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  • The OTR and GHSR are known to


    The OTR and GHSR are known to play similar roles in centrally regulated behaviours, where the OTR is implicated in mood, sociability and appetite regulation (Morton et al., 2012; Myers et al., 2014; Sala et al., 2013; Lukas et al., 2011; Slattery and Neumann, 2010), with the GHSR primarily involved in appetite and metabolism (Howick et al., 2017; Schellekens et al., 2010; De Vriese and Delporte, 2007) and also in anxiety and mood (Chuang and Zigman, 2010; Schellekens et al., 2012). Both the OTR and GHSR have the ability to form heterodimers with other GPCRs which results in alterations in receptor signalling (Schellekens et al., 2015; Wrzal et al., 2012; Terrillon et al., 2003; Romero-Fernandez et al., 2013; Kern et al., 2012; Rediger et al., 2011). Moreover, an overlap in OTR and GHSR receptor expression can be observed across multiple find molarity regions including the hippocampus find molarity (Zigman et al., 2006; Lin et al., 2017, 2018), the paraventricular and ventromedial sub regions of the hypothalamus (Yoshimura et al., 1993; Zigman et al., 2006), the basolateral amygdala and the piriform cortex (Mani et al., 2014; Marusak et al., 2015).
    Materials and methods
    Discussion The concept of heterodimerization adds a novel dimension to GPCR pharmacology and paves the way forward for new GPCR-targeting therapeutics (Hübner et al., 2016; Qian et al., 2018). The influence of heterocomplex formation on downstream GPCR signalling and functionality is vast but always directly dependent on the receptors that are present within the complexes (Terrillon and Bouvier, 2004; Romero-Fernandez et al., 2013; Scarlett et al., 2018; Schellekens et al., 2013a). Interestingly, ligands specific to one GPCR present within the complex will be able to modulate downstream signalling of the other GPCR within the heterocomplex, to which they have no innate affinity (Ward et al., 2011; Ellis et al., 2006; Borroto-Escuela et al., 2014; Wrzal et al., 2012; Schellekens et al., 2015). Therefore, the discovery of novel GPCR-GPCR interactions is poised to lead to a better understanding of the pathophysiology of disorders associated with alterations in GPCR signalling and function (Borroto-Escuela et al., 2017). Here we show, to our knowledge for the first time, compelling evidence for an interaction between the OTR and GHSR, which is likely to occur via the formation of a heterocomplex and leads to a significant attenuation of OTR-mediated Gαq signalling. This represents an exciting novel finding, which contributes to the advancement of the GPCR dimerization field overall, but in particular contributes to the intricate signalling crosstalk between oxytocinergic and ghrelinergic systems. Aberrant ghrelinergic signalling via the GHSR is associated with a multitude of disorders, spanning from obesity to depression (Howick et al., 2017; Tuncel et al., 2016; Huang et al., 2017). Likewise, oxytocinergic signalling via the OTR, plays a pivotal role in social behaviour and is involved in mood disorders, including anxiety, depression, but also addiction and obesity (Blevins et al., 2015; Ott et al., 2013; Thienel et al., 2016; Zhou et al., 2014; Brown et al., 2014; Jobst et al., 2014, 2015; Mcquaid et al., 2014). Thus, the ghrelinergic and oxytocinergic system share some interesting overlapping functionalities and play key roles in the pathophysiology of certain brain disorders, which may be explained by the formation of an OTR/GHSR heteromer. Here, we demonstrate co-localized expression of the OTR and GHSR, a positive fcFRET and dual receptor trafficking, indicative of the direct physical interaction of the OTR/GHSR pair. Interestingly, we observe a ubiquitous subcellular co-localization of GHSR and OTR under basal conditions, which is in contrast with the usually reported localization of GPCRs on the cell membrane (Mcneely et al., 2012; Nevins and Marchese, 2018) However, high ligand-independent constitutive activity under basal conditions has previously been reported to occur for both the OTR and the GHSR, which can explain a mainly cytosolic GPCR expression pattern (Di Benedetto et al., 2014; Mear et al., 2013). High constitutive expression and agonist-dependent endocytosis, associated with a ubiquitous subcellular expression, have also been shown with the β2 adrenergic and M3 muscarinic receptors (Scarselli and Donaldson, 2009). In addition, altered basal ligand-independent and agonist-mediated changes in receptor trafficking have also been reported upon heteromerization of the GHSR and the 5-HTR2C (Schellekens et al., 2013c). Here, we observe no changes in basal OTR trafficking following GHSR co-expression, but we demonstrate unique ligand-mediated trafficking of the GHSR/OTR pair upon co-expression, indicating receptor co-trafficking, which is absent under control conditions.