title: The regulated expression, intracellular trafficking, and membrane recycling of the P2Y-like receptor GPR17 in Oli-neu oligodendroglial cells.
abstract: GPR17 is a G-protein-coupled receptor that is activated by two classes of molecules: uracil-nucleotides and cysteinyl-leukotrienes. GPR17 is required for initiating the differentiation of oligodendrocyte precursors but has to be down-regulated to allow cells to undergo terminal maturation. Although a great deal has been learned about GPR17 expression and signaling, no information is currently available about the trafficking of native receptors after the exposure of differentiating oligodendrocytes to endogenous agonists. Here, we demonstrate that neuron-conditioned medium induces the transcriptionally mediated, time-regulated expression of GPR17 in Oli-neu, an oligodendrocyte precursor cell line, making these cells suitable for studying the endocytic traffic of the native receptor. Agonist-induced internalization, intracellular trafficking, and membrane recycling of GPR17 were analyzed by biochemical and immunofluorescence assays using an ad hoc-developed antibody against the extracellular N-terminal of GPR17. Both UDP-glucose and LTD(4) increased GPR17 internalization, although with different efficiency. At early time points, internalized GPR17 co-localized with transferrin receptor, whereas at later times it partially co-localized with the lysosomal marker Lamp1, suggesting that a portion of GPR17 is targeted to lysosomes upon ligand binding. An analysis of receptor recycling and degradation demonstrated that a significant aliquot of GPR17 is recycled to the cell surface. Furthermore, internalized GPR17 displayed a co-localization with the marker of the "short loop" recycling endosomes, Rab4, while showing very minor co-localization with the "long loop" recycling marker, Rab11. Our results provide the first data on the agonist-induced trafficking of native GPR17 in oligodendroglial cells and may have implications for both physiological and pathological myelination.
authors.affiliation: Consiglio Nazionale delle Ricerche-Institute of Neuroscience, Department of Medical Biotechnologies and Translational Medicine, University of Milan, Milan 20129, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Fratangeli
authors.forename: Elena
authors.initials: E
authors.lastname: Parmigiani
authors.forename: Marta
authors.initials: M
authors.lastname: Fumagalli
authors.forename: Davide
authors.initials: D
authors.lastname: Lecca
authors.forename: Roberta
authors.initials: R
authors.lastname: Benfante
authors.forename: Maria
authors.initials: M
authors.lastname: Passafaro
authors.forename: Annalisa
authors.initials: A
authors.lastname: Buffo
authors.forename: Maria P
authors.initials: MP
authors.lastname: Abbracchio
authors.forename: Patrizia
authors.initials: P
authors.lastname: Rosa
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D019070:Cell Lineage; D002462:Cell Membrane / Q000378:metabolism*; D003001:Cloning, Molecular; D004705:Endocytosis; D005786:Gene Expression Regulation; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009836:Oligodendroglia / Q000166:cytology; D034622:RNA Interference; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D058470:Receptors, Purinergic P2Y / Q000378:metabolism*
chemical_list: C121670:GPR17 protein, human; C534979:GPR17 protein, mouse; C514731:GPR17 protein, rat; D009419:Nerve Tissue Proteins; D043562:Receptors, G-Protein-Coupled; D058470:Receptors, Purinergic P2Y