Am J Physiol Cell Physiol 307: C221–C231, 2014. First published May 28, 2014; doi:10.1152/ajpcell.00139.2014.

Review

Calcium-sensing receptor 20 years later Tariq I. Alfadda,1* Ahmad M. A. Saleh,1* Pascal Houillier,3 and John P. Geibel1,2 1

Department of Surgery, Yale School of Medicine, New Haven, Connecticut; 2Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, Connecticut; and 3INSERM UMR_S1138, Paris, France; Paris Descartes University, Paris, France; Assistance Publique-Hopitaux de Paris, Hopital Europeen Georges Pompidou, Paris, France Submitted 8 May 2014; accepted in final form 23 May 2014

Alfadda TI, Saleh AM, Houillier P, Geibel JP. Calcium-sensing receptor 20 years later. Am J Physiol Cell Physiol 307: C221–C231, 2014. First published May 28, 2014; doi:10.1152/ajpcell.00139.2014.—The calcium-sensing receptor (CaSR) has played an important role as a target in the treatment of a variety of disease states over the past 20 plus years. In this review, we give an overview of the receptor at the cellular level and then provide details as to how this receptor has been targeted to modulate cellular ion transport mechanisms. As a member of the G proteincoupled receptor (GPCR) family, it has a high degree of homology with a variety of other members in this class, which could explain why this receptor has been identified in so many different tissues throughout the body. This diversity of locations sets it apart from other members of the family and may explain how the receptor interacts with so many different organ systems in the body to modulate the physiology and pathophysiology. The receptor is unique in that it has two large exofacial lobes that sit in the extracellular environment and sense changes in a wide variety of environmental cues including salinity, pH, amino acid concentration, and polyamines to name just a few. It is for this reason that there has been a great deal of research associated with normal receptor physiology over the past 20 years. With the ongoing research, in more recent years a focus on the pathophysiology has emerged and the effects of receptor mutations on cellular and organ physiology have been identified. We hope that this review will enhance and update the knowledge about the importance of this receptor and stimulate future potential investigations focused around this receptor in cellular, organ, and systemic physiology and pathophysiology. G protein-coupled receptors; calcimimetics; kidney; gastrointestinal tract; divalent ions

since the initial review on calcium-sensing receptor (CaSR) was published (17). The initial review focused on the preliminary characterization and description of the receptor and how it related to the parathyroid and whole body calcium handling and homeostasis (17). Since that time, not only has there been a great deal of new information concerning CaSR and parathyroid disease come to light, but in addition the receptor has now been identified in many other tissues and organs in the body where it has been shown to have a diverse role in cellular and organismic pathophysiology (1, 2, 24, 28). With the further identification and functional characterization of the receptor in multiple organ systems and at the cellular level (Fig. 1), we felt it was now time to provide an updated overview of the calcium-sensing receptor and its diverse functions throughout the body. Our review will give a brief historical review of the identification of the receptor in the parathyroid and its role as an important target for parathyroid disease. We include a discussion of the initial cloning studies and the ability of this receptor

IT IS NOW 20 YEARS

* T. I. Alfadda and A. M. A. Saleh contributed equally to this work. Address for reprint requests and other correspondence: J. P. Geibel, BML 238 Dept. of Surgery, 310 Cedar St., New Haven, CT 06520 (e-mail: john. [email protected]; [email protected]). http://www.ajpcell.org

to “sense” divalent and trivalent ions in the extracellular environment. We follow this with a molecular profile of the receptor based on the many years of research into the structure and function of the receptor. In terms of the physiology of the receptor, we begin with an overview of the receptor and how modulation of the receptor can play a role in both calcium homeostasis and fluid regulation via the kidney. We then provide an overview of the receptor in the gut. We present the recent findings on the receptor related to gastric function and the recent findings on intestinal fluid and salt transport and its modulation via the receptor. In the next section, we discuss the role of the receptor in skin, and finally we give a brief overview of receptor mutations that were identified with a human phenotype, and what outcomes these activating or inactivating mutations have on the pathophysiology of the individual. IDENTIFICATION OF A NOVEL CALCIUM SENSOR

Extracellular fluid (ECF) calcium concentration is maintained within a narrow range in normal individuals since any change in calcium entry into the ECF is rapidly matched by an identical change in urinary calcium excretion (95). Available evidence indicates that an adequate secretion of parathyroid hormone (PTH) is required for the minute-to-minute control of ECF calcium concentration (66). Parathyroid cells must appro-

0363-6143/14 Copyright © 2014 the American Physiological Society

C221

Review C222

CaSR REVIEW

PARATHYROID GLANDS

SKIN

Modulates PTH secretion

Promotes keratinocyte differentiation

KIDNEYS

STOMACH Modulates acid secretion

Modifies calcium reabsorption Modulates fluid reabsorption and acid secretion

BONE

COLON

Regulates the recruitment and differentiation of osteoblasts and osteoclasts

Controls toxin-mediated fluid secretion

Fig. 1. Summary of some of the organ systems in which the CaSR was identified and the identified functions it carries in these locations.

priately adapt their PTH secretion to the prevailing ECF calcium concentration. Therefore, parathyroid gland cells must have the capacity to “sense” small changes in ECF calcium concentration. A very steep inverse sigmoidal relationship between PTH secretion and ECF-free calcium has been described both in vivo (10, 94, 115) and in vitro (15, 115) in humans. In addition, the response curves to agonists in the parathyroid gland cells show a Hill coefficient of about 3, suggesting a positive cooperativity that could account for the narrow range over which ECF calcium actually controls PTH secretion (13). Upon changes in ECF calcium concentration, several intracellular signaling pathways are affected, including the cAMPprotein kinase A, phospholipase C-protein kinase C, and inositol phosphate pathways (12). This and other evidence has suggested that ECF calcium can modulate the parathyroid cell function by a receptor-like mechanism, coupled to one or more G protein-dependent pathways (26, 44). Cloning/Genetics In 1993, Brown et al. (14) isolated a 5.3 kb clone, named BoPCaR (for bovine parathyroid calcium receptor), by expression cloning in Xenopus laevis oocytes. BoPCaR had pharmacological properties very close to those of the calcium-sensing protein expressed in bovine parathyroid cells (14, 27). Partic-

ularly, it was confirmed that several other di- (e.g., Mg2⫹, Cd2⫹), tri- (e.g., Gd3⫹, La3⫹) or polyvalent (e.g., neomycin) cations can mimic the effect of free calcium on the parathyroid gland cells. Subsequently, the cloning of full-length CaSRs from various mammalian species, including human, rat and rabbit, was performed (1, 112). Primitive Receptor Found In a Variety of Animals and Fish The presence of CaSR was confirmed in all vertebrate classes. The strong structural conservation of CaSR throughout evolution is suggested by the high similarity in sequences across all the vertebral classes (58). Birds also express CaSR, as demonstrated by the cloning of a full-length calcium receptor from the chicken parathyroid (37). More unexpected, perhaps, is the expression of a gene homologous to CaSR in animals that do not have parathyroid glands, such as teleost and elasmobranch fish (65, 87). The dogfish shark CaSR is expressed in several segments of the shark kidney tubule and, importantly, in many osmoregulatory segments, including rectal gland, intestine, stomach, olfactory epithelia, and gill chloride cells. Among the major clades, both extracellular (ECD) and intracellular (ICD) domains display some differences. In the ECD, an insertion sequence is present in tetrapods, lobe-finned and elasmobranch (cartilaginous) fish, but absent in bony fish,

AJP-Cell Physiol • doi:10.1152/ajpcell.00139.2014 • www.ajpcell.org

Review CaSR REVIEW

urochordates, and cephalochordates. The length of the ICD differs markedly among the various clades. Only the cephalochordates and the tetrapods CaSR contains insertion sequences (58). By contrast, the sequence of the putative calcium-binding sites within the ECD among the vertebrates remains remarkably conserved (63). The importance of CaSR in bone development from an ontogenic standpoint was recently demonstrated (22). The role that CaSR might have played in the phylogenic development of the skeleton remains an open question for further study. The expression of CaSR is detectable in various organs (including kidney, intestine, olfactory organ, gills) of fish. Available evidence suggests that CaSR acts as a salinity sensor in fish, to inform internal organs of changes in the salinity in surrounding water (87). MOLECULAR PROFILE OF THE RECEPTOR

A major barrier to advancing our understanding of the role of calcium in regulating CaSR was the lack of adequate information about their calcium-binding locations, which was greatly hindered by the lack of a solved three-dimensional structure and the rapid off rates due to low calcium-binding affinities. Previously, Huang et al. (64) managed to identify three potential calcium-binding sites in a modeled CaSR structure using computational algorithms based on the geometric description and surface electrostatic potentials (64). They concluded that the calcium-binding site was on the ECD of the CaSR (64). The accurate location of the calcium-binding site on the receptor gives researchers a specific location to target when activation of the CaSR is required. CaSR, and the two type B receptors to ␥-aminobutyric acid and GPRC6A, belongs to the family C of GPCRs. All these receptors have a large ECD including a “Venus flytrap” (VFT) sequence that contains the dimerization sites and the orthosteric sites for binding of endogenous agonists (Fig. 2). The ECD of the human CaSR contains 612 amino acid residues. The ECD is linked to the transmembrane domains (TMD) by a cysteine-rich domain (CRD) that contains 9 out of the 17 cysteine residues characteristic of the ECD of the receptor. These nine cysteine resi-

C223

dues form four disulfide bridges within the CRD and one additional bridge with the VFT sequence. They are strictly required for the proper function of the receptor, indicating that the tertiary and quaternary structure is important for function. During its intracellular biosynthesis, the CaSR is dimerized in the endoplasmic reticulum through two disulfide bonds, between the cysteine 129 and 131 of each monomer (41). However, non-covalently bound dimers might still be expressed at the cell surface when the disulfide bonds are lacking (16). The receptor’s ECD contains nine potential sites of N-linked glycosylation, which is performed in the Golgi apparatus and is required for the normal expression of the protein at the cell surface (14, 40). The fully glycosylated monomer has a molecular mass of 150 –160 kDa, indicating a carbohydrate content of 35– 40 kDa/monomer. While it is important for the normal membrane expression of the receptor, N-linked glycosylation is probably not critical for its biological activity (16). The CaSR ECD is formed by two lobes (LB1 and LB2) separated by a cavity delineating the ligand-binding site representing the VFT (122). Calcium binds in the cleft between the two lobes of each VFT causing the lobes to close on one another and the VFT to rotate, leading to receptor activation (57) (Fig. 2). Recent studies have shown that the VFT contains five calcium-binding sites (63, 64, 122). These sites are located on the protein surface and contain clusters of neutral or negatively charged amino acids. Site 1, the main site, is situated in the cleft between the lobes of the VFT (63, 64, 122). CASR AND THE KIDNEY

Location of the Receptor Throughout the Kidney Following its initial cloning from bovine parathyroid glands (14), CaSR was cloned from the rat and human kidney (1, 112). The human kidney receptor protein was found to have more than 93% homology to bovine parathyroid and rat kidney receptor (1). Given the diversity in the functions of renal tubular cells from the various nephron segments, knowing the sites of expression of the receptor in the kidney tubule is

Fig. 2. A schematic depicting the molecular structure of the receptor. In the inactive state shown to the left, the “Venus flytrap” is interacting with calcium ions, which leads to the activation of the receptor. Activation results in conformational changes, resulting in downstream signal transduction. AJP-Cell Physiol • doi:10.1152/ajpcell.00139.2014 • www.ajpcell.org

Review C224

CaSR REVIEW

mandatory to anticipate which functions the CaSR could control. Employing immunohistochemical studies using antibodies raised against different parts of the receptor, all authors agree that intense staining was noted on the basolateral membranes of cells of the medullary and cortical thick ascending limb (TAL) (23, 77, 110, 126). Localization of the receptor to other parts of the nephron using this technique is controversial. In addition to TAL, Riccardi et al. (110) showed that the CaSR protein is expressed in the proximal convoluted tubule (PCT), proximal straight tubule (PST), distal convoluted tubule (DCT) and cortical collecting ducts (CCD) of the rat kidney. CaSR is mainly located at the apical brush-border membrane in the proximal tubule (110) and is expressed on the apical membrane of inner medullary collecting duct (IMCD) cells (23, 118). However, other studies showed no detectable expression of the protein in the PCT, DCT, or collecting duct (77, 126). Other studies utilizing reverse transcription-polymerase chain reaction (RT-PCR) have confirmed the presence of abundant transcription product in TAL in addition to significant product in DCT and CCD of rat kidney (111, 140). The initial study by Riccardi et al. (111) also reported transcription product in glomeruli, PCT, PST, and IMCD. The expression in the thin limbs and connecting tubule was not assessed in that study. Role of the Receptor in the Kidney All authors agree that CaSR is abundantly expressed in the medullary and cortical parts of the TAL of Henle’s loop (77, 110, 126). These tubular segments actively reabsorb sodium chloride. From an electrophysiological standpoint, the overall consequence of sodium chloride reabsorption in the TAL is a lumen-positive transepithelial difference that is the driving force for the passive absorption of the divalent cations Ca2⫹ and Mg2⫹ along the paracellular pathway (9, 121). Previous experiments conducted in dogs, rats, and mice demonstrated that calcium and magnesium absorption in the

TAL decrease when either peritubular calcium or magnesium concentration is increased (36, 102–105, 139). Electrophysiological studies using patch-clamp techniques demonstrated that calcium and neomycin, two nonspecific agonists of the CaSR, reduce the activity of an apical K⫹ channel via the generation of arachidonic acid metabolites by the cytochrome P-450 pathway (136, 137). Some investigators reported that calcium and neomycin decrease both the transepithelial potential difference and the chloride transport in the microperfused rat cortical TAL (34). Other studies showed alteration in calcium reabsorption without any change in transepithelial potential or NaCl transport (77, 86). In mice with a kidney-specific disruption of CaSR, claudin 14, a putative negative regulator of the paracellular pathway permeability in the TAL (53), was found to be downregulated, whereas claudin 16, a positive regulator, was upregulated (126). ECF calcium is able to alter transepithelial transport and/or transporters expression or affinity in other tubular segments. For example, ECF calcium inhibits Na⫹-K⫹-ATPase activity in the proximal tubule, which may participate in the natriuretic effect of hypercalcemia (73). In the mouse S3 segment of the proximal tubule, luminal gadolinium, a trivalent cationic agonist of CaSR, inhibits the PTH-evoked, but not the dopamineevoked, decrease in phosphate absorption (3). Maiti and Beckman reported that both ECF calcium and gadolinium are able to increase the expression of the vitamin D receptor in a proximal tubule-derived cell line (80), an effect mediated by the p38␣ MAP kinase pathway (81). Riccardi et al. (113) reported that both a chronic high-phosphate diet and an acute injection of PTH reduce the expression of both Npt2a and CaSR in the rat brush border. Recently, Capasso et al. (20) showed that both luminal calcium and the calcimimetic NPS R-568 (Table 1) increase fluid absorption and acid secretion in the PCT of both the mouse and the rat, an effect not observed in CaSR (and Gcm2) knockout mice. The DCT and the connecting tubules are short tubular segments responsible for the fine-tuning of magnesium and

Table 1. Summary of compounds targeting the CaSR Calcimimetics: stimulate CaSR Type I: direct agonists

Type II: positive allosteric modulators

Compound

Indication

Mechanism

Ca2⫹, other divalent and trivalent cations, spermine, some aminoglycosides, some amino acids, and peptides Cinacalcet

Physiological or investigational

Binds to physiological ligand site on ECD

R-467 R-568

Calcilytics: negative allosteric modulators

NPS 2143; Ronacaleret; Calhex 231

• Parathyroid carcinoma Binds to TMD, • Secondary hyperparathyroidleading to ism in patients with CKD conformational on hemodialysis change and more • Primary hyperparathyroidsensitivity to ism in patients unable to physiological ligand undergo surgery Investigational Binds to TMD, leading to conformational change and more sensitivity to physiological ligand Investigational as anabolic Binds to TMD to agents in cases of agedecrease sensitivity related osteoporosis to physiological ligand

Comments

See Ref. 89; Reported to be effective in a patient with NSHPT (46) Different mutations of CaSR altered affinity to Cinacalcet (71) See Ref. 90

See Refs. 4, 88, 97

CaSR, calcium-sensing receptor; ECD, extracellular domain; TMD, transmembrane domain; NSHPT, neonatal severe primary hyperparathyroidism. AJP-Cell Physiol • doi:10.1152/ajpcell.00139.2014 • www.ajpcell.org

Review CaSR REVIEW

calcium reabsorption. Both of these cations are actively transported along a transcellular route. Magnesium enters the cell through the apical Mg channel transient receptor potential melastatin member 6 (TRPM6) while calcium enters through the calcium channel transient receptor potential vanilloid member 5 (TRPV5). Both transports are activated by PTH and the active metabolite of vitamin D, 1,25(OH)2vitamin D (61). Some groups reported the expression of CaSR in the rat and human DCT and connecting tubule cells (110, 127). In HEK293 cells, overexpressing TRPV5 and CaSR, neomycin or phenylalanine increased the activity of TRPV5 (127), suggesting that ECF calcium might stimulate its own reabsorption in this segment. Patients with frank hypercalcemia may have polyuria due to a decrease in the urine concentrating ability (51, 124). Using in vitro microperfused rat IMCD, Sands et al. (118) showed that the nonspecific activators of CaSR, calcium and neomycin, decrease the vasopressin-elicited, transepithelial water flux by ⬃30 – 40%. In addition, hypercalcemia and/or hypercalciuria, resulting from a treatment of rats by dihydrotachysterol, reduces the abundance of aquaporin 2 in the same segment (117). This may explain why Trpv5-null mice, which are severely hypercalciuric, also have a significant polyuria (62). Trpv5-null mice have a low urinary pH, suggesting that a high urinary calcium concentration is able to promote distal urinary acidification. Actually, luminal calcium concentration increases hydrogen ion secretion by type A intercalated cells, an effect involving the H⫹-ATPase (109). All these data indicate that ECF calcium concentration is able to significantly alter several functions of the distal nephron. End-Stage Renal Disease and CaSR Chronic kidney disease (CKD) has many consequences, among which are disorders of mineral metabolism and secondary hyperparathyroidism (115). One of the mechanisms of secondary hyperparathyroidism is the decrease in parathyroid gland cells expression of CaSR; several excellent reviews have been published on this topic in recent years (33, 70). Surprisingly, much less attention has been paid to the changes in CaSR expression/function in the kidney. One study performed in experimentally induced chronic renal insufficiency in the rat showed that the abundance of both CaSR transcripts and protein is reduced (84). Whether this can play a role in the early decrease in urinary calcium excretion that accompanies CKD remains to be studied. Pharmacological Target for Renal Stone Disease Hypercalciuria is one of the main risk factors for calcium nephrolithiasis, a disease affecting ⬃10% of the Western population. The so-called idiopathic hypercalciuria is documented in about 40% of patients with calcium nephrolithiasis (74). The pathophysiology of idiopathic hypercalciuria is complex, frequently associating increased intestinal calcium absorption with decreased renal tubular calcium reabsorption. Since CaSR is expressed in the kidney where it directly controls renal tubular calcium absorption, the possible involvement of the receptor in the pathophysiology of idiopathic hypercalciuria has been investigated. No deleterious point mutations in the CaSR gene have been observed in a panel of French patients with familial calcium nephrolithiasis (72).

C225

Similarly, no linkage was observed between the CaSR locus and hypercalciuria or the risk of renal stones in a large group of Canadian brothers (98). However, other groups have reported that the risk of renal stones is greater in patients bearing the ACG haplotype at positions 986, 990, and 1011 (133). The association between the Arg990Gly single nucleotide polymorphism (SNP) and hypercalciuria and/or nephrolithiasis has been also observed in Canadian and Iranian patients (55, 120), but not in Caucasian female twins living in UK (56). In one study, heterologous expression experiments of wild-type or Arg990Gly mutant CaSR in HEK-293 cells showed a lower EC50 in cells expressing the mutant CaSR (134); in discrepancy with the latter, another study did not show any difference in EC50 of HEK-293 transfected with wild-type or Arg990Gly mutant CaSR (56). Whether common SNPs in the CaSR gene do affect urinary calcium excretion and/or the risk of calcium stone disease remains an open question. Drugs targeting the CaSR are expected to affect urinary calcium excretion and the risk of stone recurrence. Actually, a calcimimetic (Table 1) may increase urinary calcium excretion by both direct and indirect effects. The indirect effect is mediated by the decrease in PTH release amplified by the direct activation of the renal tubular CaSR in the TAL. Accordingly, a single dose of Cinacalcet (Table 1) acutely increases urinary calcium excretion in renal transplant recipients with secondary hyperparathyroidism (32). CASR AND THE GUT

Stomach Location of the receptor in the stomach. In the stomach, the receptor was first identified in the amphibian Necturus maculosus using RT-PCR. It was localized to the basolateral membrane of surface epithelial cells in the antrum and to a lesser extent the acid-secreting glands in the fundus by immunohistochemical studies (31). Later, the transcript and immunohistochemical identification of the receptor was carried out on cultured human antral gastrin cells (108). The presence of the receptor in gastric epithelial and glandular cells was confirmed in a model of isolated gastric glands of the rat (28). The same study demonstrated the receptor’s presence in cells of submucosal and myenteric plexuses (28). The receptor was again identified in mainly the basolateral and to a lesser extent the apical membranes of gastric mucous epithelial cells in humans (116). Role of the receptor in the stomach. Functional studies of the CaSR in the Necturus stomach showed hyperpolarization of basal membranes of gastric glands when subjected to high levels of calcium or to the calcimimetic NPS-467 (31) (Table 1). In rats, activation of the CaSR by gadolinium (Gd3⫹) resulted in increases in intracellular calcium concentrations (28). Moreover, stimulation of the receptor on the basolateral membrane of parietal cells resulted in increased acid secretion through apical H⫹-K⫹-ATPase while inhibition of the receptor resulted in decreased acid secretion even when glands were stimulated with classic secretagogues (50). Similar results were obtained when gastric glands were subjected to different concentrations of L-amino acids, known positive allosteric modifiers of CaSR, where increasing concentrations of these amino acids resulted in increases in acid secretion independent of secretagogue presence (19). In freshly isolated human gastric

AJP-Cell Physiol • doi:10.1152/ajpcell.00139.2014 • www.ajpcell.org

Review C226

CaSR REVIEW

glands, activation of the receptor resulted in similar effects to that seen in rats, that is, increased acid secretion (38). Inhibition of the receptor similarly resulted in decreased acid secretion despite the presence of histamine (38). In human antral gastrin cells in culture, stimulation of the CaSR by increasing ECF calcium levels and by the calcimimetic drug spermine (Table 1) resulted in increases in gastrin secretion in a dose-dependent manner (108). In addition, increasing ECF calcium in cell cultures of gastric glandular cells induced a proliferative response (116). In 17 healthy human subjects, the administration of Cinacalcet, a CaSR allosteric agonist (Table 1), over an 11-day course caused increased gastrin secretion and basal acid secretion (21). Effect of hypercalcemia on the CaSR in the stomach may explain the association of primary hyperparathyroidism with peptic ulcer disease (47). Pharmacological target for acid secretion. Acid rebound phenomenon is a well-observed finding with the use of calcium-containing antacids (49, 108). The activation of CaSR and in turn increased acid secretion may explain this observation. In this context, modulation of gastric acid secretion by targeting CaSR on acid-secreting cells as well as neighboring G and enterochromaffin-like (ECL) cells is theoretically feasible (6, 38). In most cases the acid secretion should be decreased and this entails the inhibition of CaSR by calcilytics (38, 50). Many calcilytic compounds (Table 1) have been used in research models; some are available orally but none are approved for use in humans (68). The systemic effects of CaSR-modulating drugs should also be taken into account (21). Small and Large Intestine Location of the receptor in the small and large intestine. The receptor was identified along the small and large intestine (24). It was clearly localized on the basal membranes of intestinal crypt and villi epithelial cells (24). On the apical surface of epithelial cells, the receptor was only identified in the small intestinal villi (24). Similarly, a previous study had shown products of transcription across intestinal segments with major transcript in the duodenum, jejunum, and ileum (45). The presence of the receptor in the large intestine was confirmed by RT-PCR and Northern blotting (24, 29, 45). Further immunohistochemical studies localized the receptor to both basal and apical membranes of colonic crypt cells (24, 29). Strong immunostaining was also observed in the serosa, submucosa, and nerve fibers (24). Role of the receptor in the small and large intestine. Previous studies postulated that, in the colon, surface cells were responsible for absorption and the crypts were responsible for secretion and that these two systems worked independently. Recent studies have shown that both surface and crypt cells are capable of both secretion and absorption (75, 85, 106). These studies conclude that fluid absorption and secretion are the two main roles of the colon (75, 85, 106). The CaSR has important roles in the colon such as fluid transport and colon motility. The effect on colon motility is mainly attributed to the role CaSR plays in the smooth muscle nerve plexuses (49). The effect of the receptor on fluid transport provides a new target both for constipation and more importantly for diarrhea (49). Early studies performed by Favus, Kathpalia, and Coe (42, 43) observed the presence of a functional calcium-sensing

mechanism in the rat intestine by demonstrating that the intestines can secrete or absorb calcium in response to changes in ECF calcium concentration and modulation by vitamin D (42, 43). Recent studies have proven through immunolocalization and receptor function studies that this calcium-sensing mechanism is actually the CaSR (64). Recently, Mace, Schindler, and Patel showed that CaSR could detect amino acids in the intestine to modify gut peptide secretion (79). These findings suggest that CaSR acts as an important regulator of Kand L-cell (intestinal enteroendocrine cells) activity in response to nutrient and nonnutrient stimuli (79). These recent studies establish new functions for CaSR as a regulator of gut peptide secretion that senses nutrients and provides signaling pathways for the release of glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide 1 (GLP-1), and peptide YY (PYY), which are anti-diabetic gut peptides (79). Pharmacological target for diarrhea and inflammatory bowel disease. Fluid and electrolyte loss in secretory diarrhea is cyclic nucleotide dependent. Exposure to cholera toxin (CTX) results in upregulation of cAMP, which leads to diarrhea. On the other hand, Escherichia coli heat stable toxin (STa) upregulates cGMP. This fluid and electrolyte loss occurs not only through an increase in the secretory process via chloride loss but also in the reduced absorptive capacity of the intestines via the basolateral sodium hydrogen exchanger’s subtype 2 and 3 (52, 78). Recent studies have shown that stimulation of the CaSR in the presence of cyclic nucleotides (i.e., CTX, STa, and guanylin) will suppress secretion and increase absorption (48). This occurs mainly through cyclic nucleotide degradation via phosphodiesterases (48). The reversal of CTX and STa endotoxin-induced fluid secretion by a small-molecule CaSR agonist (R-568) suggests that these compounds may provide a unique therapy for secretory diarrhea (48). Inflammatory bowel disease (IBD; secretory type; IBD-S) is characterized by a large amount of fluid loss due to diarrhea and has been a topic of importance for researchers. The significance of finding a relation between IBD-S and CaSR has been an area of interest for years. Researchers have tried to link the association between patients who have familial hypocalciuric hypercalcemia due to a CaSR mutation and Crohn’s disease, yet the link still remains unclear (60). SKIN/KERATINOCYTES

Skin although not directly involved in mineral homeostasis also expresses the CaSR (7). ECF calcium is a critical factor for differentiation in epidermal keratinocytes: increasing ECF calcium concentration evokes accumulation of inositol phosphates and elevation in free cytosolic calcium concentration (7). It also stimulates the binding of E-cadherins from adjacent cells as well as the interactions with catenins to form the core structure of adherens junctions. Keratinocyte differentiation, as well as the interaction between E-cadherin and catenins, is blocked when CaSR expression is inhibited (128). The differentiated keratinocytes display a lower responsiveness to ECF calcium than nondifferentiated keratinocytes (93). The reason might be that keratinocytes do express a splice variant of CaSR, lacking exon 5, which exerts a dominant negative effect of the full-length CaSR, and that the expression of this truncated splice variant increases as differentiation progresses (93).

AJP-Cell Physiol • doi:10.1152/ajpcell.00139.2014 • www.ajpcell.org

Review CaSR REVIEW

Pharmacological Targeting of Skin CaSR Since CaSR was identified, its effect on keratinocyte growth and differentiation was established, but no disease states of the skin were associated with mutations of the receptor (2, 129). Deletions in the CaSR resulted in abnormal ultrastructure and differentiation of the epidermis while overexpression led to accelerated hair follicle formation and increased differentiation markers (130). How this observation can be exploited in modulation of skin conditions, e.g., wound healing, is currently unknown but is of potential clinical use and worthy of further investigation. RECEPTOR MUTATIONS

Activating Mutations Autosomal dominant hypocalcemia. Since CaSR was first cloned, more than 90 gain-of-function mutations were reported (99). Most of these mutations have been described in patients with autosomal dominant hypocalcemia (99). The disease state is characterized by low calcium and below normal or inappropriately low PTH levels (39). Some patients also suffer from hypercalciuria with nephrolithiasis and nephrocalcinosis (107). In addition, intracranial calcifications and seizures have been described (18, 107, 123). The pathophysiology involves a lower set point for calcium detection by CaSR in the parathyroid glands, resulting in higher sensitivity of the receptor to circulating calcium and in turn a lower PTH production (11, 125). In most cases the responsiveness to changes in ECF calcium levels is still maintained albeit to bring calcium to the new “normal” lower level (25). Most activating mutations are found on the ECD and seem to increase the receptor’s affinity for calcium (25). Sporadic idiopathic hypoparathyroidism. Several de novo activating mutations have been reported in patients with sporadic hypoparathyroidism (5, 35, 76). Further genetic testing of the parents in these cases failed to demonstrate the mutations (5, 35, 76). Nevertheless, not all cases of sporadic idiopathic hypoparathyroidism were associated with activating mutations in the CaSR, suggesting that other mechanisms are involved (119). Bartter syndrome type V. Bartter syndrome is a condition of deficient reabsorption of sodium and chloride in the kidney, leading to hypokalemic metabolic alkalosis, hyperreninemia, and hyperaldosteronemia (138). Type V Bartter syndrome is caused by some of the activating mutations of the CaSR and is typically associated with autosomal dominant hypocalcemia (132, 138). The mutation is thought to inhibit sodium reabsorption in the TAL, leading to the clinical manifestations (132). Activating autoantibodies. Activating autoantibodies against the CaSR have also been reported (54, 67). The clinical spectrum included mainly patients with sporadic hypoparathyroidism and includes patients with autoimmune polyendocrine syndrome type 1 (54, 67). Inactivating Mutations Familial hypocalciuric hypercalcemia (familial benign hypercalcemia). Familial hypocalciuric hypercalcemia (FHH) is an autosomal dominant disease, with a high degree of penetrance. In most cases, FHH is the consequence of a heterozygous loss-of-function mutation of the CaSR (100),

C227

although recently, loss-of-function mutations of two other proteins, adaptor protein 2 ␴-subunit (AP2S1) (92) and G␣11 (91), have been involved in some kindred. A lifelong, most often asymptomatic, hypercalcemia together with a normal urinary calcium excretion are the hallmarks of the disease (82, 83). The serum PTH concentration is typically normal, inappropriate to the prevailing hypercalcemia, though it may be high in 20% of the afflicted patients, making it difficult to confirm the diagnosis of primary hyperparathyroidism (30). A few patients suffer from pancreatitis or chondrocalcinosis. Neonatal severe primary hyperparathyroidism. Neonatal severe primary hyperparathyroidism (NSHPT) is a rare disease characterized by the early onset of extreme hypercalcemia, severe hyperparathyroidism related to parathyroid hyperplasia, skeletal demineralization, respiratory distress, and hypotonia (8, 114). Physicians have often observed the familial coincidence between FHH and NSHPT. In 1994, Pollak et al. (101) reported that NSHPT is the homozygous form of FHH, the afflicted patients bearing either two identical mutations (homozygous, most often issued from consanguineous parents) or two distinct mutations (compound heterozygous). However, Pearce et al. (96) reported that NSHPT can be caused by heterozygous mutations in the CaSR gene, seemingly because some mutant proteins might exert a negative dominant effect. Signs and symptoms of patients with NSHPT are reminiscent of the phenotype of Casr-null mice (59), which have a marked elevation in blood calcium and PTH concentrations, parathyroid gland hyperplasia, bone abnormalities, failure to thrive, and premature death. In mice, the bone phenotype and lethality can be efficiently rescued by the genetic deletion of parathyroid glands, as observed in the double homozygous Casr- and Gcm2- or Pth-deficient mice (69, 131). Most of the skeletal effects of CaSR disruption can therefore be ascribed to the attendant severe hyperparathyroidism. By contrast, hypocalciuria remains in the double homozygous mice, further indicating that the renal effect of CaSR is independent of the level of PTH. As it is the case in the mouse, NSHPT can be fatal in humans if not aggressively treated. Recent observations suggest that bisphosphonates help to improve the life-threatening hypercalcemia and severe bone demineralization (135), while in the past, total parathyroidectomy was sometimes needed in the most severe cases. A recent report demonstrated good response to Cinacalcet in a neonate with NSHPT (46). SUMMARY

We have tried in this review to give an overview of the receptor and its molecular profile along with the identification of the various organ systems where it has been shown, to date, to play an important role. Over the past 20 plus years since the identification of the receptor, the roles for receptor function and interactions with cellular and organ homeostasis have expanded greatly from the initial observations in the parathyroid. We hope that this review serves two functions: provide the reader with some insights into the receptor at all levels and, secondarily, induce new interest in the receptor and its potential roles in yet to be studied cellular and organ systems. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s).

AJP-Cell Physiol • doi:10.1152/ajpcell.00139.2014 • www.ajpcell.org

Review C228

CaSR REVIEW

AUTHOR CONTRIBUTIONS T.I.A. prepared figures; T.I.A., A.M.A.S., and P.H. drafted manuscript; T.I.A., A.M.A.S., P.H., and J.P.G. edited and revised manuscript; T.I.A., A.M.A.S., P.H., and J.P.G. approved final version of manuscript.

20. 21.

REFERENCES 1. Aida K, Koishi S, Tawata M, Onaya T. Molecular cloning of a putative Ca(2⫹)-sensing receptor cDNA from human kidney. Biochem Biophys Res Commun 214: 524 –529, 1995. 2. Arabzadeh A, Troy TC, Turksen K. Insights into the role of the calcium sensing receptor in epidermal differentiation in vivo. Mol Biotechnol 43: 264 –272, 2009. 3. Ba J, Brown D, Friedman PA. Calcium-sensing receptor regulation of PTH-inhibitable proximal tubule phosphate transport. Am J Physiol Renal Physiol 285: F1233–F1243, 2003. 4. Balan G, Bauman J, Bhattacharya S, Castrodad M, Healy DR, Herr M, Humphries P, Jennings S, Kalgutkar AS, Kapinos B, Khot V, Lazarra K, Li M, Li Y, Neagu C, Oliver R, Piotrowski DW, Price D, Qi H, Simmons HA, Southers J, Wei L, Zhang Y, Paralkar VM. The discovery of novel calcium sensing receptor negative allosteric modulators. Bioorg Med Chem Lett 19: 3328 –3332, 2009. 5. Baron J, Winer KK, Yanovski JA, Cunningham AW, Laue L, Zimmerman D, Cutler GB Jr. Mutations in the Ca(2⫹)-sensing receptor gene cause autosomal dominant and sporadic hypoparathyroidism. Hum Mol Genet 5: 601–606, 1996. 6. Bevilacqua M, Dominguez LJ, Righini V, Valdes V, Toscano R, Sangaletti O, Vago T, Baldi G, Barrella M, Bianchi-Porro G. Increased gastrin and calcitonin secretion after oral calcium or peptones administration in patients with hypercalciuria: a clue to an alteration in calcium-sensing receptor activity. J Clin Endocrinol Metab 90: 1489 – 1494, 2005. 7. Bikle DD, Ratnam A, Mauro T, Harris J, Pillai S. Changes in calcium responsiveness and handling during keratinocyte differentiation. Potential role of the calcium receptor. J Clin Invest 97: 1085–1093, 1996. 8. Blair JW, Carachi R. Neonatal primary hyperparathyroidism–a case report and review of the literature. Eur J Pediatr Surg 1: 110 –114, 1991. 9. Bourdeau JE, Burg MB, Knepper MA. Voltage dependence of calcium transport in the thick ascending limb of Henle’s loop. Am J Physiol Renal Fluid Electrolyte Physiol 236: F357–F364, 1979. 10. Brent GA, LeBoff MS, Seely EW, Conlin PR, Brown EM. Relationship between the concentration and rate of change of calcium and serum intact parathyroid hormone levels in normal humans. J Clin Endocrinol Metab 67: 944 –950, 1988. 11. Brown EM. The calcium-sensing receptor: physiology, pathophysiology and CaR-based therapeutics. Subcell Biochem 45: 139 –167, 2007. 12. Brown EM. Extracellular Ca2⫹ sensing, regulation of parathyroid cell function, and role of Ca2⫹ and other ions as extracellular (first) messengers. Physiol Rev 71: 371–411, 1991. 13. Brown EM, Chen CJ, LeBoff MS, Kifor O, El-Hajj G. Mechanisms underlying the inverse control of parathyroid hormone secretion by calcium. In: Secretion and Its Control, edited by Oxford G, Armstrong CM. New York: Rockefeller Univ. Press, 1989, p. 252–268. 14. Brown EM, Gamba G, Riccardi D, Lombardi M, Butters R, Kifor O, Sun A, Hediger MA, Lytton J, Hebert SC. Cloning and characterization of an extracellular Ca(2⫹)-sensing receptor from bovine parathyroid. Nature 366: 575–580, 1993. 15. Brown EM, Gardner DG, Brennan MF, Marx SJ, Spiegel AM, Attie MF, Downs RW Jr, Doppman JL, and Aurbach CD. Calciumregulated parathyroid hormone release in primary hyperparathyroidism: studies in vitro with dispersed parathyroid cells. Am J Med 66: 923–931, 1979. 16. Brown EM, MacLeod RJ. Extracellular calcium sensing and extracellular calcium signaling. Physiol Rev 81: 239 –297, 2001. 17. Brown EM, Pollak M, Riccardi D, Hebert SC. Cloning and characterization of an extracellular Ca(2⫹)-sensing receptor from parathyroid and kidney: new insights into the physiology and pathophysiology of calcium metabolism. Nephrol Dial Transplant 9: 1703–1706, 1994. 18. Burren CP, Curley A, Christie P, Rodda CP, Thakker RV. A family with autosomal dominant hypocalcaemia with hypercalciuria (ADHH): mutational analysis, phenotypic variability and treatment challenges. J Pediatr Endocrinol Metab 18: 689 –699, 2005. 19. Busque SM, Kerstetter JE, Geibel JP, Insogna K. L-type amino acids stimulate gastric acid secretion by activation of the calcium-sensing

22. 23.

24.

25. 26.

27. 28.

29.

30.

31.

32.

33. 34. 35.

36.

37.

38.

receptor in parietal cells. Am J Physiol Gastrointest Liver Physiol 289: G664 –G669, 2005. Capasso G, Geibel PJ, Damiano S, Jaeger P, Richards WG, Geibel JP. The calcium sensing receptor modulates fluid reabsorption and acid secretion in the proximal tubule. Kidney Int 84: 277–284, 2013. Ceglia L, Harris SS, Rasmussen HM, Dawson-Hughes B. Activation of the calcium sensing receptor stimulates gastrin and gastric acid secretion in healthy participants. Osteoporos Int 20: 71–78, 2009. Chang W, Tu C, Chen TH, Bikle D, Shoback D. The extracellular calcium-sensing receptor (CaSR) is a critical modulator of skeletal development. Sci Signal 1: ra1, 2008. Chattopadhyay N, Baum M, Bai M, Riccardi D, Hebert SC, Harris HW, Brown EM. Ontogeny of the extracellular calcium-sensing receptor in rat kidney. Am J Physiol Renal Fluid Electrolyte Physiol 271: F736 –F743, 1996. Chattopadhyay N, Cheng I, Rogers K, Riccardi D, Hall A, Diaz R, Hebert SC, Soybel DI, Brown EM. Identification and localization of extracellular Ca(2⫹)-sensing receptor in rat intestine. Am J Physiol Gastrointest Liver Physiol 274: G122–G130, 1998. Chattopadhyay N, Mithal A, Brown EM. The calcium-sensing receptor: a window into the physiology and pathophysiology of mineral ion metabolism. Endocr Rev 17: 289 –307, 1996. Chen CJ, Barnett JV, Congo DA, Brown EM. Divalent cations suppress 3=,5=-adenosine monophosphate accumulation by stimulating a pertussis toxin-sensitive guanine nucleotide-binding protein in cultured bovine parathyroid cells. Endocrinology 124: 233–239, 1989. Chen TH, Pratt SA, Shoback DM. Injection of bovine parathyroid poly(A)⫹ RNA into Xenopus oocytes confers sensitivity to high extracellular calcium. J Bone Miner Res 9: 293–300, 1994. Cheng I, Qureshi I, Chattopadhyay N, Qureshi A, Butters RR, Hall AE, Cima RR, Rogers KV, Hebert SC, Geibel JP, Brown EM, Soybel DI. Expression of an extracellular calcium-sensing receptor in rat stomach. Gastroenterology 116: 118 –126, 1999. Cheng SX, Okuda M, Hall AE, Geibel JP, Hebert SC. Expression of calcium-sensing receptor in rat colonic epithelium: evidence for modulation of fluid secretion. Am J Physiol Gastrointest Liver Physiol 283: G240 –G250, 2002. Christensen SE, Nissen PH, Vestergaard P, Heickendorff L, Brixen K, Mosekilde L. Discriminative power of three indices of renal calcium excretion for the distinction between familial hypocalciuric hypercalcaemia and primary hyperparathyroidism: a follow-up study on methods. Clin Endocrinol (Oxf) 69: 713–720, 2008. Cima RR, Cheng I, Klingensmith ME, Chattopadhyay N, Kifor O, Hebert SC, Brown EM, Soybel DI. Identification and functional assay of an extracellular calcium-sensing receptor in Necturus gastric mucosa. Am J Physiol Gastrointest Liver Physiol 273: G1051–G1060, 1997. Courbebaisse M, Diet C, Timsit MO, Mamzer MF, Thervet E, Noel LH, Legendre C, Friedlander G, Martinez F, Prie D. Effects of cinacalcet in renal transplant patients with hyperparathyroidism. Am J Nephrol 35: 341–348, 2012. Cunningham J, Locatelli F, Rodriguez M. Secondary hyperparathyroidism: pathogenesis, disease progression, and therapeutic options. Clin J Am Soc Nephrol 6: 913–921, 2011. De Jesus Ferreira MC, Bailly C. Extracellular Ca2⫹ decreases chloride reabsorption in rat CTAL by inhibiting cAMP pathway. Am J Physiol Renal Physiol 275: F198 –F203, 1998. De Luca F, Ray K, Mancilla EE, Fan GF, Winer KK, Gore P, Spiegel AM, Baron J. Sporadic hypoparathyroidism caused by de novo gain-offunction mutations of the Ca(2⫹)-sensing receptor. J Clin Endocrinol Metab 82: 2710 –2715, 1997. Desfleurs E, Wittner M, Simeone S, Pajaud S, Moine G, Rajerison R, Di Stefano A, Mandon B, Roinel N, de Rouffignac C. Calcium-sensing receptor: regulation of electrolyte transport in the thick ascending limb of Henle’s loop. Kidney Blood Press Res 21: 401–412, 1998. Diaz R, Hurwitz S, Chattopadhyay N, Pines M, Yang Y, Kifor O, Einat MS, Butters R, Hebert SC, Brown EM. Cloning, expression, and tissue localization of the calcium-sensing receptor in chicken (Gallus domesticus). Am J Physiol Regul Integr Comp Physiol 273: R1008 – R1016, 1997. Dufner MM, Kirchhoff P, Remy C, Hafner P, Muller MK, Cheng SX, Tang LQ, Hebert SC, Geibel JP, Wagner CA. The calciumsensing receptor acts as a modulator of gastric acid secretion in freshly isolated human gastric glands. Am J Physiol Gastrointest Liver Physiol 289: G1084 –G1090, 2005.

AJP-Cell Physiol • doi:10.1152/ajpcell.00139.2014 • www.ajpcell.org

Review CaSR REVIEW 39. Egbuna OI, Brown EM. Hypercalcaemic and hypocalcaemic conditions due to calcium-sensing receptor mutations. Best Pract Res Clin Rheumatol 22: 129 –148, 2008. 40. Fan G, Goldsmith PK, Collins R, Dunn CK, Krapcho KJ, Rogers KV, Spiegel AM. N-linked glycosylation of the human Ca2⫹ receptor is essential for its expression at the cell surface. Endocrinology 138: 1916 –1922, 1997. 41. Fan GF, Ray K, Zhao XM, Goldsmith PK, Spiegel AM. Mutational analysis of the cysteines in the extracellular domain of the human Ca2⫹ receptor: effects on cell surface expression, dimerization and signal transduction. FEBS Lett 436: 353–356, 1998. 42. Favus MJ, Kathpalia SC, Coe FL. Kinetic characteristics of calcium absorption and secretion by rat colon. Am J Physiol Gastrointest Liver Physiol 240: G350 –G354, 1981. 43. Favus MJ, Kathpalia SC, Coe FL, Mond AE. Effects of diet calcium and 1,25-dihydroxyvitamin D3 on colon calcium active transport. Am J Physiol Gastrointest Liver Physiol 238: G75–G78, 1980. 44. Fitzpatrick LA, Brandi ML, Aurbach GD. Calcium-controlled secretion is effected through a guanine nucleotide regulatory protein in parathyroid cells. Endocrinology 119: 2700 –2703, 1986. 45. Gama L, Baxendale-Cox LM, Breitwieser GE. Ca2⫹-sensing receptors in intestinal epithelium. Am J Physiol Cell Physiol 273: C1168 –C1175, 1997. 46. Gannon AW, Monk HM, Levine MA. Cinacalcet monotherapy in neonatal severe hyperparathyroidism: a case study and review. J Clin Endocrinol Metab 99: 7–11, 2014. 47. Gasparoni P, Caroli A, Sardeo G, Maschio S, Lo Giudice C, Fioretti D. [Primary hyperparathyroidism and peptic ulcer]. Minerva Med 80: 1327–1330, 1989. 48. Geibel J, Sritharan K, Geibel R, Geibel P, Persing JS, Seeger A, Roepke TK, Deichstetter M, Prinz C, Cheng SX, Martin D, Hebert SC. Calcium-sensing receptor abrogates secretagogue-induced increases in intestinal net fluid secretion by enhancing cyclic nucleotide destruction. Proc Natl Acad Sci USA 103: 9390 –9397, 2006. 49. Geibel JP, Hebert SC. The functions and roles of the extracellular Ca2⫹-sensing receptor along the gastrointestinal tract. Annu Rev Physiol 71: 205–217, 2009. 50. Geibel JP, Wagner CA, Caroppo R, Qureshi I, Gloeckner J, Manuelidis L, Kirchhoff P, Radebold K. The stomach divalent ion-sensing receptor scar is a modulator of gastric acid secretion. J Biol Chem 276: 39549 –39552, 2001. 51. Gill JR Jr, Bartter FC. On the impairment of renal concentrating ability in prolonged hypercalcemia and hypercalciuria in man. J Clin Invest 40: 716 –722, 1961. 52. Golin-Bisello F, Bradbury N, Ameen N. STa and cGMP stimulate CFTR translocation to the surface of villus enterocytes in rat jejunum and is regulated by protein kinase G. Am J Physiol Cell Physiol 289: C708 –C716, 2005. 53. Gong Y, Renigunta V, Himmerkus N, Zhang J, Renigunta A, Bleich M, Hou J. Claudin-14 regulates renal Ca(⫹)(⫹) transport in response to CaSR signalling via a novel microRNA pathway. EMBO J 31: 1999 – 2012, 2012. 54. Goswami R, Brown EM, Kochupillai N, Gupta N, Rani R, Kifor O, Chattopadhyay N. Prevalence of calcium sensing receptor autoantibodies in patients with sporadic idiopathic hypoparathyroidism. Eur J Endocrinol 150: 9 –18, 2004. 55. Hamilton DC, Grover VK, Smith CA, Cole DE. Heterogeneous disease modeling for Hardy-Weinberg disequilibrium in case-control studies: application to renal stones and calcium-sensing receptor polymorphisms. Ann Hum Genet 73: 176 –183, 2009. 56. Harding B, Curley AJ, Hannan FM, Christie PT, Bowl MR, Turner JJ, Barber M, Gillham-Nasenya I, Hampson G, Spector TD, Thakker RV. Functional characterization of calcium sensing receptor polymorphisms and absence of association with indices of calcium homeostasis and bone mineral density. Clin Endocrinol (Oxf) 65: 598 –605, 2006. 57. Hendy GN, Canaff L, Cole DE. The CASR gene: alternative splicing and transcriptional control, and calcium-sensing receptor (CaSR) protein: structure and ligand binding sites. Best Pract Res Clin Endocrinol Metab 27: 285–301, 2013. 58. Herberger AL, Loretz CA. Vertebrate extracellular calcium-sensing receptor evolution: selection in relation to life history and habitat. Comp Biochem Physiol Part D Genomics Proteomics 8: 86 –94, 2013.

C229

59. Ho C, Conner DA, Pollak MR, Ladd DJ, Kifor O, Warren HB, Brown EM, Seidman JG, Seidman CE. A mouse model for familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Nat Genet 11: 389 –394, 1995. 60. Ho J, Fox D, Innes AM, McLeod R, Butzner D, Johnson N, Trevenen C, Kendrick V, Cole DE. Kabuki syndrome and Crohn disease in a child with familial hypocalciuric hypercalcemia. J Pediatr Endocrinol Metab 23: 975–979, 2010. 61. Hoenderop JG, Bindels RJ. Epithelial Ca2⫹ and Mg2⫹ channels in health and disease. J Am Soc Nephrol 16: 15–26, 2005. 62. Hoenderop JG, van Leeuwen JP, van der Eerden BC, Kersten FF, van der Kemp AW, Merillat AM, Waarsing JH, Rossier BC, Vallon V, Hummler E, Bindels RJ. Renal Ca2⫹ wasting, hyperabsorption, and reduced bone thickness in mice lacking TRPV5. J Clin Invest 112: 1906 –1914, 2003. 63. Huang Y, Zhou Y, Castiblanco A, Yang W, Brown EM, Yang JJ. Multiple Ca(2⫹)-binding sites in the extracellular domain of the Ca(2⫹)sensing receptor corresponding to cooperative Ca(2⫹) response. Biochemistry 48: 388 –398, 2009. 64. Huang Y, Zhou Y, Yang W, Butters R, Lee HW, Li S, Castiblanco A, Brown EM, Yang JJ. Identification and dissection of Ca(2⫹)-binding sites in the extracellular domain of Ca(2⫹)-sensing receptor. J Biol Chem 282: 19000 –19010, 2007. 65. Hubbard PC, Canario AV. Evidence that olfactory sensitivities to calcium and sodium are mediated by different mechanisms in the goldfish Carassius auratus. Neurosci Lett 414: 90 –93, 2007. 66. Jaeger P, Jones W, Kashgarian M, Baron R, Clemens TL, Segre GV, Hayslett JP. Animal model of primary hyperparathyroidism. Am J Physiol Endocrinol Metab 252: E790 –E798, 1987. 67. Kemp EH, Gavalas NG, Krohn KJ, Brown EM, Watson PF, Weetman AP. Activating autoantibodies against the calcium-sensing receptor detected in two patients with autoimmune polyendocrine syndrome type 1. J Clin Endocrinol Metab 94: 4749 –4756, 2009. 68. Kiefer L, Leiris S, Dodd RH. Novel calcium sensing receptor ligands: a patent survey. Expert Opin Ther Pat 21: 681–698, 2011. 69. Kos CH, Karaplis AC, Peng JB, Hediger MA, Goltzman D, Mohammad KS, Guise TA, Pollak MR. The calcium-sensing receptor is required for normal calcium homeostasis independent of parathyroid hormone. J Clin Invest 111: 1021–1028, 2003. 70. Kumar R, Thompson JR. The regulation of parathyroid hormone secretion and synthesis. J Am Soc Nephrol 22: 216 –224, 2011. 71. Leach K, Wen A, Cook AE, Sexton PM, Conigrave AD, Christopoulos A. Impact of clinically relevant mutations on the pharmacoregulation and signaling bias of the calcium-sensing receptor by positive and negative allosteric modulators. Endocrinology 154: 1105–1116, 2013. 72. Lerolle N, Coulet F, Lantz B, Paillard F, Houillier P, Soubrier F, Gattegno B, Jeunemaitre X, Ronco P, Rondeau E. No evidence for point mutations of the calcium-sensing receptor in familial idiopathic hypercalciuria. Nephrol Dial Transplant 16: 2317–2322, 2001. 73. Levi M, Molitoris BA, Burke TJ, Schrier RW, Simon FR. Effects of vitamin D-induced chronic hypercalcemia on rat renal cortical plasma membranes and mitochondria. Am J Physiol Renal Fluid Electrolyte Physiol 252: F267–F275, 1987. 74. Levy FL, Adams-Huet B, Pak CY. Ambulatory evaluation of nephrolithiasis: an update of a 1980 protocol. Am J Med 98: 50 –59, 1995. 75. Lin KI, Chattopadhyay N, Bai M, Alvarez R, Dang CV, Baraban JM, Brown EM, Ratan RR. Elevated extracellular calcium can prevent apoptosis via the calcium-sensing receptor. Biochem Biophys Res Commun 249: 325–331, 1998. 76. Livadariu E, Auriemma RS, Rydlewski C, Vandeva S, Hamoir E, Burlacu MC, Maweja S, Thonnard AS, Betea D, Vassart G, Daly AF, Beckers A. Mutations of calcium-sensing receptor gene: two novel mutations and overview of impact on calcium homeostasis. Eur J Endocrinol 165: 353–358, 2011. 77. Loupy A, Ramakrishnan SK, Wootla B, Chambrey R, de la Faille R, Bourgeois S, Bruneval P, Mandet C, Christensen EI, Faure H, Cheval L, Laghmani K, Collet C, Eladari D, Dodd RH, Ruat M, Houillier P. PTH-independent regulation of blood calcium concentration by the calcium-sensing receptor. J Clin Invest 122: 3355–3367, 2012. 78. Lucas ML. A reconsideration of the evidence for Escherichia coli STa (heat stable) enterotoxin-driven fluid secretion: a new view of STa action and a new paradigm for fluid absorption. J Appl Microbiol 90: 7–26, 2001.

AJP-Cell Physiol • doi:10.1152/ajpcell.00139.2014 • www.ajpcell.org

Review C230

CaSR REVIEW

79. Mace OJ, Schindler M, Patel S. The regulation of K- and L-cell activity by GLUT2 and the calcium-sensing receptor CasR in rat small intestine. J Physiol 590: 2917–2936, 2012. 80. Maiti A, Beckman MJ. Extracellular calcium is a direct effecter of VDR levels in proximal tubule epithelial cells that counter-balances effects of PTH on renal vitamin D metabolism. J Steroid Biochem Mol Biol 103: 504 –508, 2007. 81. Maiti A, Hait NC, Beckman MJ. Extracellular calcium-sensing receptor activation induces vitamin D receptor levels in proximal kidney HK-2G cells by a mechanism that requires phosphorylation of p38alpha MAPK. J Biol Chem 283: 175–183, 2008. 82. Marx SJ, Attie MF, Levine MA, Spiegel AM, Downs RW, Lasker RD. The hypocalciuric or benign variant of familial hypercalcemia: clinical and biochemical features in fifteen kindreds. Medicine (Baltimore). 60: 397–412, 1981. 83. Marx SJ, Spiegel AM, Levine MA, Rizzoli RE, Lasker RD, Santora AC, Downs RW, Aurbach GD. Familial hypocalciuric hypercalcemia: the relation to primary parathyroid hyperplasia. N Engl J Med 307: 416 –426, 1982. 84. Mathias RS, Nguyen HT, Zhang MY, Portale AA. Reduced expression of the renal calcium-sensing receptor in rats with experimental chronic renal insufficiency. J Am Soc Nephrol 9: 2067–2074, 1998. 85. McNeil SE, Hobson SA, Nipper V, Rodland KD. Functional calciumsensing receptors in rat fibroblasts are required for activation of SRC kinase and mitogen-activated protein kinase in response to extracellular calcium. J Biol Chem 273: 1114 –1120, 1998. 86. Motoyama HI, Friedman PA. Calcium-sensing receptor regulation of PTH-dependent calcium absorption by mouse cortical ascending limbs. Am J Physiol Renal Physiol 283: F399 –F406, 2002. 87. Nearing J, Betka M, Quinn S, Hentschel H, Elger M, Baum M, Bai M, Chattopadyhay N, Brown EM, Hebert SC, Harris HW. Polyvalent cation receptor proteins (CaRs) are salinity sensors in fish. Proc Natl Acad Sci USA 99: 9231–9236, 2002. 88. Nemeth EF, Delmar EG, Heaton WL, Miller MA, Lambert LD, Conklin RL, Gowen M, Gleason JG, Bhatnagar PK, Fox J. Calcilytic compounds: potent and selective Ca2⫹ receptor antagonists that stimulate secretion of parathyroid hormone. J Pharmacol Exp Ther 299: 323–331, 2001. 89. Nemeth EF, Heaton WH, Miller M, Fox J, Balandrin MF, Van Wagenen BC, Colloton M, Karbon W, Scherrer J, Shatzen E, Rishton G, Scully S, Qi M, Harris R, Lacey D, Martin D. Pharmacodynamics of the type II calcimimetic compound cinacalcet HCl. J Pharmacol Exp Ther 308: 627–635, 2004. 90. Nemeth EF, Steffey ME, Hammerland LG, Hung BC, Van Wagenen BC, DelMar EG, Balandrin MF. Calcimimetics with potent and selective activity on the parathyroid calcium receptor. Proc Natl Acad Sci USA 95: 4040 –4045, 1998. 91. Nesbit MA, Hannan FM, Howles SA, Babinsky VN, Head RA, Cranston T, Rust N, Hobbs MR, Heath H 3rd, Thakker RV. Mutations affecting G-protein subunit alpha11 in hypercalcemia and hypocalcemia. N Engl J Med 368: 2476 –2486, 2013. 92. Nesbit MA, Hannan FM, Howles SA, Reed AA, Cranston T, Thakker CE, Gregory L, Rimmer AJ, Rust N, Graham U, Morrison PJ, Hunter SJ, Whyte MP, McVean G, Buck D, Thakker RV. Mutations in AP2S1 cause familial hypocalciuric hypercalcemia type 3. Nat Genet 45: 93–97, 2013. 93. Oda Y, Tu CL, Pillai S, Bikle DD. The calcium sensing receptor and its alternatively spliced form in keratinocyte differentiation. J Biol Chem 273: 23344 –23352, 1998. 94. Papapoulos SE, Manning RM, Hendy GN, Lewin IG, O’Riordan JL. Studies of circulating parathyroid hormone in man using a homologous amino-terminal specific immunoradiometric assay. Clin Endocrinol (Oxf) 13: 57–67, 1980. 95. Parfitt AM. The actions of parathyroid hormone on bone: relation to bone remodeling and turnover, calcium homeostasis, and metabolic bone diseases. II. PTH and bone cells: bone turnover and plasma calcium regulation. Metabolism 25: 909 –955, 1976. 96. Pearce SHS, Trump D, Wooding C, Besser GM, Chew SL, Grant DB, Heath DA, Hughes IA, Paterson CR, Whyte MP, Thakker RV. Calcium-sensing receptor mutations in familial benign hypercalcemia and neonatal hyperparathyroidism. J Clin Invest 96: 2683–2692, 1995. 97. Petrel C, Kessler A, Maslah F, Dauban P, Dodd RH, Rognan D, Ruat M. Modeling and mutagenesis of the binding site of Calhex 231, a novel

98.

99.

100.

101.

102.

103.

104.

105.

106.

107.

108.

109.

110.

111.

112.

113.

114. 115.

116.

negative allosteric modulator of the extracellular Ca(2⫹)-sensing receptor. J Biol Chem 278: 49487–49494, 2003. Petrucci M, Scott P, Ouimet D, Trouve ML, Proulx Y, Valiquette L, Guay G, Bonnardeaux A, Yamamoto M, Akatsu T, Nagase T, Ogata E. Evaluation of the calcium-sensing receptor gene in idiopathic hypercalciuria and calcium nephrolithiasis. Kidney Int 58: 38 –42, 2000. Pidasheva S, D’Souza-Li L, Canaff L, Cole DE, Hendy GN. CASRdb: calcium-sensing receptor locus-specific database for mutations causing familial (benign) hypocalciuric hypercalcemia, neonatal severe hyperparathyroidism, and autosomal dominant hypocalcemia. Hum Mutat 24: 107–111, 2004. Pollak MR, Brown EM, Chou YH, Hebert SC, Marx SJ, Stelnmann B, Levi T, Seidman CE, Seidman JG. Mutations in the Ca2⫹ sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Cell 75: 1297–1303, 1993. Pollak MR, Chou YH, Marx SJ, Steinmann B, Cole DE, Brandi ML, Papapoulos SE, Menko FH, Hendy GN, Brown EM. Familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Effects of mutant gene dosage on phenotype. J Clin Invest 93: 1108 –1112, 1994. Quamme GA. Control of magnesium transport in the thick ascending limb. Am J Physiol Renal Fluid Electrolyte Physiol 256: F197–F210, 1989. Quamme GA. Effect of hypercalcemia on renal tubular handling of calcium and magnesium. Can J Physiol Pharmacol 60: 1275–1280, 1982. Quamme GA, Dirks JH. Intraluminal and contraluminal magnesium on magnesium and calcium transfer in the rat nephron. Am J Physiol Renal Fluid Electrolyte Physiol 238: F187–F198, 1980. Quamme GA, Wong NL, Dirks JH, Roinel N, De Rouffignac C, Morel F. Magnesium handling in the dog kidney: a micropuncture study. Pflügers Arch 377: 95–99, 1978. Quinn SJ, Ye CP, Diaz R, Kifor O, Bai M, Vassilev P, Brown E. The Ca2⫹-sensing receptor: a target for polyamines. Am J Physiol Cell Physiol 273: C1315–C1323, 1997. Raue F, Pichl J, Dorr HG, Schnabel D, Heidemann P, Hammersen G, Jaursch-Hancke C, Santen R, Schofl C, Wabitsch M, Haag C, Schulze E, Frank-Raue K. Activating mutations in the calcium-sensing receptor: genetic and clinical spectrum in 25 patients with autosomal dominant hypocalcaemia - a German survey. Clin Endocrinol (Oxf) 75: 760 –765, 2011. Ray JM, Squires PE, Curtis SB, Meloche MR, Buchan AM. Expression of the calcium-sensing receptor on human antral gastrin cells in culture. J Clin Invest 99: 2328 –2333, 1997. Renkema KY, Velic A, Dijkman HB, Verkaart S, van der Kemp AW, Nowik M, Timmermans K, Doucet A, Wagner CA, Bindels RJ, Hoenderop JG. The calcium-sensing receptor promotes urinary acidification to prevent nephrolithiasis. J Am Soc Nephrol 20: 1705–1713, 2009. Riccardi D, Hall AE, Chattopadhyay N, Xu JZ, Brown EM, Hebert SC. Localization of the extracellular Ca2⫹/polyvalent cation-sensing protein in rat kidney. Am J Physiol Renal Physiol 274: F611–F622, 1998. Riccardi D, Lee WS, Lee K, Segre GV, Brown EM, Hebert SC. Localization of the extracellular Ca2⫹-sensing receptor and PTH/PTHrP receptor in rat kidney. Am J Physiol Renal Fluid Electrolyte Physiol 271: F951–F956, 1996. Riccardi D, Park J, Lee WS, Gamba G, Brown EM, Hebert SC. Cloning and functional expression of a rat kidney extracellular calcium/ polyvalent cation-sensing receptor. Proc Natl Acad Sci USA 92: 131– 135, 1995. Riccardi D, Traebert M, Ward DT, Kaissling B, Biber J, Hebert SC, Murer H. Dietary phosphate and parathyroid hormone alter the expression of the calcium-sensing receptor (CaR) and the Na⫹-dependent Pi transporter (NaPi-2) in the rat proximal tubule. Pflügers Arch 441: 379 –387, 2000. Ross AJ 3rd, Cooper A, Attie MF, Bishop HC. Primary hyperparathyroidism in infancy. J Pediatr Surg 21: 493–499, 1986. Rudberg C, Akerstrom G, Ljunghall S, Grimelius L, Johansson H, Pertoft H, Wide L. Regulation of parathyroid hormone release in primary and secondary hyperparathyroidism - studies in vivo and in vitro. Acta Endocrinol 101: 408 –413, 1982. Rutten MJ, Bacon KD, Marlink KL, Stoney M, Meichsner CL, Lee FP, Hobson SA, Rodland KD, Sheppard BC, Trunkey DD, Deveney KE, Deveney CW. Identification of a functional Ca2⫹-sensing receptor

AJP-Cell Physiol • doi:10.1152/ajpcell.00139.2014 • www.ajpcell.org

Review CaSR REVIEW

117.

118.

119.

120.

121. 122.

123. 124. 125. 126.

127.

128.

in normal human gastric mucous epithelial cells. Am J Physiol Gastrointest Liver Physiol 277: G662–G670, 1999. Sands JM, Flores FX, Kato A, Baum MA, Brown EM, Ward DT, Hebert SC, Harris HW. Vasopressin-elicited water and urea permeabilities are altered in IMCD in hypercalcemic rats. Am J Physiol Renal Physiol 274: F978 –F985, 1998. Sands JM, Naruse M, Baum M, Jo I, Hebert SC, Brown EM, Harris HW. Apical extracellular calcium/polyvalent cation-sensing receptor regulates vasopressin-elicited water permeability in rat kidney inner medullary collecting duct. J Clin Invest 99: 1399 –1405, 1997. Sarin R, Tomar N, Ray D, Gupta N, Sharma YD, Goswami R. Absence of pathogenic calcium sensing receptor mutations in sporadic idiopathic hypoparathyroidism. Clin Endocrinol (Oxf) 65: 359 –363, 2006. Shakhssalim N, Kazemi B, Basiri A, Houshmand M, Pakmanesh H, Golestan B, Eilanjegh AF, Kashi AH, Kilani M, Azadvari M. Association between calcium-sensing receptor gene polymorphisms and recurrent calcium kidney stone disease: a comprehensive gene analysis. Scand J Urol Nephrol 44: 406 –412, 2010. Shareghi GR, Agus ZS. Magnesium transport in the cortical thick ascending limb of Henle’s loop of the rabbit. J Clin Invest 69: 759 –769, 1982. Silve C, Petrel C, Leroy C, Bruel H, Mallet E, Rognan D, Ruat M. Delineating a Ca2⫹ binding pocket within the Venus flytrap module of the human calcium-sensing receptor. J Biol Chem 280: 37917–37923, 2005. Sorheim JI, Husebye ES, Nedrebo BG, Svarstad E, Lind J, Boman H, Lovas K. Phenotypic variation in a large family with autosomal dominant hypocalcaemia. Horm Res Paediatr 74: 399 –405, 2010. Suki WN, Eknoyan G, Rector FC Jr, Seldin DW. The renal diluting and concentrating mechanism in hypercalcemia. Nephron 6: 50 –61, 1969. Tfelt-Hansen J, Brown EM. The calcium-sensing receptor in normal physiology and pathophysiology: a review. Crit Rev Clin Lab Sci 42: 35–70, 2005. Toka HR, Al-Romaih K, Koshy JM, DiBartolo S 3rd, Kos CH, Quinn SJ, Curhan GC, Mount DB, Brown EM, Pollak MR. Deficiency of the calcium-sensing receptor in the kidney causes parathyroid hormoneindependent hypocalciuria. J Am Soc Nephrol 23: 1879 –1890, 2012. Topala CN, Schoeber JP, Searchfield LE, Riccardi D, Hoenderop JG, Bindels RJ. Activation of the Ca2⫹-sensing receptor stimulates the activity of the epithelial Ca2⫹ channel TRPV5. Cell Calcium 45: 331– 339, 2009. Tu CL, Chang W, Xie Z, Bikle DD. Inactivation of the calcium sensing receptor inhibits E-cadherin-mediated cell-cell adhesion and calcium-

129. 130. 131. 132.

133.

134.

135.

136. 137. 138.

139. 140.

C231

induced differentiation in human epidermal keratinocytes. J Biol Chem 283: 3519 –3528, 2008. Tu CL, Oda Y, Bikle DD. Effects of a calcium receptor activator on the cellular response to calcium in human keratinocytes. J Invest Dermatol 113: 340 –345, 1999. Tu CL, Oda Y, Komuves L, Bikle DD. The role of the calcium-sensing receptor in epidermal differentiation. Cell Calcium 35: 265–273, 2004. Tu Q, Pi M, Karsenty G, Simpson L, Liu S, Quarles LD. Rescue of the skeletal phenotype in CasR-deficient mice by transfer onto the Gcm2 null background. J Clin Invest 111: 1029 –1037, 2003. Vezzoli G, Arcidiacono T, Paloschi V, Terranegra A, Biasion R, Weber G, Mora S, Syren ML, Coviello D, Cusi D, Bianchi G, Soldati L. Autosomal dominant hypocalcemia with mild type 5 Bartter syndrome. J Nephrol 19: 525–528, 2006. Vezzoli G, Tanini A, Ferrucci L, Soldati L, Bianchin C, Franceschelli F, Malentacchi C, Porfirio B, Adamo D, Terranegra A, Falchetti A, Cusi D, Bianchi G, Brandi ML. Influence of calcium-sensing receptor gene on urinary calcium excretion in stone-forming patients. J Am Soc Nephrol 13: 2517–2523, 2002. Vezzoli G, Terranegra A, Arcidiacono T, Biasion R, Coviello D, Syren ML, Paloschi V, Giannini S, Mignogna G, Rubinacci A, Ferraretto A, Cusi D, Bianchi G, Soldati L. R990G polymorphism of calcium-sensing receptor does produce a gain-of-function and predispose to primary hypercalciuria. Kidney Int 71: 1155–1162, 2007. Waller S, Kurzawinski T, Spitz L, Thakker R, Cranston T, Pearce S, Cheetham T, van’t Hoff WG. Neonatal severe hyperparathyroidism: genotype/phenotype correlation and the use of pamidronate as rescue therapy. Eur J Pediatr 163: 589 –594, 2004. Wang W, Lu M, Balazy M, Hebert SC. Phospholipase A2 is involved in mediating the effect of extracellular Ca2⫹ on apical K⫹ channels in rat TAL. Am J Physiol Renal Physiol 273: F421–F429, 1997. Wang WH, Lu M, Hebert SC. Cytochrome P-450 metabolites mediate extracellular Ca(2⫹)-induced inhibition of apical K⫹ channels in the TAL. Am J Physiol Cell Physiol 271: C103–C111, 1996. Watanabe S, Fukumoto S, Chang H, Takeuchi Y, Hasegawa Y, Okazaki R, Chikatsu N, Fujita T. Association between activating mutations of calcium-sensing receptor and Bartter’s syndrome. Lancet 360: 692–694, 2002. Wong NLM, Dirks JH, Quamme GA. Tubular reabsorptive capacity for magnesium in the dog kidney. Am J Physiol Renal Fluid Electrolyte Physiol 244: F78 –F83, 1983. Yang T, Hassan S, Huang YG, Smart AM, Briggs JP, Schnermann JB. Expression of PTHrP, PTH/PTHrP receptor, and Ca2⫹-sensing receptor mRNAs along the rat nephron. Am J Physiol Renal Physiol 272: F751–F758, 1997.

AJP-Cell Physiol • doi:10.1152/ajpcell.00139.2014 • www.ajpcell.org