ATP stimulates GRK-3 phosphorylation and β-arrestin-2-dependent internalization of P2X7 receptor
ATP stimulates GRK-3 phosphorylation and β-arrestin-2-dependent internalization of P2X7 receptor
复制标题
DOI：
10.1152/ajpcell.00315.2004
复制
发表时间：
2005-06-01
影响因子：
5.5
通讯作者：
Gorodeski, GI
中科院分区：
文献类型：
--
作者：
Feng, YH;Wang, LQ;Gorodeski, GI
本研究的目的是了解参与P2X(7)受体激活的机制。用三磷酸腺苷或P2X(7)受体特异性配体2‘，3’-O-(4-苯甲酰基苯甲酰基)腺苷5‘-三磷酸(BzATP)处理后，CaSki细胞内源性P2X(7)受体的作用慢于外源性P2X(7)受体的人胚胎肾(HEK)-293细胞(HEK-293-hP2X(7)-R)。在这两种类型的细胞中，Western blotting显示了三种形式的受体的表达：功能性的85 kDa主要存在于膜上，65 kDa和18 kDa的形式在质膜和胞浆中都有表达。用三磷酸腺苷处理后，膜上85 kDa的形式瞬时减少，18 kDa的形式增加，表明受体内化、降解和再循环。在CaSki细胞中，三磷酸腺苷刺激酪氨酸和丝氨酸残基上85 kDa形式的磷酸化。苏氨酸残基的磷酸化随着ATP的增加而增加，并且增加了酪氨酸和丝氨酸残基的磷酸化所需的ATP，这表明了显性-负效应。在CaSki和HEK-293-hP2X(7)-R细胞中，ATP还增加了85 kDa形式与G蛋白偶联受体激酶(GRK)-3、β-arrestin-2和Dynamin的结合，并刺激β-arrestin-2重新分布到细胞的膜下区域。这些结果提示了一种新的P2X(7)受体作用机制，激活涉及GRK-3-，β-arrestin-2-，并依赖于动力蛋白将受体内化到cathrin结构域，然后部分地通过受体降解以及受体循环进入质膜。
The objective of this study was to understand the mechanisms involved in P2X(7) receptor activation. Treatments with ATP or with the P2X(7) receptor-specific ligand 2', 3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP) induced pore formation, but the effect was slower in CaSki cells expressing endogenous P2X(7) receptor than in human embryonic kidney ( HEK)-293 cells expressing exogenous P2X(7) receptor (HEK-293- hP2X(7)-R). In both types of cells Western blots revealed expression of three forms of the receptor: the functional 85-kDa form present mainly in the membrane and 65- and 18-kDa forms expressed in both the plasma membrane and the cytosol. Treatments with ATP transiently decreased the 85-kDa form and increased the 18-kDa form in the membrane, suggesting internalization, degradation, and recycling of the receptor. In CaSki cells ATP stimulated phosphorylation of the 85-kDa form on tyrosine and serine residues. Phosphorylation on threonine residues increased with added ATP, and it increased ATP requirements for phosphorylation on tyrosine and serine residues, suggesting a dominant-negative effect. In both CaSki and in HEK-293- hP2X(7)-R cells ATP also increased binding of the 85-kDa form to G protein-coupled receptor kinase (GRK)-3, beta-arrestin-2, and dynamin, and it stimulated beta-arrestin-2 redistribution into submembranous regions of the cell. These results suggest a novel mechanism for P2X(7) receptor action, whereby activation involves a GRK-3-, beta-arrestin-2-, and dynamin-dependent internalization of the receptor into clathrin domains, followed in part by receptor degradation as well as receptor recycling into the plasma membrane.