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  • HEK cells stably transfected with

    2019-09-11

    HEK-293 Cytarabine stably transfected with CysLT1 were stimulated with LTD and internalization was studied by confocal microscopy, FACS or cell-surface ELISA. Cells were co-transfected with wildtype and dominant negative (DN) mutants of arrestin and dynamin. Clathrin-mediated endocytosis was investigated by pre-treatment with either hyperosmotic sucrose or Concanavalin A before stimulation with LTD. Trafficking of CysLT1 was studied by co-localization with endogenous Rab proteins using confocal microscopy. CysLT1 internalizes rapidly, with up to 60% receptors internalized within 1h of stimulation. The receptors accumulate in the peri-nuclear region and the internalization is sustained for 5 hours. However, after agonist removal, the majority of receptors are re-expressed rapidly. Co-transfection with DN constructs of Arr-2 and Arr-3 did not block the internalization of the receptor while DN dynamin (K44A) blocked internalization of CysLT1. Pretreatement with hyperosmotic sucrose and concanavalin A blocked CysLT1 internalization by 90% and 70%, respectively, indicating clathrin vesicle participation. After stimulation with LTD, CysLT1 co-localized with both Rab-5 and Rab-11. The internalization of CysLT1 is independent of arrestins but dependent on dynamin activity. CysLT1 internalizes through clathrin vesicles and is associated with Rabs5-positive early endosomes. Rab11 could control late recycling of CysLT1 or transport between endosomes and the trans-Golgi network.
    Introduction P2Y receptors are membrane bound and G-protein-coupled receptors (GPCRs) for extracellular nucleotides (Burnstock and Kennedy, 1985). Eight mammalian P2Y receptor subtypes (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14) have been identified (Ralevic and Burnstock, 1998; von Kügelgen and Wetter, 2000; Abbracchio et al., 2006; von Kügelgen and Harden, 2011; von Kügelgen and Hoffmann, 2016; Kennedy, 2017; Pérez-Sen et al., 2017; von Kügelgen, 2017). All P2Y receptors belong to the δ-subgroup of class A GPCRs (Fredriksson et al., 2003; Lagerström and Schiöth, 2008). Two subfamilies of P2Y receptors have been defined by similarities in amino acid sequences and signal transduction pathways (Abbracchio et al., 2006; von Kügelgen, 2006). The first subfamily consists of the P2Y1, P2Y2, P2Y4, P2Y6 and P2Y11 receptors. These receptors couple via Gq-proteins. The P2Y11 receptor couples in addition to Gs-proteins (Fig. 1; references in Table 1). The receptors of the second subfamily (P2Y12, P2Y13, and P2Y14 receptors) mediate cellular responses via activation of Gi-proteins (Fig. 1; references in Table 1). Some GPCRs with a published “p2y” nomenclature do not function as P2Y receptors for extracellular nucleotides. E.g., the “p2y5” receptor protein acts as a receptor for lysophosphatidic acid (Pasternack et al., 2008). P2Y receptors play important roles in physiology and pathophysiology (Ralevic and Burnstock, 1998, 2003; Müller, 2002; Leipziger, 2003; Burnstock, 2007; Burnstock et al., 2011; Burnstock and Boeynaems, 2014; Franke and Illes, 2014; Idzko et al., 2014; Oliveira et al., 2016; Burnstock, 2017; Le Duc et al., 2017; Nishimura et al., 2017). The ADP-induced platelet aggregation is mediated by activation of P2Y1 and P2Y12 receptors (Cattaneo, 2011; Liverani et al., 2014b). The nucleoside analogue ticagrelor and active metabolites of the thienopyridine compounds ticlopidine, clopidogrel and prasugrel inhibit platelet P2Y12 receptors and reduce thereby platelet aggregation. These drugs are used for the prevention and therapy of cardiovascular events (Cattaneo, 2011). The P2Y2 receptor agonist diquafosol is used for the treatment of the dry eye syndrome (Jacobson and Civan, 2016). In addition to an action via P2Y receptors, extracellular UDP activates GPCRs for cysteinyl leukotrienes (CysLT1, CysLT2; Mellor et al., 2001, 2003). GPR17 has been reported to function as a receptor for cysteinyl leukotrienes and uracil nucleotides (Boda et al., 2011; Fumagalli et al., 2011; but see Hennen et al., 2013; Qi et al., 2013).