Martin-Fernandez M L, Clarke D T, Tobin M J, Jones G R
CLRC Daresbury Laboratory, Warrington, United Kingdom.
Cell Mol Biol (Noisy-le-grand). 2000 Sep;46(6):1103-12.
The interactions of growth factors with cell surface receptors regulate fundamental cell processes, such as growth, differentiation and transformation. Understanding the nature of these interactions at the molecular level is of fundamental importance in cell biology. This is not only from the point of view of basic science, but also because of the repercussions such knowledge might have in understanding the mode of action of drugs in cells. Receptor mediated endocytosis has been implicated in the downregulation of the mitogenic signal. However, no data are thus far available on how growth factor/receptor interactions might control endocytic trafficking. Here we show that information on modes of binding and receptor conformational changes can be obtained using time-resolved fluorescence methods. We have found that fluorescent probes bound to epidermal growth factor (EGF) show dynamic fluorescence quenching when EGF is bound to internalising EGF receptors (EGFR). We propose that this dynamic quenching takes place because EGF-bound probes interact with tryptophan residues in the extracellular domain of the EGF-EGFR complex. Real-time accumulation of fluorescent decays has also allowed us to follow the time course of a conformational change in EGFR occurring during endocytosis, and correlate this information with endosomal trafficking and EGFR recycling.
生长因子与细胞表面受体的相互作用调控着诸如生长、分化和转化等基本细胞过程。在分子水平上理解这些相互作用的本质在细胞生物学中至关重要。这不仅从基础科学的角度来看是如此，还因为此类知识可能对理解药物在细胞中的作用方式产生影响。受体介导的内吞作用与促有丝分裂信号的下调有关。然而，目前尚无关于生长因子/受体相互作用如何控制内吞运输的数据。在此我们表明，使用时间分辨荧光方法可以获得有关结合模式和受体构象变化的信息。我们发现，当表皮生长因子（EGF）与内化的表皮生长因子受体（EGFR）结合时，与EGF结合的荧光探针会出现动态荧光猝灭。我们提出这种动态猝灭的发生是因为与EGF结合的探针与EGF-EGFR复合物细胞外结构域中的色氨酸残基相互作用。荧光衰减的实时积累还使我们能够追踪内吞过程中EGFR发生构象变化的时间进程，并将此信息与内体运输和EGFR再循环相关联。
