Mei Bai

A Familial Syndrome of Hypocalcemia with Hypercalciuria Due to Mutations in the Calcium-Sensing Receptor

BACKGROUND The calcium-sensing receptor regulates the secretion of parathyroid hormone in response to changes in extracellular calcium concentrations, and mutations that result in a loss of function of the receptor are associated with familial hypocalciuric hypercalcemia. Mutations involving a gain of function have been associated with hypocalcemia in two kindreds. We examined the possibility that the latter type of mutation may result in a phenotype of familial hypocalcemia with hypercalciuria. METHODS We studied six kindreds given a diagnosis of autosomal dominant hypoparathyroidism on the basis of their hypocalcemia and normal serum parathyroid hormone concentrations, a combination that suggested a defect of the calcium-sensing receptor. The hypocalcemia was associated with hypercalciuria, and treatment with vitamin D resulted in increased hypercalciuria, nephrocalcinosis, and renal impairment. Mutations in the calcium-sensing-receptor gene were identified by DNA-sequence analysis and expressed in human embryonic kidney cells (HEK-293). RESULTS Five heterozygous missense mutations (Asn118Lys, Phe128Leu, Thr151Met, Glu191Lys, and Phe612Ser) were detected in the extracellular domain of the calcium-sensing-receptor gene and shown to cosegregate with the disease. Analysis of the functional expression of three of the mutant receptors in HEK-293 cells demonstrated shifts in the dose-response curves so that the extracellular calcium concentrations needed to produce half-maximal increases in total inositol phosphate in the cells were significantly (P=0.02 to P<0.001) lower than those required for the wild-type receptor. CONCLUSIONS Gain-of-function mutations in the calcium-sensing receptor are associated with a familial syndrome of hypocalcemia with hypercalciuria that needs to be distinguished from hypoparathyroidism.

Expression and characterization of inactivating and activating mutations in the human Ca2+o-sensing receptor.

Nearly 30 mutations have been identified to date in the coding region of the extracellular calcium-sensing receptor (CaR) that are associated with inherited human hypo- and hypercalcemic disorders. To understand the mechanisms by which the mutations alter the function of the receptor may help to discern the structure-function relationships in terms of ligand-binding and G protein coupling. In the present studies, we transiently expressed eight known CaR mutations in HEK293 cells. The effects of the mutations on extracellular calcium- and gadolinium-elicited increases in the cytosolic calcium concentration were then examined. Seven inactivating mutations, which cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism, show a reduced functional activity of the receptor because they may 1) reduce its affinity for agonists; 2) prevent conversion of the receptor from a putatively immature, high mannose form into the fully glycosylated and biologically active form of the CaR, in addition to lowering its affinity for agonists; or 3) fail to couple the receptor to and/or activate its respective G protein(s). Conversely, one activating mutation, which causes a form of autosomal dominant hypocalcemia, appears to increase the affinity of the receptor for its agonists.

Dimerization of the extracellular calcium-sensing receptor (CaR) on the cell surface of CaR-transfected HEK293 cells

The extracellular calcium (Ca2+ o )-sensing receptor (CaR) is a G protein-coupled receptor that plays important roles in calcium homeostasis. In this study, we employed epitope tagging, cell-surface biotinylation, and immunoprecipitation techniques to demonstrate that the CaR is expressed mostly in the form of a dimer on the surface of transfected human embryonic kidney (HEK293) cells. Western analysis of cell-surface proteins under nonreducing conditions showed that the CaR exists in several forms with molecular masses greater than 200 kDa. Most of these high molecular mass forms of the receptor could be converted to a single monomeric species at 160 kDa under reducing conditions. This result suggests that the CaR forms dimers or even higher oligomers on the cell surface through intermolecular disulfide bonds that are sensitive to reducing agents. Consistent with this hypothesis, use of a cell-surface cross-linking agent substantially increases the proportion of the putative dimeric CaR at 280 kDa relative to the monomeric form of the receptor at 160 kDa under reducing conditions. Dimerization of the CaR in intact cells was further demonstrated when we co-transfected and co-immunoprecipitated the wild type, full-length receptor and a truncated form of the CaR lacking its cytoplasmic tail. Taken together, we conclude from these results that the functional CaR resides on the cell surface of transfected HEK293 cells in the form of a dimer.

In vivo and in vitro characterization of neonatal hyperparathyroidism resulting from a de novo, heterozygous mutation in the Ca2+-sensing receptor gene: normal maternal calcium homeostasis as a cause of secondary hyperparathyroidism in familial benign hypocalciuric hypercalcemia.

We characterized the in vivo, cellular and molecular pathophysiology of a case of neonatal hyperparathyroidism (NHPT) resulting from a de novo, heterozygous missense mutation in the gene for the extracellular Ca2+ (Ca2+(o))-sensing receptor (CaR). The female neonate presented with moderately severe hypercalcemia, markedly undermineralized bones, and multiple metaphyseal fractures. Subtotal parathyroidectomy was performed at 6 wk; hypercalcemia recurred rapidly but the bone disease improved gradually with reversion to an asymptomatic state resembling familial benign hypocalciuric hypercalcemia (FBHH). Dispersed parathyroid cells from the resected tissue showed a set-point (the level of Ca2+(o) half maximally inhibiting PTH secretion) substantially higher than for normal human parathyroid cells (approximately 1.8 vs. approximately 1.0 mM, respectively); a similar increase in set-point was observed in vivo. The probands CaR gene showed a missense mutation (R185Q) at codon 185, while her normocalcemic parents were homozygous for wild type (WT) CaR sequence. Transient expression of the mutant R185Q CaR in human embryonic kidney (HEK293) cells revealed a substantially attenuated Ca2+(o)-evoked accumulation of total inositol phosphates (IP), while cotransfection of normal and mutant receptors showed an EC50 (the level of Ca2+(o) eliciting a half-maximal increase in IPs) 37% higher than for WT CaR alone (6.3+/-0.4 vs. 4.6+/-0.3 mM Ca2+(o), respectively). Thus this de novo, heterozygous CaR mutation may exert a dominant negative action on the normal CaR, producing NHPT and more severe hypercalcemia than typically seen with FBHH. Moreover, normal maternal calcium homeostasis promoted additional secondary hyperparathyroidism in the fetus, contributing to the severity of the NHPT in this case with FBHH.

The Ca2+-sensing receptor: a target for polyamines

The Ca2+-sensing receptor (CaR) is activated at physiological levels of external Ca2+(Cao) but is expressed in a number of tissues that do not have well-established roles in the control of Cao, including several regions of the brain and the intestine. Polyamines are endogenous polyvalent cations that can act as agonists for the CaR, as shown by our current studies of human embryonic kidney (HEK-293) cells transfected with the human CaR. Cellular parameters altered by polyamines included cytosolic free Ca2+(Cai), inositol phosphate production, and the activity of a nonselective cation channel. Spermine stimulated Cai transients in CaR-transfected HEK cells, with a concentration producing a half-maximal response (EC50) of ∼500 μM in the presence of 0.5 mM Ca2+, whereas sustained increases in Cai had an EC50 of ∼200 μM. The order of potency was spermine > spermidine >> putrescine. Elevation of Cao shifted the EC50 for spermine sharply to the left, with substantial stimulation below 100 μM. Addition of subthreshold concentrations of spermine increased the sensitivity of CaR-expressing HEK cells to Cao. Parathyroid hormone secretion from bovine parathyroid cells was inhibited by 50% in the presence of 200 μM spermine, a response similar to that elicited by 2.0 mM Cao. These data suggest that polyamines could be effective agonists for the CaR, and several tissues, including the brain, may use the CaR as a target for the actions of spermine and other endogenous polycationic agonists.

Functional characterization of calcium-sensing receptor mutations expressed in human embryonic kidney cells.

The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that plays a key role in extracellular calcium ion homeostasis. We have engineered 11 CaR mutants that have been described in the disorders familial benign hypercalcemia (FBH), neonatal severe hyperparathyroidism (NSHPT), and autosomal dominant hypocalcaemia (ADH), and studied their function by characterizing intracellular calcium [Ca2+]i transients in response to varying concentrations of extracellular calcium [Ca2+]o or gadolinium [Gd3+]o. The wild type receptor had an EC50 for calcium (EC50[Ca2+]o) (the value of [Ca2+]o producing half of the maximal increase in [Ca2+]i) of 4.0 mM (+/- 0.1 SEM). However, five missense mutations associated with FBH or NSHPT, (P55L, N178D, P221S, R227L, and V817I) had significantly higher EC50[Ca2+]os of between 5.5 and 9.3 mM (all P < 0.01). Another FBH mutation, Y218S, had an EC50[Ca2+]o of > 50 mM but had only a mildly attenuated response to gadolinium, while the FBH mutations, R680C and P747fs, were unresponsive to either calcium or gadolinium. In contrast, three mutations associated with ADH, (F128L, T151M, and E191K), showed significantly reduced EC50[Ca2+]os of between 2.2 and 2.8 mM (all P < 0.01). These findings provide insights into the functional domains of the CaR and demonstrate that mutations which enhance or reduce the responsiveness of the CaR to [Ca2+]o cause the disorders ADH, FBH, and NSHPT, respectively.

Dimerization of G-protein-coupled receptors: roles in signal transduction

Recently, many G-protein-coupled receptors (GPCRs) have been demonstrated to form constitutive dimers consisting of identical or distinct monomeric subunits. The discovery of GPCR dimerization has revealed a new level of molecular cross-talk between signalling molecules and may define a general mechanism that modulates the function of GPCRs under both physiological and pathological conditions. The heterodimerization between distinct GPCRs could be responsible for the generation of pharmacologically defined receptors for which no gene has been identified so far. Elucidating the role of dimerization in the activation processes of GPCRs will lead us to develop novel pharmaceutical agents that allosterically promote activation or inhibition of GPCR signalling.

The Extracellular Calcium-sensing Receptor Dimerizes through Multiple Types of Intermolecular Interactions

Recent studies have shown that the G protein-coupled, extracellular calcium ([Ca2+] o )-sensing receptor (CaR) forms disulfide-linked dimers through cysteine residues within its extracellular domain and that dimerization of the CaR has functional implications. In this study, we have investigated which of these disulfide linkages are essential for dimerization of the CaR and whether they are required for these functional interactions. Our results confirm the key roles of Cys129 and Cys131 in CaR dimerization. However, utilizing cross-linking of the CaR or immunoprecipitation of a non-FLAG-tagged CaR with a FLAG-tagged CaR using anti-FLAG antibody, we demonstrate that CaRs with or without these two cysteines form dimers on the cell surface to a similar extent. In addition, reconstitution of CaR-mediated signaling by cotransfection of two individually inactive mutant CaRs is nearly identical in the presence or absence of both Cys129 and Cys131, showing that covalent linkage of CaR dimers is not needed for functional interactions between CaR monomers. These findings suggest that the CaR has at least two distinct types of motifs mediating dimerization and functional interactions, i.e. covalent interactions involving intermolecular disulfide bonds and noncovalent, possibly hydrophobic, interactions.

Polyvalent cation receptor proteins (CaRs) are salinity sensors in fish

To determine whether calcium polyvalent cation-sensing receptors (CaRs) are salinity sensors in fish, we used a homology-based cloning strategy to isolate a 4.1-kb cDNA encoding a 1,027-aa dogfish shark (Squalus acanthias) kidney CaR. Expression studies in human embryonic kidney cells reveal that shark kidney senses combinations of Ca2+, Mg2+, and Na+ ions at concentrations present in seawater and kidney tubules. Shark kidney is expressed in multiple shark osmoregulatory organs, including specific tubules of the kidney, rectal gland, stomach, intestine, olfactory lamellae, gill, and brain. Reverse transcriptase–PCR amplification using specific primers in two teleost fish, winter flounder (Pleuronectes americanus) and Atlantic salmon (Salmo salar), reveals a similar pattern of CaR tissue expression. Exposure of the lumen of winter flounder urinary bladder to the CaR agonists, Gd3+ and neomycin, reversibly inhibit volume transport, which is important for euryhaline teleost survival in seawater. Within 24–72 hr after transfer of freshwater-adapted Atlantic salmon to seawater, there are increases in their plasma Ca2+, Mg2+, and Na+ that likely serve as a signal for internal CaRs, i.e., brain, to sense alterations in salinity in the surrounding water. We conclude that CaRs act as salinity sensors in both teleost and elasmobranch fish. Their tissue expression patterns in fish provide insights into CaR functions in terrestrial animals including humans.

Protein Kinase C Phosphorylation of Threonine at Position 888 in Ca2+ o -Sensing Receptor (CaR) Inhibits Coupling to Ca2+ Store Release

Previous studies in parathyroid cells, which express the G protein-coupled, extracellular calcium-sensing receptor (CaR), showed that activation of protein kinase C (PKC) blunts high extracellular calcium (Ca2+ o )-evoked stimulation of phospholipase C and the associated increases in cytosolic calcium (Ca2+ i ), suggesting that PKC may directly modulate the coupling of the CaR to intracellular signaling systems. In this study, we examined the role of PKC in regulating the coupling of the CaR to Ca2+ i dynamics in fura-2-loaded human embryonic kidney cells (HEK293 cells) transiently transfected with the human parathyroid CaR. We demonstrate that several PKC activators exert inhibitory effects on CaR-mediated increases in Ca2+ i due to release of Ca2+ from intracellular stores. Consistent with the effect being mediated by activation of PKC, the inhibitory effect of PKC activators on Ca2+ release can be blocked by a PKC inhibitor. The use of site-directed mutagenesis reveals that threonine at amino acid position 888 is the major PKC site that mediates the inhibitory effect of PKC activators on Ca2+ mobilization. The effect of PKC activation can be maximally blocked by mutating three PKC sites (Thr888, Ser895, and Ser915) or all five PKC sites. In vitro phosphorylation shows that Thr888 is readily phosphorylated by PKC. Our results suggest that phosphorylation of the CaR is the molecular basis for the previously described effect of PKC activation on Ca2+ o -evoked changes in Ca2+ i dynamics in parathyroid cells.