Surgical removal of the parathyroid glands, whether intentional or inadvertent, induces acute, and in some cases catastrophic, hypocalcemia in experimental animals and in humans (e.g., MacCallum and Voegtlin, 1909; MacCallum et al., 1914; Westerdahl et al., 2000; Vasher et al., 2010; Salinger and Moore, 2013). In addition, perturbations of the plasma ionized calcium concentration in vivo by intravenous infusions of calcium salts to induce hypercalcemia or Ca²⁺ chelators such as citrate or EGTA to induce hypocalcemia provoke rapid negative and positive changes in the serum PTH concentration respectively (Fox and Heath, 1981; Conlin et al., 1989; Schwarz et al., 1992). These studies demonstrate that the gland is equipped with a Ca²⁺-sensor that suppresses PTH secretion in response to elevated Ca²⁺ concentration.

The successful preparation of bovine parathyroid cells using collagenase digestion of sliced parathyroid gland tissue provided novel opportunities to assess the cellular Ca²⁺ sensing mechanism in vitro (Brown et al., 1976) and similar observations were made for porcine (Morrissey and Cohn, 1978) and also human (Birnbaumer et al., 1977; Brown et al., 1978a, 1979a; Conigrave et al., 2004) parathyroid cells. In all these cases, mammalian parathyroid cells in primary culture supported a robust endogenous secretion of PTH that was promptly shut off upon elevation of Ca²⁺_o. In cells prepared from samples of parathyroid tissue derived from patients with primary hyperparathyroidism there was impairment but not complete loss of Ca²⁺_o sensitivity (Brown et al., 1979a,c; Mun et al., 2009). The behavior raises questions about the nature of the extracellular Ca²⁺ sensor. It also raises questions about the nature of the intrinsic/endogenous PTH secretion mechanism.

In the first description of a viable, functional parathyroid cell preparation (Brown et al., 1976) bovine parathyroid cells in primary culture in Eagle's medium (minus bicarbonate) secreted PTH linearly at a rate of 20–30 pmol cell⁻¹ h⁻¹ for up to 3 h. PTH secretion was suppressed by around 60% at a Ca²⁺_o of 1.5 mM when compared to that observed at 0.5 mM Ca²⁺_o. In the presence of 0.5 mM Ca²⁺_o, elevated extracellular Mg²⁺ concentration (Mg²⁺_o) also suppressed PTH secretion although Mg²⁺_o was less potent than Ca²⁺_o. Finally, increases in PTH secretion were observed in response to the β-adrenergic agonist isoproterenol that were partially reversed by the β-adrenergic antagonist propranolol (Brown et al., 1976). Thus, key features of the preparation included: Ca²⁺_o- and Mg²⁺_o-mediated suppression of PTH secretion, pointing to the existence of an intrinsic divalent cation sensor with a preference for Ca²⁺_o over Mg²⁺_o; and stimulation of PTH secretion by cAMP-linked GPCRs including beta-adrenergic, dopaminergic, and prostanoid receptors (Brown et al., 1977a,b; Gardner et al., 1980). These findings pointed to the existence of neuronal, hormonal, and/or local stimulatory control of PTH secretion. Although not clearly identified, the findings also demonstrated the existence of an intrinsic PTH secretion mechanism. According to one interpretation, parathyroid cells are equipped with a constitutive PTH secretion mechanism. According to an alternative interpretation, parathyroid cells respond to an autocrine/paracrine mechanism that supports PTH secretion.

The Ca²⁺-sensing mechanism in the parathyroid supports the operation of an extracellular “calciostat” in vivo. The set-point for this calciostat occurs at a plasma ionized Ca²⁺ concentration of around 1.1–1.2 mM corresponding to plasma total calcium concentrations of around 2.2–2.4 mM, of which approximately half is in an albumin-bound form. PTH secretion rates rise 2 to 4-fold as Ca²⁺_o drops toward 1.0 mM and are effectively suppressed by >50% as Ca²⁺_o rises toward 1.4 mM (review: Conigrave et al., 2000a). The changes in PTH secretion rate are reflected in consonant changes in the serum PTH level (normal range 1–6 pmol/L). This set-point behavior can be readily demonstrated in perifused parathyroid cell preparations including those prepared from human parathyroid glands (Conigrave et al., 2004; Figure 1). Ca²⁺_o-dependent inhibitory control of renal Ca²⁺ reabsorption, resulting in elevated renal calcium excretion, also contributes to the calciostat function, providing a key element of the defense against hypercalcemia (Kantham et al., 2009; Loupy et al., 2012).

Investigation of the molecular requirements for divalent cation sensing in parathyroid cell preparations led to some surprising observations. Firstly, tervalent inorganic cations of the lanthanide group including Gd³⁺ and Tb³⁺ were found to be high potency activators (EC₅₀ ≈ 5–50 μM) of parathyroid PI-PLC, suppressors of GPCR-stimulated cAMP accumulation, and inhibitors of PTH secretion (Brown et al., 1990) in a manner analogous to divalent cations. Furthermore, and even more surprisingly, organic multivalent cations including polyarginine, polylysine, and protamine (Brown et al., 1991a), the PLC inhibitor neomycin (Brown et al., 1991b), and polyamines such as spermine (Nemeth and Scarpa, 1987b) stimulated intracellular Ca²⁺ mobilization and inhibited PTH secretion.

The demonstration that the parathyroid calcium sensor coupled to the activation of PI-PLC and, at least in certain circumstances, to heterotrimeric G_i G-proteins, and was promiscuous with respect to inorganic and organic multivalent cations provided a strategy by which a putative PLC-coupled receptor might be cloned by cellular expression of pools of mRNA derived from a size-fractionated bovine parathyroid cDNA library (Brown et al., 1993). Xenopus oocytes express a large conductance Cl⁻ channel whose open probability is highly sensitive to changes in intracellular Ca²⁺ concentration (e.g., downstream of GPCR-mediated generation of IP₃ and intracellular Ca²⁺ mobilization). In this case, the successful cloning of the novel class C GPCR that is now referred to as “the calcium-sensing receptor” relied on its high degree of sensitivity to Gd³⁺, which was used to identify “active” pools of mRNA for further separation and purification. Once cloned, the receptor was readily expressed not only in Xenopus oocytes but also various mammalian cell lines including HEK-293 cells and was found to exhibit sensitivity not only to divalent inorganic cations including Ca²⁺ and Mg²⁺, and tervalent inorganic cations including Gd³⁺ but also to organic cations including the antibiotic neomycin (Brown et al., 1993), polyamines such as spermine (Quinn et al., 1997), cationic polypeptides such as polyarginine and polylysine (Ray and Northup, 2002), and cationic proteins including beta amyloid (Ye et al., 1997). The cloning of the bovine parathyroid CaSR was followed subsequently by the cloning of its orthologs from human parathyroid (Garrett et al., 1995), rat kidney (Riccardi et al., 1995), and rat brain (Ruat et al., 1995).

The CaSR is known now to be expressed widely, with various Ca²⁺_o dependent functions in cell and developmental biology as detailed elsewhere in this issue. It is also known to activate a large number of signaling pathways downstream of various G-proteins and multiple cell membrane-associated as well as cytoplasmic enzymes (review: Conigrave and Ward, 2013).

The CaSR mediates, for example, the activation of various protein kinases including protein kinase C isoforms, which negatively modulate CaSR function (Jiang et al., 2002; Davies et al., 2007; Lazarus et al., 2011; Young et al., 2014), and the mitogen activated protein (MAP) kinases ERK_1/2, p38 and JNK (Kifor et al., 2001; Tfelt-Hansen et al., 2003; review: Conigrave and Ward, 2013). The roles of protein kinases in CaSR-mediated inhibitory control of PTH secretion are not well-understood but ERK_1/2 appears to contribute (Corbetta et al., 2002) and could be activated downstream of either G_q/11 or G_i (review: Conigrave and Ward, 2013).

While the CaSR is expressed and trafficked to the plasma membrane as functional homodimers (Bai et al., 1998, 1999) that couple efficiently to G_q/11, it is also capable of forming heterodimers with other members of GPCR family C including metabotropic glutamate receptors (Gama et al., 2001) and GABA_B receptors, especially GABA_B1 (Chang et al., 2007; Cheng et al., 2007). The consequences of heterodimerization for receptor localization to specific subdomains of the plasma membrane and for signaling pathway selection in different tissues and for the parathyroid, in particular, are not yet clear.

The first reported transgenic mouse in which the CaSR was “knocked out,” was homozygous for a 20 bp insertion that disabled incorporation of CaSR exon-5 (referred to as CaSR exon-4 in the paper) into the mature, fully processed mRNA (Ho et al., 1995). CaSR exon-5 encodes residues 465–536 (http://www.casrdb.mcgill.ca) at the extreme C-terminal end of the VFT domain, immediately prior to the start of the Cysteine-rich domain. Mice with this genotype exhibited a condition comparable to NSHPT in which homozygotes were normal at birth but exhibited severe growth retardation and markedly reduced muscle power in the days after birth (Ho et al., 1995).

The results of biochemical analyses demonstrated the cardinal features of primary hyperparathyroidism including markedly elevated plasma Ca²⁺ concentration, suppressed plasma inorganic phosphate concentration, and markedly elevated serum PTH levels. In addition, the parathyroid glands were enlarged with prominent chief cell hyperplasia (Ho et al., 1995). These findings are consistent with a severe resistance syndrome arising from markedly impaired Ca²⁺-dependent feedback control of PTH secretion i.e., with loss of the parathyroid Ca²⁺ sensor.

Heterozygotes, unlike the homozygotes, were phenotypically normal in the weeks and months after birth but exhibited mild biochemical disturbances consistent with FHH in humans including mildly elevated plasma Ca²⁺ concentration, suppressed renal calcium excretion, and inappropriately normal plasma PTH levels. These findings suggest a mildly impaired but intact parathyroid Ca²⁺ sensing mechanism together with impaired extracellular Ca²⁺-dependent inhibition of renal Ca²⁺ reabsorption resulting in an increase in the setpoint of the calciostat.

While other class C GPCRs, like the CaSR, exhibit Ca²⁺-sensing properties (Kubo et al., 1998; Wise et al., 1999; Christiansen et al., 2007) it seems unlikely that the parathyroid is equipped with an alternative CaSR since, as described above, mice that are homozygous for either global (Ho et al., 1995) or tissue-selective (Chang et al., 2008) knockouts of the CaSR exhibit a severe, uncompensated form of primary hyperparathyroidism in which the plasma levels of both PTH and calcium are markedly elevated from birth. The phenotype suggests a marked impairment of Ca²⁺_o-dependent negative feedback on PTH secretion with attendant hyperparathyroidism and PTH-dependent bone resorption. Thus, if the parathyroid expresses an alternative or supplementary calcium sensor, it is unable to compensate for loss of the CaSR. It is possible that under some circumstances Ca²⁺-sensing is mediated not by CaSR homodimers but by CaSR heterodimers involving other members of GPCR family C including metabotropic glutamate receptors or GABA_B1 receptors as noted above (Gama et al., 2001; Chang et al., 2007; Cheng et al., 2007).

Previous work suggested a role for Ca²⁺-permeable channels in the control of PTH secretion based on observations that stereoisomers of the Ca²⁺ channel modulator 202–791 either inhibited (+202 to 791) or stimulated (−202 to 791) PTH secretion (Fitzpatrick et al., 1986b), and antibodies that target skeletal muscle Ca²⁺ channels also modulated PTH secretion (Fitzpatrick et al., 1988). Other Ca²⁺ channel activators, including maitotoxin (Fitzpatrick et al., 1989), and the diltiazem analog TA-3090 (Chen and Brown, 1990) were also found to inhibit PTH secretion. This work was “turned on its head” by the successful development of “calcimimetics” by structural modification of an L-type Ca²⁺ channel blocker, fendiline (Nemeth et al., 1998), and the subsequent demonstration that modulation of PTH secretion by these agents arises not from actions on Ca²⁺ channels but rather the cloned CaSR (Nemeth et al., 2004; review: Nemeth, 2006). Thus, various agents that modulate Ca²⁺ channel activity can also interact with an allosteric site in the CaSR's heptahelical domain (Leach et al., 2016). Calcimimetics, positive modulators of the CaSR, and calcilytics, negative modulators of the CaSR, are discussed in greater detail below.

Nevertheless, more recent work raises the possibility that Ca²⁺-permeable channels may indeed contribute to the control of PTH secretion. Thus, parathyroid cells express NMDA receptor subunits and NMDA inhibits PTH secretion (Parisi et al., 2009). While these receptors may contribute to the tonic control of PTH secretion, it is not known whether Ca²⁺ fluxes arising from the activation of NMDA receptors are sensitive to Ca²⁺_o concentration in parathyroid cells. In addition, various amino acids and amino acid analogs are known to interact with the CaSR (Conigrave et al., 2000b, 2004; review: Conigrave and Hampson, 2010) and it is not yet clear whether the inhibitory effect of NMDA on PTH secretion is exerted by the activation of Ca²⁺-permeable ion channels or via positive modulation of the CaSR.

As noted above, calcimimetics were developed from the Ca²⁺ channel blocker fendiline that induces Ca²⁺_i mobilization and suppresses PTH secretion from bovine parathyroid cells (Nemeth et al., 1998; review: Nemeth, 2006). Drug development resulted in a new class of pharmaceuticals, the phenylalkylamine calcimimetics, which are positive allosteric modulators of the CaSR that markedly enhance the sensitivity of CaSR-mediated intracellular signaling pathways to Ca²⁺_o (Nemeth et al., 1998). Early examples included NPS R467 and NPS R568, which together with their less potent S-isomers have been key agents for the analysis of CaSR-mediated effects in various cell and tissue systems. More recent examples include cinacalcet, an agent that is well-absorbed orally (Nemeth et al., 2004) and is effective clinically in the treatment of both secondary hyperparathyroidism due to chronic kidney disease (Moe et al., 2005; Messa et al., 2008) as well as primary hyperparathyroidism (Peacock et al., 2005, 2011; see also review: Nemeth and Shoback, 2013).

One key effect of calcimimetics is suppression of the serum PTH level. In primary hyperparathyroidism, for example, in which the plasma total calcium concentration is typically elevated from its normal upper limit of 2.6 mM to around 2.8–3.0 mM, oral therapy with cinacalcet suppressed serum PTH levels and restored the plasma calcium concentration into the normal range for up to 12 months or more (Peacock et al., 2005). Another key effect is suppression or even reversal of parathyroid hyperplasia. For example, cinacalcet suppresses parathyroid cell proliferation and reduces gland size in models of primary (Imanishi et al., 2011) and secondary (Colloton et al., 2005; Miller et al., 2012) hyperparathyroidism, and also induces apoptosis in second hyperparathyroidism (Tatsumi et al., 2013).

The demonstration that calcimimetics from the same class and across different classes exhibit different biased signaling profiles (Davey et al., 2012) is encouraging efforts to develop new generation calcimimetics in support of tissue-specific CaSR-targeted pharmacotherapy e.g., parathyroid vs. kidney vs. thyroid C-cells (review: Leach et al., 2015). Recent modeling of calcimimetic binding in the CaSR's heptahelical domain suggests that agents such as A265347 with less pronounced biased signaling profiles may bind more deeply in the allosteric pocket (Leach et al., 2016).

More recently, a peptide activator of the CaSR (AMG-416; L-Cys-AcDCys-DAla-(DArg)₂-DAla-DArgNH₂) has entered clinical practice for the treatment of patients with secondary hyperparathyroidism on hemodialysis (Bell et al., 2015). Administered intravenously it has superior pharmacokinetics including effective suppression of PTH levels beyond 24 h (Walter et al., 2013) due, presumably, to its ability to form a di-sulfide with CaSR residue C482 in its extracellular domain (Alexander et al., 2015).

Several classes of calcilytics (negative modulators of the CaSR) have been developed. These agents, in general, bind in the HH domain and suppress CaSR signaling. For this reason, they have proved useful in assessing the role of the CaSR in Ca²⁺- or L-amino acid-induced cellular or tissue responses (e.g., Dvorak et al., 2004; Daly et al., 2013). In the parathyroid, calcilytics promote PTH secretion by reversing the inhibitory action of the CaSR (Nemeth et al., 2001). As a consequence, it was hoped that these agents might prove useful in the treatment of osteoporosis by elevating serum PTH levels to emulate the action of intermittent subcutaneous injections of PTH1–34 (teriparatide). However, none of the calcilytics that have entered human clinical trials, thus far, have been successful in significantly increasing bone density or reducing fracture risk (review: Nemeth and Goodman, 2016). Two main explanations seem reasonable: (i) the maximum increase in the serum level of endogenous PTH is significantly less than that achieved by subcutaneous injections of PTH1–34 (e.g., Kimura et al., 2011); or (ii) calcilytics suppress CaSRs in cells of the osteoblast lineage to interfere with PTH-induced cell maturation and key differentiated functions including matrix synthesis and mineralization (Dvorak et al., 2004).

In addition to its regulation by Ca²⁺ ions, the CaSR also responds promiscuously to L-amino acids of various classes (Conigrave et al., 2000b), and one of the most potent, L-Trp, has been shown recently to bind in the receptor's VFT domain ligand-binding groove (Geng et al., 2016; see below). This behavior resembles that of several class C GPCRs (Conigrave and Hampson, 2006, 2010) and supports macronutrient sensing in various tissues including the gastrointestinal tract (review: Conigrave and Brown, 2006). Based on the signaling pathway analysis performed to date, however, Ca²⁺_o and L-amino acids are not equivalent activators. In particular, L-amino acids preferentially activate a Ca²⁺_i mobilizing pathway and have more limited actions on PI-PLC and ERK_1/2 (review: Conigrave and Ward, 2013). Nevertheless, L-amino acids are potent activators of Ca²⁺_i mobilization in parathyroid cells and also suppress PTH secretion at physiologically relevant concentrations (Conigrave et al., 2004). Furthermore, glutathione and various analogs (e.g., S-methylglutathione) also activate Ca²⁺_i mobilization and suppress PTH secretion, presumably by binding to the same VFT domain ligand-binding groove (Broadhead et al., 2011). These findings imply that protein nutritional state is negatively coupled to the control of PTH secretion and thus serum PTH levels. The full significance of these effects, however, is not yet known (see below).

Analysis of the promoter regions of the CaSR gene has led to the identification of two key positive modulators of expression: (i) inflammatory cytokines including IL-1β, IL-6 and TNFα (Canaff and Hendy, 2005); and (ii) hormonally active analogs of vitamin D including 1,25-dihydroxyvitamin D₃ (Canaff and Hendy, 2002), and possibly 25-hydroxyvitamin D₃, whose plasma levels are nearly 1000-fold higher. These results suggest that CaSR expression may be upregulated in the parathyroid and other CaSR-expressing tissues in response to various inflammatory conditions and in response to elevations in either serum 1,25-dihydroxyvitamin D₃ or 25-hydroxyvitamin D₃ levels.

The CaSR couples to various G-proteins (review: Conigrave and Ward, 2013). Notable from the perspective of parathyroid function are G_i, which suppresses agonist-stimulated GPCR-mediated cAMP production and contributes to the activation of ERK_1/2 at least in part via β-arrestin, and G_q/11, which activates PI-PLC and induces Ca²⁺_i mobilization, with attendant activation of several protein kinase C isoforms and ERK_1/2.

Both the G_i and G_q/11 pathways appear to be important for the inhibitory control of PTH secretion. With respect to G_q and G₁₁, it is now known that Gα_q and Gα₁₁ are required for the normal control of PTH secretion. Thus, in a transgenic mouse in which parathyroid-specific ablation of Gα_q was produced on a global Gα₁₁ null background, severe neonatal hyperparathyroidism was observed (Wettschureck et al., 2007) and resembled the phenotypes of both global (Ho et al., 1995) and parathyroid-specific (Chang et al., 2008) ablation of the CaSR. These findings demonstrate that G_q and G₁₁ are required for CaSR-mediated control of PTH secretion and thus lie at the top of a key inhibitory signaling pathway(s). Consistent with these findings, inactivating and activating mutations of the human Gα₁₁ gene have been shown respectively to underlie variant forms of FHH (FHH2) and ADH (ADH2; Nesbit et al., 2013a; Gorvin et al., 2016; Piret et al., 2016).

Under certain circumstances, the CaSR also couples to G_s (review: Conigrave and Ward, 2013) but the significance of this pathway for the control of PTH secretion is unknown. It is interesting to speculate that the “inactive” form of the receptor, which is promoted under conditions of low Ca²⁺ and high phosphate concentrations (Geng et al., 2016) might preferentially couple to G_s in the parathyroid.

Receptor trafficking studies have largely focused on cell systems in which the CaSR is expressed heterologously (reviews: Breitwieser, 2013, 2014). These studies demonstrate that trafficking of the CaSR is modulated by various binding partner proteins (review: Huang and Miller, 2007), can be promoted by allosteric modulators such as cinacalcet and NPS-2143 acting as pharmaco-chaperones (Leach et al., 2013), and is sensitive to receptor-dependent signaling (Grant et al., 2011, 2012; review: Breitwieser, 2012). In the parathyroid, the CaSR interacts with caveolin and is thus likely to localize to sub-domains of the plasma membrane known as caveolae (Kifor et al., 1998). In addition, recent findings suggest that the CaSR is processed between the plasma membrane and intracellular endosomes via clathrin-coated vesicles since mutations of Arg15 of the sigma (σ) subunit of the clathrin-binding protein AP2 have been linked to a variant form of FHH, now known as FHH3 (Nesbit et al., 2013b). The findings suggest that the formation, and/or maintenance, of CaSR signaling complexes is impaired under conditions in which clathrin-coated vesicle-mediated processing of the CaSR is impaired.

While X-ray crystal structures of class C GPCR VFT domains (Kunishima et al., 2000; Tsuchiya et al., 2002), entire extracellular (VFT-plus-Cys-rich) domains (Muto et al., 2007), and even heptahelical domains (Doré et al., 2014) have been reported over the last 15 years, crystal structures for CaSR domains have only recently become available (Geng et al., 2016; Zhang et al., 2016).

These newly described CaSR structures provide information on the inactive and active forms of its VFT domain (Geng et al., 2016; Zhang et al., 2016) and entire extracellular domain (Geng et al., 2016). While the protein conformations of the active forms of the VFT domain structures were almost identical, the identification of divalent cation, and anion binding sites were quite different in the structures reported by the two groups. Zhang et al. (2016) identified just one Ca²⁺ site in the active form of the VFT domain and relied on modeling of electron densities to ascribe it to the ligand-binding cleft, where it was closely associated with an L-amino acid-binding site. Surprisingly, however, they identified a formaldehyde derivative rather than the native form of L-Trp in the site.

In the structures described by Geng et al. (2016), on the other hand, an anomalous mapping strategy was used to identify four, previously unrecognized, Ca²⁺ binding sites, one of which (“Site 2”) was present in both the inactive and active structures and three of which were only identified in the active structure and, thus, may act to stabilize it. Interestingly, no Ca²⁺ binding site was located in the closed (active) form of the agonist-binding cleft in the structure reported by Geng et al., which was occupied instead by the amino acid L-Trp (Geng et al., 2016). In addition, Geng et al. identified several binding sites for inorganic phosphate in the inactive structure (Geng et al., 2016), raising the possibility that not only the Ca²⁺_o concentration but also the ratio of Ca²⁺_o to phosphate concentrations may control the receptor's transition between inactive and active states.

The findings that the receptor binds inorganic phosphate (P_i) as well as Ca²⁺ ions and that Ca²⁺ stabilizes the active state, whereas P_i stabilizes the inactive state have potentially important implications for understanding parathyroid function since elevated P_i concentrations stimulate PTH secretion (Slatopolsky et al., 1996) whereas elevated Ca²⁺_o inhibits it. Does the CaSR modulate its response to Ca²⁺_o according to the background level of inorganic phosphate? Does the Ca:P_i ratio determine PTH secretion rates by controlling the activation state of the CaSR? Does the CaSR act as a phosphate sensor in other tissues such as osteocytes or osteoblasts in bone?

There are several unresolved problems. Four of them are considered below in the form of sets of questions.

The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.