Photo-switchable beta arrestins to study receptor biology and signaling

细胞表面受体是一种特殊类型的蛋白质，它从细胞外部感知信息，并将其穿过细胞膜传递，以触发生物反应。G蛋白偶联受体（GPCR）是哺乳动物基因组中编码的主要类型的细胞表面受体。这些GPCR负责多种生理功能，从视觉和嗅觉到大脑和心血管功能的调节。GPCR激活后，与称为β抑制蛋白的衔接分子结合，抑制某些生物反应，同时刺激其他生物反应。由于在调节细胞反应中的这种双重作用，假定β抑制蛋白以非常动态的方式起作用，并且可以在GPCR刺激后的不同时刻实现不同的功能。然而，目前还没有允许快速（在几秒钟内）调节活细胞或生物体中β抑制蛋白活性的方法。蛋白质产生的最新发展导致了光遗传学领域的出现，光遗传学是使用特定类型的光照射来调节蛋白质活性的方法的集合。在这里，我们提出使用光遗传学方法来控制体内β抑制蛋白的活性。Pif/Phy系统允许使用光来打开或关闭来自杂草拟南芥的两个小蛋白质结构域之间的相互作用。通过将这些结构域外源地添加到其他蛋白质如β抑制蛋白中，因此可以使用这种可切换的相互作用来将蛋白质的构象从活性形式来回改变为非活性形式。我们已经产生了第一个版本的工程β抑制蛋白，可以使用Pif/Phy系统进行调节。我们现在建议用不同的GPCR和细胞表达系统进一步测试该技术。在优化这种方法之后，我们将使用缺乏内源性β抑制蛋白的遗传修饰小鼠，并产生新的动物和细胞系，其中β抑制蛋白的活性将受到光的调节。这种光遗传学方法的验证将允许在培养细胞和体内以高时间尺度分辨率研究β抑制蛋白的精确功能。

Cell surface receptors are a specialized type of protein that perceive information from the outside of the cell and transmit it across the cell membrane to trigger a biological response. G protein coupled receptors (GPCR) are the main type of cell surface receptors encoded in the mammalian genome. These GPCR are responsible for several physiological functions ranging from vision and olfaction to the regulation of brain and cardiovascular functions. Following their activation GPCR associate with adaptor molecules termed beta arrestins that inhibit some biological responses while stimulating others. Because of this dual role in modulating cellular responses, it is postulated that beta arrestins act in a very dynamic way and may fulfill different functions at different moments after GPCR stimulation. However, there is presently no method allowing to modulate rapidly (in seconds) the activity of beta arrestins in living cells or organisms. Recent development in protein engendering has led to the emergence of the field of optogenetic, which is a collection of methods to modulate protein activity using specific types of light illumination. Here we propose to use an optogenetic approach to control the activity of beta arrestins in vivo. The Pif/Phy system allows using light to turn ON or OFF and interaction between two small protein domains derived from the weed Arabidopsis thaliana. By adding these domains exogenously to other proteins like the beta arrestins it is thus possible to use this switchable interaction to change the conformation of a protein back and forth from an active to an inactive form. We have generated a first version of an engineered beta arrestins that can be regulated using the Pif/Phy system. We now propose to tests this technology further with different GPCR and cellular expression systems. Following optimization of this approach we will then use genetically modify mice lacking endogenous beta arrestins and generate new animal and cell lines in which the activity of beta arrestins will be regulated by light. The validation of this optogenetic approach will allow to study the precise functions of beta arrestin with a high time scale resolution both in cultured cells and in vivo.