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  • br Materials and methods br


    Materials and methods
    Results and discussion
    Funding This work was supported by the Canadian Institute of Health Research (CIHR) [grant number FDN-148413] to PS, the National Science and Engineering Research Council of Canada (NSERC) [grant number CRD-399680] to ÉM, and the FRQ-S funded Réseau québécois de recherche sur le médicament (RQRM). The authors declare no competing financial interests.
    Authors contribution
    Acknowledgments ÉBO was supported by a research fellowship from the Institut de pharmacologie de Sherbrooke (IPS) and Centre d’excellence en neurosciences de l’Université de Sherbrooke (CNS). PS holds a Canada Research Chair in Neurophysiopharmacology of Chronic Pain. Drs. M. Bouvier, T. Hebert, S.A. Laporte, G. Pineyro, J.-C. Tardif and E. Thorin (CQDM Team) are also acknowledged for providing us with the G-protein BRET-based biosensors.
    Introduction Up to date, nearly 87 class A G protein-coupled receptors (GPCRs) are still classified as orphan GPCRs and their ligands have not yet been identified [1]. The G protein–coupled receptor 25 (GPR25) is an example of orphan receptors in the class A GPCR family. GPR25 was first cloned in 1997 and mapped on human chromosome 1 [2]. GPR25 is expressed in human memory T-cells and NK-cells and identified as a primary causal gene associated with autoimmune diseases revealed by cis-eQTL mapping based on a genome-wide association study (GWAS) [3]. Moreover, GPR25 is reported to be associated with arterial stiffness, and presumably, it may be capable of binding an unknown ligand to regulate blood pressure (BP) [4]. Despite the progress on understanding the potential roles of GPR25, the identity of its endogenous ligand remains unknown. Within the class A GPCR family, GPR25 shares a relatively high degree of amino pacap sequence identity (29–34%) with vertebrate Apelin receptor (APLNR) [5]. It is clear that APLNR is activated by two distinct peptides, named Apelin and Apela (also known as Elabela and Toddler), which are encoded by two separate genes (APELIN and APELA), in humans [6], rats [7] and zebrafish [8,9]. Apelin is a peptide of 36 or 13 amino acids (designated as Apelin-36 and Apelin-13 respectively) and identified as the first endogenous ligand of APLNR [10]. Both Apelin and APLNR are reported to be widely expressed in the central nervous system (CNS) and peripheral tissues and involved in the regulation of many physiological/pathological processes, including the cardiovascular functions, angiogenesis, water balance and stress-induced disorders in vertebrates [[11], [12], [13]]. Apela is an alternative endogenous ligand of APLNR which was identified in zebrafish and mammals just recently. Apela is a peptide of 36 (Apela-36) or 22 (Apela-22) amino acids. Despite the low degree of amino acid sequence identity (25%) shared between Apelin and Apela peptides [14], both peptides can equipotently activate APLNR, which is functionally coupled to Gi protein and its activation decreases intracellular cAMP levels. In zebrafish, Apela is reported to be essential for cell movement during gastrulation and heart development [8,9]. In mammals, Apela is highly expressed in the kidneys of humans [6] and rats [7] and plays important roles in angiogenesis [6], fluid homeostasis [7], placental development [15] and cardiovascular development [16]. The structural similarity shared between APLNR and GPR25 led us to hypothesize that like APLNR, GPR25 could be activated by Apelin and/or Apela in vertebrates. To test this hypothesis, in this study, we cloned GPR25 from several representative vertebrate species, including zebrafish, pigeons, spotted gars and humans, and tested whether this receptor could be activated by Apelin and Apela in vitro. Our data, for the first time, demonstrated that the orphan receptor GPR25 can be activated by Apelin and Apela in non-mammalian vertebrates, and its activation can decrease intracellular cAMP levels. Our findings will help to unravel the physiological roles of GPR25 signaling in vertebrates.