Endosomal trafficking is intricately linked to G protein-coupled receptors (GPCR) fate and signaling. Extracellular uridine diphosphate (UDP) acts as a signaling molecule by selectively activating the GPCR P2Y6. Despite the recent interest for this receptor in pathologies, such as gastrointestinal and neurological diseases, there is sparse information on the endosomal trafficking of P2Y6 receptors in response to its endogenous agonist UDP and synthetic selective agonist 5-iodo-UDP (MRS2693). Confocal microscopy and cell surface ELISA revealed delayed internalization kinetics in response to MRS2693 vs. UDP stimulation in AD293 and HCT116 cells expressing human P2Y6. Interestingly, UDP induced clathrin-dependent P2Y6 internalization, whereas receptor stimulation by MRS2693 endocytosis appeared to be associated with a caveolin-dependent mechanism. Internalized P2Y6 was associated with Rab4, 5, and 7 positive vesicles independent of the agonist. We have measured a higher frequency of receptor expression co-occurrence with Rab11-vesicles, the trans-Golgi network, and lysosomes in response to MRS2693. Interestingly, a higher agonist concentration reversed the delayed P2Y6 internalization and recycling kinetics in the presence of MRS2693 stimulation without changing its caveolin-dependent internalization. This work showed a ligand-dependent effect affecting the P2Y6 receptor internalization and endosomal trafficking. These findings could guide the development of bias ligands that could influence P2Y6 signaling.
Keywords: Endosomes; G protein-coupled receptors; Internalization; Ligands; P2Y(6) receptor; Trafficking.
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