Thursday, November 21, 2024
11/21/2024
Abstract Cells sense the extracellular environment primarily using G protein-coupled receptors (GPCRs). They represent the largest family of cell surface proteins and play key physiological roles in maintaining cellular life. GPCRs employ heterotrimeric G protein to transduce signals to the cell interior. Dysfunctions in GPCR, as well as G protein signaling, contribute to some of the most prevalent human diseases and thus, GPCRs have become the largest drug target. Out of over 800 members, more than a hundred GPCRs are controlled by peptide or small protein ligands. Even one family of such GPCRs, the protease-activated receptor (PAR) family, shows an extensive physical presence throughout the body from the brain to the heart and regulates many known and possibly even more unknown physiological roles, from immune to cardiac. A fundamental limitation in making advances in PARs in human physiology is the lack of tools to control endogenously expressed receptors both in cultured cells and in vivo. Though opsins can activate G protein signaling with spatial and temporal control, they only loosely recapitulate signaling of endogenous GPCRs. Similarly, there are no optogenetic or even chemical tools available for controlling endogenous heterotrimer signaling. Therefore, in Aim 1, we plan to deliver a library of photoligands to control endogenous PAR receptors instantaneously and reversibly. The preliminary data shows optical activation of wild type PAR1 receptor by a genetically encoded photoligand and attests to the feasibility of the proposed. Though the proposal focuses on PAR family GPCRs, the broader adaptability in photoligand-design will allow optical control of other peptide or small protein activated GPCRs, expanding the future biomedical significance of Aim 1. Our photoligands will be the first of their kind to deliver such a precise regulation of subcellular, cellular, tissue, or even organ-level GPCR signaling on optical command, fulfilling the demands of future biomedical investigations. Similarly, despite the central roles of heterotrimeric G proteins in transducing signaling from all GPCRs, other than the few available inhibitors of their signaling, there are no direct routes to activate them with an appreciable spatial or temporal control. Despite the optical control, the available optogenetic regulators aim only downstream effectors of G proteins and elicit higher background signaling due to overexpressed active proteins. We use Aim 2 to gain direct access to endogenous G protein heterotrimers to control one or both G protein subunit signaling optically. Using a peptide domain derived from a native controller of G protein signaling, we show optically induced macrophage migration by the localized generation of Gβγ. Engineered optogenetic tools in Aim 2 will not only provide experimental means to bypass the limitations in chemical agents, but also inform the science on G protein subunit function and promote future molecule discovery/screening efforts to control heterotrimer and or its select-subunits.
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