As with most G protein coupled
As with most G protein-coupled receptors (GPCRs), sustained activation of APJ can cause desensitization and this has been reported to occur for APJ-mediated effects on cytoplasmic Ca2+ concentration, as well as for effects on activity of adenylyl cyclase, ERK and Akt (Ishida et al., 2004, Masri et al., 2006). APJ also undergoes agonist-induced internalization and down-regulation and so research has focused on the possible role for the canonical pathway for rapid homologous receptor desensitization and trafficking in mediating adaptive responses to APJ activation (Evans et al., 2001, Zhou et al., 2003, Lee et al., 2010). In this pathway, agonist occupied GPCRs are preferred substrates for phosphorylation by G-protein receptor kinases (GRKs) and this phosphorylation mediates binding with β-arrestins that prevent the receptors from activating their cognate G-proteins, thereby causing receptor desensitization. The β-arrestins also target the desensitized receptors for internalization via clathrin-coated vesicles (CCVs). After this the vesicles are uncoated, β-arrestins dissociate, receptors are dephosphorylated and the receptor-containing vesicles may be trafficked back to the plasma membrane (a process that can mediate resensitization to the agonist) or to lysosomes for proteolytic digestion (a process that can cause receptor down-regulation). Differing patterns of β-arrestin interaction have allowed the sorting of GPCRs into two classes: Class A receptors, that have a brief interaction with β-arrestins (at the plasma membrane) and preferentially bind β-arrestin2 over -1, and display rapid recycling; and Class B receptors, that form a stable complex with both β-arrestins with equal affinity, and which internalize with the β-arrestins into endosomes. Additional players in this process include epsin and EPS15, which act as adapter proteins for clathrin-mediated endocytosis (CME) (Wolfe and Trejo, 2007), and dynamin, a GTPase that forms a multimeric complex around the neck of nascent endocytic vesicles and mediates their p38 pathway off to form endosomes (Damke, 1996). The adaptive processes outlined above are thought to be relevant for APJ as apelin causes clathrin-mediated APJ internalization (Reaux et al., 2001, El Messari et al., 2004) and also translocation of β-arrestin1 and -2 to the cell surface, indicating translocation to phosphorylated APJ (Lee et al., 2010). Moreover, after agonist-induced internalization, APJ can either be recycled to the cell surface or be degraded in lysosomes (Lee et al., 2010). Interestingly, APJ trafficking displays ligand bias for both Class A and B β-arrestin/recycling behaviour as when internalization is stimulated by [Pyr1]apelin-13, internalized APJ is rapidly recycled to the plasma membrane with none remaining in the cytoplasm at 60 min, whereas APJ is retained within the cell for up to 120 min after apelin-36-stimulated internalization (Zhou et al., 2003). Similarly, although apelin-13 causes β-arrestin1 translocation to the plasma membrane, the internalized receptors are not associated with β-arrestin1 and are rapidly recycled to the cell surface via early endosomes (Evans et al., 2001, Lee et al., 2010), whereas after apelin-36 stimulation the internalized APJ are co-localized with β-arrestin1 and then undergo rab-7-dependent trafficking to lysosomes (Lee et al., 2010). Finally, truncation of the APJ C-terminus (in order to delete potential GRK phosphorylation sites) prevents homologous desensitization to effects of apelin-13, but not to those of apelin-36, on inhibition of adenylyl cyclase and activation of ERK and Akt (Masri et al., 2006, Lee et al., 2010). Apelin/APJ has emerged as a major signalling pathway in physiological homeostasis (O'Carroll et al., 2013) and central to ascertaining the precise function of this receptor is an understanding of the system of regulation that dynamically modulates APJ signalling. In peripheral tissues the apelinergic system appears to be down-regulated in hypertensive disease – levels of apelin immunoreactivity in plasma, and in ventricular and aortic tissues, are lower in the spontaneously hypertensive rat, a genetic model of hypertension, than in control Wistar-Kyoto normotensive rats (Zhang et al., 2006b, Zhang et al., 2006a, Zhong et al., 2005). Additionally circulating levels of apelin are decreased in patients with essential (Sonmez et al., 2010) and pulmonary (Chandra et al., 2011) hypertension, while there is a negative correlation between plasma apelin levels and blood pressure (Zhu et al., 2013). This suggests a role for decreased peripheral apelin signalling in the pathophysiology of hypertension. Receptor trafficking is a key process for regulating receptor signalling pathways and cellular functions, however in the case of APJ the mechanisms and proteins involved in agonist-induced trafficking are not well understood. To further understand the signalling and regulation of APJ, and thus the efficacy of ligands for potential therapeutic intervention, this study set out to characterize the mechanisms underlying [Pyr1]apelin-13-induced APJ desensitization and internalization, and to determine whether agonist-induced APJ internalization contributes to its functional desensitization. A stable HEK-293 cell line expressing N-terminus HA-tagged mouse APJ (mAPJ) was generated, and a semi-automated imaging protocol was used to quantitate ERK1/2 activation and APJ trafficking in this cell line following agonist activation with [Pyr1]apelin-13. The mechanisms of [Pyr1]apelin-13-induced internalization were further explored using dominant-negative mutant (DNM) cDNA constructs of GRK2 (GRKDNM), β-arrestin1 (βARRDNM), EPS15 (EPSDNM) and dynamin (DYNDNM), known effectors of CME.