Pharmacological and Physiological Studies of the Calcium-Sensing Receptor
Alex Rojas Bie Thomsen
Calcium is important in a physiological setting and its concentration is kept within a narrow range throughout the living organism. In target tissues the calcium-sensing receptor (CaSR) senses extracellular calcium, which causes the receptor to modulate activity of intracellular signaling pathways. This results in changes of calcium-regulatory mechanisms including secretion of hormones and absorption/excretion of calcium. The CaSR is a pleiotropic family C G protein-coupled receptor (GPCR) that activates several G proteins including Gq/11 protein, Gi/o protein, G12/13 protein, and in rare cases Gs protein. Several studies of other pleiotropic GPCRs show that some agonists affect activity of their signaling pathways differentially. The explanation of this “biased” agonism is that GPCRs can adopt various active conformations each of which are stabilized by different agonists. The various active conformationsfor a given GPCR affect its signaling pathways distinctly and therefore the signaling profile might vary between agonists. However, biased agonism of the CaSR is not well investigated and thus was a major research topic of the present PhD thesis.
In the first study we used HEK293-CaSR cells to investigate the ability of 12 well-known CaSR orthosteric agonists to activate three different CaSR-mediated signaling pathways including Gq/11, Gi/o, and extracellular signal-regulated kinase 1 and 2 (ERK1/2). Of the 12 CaSR agonists tested several of them turned out to be biased agonists in terms of potency and/or efficacy. The most notable biased agonists were barium, spermine, neomycin, and tobramycin. In addition, it was found that polyamines and aminoglycosides in general were biased towards ERK1/2 signaling compared to calcium and the calcium-related cation strontium.
In a second study, we sought to investigate biased agonism of the CaSR in rat medullary thyroid carcinoma 6-23 cells, which express CaSR endogenously. In these cells strontium behaved as a biased agonist by being more potent than calcium in stimulating calcitonin secretion while being less potent than calcium in stimulating IP1 accumulation. We found that the biased agonism of strontium was a result of strontium-activated CaSR coupling to a different signaling pathway than calcium-stimulated CaSR. The two studies together support the model of CaSR having several active conformations that are stabilized by different agonists. In addition, our results show that the bias effect might depend on cellular background since strontium only acted as a biased agonist in 6-23 cells.
A second major topic of this PhD thesis was the involvement of calcium and CaSR in phosphorous homeostasis with focus on the newly discovered hormone fibroblast growth factor 23 (FGF23). Calcium and phosphorous homeostasis are highly dependent on each other and are regulated by the same hormones. The role of FGF23 in phosphorous homeostasis is well characterized and FGF23 is secreted from osteoblasts/osteocytes in response to hyperphosphatemic conditions. FGF23 in turn activate FGF receptor-klotho complexes in the renal proximal tubule causing reduced reabsorption of inorganic phosphate and reduced production of active 1.25-dihydroxyvitamin D3 (1,25(OH)2D3), thus maintaining phosphorous homeostasis. The involvement of FGF23 in calcium homeostasis, however, remains poorly understood. Therefore, the last study sought to investigate the regulatory role of calcium and CaSR on FGF23 secretion. By using a knockout mouse model of CaSR we found that CaSR is involved in high phosphorous diet-induced FGF23 secretion. However, the involvement of CaSR in high phosphorous diet-induced FGF23 secretion did not seem to be direct since enhanced serum levels of calcium per se caused FGF23 secretion independent of full length CaSR. It was not found how the CaSR is involved in high phosphorous diet-induced FGF23 secretion but it might be that CaSR regulates proteins important for phosphate sensing. Collectively, our results fit with a hypothesis of FGF23 being a hormone secreted in response to high calcium/inorganic phosphate mineral load sensed by osteoblasts/osteocytes.
