Single-chain fluorescent integrators for mapping G-protein-coupled receptor agonists

GPCRs transduce the effects of many neuromodulators including dopamine, serotonin, epinephrine, acetylcholine, and opioids. The localization of synthetic or endogenous GPCR agonists impacts their action on specific neuronal pathways. In this paper, we show a series of single-protein chain integrator sensors to determine GPCR agonist localization in the whole brain. We previously engineered integrator sensors for the mu and kappa opioid receptor agonists called M- and K-SPOTIT, respectively. Here, we show a new integrator sensor design platform called SPOTall that we used to engineer sensors for the beta-2-adrenergic receptor (B2AR), the dopamine receptor D1, and the cholinergic receptor muscarinic 2 agonists. For multiplexed imaging of SPOTIT and SPOTall, we engineered a red version of the SPOTIT sensors. Finally, we used M-SPOTIT and B2AR-SPOTall to detect morphine, isoproterenol, and epinephrine in the mouse brain. The SPOTIT and SPOTall sensor design platform can be used to design a variety of GPCR integrator sensors for unbiased agonist detection of many synthetic and endogenous neuromodulators across the whole brain.