Disorders of the calcium-sensing receptor and partner proteins: insights into the molecular basis of calcium homeostasis

1Extracellular calcium (Ca2+o) is essential for the regulation of a variety of biological processes including neuromuscular excitability, blood coagulation and mineralisation of bone matrix and for providing a steady supply of calcium for intracellular functions ranging from muscle contraction to hormone synthesis and secretion. Thus, there is a requirement for Ca2+o to be tightly regulated, and a complex homeostatic system has evolved to maintain Ca2+o at near-constant concentrations (Fig. 1). The Ca2+o homeostatic system comprises four main components: (1) the parathyroid glands, which sense the prevailing Ca2+o concentrations and regulate the actions of target calcitropic organs; (2) the intestine and kidneys, which facilitate the transfer of calcium between the external environment and extracellular fluid; (3) the skeleton, which represents the major reservoir of total body calcium and is critical for buffering short-term changes in Ca2+o concentrations; and (4) calcitropic hormones such as parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3, which mediate the interactions between the parathyroid glands, bone, kidney and intestines (Fig. 1). Thus, low Ca2+o concentrations lead to PTH release from the parathyroid glands. This hormone exerts three distinct effects on Ca2+o homeostasis, which are to enhance bone resorption, urinary calcium reabsorption, and the renal synthesis of 1,25-dihydroxyvitamin D3, thereby leading to intestinal calcium absorption (Fig. 1). Low Ca2+o concentrations are also sensed by the kidneys, which increase urinary calcium reabsorption independent of the actions of PTH. These combined events mediate a rise in Ca2+o concentrations, which together with 1,25-dihydroxyvitamin D3, leads to feedback inhibition of PTH secretion (Fig. 1).

2Studies of patients and kindreds with familial hypocalciuric hypercalcaemia (FHH) or autosomal dominant hypocalcaemia (ADH) have helped to elucidate the molecular basis of Ca2+o sensing and overall regulation of Ca2+o homeostasis by calcitropic tissues such as the parathyroid glands and kidneys. These studies also revealed the involvement of a G protein-coupled receptor (GPCR) pathway that comprises the calcium-sensing receptor (CaSR), G-protein α-11 (Gα11) subunit and adaptor-related protein complex 2 sigma (AP2σ) subunit. This article will provide an overview of the role of the CaSR, Gα11 and AP2σ proteins, review the disorders caused by mutations of these proteins, outline potential targeted therapies, and describe insights gained into the molecular basis of Ca2+o homeostasis by studies of the CaSR and its partner proteins.

3The human CaSR is a 1078 amino acid dimeric cell-surface protein that belongs to class C of the GPCR superfamily, which also includes the metabotropic glutamate, gamma-aminobutyric acid type B (GABAB) and taste 1 receptors. The CaSR is highly expressed in the parathyroid glands and kidneys. It has a large (612 amino acid) extracellular domain (ECD), which was recently crystallised and shown to comprise two globular lobes that adopt a venus flytrap (VFT) conformation. The cleft region located between the two lobes of the VFT is predicted to bind Ca2+o (Fig. 2), thereby leading to the activation of multiple intracellular signalling cascades via interactions with its transmembrane and intracellular domains. In the parathyroid glands, the Gq/11 family of heterotrimeric guanine nucleotide–binding proteins (G proteins) are considered to represent the major downstream signalling partners for the CaSR. The binding of the CaSR to the Gq and G11 proteins is predicted to lead to the dissociation of the respective Gα-subunits from their obligate Gβγ heterodimer, which activate the phospholipase C-beta (PLCβ) enzyme, thereby facilitating the hydrolysis of a plasma membrane lipid constituent, known as phosphatidylinositol 4,5-bisphosphate (PIP2), to produce inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) (Fig. 2). IP3 in turn stimulates the rapid release of calcium from intracellular stores, whereas DAG activates the mitogen-activated protein kinase (MAPK) cascade. These intracellular events mediate a decrease in PTH secretion from the parathyroid chief cell and a reduction in renal tubular calcium reabsorption (Fig. 2). The level of CaSR cell-surface expression likely influences Ca2+o homeostasis and is regulated by agonist-driven insertional signalling (ADIS), which leads to enhanced anterograde trafficking of newly synthesised receptors to the plasma membrane after prolonged exposure to Ca2+o. Moreover, the β-arrestin protein together with the adaptor-related protein complex 2 (AP2), which comprises a heterotetrameric complex of α-, β-, μ- and σ-subunits is considered to facilitate CaSR internalisation from the plasma membrane by clathrin-mediated endocytosis and retrograde trafficking. Thus, AP2 and β-arrestin likely repre­sent key determinants of CaSR cell-surface expression.

4More than 230 different germline mutations of the CaSR, which is encoded by the CASR gene (Fig. 3) located on chromosome 3q21.1, have been reported. These mutations may cause a loss of CaSR function and give rise to hypercalcaemic disorders such as FHH type 1 (FHH1), neonatal severe hyperparathyroidism (NSHPT) and adult-onset primary hyperparathyroidism (PHPT); or lead to a gain of function that is associated with hypocalcaemic disorders such as ADH type 1 (ADH1) and Bartter syndrome type V (Table 1).

5FHH comprises three genetically distinct conditions, designated as FHH types 1–3 (Table 1), which are due to loss-of-function mutations affecting the CaSR, Gα11 and AP2σ proteins, respectively. FHH1 (OMIM #145980) is the most common type and accounts for ~65% of all FHH cases. FHH is a highly penetrant autosomal dominant condition characterised by lifelong non-progressive mild-to-moderate hypercalcaemia, normal (in ~80% of patients) or mildly raised serum PTH levels (in ~20% of patients), and low urinary calcium excretion (in ~95% of patients). FHH is considered to be a benign disorder, as patients are typically asymptomatic. However, an increased prevalence of chondrocalcinosis with advancing age and occasional cases of pancreatitis have been reported. Individuals with FHH have been misdiagnosed as having PHPT and undergone parathyroidectomy, which generally fails to normalise the hypercalcaemia. Mutational analysis may be required to distinguish FHH from PHPT, and to date, FHH1 has been associated with >130 different mutations of the CASR gene (Fig. 3), which are missense substitutions in >85% of cases, whereas nonsense, deletion, insertion and splice-site mutations that lead to truncated CaSR proteins have been described in <15% of cases. Studies of FHH1-associated CaSR mutations have identified critical receptor structure–function relationships and demonstrated a mutational hotspot within the ECD. Indeed, an analysis of the locations of recurrent FHH1-causing CaSR mutations or residues affected by multiple different loss-of-function CaSR mutations has revealed a clustering of mutations at the major predicted Ca2+o binding site located within the cleft region of the bilobed extracellular VFT domain of the CaSR (Fig. 3). These mutated residues may be directly involved in the binding of Ca2+o or indirectly influence alterations in receptor conformational states that occur upon Ca2+o binding. Moreover, in vitro functional expression studies have identified specific VFT domain residues, which when mutated can result in opposing effects on CaSR responses and lead to a loss or gain of function (Fig. 3). These residues may potentially act as intra-molecular switches to regulate the entry and binding of Ca2+o within the VFT cleft region (Fig. 3). FHH-causing mutations also cluster in the CaSR transmembrane domain (TMD) and may inhibit the transmission of activation signals to the intracellular environment, by impairing interactions with heterotrimeric G proteins and other components of the CaSR signal transduction pathway. Some loss-of-function CaSR mutations have been shown to cause signalling bias by switching the wild-type CaSR from preferentially coupling with intracellular Ca2+ (Ca2+i) to a mutant receptor that signals equally via the Ca2+i and MAPK pathways, or which predominantly acts via MAPK. Loss-of-function CaSR mutations causing signalling bias are located within the ECD and TMD; however, the structural motifs within these regions that determine whether the CaSR preferentially couples to Ca2+i or MAPK pathways remain to be established. Around 50% of CaSR mutations that lead to FHH1 have been shown to result in reduced cell-surface receptor expression as a consequence of defective trafficking to the plasma membrane, with mutant CaSRs being retained intracellularly and unable to exit either the endoplasmic reticulum or Golgi apparatus. Such cellular studies also provide an explanation for the benign phenotype of FHH1, which is a heterozygous condition, by demonstrating that co-expression of wild-type and mutant FHH1-causing CaSRs ameliorates the loss of function, with the co-expressed wild-type CaSRs increasing trafficking of mutant receptors to the plasma membrane via the ADIS mechanism. Whereas, cells expressing only mutant loss-of-function CaSRs have both defective membrane targeting and reduced signalling responses.

6NSHPT (OMIM #239200) is a potentially life-threatening disorder most often caused by homozygous or compound heterozygous loss-of-function CaSR mutations (Table 1). NSHPT is characterised by severe neonatal hypercalcaemia (serum calcium concentrations are typically 3.5–5.0 mmol/L), 5- to 10-fold elevations of serum PTH concentrations, marked parathyroid gland enlargement, failure to thrive, and hyperparathyroid skeletal disease leading to multiple fractures and respiratory distress. Parathyroidectomy is usually required to treat NSHPT, and bisphosphonates have been successfully used in some patients to manage marked hypercalcaemia and skeletal demineralisation before parathyroid surgery. Severe neonatal hypercalcaemia is also reported to be associated with heterozygous loss-of-function CaSR mutations, and these findings indicate that NSHPT may be due to factors other than mutant gene dosage. For example, the degree of severity of a dominant-negative mutation or maternal serum calcium concentration may play a role in the phenotypic expression of a CaSR mutation in the neonate. More than 25 different CaSR mutations have been described in association with NSHPT, of which >40% are either nonsense or frameshift mutations that are predicted to lead to a truncated CaSR.

7Loss-of-function CaSR mutations may occasionally present after the neonatal period with marked hyper­calcaemia. Indeed, homozygous loss-of function mutations, which are located at the N-terminal region of the CaSR, have been reported to lead to symptomatic hypercalcaemia in childhood or early adulthood, which required treatment with parathyroidectomy. Occasionally, heterozygous and homozygous loss-of function CaSR mutations have been detected in adult patients with PHPT caused by parathyroid adenomas or hyperplasia (Table 1). The occurrence of PHPT or severe FHH after infancy may be due to the degree of loss of function associated with the underlying CaSR mutations. Indeed, the homozygous CaSR mutations, present in these patients, have been associated with milder alterations in Ca2+i signalling than homozygous muta­tions leading to NSHPT. An analysis of CaSR mutations identified in patients with PHPT or severe FHH that presented after infancy has indicated that CaSR mutations located in the receptor ECD are associated with more severe hypercalcaemia. This hypercalcaemia is recalcitrant to treatment by parathyroidectomy, possibly due to multiple gland involvement. These findings may have some clinical utility in identifying those patients (i.e. with CaSR ECD mutations) in whom a full neck exploration is required, as they are more likely to have multi-gland disease.

8ADH comprises two genetically distinct disorders, designated as ADH types 1 and 2 (Table 1), which are caused by germline gain-of-function mutations of the CaSR and Gα11 proteins, respectively. ADH1 (OMIM #601198) accounts for ~70% of all ADH cases. ADH is characterised by mild-to-moderate hypocalcaemia, with serum calcium concentrations, adjusted for variations in serum albumin concentrations, rarely below 1.50 mmol/L, and ~50% of patients may develop hypocalcaemic symptoms such as paraesthesia, carpopedal spasms and seizures. Although ADH is associated with increased circulating phosphate concentrations and inappropriately low or normal PTH concentrations, this is considered to represent a distinct disease entity from hypoparathyroidism. This is because affected individuals generally have PTH concentrations that are detectable and within the reference range and also a relative hypercalciuria that is characterised by urinary calcium-to-creatinine ratios that are within or above the reference range. Ectopic calcification of the kidneys and basal ganglia is a common feature of ADH and affects >35% of patients. Patients with severe gain-of-function CaSR mutations may also have Bartter syndrome type 5, which is characterised by hypokalaemic alkalosis, renal salt wasting and hyperreninaemic hyperaldosteronism. Mutational analysis is commonly required for the diagnosis of ADH, and >70 different CaSR mutations have been identified to date in individuals affected with ADH1. Of these mutations, 95% are heterozygous missense substitutions. The structure–function analyses of these mutations have defined key peptide motifs of the CaSR that maintain this receptor in an inactive conformation. In particular, gain-of-function mutations cluster within the second peptide loop of the extracellular VFT domain (residues 116–136) (Fig. 3) that contributes to the interface of the dimeric CaSR. Mutations affecting this extracellular peptide loop may lead to a gain of function by promoting conformational changes such as dimer rotation that facilitates receptor activation. A second hotspot for ADH1-associated mutations is located in a region that encompasses transmembrane domains 6 and 7, and the intervening third extracellular loop of the CaSR (residues 819–837) (Fig. 3). This region is likely important for locking the ligand-unbound family C GPCRs in an inactive conformation by forming a network of interactions with other transmembrane domains that inhibits the binding of G proteins. Cellular studies have demonstrated that most ADH1-causing CaSR mutations cause a signalling bias by coupling more strongly to Ca2+i mobilisation than to the MAPK pathway, which contrasts with FHH1-causing CaSR mutations, which are biased towards MAPK signalling.

9Calcimimetics are ligands that mimic or enhance the effects of Ca2+o at the CaSR and are divided into two types: type I calcimimetics are agonists, which include naturally occurring ligands such as polyvalent cations; and type II calcimimetics are positive allosteric modulators that include l-amino acids and cinacalcet, which is a synthetic amino alcohol compound. Calcilytics are inhibitors of CaSR function, and to date, no endogenous calcilytic compounds have been identified. However, synthetic calcilytic compounds have been generated and shown to act as negative allosteric modulators of the CaSR. A recent study has identified a putative binding cavity for synthetic calcimimetic and calcilytic compounds within the extracellular portion of the CaSR TMD and delineated key glutamate residues within the binding cavity, which may facilitate the conformational changes that lead to altered receptor activity upon the binding of these compounds. Calcimimetic drugs represent a potential targeted therapy for NSHPT and symptomatic forms of FHH1. They are shown to improve the signalling by loss-of-function CaSR mutants in vitro and to act as pharmacochaperones that facilitate correct protein folding and plasma membrane targeting of mutant CaSRs. These in vitro effects of calcimimetic drugs are in keeping with reports of FHH1 patients with marked hypercalcaemia or complications such as recurrent pancreatitis who have responded to treatment with cinacalcet, which is licensed for the management of certain hyperparathyroid disorders. Cinacalcet has also been successfully used to manage life-threatening hypercalcaemia in NSHPT probands harbouring a heterozygous CaSR mutation, Arg185Gln. However, it is ineffective for NSHPT caused by biallelic deletional CaSR mutations.

10Symptomatic ADH is conventionally treated with calcium and active vitamin D preparations. However, these therapies do not rectify the underlying pathophysiological alterations in parathyroid and renal tubular function, and their use predisposes affected individuals to the development of marked hypercalciuria, nephrocalcinosis, nephrolithiasis and renal impairment. ADH1 patients have also been treated with recombinant PTH (1–34) (teriparatide); however, when administered by once- or twice-daily bolus injections, this peptide may not always prevent hypercalciuric renal complications. Calcilytic drugs, which are CaSR negative allosteric modulators, represent a potential targeted therapy for ADH1. Calcilytics comprise two main classes of compounds, which are the amino alcohols (e.g. NPS-2143, ronacaleret and JTT-305/MK-5442) and quinazolinones (e.g. ATF936 and AXT914). In vitro studies have shown that NPS-2143, a long-acting calcilytic, corrects the gain of function associated with ADH-causing CaSR mutations. However, the in vitro efficacy of NPS-2143 was reduced by mutations affecting NPS-2143-binding residues within the TMD. In contrast, the quinazolinone calcilytic drugs (ATF936 and AXT914) have been demonstrated to rectify the excessive signalling responses of all ADH mutants evaluated to date, including those mutations leading to constitutive activation and/or Bartter syndrome type 5. To assess whether calcilytics may ameliorate the hypocalcaemia associated with ADH1, these drugs have been administered to mouse models harbouring germline gain-of-function CaSR mutations. In a single-dose in vivo study, NPS-2143 was administered to Nuf mice, which have hypocalcaemia, reduced plasma PTH concentrations and ectopic calcification in association with a germline gain of function Casr mutation, Leu723Gln. Intraperitoneal injection of NPS-2143 significantly increased plasma calcium and PTH concentrations in heterozygous- and homozygous-affected Nuf mice at 1 h after administration, with values returning to baseline after 4 h. The elevations in plasma calcium induced by NPS-2143 were not associated with any increase in urinary calcium excretion. Longer-term in vivo studies involving the JTT-305/MK-5442 calcilytic compound have been undertaken in two ADH1 mouse models, which harbour germline Cys129Ser and Ala843Glu gain-of-function CaSR mutations, respectively. Administration of JTT-305/MK-5442 by daily oral gavage over a 12-week period led to sustained increases in serum calcium concentrations and a significant reduction in urinary calcium excretion in both ADH1 mouse mutants. Recently, a calcilytic compound known as NPSP795 has been evaluated in a clinical trial involving five ADH1 patients. The intravenous administration of NPSP795 significantly increased plasma PTH concentrations and reduced urinary calcium excretion. However, circulating calcium levels were not altered in this study. The optimal dosing regimen for NPSP795, which is a short-acting calcilytic compound intended to elicit a rapid and transient increase in plasma levels of PTH, remains to be established in ADH1 patients.

11Germline mutations affecting Gα11, which is encoded by the GNA11 gene (Fig. 4) on chromosome 19p13.3, have recently been identified as the genetic cause of FHH and ADH in some patients. This finding has revealed Gα11 to be a major component of the CaSR signalling pathway and highlighted its importance in Ca2+o homeostasis. Loss-of-function Gα11 mutations give rise to FHH2, whereas germline gain-of-function Gα11 mutations are associated with ADH2 (Fig. 4 and Table 1) and somatic gain-of-function Gα11 mutations cause uveal melanomas.

12The existence of a genetically distinct form of FHH was first highlighted by genetic linkage studies that mapped an FHH locus to chromosome 19p, which contains the GNA11 locus. This form of FHH was designated as FHH2 (OMIM #145981) (Table 1). GNA11 was considered a likely candidate gene for FHH2, as this encodes the Gα11 protein, which is highly expressed in the parathyroid glands and acts as a signalling partner for the CaSR. Moreover, mouse model studies have demonstrated parathyroid specific ablation of Gα11, and the related Gαq protein, to result in marked hypercalcaemia, hyperparathyroidism and parathyroid gland enlargement. DNA sequence analysis of the reported FHH2 kindred revealed a germline heterozygous GNA11 mutation that resulted in an in-frame isoleucine deletion at codon 199 or 200 (Ile199/200del) of the Gα11 protein. Mutational analysis of the GNA11 gene in additional FHH probands, who did not harbour CASR mutations, has identified heterozygous Leu135Gln and Thr54Met missense mutations in two unrelated probands. The Thr54Met, Leu135Gln and Ile199/200del Gα11 mutations are associated with a mild FHH phenotype characterised by serum adjusted calcium concentrations <2.80 mmol/L. In keeping with these clinical findings, in vitro studies have shown that these FHH2-associated Gα11 mutations lead to a mild impairment of CaSR signal transduction. Indeed, the Thr54Met, Leu135Gln and Ile199/200del FHH2 mutants were associated with around a 30% increase in the half-maximal effective concentration (EC50) of CaSR-expressing cells, whereas CaSR mutations leading to FHH1 generally cause a >50% increase in the EC50 value.

13Homology modelling revealed that FHH2-causing mutations are located within key regions of the Gα11-subunit, which consists of a GTPase domain that binds GDP and GTP and a smaller helical domain that acts as a clasp to secure these bound guanine nucleotides (Fig. 4). Thus, the Ile199/200del mutation is located within the GTPase domain and predicted to disrupt a hairpin loop, which comprises part of the Gα-GPCR interface and is also situated between flexible ‘switch’ regions (Fig. 4) that undergo substantial conformational changes upon GTP binding. In contrast, the Leu135Gln mutation is located in the Gα11 helical domain (Fig. 4). It is not predicted to influence CaSR–Gα11 coupling, but instead likely diminishes CaSR signal transduction by influencing the interaction of Gα11 with downstream effectors. The Thr54Met mutation is located at the interface between GTPase and helical domains (Fig. 4) and predicted to impair coupling and/or dissociation of Gα11 from the CaSR by influencing guanine nucleotide binding at the inter-domain interface. Thus, the identification of these FHH2-causing Gα11 mutations has revealed residues critical for Gα11-subunit function.

14After the identification of loss-of-function Gα11 mutations leading to FHH2, it was hypothesised that gain-of-function germline Gα11 mutations may have opposite effects on Ca2+o homeostasis and give rise to a disorder with an ADH-like phenotype. DNA sequence analysis of eight ADH probands, who did not harbour CaSR mutations, identified germline heterozygous Gα11 mutations in two individuals. In vitro functional studies of these mutations, which comprised Arg181Gln and Phe341Leu Gα11 missense substitutions, demonstrated cells expressing the mutant Gα11 proteins to have enhanced sensitivity to Ca2+o, consistent with a gain of function. In parallel with these studies, genetic linkage studies of two unrelated hypocalcaemic kindreds mapped the disease locus to chromosome 19p13.3, which is the location of the GNA11 locus. DNA sequence analysis revealed the occurrence of germline heterozygous Gα11 mutations, Arg60Cys and Ser211Trp. Moreover, heterozygous Arg60Leu and Val340Met Gα11 mutations have been identified by whole-exome sequencing in additional ADH kindreds, and these mutations were demonstrated to lead to enhanced CaSR-mediated signal transduction. These individuals and families with gain-of-function Gα11 mutations, who were designated as having ADH2 (OMIM #615361) (Table 1), generally had serum-adjusted calcium concentrations ranging 1.75–2.15 mmol/L. The affected individuals typically presented with hypocalcaemic symptoms such as paraesthesia, muscle cramps, carpopedal spasm and seizures. Some ADH2 patients were susceptible to treatment-related hypercalciuria, nephrocalcinosis and nephrolithiasis, although affected individuals generally had a milder urinary phenotype, with significantly reduced urinary calcium excretion compared with ADH1 patients who harbour gain-of-function CaSR mutations. Furthermore, patients with germline gain-of-function Gα11 mutations, in contrast to patients with gain-of-function CaSR mutations, harbour non-calcitropic phenotypes. For example, studies of the kindred with the Arg60Leu Gα11 mutation showed this to be associated with impaired post-natal growth, and the affected adults were significantly shorter than unaffected adult family members (height >2SD below mean of unaffected individuals). In addition, some affected members of the kindred with the Val340Met Gα11 mutation were found to have keratoconus, a corneal disorder.

15In contrast to germline gain-of-function Gα11 mutations, which affect Ca2+o homeostasis, somatic gain-of-function Gα11 mutations are reported to cause uveal melanoma, a primary intraocular tumour, by inducing constitutive upregulation of proliferative signalling involving extracellular signal-regulated kinases 1 and 2 (ERK1/2), which are components of the MAPK signalling pathway. However, in vitro studies have shown that the ADH-causing Gα11 mutations do not have such oncogenic potential, and that these ADH-causing germline mutants phosphorylated ERK1/2 only in the presence of Ca2+o, and were thus not constitutively activating. Thus, the milder disturbance of signalling associated with the ADH2 mutants may provide an explanation for their occurrence as a post-natal phenotype that can be transmitted as an autosomal dominant disorder, in contrast to the uveal melanoma-associated constitutively activating G-protein mutation, Gln209Leu, which has been shown to be cytotoxic when expressed at high levels and is likely to be embryonically lethal if present within the germline.

16An analysis of the location of ADH2-causing mutations revealed that mutated Arg60 and Arg181 residues are situated adjacent to the linker 1 and linker 2 peptides, respectively. The peptides are predicted to act as a hinge that connects the Gα11 GTPase domain with the helical domain, thereby allowing these two domains to form a clamshell around bound guanine nucleotides (Fig. 4). Thus, mutations affecting the Arg60 and Arg181 residues may lead to the opening of the Gα11 clamshell and induce G protein activation by promoting the exchange of GDP for GTP. The ADH2-causing Ser211Trp mutation is located within the region of Gα11, which binds to the Gβγ heterodimer, and this mutation may promote the dissociation of the Gα11 subunit, thus enhancing CaSR-mediated signal transduction. The mutated Phe341 residue is located at the C-terminus of the Gα-subunit (Fig. 4) and forms a part of the cluster of phenylalanine residues, which likely stabilises GTP in a conformation required for its hydrolysis, and the ADH2-causing Phe341Leu mutation is thus predicted to activate Gα11 by impairing the hydrolysis of GTP to GDP. In contrast to these predicted effects of the Phe341Leu mutation, the neighbouring Val340 residue is not involved in GDP/GTP exchange, but instead may influence the stability of Gα-GPCR interactions.

17Although calcimimetic and calcilytic compounds represented targeted therapies for patients with CaSR mutations resulting in symptomatic forms of FHH1 and ADH1, it was unclear if these CaSR allosteric modulators may rectify abnormalities of the downstream Gα11 protein, and thus, have potential benefit for FHH2 and ADH2 patients. Recent in vitro studies have revealed cinacalcet and NPS-2143 to correct the loss and gain of function associated with Gα11 mutations leading to FHH2 and ADH2, respectively. Indeed, siRNA knockdown studies showed that these CaSR allosteric modulators directly influence signalling mediated by the FHH2 and ADH2 mutant Gα11 proteins rather than by exerting indirect effects on endogenously expressed wild-type Gα11 proteins. However, some Gα11 mutations (Ile199/200del and Phe341Leu) showed diminished sensitivity to cinacalcet and NPS-2143 and these differences in the sensitivities of the mutants to CaSR-targeted drugs may be explained by an analysis of the crystal structure of the G protein alpha-s (Gαs) complexed with the β2-adrenergic receptor. The analysis showed that residues homologous to Ile199 and Phe341, in the related Gαs protein, are located within a hydrophobic pocket at the interface between GPCR and Gα-subunit (Fig. 5). Thus, Gα11 mutations located at the GPCR–Gα interface may potentially influence the efficacy of CaSR allosteric modulators. The NPS-2143 calcilytic compound was also shown to rectify the constitutive activation caused by a uveal melanoma-associated Gα11 mutation, and these findings suggest a potential therapeutic role for calcilytics in the management of this intraocular tumour.

18Genetic linkage studies of two multi-generational FHH-unrelated kindreds from Oklahoma and Northern Ireland, designated FHHOK and FHHNI, respectively, mapped the disease locus, designated FHH3, to chromosomal 19q13.3, thereby highlighting a third genetically distinct form of FHH (FHH3, OMIM #600740) (Table 1). FHH3 was associated with elevations in plasma PTH, mild hypophosphataemia and osteomalacia in affected family members aged more than 30 years. Whole-exome capture and high-throughput sequence analysis revealed that affected individuals from the unrelated FHHOK and FHHNI kindreds harbour the same heterozygous germline Arg15Cys mutation of the adaptor-related protein complex 2 sigma subunit 1 (AP2S1) gene, which encodes the σ-subunit of the ubiquitously expressed heterotetrameric AP2 complex. The AP2 complex is a central component of clathrin-coated vesicles and facilitates the endocytosis of plasma membrane proteins such as GPCRs. To date, AP2S1 mutations have been reported in >60 FHH3 patients and families. All affected individuals harbour a heterozygous missense mutation affecting the Arg15 residue of the encoded AP2σ-subunit (Fig. 6) and resulting in Arg15Cys, Arg15His or Arg15Leu. Crystallography studies have revealed that the Arg15 residue plays a key role in binding to membrane cargo proteins (Fig. 6). It is predicted that these FHH3-causing Arg15 mutations disrupt an interaction between the AP2 complex and the intracellular carboxyl terminus of the CaSR, thereby impairing endocytosis of this GPCR. This hypothesis is supported by in vitro expression studies, which have demonstrated that these FHH3-causing AP2σ mutations alter CaSR cell-surface expression and impair signal transduction in a dominant-negative manner. Thus, these studies have revealed that the AP2 endocytic complex plays a role in the regulation of Ca2+o homeostasis.

19Nucleotide substitutions affecting codon 15 of the AP2S1 gene are predicted to result in the replacement of the wild-type Arg residue with a mutant Cys, Gly, His, Leu, Pro or Ser residue. All of these potential missense AP2σ substitutions have been demonstrated to diminish CaSR signal transduction in vitro. However, to date, only Arg15Cys, Arg15His and Arg15Leu AP2σ mutations have been observed in FHH3 patients (Fig. 6), and the likely cause of this mutation bias has been revealed by cellular studies. These studies have shown that the non-observed Arg15Gly, Arg15Pro and Arg15Ser AP2σ mutants impair cell growth in vitro. Thus, a possible explanation for the absence of the Arg15Gly, Arg15Pro and Arg15Ser AP2σ mutations in patients is that these deleterious mutations are embryonically lethal, whereas the FHH3-causing AP2σ mutations (Arg15Cys, Arg15His and Arg15Leu) are tolerated and compatible with embryonic and post-natal survival.

20FHH3 may be associated with symptomatic hypercalcaemia and reduced bone mineral density, and with cognitive deficits and/or behavioural disturbances in children harbouring the Arg15Cys or Arg15Leu AP2σ mutations. FHH3 is associated with a more severe biochemical phenotype than FHH1, which is characterised by significantly higher serum calcium and magnesium concentrations and a significantly reduced fractional excretion of calcium. Furthermore, an analysis of adults and children with AP2σ mutations, by one study, has revealed a genotype–phenotype correlation with Arg15Leu probands having significantly higher serum calcium concentrations and presenting at a younger age, typically in childhood, compared with probands with Arg15Cys or Arg15His mutations. The absence of such a genotype–phenotype correlation in another study, which consisted of mainly adult FHH3 patients, suggests that the more severe hypercalcaemia associated with the Arg15Leu mutation might be age dependent.

21Cinacalcet has been evaluated as a therapy for symptomatic hypercalcaemia associated with FHH3. In vitro studies revealed that this calcimimetic drug rectifies the significantly impaired intracellular calcium and MAPK responses associated with all three FHH3-causing AP2σ mutations. The administration of cinacalcet at a dose of 30–60 mg daily to three symptomatic FHH3 probands, each harbouring an Arg15Cys, Arg15His or Arg15Leu AP2σ mutation, led to >20% reductions in serum calcium concentrations and improved symptoms in all three FHH probands. These studies show that cinacalcet-mediated allosteric modulation of the CaSR can rectify the loss-of-function and symptomatic hypercalcaemia that are associated with the three types of FHH3-causing Arg15 AP2σ mutations. Cinacalcet has also been shown to correct the hypercalcaemia in a patient with chromosome 22q11.2 deletion syndrome who also had an Arg15Leu AP2σ mutation.

22Germline coding-region mutations of the CASR and GNA11 genes, which enhance CaSR-mediated signal transduction, have been identified in around 70% of ADH cases. This raises the possibility that ADH patients who do not harbour such mutations may instead have abnormalities of the untranslated or non-coding regulatory regions of CASR and GNA11 or have mutations involving other mediators of Ca2+o homeostasis. It had been postulated that some patients with ADH might harbour AP2σ mutations, which enhance the sensitivity of CaSR-expressing cells to Ca2+o, and this putative form of ADH was designated ADH type 3 (ADH3). However, an analysis of the AP2S1 gene in 19 patients and families who were considered to have ADH but did not have mutations of CASR and GNA11, or of other genes associated with isolated hypoparathyroidism, such as PTH or GCMB, failed to identify coding-region mutations or copy number variants (CNVs). Moreover, investigations of 10 familial cases and 50 sporadic cases of isolated hypoparathyroidism for coding-region mutations or CNVs affecting AP2S1 did not identify any abnormalities. Thus, these studies indicated that AP2σ mutations are unlikely to cause hypocalcaemic disorders such as ADH.

23The identification and characterisation of gene abnormalities underlying FHH and ADH have led to the delineation of a parathyroid and renal G protein-coupled Ca2+o-sensing mechanism, which involves the CaSR, Gα11 and AP2σ proteins. The CaSR is shown to play a pivotal role in the regulation of Ca2+o concentrations, whereas the Gα11 protein appears to be a key mediator of downstream CaSR signal transduction, and the AP2σ protein is likely required for both CaSR signalling and trafficking. These studies have also provided new insights into the clinical phenotypes of Ca2+o-sensing disorders and revealed FHH3 to represent a distinct disorder of Ca2+o homeostasis, which is characterised by symptomatic hypercalcaemia, low BMD and cognitive deficits. Advances have been made in the treatment of FHH and ADH, and the calcimimetic and calcilytic drugs have been shown to be of potential benefit for managing symptomatic forms of these Ca2+o-sensing disorders.

24This review is based on the 2015 Dale Medal Lecture, presented by Prof. Rajesh Thakker at the Society for Endocrinology BES 2015, Edinburgh, UK.

25The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.

26The work in the Academic Endocrine Unit is supported by the United Kingdom Medical Research Council (MRC) programme grants – G9825289 and G1000467, and National Institute for Health Research (NIHR) Oxford Biomedical Research Centre Programme. R V Thakker is a Wellcome Trust Investigator and NIHR Senior Investigator.