Endocytosis mechanism of P2Y2 nucleotide receptor tagged with green fluorescent protein: clathrin and actin cytoskeleton dependence |
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Authors: | M E Tulapurkar R Schäfer T Hanck R V Flores G A Weisman F A González G Reiser |
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Institution: | (1) Institut für Neurobiochemie, Medizinische Fakultät, Otto-von-Guericke-Universität, Leipziger Straße 44, 39120 Magdeburg, Germany;(2) University of Puerto Rico, Río Piedras Campus, San Juan, Puerto Rico;(3) University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico;(4) Department of Biochemistry, University of Missouri-Columbia, Missouri, USA |
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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.Received 8 February 2005; received after revision 18 March 2005; accepted 11 April 2005 |
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Keywords: | Human embryonic kidney (HEK-293) cells receptor regulation translocation internalization proteasomal system |
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