Tenhumberg Stephanie, Schuster Björn, Zhu Lixin, Kovaleva Marina, Scheller Jürgen, Kallen Karl-Josef, Rose-John Stefan
Department of Biochemistry, Christian Albrechts University Kiel, Olshausenstrasse 40, D-24098 Kiel, Germany.
Biochem Biophys Res Commun. 2006 Aug 4;346(3):649-57. doi: 10.1016/j.bbrc.2006.05.173. Epub 2006 Jun 6.
It is established that cytokine receptors signal after ligand binding as homo- or hetero-dimers in heteromeric complexes, but it is unclear, when dimerization occurs. To investigate gp130 dimerization, we performed co-precipitation experiments with the endogenous cytokine receptors gp130 and leukemia inhibitory factor receptor (LIF-R) and with gp130 variants carrying two different C-terminal peptide tags. Furthermore, fluorescence resonance energy transfer (FRET) was employed to detect dimerization of two fluorescent-tagged gp130 variants. Confocal laser scanning microscopy was used for FRET detection in live cells. gp130 and LIF-R could be coprecipitated in the absence of ligand. The interaction, however, was intensified by the addition of LIF. Similar results were obtained with the gp130 variants and confirmed by FRET analysis in live cells. The present study clearly demonstrates the existence of preformed but inactive gp130/LIF-R hetero- and gp130/gp130 homo-dimers. The addition of ligand enhanced the respective dimer formation and was required for signal transduction.
已知细胞因子受体在配体结合后以异源复合物中的同二聚体或异二聚体形式发出信号，但二聚化何时发生尚不清楚。为了研究gp130的二聚化，我们对内源性细胞因子受体gp130和白血病抑制因子受体（LIF-R）以及携带两种不同C末端肽标签的gp130变体进行了共沉淀实验。此外，采用荧光共振能量转移（FRET）来检测两种荧光标记的gp130变体的二聚化。共聚焦激光扫描显微镜用于活细胞中的FRET检测。在没有配体的情况下，gp130和LIF-R可以共沉淀。然而，通过添加LIF，这种相互作用得到增强。gp130变体也获得了类似的结果，并通过活细胞中的FRET分析得到证实。本研究清楚地证明了预先形成但无活性的gp130/LIF-R异二聚体和gp130/gp130同二聚体的存在。配体的添加增强了各自的二聚体形成，并且是信号转导所必需的。
