Endocytosis mechanism of P2Y2 nucleotide receptor tagged with green fluorescent protein: clathrin and actin cytoskeleton dependence (original) (raw)

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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Authors and Affiliations

  1. Institut für Neurobiochemie, Medizinische Fakultät, Otto-von-Guericke-Universität, Leipziger Straße 44, 39120, Magdeburg, Germany
    M. E. Tulapurkar, R. Schäfer, T. Hanck & G. Reiser
  2. University of Puerto Rico, Río Piedras Campus, San Juan, Puerto Rico
    R. V. Flores & F. A. González
  3. University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico
    F. A. González
  4. Department of Biochemistry, University of Missouri-Columbia, Missouri, USA
    G. A. Weisman

Authors

  1. M. E. Tulapurkar
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  2. R. Schäfer
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  3. T. Hanck
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  4. R. V. Flores
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  5. G. A. Weisman
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  6. F. A. González
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  7. G. Reiser
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Corresponding author

Correspondence toG. Reiser.

Additional information

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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