Internalization and desensitization of a green fluorescent protein-tagged P2Y nucleotide receptor are differently controlled by inhibition of calmodulin-dependent protein kinase II.

摘要

De- and re-sensitization and trafficking of P2Y nucleotide receptors modulate physiological responses of these receptors. Here, we used the rat brain P2Y1 receptor tagged with green fluorescent protein (P2Y1-GFP receptor) expressed in HEK293 human embryonic kidney cells. Ca2+ release was used as a functional test to investigate ATP-induced receptor de- and re-sensitization. By confocal laser scanning microscopy (CLSM), endocytosis of P2Y1-GFP receptor was visualized in live cells. Stimulation of the cells with ATP induced complete receptor endocytosis within 30 min and appearance of the P2Y1 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 Ca2+ response. The decreased Ca2+ 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 Ca2+ response upon stimulation with thrombin. However, okadaic acid, an inhibitor of protein phosphatase 2A (PP2A), did not affect ATP-induced P2Y1 receptor endocytosis, but delayed the reappearance of the P2Y1 receptor on the plasma membrane after ATP withdrawal. Consistently, in okadaic acid-treated cells the ATP-induced Ca2+ response observed after the 30-min exposure to ATP recovered only partially. Thus, CaMKII seems to be involved in P2Y1 receptor internalization, but not desensitization, whereas protein phosphatase 2A might play a role in recycling of the receptor back to the plasma membrane.