In Vitro Characterization of Pig Calcium-Sensing Receptor (CaSR) Ligands and Cell Signaling Pathways Related to CaSR Activation using a Dual-Luciferase Reporter Assay

Our previous study demonstrated that pig calcium-sensing receptor (pCaSR) is widely expressed in different tissues including intestinal segments in weaned piglets and the ileum has a higher expression level of pCaSR. Further research is needed to characterize the functions of pCaSR, its potential ligands and cell signaling pathways related to CaSR activation in enteroendocrine cells and potentially in enterocytes. In the present study, a dual-luciferase reporter assay was developed to investigate the potential ligands of pCaSR and the ligand-mediated signaling pathways. The results showed that extracellular calcium activated pCaSR in a concentration-dependent manner with EC₅₀ = 4.27 mM through the Gq/11 signaling pathway, EC₅₀ = 2.76 mM through Gs signaling, EC₅₀ = 2.85 mM through ERK1/2 activation signaling, and EC₅₀ = 2.26 mM through RhoA activation signaling. Given that Gs signaling, ERK1/2 activation signaling and RhoA activation signaling pathways shared similar EC50 values by the extracellular calcium stimulation, it was possible that the activation of pCaSR might lead to changes in cAMP production, ERK1/2 and RhoA activation with similar efficacies. In contrast, EC₅₀ value through Gq/11 signaling was more than 1.5 times higher than those by the other three signaling pathways tested, indicating that pCaSR increases intracellular calcium concentration less efficiently. Meanwhile, compared with the control group, the pCaSR activation response triggered by α-casein was significantly higher in all four reporter assays (P < 0.05), which demonstrated that α-casein was an agonist for pCaSR. Moreover, it implied that α-casein could active pCaSR through four main signaling pathways. In conclusion, similar to the human CaSR, pCaSR also showed biased agonism through four main signaling pathways. Further studies are needed to investigate the agonist type of α-casein and related potencies. 

Key words: calcium-sensing receptor, pigs, α-casein, agonist, ligand.

Published in the proceedings of the Animal Nutrition Conference of Canada 2020. For information on the event, past and future editions, check out https://animalnutritionconference.ca/.