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• W2041650648 abstract "Phosphorylation plays vital roles in the regulation of G protein-coupled receptor (GPCR) functions. The apelin and apelin receptor (APJ) system is involved in the regulation of cardiovascular function and central control of body homeostasis. Here, using tandem mass spectrometry, we first identified phosphorylated serine residues in the C terminus of APJ. To determine the role of phosphorylation sites in APJ-mediated G protein-dependent and -independent signaling and function, we induced a mutation in the C-terminal serine residues and examined their effects on the interaction between APJ with G protein or GRK/β-arrestin and their downstream signaling. Mutation of serine 348 led to an elimination of both GRK and β-arrestin recruitment to APJ induced by apelin-13. Moreover, APJ internalization and G protein-independent ERK signaling were also abolished by point mutation at serine 348. In contrast, this mutant at serine residues had no demonstrable impact on apelin-13-induced G protein activation and its intracellular signaling. These findings suggest that mutation of serine 348 resulted in inactive GRK/β-arrestin. However, there was no change in the active G protein thus, APJ conformation was biased. These results provide important information on the molecular interplay and impact of the APJ function, which may be extrapolated to design novel drugs for cardiac hypertrophy based on this biased signal pathway. Phosphorylation plays vital roles in the regulation of G protein-coupled receptor (GPCR) functions. The apelin and apelin receptor (APJ) system is involved in the regulation of cardiovascular function and central control of body homeostasis. Here, using tandem mass spectrometry, we first identified phosphorylated serine residues in the C terminus of APJ. To determine the role of phosphorylation sites in APJ-mediated G protein-dependent and -independent signaling and function, we induced a mutation in the C-terminal serine residues and examined their effects on the interaction between APJ with G protein or GRK/β-arrestin and their downstream signaling. Mutation of serine 348 led to an elimination of both GRK and β-arrestin recruitment to APJ induced by apelin-13. Moreover, APJ internalization and G protein-independent ERK signaling were also abolished by point mutation at serine 348. In contrast, this mutant at serine residues had no demonstrable impact on apelin-13-induced G protein activation and its intracellular signaling. These findings suggest that mutation of serine 348 resulted in inactive GRK/β-arrestin. However, there was no change in the active G protein thus, APJ conformation was biased. These results provide important information on the molecular interplay and impact of the APJ function, which may be extrapolated to design novel drugs for cardiac hypertrophy based on this biased signal pathway. G protein-coupled receptors (GPCRs), 3The abbreviations used are: GPCRG protein-coupled receptorsGRKG protein-coupled receptor kinaseAPJapelin receptorBRETbioluminescence resonance energy transferTRITCtetramethylrhodamine isothiocyanatePTXpertussis toxinAT1ARangiotensin type 1A receptorODoptical densityRlucRenilla reniformis luciferase. which constitute the largest superfamily of cell surface receptors involved in many cellular signaling and physiological responses, are activated by binding with peptides, lipids, small molecules, and hormones (1.Bockaert J. Pin J.P. Molecular tinkering of G protein-coupled receptors: an evolutionary success.EMBO J. 1999; 18: 1723-1729Crossref PubMed Scopus (1228) Google Scholar, 2.Pierce K.L. Premont R.T. Lefkowitz R.J. Seven-transmembrane receptors.Nat. Rev. Mol. Cell Biol. 2002; 3: 639-650Crossref PubMed Scopus (2097) Google Scholar). Consequently, GPCRs are extensively researched within the context of both potential and established drug targets (3.Ma P. Zemmel R. Value of novelty?.Nat. Rev. Drug Discov. 2002; 1: 571-572Crossref PubMed Scopus (158) Google Scholar). G protein-coupled receptors G protein-coupled receptor kinase apelin receptor bioluminescence resonance energy transfer tetramethylrhodamine isothiocyanate pertussis toxin angiotensin type 1A receptor optical density Renilla reniformis luciferase. Upon stimulation by agonists, GPCRs are activated and initiate intracellular signaling events. In addition to G protein-dependent signaling, the activated GPCRs may also interact with many intracellular proteins to expand their ability to transmit signals through G protein-independent signaling pathway, such as G protein-coupled receptor kinases (GRKs) and β-arrestins. In this process, the agonist-occupied receptor is phosphorylated by GRKs, subsequently recruiting cytoplasmic β-arrestins to the GRK-phosphorylated receptor (4.Kohout T.A. Lefkowitz R.J. Regulation of G protein-coupled receptor kinases and arrestins during receptor desensitization.Mol. Pharmacol. 2003; 63: 9-18Crossref PubMed Scopus (358) Google Scholar). As intracellular adaptor and scaffolding proteins, β-arrestins play important roles in GPCRs desensitization, internalization, and intracellular trafficking and could activate signaling cascades independently of G protein activation (5.Luttrell L.M. Lefkowitz R.J. The role of beta-arrestins in the termination and transduction of G-protein-coupled receptor signals.J. Cell Sci. 2002; 115: 455-465Crossref PubMed Google Scholar, 6.DeWire S.M. Ahn S. Lefkowitz R.J. Shenoy S.K. β-Arrestins and cell signaling.Annu. Rev. Physiol. 2007; 69: 483-510Crossref PubMed Scopus (1152) Google Scholar). For example, β-arrestin2 binds Raf-1 and ERK1/2 directly and MEK-1 indirectly, leading to a β-arrestin-dependent ERK activation (7.DeFea K.A. Zalevsky J. Thoma M.S. Déry O. Mullins R.D. Bunnett N.W. β-Arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2.J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (686) Google Scholar, 8.Luttrell L.M. Roudabush F.L. Choy E.W. Miller W.E. Field M.E. Pierce K.L. Lefkowitz R.J. Activation and targeting of extracellular signal-regulated kinases by β-arrestin scaffolds.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 2449-2454Crossref PubMed Scopus (701) Google Scholar). Apelin receptor (APJ) is a member of the family A of GPCRs with a range of physiological functions including the regulation of cardiovascular function and fluid homeostasis (9.Katugampola S.D. Maguire J.J. Matthewson S.R. Davenport A.P. [125I]-(Pyr(1))Apelin-13 is a novel radioligand for localizing the APJ orphan receptor in human and rat tissues with evidence for a vasoconstrictor role in man.Br. J. Pharmacol. 2001; 132: 1255-1260Crossref PubMed Scopus (186) Google Scholar, 10.Reaux A. De Mota N. Skultetyova I. Lenkei Z. El Messari S. Gallatz K. Corvol P. Palkovits M. Llorens-Cortès C. Physiological role of a novel neuropeptide, apelin, and its receptor in the rat brain.J. Neurochem. 2001; 77: 1085-1096Crossref PubMed Scopus (316) Google Scholar). APJ remained an orphan GPCR until 1998 when the peptide apelin was isolated from bovine stomach extracts as its endogenous ligand (11.Tatemoto 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. Isolation and characterization of a novel endogenous peptide ligand for the human APJ receptor.Biochem. Biophys. Res. Commun. 1998; 251: 471-476Crossref PubMed Scopus (1338) Google Scholar). Subsequently, numerous researchers have showed that apelin has an important regulatory role in various physiological processes. Among them, apelin-13 exhibits much stronger activity than other active isoforms in regulating the cardiovascular function (11.Tatemoto 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. Isolation and characterization of a novel endogenous peptide ligand for the human APJ receptor.Biochem. Biophys. Res. Commun. 1998; 251: 471-476Crossref PubMed Scopus (1338) Google Scholar). The apelin/APJ system is currently envisaged to be an important therapeutic target for the treatment of heart failure, hypertension, and obesity-related diseases (12.Giddings A.S.R. Thomas J. Tajuba J. Bortoff K. Maitra R. Development of a functional HTS assay for the APJ receptor.Int. J. High Throughput Screening. 2010; 1: 39-47Google Scholar, 13.Castan-Laurell I. Dray C. Attané C. Duparc T. Knauf C. Valet P. Apelin, diabetes, and obesity.Endocrine. 2011; 40: 1-9Crossref PubMed Scopus (207) Google Scholar). The mechanisms of APJ signal transduction are still under active investigation. New research has found that APJ has a dual role in cardiac hypertrophy (14.Scimia M.C. Hurtado C. Ray S. Metzler S. Wei K. Wang J. Woods C.E. Purcell N.H. Catalucci D. Akasaka T. Bueno O.F. Vlasuk G.P. Kaliman P. Bodmer R. Smith L.H. Ashley E. Mercola M. Brown J.H. Ruiz-Lozano P. APJ acts as a dual receptor in cardiac hypertrophy.Nature. 2012; 488: 394-398Crossref PubMed Scopus (180) Google Scholar). Apelin activates the signals of APJ through the Gαi pathway and elicits a cardiac protective response. Although sustained overload activates APJ to induce cardiac hypertrophy via a G protein-independent fashion. Previous data suggested that the C-terminal portion of APJ are required for internalization and mediates receptor internalization through palmitoylation and phosphorylation (15.Masri B. Morin N. Pedebernade L. Knibiehler B. Audigier Y. The apelin receptor is coupled to Gi1 or Gi2 protein and is differentially desensitized by apelin fragments.J. Biol. Chem. 2006; 281: 18317-18326Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). But it is still unclear how APJ mediates its G protein-dependent and -independent signaling pathways. It has been demonstrated that mutagenesis of key serine/threonine residues at receptor phosphorylation result in diminished β-arrestin binding after agonist stimulation in receptors such as the M2 muscarinic receptor, the AT1A angiotensin receptor, and the V2 vasopressin receptor (16.Pals-Rylaarsdam R. Gurevich V.V. Lee K.B. Ptasienski J.A. Benovic J.L. Hosey M.M. Internalization of the m2 muscarinic acetylcholine receptor: arrestin-independent and -dependent pathways.J. Biol. Chem. 1997; 272: 23682-23689Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 17.Qian H. Pipolo L. Thomas W.G. Association of β-arrestin 1 with the type 1A angiotensin II receptor involves phosphorylation of the receptor carboxyl terminus and correlates with receptor internalization.Mol. Endocrinol. 2001; 15: 1706-1719PubMed Google Scholar, 18.Oakley R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. Association of β-arrestin with G protein-coupled receptors during clathrin-mediated endocytosis dictates the profile of receptor resensitization.J. Biol. Chem. 1999; 274: 32248-32257Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar). There are several putative phosphorylated amino acids at the C terminus of APJ, which has not been investigated. In the work presented here, we first identified a key C-terminal amino acid residue required for GRKs/β-arrestins recruitment to activated APJ after agonist binding. We found that the key residue for APJ regulation is serine 348. Furthermore, we assessed the role of serine 348 in APJ internalization and G protein-independent ERK1/2 activation. The results suggest that serine 348 is required for GRKs and β-arrestin-mediated biased G protein independent signaling of APJ. Taken together, these results are helpful for screening novel and existing drugs of the cardiovascular system based on this biased signaling pathway. Human apelin-13 and 125I-apelin-13 were purchased from Phoenix Pharmaceuticals (Burlingame, CA). The amino acid sequence of apelin-13 is Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe, as previously described (19.Bai B. Tang J. Liu H. Chen J. Li Y. Song W. Apelin-13 induces ERK1/2 but not p38 MAPK activation through coupling of the human apelin receptor to the Gi2 pathway.Acta Biochim. Biophys. Sin. 2008; 40: 311-318Crossref Scopus (52) Google Scholar). Lipofectamine 2000 was obtained from Invitrogen. Forskolin, PTX, and anti-FLAG antibody were obtained from Sigma. Anti-HA-agarose were obtained from Pierce Chemical Co. Anti-phospho-ERK1/2 antibody, anti-ERK1/2 antibody, anti-HA antibody, anti-β-arrestin1 antibody, anti-β-arrestin2 antibody, and anti-β-actin antibody were purchased from Cell Signaling Technology. Human embryonic kidney 293 (HEK293) cells were maintained at 37 °C and 5% CO2 in Complete Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 10% fetal calf serum (FCS). Transient transfections were carried out 24 h after seeding using Lipofectamine 2000 following the manufacturer's instructions. For mass spectrometry experiments, HEK293 cells stably expressing WT APJ receptor were treated with 100 nm apelin-13 at 37 °C for 10 min. The cells were washed and lysed for 30 min on ice with RIPA buffer containing protease and phosphatase inhibitors. After centrifugation, the supernatant was reduced with 10 mm dithiothreitol (DTT) at 56 °C for 1 h and subsequently alkylated with 55 mm iodoacetamide at room temperature for 45 min in the dark. The sample was resolved by SDS-PAGE on 10% gels and stained with colloidal Coomassie Blue. Subsequently, the protein was digested with trypsin overnight at 37 °C, and then the fractions were passed through a single TiO2 Spin Tip for phosphopeptide selective enrichment. Phosphopeptides were then purified and washed as described (20.Blethrow J.D. Tang C. Deng C. Krutchinsky A.N. Modular mass spectrometric tool for analysis of composition and phosphorylation of protein complexes.PloS One. 2007; 2: e358Crossref PubMed Scopus (15) Google Scholar, 21.Choi B.K. Cho Y.M. Bae S.H. Zoubaulis C.C. Paik Y.K. Single-step perfusion chromatography with a throughput potential for enhanced peptide detection by matrix-assisted laser desorption/ionization-mass spectrometry.Proteomics. 2003; 3: 1955-1961Crossref PubMed Scopus (47) Google Scholar). Peptide composition was determined by LC-MS/MS mass spectrometry. LC-MS/MS measurements were carried out by a Shimadzu HPLC system (Columbia, MD) coupled to an AB SCIEX Triple TOF 5600 mass spectrometer (SCIEX, Foster City, CA). The two buffers used for the reverse phase chromatography were buffer A (0.1% (v/v) formic acid in water) and buffer B (0.1% (v/v) formic acid in acetonitrile). The HPLC gradient consists of holding 0% buffer B for 3 min, followed by increasing to 5% buffer B in 5 min, to 35% buffer B in 35 min, then to 60% buffer B in 5 min. In the following 2 min, the gradient was increased to 80% for 2 min. Initial chromatographic conditions were restored in 1 min and maintained for 10 min. A flow rate of 300 nl/min was used to elute the peptides into the ionization source of the mass spectrometer. The acquired MS/MS spectra were processed with the Mascot search engine (Matrix Science, London, UK) for database correlation analysis. The search parameters through Mascot were set for phosphorylation emphasis and to search for biological modifications. Trypsin was set as the enzyme, and the IPI human database was searched. Each filtered MS/MS spectra exhibiting possible phosphorylated peptide was manually checked and validated. The pcDNA3.1(+)-APJ and pcDNA3.1(+)-HA tagged-APJ plasmids were purchased from Missouri S&T cDNA Resource Center (Rolla, MO). Rluc-β-arrestin1 and Rluc-β-arrestin2 were generated by PCR amplification with plasmids containing β-arrestin1/β-arrestin2 ORF (kindly provided by Prof. Karin Eidne (QEII Medical Centre, Nedlands, Australia)) as templates, the coding sequences of human β-arrestin without their stop codons and subcloned into pRluc-N1 (PerkinElmer Life Sciences Inc.). On the other hand, the cDNA encoding the hAPJ without its stop codon was subcloned into pEGFP-N1 (Clontech, Mountain View, CA). Plasmids GRK2-GFP2 and GRK5-GFP2 were kindly provided by Prof. Christian E. Elling, 7TM Pharma A/S, 2970 Horsholm, Denmark. FLAG-tagged β-arrestin1/β-arrestin2 was kindly provided by Prof. Vsevolod V. Gurevich (Department of Pharmacology, Vanderbilt University). For construction of the Gαi2-Rluc and Gαq-Rluc fusion proteins, the coding sequence of humanized Rluc was PCR amplified and inserted in the coding sequence of each Gα subunit. Depending on the Gα subunit, the Rluc was inserted between either residues 91 and 92 of Gαi2, or residues 97 and 98 of Gαq. A series of APJ mutants were generated by overlap extension PCR using high fidelity Pfu polymerase and mutagenic primers as described previously (22.Li Y. Chen J. Bai B. Du H. Liu Y. Liu H. Heterodimerization of human apelin and κ opioid receptors: roles in signal transduction.Cell Signal. 2012; 24: 991-1001Crossref PubMed Scopus (66) Google Scholar). The mutagenic APJ cDNA was cut sequentially with EcoRI and HindIII and then ligated back into the original pcDNA3.1(+). All mutational cDNAs were confirmed by sequence analysis of both strands. All constructs were verified by sequencing. HEK293 cells were transiently transfected with the same amount of pcDNA3.1(+) containing HA-tagged wild-type APJ or HA-tagged APJ-S335A, APJ-S345A, and APJ-S348A. Twenty-four hours after transfection, cells were fixed in 4% paraformaldehyde for 15 min at room temperature, washed, and incubated in blocking solution (3% BSA) for 1 h. Subsequently, cells were incubated with 1:1000 primary rabbit polyclonal anti-HA antibody overnight at 4 °C. After washing three times with PBS, the cells were incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) at 1:1000 dilutions for 1 h at room temperature. After extensive washing, the immunoreactivity was detected by the addition of TMB Plus substrate (Santa Cruz Biotechnology), and the reaction was stopped with 0.2 m H2SO4. The absorbance at 450 nm was measured on a microplate reader (Bio-Rad). For each experiment, mock conditions corresponding to the transfection of vector without receptor were included. The expression levels of mutational receptors were calculated as a percentage of WT APJ expression using the formula: [(ODmutant − ODmock)/(ODwt − ODmock)] × 100%. Receptor internalization was measured with 100 nm apelin-13 treatment in 60 min at 37 °C by the above cell surface ELISA procedure. The percentages of mutational receptor internalization were defined as described previously (23.Hawtin S.R. Charged residues of the conserved DRY triplet of the vasopressin V1a receptor provide molecular determinants for cell surface delivery and internalization.Mol. Pharmacol. 2005; 68: 1172-1182Crossref PubMed Scopus (22) Google Scholar) using the formula: [(ODbasal − ODmock) − (ODstimulated − ODmock)]/(ODbasal − ODmock) × 100%. HEK293 cells were transiently transfected with the same amount of WT APJ and mutational APJs. 48 h after transfection, a washed cell membrane preparation was prepared as described previously (24.Chen J. Randeva H.S. Genomic organization of mouse orexin receptors: characterization of two novel tissue-specific splice variants.Mol. Endocrinol. 2004; 18: 2790-2804Crossref PubMed Scopus (36) Google Scholar). The interactions of 125I-apelin-13 with WT APJ or mutational APJ receptors were measured using radioligand binding displacement binding assays according to a previous report (9.Katugampola S.D. Maguire J.J. Matthewson S.R. Davenport A.P. [125I]-(Pyr(1))Apelin-13 is a novel radioligand for localizing the APJ orphan receptor in human and rat tissues with evidence for a vasoconstrictor role in man.Br. J. Pharmacol. 2001; 132: 1255-1260Crossref PubMed Scopus (186) Google Scholar). HEK293 cells were plated on poly-d-lysine-coated glass coverslips in 6-well plates, grown to 60% confluence, and transiently co-transfected with constant amounts of plasmids encoding for HA-APJ and EGFP-β-arrestins. Twenty-four hours post-transfection, medium was changed to serum-free DMEM, and the cells were incubated with 100 nm apelin-13 at different time intervals. Then, the cells were fixed in 4% paraformaldehyde for 15 min, washed with PBS, and incubated with 3% BSA in PBS/Triton X-100 (0.1%) for 1 h at room temperature. For the staining, anti-HA antibody was incubated as the first antibody overnight at 4 °C. After washing the cells with PBS, cells were incubated with IgG TRITC-conjugated secondary antibody (Santa Cruz Biotechnology) for 1 h at room temperature. Following a wash step, the cells were mounted on glass slides with VECTASHIELD medium containing DAPI (Vector Laboratories Inc., Peterborough, UK). Images were observed with a ×63 oil immersion objective in a Leica model DMRE laser scanning confocal microscope (Leica, Milton Keynes, UK). HEK293 cells were transiently transfected with Rluc-tagged and various EGFP (or GFP2)-tagged constructs. Twenty-four hours after transfection, cells were then harvested in HEPES-buffered phenol red-free complete medium containing 5% FCS and seeded in poly-d-lysine-coated 96-well white microplates (Corning 3600). All BRET measurements were performed according to the donor and acceptor pairs used (Table 1) by the Mithras LB940 plate reader (Berthold Technologies, Bad Wildbad, Germany) and MicroWin 2000 software as described previously.TABLE 1Summary of substrate and filter setting used in BRET assaysMethodDonorSubstrateDonor emissionAcceptorAcceptor emissionnmnmBRET1RlucCoelenterazine h460EGFP535BRET2RlucDeep Blue C400GFP2515eBRETRlucEnduRenTM460EGFP535 Open table in a new tab Immunoprecipitations were performed as described previously (25.Wang C. Pan Y. Zhang R. Bai B. Chen J. Randeva H.S. Heterodimerization of mouse orexin type 2 receptor variants and the effects on signal transduction.Biochim. Biophys. Acta. 2014; 1843: 652-663Crossref PubMed Scopus (23) Google Scholar). HEK293 cells were transiently transfected with HA-APJ (or HA-APJ348) and FLAG-β-arrestin1 (or β-arrestin2) or the vector control. 36 h after transfection, cells were serum-starved overnight and stimulated with 100 nm apelin-13 for 15 min. For co-immunoprecipitations, cells were lysed and the supernatant fractions were collected and then incubated with anti-HA-agarose beads overnight at 4 °C with end-over-end rotation. The beads were washed three times with TBST and precipitates were eluted with SDS sample buffer containing β-mercaptoethanol. The supernatants were then analyzed with SDS-PAGE and immunoblotting for anti-FLAG antibody immunoreactivity. HEK293 cells were transiently transfected with the WT APJ and various mutational APJs (APJ-S335A, APJ-S345A, APJ-S348A) as described above. After 24 h, the cells were incubated with 3-isobutyl-1-methylxanthine (0.5 mm) and MgCl2 (10 mm) for 20 min and then stimulated for 10 min with forskolin (10 µm) in either the absence or presence of various concentrations of apelin-13 (0.1–10,000 nm) at 37 °C. Subsequently, cells were washed twice with ice-cold PBS and suspended in acetate buffer. Intracellular cAMP level was measured using the absorbance-based cAMP ELISA kits (Cell Biolabs, Inc.) according to the manufacturer's protocol. HEK293 cells expressing WT APJ or mutants were plated at 5 × 104 cells per well in a 96-well poly-d-lysine-coated black plate (Corning), respectively. In addition, un-transfected HEK293 cells were used as negative controls. Calcium fluorescences were detected using Fluo-4 NW Calcium Assay Kits (Invitrogen) according to the manufacturer's instructions. Before the detection, the cells were incubated at 37 °C for 30 min in the dye loading solution dissolved in assay buffer. Following incubation, the plates were washed twice with assay buffer, equilibrated at room temperature for an additional 30 min. Subsequently, all cells stimulated with 100 nm apelin-13 and calcium fluorescences were immediately detected with the Mithras LB940 plate reader (Berthold Technologies, Bad Wildbad, Germany). Basal readings were obtained for 5 s prior to agonist addition and subtracted from post-injection readings to obtain a ligand-induced Ca2+ response. Calcium fluorescence ratio was represented using the formula: ligand-induced Ca2+ readings/basal readings. Data were imported into GraphPad Prism 5 software for statistical analysis and graphing. HEK293 cells expressing WT APJ or APJ-S348A in six-well plates were grown to 70–80% confluences. After overnight serum starvation, the cells were treated with apelin-13. Subsequently, cells were washed, harvested, and lysed in RIPA lysis buffer. Ten micrograms of cell extracts were separated by 10% SDS-PAGE and proteins were transferred to polyvinylidene fluoride (PVDF) membranes. The phosphorylation status of ERK1/2 was detected by immunoblotting with the antibody against phospho-ERK1/2. As a control for loading, the same membranes were stripped of antibody and re-probed with anti-total ERK1/2 antibody. The band intensities were measured by densitometry analysis and the change in ERK phosphorylation in both samples was calculated as the phospho-ERK/ERK ratio (see Ref. 19.Bai B. Tang J. Liu H. Chen J. Li Y. Song W. Apelin-13 induces ERK1/2 but not p38 MAPK activation through coupling of the human apelin receptor to the Gi2 pathway.Acta Biochim. Biophys. Sin. 2008; 40: 311-318Crossref Scopus (52) Google Scholar for details). For β-arrestin1/2 shRNA experiments, HEK-293 cells stably expressing APJ or APJ-S348A were transiently transfected with either β-arrestin1 shRNA (Sigma, Clone ID NM_004041.3–828s21c1) or β-arrestin2 shRNA (Sigma, Clone ID NM_004313.3–309s21c1) and shRNA Negative Control Med GC (Sigma, pLKO.1-puro) according to the manufacturer's instructions using Lipofectamine 2000 reagent. Forty-eight hours later, cells were stimulated with apelin-13 and lysed for ERK1/2 assays. All data are shown as the mean ± S.E. Data were presented and analyzed using Prism 5.0 graphing software (GraphPad). Sigmoidal curves were fitted to the dose-response data using non-linear regression. Statistical analysis was performed using one-way analysis of variance followed by Tukey's multiple comparison post-test. Previous studies have shown that C-terminal serines of APJ are required for mediating receptor desensitization and internalization (15.Masri B. Morin N. Pedebernade L. Knibiehler B. Audigier Y. The apelin receptor is coupled to Gi1 or Gi2 protein and is differentially desensitized by apelin fragments.J. Biol. Chem. 2006; 281: 18317-18326Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). However, the key residues involved in APJ regulation have not been defined. Putative APJ phosphorylation sites were obtained from NetPhos 2.0 prediction software (26.Blom N. Gammeltoft S. Brunak S. Sequence and structure-based prediction of eukaryotic protein phosphorylation sites.J. Mol. Biol. 1999; 294: 1351-1362Crossref PubMed Scopus (2515) Google Scholar) with APJ C-terminal serine 335, 345, and 348 mutants being chosen as above. Among them, sequence analysis revealed that serine 345 and 348 are highly conserved C-terminal phosphorylation sites between human, mouse, rat, and other creatures (Fig. 1A). To identify precise phosphorylation sites, we treated the stably transfected HEK293 cells with 100 nm apelin-13 for 10 min. Cell lysis and protein extraction was resolved by SDS-PAGE and stained (Fig. 2A). Following reduction and alkylation as described under ”Experimental Procedures,“ we used mass spectrometry to better define the specific sites phosphorylated in APJ under apelin-13 stimulation. We conducted a mass spectrometric analysis of the phosphoacceptor sites on APJ and two serine phosphor-acceptor sites (Ser-345 and Ser-348) in the C-terminal tail of APJ are shown in Fig. 2B. To determine which phosphorylation site identified was most likely to be the most important for the function of APJ, we generated mammalian APJ expression plasmids by site-directed mutagenesis to construct C-terminal receptor serine to alanine mutants. After a series of mutants were constructed, we first investigated the cell surface expression level of WT APJ, APJ-S335A, APJ-S345A, and APJ-S348A in HEK293 cells. Both ELISA and BRET results showed that all three point mutants had little effect on the receptor cell surface expression compared with the WT APJ (Fig. 1, B and C). This data suggested that APJ mutants were correctly synthesized and trafficked to the plasma membrane. To characterize the binding properties of APJ, WT and mutational APJs were transiently transfected into HEK293 cells. Competitive displacement studies of 125I-apelin-13 by apelin-13 (cold), showed no differences in IC50 values of the WT APJ and mutational APJs (Table 2). Also, the Bmax values for binding 125I-apelin-13 to the membrane preparation did not differ (Table 2). The results further verified that the mutagenesis of APJ C-terminal serine residues had little influence on agonist binding. At least, serines 335, 345, and 348 were not key residues for apelin binding. Upon agonist binding, the mutants were likely to be activated and transmit downstream signaling, which can be used for subsequent experiments.TABLE 2Binding characteristics of WT and mutational APJsReceptorIC50Bmaxnmfmol/mg proteinWT APJ0.68 ± 0.088.26 ± 0.17APJ-S335A0.75 ± 0.047.92 ± 0.34APJ-S345A0.67 ± 0.058.43 ± 0.15APJ-S348A0.70 ± 0.098.37 ± 0.35 Open table in a new tab The effect of apelin-13 on cAMP signaling in HEK293 cells was previously studied (19.Bai B. Tang J. Liu H. Chen J. Li Y. Song W. Apelin-13 induces ERK1/2 but not p38 MAPK activation through coupling of the human apelin receptor to the Gi2 pathway.Acta Biochim. Biophys. Sin. 2008; 40: 311-318Crossref Scopus (52) Google Scholar, 22.Li Y. Chen J. Bai B. Du H. Liu Y. Liu H. Heterodimerization of human apelin and κ opioid receptors: roles in sign" @default.
• W2041650648 created "2016-06-24" @default.
• W2041650648 creator A5016610921 @default.
• W2041650648 creator A5048379858 @default.
• W2041650648 creator A5058412586 @default.
• W2041650648 creator A5072949609 @default.
• W2041650648 creator A5073344542 @default.
• W2041650648 date "2014-11-01" @default.
• W2041650648 modified "2023-10-16" @default.
• W2041650648 title "Identification of Serine 348 on the Apelin Receptor as a Novel Regulatory Phosphorylation Site in Apelin-13-induced G Protein-independent Biased Signaling" @default.
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