Effects Of APJ Carboxyl Terminal Phosphorylation Site On G Proein-independent Biased Signaling

BackgroudG protein-coupled receptors (GPCRs), 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. GPCRs are widely expressed in the body and the signaling pathway is closely related to a number of diseases. Now, drugs targeting GPCRs make up more than 50% of all prescribed medicines.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. As intracellular adaptor and scaffolding proteins, β-arrestins play important roles in GPCRs desensitization, internalization, intracellular trafficking and could activate signaling cascades independently of G protein activation.Apelin receptor (APJ) is a member of the family A of GPCRs with a range of physiological functions. APJ remained an orphan GPCR until 1998 when the peptide apelin was isolated from bovine stomach extracts as its endogenous ligand. Both APJ and apelin are widely distributed in the central nervous system and peripheral tissues, which play an important regulatory role in various physiological processes. The apelin/APJ system is currently envisaged to be an important therapeutic target for the treatment of heart failure, hypertension and obesity related diseases.The mechanisms of APJ signal transduction are still under active investigation. APJ has a dual role in cardiac hypertrophy through different signaling pathsway. Similar to most GPCRs, C-terminal portion of APJ are required for internalization and mediates receptor internalization through palmitoylation and phosphorylation. But it is still unclear how APJ mediates its G protein-dependent and -independent signaling pathways. There are several putative phosphorylated amino acids at the C-terminus of APJ, which has not been investigated.In this paper, we aim to clarify the precise phosphorylation sites in the carboxyl terminus of APJ and investigate the effects of these residues on receptor desensitization, internalization, resensitization and G protein-independent signaling pathway. Taken together, these results are helpful for screening novel and existing drugs of the cardiovascular system based on this biased signaling pathway.Methods1. Cell culture and transfectionHuman embryonic kidney 293 (HEK293) cells were cultured. Transient transfections were carried out using Lipofectamine 2000 following the manufacturer’s instructions.2. Protein preparation and mass spectrometryAPJ protein were lysed and extracted. After alkylation and enrichment, LC-MS/MS mass spectrometry was carried out to check the phosphorylated site of APJ. 3. Mutagenesis and plasmid constructsA series of APJ mutants were generated by overlap extension PCR. The mutagenic APJ cDNA was cut sequentially with EcoR I and Xho I 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. 4. ELISA assaysThe expression levels of wide type and mutational receptors were detected by ELISA assays. Intracellular cAMP level was measured using the absorbance-based cAMP ELISA kits according to the manufacturer’s protocol.5. Radioligand binding assaysThe interactions of 125I-apelin-13 with wt APJ or mutational APJ receptors were measured using radioligand binding displacement binding assays.6. Confocal microscopy assaysThe receptor expression and internalization were observed with laser scanning confocal microscope.7. Dose-response and real-time kinetic BRET assaysAll BRET measurements were performed to detect the interaction between wt APJ or mutational APJ receptors and GRK2/5 (or β-arrestinl/2).8. Co-immunoprecipitation and immunoblotting assaysImmunoprecipitations were performed to detect the interaction between wt APJ or mutional APJ receptor and β-arrestin1/2.9. Intracellular calcium assaysCalcium fluorescences were detected using Fluo-4 NW calcium assay kits according to the manufacturer’s instructions.10. Western blot assaysWestern blot was detected 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.11. Statistical analysisAll the data are shown as the means±SEM. Data were presented and analyzed using Prism 5.0 graphing software.Results1. Identification of phosphorylation sites on APJ by mass spectrometryPutative APJ phosphorylation sites were obtained from NetPhos 2.0 prediction software with APJ C-terminal serine 335,345 and 348 mutants being chosen. Subsequently, mass spectrometry was used to better define the specific phosphorylated sites in APJ under apelin-13 stimulation, two serine phosphor-acceptor sites (ser 345 and ser 348) were shown.2. Site-directed mutagenesis and plasmid constructsWe generated mammalian APJ expression plasmids by site-directed mutagenesis to construct C-terminal receptor serine to alanine mutants.3. Expression of wt and mutational APJsBoth ELISA and BRET results showed that all the three point mutants have little effect on the receptor cell-surface expression compared to the wt APJ.4. Binding properties of wt and mutational APJsCompetitive displacement studies of 125I-apelin-13 by apelin-13, showed no differences in binding properties of the wt APJ and mutational APJs. At least, serine 335,345 and 348 are not the key residues for apelin binding.5. Functional validation of APJ mutants by apelin-13-induced inhibition of cAMP and elevation of Ca2+In HEK293 cells expressing wt APJ or mutational APJs, the forskolin-induced intracellular cAMP production were reduced significantly by apelin-13 in a dose-dependent manner in all groups. There is no significant difference between wt and all mutational APJs. Similarly, wt APJ and three mutants resulted in significant increase of intracellular calcium.6. Serine 348 mutation of APJ is required for apelin-13 induced receptor internalizationIn HEK293 cells expressing wt APJ, apelin-13 (100 nM) caused a robust time-dependent receptor internalization at 5 min, peaking at 30 min following addition of the agonist. After 60 min,70% of receptor recycled to the membrane. Mutation of C-terminal serine 335 and 345 had no effect on apelin-13 induced receptor internalization, compared to the wt APJ. In contrast, mutation of serine 348 significantly blocked apelin-13 induced receptor internalization.These results were also confirmed by confocal microscopy. Wt APJ or APJ-S335A, APJ-S345A, APJ-S348A mutation were clearly localized at the cell surface. Upon stimulation with apelin-13,the internalization of APJ-S335A and APJ-S345A showed similar process compared to the wt APJ. In contrast, apelin-13 failed to induce a significant shift in localization of APJ-S348A and no co-localization was observed with β-arrestinl or β-arrestin2 after stimulation.7. Serine 348 mutation of APJ impairs its interaction with GRK2/5 and β-arrestinl/2Agonist dose-response curves of GRK2/5 recruitment to APJ at 10 min post-stimulation were detected using BRET2. The data showed that apelin-13 caused a robust dose-dependent increase in BRET signal for wt APJ, APJ-S335A and APJ-S345A, indicative of GRK2/5 being recruited to activated receptors. The EC50 values were as follows:6.58±0.58 nM (WT APJ),6.20±1.56 nM (APJ-S335A), 5.69±1.42 nM (APJ-S345A) with GRK2;9.45±2.58 nM (WT APJ),8.20±1.85 nM (APJ-S335A),7.34±1.17 nM (APJ-S345A) with GRK5. In contrast, APJ-S348A mutant was unable to recruit GRK2/5 at any dose tested.Similarly, agonist dose-response curves of β-arrestinl/2 recruitment to wt APJ at 15 min were also detected using BRET1. The data showed that apelin-13 caused a robust dose-dependent increase in BRET signal for wt APJ, APJ-S335A and APJ-S345A, indicative of β-arrestinl/2 being recruited to activated receptors. The EC50 values were as follows:5.69±0.71 nM (WT APJ),5.06±1.05 nM (APJ-S335A),5.55±0.94 nM (APJ-S345A) with p-arrestinl;8.29±1.25 nM (WT APJ),7.25±0.73 nM (APJ-S335A),7.55±1.03 nM (APJ-S345A)with p-arrestin2. In contrast, APJ-S348A mutant was unable to recruit β-arrestinl/2 at any dose tested.The interaction between APJ and β-arrestins has also been confirmed by cellular co-immunopprecipitation assay. The results indicated that co-immunoprecipitations could be detected in the cells transfected with both Flag-β-arrestinl or β-arrestin2 and HA-APJ plasmids with apelin-13 treatment, but not in cells transfected with either vector alone. The co-immunoprecipitations could not be detected in the cells expressing with both Flag-β-arrestinl or β-arrestin2 and HA-APJ-S348A.8. Serine 348 mutation of APJ impairs its desensitization and resensitization characteristicsFor receptor internalization, HEK293 cells expressing wt APJ pretreated with apelin-13 for 3 h, resulted in a sustained desensitization of apelin-13 induced decreases in cAMP level. However, HEK293 cells expressing APJ-S348A could not induce obvious desensitization of apelin-13 induced decreases in cAMP concentration.For receptor resensitization, after pretreatment of HEK293 cells expressing wt APJ with 100 nM apelin-13 for 3 h, the cells were washed to remove agonist and allowed to rest for various intervals. A 70% recovery of apelin-13 responsiveness was apparent after a 30 min resting period, and full recovery was achieved with cells cultured for 2 h before addition of apelin-13. In contrast, the recovery process could not be detected in the cells expressing mutational APJ-S348A.9. Serine 348 mutation of APJ does not alter its interaction with G proteinAddition of apelin-13 to cells co-expressing EGFP-tagged wt APJ (or mutants) and Rluc-tagged Gaq resulted in a rapid and significant increasing ligand-induced BRET signal. The BRET signal peaked at 5 min after treatment and decreased rapidly thereafter.Similar treatment of cells co-expressing APJ (or mutants) and Gαi2 also induced an increase in BRET signal with a similar kinetic profile. Overall, BRET data clearly show that the three mutants and Gαq (or Gαi2) interaction are similar to wt APJ.10. Serine 348 mutation of APJ decreased G protein-independent ERK activationFor time-dependent ERK1/2 signalling activation, the results showed that a significantly increased ERK1/2 phosphorylation by apelin-13 appeared after 2 min treatment in wt APJ, with the maximal level at 5 min, and then phospho-ERK 1/2 levels kept high until 60 min. In contrast, in mutational APJ-S348A, ERK1/2 phosphorylation was consistent with wt APJ in 5 min after agonist stimulation. Subsequently, the phospho-ERK1/2 level decreased significantly to the near basal level from 15 min.As for dose-dependent ERK phosphorylation in mutational APJ-S348A, we also found that apelin-13 (from 0.1～1000 nM)could not induce ERK1/2 activation as indicated for 15min. However, when duration of agonist was reduced, we observed dose-dependent ERK activation in both groups at 5 min, which has no statistical difference between HEK293 cells expressing wt APJ and APJ-S348A.Pretreatment of HEK293-APJ or APJ-S348A cells with PTX, which selectively deactivated Gai/o-protein to prevent its interaction with the receptor, mostly abrogated the apelin-13 induced ERK 1/2 activation, indicating that apelin-13 activated ERK 1/2 by G protein-dependent activation pathway is not affected by mutation at ser 348.To test the potential role of β-arrestins, we analyzed the time course of apelin-13 induced ERK 1/2 phosphorylation after depleting cellular levels of β-arrestinl or β-arrestin2 by transfecting shRNA specifically directed against each isoform. In the presence of a non-targeted control shRNA, apelin-13 induced ERK1/2 phosphorylation in cells expressing APJ is identical to that observed without any shRNA transfection. However, ERK1/2 phosphorylation level was reduced significantly by both β-arrestinl and β-arrestin2 shRNA individually on wt APJ beyond 15 min. As for the role of β-arrestin shRNA on mutational APJ-S348A, we found that apelin-13 induced ERK1/2 signalling did not differ.ConclusionIn conclusion, the most striking finding of the present study is the identification of a key phosphorylation site in the C-terminus of APJ for its binds to GRKs/β-arrestins. Mutation of serine 348 led to an elimination of receptor desensitization, internalization, resensitization and G protein-independent ERK1/2 signaling.In contrast, this mutant at serine residues had no demonstrable impact on apelin-13 induced G protein activation and its intracellular signaling. Thus, APJ-S348A conformation was biased, which may be extrapolated to design novel drugs for cardiac hypertrophy based on this biased signal pathway.Background:APJ induced Gαi activation evokes a protective response in cardiac hypertrophy while G-protein-independent signaling induces hypertrophy.Results:Serine 348 is identified as one of phosphorylation site in APJ, which is crucial for APJ interactions with GRK2/5,β-arrestin1/2 and its internalization.Conclusion:Mutation at serine 348 modulates G-independent signaling of APJ.Significance:Understanding APJ signaling and regulation could provide novel clues for the development of functionally selective APJ ligands.