Identification of peptide ligands for orphan GPCRs by measuring Ca2+-induced luminescence in transfected cells

Introduction

A large variety of molecules act as ligands for G-protein coupled receptors (GPCRs) physiologically and these receptors are targets for many drugs that are used therapeutically ¹. Genome/transcriptome sequencing has enabled the discovery of many novel genes encoding GPCRs in humans and other animals. Identification of the ligands for these ‘orphan’ receptors requires experimental testing of candidate molecules ^2,3. To accomplish this, candidate ligands are tested for their effects on cells expressing an orphan GPCR and if a ligand is identified then the orphan GPCR is said to have been ‘deorphanised’. As part of a broader investigation of the evolution of neuropeptide signalling, Ca²⁺-induced luminescence can be measured in Chinese Hamster Ovary cells (CHO-K1 cells) transfected with orphan GPCRs to test candidate peptide ligands. In recent studies, neuropeptides that act as ligands for many GPCRs in the common European starfish Asterias rubens were identified. For example, the identification of neuropeptides that act as ligands for an expanded family of GPCRs in A. rubens that are related to the human kisspeptin receptor was recently reported ⁴. One of these receptors (ArKPR6) is activated by the neuropeptide S2 (SGPYSFNSGLTF-NH2), which was one of the first neuropeptides to be identified in starfish ⁵. Using this as an example of neuropeptide receptor deorphanisation, this application note describes how, by measuring Ca²⁺-induced luminescence in CHO-K1 cells transfected with ArKPR6, the peptide S2 was identified as a ligand for the orphan GPCR ArKPR6.

Assay principle

CHO-K1-cells stably expressing a Ca²⁺-sensitive Green Fluorescent Protein (GFP) - Aequorin fusion protein (G5A) are used as an expression system ⁶. These cells are co-transfected with an orphan GPCR and a promiscuous G-protein (e.g. Gα_qi4, supplied by Luis Alfonso Yañez Guerra, University of Southampton) that couples a large variety of GPCRs to the Phospholipase C (PLC)/ Inositol-1,4,5-tri-phosphate (IP3)/Ca²⁺ signalling pathway ⁷. In the presence of the co-factor for aequorin (coelenterazine), binding of a ligand to the orphan GPCR triggers Ca²⁺-induced luminescence, which is measured. By testing the ligand at different concentrations, a concentration-response curve can be generated.

Results & Discussion

Figure 2 shows that, by comparison with 10% DMSO and basal media, S2 triggers luminescence in CHO-K1 cells co-transfected with the orphan GPCR ArKPR6 and Gα_qi4. Triton X-100 is used as a positive control in these experiments because it non-specifically triggers intracellular Ca²⁺ elevation in cells and thereby demonstrates cell viability if luminescence is observed. The luminescence measurements with S2, basal media and 10% DMSO were analysed by one-way ANOVA followed by Dunnet’s multiple comparison test, demonstrating a statistically significant increase in luminescence with S2 by comparison with basal media and 10% DMSO.