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Original Studies |
Service de Pédiatrie 2 (A.L., J.B.), Centre Hospitalier Universitaire, 87042 Limoges, France; Centre National de la Recherche Scientifique Unité Proper de Recherche 1524 (M.G., C.S.), Hôpital Saint Vincent de Paul, 75014 Paris, France; Endocrine-Hypertension Division (M.B., Z.Z., E.M.B.), Department of Medicine, Brigham and Women’s Hospital, and Harvard Medical School, Boston, Massachusetts 02115; Unité de Biologie Moléculaire (J.-P.L., M.-L.K.), Service de Biochimie Médicale, AP-HP, Hôpital Pitié-Salpétrière, 75013 Paris, France; and Service de Biochimie (A.L., M.R.), Faculté de Médecine, 87025 Limoges, France
Address correspondence and requests for reprints to: Dr. Anne Lienhardt, Service de Pédiatrie 2, Centre Hospitalier Universitaire Dupuytren, 2 avenue Martin Luther King, 87042 Limoges cedex, France. E-mail: anne.lienhardt{at}unilim.fr
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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600 amino acids) extracellular domains and long
carboxylterminal (C) intracellular tails. Soon after the cloning of the
CaSR, heterozygous activating mutations within its gene were reported
as a cause of familial hypoparathyroidism (14). The elucidation of the
molecular basis for this form of familial hypoparathyroidism with a
dominant inheritance pattern identified a distinct clinical entity
among the various forms of hypoparathyroidism, autosomal dominant
hypocalcemia (ADH) (15). ADH is a rare inherited disease that can come to clinical attention at any time of life. The clinical presentation is variable, ranging from asymptomatic forms to severe neonatal seizures. However, the biochemical features of the condition are more uniform, showing mild-to-moderate or, occasionally, more severe, hypocalcemia and hyperphosphatemia accompanied by low or normal serum levels of PTH and normal or elevated levels of urinary calcium excretion despite low serum calcium concentrations (15, 16, 17).
We now report a family with mild hypocalcemia inherited as a dominant trait that is caused by an unusual mutation in the CaSR gene (a deletion within its C-terminal tail), resulting in a substantially shortened C-tail. Furthermore, we demonstrate that the mutant receptor exhibits a gain-of-function, showing a reduction in the level of the extracellular calcium concentration (Ca2+o), producing half-maximal CaSR-elicited increases in the cytosolic calcium concentration (Ca2+i), presumably caused by receptor-mediated activation of phospholipase C (18). This naturally occurring deletion in the CaSR’s C-tail provides insights into the normal structure-function relationships of this part of the receptor and affords direct, in vivo support of previous in vitro observations that truncations within the CaSR’s C-tail can lead to increased cell surface expression and activity of this receptor (19). Finally, to our knowledge, the mutation in the CaSR gene in this family provides the first example of a homozygous, affected individual in a kindred with ADH.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Seven members from the same kindred were studied (five males and two females). Informed consent was obtained from all subjects, according to the guidelines of the French consultative committee for the protection of human studies.
Amplification of genomic DNA and sequence analysis
Samples of venous blood were obtained, and genomic DNA was extracted from leukocytes using a proteinase K-phenol-chloroform procedure (20). Exons 2–7 of the CaSR gene, encompassing the entire coding sequence, were amplified using the PCR with previously reported primers (21, 22), except for exon 7, which was amplified in two segments: 1) segment A, the 5' portion; and 2) segment B, the remainder of the exon. The sequences of the primers were as follows: 7B: 5'-GTCTGGATCTCC TTCATTCCA-3' (nucleotides 2449–2469); 7BR: 5'-TCTGGGGGATTCCTCAT CCC-3' (within the 3' non-coding, flanking region of the gene); and 7BDR: 5'-TTTCTGTAACAGTGCTGCCTC-3' (nucleotides 3220–3200). Primers 7B, 7BR, and 7BDR were designed to amplify the segment B, based on the DNA sequence deposited in GenBank (accession number X81086). The primer pair 7B and 7BR amplifies all of B, whereas the primer pair 7B and 7BDR amplifies a portion of B from the wild-type (WT) CaSR gene. PCR products were electrophoresed on 1% agarose gels, visualized with ethidium bromide, and then purified on Microcon-100 columns (AMICON, Beverly, MA). Both strands of the products were directly sequenced using the Amplitaq dye Terminator Cycle Sequencing kit and an AB PRISM 377 DNA sequencer (Perkin-Elmer Corp., Roissy, France).
Construction of the Flag-tagged mutant CaSR
The mutation identified in this family’s CaSR gene (a large deletion within the C-tail) was engineered into a reconstructed WT CaSR complementary DNA (cDNA) by using the PCR as follows: cassette 6 (23) of the reconstructed CaSR cDNA, containing the deletion in this family’s CaSR, was amplified using a pair of primers with the sequences, 5'-CGGGGTACCTCGAGGATGAGATCATCTTCAT-3' and 5'-GCTCTAGATTATG AATTCACTACGTTGCTGCGGCGCAG-3'. The PCR product was subsequently digested with XhoI and XbaI and ligated to the larger of the digested fragments of the reconstructed, Flag-tagged, WT receptor in pcDNA3 to produce the mutant receptor in a form suitable for expression studies (23). The presence of the mutation was confirmed by direct sequencing.
Transient expression of the WT CaSR and the mutated CaSR harboring the deletion within the C-tail in HEK293 cells
DNA was prepared using the Midi Plasmid kit (QIAGEN, Chatsworth, CA). Lipofectamine (Life Technologies, Gaithersburg, MD) was employed as the DNA carrier for transfection. Human embryonic kidney (HEK293) cells (provided by NPS Pharmaceuticals, Inc., Salt Lake City, UT) were cultured in DMEM (Life Technologies). Transient transfection was performed, as previously described, by adding a DNA-lipofectamine mixture diluted with OPTI-MEM 1 Reduced Serum Medium (Life Technologies) to 90% confluent HEK293 cells plated in 13.5 x 20.1-mm glass coverslips for measurement of CaSR-mediated changes in Ca2+i or in six-well plates for preparation of cellular proteins for Western analysis using 0.625 µg cDNA (23). After 5 h incubation at 37 C, an amount of OPTI-MEM 1 Reduced Serum Medium with 20% FCS equal to that present in the wells was added to the transfected cells, which was then replaced with fresh DMEM and 10% FCS at 24 h after the start of transfection. The expressed CaSR protein was assayed at 48 h after transfection. To perform coexpression of the WT and mutant receptors, 0.625 µg of each cDNA were mixed and used to transfect HEK293 cells, as described above.
Measurement of Ca2+i by fluorimetry in cell populations
Coverslips with nearly confluent HEK293 cells previously transfected with the appropriate CaSR cDNAs were loaded for 2 h at room temperature with fura-2/AM (Molecular Probes, Inc., Eugene, OR) in 20 mmol/L HEPES (pH 7.4) containing 125 mmol/L NaCl, 4 mmol/L KCl, 1.25 mmol/L CaCl2, 1 mmol/L MgSO4, 1 mmol/L NaH2PO4, 0.1% BSA, and 0.1% dextrose and were washed once with bath solution (20 mmol/L HEPES (pH 7.4) containing 125 mmol/L NaCl, 4 mmol/L KCl, 0.5 mmol/L CaCl2, 0.5 mmol/L MgCl2, 0.1% dextrose, and 0.1% BSA) at 37 C for 20 min. The coverslips were then placed diagonally in a thermostatted quartz cuvette containing the bath solution using a modification of the technique employed previously in this laboratory (23). Extracellular calcium was increased stepwise to give the desired final concentrations with additions of Ca2+o in increments of 1 mmol/L, which were followed by 5 mmol/L increments after achieving a level of 5.5 mmol/L Ca2+o. Excitation monochrometers were centered at 340 nm and 380 nm, and emitted light was collected at 510 ± 40 nm through a wide-band emission filter. The 340/380 excitation ratio of emitted light was used to evaluate changes in Ca2+i, as described previously (23).
Western analysis of CaSRs expressed on the cell surface and in whole cell lysates
Before preparing whole-cell lysates, intact HEK293 cells transiently transfected with the Flag-tagged WT or mutated CaSR were labeled with ImmunoPure Sulfo-NHS-Biotin (Pierce Chemical Co., Rockford, IL) (23). The whole cell lysate was prepared in a nondenaturing buffer [1% Triton X-100, 0.5% NP-40, 150 mmol/L NaCl, 10 mmol/L Tris-HCl (pH 7.4), 2 mmol/L EDTA, 1 mmol/L EGTA, 100 µM iodoacetamide, and a cocktail of protease inhibitors including 83 µg/mL aprotinin, 30 µg/mL leupeptin, 1 mg/mL pefabloc, 50 µg/mL calpain inhibitor, 50 µg/mL bestatin, and 5 µg/mL pepstatin]. Flag-tagged CaSRs were solubilized and immunoprecipitated with anti-Flag M2 monoclonal antibody (VWR Scientific, Bridgeport, NJ), resolved by SDS-polyacrylamide gel electrophoresis (PAGE, 5%–9% gradient gel) under reducing conditions, and blotted on nitrocellulose membranes. After blocking with 5% milk, the forms of the receptor present on the cell surface were detected using an avidin-horseradish peroxidase conjugate (Bio-Rad Laboratories, Inc., Rockville Center, NY), followed by visualizing the biotinylated bands with an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Piscataway, NJ). After removal of the avidin, using the recommended procedure for stripping the blots (Amersham Pharmacia Biotech), all forms of the CaSR on the same blot were detected using anti-CaSR antiserum, 4641, a polyclonal antiserum raised against a peptide within the extracellular domain of the CaSR (corresponding to residues 214–236 of the human CaSR; kindly provided by Drs. Forrest Fuller and Rachel Simin at NPS Pharmaceuticals, Inc.), followed by a secondary, horseradish peroxidase-conjugated goat antirabbit antibody and then an enhanced chemiluminescence system (Amersham Pharmacia Biotech).
Statistical analysis
The mean EC50 (the effective concentration of Ca2+o giving one half of the maximal response) for the WT or mutant receptors in response to increasing concentrations of Ca2+o were calculated from the EC50 s for all of the individual experiments and were expressed with the SEM as the index of dispersion. Comparison of the EC50 s was performed using ANOVA. A P value of less than or equal to 0.05 was considered to indicate a statistically significant result.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Seven members of the family were studied, and the family pedigree
is shown in Fig. 1A
: two family members
are normal, with respect to calcium metabolism (II1 and III3), and five
are affected (I2, II2, III1, III2, and III4). Pertinent clinical
features of the affected patients are reported in Table 1, and Table 2 summarizes the biological data
including those of subject III3. None had severe clinical signs or
symptoms of hypocalcemia. The oldest was discovered to have
hypocalcemia during a hospitalization after an accidental fall; later,
he reported mild muscle cramps of many years duration. After the
discovery of this individual’s hypocalcemia, no further family studies
were carried out at that time. Eight years later, his daughter (subject
II2) presented with muscle cramps during her second pregnancy and was
found to be hypocalcemic. Both of these family members felt otherwise
well. A family survey was then performed. A total of four affected
individuals have experienced transient signs of neuromuscular
irritability. All affected members had, before treatment, total and
ionized hypocalcemia and showed normal or elevated rates of urinary
calcium excretion despite their low serum calcium concentrations.
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An initial screening was performed of the PCR products amplified
from the various regions of the coding sequence of the CaSR gene using
agarose gel electrophoresis. Within the distal half of the seventh
exon, affected family members exhibited a PCR product that differed
from that amplified from unaffected members, as shown in Fig. 1B
. One
band at the predicted size of 868 bp was detected in the products
amplified from the 2 unaffected members of the family (II1 and III3),
whereas 2 bands were detected in 4 of the affected individuals (II2,
III1, III2, and III4). The larger of the 2 bands was of the expected
size, and the smaller was about 325 bp in length. Only the smaller band
was amplified from genomic DNA of patient I2. Direct sequencing of the
smaller band revealed a deletion of 543 bp within exon 7, beginning at
nucleotide 2682 and ending at nucleotide 3224 (Fig. 2). The messenger RNA species generated
as a result of the deletion is predicted to encode a protein of only
897 amino acids, with a deletion of 181 residues within the cytoplasmic
tail, from Serine 895 to Valine 1075, and then terminating with the 3
residues normally present at the end of the CaSR’s C-tail (also see
Fig. 3). The smaller band observed on gel
electrophoresis corresponds to the PCR product amplified from the
mutant allele, whereas the larger band corresponds to that amplified
from the normal allele. The deletion was inherited as a heterozygous
trait in affected family members II2, III1, III2, and III4, which is
compatible with the diagnosis of ADH in this family. Using primers 7B
(designed within the seventh exon) and 7BDR (designed within the
deletion), only one band at the predicted size of 771 bp was found
after PCR amplification of the DNA extracted from all members of the
family, normal and affected, except patient I2. No band was visualized
for patient I2, confirming his homozygous genotype, with respect to the
deletion (data not shown). Moreover, we screened DNA from 50 normal
unrelated individuals using gel electrophoresis of PCR products
amplified from the second part of the seventh exon; all had a single
band at the expected size for a product amplified from the normal CaSR
gene (data not shown).
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Figure 4 shows typical dose-response
curves for the WT and mutated receptors, with respect to the elevations
in Ca2+i elicited by increasing
levels of Ca2+o when expressed transiently in
HEK293 cells. Compared with the WT CaSR, the mutant receptor exhibited
a statistically significantly left-shifted dose-response curve with a
reduced EC50 for
Ca2+o-induced changes in
Ca2+i (2.2 mmol/L vs.
3.3 mmol/L). When the mutant receptor was cotransfected with the WT
CaSR, to mimic the in vivo heterozygous state, the resultant
EC50 did not differ statistically from that of
the mutant receptor expressed by itself.
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Figure 5A shows that the mutant
receptor was expressed on the cell surface at a substantially higher
level than that of the WT CaSR. In addition, the ratio of the mature
species of the receptor (the higher of the two bands in the doublet at
140–160 kDa) (23) to the immature one (the lower band of the doublet)
is much greater for the mutant than for the WT CaSR (Fig. 5B),
suggesting that the mutant receptor CaSR is processed much more
efficiently than the WT receptor.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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). We have now
identified a large, in-frame deletion of 181 amino acids within the
CaSR’s C-tail between Serine 895 (S895) to Valine 1075 (V1075) that is
followed by a short sequence of three amino acid residues that are
normally present at the CaSR’s extreme C-terminus. In vitro
functional expression of a mutant receptor, engineered to include this
deletion, confirms that it exhibits the expected gain-of-function. That
is, the dose-response curve for the mutant receptor showed a
significant leftward shift for its activation by elevated levels of
Ca2+o, the CaSR’s principal
physiological agonist, which reduced the EC50
from 3.3 mmol/L for the WT to 2.2 mmol/L for the mutant receptor. In
accord with previous results observed with a CaSR engineered to include
a truncated C-tail (S892stop) (30), this large deletion of the tail is
characterized by a higher biological activity (i.e. reduced
EC50) than that of the WT receptor, which could
readily account for the clinical and biochemical features of affected
family members. Therefore, we can conclude that this family represents
another example of ADH, although it represents one caused by a novel
form of CaSR mutation that has not be described previously,
i.e. a truncation within the C-tail. To date, gain-of-function mutations in GPCRs have been described in the heterozygous state, as is also the case for the previously described families with ADH and sporadic cases of activating mutations causing the same biochemical phenotype (14, 17, 23, 24, 25, 26, 27, 28). The first-generation patient of the present family seems to be the first case of ADH caused by an activating mutation of the CaSR present in the homozygous state. Perhaps surprisingly, this patient’s clinical and biochemical features were no more severe than those of family members heterozygous for the same mutation. To explore further the basis for the similar clinical features in this homozygous individual and in heterozygous family members, we compared the EC50 for Ca2+o-evoked increases in Ca2+i in HEK293 cells transfected with 1) the WT CaSR cDNA alone; 2) the mutated CaSR cDNA receptor alone (to reproduce the homozygous state); or 3) both the WT and the mutated CaSR cDNAs (to mimic the heterozygous state). The EC50 of the M/M and the M/WT transfected cells were not significantly different (2.2 mmol/L vs. 2.2 mmol/L). Therefore, the homozygous state of this deletion mutation does not produce a greater gain-of-function than observed for the heterozygous state.
Recent in vitro studies have shown that the CaSR is expressed on the cell surface principally as a dimer, and the cytoplasmic tail of the receptor is not required for dimerization (30). The dimerization of the CaSR, however, seems to be functionally significant, because heterodimeric CaSR comprising two different, individually inactive mutated CaSRs can reconstitute signal transduction (31). This result strongly suggests that the two monomers within the dimeric CaSR can interact in some manner to produce optimal activation of signal transduction. Moreover, one inactivating missense CaSR mutation (R185Q) exhibits a dominant negative effect on the cotransfected WT monomer, leading to substantial loss of function when coexpressed in HEK293 cells (23, 32). The mutant CaSR identified in the family with ADH described here is expressed at more robust levels on the cell surface than the WT CaSR. Moreover, functional studies in vitro showed that it exhibits similar Ca2+o-evoked Ca2+i responses when transfected alone or when cotransfected with the WT receptor. Therefore, this mutated allele may exert a dominant positive effect on the WT CaSR allele, just the opposite of the dominant negative action of R185Q (23, 32). This postulated mechanism is in accord with the similar clinical features of affected members of the family described here, despite differing gene dosages. That is, the homozygous patient remained asymptomatic into late adulthood, and his phenotype is no more severe than those of the other affected members of the family who are heterozygous for the mutation.
What are the mechanisms through which this mutation could produce gain-of-function? The CaSR belongs to a recently identified and growing family of GPCRs (the family C GPCRs) characterized by very large extracellular domains and long cytoplasmic tails (218 residues in the case of the human CaSR). The introduction of truncations and point mutations within the CaSR’s C-tail have recently shown that the tail modulates several aspects of the receptor’s function, including signal transduction, intracellular receptor trafficking, and the level of cell surface expression (30, 33, 34, 35). Residues from 868 to 886 have been identified as crucial for normal signal transduction (30, 33, 34, 35). Receptors with deletions of more than 200 amino acids (e.g. those truncated at residues 863, 865, 874, or 877) are inactive despite exhibiting levels of cell surface expression equivalent to that of the WT receptor, if not more, whereas those with truncations of a lesser degree vary in their behavior (30, 33, 34, 35). Truncation at residue 892 results in a CaSR that is not only functional but supranormal in its level of activity, as evidenced by its lower-than-normal EC50 (3.2 ± 0.1 mmol/L vs. 4 ± 0.2 mmol/L for the WT CaSR) (30). A CaSR truncated at residue 903 also exhibits a greater level of cell surface expression than the WT CaSR (33), although its EC50 for Ca2+o-evoked increases in inositol phosphates are similar to that of the WT receptor.
The naturally occurring deletion mutant identified in this study is located between residues 892 and 903. Similar to the latter two truncated receptors, the receptor studied here is also expressed on the cell surface at a greater level than the WT CaSR, as assessed by Western blot analysis and cell surface labeling. Therefore, one possible mechanism underlying its left-shifted EC50 could be a greater cell surface density of the mutant receptor. This mechanism is the converse of what is observed in mice heterozygous or homozygous for targeted disruption of the CaSR gene. Heterozygotes, in which there is about a 50% reduction in the level of the receptor in parathyroid gland (as assessed by immunohistochemistry), show a modest rightward shift in the set-point of the parathyroid gland for Ca2+o-induced inhibition of PTH secretion, whereas homozygotes, exhibiting an essentially complete loss of receptor from parathyroid gland, show a severe increase in set-point (36). The gain-of-function of this mutant receptor could also be explained by additional mechanisms. Indeed, two different mechanisms unrelated to cell surface expression might lead to a gain-of-function of a GPCR. First, numerous naturally occurring mutations of several different GPCRs are thought to mimic the conformational change(s) associated with normal ligand-induced activation of the GPCR and produce constitutively increased receptor activity that then modulates intracellular signaling pathways at lower-than-normal concentrations of agonist (37, 38, 39, 40, 41, 42, 43, 44, 45, 46). In contrast to these mutations localized along the extracellular, transmembrane, or loops domains, the CaSR mutation described here is a large deletion occurring in the cytoplasmic C-tail between S895 to V1075. Though it is not clear how this mutation would produce conformational changes similar to those caused by the CaSR’s agonists, we cannot rule out that such a mechanism occurs. Second, there might be deficient attenuation of signal transduction after initiation of signaling by the mutant CaSR, given that the C-tails of numerous GPCRs have been shown to play critical roles in attenuating signal transduction through diverse mechanisms, which include receptor desensitization, endocytosis, and down-regulation (47). For example, the rat gonadotropin releasing hormone receptor, which lacks a cytoplasmic tail, undergoes acute desensitization and accelerated internalization when a functional intracellular C-terminal tail is added (48). Thus, the in vivo and in vitro results in our study could also be consistent with a potential role for the portion of the CaSR’s C-tail between residues 895 and 1075 in endocytosis and/or down-regulation of this receptor, leading to its higher cell-surface expression and allowing more ligand-binding domains. Further in vitro studies will be necessary to explore the possible role of the CaSR’s C-tail in these processes.
In summary, we have identified a family with ADH caused by a deletion of 181 amino acids within the C-terminal cytoplasmic tail of the CaSR. This large deletion leads to a greater cell surface expression of the CaSR and causes a gain-of-function that is responsible for the disease. Surprisingly, the presence of the mutation in the homozygous state in one affected family member does not cause a more severe phenotype, possibly because of a dominant positive effect of the mutant CaSR on the WT receptor when expressed together. This naturally occurring, large deletion of the CaSR’s C-tail confirms the conclusions drawn from several previous in vitro studies. That is, a large portion of the CaSR’s C-terminal tail is not indispensable for its biological activity but contains determinants that may constrain the receptor’s expression and/or its level of intrinsic activity or participate in the attenuation of signal transduction. To our knowledge, this is the first report of an activating mutation resulting from a large, naturally occurring deletion of the cytoplasmic tail of a GPCR.
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Acknowledgments |
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Footnotes |
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Received October 1, 1999.
Revised January 5, 2000.
Accepted January 6, 2000.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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X174 with
HaeIII (Bioprobe, Quantum, Montreuil, France); T, PCR
control without added DNA.
