Optogenetic interrogation reveals separable G-protein-dependent and -independent signalling linking G-protein-coupled receptors to the circadian oscillator

Abstract

BackgroundEndogenous circadian oscillators distributed across the mammalian body are synchronised among themselves and with external time via a variety of signalling molecules, some of which interact with G-protein-coupled receptors (GPCRs). GPCRs can regulate cell physiology via pathways originating with heterotrimeric G-proteins or β-arrestins. We applied an optogenetic approach to determine the contribution of these two signalling modes on circadian phase.ResultsWe employed a photopigment (JellyOp) that activates Gαs signalling with better selectivity and higher sensitivity than available alternatives, and a point mutant of this pigment (F112A) biased towards β-arrestin signalling. When expressed in fibroblasts, both native JellyOp and the F112A arrestin-biased mutant drove light-dependent phase resetting in the circadian clock. Shifts induced by the two opsins differed in their circadian phase dependence and the degree to which they were associated with clock gene induction.ConclusionsOur data imply separable G-protein and arrestin inputs to the mammalian circadian clock and establish a pair of optogenetic tools suitable for manipulating Gαs- and β-arrestin-biased signalling in live cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-017-0380-8) contains supplementary material, which is available to authorized users.

A live cell assay of GPCR coupling allows identification of optogenetic tools for controlling Go and Gi signaling

“…These negative control results allow us to conclude that positive light responses observed with GsX chimeras are driven solely by interaction between opsin and those particular GsX constructs, specifically their C-terminal tails, since that is the only aspect of the experiment that has been varied. Importantly, although rod opsin-driven light responses (~40-fold increase in GloSensor signal when coupled to Gsi) were the largest recorded amongst this set of GPCRs, they were still substantially smaller than those produced by a natively Gs-coupled opsin, JellyOp [ 27 , 43 ] (~200-fold, Additional file 2 : Figure S1c). This indicates that the responses we observe in the GsX assay are far from saturating the downstream components of this signaling system, including adenylyl cyclases and the GloSensor reporter itself.…”

Convergent evolution of tertiary structure in rhodopsin visual proteins from vertebrates and box jellyfish

“…JellyOp expresses efficiently in HEK293 cells and upon light activation causes a robust induction of cAMP through activation of the Gαs pathway that can be tracked in live cells with a luminescent cAMP reporter ( Fig. 2 A ) ( 16 – 18 ). We therefore attempted to use the relative spectral sensitivity of the JellyOp-driven second messenger response (action spectrum) to test the hypothesis that the E94Q substitution had resulted in counterion neutralization.…”

“…JellyOp activation in HEK293 cells was measured using the bioluminescent GloSensor22F reporter as described previously ( 17 , 18 ). Relative sensitivity was determined from second messenger irradiance response curves at eight near-monochromatic wavelength bands.…”

“…For this purpose, we chose JellyOp, a visible-light–sensitive opsin from the box jellyfish Carybdea rastonii ( 16 ). JellyOp is expressed in the upper and lower lens eyes of this organism and has been applied as an optogenetic tool to provide light-driven activation of Gαs signaling with high sensitivity and signal:noise ( 16 – 18 ). JellyOp thus represents a good system to explore the evolution of the position of the counterion along the development of complex visual structures in radiata, and also to gain structural information on the retinal-binding site that could facilitate rational engineering of improved optogenetic tools.…”

Selective optogenetic control of Gq signaling using human Neuropsin

“…The advantages of optogenetic stimulation are unprecedented temporal and spatial precision, cell-type-specific manipulations, and subcellular targeting. Thus optogenetics has been used to investigate GPCR signaling in cells 18 – 20 , the heart 21 , 22 , the eye 23 – 25 , and the brain 7 , 19 , 26 – 29 . Ideally, optogenetic GPCRs should be selective for only one of the four major G protein classes.…”