Fan G H, Yang W, Sai J, Richmond A
Department of Veterans Affairs, Nashville, Tennessee 37212-2637, USA.
J Biol Chem. 2001 May 18;276(20):16960-8. doi: 10.1074/jbc.M009292200. Epub 2001 Feb 26.
Protein phosphatase 2A (PP2A) is postulated to be involved in the dephosphorylation of G protein-coupled receptors. In the present study, we demonstrate that the carboxyl terminus of CXCR2 physically interacts with the PP2A core enzyme, a dimer formed by PP2Ac and PR65, but not with the PP2Ac monomer, suggesting direct interaction of the receptor with PR65. The integrity of a sequence motif in the C terminus of CXCR2, KFRHGL, which is conserved in all CC and CXC chemokine receptors, is required for the receptor binding to the PP2A core enzyme. CXCR2 co-immunoprecipitates with the PP2A core enzyme in HEK293 cells and in human neutrophils. Overexpression of dominant negative dynamin 1 (dynamin 1 K44A) in CXCR2-expressing cells blocks the receptor association with the PP2A core enzyme, and an internalization-deficient mutant form of CXCR2 (I323A,L324A) also exhibits impaired association with the PP2A core enzyme, suggesting that the receptor internalization is required for the receptor binding to PP2A. A phosphorylation-deficient mutant of CXCR2 (331T), which has previously been shown to undergo internalization in HEK293 cells, binds to an almost equal amount of the PP2A core enzyme in comparison with the wild-type CXCR2, suggesting that the interaction of the receptor with PP2A is phosphorylation-independent. The dephosphorylation of CXCR2 is reversed by treatment of the cells with okadaic acid. Moreover, pretreatment of the cells with okadaic acid increases basal phosphorylation of CXCR2 and attenuates CXCR2-mediated calcium mobilization and chemotaxis. Taken together, these data indicate that PP2A is involved in the dephosphorylation of CXCR2. We postulate that this interaction results from direct binding of the regulatory subunit A (PR65) of PP2A to the carboxyl terminus of CXCR2 after receptor sequestration and internalization.
蛋白磷酸酶2A（PP2A）被认为参与G蛋白偶联受体的去磷酸化过程。在本研究中，我们证明CXCR2的羧基末端与PP2A核心酶发生物理相互作用，PP2A核心酶是由PP2Ac和PR65形成的二聚体，而非与PP2Ac单体相互作用，这表明受体与PR65存在直接相互作用。CXCR2 C末端的序列基序KFRHGL的完整性是受体与PP2A核心酶结合所必需的，该基序在所有CC和CXC趋化因子受体中均保守。CXCR2在HEK293细胞和人类中性粒细胞中与PP2A核心酶共免疫沉淀。在表达CXCR2的细胞中过表达显性负性发动蛋白1（发动蛋白1 K44A）会阻断受体与PP2A核心酶的结合，并且CXCR2的内化缺陷突变形式（I323A，L324A）也表现出与PP2A核心酶的结合受损，这表明受体内化是受体与PP2A结合所必需的。CXCR2的磷酸化缺陷突变体（331T）先前已被证明在HEK293细胞中发生内化，与野生型CXCR2相比，它与几乎等量的PP2A核心酶结合，这表明受体与PP2A的相互作用不依赖于磷酸化。用冈田酸处理细胞可逆转CXCR2的去磷酸化。此外，用冈田酸预处理细胞会增加CXCR2的基础磷酸化，并减弱CXCR2介导的钙动员和趋化作用。综上所述，这些数据表明PP2A参与CXCR2的去磷酸化过程。我们推测这种相互作用是由于PP2A的调节亚基A（PR65）在受体隔离和内化后与CXCR2的羧基末端直接结合所致。
