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Novel Inactivating Mutations of the Calcium-Sensing Receptor: The Calcimimetic NPS R-568 Improves Signal Transduction of Mutant Receptors

Division of Neuroendocrinology (R.R., B.M., C.S.), Department of Neurosurgery, Friedrich-Alexander University Erlangen-Nuremberg, 91054 Erlangen, Germany; Department of Endocrinology, Diabetes and Nutrition (R.R., C.B.-V., V.B., M.M., C.S.), Charité-University Medicine Berlin, Campus Benjamin Franklin, 12200 Berlin, Germany; and Endocrine Practice (C.H., E.S., K.F.-R., F.R.), 69120 Heidelberg, Germany

Address all correspondence and requests for reprints to: Christof Schöfl, M.D., Division of Neuroendocrinology, Department of Neurosurgery, Friedrich-Alexander University Erlangen-Nuremberg, Schwabachanlage 6, 91054 Erlangen, Germany. E-mail: christof.schoefl{at}uk-erlangen.de.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Context and Objective: Inactivating mutations in the calcium-sensing receptor (CaSR) gene cause neonatal severe hyperparathyroidism and familial hypocalciuric hypercalcemia (FHH). The aims of the present study were the functional characterization of novel mutations of the CaSR found in FHH patients, the comparison of in vitro receptor function with clinical parameters, and the effect of the allosteric calcimimetic NPS R-568 on the signaling of mutant receptors.

Methods: Wild-type and mutant CaSRs (W530G, C568Y, W718X, M734R, L849P, Q926R, and D1005N) were expressed in human embryonic kidney 293 cells. Receptor signaling was studied by measuring intracellular free calcium in response to different concentrations of extracellular calcium ([Ca²⁺]_o).

Results: Four CaSR mutations (C568Y, W718X, M734R, and L849P) demonstrated a complete lack of a [Ca²⁺]_o-induced cytosolic Ca²⁺ response up to 30 mM [Ca²⁺]_o, whereas the CaSR mutants W530G, Q926R, and D1005N retained some sensitivity to [Ca²⁺]_o. There was no significant relation between the in vitro calcium sensitivity, serum calcium, and intact PTH levels in the patients. Patients with C-terminal CaSR mutations had a calcium to creatine ratio above the established diagnostic threshold of 0.01 for FHH. The calcimimetic NPS R-568 enhanced the responsiveness to [Ca²⁺]_o in CaSR mutants of the extracellular domain (W530G and C568Y) as well as the intracellular C-terminal domain (Q926R and D1005N).

Conclusion: Therefore, calcimimetics might offer medical treatment for symptomatic FHH patients, and more important, for patients with neonatal severe hyperparathyroidism that harbor calcimimetic-sensitive CaSR mutants.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The extracellular calcium-sensing receptor (CaSR) plays a central role in maintaining calcium homeostasis through its ability to regulate PTH release and renal tubular calcium reabsorption. It was cloned from the parathyroid gland, and subsequently identified in tissues that participate in the regulation of calcium homeostasis, including calcitonin-secreting C cells, kidney cells, bone and intestinal cells, where it allows the detection of minute changes in the extracellular calcium ([Ca²⁺]_o) concentration (1, 2).

Inactivating mutations of the CaSR, characterized by resistance to [Ca²⁺]_o, give rise to disorders of calcium homeostasis, neonatal severe hyperparathyroidism (NSHPT), and familial hypocalciuric hypercalcemia (FHH), which are caused by homozygous and heterozygous mutations, respectively (3). NSHPT is a severe hypercalcemic disease developing during the early days of life and may be fatal unless parathyroidectomy is performed. There is currently no definitive medical treatment available, although pamidronate can be used to stabilize these patients before surgery. FHH patients are frequently asymptomatic and discovered by chance. Typically, they exhibit mild to moderate hypercalcemia, unsuppressed intact PTH (iPTH) levels, and reduced renal calcium excretion (4). Although FHH patients generally do not require medical treatment to correct hypercalcemia, there is accumulating evidence that FHH may be associated with an increased risk of pancreatitis (5). In addition, polymorphisms in the C-terminal domain of the CaSR are associated with slightly elevated serum calcium and an increased risk of coronary heart disease (6). This suggests that FHH patients, whose CaSR mutations have a stronger effect on calcium homeostasis than the polymorphisms, may be exposed to, so far, unrecognized risks, which would require broader and more rigorous treatment. Therefore, the correction of reduced calcium sensitivity of mutant CaSR would be desirable.

The CaSR belongs to the G protein-coupled receptor superfamily, and is closely related to the metabotropic glutamate receptor, -aminobutyric acid-_B receptor, and certain taste, odorant, and pheromone receptors (1). It is a 1078-amino acid glycoprotein, encoded by the CaSR gene located on chromosome 3q13.3–21 (7), and comprises a large extracellular domain (ECD), a seven transmembrane-spanning region and an intracellular C-terminal tail (2). The active cell surface form of the receptor is a dimer (8, 9, 10), allowing interaction between the two receptors’ extracellular ligand binding domains called Venus-flytrap domains upon calcium binding (11, 12). This leads to conformational changes of the intracellular loops (ICLs), G protein binding (13), and activation of several different second messengers. The best-characterized pathway involves G_q11, which activates phospholipase C, thereby increasing intracellular free calcium ([Ca²⁺]_i) and activating protein kinase C activity. Mutant and wild-type (wt) CaSRs can form heterodimers in vitro that could interfere with normal receptor function, resulting in impaired transmembrane signaling even in the presence of wt CaSRs, like in patients with FHH (12).

Calcimimetics like NPS R-568 or cinacalcet bind to the transmembrane domain (TMD) of the CaSR and allosterically increase its sensitivity to [Ca²⁺]_o, thereby enhancing signal transduction. Cinacalcet is currently used to treat secondary hyperparathyroidism in kidney disease (14, 15). However, there is only limited information as to whether these drugs could restore or improve the function of mutant CaSRs and, therefore, could offer a treatment option for symptomatic FHH or NSHPT patients.

In the present study, we functionally characterized seven novel mutations of the CaSR gene found in FHH patients, compared in vitro function with clinical parameters in the patients, and determined whether NPS R-568 could improve the signal transduction of mutant CaSRs.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subjects

Patients were referred for endocrine evaluation of incidentally detected elevated serum calcium levels. The diagnosis of FHH was based on elevated serum calcium, elevated or inappropriately normal iPTH, and inappropriately low urinary calcium excretion (Table 1). Informed consent for genomic testing and in vitro studies was obtained, and mutations in the CaSR gene were found in all patients. Except for the C568Y mutation, all patients have been described in an abstract presented at the Annual Meeting of The Endocrine Society in 2005 (16).

The cDNA for human CaSR cloned into the HindIII and XhoI restriction sites of the pCR3.1 expression vector (pCR3.1/hCaSR) was kindly provided by Dr. Allen Spiegel (Albert Einstein College of Medicine, New York, NY). Site-directed mutagenesis to generate CaSR mutants was performed by the overlap extension method as described (17) using primers listed in supplemental Table 1, which is published as supplemental data on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org) and Pfu-Polymerase (Roche, Mannheim, Germany). The accuracy of all constructs was checked by sequencing.

Expression of CaSR in human embryonic kidney (HEK) 293 cells

HEK293 cells were propagated in 5% CO₂ at 37 C in 75 cm² flasks in MEM with Earle’s Salts medium (PAA Laboratories GmbH, Cölbe, Germany) supplemented with 2 mM L-glutamine and 10% heat-inactivated fetal calf serum (Invitrogen, Karlsruhe, Germany). For transfection, 1 µg wt or mutant pCR3.1/hCaSR plasmid DNA or 0.5 µg each for cotransfection experiments was diluted in 100 µl Opti-MEM (Invitrogen), mixed with 2 µl Fugene HD (Roche), incubated at room temperature for 30 min, and added to HEK293 cells seeded on 22-mm glass coverslips in 35-mm dishes for [Ca²⁺]_i measurement or in six-well plates for protein extraction. Measurement of [Ca²⁺]_i and total protein extraction for Western blots were performed 48 h after transfection.

Measurement of [Ca²⁺]_i

HEK293 cells cultured on coverslips and transfected with wt or mutant CaSRs were loaded with 5 µM fura-2/AM (Invitrogen) in loading buffer containing 20 mM HEPES (pH 7.4), 130 mM NaCl, 4.7 mM KCl, 1.25 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 10 mM glucose, 0.2% BSA, and 0.025% pluronic acid (Invitrogen) for 30 min at 37 C. After washing three times with the same buffer as used for fura-2 loading but without pluronic acid and with 0.1% BSA (weight/vol) and only 0.5 mM CaCl₂ (superfusion buffer), the cover glass was mounted in a temperature-controlled superfusion chamber at 37 C, placed on the stage of an Axiovert IM 135 microscope equipped with a 40x/1.3 Achrostigmat oil immersion objective (Carl Zeiss, Göttingen, Germany), and superfused (1 ml/min) with superfusion buffer. Fura-2 fluorescence at 510 nm during excitation with 340 and 380 nm was recorded from single cells of average size and healthy appearance with a dual wavelength excitation microfluorometry system (Deltascan 4000; Photon Technology Instruments, Seefeld, Germany). [Ca²⁺]_i was calculated according to the formula (18): [Ca²⁺]_i = K_dB(R – R_min)/(R_max – R), where K_d = 225 nM, and R_max, R_min, and B are constants that were determined in the superfusion chamber from solutions containing fura-2 free acid (1 µM) and various concentrations of free Ca²⁺. Dose-response curves were determined by stepwise increasing [Ca²⁺]_o to 1–5, 10, 15, 20, 25, and 30 mM. Between steps cells were washed with superfusion buffer containing 0.5 mM CaCl₂ for 5 min to avoid receptor or pathway desensitization. The magnitude of the [Ca²⁺]_i response at each concentration of [Ca²⁺]_o was determined as the maximal increase from baseline. To study the effect of NPS R-568, [Ca²⁺]_o was increased stepwise as described previously, and 1 µM NPS R-568 was added when cells were superfused with 3 or 10 mM [Ca²⁺]_o. The EC₅₀ for [Ca²⁺]_o was determined from dose-response curves created with GraphPad Prism 4.0 software (GraphPad Software Inc., San Diego, CA).

Western blot

Cells in six-well plates were rinsed with ice-cold PBS and scraped on ice in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate, 5 mM EDTA, freshly added protease inhibitors mixture (Roche), and 100 mM phenylmethanesulfonylfluoride. Protein concentration was determined using the BC assay (Interchim, Montluçon, France). Seventy-five micrograms of protein were denatured in LDS buffer (Invitrogen) under nonreducing and reducing conditions (2.5% β-mercaptoethanol, 100 mM dithiothreitol), separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels as indicated in the figure legends, and blotted onto polyvinylidene fluoride membrane. After incubation with mouse antibody against hCaSR aa214–235 (Acris, Hiddenhausen, Germany) and horseradish peroxidase-coupled sheep antimouse secondary antibody (Amersham, Freiburg, Germany), signals were developed using an enhanced chemiluminescence system (Amersham) and imaging on a Chemi-Smart 5000 chemiluminescence detection system (Vilber Lourmat, Eberhardzell, Germany).

Statistics

Statistical analysis was performed with SPSS software (version 14.0; SPSS, Inc., Chicago, IL). Values are reported as mean ± SEM. A two-sided of less than 0.05 was considered significant. Normal distribution was tested by the Kolmogorov-Smirnov test. Mean EC₅₀ values and clinical parameters were compared using the unpaired Student’s t test. Correlation analyses were done using the Pearson correlation coefficient. The relation between the EC₅₀ values (as derived from the in vitro experiments), serum calcium, and iPTH was modeled by multiple linear regression analysis with iPTH as the dependent variable. Furthermore, interaction terms (either multiplicative or additive) were included to test for putative interactions between EC₅₀ and serum calcium with respect to iPTH.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Patients and mutations

We found seven novel mutations in the coding region of the CaSR gene in patients presenting with the clinical diagnosis of FHH, whose clinical parameters are given in Table 1. Two mutations were missense mutations in the Cys-rich region of the ECD, one was a nonsense mutation in the second ICL, two were missense mutations in the TMD, and two mutations in the intracellular C-terminal tail were also missense mutations (Table 1).

Transmembrane signaling of mutant CaSRs

The functional consequences of these mutations in the CaSR protein were assessed by the expression of wt or mutant CaSRs and by measuring changes in [Ca²⁺]_i in response to increasing [Ca²⁺]_o in fura-2 loaded in HEK293 cells (Fig. 1A). All seven mutations resulted in a significantly impaired function of the CaSR, confirming the clinical diagnosis of FHH. Four mutations (C568Y, W718X, M734R, and L849P) rendered the CaSR completely unresponsive to [Ca²⁺]_o up to 30 mM (Fig. 1, B–D, closed symbols). The remaining three mutations (W530G, Q926R, and D1005N) resulted in a right-shifted dose-response curve with an increased EC₅₀ for [Ca²⁺]_o-induced changes in [Ca²⁺]_i, and a reduced maximum [Ca²⁺]_i response compared with the wt receptor (Fig. 1, B–D, and Table 2). wt and mutant CaSRs were coexpressed to mimic the heterozygous situation in FHH patients. This attenuated the functional impairment of all mutants (Fig. 1, B–D, open symbols). A maximum [Ca²⁺]_i response comparable to the wt CaSR was observed, however, only in cells that coexpressed wt and Q926R and D1005N CaSR mutants (Fig. 1D and Table 2). Untransfected HEK293 cells were completely unresponsive to [Ca²⁺]_o (data not shown).

View larger version (40K): [in this window] [in a new window]   FIG. 1. Sensitivity of wt and mutant CaSRs to [Ca2+]o. A, Typical response of [Ca2+]i to increasing levels of [Ca2+]o in single HEK293 cells as determined by dual wavelength excitation microfluorometry. Cells transfected with wt, mutant (M734R), and cotransfected with wt and mutant CaSR (M734R/WT) were stimulated with 1–15 mM [Ca2+]o as indicated. B–D, Dose-response curves of [Ca2+]i in response to a stepwise increase of [Ca2+]o. Average relative responses (± SEM) of CaSRs with mutations in the extracellular (B), transmembrane (C), and C-terminal domains (D) expressed alone (closed symbols) or coexpressed with wt (open symbols) in comparison to wt CaSR (dashed line).

Receptor expression in HEK293 cells was analyzed by Western blotting to determine whether impaired receptor function was caused by altered CaSR protein expression. wt CaSR and four of the mutations (W530G, C568Y, Q926R, and D1005N) showed discernible expression of 130 and 150-kDa bands under reducing conditions (Fig. 2A) that represent the high mannose form expressed intracellularly and the fully glycosylated variant expressed on the cell surface. Two mutations (M734R and L849P) showed only the 130-kDa band consistent with poor surface expression. The nonsense mutation (W718X), which predicts a truncated CaSR polypeptide of 79 kDa lacking the last four TMDs and the intracellular tail, displayed a single smaller band at about 100 kDa. Under nonreducing conditions (Fig. 2B), bands at approximately 300 kDa were visible for both wt and mutant proteins except the W718X truncation mutant, which showed bands of approximately 200 kDa. This suggests that all mutants are able to form dimers or polymers that can also occur in the endoplasmic reticulum and does not necessarily indicate surface expression (11). Untransfected HEK293 cells show no immunostainable bands.

View larger version (104K): [in this window] [in a new window]   FIG. 2. Protein expression of wt and mutant CaSRs. Western blot analysis of extracts of HEK293 cells expressing the indicated wt and mutant CaSR proteins using anti-CaSR antibody. Proteins were denatured under reducing conditions and resolved on an 8% linear Tris-glycine gel (A), and denatured under nonreducing conditions and resolved on a 3–8% gradient Tris-acetate gel (B). The 130 and 150-kDa bands represent the high mannose intracellular and fully glycosylated cell surface-expressed form, respectively.

The clinical and functional in vitro data are summarized in Tables 1 and 2. There was no significant relation among the in vitro calcium sensitivity (expressed by the EC₅₀ mutant/wt obtained from cells coexpressing the mutant and wt CaSRs), serum calcium, and iPTH levels in the patients. None of the correlations between these parameters was significant. Likewise, neither the EC₅₀ nor serum calcium was significantly associated with iPTH in a multivariate regression model, and further inclusion of either an additive or a multiplicative interaction term did not yield any significant interaction with respect to iPTH. In FHH patients harboring CaSR mutants that were either truncated or poorly expressed on the cell surface of HEK293 cells (W718X, M734R, and L849P), iPTH tended to be lower (53 ± 9 vs. 140 ± 35 pg/ml; P = 0.09), whereas serum calcium was similar compared with patients with mutant CaSRs that are well expressed on the cell surface (2.78 ± 0.04 vs. 2.90 ± 0.08; P = 0.35). Furthermore, the two FHH patients with mutations in the C terminus of the CaSR (Q926R and D1005N) had a urinary calcium to creatinine ratio above the established diagnostic threshold of 0.01 (19), whereas patients with other mutations (W530G, W718X, and L849P) were clearly below (Table 1). However, due to the low number of cases, this difference was not statistically significant (mean urinary calcium to creatinine ratio 0.025 vs. 0.0042; P = 0.15).

Rescue of some mutant CaSRs by the calcimimetic NPS R-568

To test whether the calcimimetic NPS R-568 could improve the signaling function of inactivating CaSR mutants, the [Ca²⁺]_i response to 3 and 10 mM [Ca²⁺]_o in the presence of 1 µM NPS R-568 was investigated. In HEK293 cells expressing wt CaSR or the W530G, Q926R, and D1005N CaSR mutants, NPS R-568 (1 µM) significantly enhanced the [Ca²⁺]_i response to 3 mM [Ca²⁺]_o (P < 0.01), as depicted in Fig. 3. Furthermore, NPS R-568 restored the [Ca²⁺]_i response of these mutants to levels that were comparable to the ones observed at 3 mM [Ca²⁺]_o in cells expressing wt CaSR without NPS R-568 treatment. The CaSR mutant C568Y, which did not respond to [Ca²⁺]_o up to 30 mM (Fig. 2B), showed a significant increase in [Ca²⁺]_i to 10 mM but not 3 mM [Ca²⁺]_o in the presence of 1 µM NPS R-568. However, in cells expressing the W718X, M734R, and L849P mutants, even in the presence of NPS R-568, no [Ca²⁺]_o-induced cytosolic calcium response could be observed.

View larger version (23K): [in this window] [in a new window]   FIG. 3. Effect of R-568 on the sensitivity of wt and CaSRs. Average response (± SEM) in [Ca2+]i in the absence and presence of 1 µM NPS R-568 during stimulation with 3 mM (A) and 10 mM (B) [Ca2+]o. Cells were stepwise stimulated as in Fig. 1, and NPS R-568 was added only during stimulation with 3 or 10 mM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 indicate the significance of the difference in absolute [Ca2+]i increase in the presence or absence of NPS R-568.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

However, not all sequence changes in the CaSR gene are diagnostic. There are a couple of frequently occurring polymorphisms that influence calcium homeostasis but do not inactivate the CaSR to an extent to cause FHH or NSHPT in the heterozygous and homozygous states (20). Therefore, novel mutations in the CaSR gene require functional testing to show an impaired receptor function before a definitive diagnosis of FHH or NSHPT can be made.

Here, we show that all seven novel mutations we found in patients with presumed FHH indeed inactivate the CaSR, and, thus, patients carrying these mutations can be diagnosed with FHH.

However, not all mutations in CaSRs lead to the same phenotype. There is considerable variation in the residual in vitro activity of the CaSR as well as the patients’ serum calcium levels, serum iPTH levels, and the degree of hypocalciuria. This has been attributed to different effects of these mutations in the kidney and the parathyroid gland. This is exemplified by the F881L mutation in the C-terminal tail that leads to hypercalcemia and increased urinary calcium excretion rather than hypocalciuria, thus resembling the classical presentation of primary hyperparathyroidism (21). Two of our patients had mutations in the same region (Q926R, D1005N). Both mutations in the C-terminal tail (Q926R, D1005N) had the lowest EC₅₀ and the highest maximum responses of the seven mutations studied. Interestingly, both patients carrying these mutations had unexpected high urinary calcium excretion in the lower normal range. Whether this is due to the location of the mutations in the C-terminal tail or due to the fact that all three mutants have a relatively mild impairment of receptor function (Ref. 21 and Fig. 1) is currently unknown. Furthermore, the three patients with mutant receptors that prevented proper surface expression had rather normal levels of iPTH, despite a similar degree of hypercalcemia as patients with mutant CaSRs expressed at the cell surface. This may suggest that the presence of mutant CaSRs on the cell surface impairs the function of the wt protein. Heterodimerization of mutant with wt receptor would predict a dominant negative effect. Although cotransfection of the wt with mutant receptors in all cases shifted the EC₅₀ rightward relative to wt, in four of seven cases, this was not statistically significant, likely because of the relatively small shifts observed. Nevertheless, our data are compatible with most of the mutant receptors producing a modest, and in some cases more marked, dominant negative effect (Fig. 1), but alternative mechanisms cannot be excluded. However, there are other factors, such as nutritional status or general health of the patients, that we have not studied but are likely to play a role in the clinical presentation as illustrated by others (20, 22, 23).

The progress in the molecular understanding of CaSR disorders has substantially improved diagnosis in hypercalcemic patients but has not yet translated into significant changes in the treatment of these patients. FHH patients are most often not treated at all, whereas patients with NSHPT usually undergo total or subtotal parathyroidectomy soon after birth. Surgery vastly improves the prognosis of NSHPT patients by reducing excess iPTH, but the impaired calcium sensitivity in other organs such as the kidney persists. Postnatal parathyroidectomy is also unable to prevent possible prenatal damage to the skeletal system, such as multiple fractures and generalized bony demineralization (24). FHH patients in contrast are usually asymptomatic and do not require such an aggressive therapy. However, there is accumulating evidence that FHH and elevated calcium levels might not be as benign as hitherto thought. FHH seems to be associated with pancreatitis (5), and may confer an elevated cardiovascular risk because polymorphisms in the CaSR gene that only mildly affect receptor function and elevate serum calcium appear to increase the risk for coronary heart disease (6).

All these clinical problems could be addressed if the function of mutant CaSRs could be restored. Calcimimetics are highly specific allosteric activators of the CaSR without intrinsic activity in the absence of calcium. They have been shown to augment the effect of calcium on wt CaSRs, and the substance cinacalcet is available in an oral formulation for the treatment of primary and secondary hyperparathyroidism (14, 15). However, the effect on mutant CaSR is not well studied, and, therefore, their therapeutic potential for patients’ FHH or NSHPT is unclear.

In one anecdotal report about a patient with an R220W mutation and recurrent pancreatitis, administration of cinacalcet corrected serum calcium, phosphate and iPTH levels, but no in vitro analysis of the effect of cinacalcet on CaSR signaling was performed (5). In an in vitro study, R-568 was able to rescue the surface expression of misfolded CaSR mutants (25), and in another in vitro study, the calcimimetics L-phenylalanine and NPS R-467 showed positive effects on a variety of mutations in the extracellular part of the CaSR but could not show positive effects on mutations of the C-terminal tail (26).

However, our results indicate that the calcimimetic NPS R-568 can sensitize receptors with mutants of the ECD (W530G and C568Y) as well as mutations in the C-terminal tail (Q926R, D1005N). We did not test whether the patients harboring these mutations would respond to cinacalcet treatment because there were no clinical problems yet requiring treatment. However, our findings that NPS R-568 restores intracellular signaling of mutant receptors at 3 mM [Ca²⁺]_i to wt levels suggest that calcimimetics would work in patients with FHH or NSHPT, and we will consider administration of cinacalcet if these patients or their offspring at risk for NSHPT should require medical treatment in the future. Therefore, functional studies of the in vitro effect of calcimimetics on certain mutants may provide important information on whether patients may respond to cinacalcet or not.

Interestingly, all four mutants that were expressed on the cell surface as judged by Western blot showed at least some improvement in receptor function in response to NPS R-568. If this holds true for more of the about 130 different mutations currently present in the CaSR database (www.casrdb.mcgill.ca), patients with inactivating mutations in the CaSR might soon have more treatment options for FHH and NSHPT.

	Acknowledgments

 
We thank Jianxin Hu and Allen Spiegel for providing the human calcium-sensing receptor expression vector pCR3.1/hCaSR, Amgen for providing NPS R-568, and M. Schaefer for providing human embryonic kidney 293 cells. We also thank Susanne Schnell, Janin Andres, and especially Anja Völzke for technical and methodological assistance.

	Footnotes

 
This work was supported by institutional grants from the Charité-University Medicine Berlin and from the Friedrich-Alexander University Erlangen-Nuremberg.

Disclosure Statement: The authors have nothing to disclose.

First Published Online September 16, 2008

Abbreviations: CaSR, Calcium-sensing receptor; [Ca²⁺]_o, extracellular calcium; ECD, extracellular domain; FHH, familial hypocalciuric hypercalcemia; HEK, human embryonic kidney; ICL, intracellular loop; iPTH, intact PTH; [Ca²⁺]_i, intracellular free calcium; NSHPT, neonatal severe hyperparathyroidism; TMD, transmembrane domain; wt, wild type.

Received May 19, 2008.

Accepted September 9, 2008.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

1. Brown EM, Gamba G, Riccardi D, Lombardi M, Butters R, Kifor O, Sun A, Hediger MA, Lytton J, Hebert SC 1993 Cloning and characterization of an extracellular Ca(2+)-sensing receptor from bovine parathyroid. Nature 366:575–580[CrossRef][Medline]
2. Brown EM, MacLeod RJ 2001 Extracellular calcium sensing and extracellular calcium signaling. Physiol Rev 81:239–297[Abstract/Free Full Text]
3. Pearce SH, Brown EM 1996 Disorders of calcium ion sensing. J Clin Endocrinol Metab 81:2030–2035[CrossRef][Medline]
4. Gunn IR, Gaffney D 2004 Clinical and laboratory features of calcium-sensing receptor disorders: a systematic review. Ann Clin Biochem 41:441–458[Abstract/Free Full Text]
5. Festen-Spanjer B, Haring CM, Koster JB, Mudde AH 2008 Correction of hypercalcaemia by cinacalcet in familial hypocalciuric hypercalcaemia. Clin Endocrinol (Oxf) 68:324–325[Medline]
6. Marz W, Seelhorst U, Wellnitz B, Tiran B, Obermayer-Pietsch B, Renner W, Boehm BO, Ritz E, Hoffmann MM 2007 Alanine to serine polymorphism at position 986 of the calcium-sensing receptor associated with coronary heart disease, myocardial infarction, all-cause, and cardiovascular mortality. J Clin Endocrinol Metab 92:2363–2369[Abstract/Free Full Text]
7. Janicic N, Soliman E, Pausova Z, Seldin MF, Riviere M, Szpirer J, Szpirer C, Hendy GN 1995 Mapping of the calcium-sensing receptor gene (CASR) to human chromosome 3q13.3–21 by fluorescence in situ hybridization, and localization to rat chromosome 11 and mouse chromosome 16. Mamm Genome 6:798–801[CrossRef][Medline]
8. Bai M, Trivedi S, Brown EM 1998 Dimerization of the extracellular calcium-sensing receptor (CaR) on the cell surface of CaR-transfected HEK293 cells. J Biol Chem 273:23605–23610[Abstract/Free Full Text]
9. Ward DT, Brown EM, Harris HW 1998 Disulfide bonds in the extracellular calcium-polyvalent cation-sensing receptor correlate with dimer formation and its response to divalent cations in vitro. J Biol Chem 273:14476–14483[Abstract/Free Full Text]
10. Zhang Z, Sun S, Quinn SJ, Brown EM, Bai M 2001 The extracellular calcium-sensing receptor dimerizes through multiple types of intermolecular interactions. J Biol Chem 276:5316–5322[Abstract/Free Full Text]
11. Pidasheva S, Grant M, Canaff L, Ercan O, Kumar U, Hendy GN 2006 Calcium-sensing receptor dimerizes in the endoplasmic reticulum: biochemical and biophysical characterization of CASR mutants retained intracellularly. Hum Mol Genet 15:2200–2209[Abstract/Free Full Text]
12. Bai M, Trivedi S, Kifor O, Quinn SJ, Brown EM 1999 Intermolecular interactions between dimeric calcium-sensing receptor monomers are important for its normal function. Proc Natl Acad Sci USA 96:2834–2839[Abstract/Free Full Text]
13. Hu J, Spiegel AM 2003 Naturally occurring mutations of the extracellular Ca2+-sensing receptor: implications for its structure and function. Trends Endocrinol Metab 14:282–288[CrossRef][Medline]
14. Nemeth EF, Steffey ME, Hammerland LG, Hung BC, Van Wagenen BC, DelMar EG, Balandrin MF 1998 Calcimimetics with potent and selective activity on the parathyroid calcium receptor. Proc Natl Acad Sci USA 95:4040–4045[Abstract/Free Full Text]
15. Cohen A, Silverberg SJ 2002 Calcimimetics: therapeutic potential in hyperparathyroidism. Curr Opin Pharmacol 2:734–739[CrossRef][Medline]
16. Haag C, Schulze E, Lorenz A, Frank-Raue K, Raue F Novel mutations of the calcium-sensing receptor in familial hypocalciuric hypercalcemia and autosomal dominant hypocalcemia. Program of the 87^th Annual Meeting of The Endocrine Society, San Diego, CA, 2005 (Abstract P2-407)
17. Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR 1989 Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59[CrossRef][Medline]
18. Grynkiewicz G, Poenie M, Tsien RY 1985 A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450[Abstract/Free Full Text]
19. Wills MR 1969 The urinary calcium-creatinine ratio as a measure of urinary calcium excretion. J Clin Pathol 22:287–290[Abstract/Free Full Text]
20. Scillitani A, Guarnieri V, De Geronimo S, Muscarella LA, Battista C, D'Agruma L, Bertoldo F, Florio C, Minisola S, Hendy GN, Cole DE 2004 Blood ionized calcium is associated with clustered polymorphisms in the carboxyl-terminal tail of the calcium-sensing receptor. J Clin Endocrinol Metab 89:5634–5638[Abstract/Free Full Text]
21. Carling T, Szabo E, Bai M, Ridefelt P, Westin G, Gustavsson P, Trivedi S, Hellman P, Brown EM, Dahl N, Rastad J 2000 Familial hypercalcemia and hypercalciuria caused by a novel mutation in the cytoplasmic tail of the calcium receptor. J Clin Endocrinol Metab 85:2042–2047[Abstract/Free Full Text]
22. Zajickova K, Vrbikova J, Canaff L, Pawelek PD, Goltzman D, Hendy GN 2007 Identification and functional characterization of a novel mutation in the calcium-sensing receptor gene in familial hypocalciuric hypercalcemia: modulation of clinical severity by vitamin D status. J Clin Endocrinol Metab 92:2616–2623[Abstract/Free Full Text]
23. Canaff L, Hendy GN 2005 Calcium-sensing receptor gene transcription is up-regulated by the proinflammatory cytokine, interleukin-1β. Role of the NF-B PATHWAY and B elements. J Biol Chem 280:14177–14188[Abstract/Free Full Text]
24. Spiegel AM, Harrison HE, Marx SJ, Brown EM, Aurbach GD 1977 Neonatal primary hyperparathyroidism with autosomal dominant inheritance. J Pediatr 90:269–272[CrossRef][Medline]
25. Huang Y, Breitwieser GE 2007 Rescue of calcium-sensing receptor mutants by allosteric modulators reveals a conformational checkpoint in receptor biogenesis. J Biol Chem 282:9517–9525[Abstract/Free Full Text]
26. Zhang Z, Jiang Y, Quinn SJ, Krapcho K, Nemeth EF, Bai M 2002 L-phenylalanine and NPS R-467 synergistically potentiate the function of the extracellular calcium-sensing receptor through distinct sites. J Biol Chem 277:33736–33741[Abstract/Free Full Text]

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