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

Edward R Ballister,Robert J Lucas,Franck Martial,Jessica Rodgers

doi:10.1186/s12915-017-0475-2

Abstract

BackgroundAnimal opsins are light-sensitive G-protein-coupled receptors (GPCRs) that enable optogenetic control over the major heterotrimeric G-protein signaling pathways in animal cells. As such, opsins have potential applications in both biomedical research and therapy. Selecting the opsin with the best balance of activity and selectivity for a given application requires knowing their ability to couple to a full range of relevant Gα subunits. We present the GsX assay, a set of tools based on chimeric Gs subunits that transduce coupling of opsins to diverse G proteins into increases in cAMP levels, measured with a real-time reporter in living cells. We use this assay to compare coupling to Gi/o/t across a panel of natural and chimeric opsins selected for potential application in gene therapy for retinal degeneration.ResultsOf the opsins tested, wild-type human rod opsin had the highest activity for chimeric Gs proxies for Gi and Gt (Gsi and Gst) and was matched in Go proxy (Gso) activity only by a human rod opsin/scallop opsin chimera. Rod opsin drove roughly equivalent responses via Gsi, Gso, and Gst, while cone opsins showed much lower activities with Gso than Gsi or Gst, and a human rod opsin/amphioxus opsin chimera demonstrated higher activity with Gso than with Gsi or Gst. We failed to detect activity for opsin chimeras bearing three intracellular fragments of mGluR6, and observed unexpectedly complex response profiles for scallop and amphioxus opsins thought to be specialized for Go.ConclusionsThese results identify rod opsin as the most potent non-selective Gi/o/t-coupled opsin, long-wave sensitive cone opsin as the best for selectively activating Gi/t over Go, and a rod opsin/amphioxus opsin chimera as the best choice for selectively activating Go over Gi/t.

Highlights

Animal opsins are light-sensitive G-protein-coupled receptors (GPCRs) that enable optogenetic control over the major heterotrimeric G-protein signaling pathways in animal cells
Validating the GsX assay with MOR, BKB2R, and rod opsin To confirm that profiles of GPCR-GsX chimera signaling replicate known profiles of interaction with native Gα subunits, we first tested the GsX chimera panel with two well-characterized non-light-sensitive GPCRs: bradykinin B2 receptor (BKB2R) and mu-opioid receptor (MOR)
A b c d produces an increase in cAMP when co-transfected with Gsz, Gsq, or Gs12, but not Gsi or wild-type Gs. c,d HEK293T cells treated with pertussis toxin and transfected with GloSensorTM 22 F (Glo22F) cAMP reporter and mu-opioid receptor (MOR), bradykinin receptor B2 (BKB2R), or rod opsin, without exogenous Gα (-), + wild-type Gs (s), or + GsX were stimulated with endomorphin-1 (EM), bradykinin (BK), or 470 nm light

Summary

Introduction

Animal opsins are light-sensitive G-protein-coupled receptors (GPCRs) that enable optogenetic control over the major heterotrimeric G-protein signaling pathways in animal cells. We present the GsX assay, a set of tools based on chimeric Gs subunits that transduce coupling of opsins to diverse G proteins into increases in cAMP levels, measured with a real-time reporter in living cells. We use this assay to compare coupling to Gi/o/t across a panel of natural and chimeric opsins selected for potential application in gene therapy for retinal degeneration. A family of light-activated G-protein-coupled receptors (GPCRs), offer the potential for optogenetic control of a wide range of processes, analogous to the optogenetic revolution enabled by ionophoric tools such as microbial channelrhodopsin, but relevant for a much broader range of cell types. As part of our ongoing investigation into the basic biology of opsins and their use as optogenetic activators for research and therapy, we sought to develop a simple, robust assay to compare the functional coupling of many opsins toward one or several diverse G proteins

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