br Methods and materials br

Methods and materials
Results
Discussion GPCR regulation in response to agonist stimulation is common to nearly all GPCRs and is essential in physiological systems to limit persistent signalling. In this study we have investigated the [Pyr1]apelin-13-induced trafficking and desensitization of mAPJ in mAPJ-HEK293 cells using a semi-automated imaging protocol and clearly show that HA-mAPJ internalization is a GRK2-, dynamin- and EPS15-mediated event. A stable HA-mAPJ expressing cell line was generated and was used to quantify the proportion of APJ at the cell surface and within whole dmh express using semi-automated acquisition and analysis of digital fluorescence images. While the majority of epitope-tagged mAPJ was localized to the cell surface in these cells, a proportion of tagged APJ was distributed within the cell. This is in contrast to earlier studies that reported enhanced green fluorescent protein (eGFP)-APJ localization, under basal conditions, to be confined to the plasma membrane (El Messari et al., 2004). APJ acts primarily via Gi to inhibit adenylyl cyclase but has also been reported to activate other effectors including PKC, PI3K and ERK (Masri et al., 2002, Masri et al., 2004). As positioning of differing tags into the native receptor may have implications for receptor trafficking, we verified that the functional integrity of the receptor in our cell line remained intact and that these HA-mAPJ-HEK293 cells, like their non-tagged counterparts, mediate ERK activation. Significant and similar [Pyr1]apelin-13-induced stimulation of ERK1/2 was seen in both HA-tagged and untagged mAPJ transfected HEK293 cells. Many GPCRs show ligand bias (where different agonists bias signalling toward different effectors) and there is evidence that this may occur for APJ (Masri et al., 2006, Brame et al., 2015). It has been shown recently that the cyclic apelin analogue MM07 displays bias towards stimulation of a beneficial G-protein-dependent pathway, stimulating vasodilation and inotropic actions, over a more damaging G-protein-independent β-arrestin-dependent pathway that results in cardiac hypertrophy (Brame et al., 2015). In this regard, it is also of interest that APJ is most closely related to angiotensin 1 receptors (AT1), for which ligand bias has been extensively explored. AT1A receptors are Gq/11 coupled GPCRs that also activate ERK. They undergo a process of rapid homologous receptor desensitization in which arrestins bind to the activated receptors preventing them from activating their cognate G proteins and targeting them for internalization via CCVs. The arrestins can also act as scaffolds for MAPK cascade components and mediate signalling to ERK. Activation of AT1A receptors can cause an initial phase of G protein-mediated ERK activation followed by a switch to a second phase of arrestin-mediated ERK activation and ligand bias is seen when angiotensin II activates both pathways whereas analogues (such as [Sar(1),Ile(4),Ile(8)]AngII (SII)) engage only the latter (Lefkowitz and Shenoy, 2005, Ahn et al., 2004, Shenoy et al., 2006). We were interested in the possibility that APJ might also mediate such a biphasic response. We established however that when mAPJ-HEK293 cells were treated for varied times with [Pyr1]apelin-13, the ppERK response instead was rapid and transient, with an EC50 value of ∼3 nM at 5 min, showing no indication of arrestin-mediated activation of ERK1/2. Having established that the ppERK response to [Pyr1]apelin-13 desensitizes rapidly during sustained stimulation in this model, we explored possible mechanisms. Numerous adaptive mechanisms shape ERK responses and these include inhibitory phosphorylation of Ras by ERK (Dumaz and Marais, 2005) or ERK-driven expression of nuclear-inducible dual specificity phosphatases (DUSP) (Caunt et al., 2008). However, neither of these down-stream mechanisms seems likely here as APJ-mediated ERK activation is Ras-independent in CHO cells (Masri et al., 2002) and the desensitization is too fast to be mediated by DUSP neosynthesis (Caunt et al., 2008). We therefore suspected that this rapid desensitization of the response to [Pyr1]apelin-13 was due to upstream APJ-specific (rather than down-stream ERK-specific) adaptive mechanisms and explored this by looking at homologous and heterologous desensitization of this response. Desensitization can be homologous or heterologous in nature; homologous desensitization occurs when there is a loss of response solely to an agonist that is acting at one particular GPCR subtype, whereas heterologous desensitization is agonist-non-specific and involves a broad pattern of unresponsiveness at multiple GPCR subtypes. Homologous desensitization is thought to involve adaptive changes at the level of the GPCR itself, whereas heterologous desensitization may also involve altering the efficiency of downstream signalling components. Adrenaline and EGF are known activators of ERK in HEK293 cells, likely acting via the α1b adrenergic (Schonbrunn and Steffen, 2012) and EGF receptors (Kramer et al., 2002) respectively, and as expected, both caused robust increases in ppERK in PBS pre-incubated mAPJ-HEK293 cells, that were not different to the increases seen after pre-incubation with [Pyr1]apelin-13. The ppERK response to a subsequent [Pyr1]apelin-13 stimulation was however completely abrogated by pre-incubation with [Pyr1]apelin-13. These data suggest that the desensitization of [Pyr1]apelin-13-induced ERK1/2 phosphorylation was not due to a requirement to reset the intracellular signalling pathway or other post-receptor modifications, but to upstream APJ-specific adaptive changes. These could include receptor internalization, as APJ undergoes agonist-induced internalization via CCVs (Reaux et al., 2001, El Messari et al., 2004), and/or rapid homologous receptor desensitization, as APJ has been shown to cause translocation of β-arrestin to the cell surface in other systems (Lee et al., 2010). We undertook therefore to monitor APJ compartmentalization, and found that incubation of HA-mAPJ-HEK293 cells with [Pyr1]apelin-13 decreased both cell surface and whole cell expression of APJ in a time-dependent manner. These data are consistent with agonist-induced receptor internalization followed by degradation of a proportion of the internalized receptors, such that down-regulation follows the reduction in cell surface expression.