Optical functionalization of human Class A orphan G-protein-coupled receptors

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.

Light-activated Frizzled7 reveals a permissive role of non-canonical wnt signaling in mesendoderm cell migration

“…To investigate whether spatially restricted activation of Fz7 signaling has the potential to drive ppl cell polarization and direct their migration, we developed a light-activated version of Fz7. The rationale underlying our protein engineering approach was that the functional domains of seven-pass transmembrane (TM) receptors can be delineated in their canonical topology (Airan et al, 2009; Siuda et al, 2015; Li et al, 2015; Gunaydin et al, 2014; Barish et al, 2013; van Wyk et al, 2015; Morri et al, 2018). In particular, the N-terminus, three extracellular loops and seven TM domains are typically associated with ligand binding and receptor activation, whereas the three intracellular loops (ICL; ICL 1–3) and the C-terminus are typically associated with downstream signal transmission.…”

Mutations in G Protein–Coupled Receptors: Mechanisms, Pathophysiology and Potential Therapeutic Approaches

“…Therefore, GPR33 belongs to a class of rare pseudogenes that have disabling mutations in the reference genome but are intact in some individuals (Cheetham et al, 2020). GPR33 seems to be involved in innate immunity, as supported by its expression in immune-relevant tissues and functional studies (Römpler et al, 2005;Bohnekamp et al, 2010;Morri et al, 2018). The coincidental inactivation and its fixation in several species of distantly related mammalian orders (Römpler et al, 2005) suggest a selective immune response-related pressure on this chemoattractant receptor gene.…”

Innovative Optogenetic Strategies for Vision Restoration

“…Recently, optically controlled GPCRs have been engineered by multiple groups (Airan et al, 2009 ; Karunarathne et al, 2013 ; Levitz et al, 2013 ; Broichhagen et al, 2015 ; van Wyk et al, 2015 ; Morri et al, 2018 ). Unlike melanopsin or rhodopsin, these GPCRs have been constructed or modified to become light sensitive.…”