Download A Large Homozygous or Heterozygous In

0021-972X/00/$03.00/0
The Journal of Clinical Endocrinology & Metabolism
Copyright © 2000 by The Endocrine Society
Vol. 85, No. 4
Printed in U.S.A.
A Large Homozygous or Heterozygous In-Frame Deletion
within the Calcium-Sensing Receptor’s Carboxylterminal
Cytoplasmic Tail That Causes Autosomal
Dominant Hypocalcemia*
ANNE LIENHARDT, MICHÈLE GARABÉDIAN, MEI BAI, CHRISTIANE SINDING,
ZAIXIANG ZHANG, JEAN-PIERRE LAGARDE, JEAN BOULESTEIX,
MICHEL RIGAUD, EDWARD M. BROWN, AND MARIE-LAURE KOTTLER
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
ABSTRACT
Autosomal dominant hypocalcemia (ADH) can result from heterozygous missense activating mutations of the calcium-sensing receptor (CaSR) gene, a G-protein-coupled receptor playing key roles in
mineral ion metabolism. We now describe an ADH kindred of three
generations caused by a novel CaSR mutation, a large in-frame deletion of 181 amino acids within its carboxylterminal-tail from S895
to V1075. Interestingly, the affected grandfather is homozygous for
the deletion but no more severely affected than heterozygous affected
individuals. Functional properties of mutant and wild-type (WT)
CaSRs were studied in transiently transfected, fura-2-loaded human
embryonic kidney (HEK293) cells. The mutant receptor exhibited a
I
SOLATED hypoparathyroidism is a disorder of calcium metabolism characterized by hypocalcemia associated with a
low level of serum PTH. Although most cases are sporadic,
some have autosomal dominant, autosomal recessive or Xlinked modes of inheritance (1). Linkage analysis performed in
large families with autosomal dominant hypoparathyroidism
mapped a candidate gene to the locus 3q13, corresponding to
the region of chromosome 3 harboring the gene encoding the
human calcium-sensing receptor (CaSR) (2– 4). The bovine receptor was first cloned in 1993 (5) and then the human CaSR in
1995 (6); it belongs to a new subfamily of the G-protein-coupled
receptors (GPCR) that includes the metabotropic glutamate receptors (7, 8), gamma aminobutyric acid (GABAB) receptors (9),
putative pheromone receptors (10 –12), and other recently
cloned taste receptors (13). This subfamily of GPCRs is charReceived October 1, 1999. Revision received January 5, 2000. Accepted January 6, 2000.
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: [email protected].
* Generous grant support for this work was provided by The Faculté
de Médecine de Limoges, L’Assistance Publique-Hôpitaux de Paris,
France and by The St. Giles Foundation (to E.M.B.) and NIH Grants
DK52005 (to E.M.B.), DK48330 (to E.M.B.), and DK54934 (to M.B.).
gain-of-function, but there was no difference between cells transfected
with mutant complementary DNA alone or cotransfected with mutant
and WT complementary DNAs, consistent with the similar phenotypes of heterozygous and homozygous family members. Therefore,
this activating deletion may exert a dominant positive effect on the
WT CaSR. The mutant receptor’s cell surface expression was greater
than that of the WT CaSR, potentially contributing to its gain-offunction. This novel mutation in the CaSR gene provides the first
known examples of a large naturally occurring deletion within a
G-protein-coupled receptor’s carboxylterminal-tail and of a homozygous, affected individual with ADH. (J Clin Endocrinol Metab 85:
1695–1702, 2000)
acterized by very large (⬃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 (15–17).
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
1695
1696
LIENHARDT ET AL.
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 structurefunction 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.
Subjects and Methods
Patients
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 (PerkinElmer 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
JCE & M • 2000
Vol 85 • No 4
(Life Technologies) to 90% confluent HEK293 cells plated in 13.5 ⫻
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 ␮g 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 ␮g 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 ␮m iodoacetamide, and a cocktail
of protease inhibitors including 83 ␮g/mL aprotinin, 30 ␮g/mL leupeptin,
1 mg/mL pefabloc, 50 ␮g/mL calpain inhibitor, 50 ␮g/mL bestatin, and 5
␮g/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.
LARGE DELETION WITHIN THE CaSR TAIL
Results
Clinical features of affected and unaffected family members
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.
PCR and DNA sequence analysis of the mutation
An initial screening was performed of the PCR products
amplified from the various regions of the coding sequence of
FIG. 1. A, Pedigree of the family with dominant hypocalcemia. Patients I2, II2, III1, III2, and III4 are affected; hatching indicates
affected family members with the heterozygous state, and the filled
symbol denotes a family member with the homozygous state. B, Analysis of PCR products amplified from the distal half of Exon 7 from
affected and unaffected family members. PCR products amplified
from genomic DNA, using primers 7B/7BR, were analyzed by electrophoresis on a 1% agarose gel. M, Molecular weight markers generated by digestion of DNA from bacteriophage ⌽X174 with HaeIII
(Bioprobe, Quantum, Montreuil, France); T, PCR control without
added DNA.
1697
TABLE 1. Clinical features of the 5 hypocalcemic patients in the
untreated state
Patient
Age at diagnosis
(years)
I2
II2
III1
50
28
5
III2
III4
Mode of presentation at diagnosis
Muscle cramps, general fatigue
Muscle cramps during pregnancy
Febrile neuromuscular irritability
(Normocalcemic at birth)
Birth (familial survey) Asymptomatic neonatal
hypocalcemia
Paresthesias at 8
Birth (familial survey) Asymptomatic neonatal
hypocalcemia
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).
Functional study of the mutant CaSR containing the
deletion in the C-tail
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 leftshifted dose-response curve with a reduced EC50 for Ca2⫹oinduced changes in Ca2⫹i (2.2 mmol/L vs. 3.3 mmol/L).
1698
JCE & M • 2000
Vol 85 • No 4
LIENHARDT ET AL.
TABLE 2. Biological features of the patients before treatment
Patient
I2
II2
III1
III2
III3
(Unaffected)
III4
Serum calcium (mg/dL)
b
6.6–7.6
[9.0–10.2]
6.5–7.1b
[9.0–10.2]
7.1–7.3b
[9.0–10.2]
7.1–7.6b
[8.8–10.4]
8.7–9.9
[8.8–10.4]
7.4– 8.8b
[8.8–10.4]
Ionized Ca (mg/dL)
Serum PO4 (mg/dL)
Serum PTHa (pg/mL)
Urinary Ca (mg/kg䡠day)
ND
3.6– 4.2
[2.5– 4.6]
4.1– 4.9
[2.5– 4.6]
7.0–7.6b
[3.7–5.3]
6.3–7.0
[5.6– 6.8]
5.8– 6.1
[5.6– 6.8]
5.4– 6.8
[5.6– 6.8]
0.5 ng/mlb
[1– 6]
5–12b
[15– 60]
7b
[15– 60]
8b
[15– 60]
17–22
[15– 60]
2– 6b
[15– 60]
245 mg/dc
[⬍250]
148 mg/dc
[⬍250]
1 –5b
[⬍4]
4 – 6b
[⬍4]
0.69
[⬍4]
4
[⬍4]
3.4–3.6b
[4.6–5.2]
3.3–3.9b
[4.8–5.4]
3.4– 4.2b
[4.8–5.4]
ND
3.7b
[4.8–5.4]
ND, Not determined. Reference values according to age are given in brackets for each parameter.
a
An immunoradiometric assay specific for intact PTH (1– 84) was used for patients in the 2nd and 3rd generations, whereas a C-terminal
PTH assay was used for patient I2.
b
Abnormal values.
c
Urinary calcium excretion is given as mg/d for the adult patients.
FIG. 2. Direct sequencing of the mutated CaSR allele shows a large deletion
within the distal portion of exon 7 from
nucleotide (NT) 2682 to 3224. The corresponding amino acid (AA) sequence is
indicated below. International abbreviations have been used for the nucleotides and amino acids.
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.
Western analysis of the transiently expressed WT and
mutant CaSRs
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.
Discussion
In the present study, we have identified a novel mutation
in the CaSR gene associated with familial ADH. The clinical
and biochemical features of the affected members of this
LARGE DELETION WITHIN THE CaSR TAIL
1699
FIG. 3. Schematic diagram of the human CaSR. The shaded areas depict the
transmembrane domains (TM); gainof-function mutations previously described are identified by asterisks, and
carboxylterminal truncations that have
been produced by directed mutagenesis
are also indicated. The two arrows show
the location within the receptor’s C-tail
of the present deletion.
family are similar to those reported previously in ADH,
including transient signs of neuromuscular irritability and
asthenia, as well as hypocalcemia associated with inappropriately low serum levels of PTH and inappropriately normal
or elevated levels of urinary calcium excretion, given their
hypocalcemia. All cases of ADH identified to date have been
caused by missense activating mutations of the CaSR gene.
After the first report of such a CaSR mutation in a family with
ADH, 13 additional, unique mutations in the CaSR gene have
been described associated with either familial or sporadic
hypocalcemia (14, 17, 23–28). All of these mutations have
been missense mutations, and most (9 of 13) are localized
within the CaSR’s extracellular domain, which is the ligandbinding domain (29). The other mutations are present within
the first extracellular loop or in the fifth or sixth transmembrane domains (see Fig. 3). 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, 23–28). 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 ho-
1700
LIENHARDT ET AL.
FIG. 4. High Ca2⫹o-evoked increases in Ca2⫹i in fura-2-loaded
HEK293 cells transiently transfected with the WT or mutant CaSR
alone or cotransfected with the WT and mutant receptors. Cells were
transfected with the various receptor constructs, and Ca2⫹o-induced
changes in Ca2⫹i were determined as described in Subjects and Methods. Each data point is the mean value of 4 – 6 measurements. The
responses are normalized to the maximum response of the WT receptor. The SEM is indicated with a vertical bar through each point.
The EC50 for each curve is presented as mean ⫾ SEM. The mean EC50
for the mutant receptor alone and those for the cotransfected WT and
mutant receptors were not significantly different (P ⬎ 0.05). In this
experiment, 0.625 ␮g of each cDNA was employed to transfect
HEK293 cells plated on rectangular coverslips (within individual
wells of 12-well plates).
mozygous 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
JCE & M • 2000
Vol 85 • No 4
FIG. 5. Western analysis of WT and mutant receptors. Proteins were
isolated from HEK293 cells transiently transfected with the WT or
mutant CaSR cDNAs and were subjected to SDS-PAGE on a linear
gradient running gel of 5–9%, followed by analysis for cell surface
expression of the CaSR (A) or Western blotting (B), as described in
Subjects and Methods. In each panel, lane 1 represents WT receptor,
and lane 2 shows the mutant CaSR.
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, 33–35). Residues from 868 to 886 have been identified as crucial for
normal signal transduction (30, 33–35). 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, 33–35). Truncation
LARGE DELETION WITHIN THE CaSR TAIL
at residue 892 results in a CaSR that is not only functional but
supranormal in its level of activity, as evidenced by its lowerthan-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– 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
1701
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 Cterminal 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.
Acknowledgments
We thank Dr. F. Bonnet-Boutillon (Sancheville, France) for her clinical
support and L. Zekraoui (Service de Biochimie A, Hôpital Pitié Salpétrière, Paris, France) and Shu Yi Sun (Brigham and Women’s’ Hospital,
Boston, MA) for their excellent technical assistance.
References
1. Bilezikian JP, Thakker RV. 1997 Hypoparathyroidism. Curr Opin Endocrinol
Diabetes. 4:427– 432.
2. Finegold DN, Armitage MM, Galiani M, et al. 1994 Preliminary localization
of a gene for autosomal dominant hypoparathyroidism to chromosome 3q13.
Pediatr Res. 36:414 – 417.
3. Lovlie R, Eiken HG, Sorheim JI, Boman H. 1996 The Ca2⫹-sensing receptor
gene (PCAR1) mutation T151 M in isolated autosomal dominant hypoparathyroidism. Hum Genet. 98:129 –133.
4. Janicic N, Soliman E, Pausova Z, et al. 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 16. Mamm Genome.
6:798 – 801.
5. Brown EM, Gamba G, Riccardi D, et al. 1993 Cloning and characterization of
an extracellular Ca2⫹-sensing receptor from bovine parathyroid. Nature.
366:575–580.
6. Garrett JE, Capuano IV, Hammerland LG, et al. 1995 Molecular cloning and
functional expression of human parathyroid calcium receptor cDNAs. J Biol
Chem. 270:12919 –12925.
7. Masu M, Tanabe Y, Tsuchida K, Shigemoto R, Nakanishi S. 1991 Sequence
and expression of a metabotropic glutamate receptor. Nature. 349:760 –765.
8. Houamed KM, Kuijper JL, Gilbert TL, et al. 1991 Cloning, expression, and
gene structure of a G-protein-coupled receptor from rat brain. Science.
252:1318 –1321.
9. Kaupmann K, Huggel K, Heid J, et al. 1997 Expression cloning of GABA(B)
receptors uncovers similarity to metabotropic glutamate receptors. Nature.
386:239 –246.
10. Ryba NJ, Tirindelli R. 1997 A new multigene family of putative pheromone
receptors. Neuron. 19:371–379.
11. Herrada G, Dulac C. 1997 A novel family of putative pheromone receptors in
mammals with a topographically organized and sexually dimorphic distribution. Cell. 90:763–773.
12. Matsunami H, Buck LB. 1997 A multigene family encoding a diverse array of
putative pheromone receptors in mammals. Cell. 90:775–784.
13. Hoon MA, Adler E, Lindemeier J, Battey JF, Ryba NJ, Zuker CS. 1999 Putative
mammalian taste receptors: a class of taste-specific GPCRs with distinct topographic selectivity. Cell. 96:541–551.
14. Pollak MR, Brown EM, Estep HL, et al. 1994 Autosomal dominant hypocalcemia caused by a Ca2⫹-sensing receptor gene mutation. Nat Genet. 8:303–307.
15. Brown EM, Vassilev PM, Quinn S, Hebert SC. 1999 G-protein-coupled, extracellular Ca2⫹-sensing receptor: a versatile regulator of diverse cellular functions. Vitam Horm. 55:1–71.
16. Lelong M, Canlorbe P, Lagrue G, Bader JC. 1968 Familial hypoparathyroidism
with hypercalciuria. Ann Pediatr (Paris). 16:253–268.
17. Watanabe T, Bai M, Lane CR, et al. 1998 Familial hypoparathyroidism: identification of a novel gain-of-function mutation in transmembrane domain 5 of
the calcium-sensing receptor. J Clin Endocrinol Metab. 83:2497–2502.
18. Kifor O, Diaz R, Butters RR, Brown EM. 1997 The Ca2⫹-sensing receptor
activates phospholipases C, A2, and D by high extracellular Ca2⫹ in bovine
1702
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
LIENHARDT ET AL.
parathyroid and CaR-transfected, human embryonic kidney (HEK293) cells.
J Bone Miner Res. 12:715–725.
Bai M, Janicic N, Trivedi S, et al. 1997 Markedly reduced activity of mutant
calcium-sensing receptor with an inserted Alu element from a kindred with
familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. J Clin Invest. 99:1917–1925.
Sambrook J, Frisch EF, Maniatis T. 1989 Isolation of high-molecular-weight
DNA from mammalian cells: protocol I. In: Sambrook J, Frisch EF, Maniatis T,
eds. Molecular cloning. A laboratory manual. 2nd ed. New York: Cold Spring
Harbor Laboratory Press; 916 –919.
Pollak MR, Brown EM, Wu Chou YH, et al. 1993 Mutations in the human
Ca2⫹-sensing receptor gene cause familial hypocalciuric hypercalcemia and
neonatal severe hyperparathyroidism. Cell. 75:1297–1303.
Pearce SHS, Trump D, Wooding C, et al. 1995 Calcium-sensing receptor
mutations in familial benign hypercalcemia and neonatal hyperparathyroidism. J Clin Invest. 96:2683–2692.
Bai M, Quinn S, Trivedi S, et al. 1996 Expression and characterization of
inactivating and activating mutations in the human Ca2⫹-sensing receptor.
J Biol Chem. 271:19537–19545.
Perry Y, Finegold D, Armitage M, Ferrell R. 1994 A missense mutation in the
Ca sensing receptor causes familial autosomal dominant hypoparathyroidism.
Am J Hum Genet. [Suppl] 55:A17.
Pearce SHS, Williamson C, Kifor O, et al. 1996 A familial syndrome of
hypocalcemia with hypercalciuria due to mutations in the calcium-sensing
receptor. N Engl J Med. 335:1115–1122.
Baron J, Winer KK, Yanovski JA, et al. 1996 Mutations in the Ca2⫹-sensing
receptor gene cause autosomal dominant and sporadic hypoparathyroidism.
Hum Mol Genet. 5:601– 606.
De Luca F, Ray K, Mancilla EE, et al. 1997 Sporadic hypoparathyroidism
caused by de novo gain-of-function mutations of the Ca2⫹-sensing receptor.
J Clin Endocrinol Metab. 82:2710 –2715.
Okazaki R, Chikatsu N, Nakatsu M, et al. 1999 A novel activating mutation
in calcium-sensing receptor gene associated with a family of autosomal dominant hypocalcemia. J Clin Endocrinol Metab. 84:363–366.
Brauner-Osborne H, Jensen AA, Sheppard PO, O’Hara P, Krogsgaard-Larsen
P. 1999 The agonist-binding domain of the calcium-sensing receptor is located
at the amino-terminal domain. J Biol Chem. 274:18382–18386.
Bai M, Trivedi S, Brown EM. 1998 Dimerization of the extracellular calciumsensing receptor (CaR) on the cell surface of CaR-transfected HEK293 cells.
J Biol Chem. 273:23605–23610.
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.
Bai M, Pearce SHS, Kifor O, et al. 1997 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. J Clin Invest. 99:88 –96.
Ray K, Fan GF, Goldsmith PK, Spiegel AM. 1997 The carboxyl terminus of
the human calcium receptor. J Biol Chem. 272:31355–31361.
JCE & M • 2000
Vol 85 • No 4
34. Gama L, Breitwieser GE. 1998 A carboxylterminal domain controls the cooperativity for extracellular Ca2⫹ activation of the human calcium-sensing
receptor. A study with receptor-green fluorescent protein fusions. J Biol Chem.
273:29712–29718.
35. Bai M, Trivedi S, Lane CR, Yang Y, Quinn SJ, Brown EM. 1998 Protein kinase
C phosphorylation of threonine at position 888 in Ca2⫹o-sensing receptor (CaR)
inhibits coupling to Ca2⫹ store release. J Biol Chem. 273:21267–21275.
36. Ho C, Conner DA, Pollak MR, et al. 1995 A mouse model for familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Nat
Genet. 11:389 –394.
37. Gether U, Kobilkas K. 1998 G-protein-coupled receptors. II. Mechanism of
agonist activation. J Biol Chem. 273:17979 –17982.
38. Shenker A, Laue L, Kosugi S, Merendino Jr JJ, Minegishi T, Cutler Jr GB.
1993 A constitutively activating mutation of the luteinizing hormone receptor
in familial male precocious puberty. Nature. 365:652– 654.
39. Laue L, Chan WY, Hsueh AJW, et al. 1995 Genetic heterogeneity of constitutively activating mutations of the human luteinizing hormone receptor in
familial male-limited precocious puberty. Proc Natl Acad Sci USA. 92:1906 –
1910.
40. Gromoll J, Partsch CJ, Simoni M, et al. 1998 A mutation in the first transmembrane domain of the lutropin receptor causes male precocious puberty.
J Clin Endocrinol Metab. 83:476 – 480.
41. Parma J, Duprez L, Van Sande J, et al. 1993 Somatic mutations in the thyrotropin receptor gene cause hyperfunctioning thyroid adenomas. Nature.
365:649 – 651.
42. Parma J, Van Sande J, Swillens S, Tonacchera M, Dumont J, Vassart G. 1995
Somatic mutations causing constitutive activity of the thyrotropin receptor are
the major cause of hyperfunctioning thyroid adenomas: identification of additional mutations activating both the cyclic adenosine 3⬘,5⬘-monophosphate
and inositol phosphate- Ca2⫹ cascades. Mol Endocrinol. 9:725–733.
43. Duprez L, Parma J, Costagliola S, et al. 1997 Constitutive activation of the TSH
receptor by spontaneous mutations affecting the N-terminal extracellular domain. FEBS Lett. 409:469 – 474.
44. Holzapfel HP, Führer D, Wonerow P, Weinland G, Scherbaum WA, Paschke
R. 1997 Identification of constitutively activating somatic thyrotropin receptor
mutations in a subset of toxic multinodular goiters. J Clin Endocrinol Metab.
82:4229 – 4233.
45. Führer D, Wonerow P, Willgerodt H, Paschke R. 1997 Identification of a new
thyrotropin receptor germline mutation (Leu629Phe) in a family with neonatal
onset of autosomal dominant nonautoimmune hyperthyroidism. J Clin Endocrinol Metab. 82:4234 – 4238.
46. Jüppner H, Schipani E. 1997 The parathyroid hormone/parathyroid hormone-related peptide receptor in Jansen’s metaphyseal chondrodysplasia.
Curr Opin Endocrinol Diabetes. 4:433– 442.
47. Böhm SK, Grady EF, Bunnett NW. 1997 Regulatory mechanisms that modulate signaling by G-protein-coupled receptors. Biochem J. 322:1–18.
48. Heding A, Vrecl M, Bogerd J, et al. 1998 Gonadotropin-releasing hormone
receptors with intracellular carboxylterminal tails undergo acute desensitization of total inositol phosphate production and exhibit accelerated internalization kinetics. J Biol Chem. 273:11472–11477.