Abstract
Despite the progress in deorphanization of G Protein-Coupled Receptors (GPCRs), ≈100 GPCRs are still classified as orphan receptors without identified endogenous ligands and with unknown physiological functions. The lack of endogenous ligands triggering GPCR signaling has hampered the study of orphan GPCR functions. Using GPR37 as an example, we provide here the first demonstration of the channelrhodopsin 2 (ChR2)-GPCR approach to bypass the endogenous ligand and selectively activate the orphan GPCR signal by optogenetics. Inspired by the opto-XR approach, we designed the ChR2-GPR37 chimera, in which the corresponding parts of GPR37 replaced the intracellular portions of ChR2. We showed that optogenetic activation of ChR2/opto-GPR37 elicited specific GPR37 signaling, as evidenced by reduced cAMP level, enhanced ERK phosphorylation and increased motor activity, confirming the specificity of opto-GPR37 signaling. Besides, optogenetic activation of opto-GPR37 uncovered novel aspects of GPR37 signaling (such as IP-3 signaling) and anxiety-related behavior. Optogenetic activation of opto-GPR37 permits the causal analysis of GPR37 activity in the defined cells and behavioral responses of freely moving animals. Importantly, given the evolutionarily conserved seven-helix transmembrane structures of ChR2 and orphan GPCRs, we propose that opto-GPR37 approach can be readily applied to other orphan GPCRs for their deorphanization in freely moving animals.
Highlights
G Protein-Coupled Receptors (GPCRs) are the most abundant family of cell membrane proteins in the human genome, which regulates almost all cellular signaling pathways and plays a vital role in numerous physiological and pathological processes (Bockaert and Pin, 1999; Rosenbaum et al, 2009)
We found that activation of opto-GPR37 decreased the concentration of cAMP in the presence of 25 μM retinal, whereas there were no changes in control samples (Figure 2C)
Given the evolutionarily conserved seven-helix transmembrane structure of orphan GPCRs and well-defined channelrhodopsin family, we propose that the opto-GPR37 technique represents an entirely new approach for deorphanization of GPCRs in freely moving animals by optogenetics
Summary
G Protein-Coupled Receptors (GPCRs) are the most abundant family of cell membrane proteins in the human genome, which regulates almost all cellular signaling pathways and plays a vital role in numerous physiological and pathological processes (Bockaert and Pin, 1999; Rosenbaum et al, 2009). To overcome this significant limitation in this study, we proposed a novel opto-XR approach to by-pass the endogenous ligand and trigger orphan GPCR signaling by light for probing GPR37 functions in intact animals This approach is based on the ‘‘opto-XR’’ that was first demonstrated in the cultured cells (Kim et al, 2005) and implemented recently in intact animals to study the function of GPCRs (opto-α1AR and opto-β2AR) with high spatiotemporal precision (Airan et al, 2009). We created an opto-GPR37 chimera by fusing the N-terminus, extracellular and transmembrane portions of channelrhodopsin with the ILs and C-terminus of GPR37 This opto-GPR37 approach allowed us to bypass endogenous ligands and directly activate orphan GPCR signaling by the light. We propose that ChR2-orphan GPCR represents a novel approach to provide new insights on functions of orphan GPCRs in intact animals
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