected with Lipofectamine 2000 (Invitrogen) containing equal amounts of DNA including reporter gene, expression vector CAML-PEGFPN1, or ATRAP-PCDNA3.1 with appropriate amounts of empty vector pGEFPN1. The ratio of DNA to Lipofectamine was 1 μg to 3 μl. Reporter gene NFAT-Luc (Clontech) contains three tandem copies of the NFAT consensus sequence (18.Fiering S. Northrop J.P. Nolan G.P. Mattila P.S. Crabtree G.R. Herzenberg L.A. Genes Dev. 1990; 4: 1823-1834Crossref PubMed Scopus (230) Google Scholar) located upstream of the minimal thymidine kinase (TK) promoter and the TATA box from the herpes simplex virus TK promoter. Located downstream of TK is the firefly luciferase reporter gene (luc) followed by the SV40 late polyadenylation signal. After endogenous NFAT transcription factors bind to the cis-acting enhancer element, transcription is induced, and the reporter gene is activated. The negative control construct TK-luciferase, in which the reporter gene is controlled by thymidine kinase minimal promoter without upstream NFAT binding sites, was used as described (19.Guo S. Rena G. Cichy S. He X. Cohen P. Unterman T. J. Biol. Chem. 1999; 274: 17184-17192Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar). 48 h after transfection, the cells were incubated in serum-free Dulbecco's modified Eagle's medium for 16 h, and quiescent cells were pretreated with 100 nm Ang II (Sigma) for 18 h. The cells were lysed with 50 μl of passive lysis buffer (Promega, Madison, WI) for 30 min. 10 μl of the cell extract was mixed with 100 μl of luciferase reagent, and the light produced was measured for 10 s with a Victor microplate reader (PerkinElmer Life Sciences). Results were normalized to the CMV-Renilla-luciferase internal control. RNA Interference and Western Blot—Human ATRAP small interference RNA with 19 nucleotides overhung by 3′ dTdT was synthesized from Invitrogen. The sense oligonucleotide is 5′-CCU GAA GGU GAU UCU CCU AdTdT-3′, and the antisense oligonucleotide is 5′-UAG GAG AAU CAC CUU CAG GdTdT-3′. The annealed double-stranded small interference RNA (siRNA) was introduced into HEK293 cells with the help of Lipofectamine 2000 (Invitrogen). 48 h after transfection, the cells were lysed and subjected to analysis of luciferase activities. For Western blot analysis, 107 HEK293 cells were plated on 6-well plates and transfected the next day by siRNA with 9 μl of Lipofectamine/well. 24 h after transfection, the cells were washed in cold PBS buffer twice and harvested by scraping in 20 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 20 mm β-glycerophosphate, 10 mm sodium pyrophosphate, 10 mm sodium vanadate, 1 μl/ml leupeptin, 1 mm NaF, 1 mm phenylmethylsulfonyl fluoride, and protease inhibitor mixture (Sigma). The insoluble protein was dissolved in 1× NuPAGE SDS sample buffer, and equal amounts of proteins were loaded onto 12% NuPAGE bis-tris gel and transferred to a nitrocellulose membrane. The membrane was blocked with PBST (PBS, 0.1% Tween 20) containing 5% nonfat milk for 1 h and then incubated with ATRAP antibody for 2 h at room temperature. After three washes with PBST, the membrane was incubated with secondary antibody for 1 h at room temperature. Enhanced chemiluminescence (ECL) (Pierce) was used for detection. The membrane was stripped with Restore Western blot stripping buffer (Pierce) for 15 min at room temperature, and β-actin antibody was used to determine equal protein loading. Secondary antibodies were either anti-rabbit IgG/horseradish peroxidase conjugate or anti-mouse IgG/horseradish peroxidase conjugate (1:1000 dilution, Amersham Biosciences) as required. Our laboratory previously identified the novel protein ATRAP that interacts with the C-terminal end of mouse AT1 receptor. The functional significance of ATRAP in AT1 receptor signaling remains unclear. To better understand the molecular mechanisms of ATRAP-mediated AT1 signaling, we employed the yeast two-hybrid screen to identify candidate proteins that interact with mouse ATRAP. Screening of 3 × 106 transformants from a mouse testis primary cDNA library resulted in the isolation of several independent clones that have the potential to interact with ATRAP. Sequence analysis identified 12 clones that encoded for CAML. On the basis of information from the yeast screening, mouse CAML full-length cDNA was cloned into the yeast two-hybrid expression vector pGADT7. As shown in Fig. 1, A and B, CAML expressed in the reporter strain AH109 does not interact with lamin C, but CAML expressed in yeast can interact with ATRAP by activating the reporter gene α-galactosidase. Furthermore, using a series of deletions of ATRAP, we were able to determine that the N-terminal domain of ATRAP (aa 40–80) primarily mediates this interaction. No interaction between CAML and AT1 or AT2 was observed. To determine the CAML structural domain involved in the interaction with ATRAP, the N-terminal end of CAML spanning aa 1–189 and C-terminal end of CAML spanning aa 189–296 were cloned into two-hybrid expression vector pGADT7. As shown in Fig. 1B, a strong interaction between ATRAP and the N-terminal end of CAML (aa 1–189) was observed; in contrast, the C-terminal end of CAML (aa 189–296) does not interact with ATRAP. We next validated the interaction of ATRAP and CAML in cells by immunoprecipitation assays. We transfected HEK293 cells with the expression vector pCMV-HA encoding CAML aa 1–189, CAML aa 189–296, or no insert. As shown in Fig. 1C, the N-terminal end of CAML (aa 1–189) but not the C-terminal end of CAML (aa 189–296) was able to interact with endogenous ATRAP, which is consistent with the result from a yeast assay, as shown in Fig. 1B. Moreover, we confirmed the interaction of CAML with ATRAP in intact cells by the use of BRET assays. BRET is a sensitive technology developed for detecting protein-protein interactions (20.Xu Y. Piston D.W. Johnson C.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 151-156Crossref PubMed Scopus (501) Google Scholar) in living cells. As shown in Fig. 1D, HEK293 cells expressing chimeric forms of ATRAP fused to GFP and CAML fused to luciferase provided a significant BRET signal only when ATRAP was tagged with GFP at the C-terminal end and when luciferase was tagged at the N-terminal end of CAML. We next tested whether the subcellular distribution of ATRAP colocalizes with that of CAML. Immunohistochemistry of endogenous ATRAP and CAML has shown these proteins located in endoplasmic reticulum vesicular structures (14.Lopez-Ilasaca M. Liu X. Tamura K. Dzau V.J. Mol. Biol. Cell. 2003; 14: 5038-5050Crossref PubMed Scopus (91) Google Scholar, 21.Holloway M.P. Bram R.J. J. Biol. Chem. 1998; 273: 16346-16350Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 22.Holloway M.P. Bram R.J. J. Biol. Chem. 1996; 271: 8549-8552Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). As shown in the Fig. 2C, cotransfection of GFP·CAML and RFP·ATRAP in HEK293 cells shows a sharp colocalization of both proteins at perinuclear endoplasmic reticulum structures, in a pattern similar to the distribution of endogenous ATRAP (14.Lopez-Ilasaca M. Liu X. Tamura K. Dzau V.J. Mol. Biol. Cell. 2003; 14: 5038-5050Crossref PubMed Scopus (91) Google Scholar). Furthermore, we determined the distribution of N-terminal end and C-terminal deletion mutants of CAML. As expected, the N-terminal end of CAML (aa 1–189) projects the protein into cytoplasm, whereas the C-terminal end of CAML (aa 189–296) is membrane-associated, showing a pattern similar to the full-length CAML (data not shown). These results provide evidence for ATRAP and CAML interaction both in yeast and in mammalian cells. We next sought to determine the functional significance of the interaction between ATRAP and CAML. CAML protein has been shown to participate in the activation of NFAT transcription factor in T cells by stimulating calcium release and then activating calcineurin (23.Bram R.J. Crabtree G.R. Nature. 1994; 371: 355-358Crossref PubMed Scopus (143) Google Scholar). Moreover, it has been reported that Ang II is an activator for the calcineurin/NFAT pathway in vascular smooth muscle cells and cardiac myocytes, where it contributes to cardiac hypertrophy and heart failure (25.Suzuki E. Nishimatsu H. Satonaka H. Walsh K. Goto A. Omata M. Fujita T. Nagai R. Hirata Y. Circ. Res. 2002; 90: 1004-1011Crossref PubMed Scopus (66) Google Scholar, 26.Molkentin J.D. Lu J.R. Antos C.L. Markham B. Richardson J. Robbins J. Grant S.R. Olson E.N. Cell. 1998; 93: 215-228Abstract Full Text Full Text PDF PubMed Scopus (2197) Google Scholar). We hypothesized that CAML mediated the effect of Ang II in regulation of NFAT signaling. As shown in Fig. 3A, overexpression of CAML mimics the effect of Ang II; however, there was little additive effect of Ang II in cells transfected with CAML expression vector. Deletion of the NFAT consensus sequence on the reporter gene abolished the effect of Ang II or CAML (data not shown). Previous studies have suggested that the N-terminal end of CAML is responsible for mediating protein interactions, whereas the C-terminal acts as an effector domain (17.von Bulow G.U. Bram R.J. Science. 1997; 278: 138-141Crossref PubMed Scopus (310) Google Scholar, 21.Holloway M.P. Bram R.J. J. Biol. Chem. 1998; 273: 16346-16350Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar); therefore, we further tested whether the expression of truncated forms of CAML could modulate the effect of Ang II on NFAT promoter activity. As shown in Fig. 3B, overexpression of ATRAP-interacting domain of CAML (aa 1–189) sensitized the effect of Ang II on NFAT promoter activity by increasing its activation by 12-fold over the untreated control, whereas overexpression of the C-terminal end of CAML (aa 189–296) disrupted the effect of Ang II. The expression of CAML truncations did not significantly change the basal level of the reporter gene activity. These data suggest that CAML is actively involved the Ang II-induced NFAT signal pathway. We next determined whether ATRAP is a critical mediator for the Ang II-CAML-NFAT pathway. As shown in Fig. 4A, treatment of cells with Ang II or overexpression of CAML induced the NFAT reporter gene by 4-fold. In contrast, overexpression of ATRAP decreased the basal level of NFAT reporter gene expression and disrupted Ang II-induced promoter activity. Moreover, overexpression of ATRAP blocked CAML-induced promoter activity. These results indicate that ATRAP is involved in the NFAT pathway activated by Ang II and may be a negative regulator in the AT1 signaling pathway. To confirm the putative role of ATRAP in the NFAT pathway, we examined the effect of RNA interference ATRAP knockdown in the regulation of promoter activity in HEK293 cells. As shown in Fig. 4B, transfection of 40 nm ATRAP siRNA significantly decreased ATRAP protein levels, whereas the control glyceraldehyde-3-phosphate dehydrogenase siRNA had no effect on the protein levels of ATRAP. Furthermore, transfection of 40 nm ATRAP siRNA increased the basal level of NFAT promoter activity by 2-fold, and knockdown of ATRAP sensitized the effect of Ang II on promoter activity, increasing it by 6-fold (Fig. 4C). Thus, CAML is actively involved in the Ang II-induced NFAT signaling pathway by interacting with ATRAP. We explored the signaling cascade of AT1 receptors by identifying candidate proteins that can interact with ATRAP. Discovering that CAML as a partner for ATRAP enabled us to examine the molecular mechanisms by which ATRAP is involved in AT1 receptor-mediated cellular actions. CAML was initially identified as an important regulator of the T cell receptor signaling pathway (21.Holloway M.P. Bram R.J. J. Biol. Chem. 1998; 273: 16346-16350Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 22.Holloway M.P. Bram R.J. J. Biol. Chem. 1996; 271: 8549-8552Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 23.Bram R.J. Crabtree G.R. Nature. 1994; 371: 355-358Crossref PubMed Scopus (143) Google Scholar), where it causes increase of Ca2+ influx, which can in turn activate the phosphatase calcineurin, leading to dephosphorylation of NFAT transcription factor. CAML has been implicated in the signal transduction cascade from a member of the tumor necrosis receptor family (transmembrane activator and CAML interactor (TACI)) to activation of NFAT, AP-1, and NFκB transcription factor (17.von Bulow G.U. Bram R.J. Science. 1997; 278: 138-141Crossref PubMed Scopus (310) Google Scholar) and also has been reported to interact with epidermal growth factor receptor (24.Tran D.D. Russell H.R. Sutor S.L. van Deursen J. Bram R.J. Dev. Cell. 2003; 5: 245-256Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). CAML is thought to be a signal intermediate in T cell receptor signaling and may act upstream of calcineurin/NFAT and downstream of T cell receptor. Several other lines of evidence have shown that Ang II can activate the NFAT pathway, which contributes to cell growth and cardiac hypertrophy (25.Suzuki E. Nishimatsu H. Satonaka H. Walsh K. Goto A. Omata M. Fujita T. Nagai R. Hirata Y. Circ. Res. 2002; 90: 1004-1011Crossref PubMed Scopus (66) Google Scholar, 26.Molkentin J.D. Lu J.R. Antos C.L. Markham B. Richardson J. Robbins J. Grant S.R. Olson E.N. Cell. 1998; 93: 215-228Abstract Full Text Full Text PDF PubMed Scopus (2197) Google Scholar, 27.Wilkins B.J. Dai Y.S. Bueno O.F. Parsons S.A. Xu J. Plank D.M. Jones F. Kimball T.R. Molkentin J.D. Circ. Res. 2004; 94: 110-118Crossref PubMed Scopus (607) Google Scholar). The finding of CAML associated with the AT1/ATRAP complex may help us to elucidate a molecular link between AT1 receptor signaling and the downstream activation of NFAT signaling. Our previous studies of the association of ATRAP with AT1 receptor suggest that ATRAP reduces Ang II-induced inositol-1,4,5-triphosphate generation (14.Lopez-Ilasaca M. Liu X. Tamura K. Dzau V.J. Mol. Biol. Cell. 2003; 14: 5038-5050Crossref PubMed Scopus (91) Google Scholar, 15.Daviet L. Lehtonen J.Y. Tamura K. Griese D.P. Horiuchi M. Dzau V.J. J. Biol. Chem. 1999; 274: 17058-17062Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). In the current study, we employed gain-of-function and loss-of-function approaches and demonstrated that ATRAP negatively regulates Ang II-induced NFAT signaling. Overexpression of ATRAP in cells not only blocked the Ang II-induced NFAT reporter gene activity but also impaired the CAML-mediated transactivation of the NFAT reporter gene, suggesting that ATRAP disrupts the Ang II effect on NFAT by interacting with CAML, leading to down-regulation of cell signaling. Conversely, the ATRAP knockdown by siRNA increased the NFAT transcriptional activity and sensitized the effect of Ang II on gene expression. Interestingly, overexpression of the N-terminal end (aa 1–189) of CAML, which can interact with ATRAP, sensitized the effect of Ang II on NFAT reporter gene activity, suggesting that CAML N-terminal end might act in a dominant negative fashion for ATRAP, and the interaction between ATRAP and N-terminal end of CAML releases the inhibition of ATRAP for downstream signaling. On the basis of our current understanding of ATRAP and CAML interaction, we proposed a model to interpret the interaction between ATRAP and CAML in cells, as shown in Fig. 5. In this model, the AT1 receptor complex consists of different protein partners, of which CAML acts as a mediator and ATRAP acts as a desensitizer. ATRAP could act as a brake for the AT1 receptor activation toward CAML-NFAT signaling. On the other hand, CAML could also be able to regulate the ATRAP inhibitory role since overexpression of an ATRAP-interacting domain of CAML (aa 1–189) increased NFAT activity, probably by interfering with ATRAP actions. There may also be an additional not yet identified X factor that could act as a modulator of ATRAP; this factor could be displaced by overexpression of the N-terminal domain of CAML (aa 1–189). On the other hand, CAML has been shown to interact with transmembrane activator and CAML interactor in cell membranes and/or intracellular Ca2+ pools, such as the sarcoplasmic reticulum (17.von Bulow G.U. Bram R.J. Science. 1997; 278: 138-141Crossref PubMed Scopus (310) Google Scholar, 21.Holloway M.P. Bram R.J. J. Biol. Chem. 1998; 273: 16346-16350Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 22.Holloway M.P. Bram R.J. J. Biol. Chem. 1996; 271: 8549-8552Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 23.Bram R.J. Crabtree G.R. Nature. 1994; 371: 355-358Crossref PubMed Scopus (143) Google Scholar). Considering the colocalization of CAML and ATRAP, we speculate that their interaction in the endoplasmic reticulum may also regulate intracellular Ca2+ fluxes, dependent on or independent of AT1 signaling. The pools of endogenous ATRAP and CAML provide a regulatory system for the Ang II signaling onto NFAT. In this study, we provide evidence that the interaction of ATRAP and CAML mediates the action of Ang II on NFAT activation. However, several important issues remain unclear. For example, to what extent does the interaction of ATRAP and CAML control the physiological function of Ang II? What is the role of this pathway in regulating vascular contraction in which Ca2+ metabolism is actively involved? What is the functional significance of the ATRAP pathway in regulating cardiac hypertrophy? Molkentin's group (26.Molkentin J.D. Lu J.R. Antos C.L. Markham B. Richardson J. Robbins J. Grant S.R. Olson E.N. Cell. 1998; 93: 215-228Abstract Full Text Full Text PDF PubMed Scopus (2197) Google Scholar, 27.Wilkins B.J. Dai Y.S. Bueno O.F. Parsons S.A. Xu J. Plank D.M. Jones F. Kimball T.R. Molkentin J.D. Circ. Res. 2004; 94: 110-118Crossref PubMed Scopus (607) Google Scholar, 28.Wilkins B.J. Molkentin J.D. Biochem. Biophys. Res. Commun. 2004; 322: 1178-1191Crossref PubMed Scopus (365) Google Scholar) reported that the calcineurin/NFAT pathway is activated during pathological cardiac hypertrophy and failure. Our preliminary data show that CAML is up-regulated, whereas ATRAP is down-regulated in a mouse model of cardiac hypertrophy. 2S. Guo, M. Lopez-Ilasaca, and V. J. Dzau, unpublished observations. The elucidation of the molecular mechanisms of Ang II actions and the role of ATRAP and CAML in calcineurin and NFAT activation may lead to the identification of novel drug targets for the treatment of pathological cardiac and vascular hypertrophy and remodeling. We thank Dr. Richard E. Pratt for useful discussions." @default.
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