Abstract.
Extracellular nucleotides exert a large number of physiological effects through activation of P2Y receptors. We expressed rat P2Y2 (rP2Y2) receptor, tagged with green fluorescent protein (GFP) in HEK-293 cells and visualized receptor translocation in live cells by confocal microscopy. Functional receptor expression was confirmed by determining [Ca2+]i responses. Agonist stimulation caused a time-dependent translocation of the receptor from the plasma membrane to the cytoplasm. Rearrangement of the actin cytoskeleton was observed during agonist-mediated rP2Y2-GFP receptor internalization. Colocalization of the internalized receptor with early endosomes, clathrin and lysosomes was detected by confocal microscopy. The inhibition of receptor endocytosis by either high-density medium or chlorpromazine in the presence of UTP indicates that the receptor was internalized by the clathrin-mediated pathway. The caveolin- mediated pathway was not involved. Targeting of the receptor from endosomes to lysosomes seems to involve the proteasome pathway, because proteasomal inhibition increased receptor recycling back to the plasma membrane.
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Received 8 February 2005; received after revision 18 March 2005; accepted 11 April 2005
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Tulapurkar, M.E., Schäfer, R., Hanck, T. et al. Endocytosis mechanism of P2Y2 nucleotide receptor tagged with green fluorescent protein: clathrin and actin cytoskeleton dependence. CMLS, Cell. Mol. Life Sci. 62, 1388–1399 (2005). https://doi.org/10.1007/s00018-005-5052-0
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DOI: https://doi.org/10.1007/s00018-005-5052-0