The extracellular calcium concentration is tightly regulated, given its involvement in many key biological processes such as muscle and heart contraction, neuronal excitability, bone mineralization, and hormone secretion. The CaSR plays a key role in calcium homeostasis by regulating the secretion of PTH from the parathyroid glands and the reabsorption of urinary calcium by the kidney. The transfer of calcium between the environment and extracellular fluid takes place in the intestine. Bone constitutes the main calcium reservoir. Calciotropic hormones such as PTH and 1,25- hydroxyvitamin D3 mediate changes in calcium homeostasis via actions in bone, kidney, intestine and parathyroid gland. The relationship between extracellular calcium and PTH levels is represented by an inverse sigmoidal curve (Figure 1). The activity and/ or the expression level of parathyroid CaSRs predicts the cal cium set- point, defined at which the extracellular calcium level at which PTH secretion is half- maximal.
Fig1. Relationship between levels of serum calcium and plasma PTH. Depicted is the inverse sigmoid relationship between levels of serum calcium and plasma PTH in patients with familial hypocalciuric hypercalcaemia (FHH; dotted line) and normal individuals (solid line). The calcium set- point is defined as the extracellular calcium level which produces half- maximal inhibition of PTH secretion. FHH results in a shift of the curve to the right. Increased levels of serum calcium are needed to suppress plasma PTH levels in FHH. The therapeutic effect of calcimimetic leads to a shifting of the set- point to the left.
CaSRs serve as the calcium- sensing mechanism in vivo. CaSRs are predominantly expressed in the chief cells of the parathyroid gland and along the whole nephron and are also found in other tis sues such as pancreatic islet, intestine, bone, breast and skin and in some areas of the brain. The CaSR is a G- protein coupled receptor (GPCR) and belongs to the Family C of this receptor super family. The CaSR belongs to the group II subfamily, which contains the CaSR, the vomeronasal receptors (VRs) and odorant receptors, and has more than 20% amino acid identity over their transmembrane domains (TMD) comprising the 7- TM- spanning region.
The human CASR gene maps to 3q13.3- 21 and has eight exons. Six exons, exon 2– 6, encode the CaSR protein which is made up of 1078 amino acids. The large extracellular domain (ECD) of the CaSR has approximately 600 amino acids including the bi- lobed Venus flytrap- like domain (VFT). The VFT is responsible for ligand binding and connects via cysteine- rich region and peptide linker to the seven TMD. The intracellular COOH- terminal tail is 216 amino acids long. The CaSR functions as a dimer on the cell surface, which is in part due to intermolecular disulphide bonds in the ECD. Three to five calcium ions bind to the cleft of each VFT and cause closure of the VFT lobes with ensuing rotation of the VFT and a transfer of a conformational change to the cysteine- rich region of a CaSR dimer. Upon activation, the CaSR forms a novel dimer inter face between subunits. The conformational change reconfigures some TM- helices such that intracellular loops contact G proteins and initiate cell signalling. In addition to calcium, other polycations such as magnesium and barium, charged polyvalent molecules such as spermine, and β- amyloid peptides can activate the CaSR. The sensitivity to calcium can also be positively modulated by pH, ionic strength, and aromatic L- α- amino acids such as phenyl alanine, tryptophan, and histidine. According to the crystal structures of the CaSR, there are not only multiple binding sites for calcium, but unexpectedly also for phosphate ions. While calcium ions promote the active state, phosphate ions stabilize the inactive confirmation.
After ligand binding and likely dimerization of individual receptor molecules and conformational changes, the CaSR couples to multiple intracellular signalling pathways, depending on the target cell. CaSRs can couple with several G proteins (Gi, Gq/ 11, G 12/ 13). The CaSR stimulates activation of phospholipase A2, C, and D as well as various mitogen- activated protein kinases (MAPKs) and inhibits adenylate cyclase. In particular, G- protein subunit α11 stimulates phospholipase C- β (PLC- β) activity (Figure 2). PLC- β increases inositol 1,4,5- triphosphate, a mediator of rapid mobilization of calcium from intracellular stores, whereas the diacylglycerol (DAG) produced activates protein kinase C (PKC). PKC in turn initiates the MAPK pathway (Figure 2).
Fig2. Role of the CaSR, Gα11 and AP2 complex in regulation of PTH secretion and renal calcium reabsorption. Multiple calcium ions bind to the extracellular bi- lobed Venus fly trap (VFT) domain of the CaSR. Upon activation, CaSR binds to the G11 and Gq (not shown) proteins which dissociate into the respective Gα- subunits and Gβγ heterodimers. Gα11 stimulates phospholipase C (PLC). PLC catalyses the formation of inositol 1,4,5- triphosphate (IP3) and diacylglycerol (DAG) from phosphatidylinositol 4,5- biphosphate (PIP2). IP3 accumulates and leads to a rapid release of calcium intro the cytosol from intracellular stores. DAG activates protein kinase C (PKC) which in turn initiates the mitogen- activated protein kinase (MAPK) pathway. Ultimately, these intracellular signalling pathways decrease PTH secretion from parathyroid chief cells and reduce renal tubular calcium absorption. CaSR cell surface expression is dependent on the endocytic complex, which consists of clathrin, β- arrestin and the AP2 complex, and on agonist- driven insertional signalling (ADIS) (not shown). The endocytic complex transports the CaSR to the endosomal- lysosomal degradation pathway or recycles it back to the cell surface. Loss- of- function mutations in CASR lead to FHH type 1, whereas loss- of- function mutations of GNA11 are associated with FHH type 2. FHH type 3 is caused by loss- of- function mutations of AP2S1.
Current thinking, based on work in model in vitro systems with high levels of receptor expression, is that cell surface CaSR expression is regulated by two main mechanisms. The first mechanism has been called agonist- driven insertional signalling or ADIS. ADIS leads to increased anterograde trafficking of newly synthesized receptors to the plasma membrane after prolonged exposure to calcium. The second mechanism depends on an endocytic complex comprising clathrin, β- arrestin and the AP2 complex. The endocytic complex transports CaSRs to the endosomal- lysosomal degradation pathway or recycles them to the cell surface through retrograde trafficking (Figure 2).
Elevated serum calcium levels are sensed by CaSRs, and activation of downstream signalling leads to downregulation of PTH secretion. The expression of the CaSR on the cell sur face seems to be upregulated by extracellular calcium and 1,25- dihyroxyvitamin D3. Decreased PTH secretion will reduce bone resorption, urinary calcium reabsorption, and renal synthesis of 1,25- hydroxyvitamin D3, thereby decreasing intestinal calcium absorption. CaSRs respond to elevated calcium levels in the kidney, and urinary calcium reabsorption by PTH in the cortical thick ascending limb decreases by interfering directly with sodium/ chloride reabsorption. The interference with sodium/ chloride reabsorption impairs the generation of the medullary osmotic gradient which is critical for urinary concentration. In the proximal tubule, CaSRs reduce the inhibitory effect of PTH on renal phosphate reabsorption. CaSRs also reduce the urinary concentrating ability in the inner medullary collecting duct by counteracting the action of vasopressin. A decrease in serum or extracellular calcium reduces CaSR activation.
