Agonist-independent Nuclear Localization of the Apelin, Angiotensin AT1, and Bradykinin B2 Receptors
2004; Elsevier BV; Volume: 279; Issue: 9 Linguagem: Inglês
10.1074/jbc.m306377200
ISSN1083-351X
AutoresDennis K. Lee, A.J. Lança, Regina Cheng, Tuan Nguyen, Xiao Dong Ji, Fernand Gobeil, Sylvain Chemtob, Susan R. George, Brian F. O’Dowd,
Tópico(s)Cardiovascular, Neuropeptides, and Oxidative Stress Research
ResumoSignaling of the apelin, angiotensin, and bradykinin peptides is mediated by G protein-coupled receptors related through structure and similarities of physiological function. We report nuclear expression as a characteristic of these receptors, including a nuclear localization for the apelin receptor in brain and cerebellum-derived D283 Med cells and the AT1 and bradykinin B2 receptors in HEK-293T cells. Immunocytochemical analyses revealed the apelin receptor with localization in neuronal nuclei in cerebellum and hypothalamus, exhibiting expression in neuronal cytoplasm or in both nuclei and cytoplasm. Confocal microscopy of HEK-293T cells revealed the majority of transfected cells displayed constitutive nuclear localization of AT1 and B2 receptors, whereas apelin receptors did not show nuclear localization in these cells. The majority of apelin receptor-transfected cerebellum D283 Med cells showed receptor nuclear expression. Immunoblot analyses of subcellular-fractionated D283 Med cells demonstrated endogenous apelin receptor species in nuclear fractions. In addition, an identified nuclear localization signal motif in the third intracellular loop of the apelin receptor was disrupted by a substituted glutamine in place of lysine. This apelin receptor (K242Q) did not exhibit nuclear localization in D283 Med cells. These results demonstrate the following: (i) the apelin receptor exhibits nuclear localization in human brain; (ii) distinct cell-dependent mechanisms for the nuclear transport of apelin, AT1, and B2 receptors; and (iii) the disruption of a nuclear localization signal sequence disrupts the nuclear translocation of the apelin receptor. This discovery of apelin, AT1, and B2 receptors with agonist-independent nuclear translocation suggests major unanticipated roles for these receptors in cell signaling and function. Signaling of the apelin, angiotensin, and bradykinin peptides is mediated by G protein-coupled receptors related through structure and similarities of physiological function. We report nuclear expression as a characteristic of these receptors, including a nuclear localization for the apelin receptor in brain and cerebellum-derived D283 Med cells and the AT1 and bradykinin B2 receptors in HEK-293T cells. Immunocytochemical analyses revealed the apelin receptor with localization in neuronal nuclei in cerebellum and hypothalamus, exhibiting expression in neuronal cytoplasm or in both nuclei and cytoplasm. Confocal microscopy of HEK-293T cells revealed the majority of transfected cells displayed constitutive nuclear localization of AT1 and B2 receptors, whereas apelin receptors did not show nuclear localization in these cells. The majority of apelin receptor-transfected cerebellum D283 Med cells showed receptor nuclear expression. Immunoblot analyses of subcellular-fractionated D283 Med cells demonstrated endogenous apelin receptor species in nuclear fractions. In addition, an identified nuclear localization signal motif in the third intracellular loop of the apelin receptor was disrupted by a substituted glutamine in place of lysine. This apelin receptor (K242Q) did not exhibit nuclear localization in D283 Med cells. These results demonstrate the following: (i) the apelin receptor exhibits nuclear localization in human brain; (ii) distinct cell-dependent mechanisms for the nuclear transport of apelin, AT1, and B2 receptors; and (iii) the disruption of a nuclear localization signal sequence disrupts the nuclear translocation of the apelin receptor. This discovery of apelin, AT1, and B2 receptors with agonist-independent nuclear translocation suggests major unanticipated roles for these receptors in cell signaling and function. The apelin receptor was discovered as an orphan G protein-coupled receptor (GPCR) 1The abbreviations used are: GPCR(s), G protein-coupled receptor(s); GFP, green fluorescent protein; IR, immunoreactivity; MEM, minimal essential medium; mRFP, monomeric red fluorescent protein; NLS, nuclear localization signal; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride. known as APJ, sharing highest identity with the angiotensin II AT1 receptor, although no binding to the receptor was observed with angiotensin II (1O'Dowd B.F. Heiber M. Chan A. Heng H.H. Tsui L.C. Kennedy J.L. Shi X. Petronis A. George S.R. Nguyen T. Gene (Amst.). 1993; 136: 355-360Crossref PubMed Scopus (697) Google Scholar). The apelin peptide was subsequently discovered as the endogenous ligand for this receptor (2Tatemoto K. Hosoya M. Habata Y. Fujii R. Kakegawa T. Zou M.X. Kawamata Y. Fukusumi S. Hinuma S. Kitada C. Kurokawa T. Onda H. Fujino M. Biochem. Biophys. Res. 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Overall, the apelinergic system is most closely related to the angiotensin and bradykinin systems, as observed through peptide and receptor structural and sequence similarities, expression patterns, and physiology (6Lee D.K. Cheng R. Nguyen T. Fan T. Kariyawasam A.P. Liu Y. Osmond D.H. George S.R. O'Dowd B.F. J. Neurochem. 2000; 74: 34-41Crossref PubMed Scopus (584) Google Scholar, 20AbdAlla S. Lother H. Quitterer U. Nature. 2000; 407: 94-98Crossref PubMed Scopus (441) Google Scholar). Apelin, angiotensin II, and bradykinin all have blood pressure-modulating effects, expression in cardiovascular tissue, and are cleaved by the human endothelial angiotensin I-converting enzymes (21Vickers C. Hales P. Kaushik V. Dick L. Gavin J. Tang J. Godbout K. Parsons T. Baronas E. Hsieh F. Acton S. Patane M. Nichols A. Tummino P. J. Biol. Chem. 2002; 277: 14838-14843Abstract Full Text Full Text PDF PubMed Scopus (1156) Google Scholar). 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Whereas GPCRs have been best characterized as cell-surface mediators of signal transudation, there are few reports of GPCRs capable of nuclear translocation. In the large family I rhodopsin-like family of GPCRs, the AT1 receptor has been best characterized to have nuclear localization. Early studies revealed angiotensin II-binding sites were present in nuclei with angiotensin II-induced transcription of renin and angiotensinogen mRNA (25Eggena P. Zhu J.H. Clegg K. Barrett J.D. Hypertension. 1993; 22: 496-501Crossref PubMed Scopus (126) Google Scholar), and the nuclear localization of the AT1 receptor was reported to be induced by angiotensin II (26Chen R. Mukhin Y.V. Garnovskaya M.N. Thielen T.E. Iijima Y. Huang C. Raymond J.R. Ullian M.E. Paul R.V. Am. J. Physiol. 2000; 279: F440-F448Crossref PubMed Google Scholar, 27Lu D. Yang H. Shaw G. Raizada M.K. Endocrinology. 1998; 139: 365-375Crossref PubMed Google Scholar). In addition, a family II GPCR, the parathyroid hormone receptor, was observed to localize to the nucleus both in tissues and cultured cells (28Watson P.H. Fraher L.J. Natale B.V. Kisiel M. Hendy G.N. Hodsman A.B. Bone (NY). 2000; 26: 221-225Crossref PubMed Scopus (61) Google Scholar), and a recent study observed a family III GPCR, the metabotropic glutamate mGluR5 receptor, in nuclear membranes (29O'Malley K.L. Jong Y.J. Gonchar Y. Burkhalter A. Romano C. J. Biol. Chem. 2003; 278: 28210-28219Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). The prostaglandin EP1 (30Bhattacharya M. Peri K.G. Almazan G. Ribeiro-da-Silva A. Shichi H. Durocher Y. Abramovitz M. Hou X. Varma D.R. Chemtob S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15792-15797Crossref PubMed Scopus (218) Google Scholar), EP3, and EP4 receptors (31Bhattacharya M. Peri K. Ribeiro-da-Silva A. Almazan G. Shichi H. Hou X. Varma D.R. Chemtob S. J. Biol. Chem. 1999; 274: 15719-15724Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar) were observed to have nuclear membrane localization, whereas a recent report (32Boivin B. Chevalier D. Villeneuve L.R. Rousseau E. Allen B.G. J. Biol. Chem. 2003; 278: 29153-29163Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) described the endothelin ETA and ETB receptors to be localized in the nuclear membrane, with ETB receptors present in the interior of the nucleus as well. In the present study we report for the first time a nuclear localization of the apelin receptor in brain. Following our observations of immunocytochemical detection of apelin receptors in cell nuclei, we carried out experiments involving immunoblot analyses of subcellular fractionated cells and confocal microscopy of cells to verify the nuclear distribution. Previously, the AT1 receptor was reported to traffic to cell nuclei by the presence of a nuclear localization signal (NLS) sequence (27Lu D. Yang H. Shaw G. Raizada M.K. Endocrinology. 1998; 139: 365-375Crossref PubMed Google Scholar). Identification of a similar sequence in the closely related B2 receptor suggested comparable nuclear localization of this receptor. In addition, the presence of an NLS-like sequence in an alternate position in the apelin receptor suggested distinctive patterns of nuclear localization of the apelin receptor compared with the AT1 receptor. We have utilized confocal microscopy analyses to (i) compare subcellular distributions of the apelin, AT1, and B2 receptors in various cell lines and (ii) to confirm the identity of the apelin receptor NLS sequence using a site-directed mutagenesis strategy. Together, our results provide evidence of an additional commonality between the apelin, AT1, and B2 receptors which is that of nuclear expression due to the presence of NLS motifs. Construction of cDNA Encoding Apelin, AT1, and B2 Receptors—DNA encoding the human and rat apelin receptors were obtained as described previously (1O'Dowd B.F. Heiber M. Chan A. Heng H.H. Tsui L.C. Kennedy J.L. Shi X. Petronis A. George S.R. Nguyen T. Gene (Amst.). 1993; 136: 355-360Crossref PubMed Scopus (697) Google Scholar, 6Lee D.K. Cheng R. Nguyen T. Fan T. Kariyawasam A.P. Liu Y. Osmond D.H. George S.R. O'Dowd B.F. J. Neurochem. 2000; 74: 34-41Crossref PubMed Scopus (584) Google Scholar). The cDNA encoding AT1 receptor was a gift from Dr. Sylvain Meloche (University of Montreal, Montreal, Québec, Canada). The cDNA encoding the B2 receptor was a gift from Dr. Fred Hess (Merck). The cDNA encoding monomeric red fluorescent protein mRFP1 was a gift from Dr. Roger Tsien (University of California, San Diego). The cDNA encoding arrestin1-GFP was a gift from Dr. Stephen Ferguson (University of Western Ontario, London, Ontario, Canada). The stop codons of cDNAs encoding the human apelin, angiotensin II AT1, bradykinin B2, and dopamine D1 receptors were modified to contain BamHI or KpnI sites by PCR amplification from vectors containing these full-length coding regions. These fragments were subsequently cloned in-frame into the pEGFP-N1 vector (BD Biosciences Clontech, Palo Alto, CA) or the mRFP-1 vector. For epitope-tagged human and rat apelin receptors, a C9 epitope tag sequence (encoding the nine carboxyl-terminal amino acids of rhodopsin) was inserted just prior to the stop codons by PCR mutagenesis. Cell Culture and Transfection of Cells—COS-7 monkey kidney, human embryonic kidney (HEK-293T), and human cerebellum D283 Med cells (American Type Culture Collection, Manassas, VA) were maintained as monolayer cultures at 37 °C. COS-7 and HEK-293T cells were maintained in minimum essential medium (MEM) and the D283 Med cells in MEM with 2 mm l-glutamine, Earle's balanced salt solution with 1.5 g/liter sodium bicarbonate, 0.1 mm non-essential amino acids, and 1.0 mm sodium pyruvate. All media were supplemented with 10% fetal bovine serum and antibiotics. COS-7 and HEK-293T cells were transiently transfected with cDNA by the use of LipofectAMINE reagent (Invitrogen), whereas D283 Med cells were transiently transfected using FuGENE 6 transfection reagent (Roche Applied Science). Confocal Microscopy—For the live cell βarrestin1 activation assay, HEK-293T cells were transfected with DNA encoding βarrestin1-GFP and GRK2 with or without DNA encoding the apelin receptor-mRFP constructs for 48 h. 10 μm apelin-13 was administered to these cells, and confocal images were taken every minute. HEK-293T cells were co-transfected with a nuclear marker, DsRed2-Nuc (BD Biosciences Clontech), and either the apelin receptor-GFP, AT1 receptor-GFP, or B2 receptor-GFP construct for 48 h. D283 Med cells were transfected with either apelin receptor-GFP, apelin receptor (K242Q)-GFP, AT1 receptor-GFP, or the dopamine D1 receptor-GFP. Live cell confocal microscopy was performed with a Zeiss LSM 510 microscope. Cell counts for nuclear localization were obtained from at least 100 and 250 cells for D283 Med cells and HEK-293T, respectively. Images were acquired and processed with Zeiss LSM Image Browser software. Membrane Preparation—COS-7 cells were washed extensively with PBS. P2 pellets were prepared by Polytron disruption in ice-cold 5:2 lysis buffer (containing 5 mm Tris-HCl, 2 mm EDTA buffer, containing 5 μg/ml leupeptin, 10 μg/ml benzamidine, and 5 μg/ml soybean trypsin inhibitor) as described previously (33Ng G.Y. O'Dowd B.F. Lee S.P. Chung H.T. Brann M.R. Seeman P. George S.R. Biochem. Biophys. Res. Commun. 1996; 227: 200-204Crossref PubMed Scopus (246) Google Scholar). Subcellular Fractionation—Cell fractionation of D283 Med cells was performed by a modified method of the previously reported hypotonic/Nonidet P-40 lysis (34Gobeil Jr., F. Dumont I. Marrache A.M. Vazquez-Tello A. Bernier S.G. Abran D. Hou X. Beauchamp M.H. Quiniou C. Bouayad A. Choufani S. Bhattacharya M. Molotchnikoff S. Ribeiro-Da-Silva A. Varma D.R. Bkaily G. Chemtob S. Circ. Res. 2002; 90: 682-689Crossref PubMed Scopus (116) Google Scholar). Briefly, washed and pelleted cells were resuspended in lysis buffer (10 mm Tris-HCl, pH 7.4, 10 mm NaCl, 3 mm MgCl2, 5 μg/ml leupeptin, 10 μg/ml benzamidine, and 5 μg/ml soybean trypsin inhibitor), homogenized by several short bursts of low-force Polytron disruption on ice, and then centrifuged at 700 × g for 10 min at 4 °C. The supernatant and nuclei pellets were separated for recovery of P2 and nuclear fractions, respectively. The supernatant was centrifuged at 35,000 × g for 15 min at 4 °C to collect the P2 fraction. The nuclear pellet was resuspended in lysis buffer, layered over a discontinuous sucrose gradient of 4.5 ml of 2.0 and 1.6 m sucrose containing 1 mm MgCl2, and centrifuged at 100,000 × g for 1 h at 4 °C. The morphological integrity and purity of the samples were assessed by light microscopy after cresyl violet staining and with a plasma membrane marker 5′-nucleotidase assay kit (Sigma). Protein levels were determined by the Bradford assay according to the manufacturer's instructions (Bio-Rad). Nuclear fractionation of unfixed human cerebellum tissue of a control 22-year-old Caucasian male (Brain and Tissue Banks for Developmental Disorders, University of Maryland, Baltimore, MD) was performed by a modified version of the Gorski method (35Waxman D.J. Ram P.A. Park S.H. Choi H.K. J. Biol. Chem. 1995; 270: 13262-13270Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Briefly, 4 g of cerebellum tissue was minced in ice-cold homogenization buffer containing sodium fluoride (10 mm), sodium orthovanadate (1 mm), 5 μg/ml aprotinin, 0.1 mm phenylmethylsulfonyl fluoride, 2 μg/ml antipain, chymostatin, pepstatin, 5 μg/ml leupeptin, and 10 μg/ml trypsin inhibitor. Samples were homogenized on ice using a motor-driven Teflon-glass homogenizer (5 strokes), diluted to 75 ml with homogenization buffer, and layered over cushions of homogenization buffer. These samples were then centrifuged at 76,000 × g for 30 min at -2 °C. The nuclear pellet was resuspended in 2 ml of nuclear lysis buffer, left on ice for 30 min, gently homogenized (5 strokes), and lysed further for 30 min. The sample was further diluted to yield ∼0.5 mg/ml DNA concentration (determined by UV absorbance at 260 nm), and 0.1 times the total volume of 4.0 m (NH4)2SO4 was added dropwise. The sample was gently shaken for 30 min on ice and then centrifuged at 90,000 × g for 1hat0 °C. 0.3 g of (NH4)2SO4 was added per ml of supernatant, shaken for 1 h on ice, centrifuged at 90,000 × g for 25 min at 0 °C, and pellets stored overnight at 4 °C. Sample purity and protein levels were determined as for D283 Med cells. Immunoblot Analyses—Nuclear fractions were treated with DNase and RNase (0.5 μg/μl each, Marligen Biosciences, Inc., Ijamsville, MD) for 20 min at 37 °C. The protein samples were solubilized in sample buffer consisting of 50 mm Tris-HCl, pH 6.5, 1% SDS, 10% glycerol, 0.003% bromphenol blue, and 10% 2-mercaptoethanol. The samples were subjected to PAGE with 12% acrylamide gels and electroblotted onto PVDF transfer membrane as described previously (33Ng G.Y. O'Dowd B.F. Lee S.P. Chung H.T. Brann M.R. Seeman P. George S.R. Biochem. Biophys. Res. Commun. 1996; 227: 200-204Crossref PubMed Scopus (246) Google Scholar). Apelin receptor immunoreactivity (IR) was revealed with a mouse monoclonal antibody at a dilution of 1:1000 (catalogue number MAB856, R&D Systems, Inc., Minneapolis, MN). This antibody was raised using membrane-embedded apelin receptors (36Puffer B.A. Sharron M. Coughlan C.M. Baribaud F. McManus C.M. Lee B. David J. Price K. Horuk R. Tsang M. Doms R.W. Virology. 2000; 276: 435-444Crossref PubMed Scopus (38) Google Scholar). Initial experiments with this antibody were made possible by a generous gift from Dr. Robert W. Doms (University of Pennsylvania Philadelphia, PA). C9 epitope-tagged receptor-IR was detected with the 1D4 antibody (National Cell Culture Center, Minneapolis, MN). In addition, immunoblot analyses to test the purity of the subcellular fractionated samples were performed using antibodies specific for nucleoporin p62 (nuclear fraction, number N43620), annexin II (plasma membrane and intracellular vesicles, number A14020), and GM130 (Golgi apparatus, number G65120) from BD Transduction Laboratories (Lexington, KY). Immunocytochemistry—Fixed cerebellum and hypothalamic samples from the same human individual used in the subcellular fractionation immunoblot analysis as well as samples from an additional human individual (Brain and Tissue Banks for Developmental Disorders) were cut on a cryostat 32 μm thick and mounted on gelatin-coated slides for immunocytochemical analysis as reported previously (37Lanca A.J. Wu P.H. Jung B. Liu J.F. Ng V. Kalant H. Neuroscience. 1999; 91: 1331-1341Crossref PubMed Scopus (8) Google Scholar). Briefly, fixed brain sections were pre-treated with 0.3% hydrogen peroxide in PBS for 30 min at room temperature, rinsed for 10 min in PBS, and incubated for 1 h at room temperature in 1% normal goat serum in PBS and 0.3% Triton X-100. The sections were washed (2 times for 10 min) in PBS/Triton X-100 and incubated with the apelin receptor antibody (1:1000 dilution) for 48 h at 4 °C. The sections were then washed (2 times for 10 min) in PBS/Triton X-100 and processed with the avidin-biotin complex method (Vectastain Elite Kit, Vector Laboratories, Burlingame, CA). Some of the previously immunostained sections were subsequently stained with cresyl violet (Nissl staining). Sections were dehydrated and mounted in Permount mounting medium via xylenes, viewed under a Zeiss Axioskop microscope, and photographed with Kodak ASA-160 film. Control experiments were also performed by using rat versus human cerebellum tissue or by the exclusion of primary antibody. Nuclear Localization of the Apelin Receptor in Human Brain—The similarities between the apelin, angiotensin, and bradykinin systems are observed from the shared sequence identities between their respective receptors and ligands, expression distribution, and roles in comparable physiological functions. Our studies observed levels of sequence identity between the apelin and angiotensin receptors (1O'Dowd B.F. Heiber M. Chan A. Heng H.H. Tsui L.C. Kennedy J.L. Shi X. Petronis A. George S.R. Nguyen T. Gene (Amst.). 1993; 136: 355-360Crossref PubMed Scopus (697) Google Scholar), as well as structural similarities between the apelin and angiotensin II peptides (6Lee D.K. Cheng R. Nguyen T. Fan T. Kariyawasam A.P. Liu Y. Osmond D.H. George S.R. O'Dowd B.F. J. Neurochem. 2000; 74: 34-41Crossref PubMed Scopus (584) Google Scholar). We also reported comparable expression distribution patterns between the two systems, particularly in the choroid plexus, hippocampus, and hypothalamus in the brain and associated roles in the modulation of blood pressure and water consumption behavior (6Lee D.K. Cheng R. Nguyen T. Fan T. Kariyawasam A.P. Liu Y. Osmond D.H. George S.R. O'Dowd B.F. J. Neurochem. 2000; 74: 34-41Crossref PubMed Scopus (584) Google Scholar). In this study, we continued to characterize the apelinergic system by investigating the distribution of the receptor. By using a specific antibody for the human apelin receptor (36Puffer B.A. Sharron M. Coughlan C.M. Baribaud F. McManus C.M. Lee B. David J. Price K. Horuk R. Tsang M. Doms R.W. Virology. 2000; 276: 435-444Crossref PubMed Scopus (38) Google Scholar), apelin receptor IR was observed abundantly in the cerebellum and paraventricular nucleus of the hypothalamus (Fig. 1). Apelin receptor distributions were performed on these two regions as they were determined previously (6Lee D.K. Cheng R. Nguyen T. Fan T. Kariyawasam A.P. Liu Y. Osmond D.H. George S.R. O'Dowd B.F. J. Neurochem. 2000; 74: 34-41Crossref PubMed Scopus (584) Google Scholar) to express high levels of apelin receptor mRNA. Virtually all neuronal cell nuclei in the granular layer of the cerebellum was densely labeled by the apelin receptor antibody, with abundant levels also observed in the cellular nuclei of the molecular layer (Fig. 1A). Purkinje cells displayed apelin receptor-IR in both the nucleus and cytoplasm. Cerebellar sections labeled for apelin receptor-IR (brown stain) were subsequently Nissl-stained with cresyl violet (purple stain), which confirmed the dense nuclear localization of the apelin receptor in the granular layer and demonstrated a heterogeneous population of nuclei with some nuclei negative for apelin receptor-IR in the molecular layer (Fig. 1B). In addition, some neurons had an exclusive cytoplasmic distribution of apelin receptor-IR, as seen in large pyramidal cells (Fig. 1C). In the paraventricular nucleus of the hypothalamus, there were two populations of apelin receptor-IR neurons, smaller neurons (parvocellular) with dense labeling in the nucleus and larger neurons (magnocellular) with labeling in the nucleus and cytoplasm (Fig. 1D). Control sections of human cerebellum processed without the apelin receptor antibody (Fig. 1E) or from rat cerebellum processed with both apelin receptor and secondary antibodies (Fig. 1F) were absent of immunoreactive signals. These results revealed specificity of the unique nuclear expression of the apelin receptor between different cell types in human brain. The specificity of the apelin receptor antibody was verified. DNA encoding the human or rat apelin receptors or empty vector DNA was transfected into COS-7 cells. Immunoblot analyses of P2 membrane fractions revealed specific bands in cells expressing the human apelin receptor, correlating to an expected molecular mass of ∼42 kDa for the unglycosylated, monomeric receptor as well as bands of higher molecular mass consistent with glycosylated, monomeric (∼45 kDa (36Puffer B.A. Sharron M. Coughlan C.M. Baribaud F. McManus C.M. Lee B. David J. Price K. Horuk R. Tsang M. Doms R.W. Virology. 2000; 276: 435-444Crossref PubMed Scopus (38) Google Scholar)), and dimeric receptor species (80–90 kDa) (Fig. 2A). Apelin receptor-specific bands were not detected in COS-7 cells expressing the rat apelin receptor, indicating the antibody to be species-specific despite the receptors having a high degree of sequence similarity (>90%). To confirm expression of rat apelin receptors, human and rat apelin receptors were fused with a rhodopsin-derived C9 epitope tag at their carboxyl-terminal ends and detected by immunoblot analyses using the 1D4 antibody. Immunoblot analyses of P2 membrane fractions revealed specific bands in cells expressing either the human or rat apelin receptors correlating to the unglycosylated (42 kDa) and glycosylated (∼45 kDa) monomers and dimers (80–90 kDa) (Fig. 2B), confirming that the apelin receptor antibody was highly specific for the human apelin receptor. The dis
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