Adinolfi Elena, Kim Miran, Young Mark T, Di Virgilio Francesco, Surprenant Annmarie
Institute of Molecular Physiology, Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, United Kingdom.
J Biol Chem. 2003 Sep 26;278(39):37344-51. doi: 10.1074/jbc.M301508200. Epub 2003 Jul 17.
The purinergic P2X7 receptor not only gates the opening of a cationic channel, but also couples to several downstream signaling events such as rapid membrane blebbing, microvesicle shedding, and interleukin-1beta release. Protein-protein interactions are likely to be involved in most of these signaling cascades; and recently, a P2X7 receptor-protein complex comprising at least 11 distinct proteins has been identified. We have studied one of these interacting proteins, HSP90, in human embryonic kidney cells expressing either human or rat P2X7 receptors as well as in rat peritoneal macrophages using biochemical (immunoprecipitation and Western blotting) and functional (membrane blebbing and currents) assays. We found that HSP90 was tyrosine-phosphorylated in association with the P2X7 receptor complex, but not in the cytosolic compartment. The HSP90 inhibitor geldanamycin decreased tyrosine phosphorylation of HSP90 and produced a 2-fold increase in the sensitivity of P2X7 receptors to agonist. Protein expression and tyrosine phosphorylation of a mutant P2X7 receptor in which a tyrosine in the C-terminal domain was substituted with phenylalanine (Y550F) were not changed, but tyrosine phosphorylation of HSP90 associated with this mutant P2X7 receptor complex was significantly greater than that associated with the wild-type complex. P2X7-Y550F receptors showed a 15-fold lower sensitivity to agonist, which was reversed by geldanamycin. We conclude that selective tyrosine phosphorylation of P2X7 receptor-associated HSP90 may act as a negative regulator of P2X7 receptor complex formation and function.
嘌呤能P2X7受体不仅控制阳离子通道的开放，还与多个下游信号事件相关联，如快速的细胞膜起泡、微囊泡脱落以及白细胞介素-1β释放。蛋白质-蛋白质相互作用可能参与了大多数这些信号级联反应；最近，已鉴定出一种包含至少11种不同蛋白质的P2X7受体-蛋白质复合物。我们使用生化方法（免疫沉淀和蛋白质印迹）和功能方法（细胞膜起泡和电流），对表达人或大鼠P2X7受体的人胚肾细胞以及大鼠腹腔巨噬细胞中的一种相互作用蛋白HSP90进行了研究。我们发现，HSP90与P2X7受体复合物相关联时发生酪氨酸磷酸化，但在胞质区室中未发生。HSP90抑制剂格尔德霉素降低了HSP90的酪氨酸磷酸化，并使P2X7受体对激动剂的敏感性提高了2倍。一种C末端结构域中的酪氨酸被苯丙氨酸取代（Y550F）的突变型P2X7受体的蛋白质表达和酪氨酸磷酸化没有变化，但与该突变型P2X7受体复合物相关联的HSP90的酪氨酸磷酸化明显高于与野生型复合物相关联的情况。P2X7-Y550F受体对激动剂的敏感性降低了15倍，而格尔德霉素可逆转这种情况。我们得出结论，P2X7受体相关的HSP90的选择性酪氨酸磷酸化可能作为P2X7受体复合物形成和功能的负调节因子。
