Photo-switchable beta arrestins to study receptor biology and signaling

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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.