Abstract
G-protein-coupled receptors (GPCRs) form the largest receptor family, relay environmental stimuli to changes in cell behavior and represent prime drug targets. Many GPCRs are classified as orphan receptors because of the limited knowledge on their ligands and coupling to cellular signaling machineries. Here, we engineer a library of 63 chimeric receptors that contain the signaling domains of human orphan and understudied GPCRs functionally linked to the light-sensing domain of rhodopsin. Upon stimulation with visible light, we identify activation of canonical cell signaling pathways, including cAMP-, Ca2+-, MAPK/ERK-, and Rho-dependent pathways, downstream of the engineered receptors. For the human pseudogene GPR33, we resurrect a signaling function that supports its hypothesized role as a pathogen entry site. These results demonstrate that substituting unknown chemical activators with a light switch can reveal information about protein function and provide an optically controlled protein library for exploring the physiology and therapeutic potential of understudied GPCRs.
Highlights
G-protein-coupled receptors (GPCRs) form the largest receptor family, relay environmental stimuli to changes in cell behavior and represent prime drug targets
We established a platform for the design and functional evaluation of large numbers of light-activated chimeric receptors derived from human orphan and understudied Class A GPCRs (Fig. 1b, c)
We engineered the chimeric genes using a high-throughput genetic engineering method and expressed the proteins in cultured human embryonic kidney 293 (HEK293) cells. The goal of this experiment was to test if cell signaling responses induced by light stimulation of the chimeric receptors (Supplementary Data 1, 2) recapitulate those of the corresponding reference GPCRs stimulated by agonists (Supplementary Tables 1 and 2)
Summary
G-protein-coupled receptors (GPCRs) form the largest receptor family, relay environmental stimuli to changes in cell behavior and represent prime drug targets. We designed and engineered a library of chimeric receptors that consist of the extracellular and transmembrane elements of rhodopsin and the intracellular elements of 63 human orphan and understudied GPCRs. When expressed heterologously, we detected coupling to canonical cellular signaling pathways including cAMP-, Ca2+-, MAPK/ERK-, and Rhodependent pathways for a subset of these receptors. We detected coupling to canonical cellular signaling pathways including cAMP-, Ca2+-, MAPK/ERK-, and Rhodependent pathways for a subset of these receptors This receptor library complements the genome-wide experimental and computational resources for GPCR interrogation[30,31], enables novel optogenetic experiments and provides diverse candidate proteins for structure–function studies
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