Endocytosis mechanism of P2Y2 nucleotide receptor tagged with green fluorescent protein: clathrin and actin cytoskeleton dependence.
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.
Tulapurkar
M E
ME
Institut für Neurobiochemie, Medizinische Fakultät, Otto-von-Guericke-Universität, Leipziger Strasse 44, 39120 Magdeburg, Germany.
Schäfer
R
R
Hanck
T
T
Flores
R V
RV
Weisman
G A
GA
González
F A
FA
Reiser
G
G
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002746:Chlorpromazine / Q000494:pharmacology; D002966:Clathrin / Q000378:metabolism*; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D004705:Endocytosis; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D061988:Proteasome Inhibitors; D021381:Protein Transport; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058473:Receptors, Purinergic P2Y2; D014544:Uridine Triphosphate / Q000378:metabolism
D000199:Actins; C495540:CAV1 protein, human; C495537:Cav1 protein, rat; D051242:Caveolin 1; D022461:Caveolins; D002966:Clathrin; C546385:P2RY2 protein, human; C546387:P2ry2 protein, rat; D061988:Proteasome Inhibitors; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D002118:Calcium; D002746:Chlorpromazine; D014544:Uridine Triphosphate