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ACS Synth Biol. 2018 Jun 15;7(6):1488-1495. doi: 10.1021/acssynbio.8b00065. Epub 2018 Jun 4.

Optogenetic Inhibition of Gαq Protein Signaling Reduces Calcium Oscillation Stochasticity.

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1
Department of Bioengineering , University of Pennsylvania , Philadelphia , Pennsylvania 19104 , United States.

Abstract

As fast terminators of G-protein coupled receptor (GPCR) signaling, regulators of G-protein signaling (RGS) serve critical roles in fine-tuning second messenger levels and, consequently, cellular responses to external stimuli. Here, we report the creation of an optogenetic RGS2 (opto-RGS2) that suppresses agonist-evoked calcium oscillations by the inactivation of Gαq protein. In this system, cryptochrome-mediated heterodimerization of the catalytic RGS2-box with its N-terminal amphipathic helix reconstitutes a functional membrane-localized complex that can dynamically suppress store-operated release of calcium. Engineered opto-RGS2 cell lines were used to establish the role of RGS2 as a key inhibitory feedback regulator of the stochasticity of the Gαq-mediated calcium spike timing. RGS2 reduced the stochasticity of carbachol-stimulated calcium oscillations, and the feedback inhibition was coupled to the global calcium elevation by calmodulin/RGS2 interactions. The identification of a critical negative feedback circuit exemplifies the utility of optogenetic approaches for interrogating RGS/GPCR biology and calcium encoding principles through temporally precise molecular gain-of-function.

KEYWORDS:

calcium signaling; optogenetics; regulators of G-protein signaling (RGS); stochasticity

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