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，并通过细胞膜传递信息，以触发生物反应。G蛋白偶联受体是哺乳动物基因组中编码的主要细胞表面受体类型。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。在它们被激活后，GPCR与称为β拦阻蛋白的接头分子相联系，这种分子抑制一些生物反应，同时刺激另一些生物反应。由于这种调节细胞反应的双重作用，推测β-拦阻蛋白以一种非常动态的方式发挥作用，并可能在GPCR刺激后的不同时刻发挥不同的功能。然而，目前还没有一种方法可以快速(在几秒钟内)调节活细胞或生物体中的β-受体阻滞剂的活性。最近在蛋白质产生方面的发展导致了光遗传领域的出现，这是一种利用特定类型的光照明来调节蛋白质活性的方法的集合。在这里，我们建议使用光遗传学的方法来控制活体中的β-拦截素的活性。Pif/Phy系统允许利用光来开启或关闭，以及来自杂草拟南芥的两个小蛋白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中，如β拦阻蛋白，因此有可能利用这种可切换的相互作用来来回改变蛋白质的构象，从活性形式到非活性形式。我们已经产生了第一个版本的工程化Beta捕捉剂，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这项技术。在对这一方法进行优化之后，我们将使用缺乏内源性β-拦截素的小鼠进行基因改造，并产生新的动物和细胞系，在这些新的动物和细胞系中，β-拦阻蛋白的活性将受光调控。这种光遗传学方法的验证将允许以高时间尺度分辨率研究β-arrestin在培养细胞和体内的精确功能。

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.