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Internalization and desensitization of a green fluorescent protein‐tagged P2Y 1 nucleotide receptor are differently controlled by inhibition of calmodulin‐dependent protein kinase II
Author(s) -
Tulapurkar Mohan E.,
Zündorf Gregor,
Reiser Georg
Publication year - 2006
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.2005.03594.x
Subject(s) - p2y receptor , receptor , microbiology and biotechnology , internalization , biology , endocytosis , homologous desensitization , okadaic acid , phosphatase , biochemistry , chemistry , agonist , phosphorylation
De‐ and re‐sensitization and trafficking of P2Y nucleotide receptors modulate physiological responses of these receptors. Here, we used the rat brain P2Y 1 receptor tagged with green fluorescent protein (P2Y 1 ‐GFP receptor) expressed in HEK293 human embryonic kidney cells. Ca 2+ release was used as a functional test to investigate ATP‐induced receptor de‐ and re‐sensitization. By confocal laser scanning microscopy (CLSM), endocytosis of P2Y 1 ‐GFP receptor was visualized in live cells. Stimulation of the cells with ATP induced complete receptor endocytosis within 30 min and appearance of the P2Y 1 receptor in small vesicles. Removal of the agonist resulted in reappearance of the receptor after 60 min on the plasma membrane. Exposure of the cells to KN‐62 and KN‐93, inhibitors of the calmodulin dependent protein kinase II (CaMKII), prevented receptor internalization upon stimulation with ATP. However, the receptor which was still present on the plasma membrane was desensitized, seen by decreased Ca 2+ response. The decreased Ca 2+ response after 30‐min exposure to ATP can be attributed to desensitization and is not as a result of depletion of internal stores, as the cells exposed to ATP for 30 min exhibited a normal Ca 2+ response upon stimulation with thrombin. However, okadaic acid, an inhibitor of protein phosphatase 2A (PP2A), did not affect ATP‐induced P2Y 1 receptor endocytosis, but delayed the reappearance of the P2Y 1 receptor on the plasma membrane after ATP withdrawal. Consistently, in okadaic acid‐treated cells the ATP‐induced Ca 2+ response observed after the 30‐min exposure to ATP recovered only partially. Thus, CaMKII seems to be involved in P2Y 1 receptor internalization, but not desensitization, whereas protein phosphatase 2A might play a role in recycling of the receptor back to the plasma membrane.