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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.
Highlights
GPR17 is a key player in oligodendrocyte differentiation
Endogenous Expression and Biochemical Characterization of GPR17 in Oli-neu—To investigate whether Oli-neu cells express GPR17, cell cultures maintained in normal conditions (Sato medium) were analyzed by confocal immunofluorescence microscopy after immunolabeling with antibodies directed against the C terminus of GPR17 or protein markers typical of different stages of oligodendrocyte differentiation
To investigate the intracellular trafficking of GPR17 in a native expression model, we have first characterized its expression in Oli-neu cells, an immortalized oligodendrocyte precursor cell (OPC) cell line, and showed that medium derived from cortical neuron primary cultures stimulates the transient expression of GPR17, as previously observed in maturing cultured OPCs [2, 7, 10, 11]
Summary
GPR17 is a key player in oligodendrocyte differentiation. By regulating the availability of receptors at the cell surface, agonist-induced GPR17 trafficking may influence terminal cell fate. These studies indicate that GPR17 is an integral signaling component controlling oligodendrocyte ontogenesis and suggest that the appropriate activation and deactivation of GPR17 are crucial steps in OPC maturation As it has been reported for many GPCRs, after ligand binding, GPR17 may undergo endocytosis and subsequent sorting into lysosomes for degradation and/or into recycling endosomes for re-incorporation into the plasma membrane. The balance of this dynamic intracellular trafficking is physiologically relevant because it modulates receptor levels at the cell surface. The receptor is delivered into early endosomes and sorted either to lysosomes for degradation or recycled to the cell surface via the small G-protein Rab4-dependent pathway
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