Constitutive interaction of the P2Y2 receptor with the hematopoietic cell-specific G protein Gα16 and evidence for receptor oligomers
Constitutive interaction of the P2Y2 receptor with the hematopoietic cell-specific G protein Gα16 and evidence for receptor oligomers
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DOI：
10.1016/j.cellsig.2004.11.011
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发表时间：
2005-07-01
影响因子：
4.8
通讯作者：
Baltensperger, K
中科院分区：
文献类型：
--
作者：
Kotevic, I;Kirschner, KM;Baltensperger, K
造血细胞独特地表达G（α 16），G（q）型的G蛋白α亚基。G（alpha 16）是人红白血病细胞中P2Y（2）受体依赖性Ca 2+动员的必需因子，并诱导造血细胞分化。我们测试了P2Y（2）受体是否与G（α 16）发生物理相互作用。受体和G蛋白分别与绿色荧光蛋白（GFP）的青色（CFP）和黄色（YFP）变体融合。当在K562白血病细胞中表达时，融合蛋白能够在受体刺激后触发Ca2+信号，证明其功能完整性。在使用共聚焦显微镜进行的荧光共振能量转移（FRET）测量中，当受体与作为FRET受体的G蛋白共表达时，以及当CFP标记的受体与融合到YFP的受体共表达时，检测到来自固定的静息细胞的质膜区域的强烈FRET信号。我们的结论是，在静息条件下，G（α 16）和P2Y（2）受体形成组成型复合物，P2Y（2）受体作为寡聚体存在。(c)2004 Elsevier B.V保留所有权利。
Hematopoietic cells uniquely express G(alpha 16), a G protein alpha-subunit of the G(q)-type. G(alpha 16) is obligatory for P2Y(2) receptor-dependent Ca2+-mobilization in human erythroleukemia cells and induces hematopoietic cell differentiation. We tested whether P2Y(2) receptors physically interact with G(alpha 16). Receptor and G protein were fused to cyan (CFP) and yellow (YFP) variants of the green fluorescent protein (GFP), respectively. When expressed in K562 leukemia cells, the fusion proteins were capable of triggering a Ca2+-signal upon receptor stimulation, demonstrating their functional integrity. In fluorescence resonance energy transfer (FRET) measurements using confocal microscopy, a strong FRET signal from the plasma membrane region of fixed, resting cells was detected when the receptor was co-expressed with the G protein as the FRET acceptor, as well as when the CFP-tagged receptor was co-expressed with receptor fused to YFP. We conclude that, under resting conditions, G(alpha 16) and P2Y(2) receptors form constitutive complexes, and that the P2Y(2) receptor is present as an oligomer. (c) 2004 Elsevier B.V All rights reserved.