Abstract
Apelin receptor(s) (APLNR) are suggested to mediate the actions of apelin and Elabela (ELA) peptides in many physiological processes, including cardiovascular development and food intake in vertebrates. However, the functionality of APLNR has not been examined in most vertebrate groups. Here, we characterized two APLNRs APLNR1, APLNR2) in chickens and red-eared sliders, and three APLNRs in zebrafish (APLNR2a, APLNR2b, APLNR3a), which are homologous to human APLNR. Using luciferase-reporter assays or Western blot, we demonstrated that in chickens, APLNR1 (not APLNR2) expressed in HEK293 cells was potently activated by chicken apelin-36 and ELA-32 and coupled to Gi-cAMP and MAPK/ERK signaling pathways, indicating a crucial role of APLNR1 in mediating apelin/ELA actions; in red-eared sliders, APLNR2 (not APLNR1) was potently activated by apelin-36/ELA-32, suggesting that APLNR2 may mediate apelin/ELA actions; in zebrafish, both APLNR2a and APLNR2b were potently activated by apelin-36/ELA-32 and coupled to Gi-cAMP signaling pathway, as previously proposed, whereas the novel APLNR3a was specifically and potently activated by apelin. Similarly, an apelin-specific receptor (APLNR3b) sharing 57% sequence identity with zebrafish APLNR3a was identified in Nile tilapia. Collectively, our data facilitates the uncovering of the roles of APLNR signaling in different vertebrate groups and suggests a key functional switch between APLNR1 and APLNR2/3 in mediating the actions of ELA and apelin during vertebrate evolution.
Highlights
Apelin receptor (APLNR) has been identified as a class A G protein-coupled receptor (GPCR) and originally named APJ, which shares a relatively high amino acid sequence identity (31%) with angiotensin II receptor (AT1), but displays no specific binding toward angiotensin II peptide [1, 2]
Chicken APLN cDNA is 878 bp in length (KX017222) and predicted to encode a 78-amino acid (a.a.) precursor, which shows 4273% a.a. sequence identities with preproapelin of other vertebrate species, including humans, turtles, X. tropicalis, spotted gars, and zebrafish
Like zebrafish APLNR3a, tilapia APLNR3b could be potently activated by apelin-36 (EC50: 1.36 ± 0.96 nM), but not by ELA-32, suggesting that APLNR3b may function as an apelin-specific receptor (Figure 5). In support of this notion, we further demonstrated that zebrafish APLNR3a or tilapia APLNR3b expressed in human embryonic kidney 293 (HEK293) cells could be potently activated by synthetic zebrafish apelin36, but not by zebrafish ELA-36/ELA-22, whereas zebrafish APLNR2a/APLNR2b could be activated potently by both apelin and ELA (Figure 5; Table 1)
Summary
Apelin receptor (APLNR) has been identified as a class A (rhodopsin-like) G protein-coupled receptor (GPCR) and originally named APJ, which shares a relatively high amino acid sequence identity (31%) with angiotensin II receptor (AT1), but displays no specific binding toward angiotensin II peptide [1, 2]. The wide tissue expression of APLN-APLNR suggests their involvement in the regulation of many physiological processes, such as cardiovascular development and function [15, 16], blood pressure [17, 18], fluid homeostasis [19], angiogenesis [20], neuroendocrine activity [21], heart development [22,23,24], food intake [25, 26], drinking behavior [27, 28], pituitary hormone secretion [27, 29], metabolism [30], and neuroprotection [31] in the central nervous system (CNS) and peripheral tissues, some of these findings are contradictory [5]. Similar to these mammalian findings, APLN/APLNR knockdown in zebrafish or Xenopus laevis causes abnormalities in vascular development [32, 33], myocardial cell specification and heart formation [24, 34, 35], further highlighting the ancient and conserved roles of APLN-APLNR signaling in the regulation of cardiovascular development in vertebrates [36]
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