Subsequently, methods were found to purify the protein in high concentration for crystallization and cryoEM-based studies of 3D protein structure. As predicted, these studies demonstrated that the receptor protein is a homodimer. They also led to a finding that surprised many scientists working in the field: Ca2+ binds in allosteric sites distinct from the canonical agonist binding site located in the bilobed cleft of the receptor’s extracellular Venus Flytrap domain. At least one of these sites is an allosteric agonist site, which in the presence of bound Ca2+ stabilises receptor dimers in the active state. More recent protein structure studies have defined the mechanisms of action of calcimimetics and calcilytics that bind in the receptor’s heptahelical transmembrane domain, and the molecular bases for engagement between the receptor and distinct G protein subtypes and also the molecular origins of receptor-enabled promiscuity.

The CaSR doesn’t always play by the rules

However, several further surprises were in store for the CaSR research community.

Firstly, the receptor was found to be widely distributed in mammalian tissues when they were probed with CaSR-reacting antibodies and/or in RT-PCR reactions or by in situ hybridisation, testing for the presence of CaSR mRNA. While expression in some of these tissues was low, expression in some other tissues was unexpectedly high including: (i) various organs of the brain, including the hippocampus, hypothalamus, and choroid plexus; (ii) endocrine tissue of the GI tract and pancreas; (iii) in growth plate chondrocytes and in cells of both the osteoblast and osteoclast lineages; (iv) immune cells in structures including the GI tract, lung, and synovial joints; and (v) the placenta.

In the case of the brain, the CaSR has also been cloned from whole rat brain and its expression in astrocytes and oligodendrocytes as well as neurones has led to interest in the ideas that the CaSR might be a target in the treatment of neurodevelopmental disorders or degenerative disorders such as Alzheimer disease. While some of these tissues were known to be sensitive to changes in Ca2+o, others were found to demonstrate previously unrecognised Ca2+-stimulated autocrine-paracrine or endocrine functions.

Secondly, the receptor harboured binding sites for other nutrients including L-amino acids, g-glutamyl peptides including glutathione, and inorganic phosphate. Each of these findings provided new perspectives on the physiological roles of the receptor in nutrition and provided new insights into the ways in which the metabolisms of diverse nutrients might be coupled. Amino acids (AAs), for example, in the presence of fixed Ca2+ concentrations, stimulate CaSR-mediated release of key gut hormones including GLP-1 and PYY, demonstrating, at least in part, that protein breakdown couples to gut hormone responses via the nutrient-sensing function of the CaSR, and that calcium and AAs act synergistically. They also provided new opportunities for pharmacotherapy leading to the development of distinct classes of calcimimetics, which target: (i) the pocket between TMH-6 and -7 (Cinacalcet, Evocalcet); (ii) the bilobed cleft of the receptors’ extracellular Venus FlyTrap domain (Upacicalcet); and (iii) a distinct site close to the VFT hinge region, dependent on the free SH residue at Cys-482 (Etelcalcetide).

Thirdly, the receptor couples to multiple G proteins (of the Gq/11, Gi, Gs, and G12/13 families) enabling it: (i) to operate effectively in different cell types by taking advantage of one specific G protein or a specific combination of G proteins upstream of a coordinated signaling network; (ii) to control diverse bodily physiological functions; and (iii) to exhibit biased signaling, whereby preferential activation of one signaling pathway arises either physiologically or pathophysiologically from a particular biochemical, cellular or receptor-dependent context. One chemical context that can drive a specific signaling outcome arises when the concentration of one activator (e.g., Ca2+) is stable but the concentration of another activator (e.g., total AAs) rises. Receptor dependent biased signaling can be observed under conditions in which the biased signaling balance changes e.g., when the wild-type receptor and a receptor variant are compared and the variant is found to either exaggerate or reduce the differences between the relative activations of distinct signaling responses.

Finally, startlingly different phenotypes have been described in mice that are homozygous for distinct CaSR null genotypes. Thus, the first such CaSR null mouse developed by Crystal Ho and colleagues was generated by the insertion of a Neomcyin resistance cassette into Exon-5, which encodes a 77-residue peptide at the C-terminal end of the receptor’s extracellular Cys-rich domain. These mice exhibited phenotypes consistent with FHH (heterozygotes) and neonatal severe hyperparathyroidism (homozygotes). Subsequently, an alternative CaSR null mouse was generated by Cre-lox mediated deletion of Exon-7, which encodes a substantial component of the receptor’s heptahelical domain and intracellular C-terminus. Interestingly, this genetic defect was embryonic lethal leading Chang et al. to investigate the effects of tissue-specific deletion. While deletion in the parathyroid gland led to severe primary hyperparathyroidism, and deletion in osteoblasts resulted in osteopenia/osteoporosis, deletion in chondrocytes resulted in a severe phenotype with embryonic death before Day-13. The severity of the phenotype in Exon-7 null mice supports the conclusion that the CaSR has important developmental and physiological roles outside of its primary physiological role in determining Ca2+ set-points in the parathyroid and kidney.