Full text of DOI:10.1359/jbmr.2002.17.8.1461

JOURNAL OF BONE AND MINERAL RESEARCH
Volume 17, Number 8, 2002
© 2002 American Society for Bone and Mineral Research
Autosomal Dominant Hypocalcemia Caused by a Novel
Mutation in the Loop 2 Region of the Human Calcium
Receptor Extracellular Domain
JIANXIN HU,1 STEFANO MORA,2 GIACOMO COLUSSI,3,4 MARIA CARLA PROVERBIO,2
KENDRA A. JONES,1 LAURA BOLZONI,2 MARIA E. DE FERRARI,3 GIOVANNI CIVATI,3
and ALLEN M. SPIEGEL1
ABSTRACT
We report a novel missense mutation N124K in the extracellular calcium receptor (CaR) identified in two
related subjects with the phenotypic features of autosomal dominant hypocalcemia (ADH). Expression of the
N124K mutant receptor created by site-directed mutagenesis and transfected into HEK-293 cells was com-
parable with that of the wild-type (WT) receptor and two other mutant receptors N118K and L125P identified
in subjects with ADH. Functional characterization by the extracellular Ca2؉ ion ([Ca2؉]0)-stimulated phos-
phoinositide (PI) hydrolysis in transfected HEK-293 cells showed that the N124K mutant receptor was
left-shifted in Ca2؉ sensitivity. This biochemical gain-of-function is comparable with that seen in other
missense mutations of the CaR identified in subjects with ADH. We tested a series of missense substitutions
(R, Q, E, and G) in addition to K for N124 and found that only the N124K mutation and to a much lesser extent
N124R caused a left shift in Ca2؉ sensitivity. Thus, a specific substitution, not merely a mutation of the N124
residue, is required for receptor activation. The N124K mutation is one of eight naturally occurring mutations
in subjects with ADH identified in a short segment A116-C129 of the CaR extracellular domain (ECD). We
present a hypothesis to explain receptor activation by mutations in this region based on the recently described
three-dimensional structure of the related metabotropic glutamate type 1 receptor (mGluR1). (J Bone Miner
Res 2002;17:1461–1469)
Key words: autosomal dominant hypocalcemia, hypoparathyroidism, human calcium receptor, mutation,
extracellular domain
ing and activating mutations have been identified in the
CaRs that are associated with familial hypocalciuric hyper-
calcemia and autosomal dominant hypocalcemia (ADH),
respectively. With one exception, the A843E mutation that
causes constitutive activation,(3,4) naturally occurring muta-
tions identified in subjects with ADH have been shown to
INTRODUCTION
HE EXTRACELLULAR calcium receptor (CaR) is a G
protein–coupled receptor (GPCR) that plays a key role
T
in [Ca2ϩ]0 homeostasis by regulating parathyroid hormone
(PTH) secretion and renal Ca2ϩ reabsorption.(1,2) Inactivat-
cause an increase in sensitivity to [Ca2ϩ
] (“left-shift”)
0
rather than cause constitutive activation.(1,2) We describe a
The authors have no conflict of interest.
1Molecular Pathophysiology Section, National Institute on Deafness and Other Communication Disorders, National Institutes of Health,
Bethesda, Maryland, USA.
2Laboratory of Pediatric Endocrinology, IRCCS H S. Raffaele, University of Milan, Milan, Italy.
3Renal Unit, Niguarda Ca’ Granda Hospital, Milan, Italy.
4Present address: Renal Unit, Ospedale di Circolo e Fondazione Macchi, Varese, Italy.
1461

1462
HU ET AL.
novel CaR missense mutation N124K identified in a mother salts, vitamin
D
{usually 1,25-dihydroxyvitamin D3
and daughter with the phenotypic features of ADH and [1,25(OH)2D3], 0.25–1 ␮g/day}, aluminum-hydroxide (for
show that this mutation, but not other substitutions for
residue N124, causes a substantially increased sensitivity to
[Ca2ϩ]0. With N124K, a total of eight activating mutations
have been identified in subjects with ADH in a short region
A116-C129 of the CaR extracellular domain (ECD).(3,5–11)
We present a possible explanation for mutational activation
of the CaR by substitutions in this short region based on the
recently described three-dimensional structure of the ECD
of the related metabotropic glutamate type 1 receptor
(mGluR1).(12)
the control of hyperphosphatemia), and a thiazide diuretic.
Healthy control subjects (“controls”) also were used for the
analysis of PCa-PTH relationship; they were 13 subjects,
whose data were already reported in another study.(13) In-
dividual calcium and PTH data were spontaneous random
fasting values. Samples from all subjects were assayed
using the same methods for calcium and PTH assay (N-tact
PTH; Incstar Co., Stillwater, MN, USA). Informed consent
was obtained for all studies.
DNA amplification and sequence analysis
MATERIALS AND METHODS
Genomic DNA was isolated from white blood cells (Wiz-
ard, Genomic DNA Purification Kit; Promega, Madison,
WI, USA), and the coding region of the CaR gene was
polymerase chain reaction (PCR)-amplified the using
primer pairs previously described.(14,15) PCR was performed
in a 100-␮l reaction solution containing 400 ng of genomic
DNA, 1.5 mmol of MgCl2, 40 pmol of each primer, and 5
U of Eurobiotaq ADN Polymerase (Laboratoires EURO-
BIO, Les Ulis Cedex, France). PCR products were purified
using GFX PCR DNA and a Gel Band Purification Kit
(Amersham Pharmacia Biotech Italia, Milan, Italy).
Both manual and automatic sequencing were performed
using the same primers used for PCR.
Patients
Two related women have been studied. The proband, a
white woman, experienced generalized nocturnal seizures
due to severe hypocalcemia when she was 42 years old.
Inappropriately low levels of PTH were concomitantly
present. A computed tomography (CT) scan performed dur-
ing hospitalization disclosed bilateral basal ganglia calcifi-
cations. The patient was treated with oral calcium salts (1
g/day of elementary calcium) and calcitriol (0.7 ␮g/day).
During treatment, her plasma calcium levels ranged from
1.95 to 2.12 mmol/liter (7.8–8.5 mg/dl; normal range, 2.12–
2.55 mmol/liter or 8.6–10.2 mg/dl). Ten years later, she had
a hypercalcemic crisis with dehydration, renal failure, hy-
For manual sequence, the purified DNA fragments were
potension, and paroxysmal atrial fibrillation. She was ad- sequenced by the Thermo Sequenase Radiolabeled Termi-
mitted at the Renal Unit, Niguarda Ca’ Granda Hospital nator Cycle Sequencing Kit (Amersham Pharmacia Biotech
(Milan, Italy), and her plasma calcium was 3.74 mmol/liter Italia) and reaction products were run on 6% polyacryl-
(15 mg/dl) and plasma phosphorus was 1.77 mmol/liter (5.5 amide gel containing 8% urea and visualized by autoradiog-
mg/dl; normal range, 0.87–1.42 mmol/liter or 2.7–4.4 mg/
dl). During the last 6 years of follow-up, plasma calcium
levels ranged from 1.95 to 2.25 mmol/liter (7.8–9 mg/dl),
with urine calcium levels of 5.73–5.88 mmol/day (230–240
mg/24 h). PTH levels remained inappropriately low, com-
pared with plasma calcium concentrations (7.1–37 pg/ml;
normal range, 10–60 pg/ml).
The other patient is the second daughter of the proband.
At 25 years of age, she experienced abrupt generalized
seizures. Her ionized calcium levels were 0.76 mmol/liter
(3.12 mg/dl). A CT scan disclosed faint basal ganglia cal-
cification. During hospitalization, her plasma calcium levels
were 2.0 mmol/liter (8.3 mg/dl), plasma phosphorus level
was 1.48 mmol/liter (4.6 mg/dl). Intact serum PTH concen-
tration was 30 pg/ml, and urinary calcium was 6.74 mmol/
day (270 mg/24 h), with a calcium/creatinine ratio of 0.26
mg/mg.
raphy.
Automatic sequencing was performed using fluorescence-
labeled dideoxyterminators (Big Dye Terminator Cycle
Sequencing Ready Reaction Kit, ABI PRISM; Applied Bio-
systems Foster City, CA, USA) according to the manufac-
turer’s instructions and resolved by capillary electrophoresis
on the ABI PRISM 310 Automated Sequencer (Applied
Biosystems). Sequencing was performed in both strands.
Site-directed mutagenesis of the human CaR
The human CaR (hCaR) cDNA cloned in the pCR3.1
expression vector was described previously.(16) Site-directed
mutagenesis was performed using the Quickchange site-
directed mutagenesis kit (Stratagene, Inc., La Jolla, CA,
USA), according to the manufacturer’s instructions. Paren-
tal hCaR cDNA in pCR3.1 vector was amplified using pfu
Turbo DNA polymerase with mutagenic oligonucleotide
primers (sequences available on request) for 16 cycles in a
DNA thermal cycler (Perkin-Elmer, Norwalk, CT, USA).
After digestion of the parental DNA with DpnI for 1 h, the
amplified DNA with incorporated nucleotide substitution
was transformed into Escherichia coli (DH-5␣ strain). The
Hypoparathyroid patients and healthy control subjects
Data from 18 patients with primary hypoparathyroidism
were compared with data in ADH patients; 11 patients had
postsurgical hypoparathyroidism, 5 patients had idiopathic
hypoparathyroidism, and 2 patients had thalassemia with
chronic transfusion-induced hemosiderosis. They were mutations were confirmed by automated DNA sequencing
evaluated from once to up to 7 times, both off and on using a dRhodamine Terminator Cycle Sequencing kit and
therapy for hypocalcemia, which included oral calcium ABI PRISM-373A DNA sequencer (Applied Biosystems).

NOVEL CaR-ACTIVATING MUTATION
1463
Transient transfection of wild-type and mutant
receptors in HEK-293 cells
Transfections were performed using 12 ␮g (unless other-
wise indicated) of plasmid DNA for each transfection in a
75-cm2 flask of HEK-293 cells. DNA was diluted in serum-
free DMEM (BioFluids, Inc., Rockville, MD, USA), mixed
with diluted Lipofectamine (Gibco BRL, Grand Island, NY,
USA) and the mixture was incubated at room temperature
for 30 minutes. The DNA-Lipofectamine complex was di-
luted further in 6 ml of serum-free DMEM and was added
to 80% confluent HEK-293 cells plated in 75-cm2 flasks.
After 5 h of incubation, 15 ml of complete DMEM contain-
ing 10% FBS (BioFluids, Inc.) was added. Twenty-four
hours after transfection, transfected cells were split and
cultured in complete DMEM.
Phosphoinositide hydrolysis assay
Phosphoinositide (PI) hydrolysis assay has been de-
scribed previously.(16) Briefly, 24 h after transfection, trans-
fected cells from a confluent 75-cm2 flask were split. Typ-
ically, one-eighth of cells were plated in one well in a 6-well
plate and whole cell lysate was prepared 48 h posttransfec-
tion for Western blot assay. The remaining cells were plated
in two 12-well plates in complete DMEM medium contain-
ing 3.0 ␮Ci/ml of [3H]myoinositol (New England Nuclear,
Beverly, MA, USA) and cultured for another 24 h. Culture
medium was replaced by 1ϫ PI buffer (120 mM of NaCl, 5
mM of KCl, 5.6 mM of glucose, 0.4 mM of MgCl2, and
20 mM of LiCl in 25 mM of piperazine-N,N-bis[2-
ethanesulfonic acid] (PIPES) buffer, pH 7.2) and incubated
for 1 h at 37°C. After removal of PI buffer, cells were
incubated for an additional 1 h with different concentrations
of Ca2ϩ in 1ϫ PI buffer. The reactions were terminated by
addition of 1 ml of acid methanol (1:1000 vol/vol) per well.
Total inositol phosphates were purified by chromatography
on Dowex 1-X8 columns and radioactivity for each sample
was counted with a liquid scintillation counter. Graphs of
concentration dependence for stimulation of PI hydrolysis
FIG. 1. The relationship between plasma calcium and PTH in ADH
(hatched dots, case 1 and case 2), primary hypoparathyroidism (full
triangles, P.H.), and in 13 healthy subjects (open squares, controls).
Correlation lines are shown in ADH patients (y ϭ Ϫ25x ϩ 233; p Ͻ
0.001) and in control subjects (y ϭ Ϫ19x ϩ 218; p Ͻ 0.001).
proteins on the gel were electrotransferred onto a nitrocel-
lulose membrane and incubated with 0.1 ␮g/ml of protein
A–purified mouse monoclonal anti-hCaR antibody ADD
(raised against a synthetic peptide corresponding to residues
214–235 of hCaR protein). Subsequently, the membrane
was incubated with a secondary goat anti-mouse antibody
conjugated to horseradish peroxidase (Kirkegaard and Perry
Laboratories, Gaithersburg, MD, USA) at a dilution of
1:5000. The hCaR protein was detected with an enhanced
chemiluminescence system (ECL; Amersham Corp., Ar-
lington Heights, IL, USA).
by [Ca2ϩ
] for each transfection were drawn by using
0
RESULTS
GraphPad Prism version 2.0 software (GraphPad Software,
Inc., San Diego, CA, USA). Each value on a curve is the
mean of duplicate determinations. Graphs shown in this
study were representative ones from at least five indepen-
dent experiments.
Relationship between plasma calcium and PTH
Over the range of plasma calcium values, there was a
significant correlation between plasma calcium and PTH
(p Ͻ 0.001) in patients with ADH but not in those with
hypoparathyroidism (Fig. 1). The equation describing the
relationship between plasma calcium and PTH in patients
with ADH had a similar slope but different intercept from
that in 13 healthy controls previously studied.(13)
Immunoblotting analysis with detergent-solubilized
whole cell extracts
Confluent cells in 6-well plates were rinsed with ice- PBS
and scraped on ice in lysis buffer containing 20 mM of
Tris-HCl (pH 6.8), 150 mM of NaCl, 10 mM of EDTA, 1
mM of EGTA, 1% Triton X-100, and freshly added protease
inhibitors cocktail (Boehringer Mannheim, Indianapolis,
IN, USA). The protein content in each sample was deter-
Detection of a point mutation in the CaR gene
The sequence analysis of PCR-amplified genomic DNA
mined by the modified Bradford method (Bio-Rad Labora- revealed in both patients a heterozygous C to A transition at
tories, Hercules, CA, USA) and 50 ␮g of protein per lane position 372 in exon 2 of the CaR gene (Fig. 2), causing a
was separated on 5% SDS-PAGE gel under reducing con- missense mutation N124K in the amino-terminal ECD of
ditions with 300 mM of ␤-mercaptoethanol added. The the receptor (Fig. 3).

1464
HU ET AL.
hCaRs, the N124K mutant retains slightly greater expres-
sion seen on immunoblot (Fig. 4) and higher maximal
calcium response. To determine if the increased sensitivity
of the N124K mutant was caused by altered expression, we
transfected cells with varying quantities of N124K mutant
and WT hCaR cDNA and measured receptor expression by
immunoblot and function by [Ca2ϩ]0-stimulated PI hydro-
lysis assay. Figure 5 shows that receptor expression in-
creased with transfection of increasing amounts of DNA,
but this affected maximal response to [Ca2ϩ]0 and not EC50
values. Even when N124K mutant hCaR was expressed at a
much lower level than the WT hCaR (compare 3 ␮g of
N124K vs. 12 ␮g of WT hCaR DNA transfected), it still
exhibited significantly increased sensitivity to [Ca2ϩ]0 (Fig. 5).
Dominant effect of N124K mutation
The fact that the 2 patients we report here are heterozy-
gotes with both WT and N124K mutant hCaR alleles sug-
gests that the N124K mutation has a dominant effect in cells
expressing both WT and N124K mutant hCaRs. Our in vitro
study confirmed this. HEK-293 cells were cotransfected
with equal amounts of WT and N124K mutant hCaR cDNA,
and function was determined by [Ca2ϩ]0-stimulated PI hy-
drolysis assay. Figure 6 shows that sensitivity to [Ca2ϩ]0 of
cells transfected with equal amounts of N124K mutant and
WT hCaR cDNA was intermediate between that of cells
transfected with either the N124K mutant or the WT hCaR
cDNA alone.
FIG. 2. Identification of the CaR mutation in a mother and daughter
with ADH. Automated sequence analysis (reverse) of PCR-amplified
genomic DNA from the affected subjects shows a heterozygous C to A
transition at position 372 in exon 2 of the CaR gene, changing Asn to
Lys.
Functional characterization of the N124K mutant
hCaR
Comparison of function of mutant hCaRs with N124
substituted by K, R, Q, E, or G
By site-directed mutagenesis, we constructed a mutant
hCaR with N124K mutation using wild-type (WT) hCaR
cDNA as a template. We transfected hCaR cDNA into
HEK-293 cells and analyzed receptor expression on immu-
noblots stained with anti-hCaR monoclonal antibody ADD
to detect total CaR immunoreactive species. We compared
the N124K mutant CaR with WT and two other mutant
CaRs N118K and L125P, which are adjacent to residue
N124. Both the N118K and the L125P mutations have been
identified in subjects with ADH.(3,6–8) Under reducing con-
ditions, ADD antibody detected two major bands of ϳ130
kDa and 150 kDa at comparable expression levels for the
WT hCaR and the three mutant hCaRs (Fig. 4). Previous
studies have shown that the monomeric ϳ150-kDa band
represents hCaR forms expressed at the cell surface and
modified with N-linked, complex carbohydrates; the ϳ130-
kDa band represents high mannose-modified forms, trapped
intracellularly and sensitive to Endo-H digestion.(9,16,17) We
tested the function of the mutant receptor by using the intact
cell [Ca2ϩ]0-stimulated PI hydrolysis assay. Figure 4 shows
that HEK-293 cells transfected with vector only had no
calcium response. The N124K mutant hCaR not only re-
tained responsiveness to [Ca2ϩ]0, but its Ca2ϩ sensitivity
was increased also compared with WT hCaR. The effective
concentration for 50% maximal response (EC50) value for
the N124K mutant hCaR was 1.58 Ϯ 0.06 mM (mean Ϯ SE;
n ϭ 5) compared with a value of 3.08 Ϯ 0.04 mM for the
WT hCaR. EC50 values for N118K and L125P mutant
hCaRs were 0.99 Ϯ 0.05 mM and 0.71 Ϯ 0.03 mM, respec-
To determine if CaR activation is solely a function of
substitution of N124 or if activation depends on substitution
of N124 by specific amino acids, we constructed by site-
directed mutagenesis four additional mutant hCaRs with
residue N124 replaced individually by R, Q, E, or G. Figure
7 shows the results of expression and functional assay of
these mutants. Neither N124Q mutations (a conservative
amino acid substitution) nor N124E (a replacement by an
acidic residue) mutations affect the receptor’s expression or
function compared with the WT hCaR. The N124R muta-
tion caused modest activation as evidenced by its response
at 2 mM of [Ca2ϩ]0, although its activating effect is much
weaker than that of N124K, suggesting that a positive
charge at residue 124 in hCaR by itself is insufficient to
cause a substantial increase in sensitivity of the receptor to
[Ca2ϩ]0. Interestingly, N124G (a replacement by a neutral
residue) shows a significant inactivating effect on the re-
ceptor. The N124G mutation also reduced the receptor’s cell
surface expression, further highlighting the unique func-
tional consequences of substituting different amino acids for
residue N124
.
DISCUSSION
Since the initial description of an activating hCaR muta-
tively. Compared with the N118K and L125P mutant tion in a subject with ADH,(18) a total of 26 mutations in the

NOVEL CaR-ACTIVATING MUTATION
1465
FIG. 3. Schematic diagram showing amino acid sequence of the hCaR ECD. The location of signal peptide, glycosylation sites, and the sequence
of synthetic polypeptide used to raise monoclonal antibody ADD are indicated. All 19 cysteines are shown in black. The beginning and end of
the VFT domain, and the four loops in lobe 1 of the VFT (see Discussion section) are indicated. Naturally occurring activating mutations identified
in the ECD are indicated also.
hCaR have been reported in subjects with ADH.(19,20) With creases sensitivity to [Ca2ϩ]0 effectively can have the same
the exception of a missense mutation A843E in the seventh consequence as gain-of-function mutations that can activate
transmembrane domain of the hCaR, which causes apparent a given receptor in the absence of agonist. One might
constitutive receptor activation,(3,4) functional characteriza- speculate that the degree of increase in sensitivity to [Ca2ϩ
]
0
tion of other hCaR mutations identified in subjects with caused by a particular hCaR mutation would correlate with
ADH has revealed an increase in Ca2ϩ sensitivity. Given the severity of the clinical phenotype. There is limited
that the receptor agonist ionized Ca2ϩ is always present at a evidence supporting this suggestion. The A843E mutation
finite concentration in the circulation, a mutation that in- that causes “true” constitutive activation and the L125P

1466
HU ET AL.
FIG. 4. (A) Concentration dependence for [Ca2ϩ]0 stimulation of PI hydrolysis and (B) immunoblot of CaR in transiently transfected HEK-293
cells expressing WT hCaR, empty vector, N124K, N118K, and L125P mutant hCaRs. Transfection, PI assay, SDS-PAGE, and immunoblot with
monoclonal anti-hCaR ADD were performed as described in the Materials and Methods section. Twelve micrograms of DNA each of the CaRs
or empty vector was used in transfection. Molecular mass standards are indicated at the right of the blots. (A) Results of PI assay are expressed
as percent of maximal response (WT hCaR at 8 mM). (B) Immunoblot of whole cell lysate of transfected HEK-293 cells. The immunoblots shown
here and in Figs. 5–7 were done using cells from the same transfection as the cells used for the PI hydrolysis assay.
FIG. 5. (A) Concentration de-
pendence for [Ca2ϩ]0 stimulation
of PI hydrolysis and (B) immuno-
blot of CaR (right) in transiently
transfected HEK-293 cells ex-
pressing WT hCaR (12 ␮g and 6
␮g of DNA transfected) and
N124K mutant hCaR (12, 9, 6,
and 3 ␮g of DNA transfected).
Methods and format for presenta-
tion of results are as in legend to
Fig. 4 except that maximal re-
sponse is WT hCaR at 12 mM.
mutation that causes perhaps the most pronounced left shift with a less severe phenotype, it is likely that hypocalcemia
in Ca2ϩ sensitivity, based on in vitro studies, were identified had been present for a significant period before diagnosis,
in subjects with particularly severe and early (age, Ͻ10 particularly because basal ganglion calcification was al-
years) manifestations of hypocalcemia.(3) In the present ready present at the time of clinical diagnosis. Relative
subjects, as in a number of previously reported cases of hypercalciuria, a specific feature of ADH reflecting the
ADH,(19) clinical manifestations were not apparent until effect of CaR activation on renal reabsorption of [Ca2ϩ]0,
adulthood. We found that the inverse relationship between was of particular clinical significance in 1 of our subjects,
plasma calcium and PTH is preserved in our subjects with leading to renal failure during attempted correction of hy-
ADH but that this relationship is shifted to the left by pocalcemia with calcium and calcitriol.
comparison with healthy control subjects.
Of the total of 26 activating mutations identified in the
We found that the N124K mutation caused a significant hCaR in subjects with ADH, the majority (16 activation
increase in CaR sensitivity to [Ca2ϩ]0, but one that was less mutations) occur in the ECD (Fig. 3). The CaR is a member
marked than either N118K or L125P mutations. In the of family 3 of the GPCR superfamily.(21) Members of fam-
mother and daughter reported here, hypocalcemia was first ily 3 are distinguished by having a particularly large amino-
noted clinically at the ages of 42 years and 25 years, terminal ECD in addition to the seven transmembrane do-
respectively. Although the age at diagnosis is consistent main characteristic of the GPCR superfamily. The ECD

NOVEL CaR-ACTIVATING MUTATION
1467
FIG. 6. (A) Concentration de-
pendence for [Ca2ϩ]0 stimulation
of PI hydrolysis and (B) immuno-
blot of CaR in transiently trans-
fected HEK-293 cells expressing
WT hCaR (12 ␮g of DNA trans-
fected), N124K mutant hCaR (12
␮g of DNA transfected), and WT
hCaR plus N124K mutant hCaR
(6 ␮g of DNA each transfected).
Methods and format for presenta-
tion of results are as in legend to
Fig. 4 except that maximal re-
sponse is WT hCaR at 12 mM.
FIG. 7. (A) Concentration de-
pendence for [Ca2ϩ]0 stimulation
of PI hydrolysis and (B) immuno-
blot of CaR in transiently trans-
fected HEK-293 cells expressing
WT hCaR, N124K, N124R,
N124G, N124E, and N124Q mu-
tant hCaRs. Methods and format
for presentation of results are as
in legend to Fig. 4 except that
maximal response is WT hCaR at
12 mM. Twelve micrograms of
DNA each of the CaRs was used
in transfection.
of the CaR, the mGluRs and other members of family the lobe itself.(22) Of these loops, loop 2 is of particular
3 consists of a Venus-flytrap (VFT) domain followed significance and is depicted in Fig. 8. We had shown earlier
by a cysteine-rich domain that merges with the first that cysteines 129 and 131 within loop 2 of the hCaR are
transmembrane-spanning portion of the seven transmem- involved in intermolecular disulfide bonds that are the basis
brane domain.(22–24) The VFT domain is so named because, for covalent dimerization of the CaR ECD.(22) Homologous
as first shown for the bacterial periplasmic binding pro- cysteines are involved in intermolecular disulfide-linked
teins,(25) it consists of two “lobes” that are open in the dimers in other family 3 members including mGluRs.
unliganded state and that close on ligand binding. Recently,
Comparison of the three-dimensional structures of the
the three-dimensional structure of a portion of the ECD open versus closed conformations of the mGluR1 VFT
(lacking the cysteine-rich domain) of the rat mGluR1 in shows an ϳ70° rotation about an axis through the VFT
both the unliganded and the glutamate-bound conformations dimer interface (Fig. 8). Loop 2 is involved not only in
was determined by X-ray crystallography.(12) The structure covalent disulfide-linked dimerization, but it also forms a
was that of a bilobed VFT that was open in the unliganded portion of the dimer interface. Thus, it is particularly inter-
form and closed with glutamate-bound form. Based on the esting that of 16 activating mutations in the hCaR ECD, 8
three-dimensional structure of the mGluR1 VFT domain, a including the presently reported N124K mutant occur in the
schematic diagram of both the free form (without binding of proximal portion of loop 2 between residues A116 and C129
.
Ca2ϩ) and the complex form (with binding of Ca2ϩ) of the A saturation mutagenesis study of the A116-P136 region of
dimeric hCaR VFT domains is shown in Fig. 8. One lobe of the hCaR also showed that mutation of this region fre-
the VFT (the amino-terminal lobe we have termed “lobe 1”) quently leads to receptor activation.(26) Unfortunately, the
shows four loops of varying length (Fig. 3 shows the se- structure of the majority of loop 2 was indistinct in the
quence and length of each of these loops) protruding from mGluR1 crystal structure, but in the amino-terminal portion

1468
HU ET AL.
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FIG. 8. Diagram of free (no ligand bound) and complex (agonist-
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of loop 2 there was a notable difference observed between the
open and closed conformations.(12) In the former, an ␣-helix
forming a portion of the dimer interface was extended for
several residues into loop 2. In the glutamate-bound, closed
conformation, this ␣-helical extension was not present.
caused by de novo gain-of-function mutations of the Ca2ϩ
-
The clustering of activating mutations in loop 2 of the
hCaR leads us to propose the following mechanism for CaR
activation by such mutations, based on the crystal structure
of the related mGluR1 VFT. Without Ca2ϩ bound to it, the
CaR VFT dimer remains in the open position (Fig. 8, free
form). Ca2ϩ binding promotes closing of the VFT that is
accompanied by a significant rotation of one VFT monomer
relative to the other (Fig. 8, complex form). This rotation
involves a change in structure in at least the proximal
portion of loop 2 from an ␣-helix to a more disordered loop.
We suggest that activating mutations in loop 2 change its
structure and thereby decrease the normal constraints to
rotation of the VFT dimer. This would have the effect of
facilitating VFT closure at any given Ca2ϩ concentration,
hence increasing sensitivity of the receptor to activation by
Ca2ϩ. Such a mechanism is consistent with our previous
results(22) showing that mutations of cysteines 129 and 131
that prevent covalent (but not noncovalent) dimerization do
not abolish CaR response but rather increase sensitivity to
Ca2ϩ. However, not every mutation in loop 2 results in CaR
activation as seen with the various substitutions at N124 that
either caused no change or in the case of N124G actually
reduced receptor expression and activation. Ultimately, de-
termination of the structure of the CaR VFT in unliganded
and Ca2ϩ-bound conformations, including loop 2 in native
and mutated form, will be necessary to validate this hypoth-
esis. Nonetheless, further identification and study of natu-
rally occurring activating mutations such as those clustered
in loop 2, as well as those in other locations, should offer
unique insights into the structure and function of the CaR.
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ACKNOWLEDGMENTS
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We are grateful to Stephen Marx for careful reading of
this paper. L. Bolzoni was supported by a Fellowship from
Pharmacia & Upjohn Italy.

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Address reprint requests to:
Jianxin Hu, Ph.D.
Building 10, Room 8C-101
National Institutes of Health
9000 Rockville Pike
Bethesda, MD 20892, USA
Received in original form October 16, 2001; in revised form
January 24, 2002; accepted March 8, 2002.