The Experts below are selected from a list of 1677 Experts worldwide ranked by ideXlab platform
Bo Bai - One of the best experts on this subject based on the ideXlab platform.
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Temporal Expression of Apelin/Apelin Receptor in Ischemic Stroke and its Therapeutic Potential.
Frontiers in molecular neuroscience, 2017Co-Authors: Xin Wang, Xuan Zhou, Baohua Cheng, Bo BaiAbstract:Stroke is one of the leading causes of death and disability worldwide, and ischemic stroke accounts for approximately 87% of cases. Improving post-stroke recovery is a major challenge in stroke treatment. Accumulated evidence indicates that the Apelinergic system, consisting of Apelin and Apelin Receptor (APLNR), is temporally dysregulated in ischemic stroke. Moreover, the Apelinergic system plays a pivotal role in post-stroke recovery by inhibiting neuronal apoptosis and facilitating angiogenesis through various molecular pathways. In this review article, we summarize the temporal expression of Apelin and APLNR in ischemic stroke and the mechanisms of their dysregulation. In addition, the protective role of the Apelinergic system in ischemic stroke and the underlying mechanisms of its protective effects are discussed. Furthermore, critical issues in activating the Apelinergic system as a potential therapy will also be discussed. The aim of this review article is to shed light on exploiting the activation of the Apelinergic system to treat ischemic stroke.
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temporal expression of Apelin Apelin Receptor in ischemic stroke and its therapeutic potential
Frontiers in Molecular Neuroscience, 2017Co-Authors: Xin Wang, Xuan Zhou, Baohua Cheng, Bo BaiAbstract:Stroke is one of the leading causes of death and disability worldwide, and ischemic stroke accounts for approximately 87% of cases. Improving post-stroke recovery is a major challenge in stroke treatment. Accumulated evidence indicates that the Apelinergic system, consisting of Apelin and Apelin Receptor (APLNR), is temporally dysregulated in ischemic stroke. Moreover, the Apelinergic system plays a pivotal role in post-stroke recovery by inhibiting neuronal apoptosis and facilitating angiogenesis through various molecular pathways. In this review article, we summarize the temporal expression of Apelin and APLNR in ischemic stroke and the mechanisms of their dysregulation. In addition, the protective role of the Apelinergic system in ischemic stroke and the underlying mechanisms of its protective effects are discussed. Furthermore, critical issues in activating the Apelinergic system as a potential therapy will also be discussed. The aim of this review article is to shed light on exploiting the activation of the Apelinergic system to treat ischemic stroke.
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Apelin Receptor homodimer-oligomers revealed by single-molecule imaging and novel G protein-dependent signaling
Scientific reports, 2017Co-Authors: Xin Cai, Bo Bai, Rumin Zhang, Chunmei Wang, Jing ChenAbstract:The Apelin Receptor (APJ) belongs to family A of the G protein-coupled Receptors (GPCRs) and is a potential pharmacotherapeutic target for heart failure, hypertension, and other cardiovascular diseases. There is evidence APJ heterodimerizes with other GPCRs; however, the existence of APJ homodimers and oligomers remains to be investigated. Here, we measured APJ monomer-homodimer-oligomer interconversion by monitoring APJ dynamically on cells and compared their proportions, spatial arrangement, and mobility using total internal reflection fluorescence microscopy, resonance energy transfer, and proximity biotinylation. In cells with
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Apelin Receptor homodimer oligomers revealed by single molecule imaging and novel g protein dependent signaling
Scientific Reports, 2017Co-Authors: Xin Cai, Bo Bai, Jing Chen, Rumin Zhang, Chunmei WangAbstract:The Apelin Receptor (APJ) belongs to family A of the G protein-coupled Receptors (GPCRs) and is a potential pharmacotherapeutic target for heart failure, hypertension, and other cardiovascular diseases. There is evidence APJ heterodimerizes with other GPCRs; however, the existence of APJ homodimers and oligomers remains to be investigated. Here, we measured APJ monomer-homodimer-oligomer interconversion by monitoring APJ dynamically on cells and compared their proportions, spatial arrangement, and mobility using total internal reflection fluorescence microscopy, resonance energy transfer, and proximity biotinylation. In cells with <0.3 Receptor particles/μm2, approximately 60% of APJ molecules were present as dimers or oligomers. APJ dimers were present on the cell surface in a dynamic equilibrium with constant formation and dissociation of Receptor complexes. Furthermore, we applied interference peptides and MALDI-TOF mass spectrometry to confirm APJ homo-dimer and explore the dimer-interfaces. Peptides corresponding to transmembrane domain (TMD)1, 2, 3, and 4, but not TMD5, 6, and 7, disrupted APJ dimerization. APJ mutants in TMD1 and TMD2 also decreased bioluminescence resonance energy transfer of APJ dimer. APJ dimerization resulted in novel functional characteristics, such as a distinct G-protein binding profile and cell responses after agonist stimulation. Thus, dimerization may serve as a unique mechanism for fine-tuning APJ-mediated functions.
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dynamics of Apelin Receptor g protein coupling in living cells
Experimental Cell Research, 2014Co-Authors: Bo Bai, Yunlu Jiang, Xin Cai, Jing ChenAbstract:During our research on Apelin Receptor (APJ) signalling in living cells with BRET and FRET, we demonstrated that Apelin-13 stimulation can lead to the activation of Gαi2 or Gαi3 through undergoing a molecular rearrangement rather than dissociation in HEK293 cells expressing APJ. Furthermore, Gαo and Gαq also showed involvement in APJ activation through a classical dissociation model. However, both FRET signal and BRET ratio between fluorescent Gαi1 subunit and Gβγ subunits demonstrated little change after Apelin-13 stimulation. These results demonstrated that stimulation of APJ with Apelin-13 causes activation of Gαi2, Gαi3, Gαo, Gαq; among which Gαi2, Gαi3 were activated through a novel rearrangement process. These results provide helpful data for understanding APJ mediated G-protein signalling.
Anthony P. Davenport - One of the best experts on this subject based on the ideXlab platform.
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international union of basic and clinical pharmacology cvii structure and pharmacology of the Apelin Receptor with a recommendation that elabela toddler is a second endogenous peptide ligand
Pharmacological Reviews, 2019Co-Authors: Cai Read, Robyn Macrae, Duuamene Nyimanu, Janet J. Maguire, Thomas L. Williams, Peiran Yang, David J Huggins, Petra Sulentic, Robert C Glen, Anthony P. DavenportAbstract:The predicted protein encoded by the APJ gene discovered in 1993 was originally classified as a class A G protein-coupled orphan Receptor but was subsequently paired with a novel peptide ligand, Apelin-36 in 1998. Substantial research identified a family of shorter peptides activating the Apelin Receptor, including Apelin-17, Apelin-13, and [Pyr1]Apelin-13, with the latter peptide predominating in human plasma and cardiovascular system. A range of pharmacological tools have been developed, including radiolabeled ligands, analogs with improved plasma stability, peptides, and small molecules including biased agonists and antagonists, leading to the recommendation that the APJ gene be renamed APLNR and encode the Apelin Receptor protein. Recently, a second endogenous ligand has been identified and called Elabela/Toddler, a 54-amino acid peptide originally identified in the genomes of fish and humans but misclassified as noncoding. This precursor is also able to be cleaved to shorter sequences (32, 21, and 11 amino acids), and all are able to activate the Apelin Receptor and are blocked by Apelin Receptor antagonists. This review summarizes the pharmacology of these ligands and the Apelin Receptor, highlights the emerging physiologic and pathophysiological roles in a number of diseases, and recommends that Elabela/Toddler is a second endogenous peptide ligand of the Apelin Receptor protein.
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Apelin Receptor (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database
IUPHAR BPS Guide to Pharmacology CITE, 2019Co-Authors: Anthony P. Davenport, Duuamene Nyimanu, Janet J. Maguire, Matthias J. Kleinz, Thomas L. Williams, Robyn G. C. Macrae, Peiran YangAbstract:The Apelin Receptor (nomenclature as agreed by the NC-IUPHAR Subcommittee on the Apelin Receptor [68]) responds to Apelin, a 36 amino-acid peptide derived initially from bovine stomach. Apelin-36, Apelin-13 and [Pyr1]Apelin-13 are the predominant endogenous ligands which are cleaved from a 77 amino-acid precursor peptide (APLN, Q9ULZ1) by a so far unidentified enzymatic pathway [80]. A second family of peptides discovered independently and named Elabela [11] or Toddler, that has little sequence similarity to Apelin, is present, and functional at the Apelin Receptor in the adult cardiovascular system [87, 67]. Structure-activity relationship Elabela analogues have been described [61].
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Apelin-36-[L28A] and Apelin-36-[L28C(30kDa-PEG)] peptides that improve diet induced obesity are G protein biased ligands at the Apelin Receptor.
Peptides, 2019Co-Authors: Duuamene Nyimanu, Rhoda E. Kuc, Janet J. Maguire, Thomas L. Williams, Maria Bednarek, Philip D Ambery, Lutz Jermutus, Anthony P. DavenportAbstract:Abstract Background Apelin signalling pathways have important cardiovascular and metabolic functions. Recently, Apelin-36-[L28A] and Apelin-36-[L28C(30kDa-PEG)], were reported to function independent of the Apelin Receptor in vivo to produce beneficial metabolic effects without modulating blood pressure. We aimed to show that these peptides bound to the Apelin Receptor and to further characterise their pharmacology in vitro at the human Apelin Receptor. Methods [Pyr1]Apelin-13 saturation binding experiments and competition binding experiments were performed in rat and human heart homogenates using [125I]Apelin-13 (0.1 nM), and/or increasing concentrations of Apelin-36, Apelin-36-[L28A] and Apelin-36-[L28C(30kDa-PEG)] (50pM-100μM). Apelin-36 and its analogues Apelin-36-[F36A], Apelin-36-[L28A], Apelin-36-[L28C(30kDa-PEG)], Apelin-36-[A28 A13] and [40kDa-PEG]-Apelin-36 were tested in forskolin-induced cAMP inhibition and β–arrestin assays in CHO-K1 cells heterologously expressing the human Apelin Receptor. Bias signaling was quantified using the operational model for bias. Results In both species, [Pyr1]Apelin-13 had comparable subnanomolar affinity and the Apelin Receptor density was similar. Apelin-36, Apelin-36-[L28A] and Apelin-36-[L28C(30kDa-PEG)] competed for binding of [125I]Apelin-13 with nanomolar affinities. Apelin-36-[L28A] and Apelin-36-[L28C(30kDa-PEG)] inhibited forskolin-induced cAMP release, with nanomolar potencies but they were less potent compared to Apelin-36 at recruiting β-arrestin. Bias analysis suggested that these peptides were G protein biased. Additionally, [40kDa-PEG]-Apelin-36 and Apelin-36-[F36A] retained nanomolar potencies in both cAMP and β-arrestin assays whilst Apelin-36-[A13 A28] exhibited a similar profile to Apelin-36-[L28C(30kDa-PEG)] in the β–arrestin assay but was more potent in the cAMP assay. Conclusions Apelin-36-[L28A] and Apelin-36-[L28C(30kDa-PEG)] are G protein biased ligands of the Apelin Receptor, suggesting that the Apelin Receptor is an important therapeutic target in metabolic diseases.
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BS49 Human embryonic stem cell derived cardiomyocytes express functional Receptors for the cardiovascular peptide Apelin
Basic Science, 2019Co-Authors: Robyn Macrae, William G. Bernard, Rhoda E. Kuc, Maria Colzani, Tom Williams, Duuamene Nyimanu, Janet J. Maguire, Sanjay Sinha, Anthony P. DavenportAbstract:Introduction The Apelin Receptor is expressed throughout the cardiovascular system, including in cardiomyocytes. Receptor activation by either of its endogenous peptide ligands, Apelin or Elabela, has a positive inotropic effect and promotes vasodilatation. Human embryonic stem cell (hESC)-derived cells have the potential for use in translational research to investigate cellular signalling, disease pathogenesis and potential novel treatments. Our aim was to determine if hESC-derived cardiomyocytes express Apelin Receptor protein and to quantify Receptor density to ascertain if this cell type can be used as a phenotypic model for human diseases associated with Apelin Receptor mutations. Methods H9 hESCs were cultured to induce differentiation to beating cardiomyocytes. Saturation radioligand binding experiments were performed using [Glp65,Nle75,Tyr77][125I]Apelin-13 and [Pyr1]Apelin-13 to define non-specific binding. Bound radioactivity was counted and data analysed using iterative curve fitting programs to obtain values of Receptor affinity (KD) and density (BMAX). Immunocytochemistry was carried out using anti-Apelin Receptor or anti-cardiac cell marker antibodies. Results Previous work demonstrated expression of the Apelin Receptor in hESC-derived cardiomyocytes at the gene level by qRT-PCR at similar levels to adult cells (figure 1A). Here, radioligand binding studies have confirmed Receptor protein expression in beating hESC-derived cardiomyocytes. Binding was saturable and [125I]Apelin-13 bound with sub-nanomolar affinity (0.12 nM) and Receptor density found to be 21 fmol/mg (figure 1B), comparable to that found in human adult heart. Hill slope was close to 1 consistent with a single binding site for Apelin in these cells. Furthermore, beating hESC-derived cardiomyocytes stained positive for Apelin Receptor, in addition to the standard cardiac markers including cardiac troponin T (figure 2). Conclusion These data importantly confirm that hESC-derived cardiomyocytes express Apelin Receptor protein at similar levels to adult human heart. Apelin Receptor mutations have been identified in the 100,000 Genomes Bridge Project that are associated with rare diseases, including pulmonary arterial hypertension. Our ongoing experiments aim to pharmacologically characterise the Apelinergic signalling pathway in the beating hESC-derived cardiomyocytes and we propose to generate hESC-derived phenotypic models by introducing selected Apelin Receptor mutations via CRISPR/Cas-9 gene editing. Conflict of interest None
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BS16 A novel fluorescent Apelin ligand tracks Apelin Receptor internalisation
Basic Science, 2019Co-Authors: Elisabeth O’flaherty, Robyn Macrae, Tom Williams, Duuamene Nyimanu, Janet J. Maguire, Heather Currinn, Jason Brown, Alastair J H Brown, Gregory Strachan, Anthony P. DavenportAbstract:Introduction Fluorescent ligands are a promising tool in the study of GPCR pharmacology as they are safe and more cost effective than conventional probes such as radioligands. In this study, we investigated the use of a novel fluorescent ligand, Apelin-488, to successfully show qualitative and quantitative Apelin Receptor internalisation, which may help further our understanding of Apelin Receptor pharmacology. The Apelin Receptor, a class A GPCR that unusually binds two endogenous peptide ligands, Apelin and elabela/toddler, is expressed and functional in the cardiovascular system. Mutations in the human Apelin Receptor have been identified in rare diseases and our aim is to identify tool compounds to explore the potential functional consequence of these mutations. Methods CHO-K1 cells stably expressing the wild-type Apelin Receptor were treated with Apelin-488, fixed, and stained with Hoechst 33342 and a wheat germ agglutinin-AlexaFluor-594 conjugate as nuclear and membrane markers respectively. Triple fluorescence confocal images were generated using Opera PhenixTM High Content Screening System and data were quantified using Harmony High Content Imaging and Analysis Software. Fluorescent ligand intensity at the membrane or in the cytoplasm was determined, providing cellular localisation for the Apelin Receptor following ligand binding. Activity of the fluorescent ligand at the Apelin Receptor in comparison to the endogenous ligand, [Pyr1]Apelin-13 or CMF-019, a G protein biased small molecule Apelin Receptor ligand, was determined using a dynamic mass redistribution assay and radioligand binding assays. Results The fluorescent ligand, Apelin-488 binds specifically to the Apelin Receptor in stably transfected CHO-K1 cells. High content screening revealed that in these cells, the Apelin Receptor is internalised over time and was still present at the cell membrane at an early time point, 30 secs (fig 1A). However, by 60 mins, Apelin-488 membrane localisation was reduced while internal localisation had increased, suggesting substantial Apelin Receptor internalisation had occurred. (fig 1B). Untransfected CHO-K1 cells showed no Apelin-488 signal, supporting specific binding of the fluorescent ligand to the Apelin Receptor. A dynamic mass redistribution assay demonstrated that Apelin-488 is functional at the Apelin Receptor, at a similar potency as the endogenous ligand [Pyr1]Apelin-13 and the G protein biased small molecule Apelin Receptor ligand CMF-019 (Fig 2). Conclusion Apelin-488 shows specific binding to the Apelin Receptor while retaining activity in a dynamic mass redistribution and binding assays. This work provides evidence for Apelin-488 as a novel fluorescent tool for assessing internalisation of the Apelin Receptor following Receptor binding. A new method for studying Apelin Receptor internalisation is particularly interesting due to the implication of Apelin Receptor desensitisation in pulmonary arterial hypertension, mediated by arrestin and internalisation pathways. Additionally, Apelin-488 may provide insight into binding and Receptor trafficking in mutated Apelin Receptors previously identified in patients with rare cardiovascular diseases. As such, Apelin-488 and other fluorescent ligands may help streamline development of drugs that could be beneficial in clinical settings. Conflict of interest none
Jan K Rainey - One of the best experts on this subject based on the ideXlab platform.
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Structure, amphipathy, and topology of the membrane-proximal helix 8 influence Apelin Receptor plasma membrane localization.
Biochimica et biophysica acta. Biomembranes, 2019Co-Authors: Aditya Pandey, Muzaddid Sarker, Xiang-qin Liu, Danielle M. Leblanc, Hirendrasinh B. Parmar, Trần Thanh Tâm Phạm, Roy Duncan, Jan K RaineyAbstract:Abstract G-protein coupled Receptors (GPCRs) typically have an amphipathic helix (“helix 8”) immediately C-terminal to the transmembrane helical bundle. To date, a number of functional roles have been associated with GPCR helix 8 segments, but structure-function analysis for this region remains limited. Here, we examine helix 8 of the Apelin Receptor (AR or APJ), a class A GPCR with wide physiological and pathophysiological relevance. The 71 residue C-terminal tail of the AR is primarily intrinsically disordered, with a detergent micelle-induced increase in helical character. This helicity was localized to the helix 8 region, in good agreement with the recent AR crystal structure. A series of helix 8 mutants were made to reduce helicity, remove amphipathy, or flip the hydrophobic and hydrophilic faces. Each mutant AR was tested both biophysically, in the isolated C-terminal tail, and functionally in HEK 293 T cells, for full-length AR. In all instances, micelle interactions were maintained, and steady-state AR expression was efficient. However, removal of amphipathy or helical character led to a significant decrease in cell surface localization. Flipping of helix 8 amphipathic topology restored cell surface localization to some degree, but still was significantly reduced relative to wild-type. Structural integrity, amphipathy to drive membrane association, and correct topology of helix 8 membrane association all thus appear important for cell surface localization of the AR. This behavior correlates well to GPCR C-terminal tail sequence motifs, implying that these serve to specify key topological features of helix 8 and its proximity to the transmembrane domain.
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Bioactivity of the putative Apelin proprotein expands the repertoire of Apelin Receptor ligands.
Biochimica et biophysica acta. General subjects, 2017Co-Authors: Kyungsoo Shin, Nigel A. Chapman, Denis J. Dupré, Aditya Pandey, Muzaddid Sarker, Calem Kenward, Shuya K. Huang, Nathan Weatherbee-martin, Jan K RaineyAbstract:Abstract Background Apelin is a peptide ligand for a class A G-protein coupled Receptor called the Apelin Receptor (AR or APJ) that regulates angiogenesis, the adipoinsular axis, and cardiovascular functions. Apelin has been shown to be bioactive as 13, 17, and 36 amino acid isoforms, C-terminal fragments of the putatively inactive 55-residue proprotein (proApelin or Apelin-55). Although intracellular proprotein processing has been proposed, isolation of Apelin-55 from colostrum and milk demonstrates potential for secretion prior to processing and the possibility of proApelin-AR interaction. Methods Apelin isoform activity and potency were compared by an In-Cell Western™ assay for ERK phosphorylation using a stably AR-transfected HEK293A cell line. Conformational comparison of Apelin isoforms was carried out by circular dichroism and heteronuclear solution-state nuclear magnetic resonance spectroscopy. Results Apelin-55 is shown to activate the AR, with similar maximum ERK phophorylation response and potency to the shorter isoforms except for Apelin-13, which exhibited a greater potency. Correlating to this shared activity, highly similar conformations are exhibited in all Apelin isoforms for the shared C-terminal region responsible for Receptor binding and activation. Conclusions AR activation by all Apelin isoforms likely hinges upon shared conformation and dynamics in the C-terminus, with Apelin-55 providing an alternative bioactive isoform despite the addition of 19 N-terminal residues relative to Apelin-36. General significance Beyond providing novel insight into the physiology of this system, re-annotation of proApelin to the bioactive Apelin-55 isoform adds to the molecular toolkit for dissection of Apelin-AR interactions and expands the repertoire of therapeutic targets for the Apelinergic system.
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The Apelin Receptor: physiology, pathology, cell signalling, and ligand modulation of a peptide-activated class A GPCR.
Biochemistry and Cell Biology, 2014Co-Authors: Nigel A. Chapman, Denis J. Dupré, Jan K RaineyAbstract:The Apelin Receptor (AR or APJ) is a class A (rhodopsin-like) G-protein-coupled Receptor with wide distribution throughout the human body. Activation of the AR by its cognate peptide ligand, Apelin, induces diverse physiological effects including vasoconstriction and dilation, strengthening of heart muscle contractility, angiogenesis, and regulation of energy metabolism and fluid homeostasis. Recently, another endogenous peptidic activator of the AR, Toddler/ELABELA, was identified as having a crucial role in zebrafish (Danio rerio) embryonic development. The AR is also implicated in pathologies including cardiovascular disease, diabetes, obesity, and cancer, making it a promising therapeutic target. Despite its established importance, the precise roles of AR signalling remain poorly understood. Moreover, little is known about the mechanisms of peptide–AR activation. Additional complexity arises from modulation of the AR by 2 endogenous peptide ligands, both with multiple bioactive isoforms of variable le...
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Small expression tags enhance bacterial expression of the first three transmembrane segments of the Apelin Receptor
Biochemistry and cell biology = Biochimie et biologie cellulaire, 2014Co-Authors: Aditya Pandey, Muzaddid Sarker, Xiang-qin Liu, Jan K RaineyAbstract:G-protein coupled Receptors (GPCRs) are inherently dynamic membrane protein modulators of various important cellular signaling cascades. The Apelin Receptor (AR or APJ) is a class A GPCR involved i...
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Probing Binding of Apelin to the Extracellular Loops of the Apelin Receptor (APJ) in Lipid Mimetic Environments
Biophysical Journal, 2011Co-Authors: Pascaline Ngweniform, Jan K RaineyAbstract:a Department of Biochemistry & Molecular Biology and b Department of Chemistry, Dalhousie University, Halifax, NS B3H 1X5.The Apelin Receptor, previously called APJ, is a G protein-coupled Receptor highly expressed in the central nervous system (CNS), cardiovascular system, the adipoinsular axis and the mammary glands, among other tissues and organ systems. The Apelinergic system plays important biological functions in the regulation of blood pressure, blood glucose, drinking behavior and food intake. The action of this system is also implicated in tumour angiogenesis, diabetes and cardiovascular diseases. In addition, the Apelin Receptor is a co-Receptor for human immunodeficiency virus type 1 (HIV1) and simian immunodeficiency virus (SIV). Despite these roles, the mechanism of activation of Apelin Receptor by its cognate ligand has not been studied at the molecular level. Following the “divide and conquer” approach for membrane protein characterization, we present the biophysical characterization of the interaction of two extracellular loops of the Apelin Receptor (extracellular loops 1, EL1 and 3, EL3) with a fluorescently-tagged Apelin analogue in lipid environment. Peptides were synthesized by solid phase peptide synthesis and purified by high-performance liquid chromatography. Characterization both of the peptides in isolation and of the binding between Apelin and EL peptides is provided using circular dichroism spectroscopy, fluorescence resonance energy transfer and nuclear magnetic resonance spectroscopy. These results provide insight into understanding of the Apelinergic system at the molecular level and provide the first structural information on the Apelin Receptor for the development of therapeutics targeting this system.
Jing Chen - One of the best experts on this subject based on the ideXlab platform.
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Individual phosphorylation sites at the C-terminus of the Apelin Receptor play different roles in signal transduction
Redox biology, 2020Co-Authors: Jing Chen, Yunlu Jiang, Xin Cai, Rumin Zhang, Xiaoyu Chen, Huiling Mao, Maocai Yan, Chunmei WangAbstract:The Apelin and Elabela proteins constitute a spatiotemporal double-ligand system that controls Apelin Receptor (APJ) signal transduction. Phosphorylation of multiple sites within the C-terminus of APJ is essential for the recruitment of β-arrestins. We sought to determine the precise mechanisms by which Apelin and Elabela promote APJ phosphorylation, and to elucidate the influence of β-arrestin phosphorylation on G-protein-coupled Receptor (GPCR)/β-arrestin-dependent signaling. We used techniques including mass spectrometry (MS), mutation analysis, and bioluminescence resonance energy transfer (BRET) to evaluate the role of phosphorylation sites in APJ-mediated G-protein-dependent and β-dependent signaling. Phosphorylation of APJ occurred at five serine residues in the C-terminal region (Ser335, Ser339, Ser345, Ser348 and Ser369). We also identified two phosphorylation sites in β-arrestin1 and three in β-arrestin2, including three previously identified residues (Ser412, Ser361, and Thr383) and two new sites, Tyr47 in β-arrestin1 and Tyr48 in β-arrestin2. APJ mutations did not affect the phosphorylation of β-arrestins, but it affects the β-arrestin signaling pathway, specifically Ser335 and Ser339. Mutation of Ser335 decreased the ability of the Receptor to interact with β-arrestin1/2 and AP2, indicating that APJ affects the β-arrestin signaling pathway by stimulating Elabela. Mutation of Ser339 abolished the capability of the Receptor to interact with GRK2 and β-arrestin1/2 upon stimulation with Apelin-36, and disrupted Receptor internalization and β-arrestin-dependent ERK1/2 activation. Five peptides act on distinct phosphorylation sites at the APJ C-terminus, differentially regulating APJ signal transduction and causing different biological effects. These findings may facilitate screening for drugs to treat cardiovascular and metabolic diseases.
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Apelin Receptor homodimer-oligomers revealed by single-molecule imaging and novel G protein-dependent signaling
Scientific reports, 2017Co-Authors: Xin Cai, Bo Bai, Rumin Zhang, Chunmei Wang, Jing ChenAbstract:The Apelin Receptor (APJ) belongs to family A of the G protein-coupled Receptors (GPCRs) and is a potential pharmacotherapeutic target for heart failure, hypertension, and other cardiovascular diseases. There is evidence APJ heterodimerizes with other GPCRs; however, the existence of APJ homodimers and oligomers remains to be investigated. Here, we measured APJ monomer-homodimer-oligomer interconversion by monitoring APJ dynamically on cells and compared their proportions, spatial arrangement, and mobility using total internal reflection fluorescence microscopy, resonance energy transfer, and proximity biotinylation. In cells with
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Apelin Receptor homodimer oligomers revealed by single molecule imaging and novel g protein dependent signaling
Scientific Reports, 2017Co-Authors: Xin Cai, Bo Bai, Jing Chen, Rumin Zhang, Chunmei WangAbstract:The Apelin Receptor (APJ) belongs to family A of the G protein-coupled Receptors (GPCRs) and is a potential pharmacotherapeutic target for heart failure, hypertension, and other cardiovascular diseases. There is evidence APJ heterodimerizes with other GPCRs; however, the existence of APJ homodimers and oligomers remains to be investigated. Here, we measured APJ monomer-homodimer-oligomer interconversion by monitoring APJ dynamically on cells and compared their proportions, spatial arrangement, and mobility using total internal reflection fluorescence microscopy, resonance energy transfer, and proximity biotinylation. In cells with <0.3 Receptor particles/μm2, approximately 60% of APJ molecules were present as dimers or oligomers. APJ dimers were present on the cell surface in a dynamic equilibrium with constant formation and dissociation of Receptor complexes. Furthermore, we applied interference peptides and MALDI-TOF mass spectrometry to confirm APJ homo-dimer and explore the dimer-interfaces. Peptides corresponding to transmembrane domain (TMD)1, 2, 3, and 4, but not TMD5, 6, and 7, disrupted APJ dimerization. APJ mutants in TMD1 and TMD2 also decreased bioluminescence resonance energy transfer of APJ dimer. APJ dimerization resulted in novel functional characteristics, such as a distinct G-protein binding profile and cell responses after agonist stimulation. Thus, dimerization may serve as a unique mechanism for fine-tuning APJ-mediated functions.
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dynamics of Apelin Receptor g protein coupling in living cells
Experimental Cell Research, 2014Co-Authors: Bo Bai, Yunlu Jiang, Xin Cai, Jing ChenAbstract:During our research on Apelin Receptor (APJ) signalling in living cells with BRET and FRET, we demonstrated that Apelin-13 stimulation can lead to the activation of Gαi2 or Gαi3 through undergoing a molecular rearrangement rather than dissociation in HEK293 cells expressing APJ. Furthermore, Gαo and Gαq also showed involvement in APJ activation through a classical dissociation model. However, both FRET signal and BRET ratio between fluorescent Gαi1 subunit and Gβγ subunits demonstrated little change after Apelin-13 stimulation. These results demonstrated that stimulation of APJ with Apelin-13 causes activation of Gαi2, Gαi3, Gαo, Gαq; among which Gαi2, Gαi3 were activated through a novel rearrangement process. These results provide helpful data for understanding APJ mediated G-protein signalling.
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heterodimerization of Apelin Receptor and neurotensin Receptor 1 induces phosphorylation of erk1 2 and cell proliferation via gαq mediated mechanism
Journal of Cellular and Molecular Medicine, 2014Co-Authors: Bo Bai, Yunlu Jiang, Xin Cai, Jing Chen, Emmanouil KarterisAbstract:Dimerization of G protein-coupled Receptors (GPCRs) is crucial for Receptor function including agonist affinity, efficacy, trafficking and specificity of signal transduction, including G protein coupling. Emerging data suggest that the cardiovascular system is the main target of Apelin, which exerts an overall neuroprotective role, and is a positive regulator of angiotensin-converting enzyme 2 (ACE2) in heart failure. Moreover, ACE2 cleaves off C-terminal residues of vasoactive peptides including Apelin-13, and neurotensin that activate the Apelin Receptor (APJ) and neurotensin Receptor 1 (NTSR1) respectively, that belong to the A class of GPCRs. Therefore, based on the similar mode of modification by ACE2 at peptide level, the homology at amino acid level and the capability of forming dimers with other GPCRs, we have been suggested that APJ and NTSR1 can form a functional heterodimer. Using co-immunoprecipitation, BRET and FRET, we provided conclusive evidence of heterodimerization between APJ and NTSR1 in a constitutive and induced form. Upon agonist stimulation, hetrodimerization enhanced ERK1/2 activation and increased proliferation via activation of Gq α-subunits. These novel data provide evidence for a physiological role of APJ/NTSR1 heterodimers in terms of ERK1/2 activation and increased intracellular calcium and induced cell proliferation and provide potential new pharmaceutical targets for cardiovascular disease.
Janet J. Maguire - One of the best experts on this subject based on the ideXlab platform.
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international union of basic and clinical pharmacology cvii structure and pharmacology of the Apelin Receptor with a recommendation that elabela toddler is a second endogenous peptide ligand
Pharmacological Reviews, 2019Co-Authors: Cai Read, Robyn Macrae, Duuamene Nyimanu, Janet J. Maguire, Thomas L. Williams, Peiran Yang, David J Huggins, Petra Sulentic, Robert C Glen, Anthony P. DavenportAbstract:The predicted protein encoded by the APJ gene discovered in 1993 was originally classified as a class A G protein-coupled orphan Receptor but was subsequently paired with a novel peptide ligand, Apelin-36 in 1998. Substantial research identified a family of shorter peptides activating the Apelin Receptor, including Apelin-17, Apelin-13, and [Pyr1]Apelin-13, with the latter peptide predominating in human plasma and cardiovascular system. A range of pharmacological tools have been developed, including radiolabeled ligands, analogs with improved plasma stability, peptides, and small molecules including biased agonists and antagonists, leading to the recommendation that the APJ gene be renamed APLNR and encode the Apelin Receptor protein. Recently, a second endogenous ligand has been identified and called Elabela/Toddler, a 54-amino acid peptide originally identified in the genomes of fish and humans but misclassified as noncoding. This precursor is also able to be cleaved to shorter sequences (32, 21, and 11 amino acids), and all are able to activate the Apelin Receptor and are blocked by Apelin Receptor antagonists. This review summarizes the pharmacology of these ligands and the Apelin Receptor, highlights the emerging physiologic and pathophysiological roles in a number of diseases, and recommends that Elabela/Toddler is a second endogenous peptide ligand of the Apelin Receptor protein.
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Apelin Receptor (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database
IUPHAR BPS Guide to Pharmacology CITE, 2019Co-Authors: Anthony P. Davenport, Duuamene Nyimanu, Janet J. Maguire, Matthias J. Kleinz, Thomas L. Williams, Robyn G. C. Macrae, Peiran YangAbstract:The Apelin Receptor (nomenclature as agreed by the NC-IUPHAR Subcommittee on the Apelin Receptor [68]) responds to Apelin, a 36 amino-acid peptide derived initially from bovine stomach. Apelin-36, Apelin-13 and [Pyr1]Apelin-13 are the predominant endogenous ligands which are cleaved from a 77 amino-acid precursor peptide (APLN, Q9ULZ1) by a so far unidentified enzymatic pathway [80]. A second family of peptides discovered independently and named Elabela [11] or Toddler, that has little sequence similarity to Apelin, is present, and functional at the Apelin Receptor in the adult cardiovascular system [87, 67]. Structure-activity relationship Elabela analogues have been described [61].
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Apelin-36-[L28A] and Apelin-36-[L28C(30kDa-PEG)] peptides that improve diet induced obesity are G protein biased ligands at the Apelin Receptor.
Peptides, 2019Co-Authors: Duuamene Nyimanu, Rhoda E. Kuc, Janet J. Maguire, Thomas L. Williams, Maria Bednarek, Philip D Ambery, Lutz Jermutus, Anthony P. DavenportAbstract:Abstract Background Apelin signalling pathways have important cardiovascular and metabolic functions. Recently, Apelin-36-[L28A] and Apelin-36-[L28C(30kDa-PEG)], were reported to function independent of the Apelin Receptor in vivo to produce beneficial metabolic effects without modulating blood pressure. We aimed to show that these peptides bound to the Apelin Receptor and to further characterise their pharmacology in vitro at the human Apelin Receptor. Methods [Pyr1]Apelin-13 saturation binding experiments and competition binding experiments were performed in rat and human heart homogenates using [125I]Apelin-13 (0.1 nM), and/or increasing concentrations of Apelin-36, Apelin-36-[L28A] and Apelin-36-[L28C(30kDa-PEG)] (50pM-100μM). Apelin-36 and its analogues Apelin-36-[F36A], Apelin-36-[L28A], Apelin-36-[L28C(30kDa-PEG)], Apelin-36-[A28 A13] and [40kDa-PEG]-Apelin-36 were tested in forskolin-induced cAMP inhibition and β–arrestin assays in CHO-K1 cells heterologously expressing the human Apelin Receptor. Bias signaling was quantified using the operational model for bias. Results In both species, [Pyr1]Apelin-13 had comparable subnanomolar affinity and the Apelin Receptor density was similar. Apelin-36, Apelin-36-[L28A] and Apelin-36-[L28C(30kDa-PEG)] competed for binding of [125I]Apelin-13 with nanomolar affinities. Apelin-36-[L28A] and Apelin-36-[L28C(30kDa-PEG)] inhibited forskolin-induced cAMP release, with nanomolar potencies but they were less potent compared to Apelin-36 at recruiting β-arrestin. Bias analysis suggested that these peptides were G protein biased. Additionally, [40kDa-PEG]-Apelin-36 and Apelin-36-[F36A] retained nanomolar potencies in both cAMP and β-arrestin assays whilst Apelin-36-[A13 A28] exhibited a similar profile to Apelin-36-[L28C(30kDa-PEG)] in the β–arrestin assay but was more potent in the cAMP assay. Conclusions Apelin-36-[L28A] and Apelin-36-[L28C(30kDa-PEG)] are G protein biased ligands of the Apelin Receptor, suggesting that the Apelin Receptor is an important therapeutic target in metabolic diseases.
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BS49 Human embryonic stem cell derived cardiomyocytes express functional Receptors for the cardiovascular peptide Apelin
Basic Science, 2019Co-Authors: Robyn Macrae, William G. Bernard, Rhoda E. Kuc, Maria Colzani, Tom Williams, Duuamene Nyimanu, Janet J. Maguire, Sanjay Sinha, Anthony P. DavenportAbstract:Introduction The Apelin Receptor is expressed throughout the cardiovascular system, including in cardiomyocytes. Receptor activation by either of its endogenous peptide ligands, Apelin or Elabela, has a positive inotropic effect and promotes vasodilatation. Human embryonic stem cell (hESC)-derived cells have the potential for use in translational research to investigate cellular signalling, disease pathogenesis and potential novel treatments. Our aim was to determine if hESC-derived cardiomyocytes express Apelin Receptor protein and to quantify Receptor density to ascertain if this cell type can be used as a phenotypic model for human diseases associated with Apelin Receptor mutations. Methods H9 hESCs were cultured to induce differentiation to beating cardiomyocytes. Saturation radioligand binding experiments were performed using [Glp65,Nle75,Tyr77][125I]Apelin-13 and [Pyr1]Apelin-13 to define non-specific binding. Bound radioactivity was counted and data analysed using iterative curve fitting programs to obtain values of Receptor affinity (KD) and density (BMAX). Immunocytochemistry was carried out using anti-Apelin Receptor or anti-cardiac cell marker antibodies. Results Previous work demonstrated expression of the Apelin Receptor in hESC-derived cardiomyocytes at the gene level by qRT-PCR at similar levels to adult cells (figure 1A). Here, radioligand binding studies have confirmed Receptor protein expression in beating hESC-derived cardiomyocytes. Binding was saturable and [125I]Apelin-13 bound with sub-nanomolar affinity (0.12 nM) and Receptor density found to be 21 fmol/mg (figure 1B), comparable to that found in human adult heart. Hill slope was close to 1 consistent with a single binding site for Apelin in these cells. Furthermore, beating hESC-derived cardiomyocytes stained positive for Apelin Receptor, in addition to the standard cardiac markers including cardiac troponin T (figure 2). Conclusion These data importantly confirm that hESC-derived cardiomyocytes express Apelin Receptor protein at similar levels to adult human heart. Apelin Receptor mutations have been identified in the 100,000 Genomes Bridge Project that are associated with rare diseases, including pulmonary arterial hypertension. Our ongoing experiments aim to pharmacologically characterise the Apelinergic signalling pathway in the beating hESC-derived cardiomyocytes and we propose to generate hESC-derived phenotypic models by introducing selected Apelin Receptor mutations via CRISPR/Cas-9 gene editing. Conflict of interest None
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BS16 A novel fluorescent Apelin ligand tracks Apelin Receptor internalisation
Basic Science, 2019Co-Authors: Elisabeth O’flaherty, Robyn Macrae, Tom Williams, Duuamene Nyimanu, Janet J. Maguire, Heather Currinn, Jason Brown, Alastair J H Brown, Gregory Strachan, Anthony P. DavenportAbstract:Introduction Fluorescent ligands are a promising tool in the study of GPCR pharmacology as they are safe and more cost effective than conventional probes such as radioligands. In this study, we investigated the use of a novel fluorescent ligand, Apelin-488, to successfully show qualitative and quantitative Apelin Receptor internalisation, which may help further our understanding of Apelin Receptor pharmacology. The Apelin Receptor, a class A GPCR that unusually binds two endogenous peptide ligands, Apelin and elabela/toddler, is expressed and functional in the cardiovascular system. Mutations in the human Apelin Receptor have been identified in rare diseases and our aim is to identify tool compounds to explore the potential functional consequence of these mutations. Methods CHO-K1 cells stably expressing the wild-type Apelin Receptor were treated with Apelin-488, fixed, and stained with Hoechst 33342 and a wheat germ agglutinin-AlexaFluor-594 conjugate as nuclear and membrane markers respectively. Triple fluorescence confocal images were generated using Opera PhenixTM High Content Screening System and data were quantified using Harmony High Content Imaging and Analysis Software. Fluorescent ligand intensity at the membrane or in the cytoplasm was determined, providing cellular localisation for the Apelin Receptor following ligand binding. Activity of the fluorescent ligand at the Apelin Receptor in comparison to the endogenous ligand, [Pyr1]Apelin-13 or CMF-019, a G protein biased small molecule Apelin Receptor ligand, was determined using a dynamic mass redistribution assay and radioligand binding assays. Results The fluorescent ligand, Apelin-488 binds specifically to the Apelin Receptor in stably transfected CHO-K1 cells. High content screening revealed that in these cells, the Apelin Receptor is internalised over time and was still present at the cell membrane at an early time point, 30 secs (fig 1A). However, by 60 mins, Apelin-488 membrane localisation was reduced while internal localisation had increased, suggesting substantial Apelin Receptor internalisation had occurred. (fig 1B). Untransfected CHO-K1 cells showed no Apelin-488 signal, supporting specific binding of the fluorescent ligand to the Apelin Receptor. A dynamic mass redistribution assay demonstrated that Apelin-488 is functional at the Apelin Receptor, at a similar potency as the endogenous ligand [Pyr1]Apelin-13 and the G protein biased small molecule Apelin Receptor ligand CMF-019 (Fig 2). Conclusion Apelin-488 shows specific binding to the Apelin Receptor while retaining activity in a dynamic mass redistribution and binding assays. This work provides evidence for Apelin-488 as a novel fluorescent tool for assessing internalisation of the Apelin Receptor following Receptor binding. A new method for studying Apelin Receptor internalisation is particularly interesting due to the implication of Apelin Receptor desensitisation in pulmonary arterial hypertension, mediated by arrestin and internalisation pathways. Additionally, Apelin-488 may provide insight into binding and Receptor trafficking in mutated Apelin Receptors previously identified in patients with rare cardiovascular diseases. As such, Apelin-488 and other fluorescent ligands may help streamline development of drugs that could be beneficial in clinical settings. Conflict of interest none
