Spatio-temporal kinetics of growth hormone receptor signaling in single cells using FRET microscopy
Spatio-temporal kinetics of growth hormone receptor signaling in single cells using FRET microscopy
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DOI：
10.1016/j.ghir.2006.06.001
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发表时间：
2006-08-01
影响因子：
1.4
通讯作者：
Gertler, Arieh
中科院分区：
文献类型：
--
作者：
Biener-Ramanujan, Eva;Ramanujan, V. Krishnan;Gertler, Arieh
生长激素（GH）受体（R）介导的JAK 2（Janus激酶-2）-STAT 5（信号转导和转录激活因子-5）途径涉及蛋白质-蛋白质相互作用和酪氨酸磷酸化的级联反应，这些反应在细胞中以空间和时间敏感的方式发生。为了研究完整细胞中GHR二聚化或GH诱导的预二聚化GHR的构象变化和STAT 5活化动力学，采用荧光共振能量转移（FRET）和活细胞成像方法。在HEK-293 T细胞的膜处的FRET测量揭示了持续2-3分钟的瞬时GHR二聚化，在GH刺激后3分钟达到最大值，这足以诱导STAT 5活化，所述HEK-293 T细胞在C末端用青色（C）和黄色（Y）荧光蛋白（FP）标记的GHR共表达。的二聚化或GH诱导的构象变化的预二聚GHRs动力学的瞬时性质是不是GHR内化的结果，既不钾，也不胆固醇耗尽治疗延长FRET信号。YFP标记的STAT 5募集到膜，结合GHR-CFP，和磷酸化，发生在GH刺激的几分钟内。活化的STAT 5a-YFP没有显示核积累，尽管核pSTAT 5增加，表明STAT 5核穿梭的高周转。虽然GHR二聚化和STAT 5激活以前已经报道，这是第一个空间分辨证明GHR信号动力学在完整的细胞。(c)2006爱思唯尔有限公司保留所有权利。
The growth hormone (GH) receptor (R)-mediated JAK2 (Janus kinase-2)-STAT5 (signaling transducer and activator of transcription-5) pathway involves a cascade of protein-protein interactions and tyrosine phosphorylations that occur in a spatially and temporally sensitive manner in cells. To study GHR dimerization or GH-induced conformational change of predimerized GHRs and STAT5 activation kinetics in intact cells, fluorescence resonance energy transfer (FRET) and live-cell imaging methods were employed. FRET measurements at the membrane of HEK-293T cells co-expressing GHRs tagged at the C-terminus with cyan (C) and yellow (Y) fluorescent proteins (FPs) revealed transient GHR dimerization lasting 2-3 min, with a maximum at 3 min after GH stimulation, which was sufficient to induce STAT5 activation. The transient nature of the dimerization or GH-induced conformational change of predimerized GHRs kinetics was not a result of GHR internalization, as neither potassium- nor cholesterol-depletion treatments prolonged the FRET signal. YFP-tagged STAT5 recruitment to the membrane, binding to GHR-CFP, and phosphorylation, occurred within minutes of GH stimulation. Activated STAT5a-YFP did not show nuclear accumulation, despite nuclear pSTAT5 increase, suggesting high turnover of STAT5 nuclear shuttling. Although GHR dimerization and STAT5 activation have been reported previously, this is the first spatially resolved demonstration of GHR-signaling kinetics in intact cells. (c) 2006 Elsevier Ltd. All rights reserved.