Abstract
Heterotrimeric G-proteins are signal transducers involved in mediating the action of many natural extracellular stimuli and many therapeutic agents. Non-invasive approaches to manipulate the activity of G-proteins with high precision are crucial to understand their regulation in space and time. Here, we developed LOV2GIVe, an engineered modular protein that allows the activation of heterotrimeric G-proteins with blue light. This optogenetic construct relies on a versatile design that differs from tools previously developed for similar purposes, that is metazoan opsins, which are light-activated G-protein-coupled receptors (GPCRs). Instead, LOV2GIVe consists of the fusion of a G-protein activating peptide derived from a non-GPCR regulator of G-proteins to a small plant protein domain, such that light uncages the G-protein activating module. Targeting LOV2GIVe to cell membranes allowed for light-dependent activation of Gi proteins in different experimental systems. In summary, LOV2GIVe expands the armamentarium and versatility of tools available to manipulate heterotrimeric G-protein activity.
Highlights
Heterotrimeric G-proteins are critical transducers of signaling triggered by a large family of G-protein-coupled receptors (GPCRs)
This tool is based on a modular design that combines the properties of the light-sensitive LOV2 domain with a motif present in a non-GPCR activator of G-proteins of the Gi family
We propose that the versatility of the LOV2GIVe design could help overcome some limitations of currently available optogenetic tools that are based on GPCR-like proteins
Summary
Heterotrimeric G-proteins are critical transducers of signaling triggered by a large family of G-protein-coupled receptors (GPCRs). GPCRs are Guanine nucleotide Exchange Factors (GEFs) that activate G-proteins by promoting the exchange of GDP for GTP on the Ga-subunits (Gilman, 1987). This signaling axis regulates a myriad of (patho)physiological processes and represents the target for >30% of drugs on the market (Sriram and Insel, 2018), which fuels the interest in developing tools to manipulate G-protein activity in cells with high precision. Metazoan opsins are light-activated GPCRs that have been repurposed as optogenetic tools (Airan et al, 2009; Bailes et al, 2012; Karunarathne et al, 2013; Oh et al, 2010; Siuda et al, 2015), albeit with limitations. Opsins are relatively large, post-translationally modified transmembrane proteins, which makes them inherently difficult to modify for optimization and/or customization for specific applications
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