APJ receptors have a amino acid sequence with a

APJ receptors have a 380 amino tachykinin receptor sequence with a characteristic G-protein structure, including seven transmembrane domains and post-translation modification sites for phosphorylation, palmitoylation and glycosylation along with association sites for β-arrestin (O'Dowd et al., 1993). APJ receptor signaling involves multiple G-protein subunits. Initial work suggested the involvement of pertussis toxin sensitive Gαi/o subunits that inhibit forskolin-induced cyclic adenosine monophosphate (cAMP) formation in CHO cells (Tatemoto et al., 1998). This signaling pathway is also present in blood vessels, inasmuch as apelin inhibits large conductance, calcium-activated potassium (BKCa) channel currents in cerebral vascular smooth muscle cells in a pertussis toxin-sensitive manner (Modgil, Guo, O'Rourke, & Sun, 2013). Apelin was also found to increase Ca2+ mobilization in neuronal cells (Choe et al., 2000), suggesting the involvement of other G-protein subunits. Subsequent studies demonstrated that apelin-induced cardiac and smooth muscle contractile responses were attenuated by inhibitors of phospholipase C and PKC, suggesting a role for Gαq/11 subunits in APJ receptor signaling (Hashimoto et al., 2006; Szokodi et al., 2002). Moreover, apelin was shown to increase phosphorylation of myosin light chain (MLC) in vascular smooth muscle cells via activation of Gαq/11 subunits and a PKC-dependent mechanism (Hashimoto et al., 2006). In addition, a functional role for Gα13 subunits has been identified in apelin-induced cytoplasmic translocation of histone deacetylase during cardiac and vascular development (Kang et al., 2013). Various G-protein subunits, e.g. Gαi/o, Gαq and βγ dimers, can activate PI3K/Akt signaling (Murga, Laguinge, Wetzker, Cuadrado, & Gutkind, 1998), which is indeed involved in apelin-induced vascular responses. For example, apelin inhibits calcification of vascular smooth muscle cells via a PI3K/Akt signaling mechanism (Shan et al., 2011). Similarly, apelin attenuates apoptosis in vascular smooth muscle cells by activating PI3K/Akt and extracellular signal-regulated kinase (ERK) signaling (Cui et al., 2010; Tang et al., 2007). Apelin also inhibits BKCa channel currents by activation of PI3K/Akt pathways (Modgil, Guo, O'Rourke, & Sun, 2013). Apelin-induced PI3K/Akt signaling can increase phosphorylation of endothelial nitric oxide synthase (eNOS) to modulate aortic vascular tone (Zhong, Yu, et al., 2007). Likewise, Elabela improves self-renewal and regenerative ability of human progenitor stem cells via PI3K/Akt activation (Ho et al., 2015). These latter actions of Elabela might be mediated by a cell surface receptor different from the APJ receptor in human embryonic stem cells (Ho et al., 2015), suggesting the possible existence of another subtype or novel type of receptor; however, the bulk of evidence to date continues to support the view that the peptide is acting solely on the currently identified APJ receptor. Elabela also inhibits forskolin-induced cAMP formation by activating ERK1/2 signaling pathways in CHO cells (Wang, Yu, et al., 2015). In addition to these intracellular events, APJ receptors also have the ability to form homo- and heterodimers. APJ receptors are reported to hetero-dimerize with angiotensin (AT1) receptors (Siddiquee, Hampton, McAnally, May, & Smith, 2013), bradykinin (B1) receptors (Bai et al., 2014), neurotensin receptor-1 (NTSR1) (Bai, Cai, Jiang, Karteris, and Chen, 2014) and κ-opioid receptors (KOR) (Li et al., 2012). These receptors are involved in cardiovascular regulation and their ligands (apelin, des-Arg(9)-bradykinin, neurotensin and dynorphin) are sensitive to ACE-2 proteolysis (Vickers et al., 2002), suggesting the importance of these heterodimers in cardiovascular pharmacology. For example, APJ receptors can allosterically modify the functional state of AT1-receptors to a low affinity state towards angiotensin II (Siddiquee, Hampton, McAnally, May, & Smith, 2013; Sun et al., 2011) and these actions can be independent of its putative ligand, apelin (Siddiquee, Hampton, McAnally, May, & Smith, 2013). Similarly, constitutive heterodimers of APJ- and bradykinin (B1)-receptors are shown to generate higher intracellular Ca2+ and upregulate eNOS phosphorylation, possibly by strengthening the association of the dimer with Gαq subunits (Bai et al., 2014b). APJ-NTSR1 or APJ-KOR heterodimers also cause significant increases in phosphorylated ERK1/2 levels by increasing intracellular Ca2+ (Li et al., 2012; Bai et al., 2014a). A recent report demonstrated the existence of homodimers-oligomers of the human APJ receptor, which could possibly mediate different signaling events in comparison to APJ monomers (Cai, Bai, Zhang, Wang, & Chen, 2017).