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INTRODUCTION |
The extracellular calcium ([Ca2+]o)-sensing
receptor (CaR)1 is a G
protein-coupled receptor that plays a key role in mineral ion
homeostasis by sensing small perturbations in the level of [Ca2+]o and modulating the functions of
parathyroid and kidney so as to restore [Ca2+]o
to its normal level (1). Like some G protein-coupled receptors (2, 3),
recent studies have shown that the CaR on the surface of
receptor-transfected cells forms disulfide-linked dimers (4) through
cysteines within its extracellular domain (5, 6). However, one study
suggested that two cysteines at positions 101 and 236 mediate
intermolecular disulfide linkages (5), whereas another showed that two
cysteines at positions 129 and 131 are involved in disulfide-linked
dimerization (6). The resultant mutant receptors lacking either of
these two pairs of cysteine residues were suggested to form monomers
rather than dimers in transfected cells (5, 6). These putatively
monomeric CaRs had no biological activity in one case (5) or exhibited increased sensitivity to [Ca2+]o in the other
(6). Thus there is currently no consensus as to the key cysteine
residues within the CaR extracellular domain that mediate dimerization.
Our earlier studies indicated that the CaR may dimerize through
noncovalent interactions between transmembrane domains in addition to
doing so via covalent disulfide bonds within its extracellular domain
(5, 6), as suggested by our studies on naturally occurring inactivating
mutations causing familial hypocalciuric hypercalcemia (7, 8). We
showed that under reducing conditions, most mutant receptors, including
those with almost undetectable mature forms such as R66C and R680C,
show substantial amounts of SDS-resistant dimeric forms in addition to
monomeric forms in SDS-containing polyacrylamide gels. In contrast,
P747frameshift, a single-base deletion in codon 747 resulting in a
truncated receptor lacking the second extracellular loop and the
remainder of the C terminus, including the fifth, sixth, and seventh
transmembrane domains, shows only a single monomeric species and no
detectable higher molecular weight forms. Interestingly, a consensus
dimerization motif (9) for noncovalent hydrophobic interactions is
present in the CaR within TM5, that is missing in the P747frameshift.
In the present studies, we have examined the roles of
Cys101, Cys129, Cys131, and
Cys236 in mediating disulfide-linked dimerization of the
CaR and of covalent dimerization in the functional interactions between
CaR monomers within the dimeric CaR. Mutating both Cys129
and Cys131 slightly reduces cell surface expression of the
CaR and abolishes most of the sulfhydryl-sensitive CaR dimerization on
the cell surface. In contrast, mutating Cys101 and
Cys236 severely reduces surface and overall expression of
the receptor with no substantial conversion of dimers to monomers
detected under nonreducing conditions. Furthermore, we show that the
CaR still forms functional dimers even in the absence of these
intermolecular disulfide linkages.
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EXPERIMENTAL PROCEDURES |
Site-directed Mutagenesis--
Site-directed mutagenesis was
performed using the approach described by Kunkel (10) to produce
mutated receptors in which one or two cysteine residues present in the
extracellular domain of the human CaR were mutated to serines. The
dut-1 ung-1 strain of Escherichia coli, CJ236, was
transformed separately with mutagenesis cassettes, as described
previously (7). Uracil-containing, single-stranded DNA was produced by
infecting the cells with the helper phage, VCSM13 (Stratagene, La
Jolla, CA). The single-stranded DNA was then annealed to a mutagenesis
primer that contained the desired nucleotide change encoding a single
point mutation flanked on both sides by wild type sequences. The primer
was subsequently extended around the entire single-stranded DNA and
ligated to generate closed circular heteroduplex DNA. DH5
- or
DH10B-competent cells were transformed with these DNA heteroduplexes,
and incorporation of the desired mutations was confirmed by sequencing
the mutated cassettes. The resultant mutated cassettes were cloned into
the FLAG-tagged or nontagged receptor in pcDNA3 (Invitrogen), as
described previously (7).
Construction of Mutant CaRs with Double Mutations of
C101S/C236S--
Cassette 1, carrying C101S, was doubly digested with
BspEI and NheI, and the larger fragment obtained
from the above digestions was ligated to the smaller fragment resulting
from digestion of the CaR carrying C236S with BspEI and
NheI.
Construction of C129S/C131S-containing CaRs with Inactivating
Mutations--
Cassette 1 carrying C129S/C131S was doubly digested
with KpnI and BspEI, and the smaller fragment
obtained from the above digestion was ligated to the larger fragment
resulting from digestion of the CaR carrying E297K or A877Stop with
KpnI and BspEI.
Transient Expression of CaRs in HEK293 Cells--
The DNA for
transfection was prepared using the Midi Plasmid Kit (Qiagen).
LipofectAMINE (Life Technologies, Inc.) was employed as a DNA carrier
for transfection (11). The human embryonic kidney (HEK293) cells used
for transient transfection were provided by NPS Pharmaceuticals, Inc.
(Salt Lake City, UT) and cultured in Dulbecco's modified Eagle's
medium (Life Technologies, Inc.) with 10% fetal bovine serum
(Hyclone). The DNA-liposome complex was prepared by mixing DNA and
LipofectAMINE in OPTI-MEM I reduced serum medium (Life Technologies,
Inc.) and incubating the mixture at room temperature for 30 min. The
DNA-lipofectAMINE mixture was then diluted with OPTI-MEM I reduced
serum medium and added to 90% confluent HEK293 cells plated on
13.5 × 20.1-mm glass coverslips using 2.5 µg of DNA. After a
5 h incubation at 37 °C, equivalent amounts of OPTI-MEM I
reduced serum medium with 20% fetal bovine serum were added to the
medium overlying the transfected cells, and the latter was replaced
with fresh Dulbecco's modified Eagle's medium containing 10% fetal
bovine serum at 24 h after transfection. The expressed
[Ca2+]o-sensing receptor protein was assayed
48 h after the start of transfection. To perform coexpression of
two receptors, 1.25 µg of each of the two cDNAs were mixed and
used to transfect HEK293 cells.
Biotinylation of Cell Surface Forms of the CaR and Cross-linking
of Multimeric Receptors--
Before preparing whole cell lysates,
intact HEK293 cells transiently transfected with FLAG-tagged CaR were
washed twice with phosphate-buffered saline and treated with 1 mM ImmunoPure Sulfo-NHS-Biotin (Pierce), a
membrane-impermeant biotinylation reagent, at room temperature with
constant agitation for 30 min to biotinylate the proteins on the cell
surface. The reaction was then quenched by incubating the cells in 1 M Tris-HCl, pH 7.5, for 5 min. For cross-linking
experiments, we added an appropriate amount of bis(sulfosuccinimidyl) suberate (BS3), a noncleavable, membrane-impermeant
cross-linker, into the labeling solution with ImmunoPure
Sulfo-NHS-Biotin.
Preparation of Whole Cell Lysates--
The surface-biotinylated
and/or cross-linked HEK293 cells were rinsed twice with
phosphate-buffered saline and solubilized with 1% Triton X-100, 0.5%
Igepal CA-630, 150 mM NaCl, 10 mM Tris, pH 7.4, 2 mM EDTA, 1 mM EGTA, and protease inhibitors
including 83 µg/ml aprotinin, 30 µg/ml leupeptin, 1 mg/ml Pefabloc
(Roche Diagnostics, Indianapolis, IN), 50 µg/ml calpain inhibitor, 50 µg/ml bestatin, and 5 µg/ml pepstatin (1×
immunoprecipitation buffer) at room temperature. Insoluble material was
removed by centrifuging the cell lysates at 15,000 rpm for 15 min at
4 °C. The supernatants were collected as total cell lysates. The
protein concentration was determined using the Pierce BCA protein assay (Pierce).
Immunoprecipitation of FLAG-tagged CaRs--
To a
microcentrifuge tube, 5 µg of anti-FLAG M2 monoclonal antibody, 400 µl of H2O, 500 µl of 2× immunoprecipitation buffer (see above), and 100 µl of total lysate containing ~250 or 500 µg
of total proteins were added. The mixture was incubated at 4 °C for
1 h. To the mixture was then added 5 µl of an anti-mouse IgG
(Sigma). The incubation was continued for an additional 30 min at
4 °C. To the mixture was subsequently added 50 µl of 10% protein
A agarose (Life Technologies, Inc.) for a further 30-min incubation at
4 °C. The protein A-agarose was washed three times with 1×
immunoprecipitation buffer, and the immunoreactive species were
subsequently eluted in 45 or 60 µl of 2× electrophoresis sample
buffer at 65 °C for 30 min. In a given experiment, the same amount
of total protein for each sample was used for immunoprecipitation and
was eluted with the same volume of SDS-sample buffer. The receptor of
interest was detected by Western analysis.
Western Analysis of the Human CaR Expressed in Whole Cells and on
the Cell Surface--
If not specified, 15 µl of eluted,
immunoprecipitated sample was subjected to SDS-containing
polyacrylamide gel electrophoresis (PAGE) (12) using a linear gradient
of polyacrylamide (3-10%). The proteins on the gel were subsequently
electrotransferred to a nitrocellulose membrane. 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)
followed by visualization of the biotinylated bands with an enhanced
chemiluminescence (ECL) system (PerkinElmer Life Sciences).
After removal of the avidin using the recommended procedure for
stripping the blots (Amersham Pharmacia Biotech), all forms of the CaR
on the same blot were detected using an anti-CaR antiserum (4641 or
4637, polyclonal antisera raised against two peptides within the
extracellular domain of the CaR, kindly provided by Drs. Forrest Fuller
and Rachel Simin at NPS Pharmaceuticals, Salt Lake City, UT)
followed by a secondary, horseradish peroxidase-conjugated goat
anti-rabbit antibody and then an ECL system (Amersham Pharmacia Biotech).
Measurement of [Ca2+]i by Fluorimetry in
Cell Populations--
Coverslips with HEK293 cells previously
transfected with the appropriate CaR cDNAs were loaded for 2 h
at room temperature with fura-2/AM in 20 mM HEPES, pH 7.4, containing 125 mM NaCl, 4 mM KCl, 1.25 mM CaCl2, 1 mM MgSO4, 1 mM NaH2PO4, 0.1% bovine serum
albumin, and 0.1% dextrose and then washed once with a bath solution
(20 mM HEPES, pH 7.4, containing 125 mM NaCl, 4 mM KCl, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% dextrose, and 0.1% bovine
serum albumin) 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 (13). Extracellular calcium was increased stepwise to
give the desired final concentrations with additions of
[Ca2+]o in increments of 1 mM, which
were followed by 5 mM increments after achieving a level of
5.5 mM [Ca2+]o and 10 mM
increments after reaching a level of 20 mM
[Ca2+]o. Excitation monochrometers were centered
at 340 and 380 nm, and emission light was collected at 510 ± 40 nm through a wide-band emission filter. The 340/380 excitation ratio of
emitted light was used to calculate [Ca2+]i as
described previously (13).
Statistics--
The mean EC50 values for the various
wild type or mutant receptors determined in response to increasing
concentrations of [Ca2+]o were calculated from
the EC50 values for all of the individual experiments and
were expressed with the S.E. as the index of dispersion. Comparison of
the EC50 values was performed using analysis of variance or
Duncan's multiple comparison test (14) (p
0.05).
Each of the experiments described above in the experimental protocols
was generally performed at least three times.
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RESULTS |
Characterization of CaRs Carrying Single Cys to Ser Mutations in
the Extracellular Domain--
To examine the role of cysteines in the
formation of functional CaR dimers, we first looked at single Cys
mutations of the CaR. We constructed CaRs in which one of nine
cysteines in the extracellular domain (Cys60,
Cys101, Cys129, Cys131,
Cys236, Cys437, Cys449,
Cys482, or Cys598) was mutated to Ser.
Transient expression of these CaRs in HEK293 cells showed that the
mutations have varied effects on [Ca2+]o-elicited
[Ca2+]i responses (Table
I). Two of the mutant receptors, C129S
and C131S, become substantially more sensitive to
[Ca2+]o, with EC50 values that are
0.6 and 1.2 mM lower than the EC50 of wt,
respectively, but with significant reductions in their maximal
responses, which are 77 ± 4% and 54 ± 3% that of wt,
respectively. One of the mutations, C482S, does not significantly affect receptor function. Others become less sensitive to
[Ca2+]o than wt or are completely inactive (Table
I). Most of the mutant receptors that have decreased sensitivities to
[Ca2+]o also exhibit reduced maximal responses.
The mutant receptors containing C236S and C598S have no detectable
activity.
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Table I
[Ca2+]o-elicited [Ca2+]i responses
in cells transfected with CaRs carrying one or more cysteine to serine
mutations
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Consistent with the results of a previous study (15), one group of
mutant receptors carrying C129S, C131S, C437S, C449S, or C482S (Group I
mutations in Fig. 1), exhibit nearly
normal levels of expression on the cell surface and in whole cell
lysates (Fig. 1, lanes 2, 3, and
5-7). Of these receptors, C437S and C482S (Fig. 1A,
lanes 5 and 7) have slightly higher cell surface
expression than wt (Fig. 1A, lane 1), whereas
C129S, C131S, and C449S (Fig. 1A, lanes 2, 3, and
6) have somewhat lower cell surface expression than wt.

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Fig. 1.
Determination of cell surface and overall
expression of the mutant CaRs carrying one or two of the Group I
mutations. HEK293 cells were transfected with the relevant
FLAG-tagged wt or mutant CaRs. Proteins on the cell surface were
treated with Sulfo-NHS-Biotin before lysing the cells in the presence
of 100 mM iodoacetamide. The same amount of total proteins,
500 µg, were immunoprecipitated with anti-FLAG antibody in each case.
The immunopurified protein samples were eluted with 60 µl of SDS
sample buffer containing 100 mM dithiothreitol (DTT), and
15 µl of each was subjected to SDS-PAGE (3-10%). The surface
expression of the CaRs was detected with avidin (panel A).
Both surface and intracellular forms of the CaRs were then detected
with an anti-CaR antibody (4641) after removal of the avidin
(panel B). The samples in lanes 1-7 are the
following: lane 1, wt; lane 2, C129S; lane
3, C131S; lane 4, C129S/C131S; lane 5,
C437S; lane 6, C449S; and lane 7, C482S. The
monomeric species N-glycosylated with complex carbohydrates
or high mannose are indicated as C and H,
respectively, on the right hand side of the figure and are marked with
arrows.
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In contrast, another group of mutant receptors carrying C60S, C101S,
C236S, or C598S (Group II mutations in Fig.
2), have marked reductions in their
surface expression (Fig. 2A). In Fig. 2, lanes
3-6, twice as much of the samples were loaded as those in
lanes 1 and 2. The low apparent immunoreactivity
of C236S toward the anti-CaR antibody, 4641, in Fig. 2B,
lane 5, is likely due to the fact that Cys236 is
the last amino acid in the peptide
(Ala214-Cys236) against which 4641 was raised
and may be important for the binding of 4641 to the receptor. As shown
below, C236S has a similar level of overall expression to that of C598S
when it was detected with another anti-CaR antibody, 4637, which was
raised against a peptide consisting of amino acids 344-358 in the
extracellular domain of the CaR. Whereas C598S (Fig. 2B,
lane 2) had a much lower mature form than wt, two other
mutant receptors, C60S and C101S, were even lower (Fig. 2B,
lanes 3 and 4). Note that twice the amount of
eluted, immunoprecipitated samples were loaded in Fig. 2, lanes 3 and 4, as in Fig. 2, lanes 1 and
2. The mature forms of these mutant receptors are
N-glycosylated with complex carbohydrates, corresponding to
the upper bands of the doublets between 127 and 200 kDa (7).

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Fig. 2.
Determination of cell surface and overall
expression of the mutant CaRs carrying one or two of the Group II
mutations. The samples were prepared as in Fig. 1. The same amount
of total proteins, 500 µg, were immunoprecipitated in each case and
eluted with 60 µl of SDS sample buffer containing 100 mM
DTT. The CaRs on the cell surface (panel A) and in the whole
cell lysates (panel B) were detected as in Fig. 1. The
samples in lanes 1-6 are wt (lane 1), C598S
(lane 2), C60S (lane 3), C101S (lane
4), C236S (lane 5), and C101S/C236S (lane
6). Lanes 3-6 were loaded with 30 µl of the eluted,
immunoprecipitated proteins, whereas the other lanes were loaded with
15 µl of the eluted, immunoprecipitated proteins. The monomeric
species N-glycosylated with complex carbohydrates or high
mannose are indicated as C and H, respectively,
on the right hand side of the figure and are marked with
arrows.
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Under nonreducing conditions, all of the mutant receptors with a single
point mutation, behaving like wt, were present mostly as dimers or
higher multimeric forms on SDS-PAGE (Fig.
3 and 4) when we detect either only the surface forms or all forms of the receptor. However, longer exposure reveals trace amounts of monomeric CaRs for some of the mutant receptors (e.g. C129S, C131S,
and C482) on blots stained for cell surface receptor proteins (Fig. 3A, lanes 2, 3, and 6). In
contrast to previous studies with C60S, C101S, and C236S (5, 15), no
significant amounts of the monomeric species of these three mutant
receptors were detected either on the cell surface or in the whole cell
lysates (Fig. 4, lanes 2-4). As shown in Fig.
4B, lanes 4 and 5, C236S, has a
similar level of overall expression to that of C598S when it was
detected with 4637. Thus dimerization of wt appears to involve
more than one intermolecular disulfide bond, necessitating mutation of
two or more cysteines to identify those forming intermolecular
disulfide bonds.

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Fig. 3.
Detection of intermolecular disulfide-linked
dimeric CaRs carrying one of the Group I mutations under nonreducing
conditions. The samples were prepared as described in Fig. 1,
except that the immunopurified protein samples were eluted with SDS
sample buffer containing no DTT. The CaRs on the cell surface
(panel A) and in the whole cell lysates (panel B)
were detected as in Fig. 1. The same amount of total proteins, 500 µg, were immunoprecipitated in each case and eluted with 60 µl of
SDS sample buffer containing no DTT, and 15 µl of each was subjected
to SDS-PAGE (3-10%). The samples in lanes 1-6 are wt
(lane 1), C129S (lane 2), C131S (lane
3), C437S (lane 4), C449S (lane 5), and
C482S (lane 6). The monomeric and dimeric CaRs are indicated
as M and D, respectively, on the right hand side
of the figure and are marked with arrows.
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Fig. 4.
Detection of intermolecular disulfide-linked
dimeric CaRs carrying one or two of the Group II mutations under
nonreducing conditions. The samples were prepared as in Fig. 3.
The same amount of total proteins, 250 µg, were immunoprecipitated
and eluted with 45 µl of SDS sample buffer containing 100 mM DTT in each case. The CaRs on the cell surface were
determined using avidin (panel A) as in Fig. 1. The CaRs in
the whole cell lysates were then detected with an anti-CaR antibody
(4637) recognizing a different epitope from antibody 4641 after removal
of the avidin (panel B). The samples in lanes
1-6 are wt (lane 1), C60S (lane 2), C101S
(lane 3), C236S (lane 4), C598S (lane
5), and C101S/C236S (lane 6). Lanes 2-6
were loaded with 30 µl of the eluted, immunoprecipitated proteins,
whereas lane 1 was loaded with 15 µl of the eluted,
immunoprecipitated proteins. The monomeric and dimeric CaRs are
indicated as M and D, respectively, on the right
hand side of the figure and are marked with arrows.
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Construction and Characterization of Mutant CaRs with More than One
Cys to Ser Mutation in the Extracellular Domain--
To identify
cysteines forming intermolecular disulfide bonds, we constructed mutant
receptors with two or more mutations of cysteines to serines but
focused on those that had no detrimental effects on either receptor
expression or function when studied as single mutations. We found that
the mutant receptor, C129S/C131S, had a substantial level of expression
on the cell surface (Fig. 1A, lane 4), although it was
slightly lower than those of the mutant receptors with C129S or C131S
alone (Fig. 1A, lanes 2 and 3). The
activity of the mutant receptor, C129S/C131S, was also affected more
than those of the two singly mutated receptors, C129S and C131S (Table
I). C129S/C131S became even more sensitive to
[Ca2+]o, with a lower EC50 than that
of C129S and similar to that of C131S. The cumulative maximal response
of C129S/C131S was significantly lower than those of the singly mutated receptors.
Under nonreducing conditions, a substantial amount of monomeric
C129S/C131S was detected when assessed by SDS-PAGE (Fig. 5, lane
2). Detection of the cell surface form of this receptor (Fig. 5A, lane 2) revealed that the monomeric receptor
is the major species, unlike wt (Fig. 5A, lane
1). In contrast, reprobing of the same blot with an anti-receptor
antibody, detecting both cell surface and intracellular forms of the
receptor (Fig. 5B, lane 2), showed that the
monomeric form of the receptor is a minor species. Thus, whereas a
large fraction of the mutant receptor, C129S/C131S, no longer forms
intermolecular disulfide bonds on the cell surface, the majority of
intracellular receptor species either still forms intermolecular
disulfide bonds or is resistant to dissociation of dimers to monomers
during SDS-PAGE despite the lack of intermolecular disulfide linkages.
When we examined mutant receptors carrying triple, quadruple, or
quintuple mutations, we found that, like C129S/C131S (Fig. 5A, lane 2), substantial amounts of receptors
containing both C129S and C131S no longer form intermolecular
disulfide-linked dimers on the cell surface (Fig. 5A,
lanes 3 and 4). However, the major species in the
whole cell lysates are still multimers (Fig. 5B, lanes
3 and 4). Other receptors with multiple Cys to Ser
mutations, containing either Cys129 or Cys131,
still form exclusively disulfide-linked dimers on the cell surface and
in whole cell lysates (data not shown).
We also examined the properties of the mutant receptor with the two
mutations, C101S and C236S (Figs. 2 and 4, lane 6). In contrast to a previously published study (5), the overall expression level of C101S/C236S (Fig. 4B, lane 6) is
equivalent to that of C101S (Fig. 4B, lane 3) and
much less than that of C236S (Fig. 4B, lane 4).
In addition, we found the surface expression level of C101S/C236S (Fig.
2A, lane 6), although detectable, much less than
the mutant receptor carrying either mutation alone (Fig. 2A,
lanes 4 and 5). Under nonreducing conditions,
there are no detectable mature monomeric species of the CaR in whole
cell lysates even though we detected a trace amount of its immature
form when the blot for Fig. 4B was exposed for a much longer
period of time (data not shown). This mutant receptor does show slight
activity (Table I).
Thus Cys129 and Cys131 appear essential for
formation of intermolecular disulfide bonds in most of the cell surface
forms of the CaR, similar to the result of Ray et al. (6).
However, we still detect a substantial amount of dimeric C129S/C131S on
the cell surface (Fig. 5A,
lane 2), even though it is a minor species relative to its
monomeric counterpart. Next we examined whether the mutant receptor,
C129S/C131S, which appears as a monomeric receptor in SDS-containing
buffer without any dithiothreitol, may exist on the cell surface as
noncovalently linked dimers. We utilized cross-linking of cell surface
receptors and coimmunoprecipitation of cotransfected receptors as
described previously (4).

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Fig. 5.
Detection of intermolecular disulfide-linked
dimeric CaRs carrying two or more of the Group I mutations under
nonreducing conditions. The samples were prepared as in Fig. 3.
The CaRs on the cell surface (panel A) and in the whole cell
lysates (panel B) were detected as in Fig. 1. The samples in
lanes 1-4 are wt (lane 1), C129S/C131S
(lane 2), C129S/C131S/C482S (lane 3), and
C129S/C131S/C437S/C449S/C482S (lane 4). The monomeric and
dimeric CaRs are indicated as M and D,
respectively, on the right hand side of the figure and are marked with
arrows.
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Demonstration of Dimer Formation of the CaR on the Cell Surface in
the Absence of Intermolecular Disufide Bonds--
To determine whether
the mutant receptor, C129S/C131S, forms cell surface dimers, we
stabilized any preexisting dimeric or higher multimeric forms of the
receptor by covalently linking them with BS3, a
noncleavable, membrane impermeant cross-linker, and surface-labeled the
cells with Sulfo-NHS-Biotin. The surface-biotinylated and cross-linked
cells were then lysed in the presence of iodoacetamide. The FLAG-tagged
C129S/C131S and wt were then immunoprecipitated, and the immunopurified
CaRs were eluted with DTT-containing SDS sample buffer. Increasing the
concentration of cross-linker causes a progressive rise in the ratio of
dimer to monomer for both wt (Fig. 6,
lanes 1-3) and C129S/C131S (Fig. 6, lanes 4-6).
In the absence of the cross-linker, the surface form of the mutant
receptor is mostly detected as monomer (Fig. 6, lane 4),
whereas the dimeric species becomes the major form of the receptor with
5 mM BS3 (Fig. 6, lane 6). This
result suggests that the dimeric receptor is still the principal form
present on the cell surface for C129S/C131S. In Fig. 6, twice as much
volume of the immunoprecipitated samples in lanes 1 and
4 were loaded in lanes 2 and 5,
whereas four times as much in lanes 1 and 4 were
loaded in lanes 3 and 6. This increased loading
is necessary because the inclusion of BS3 reduces
biotinylation for surface detection since both sulfo-NHS-Biotin and
BS3 form covalent bonds with the same pool of primary
amines on the receptor.

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Fig. 6.
Stabilization of oligomers of the CaR on the
cell surface using a noncleavable, membrane-impermeant
cross-linker. HEK293 cells were transfected with FLAG-tagged wt
(lanes 1-3) or C129S/C131S (lanes 4-6).
Proteins on the cell surface were treated with both Sulfo-NHS-Biotin
and varying concentrations of BS3, a cross-linker, before
lysing the cells. The CaR was then immunoprecipitated with anti-FLAG
antibody from the same amount of total proteins. The immunopurified
protein samples were eluted with DTT-containing SDS buffer and
subjected to SDS-PAGE (3-10%). Surface expression of the CaR was
detected with avidin. The concentrations of BS3 were 0 mM for the samples in lanes 1 and 4,
1 mM for those in lanes 2 and 5, and
5 mM for those in lanes 3 and 6,
respectively. The monomeric and dimeric CaRs are indicated as
M and D, respectively, on the right hand side of
the figure and are marked with arrows.
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To further demonstrate that C129S/C131S forms noncovalent dimers on the
cell surface, we cotransfected a truncated C129S/C131S-containing receptor with the full-length C129S/C131S-containing receptor, one of
which is FLAG-tagged, and immunoprecipitated with anti-FLAG antibody
(Fig. 7). If the nontagged and tagged
receptors formed heterodimers, we would be able to coimmunoprecipitate
the nontagged receptor with the tagged receptor. Since the monomeric
full-length and cytoplasmic tail-truncated receptors can be resolved
under reducing conditions on SDS-PAGE because of their differences in size, it should be possible to determine the relative amounts of the
tagged and nontagged receptors on the cell surface using avidin as a
probe for surface-biotinylated receptors on the blot.

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Fig. 7.
Coimmunoprecipitation of nontagged and
FLAG-tagged receptors with cysteine mutations. HEK293 cells
transfected singly or doubly with receptors either carrying or not
carrying the double mutations, C129S and C131S, were biotinylated
before lysing the cells in the presence of 100 mM
iodoacetamide. After immunoprecipitation with anti-FLAG antibody
and elution with SDS sample buffer containing DTT (panel
A) or no DTT (panel B) and SDS-PAGE (3-10%), CaR
surface expression was detected with avidin. The samples in lanes
1-7 are tagged wt alone (lane 1), tagged wt and
nontagged A877Stop (lane 2), tagged wt and nontagged
C129S/C131S/A877Stop (lane 3), nontagged wt and tagged
C129S/C131S/A877Stop (lane 4), tagged C129S/C131S/A877Stop
alone (lane 5), tagged C129S/C131S alone (lane
6), and tagged C129S/C131S and nontagged C129S/C131S/A877Stop
(lane 7). The monomeric species of the full-length and
truncated receptors are indicated as F and T,
respectively, on the right hand side of the figure and are marked with
arrows.
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Fig. 7A, lane 7, shows that the nontagged
truncated receptor, C129S/C131S/A877Stop, was coimmunoprecipitated in
cotransfected cells with the FLAG-tagged full-length receptor,
C129S/C131S, to a similar extent as the nontagged receptor, A877Stop,
when it was cotransfected with FLAG-tagged wt (Fig. 7A,
lane 2). If one of the molecular partners in cotransfected
cells has C129S/C131S and the other does not, they apparently still
associate with one another equally well. For instance, nontagged
C129S/C131S/A877Stop can be coimmunoprecipitated with tagged wt (Fig.
7A, lane 3), and nontagged wt can be
coimmunoprecipitated with tagged C129S/C131S/A877Stop (Fig.
7A, lane 4). As a control, none of the nontagged
receptors isolated from singly transfected cells could be
immunoprecipitated by anti-FLAG antibody (data not shown).
In contrast to the results observed with cotransfection of A877Stop and
wt (Fig. 7B, lane 2), a substantial amount of the coimmunoprecipitated complex, consisting of one or both of the C129S/C131S-containing receptors, can be dissociated by the addition of
SDS sample buffer alone in the absence of reducing agents (Fig. 7B, lanes 3, 4, and 7). For
instance, the coimmunoprecipitated complex of FLAG-tagged wt and
nontagged C129S/C131S/A877Stop can be detected as a monomeric species
in a 1:1 ratio (Fig. 7B, lane 3) under
nonreducing conditions, even though most of the FLAG-tagged wt (Fig.
7B, lane 3) in the cotransfected cells is still
detected as disulfide-linked homodimers, similar to that seen for wt
transfected alone (Fig. 7B, lane 1). In other
words, the monomeric species of wt observed in Fig. 7B,
lanes 3 and 4 is actually associated noncovalently with C129S/C131S/A877Stop before the treatment with SDS
sample buffer. The inclusion of iodoacetamide in the lysis buffer
prevents the monomeric wt from associating with itself through
disulfide linkages, specifically or nonspecifically, after it is
dissociated from C129S/C131S/A877Stop in the SDS-sample buffer. Thus,
our results suggest that the CaR can form dimers through interactions
other than intermolecular disulfide bonds.
Reconstitution through Heterodimerization of
[Ca2+]o-elicited [Ca2+]i
Responses in Cells Cotransfected with Two Inactive Mutant CaRs, Both
Carrying C129S/C131S--
One test for dimerization of CaR monomers is
the recovery of function observed for some inactive mutant pairs when
they were cotransfected together in HEK293 cells (16). Similar rescue experiments were performed with mutant CaRs carrying C129S/C131S, which
were, therefore, incapable of forming covalently linked dimers. HEK293
cells were transiently cotransfected with pairs of mutant CaRs carrying
one of the two inactivating mutations, E297K or A877Stop, with or
without C129S/C131S. As a control, cells were also transfected with wt
or mutant CaRs alone. CaR-mediated biological responses were examined
by measuring [Ca2+]o-elicited increases in
[Ca2+]i. As reported earlier (16), cells
cotransfected with the individually inactive mutant CaRs, A877Stop and
E297K, exhibited much greater responses than did cells transfected with
either A877Stop or E297K alone. Introduction of the double mutations, C129S/C131S, into E297K and A877Stop did not significantly alter the
functions of the individual mutant receptors when transfected by
themselves (Fig. 8, B and
C; data are not shown for mutant receptors carrying
A877Stop, which are completely inactive). Cotransfection of
C129S/C131S/E297K with C129S/C131S/A877Stop reconstituted CaR-mediated signaling to an extent almost identical to that observed in cells cotransfected with A877Stop and E297K (Fig. 8). Furthermore, Fig. 9 shows that C129S/C131S/E297K and
C129S/C131S/A877Stop readily form heterodimers when cotransfected. For
instance, nontagged C129S/C131S/A877Stop can be coimmunoprecipitated
with FLAG-tagged C129S/C131S/E297K to an extent similar to that
observed with nontagged A877Stop and tagged E297K (Fig. 9A,
lanes 3 and 4). However, a substantial amount of
C129S/C131S/A877Stop and C129S/C131S/E297K are not associated with one
another through disulfide linkages (Fig. 9B, lanes
4 and 5), unlike A877Stop/E297K (Fig. 9B,
lanes 3). As a control, the nontagged C129S/C131S/E297K and
C129S/C131S/A877Stop cannot be immunoprecipitated when transfected
alone (Fig. 9, lanes 7 and 8).

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Fig. 8.
Cotransfection of inactive CaRs reconstitutes
CaR-mediated, [Ca2+]o-elicited
[Ca2+]i responses in HEK293 cells. Responses
are normalized to the maximal cumulative [Ca2+]i
responses of cells transfected with wt alone. HEK293 cells were
transfected with wt and mutant CaRs alone or cotransfected with
A877Stop and E297K. Some of the mutant receptors carry the additional
double mutations, C129S/C131S. Points are mean values ± S.E.
(n = 3 to 9). Error bars are smaller than
the symbols. A-G show wt alone (A), E297K alone
(B), C129S/C131S/E297K alone (C), E297K and
A877Stop (D), C129S/C131S/E297K and A877Stop (E),
E297K and C129S/C131S/A877Stop (F), and C129S/C131S/E297K
and C129S/C131S/A877Stop (G).
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Fig. 9.
Coimmunoprecipitation of a nontagged
inactivating mutant with a FLAG-tagged inactivating mutant
receptor. HEK293 cells transfected singly or doubly with
inactivating mutant receptors either carrying or not carrying the
additional double mutations, C129S and C131S, were biotinylated before
lysing the cells in the presence of 100 mM iodoacetamide.
After immunoprecipitation with anti-FLAG antibody and elution
with SDS sample buffer containing DTT (panel A) or no DTT
(panel B) and SDS-PAGE (3-10%), CaR surface expression was
detected with avidin. Samples in lanes 1-8 are tagged wt
alone (lane 1), tagged C129S/C131S/E297K (lane
2), tagged E297K and nontagged A877Stop (lane 3),
tagged C129S/C131S/E297K and nontagged C129S/C131S/A877Stop (lane
4), nontagged C129S/C131S/E297K and tagged C129S/C131S/A877Stop
(lane 5), tagged C129S/C131S/A877Stop alone (lane
6), nontagged C129S/C131S/E297K alone (lane 7), and
nontagged C129S/C131S/A877Stop alone (lane 8). The monomeric
species of the full-length and truncated receptors are indicated as
F and T, respectively, on the right hand side of
the figure and are marked with arrows.
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DISCUSSION |
The CaR principally exists as a dimer on the cell surface of
CaR-transfected HEK293 cells, which is covalently linked by disulfide bonds (4). In previous attempts to identify the cysteines in the
receptor extracellular domain responsible for CaR dimerization, two
groups obtained discrepant results. Both groups identified two
cysteines in the N-terminal extracellular domain that may be involved
in intermolecular disulfide linkages, similarly to mGluR5 (2). However,
one group proposed that Cys101 and Cys236 form
intermolecular disulfide linkages, whereas the other showed that
Cys129 and Cys131 mediate intermolecular
disulfide linkages.
In the present studies, we initially focused on cysteines that are not
very critical for receptor expression on the cell surface (15),
assuming that the cysteines that participate in the intermolecular disulfide bonds are not critical for receptor protein folding. We found
that mutating Cys129 and Cys131, individually
or in combination, has little effect on receptor protein expression.
Consistent with the results of the study performed by Ray et
al. (6), we found that the presence of the double mutations,
C129S/C131S, eliminated most interomolecular disulfide linkages in the
receptor proteins expressed on the cell surface. In contrast to the
other study (5), however, we found that the double mutations, C101S and
C236S, which interfere substantially with receptor expression when
present either individually or in combination, do not disrupt
disulfide-mediated dimerization.
The dimerization of many other G protein-coupled receptors, including
the
2-adrenergic receptor (9), the dopamine D2 receptor (17), the
opioid receptor (3, 18), and the bradykinin B2 receptor (19), are not mediated by intermolecular
disulfide bonds. For instance, constitutive dimerization of the
2-adrenergic and the dopamine D receptors occurs via
their transmembrane regions, whereas agonist-induced dimerization of
the bradykinin B2 receptor requires noncovalent
interactions of the N terminus of the receptor. In this study, we have
shown both biochemically and functionally that the removal of the
intermolecular disulfide linkages normally present in the CaR does not
affect its dimerization. As shown in Fig. 7A, lanes
2 and 7, pairs of CaRs with the double mutations, C129S
and C131S, can associate with one another to an extent similar to those
in which Cys129 and Cys131 are intact, even
though the majority of the receptor species are no longer linked with
one another through intermolecular disulfide bonds (Fig. 7B,
lanes 3, 4, and 7). Functionally,
coexpression of the inactive mutant CaRs, E297K and A877Stop, in which
these double cysteine mutations were present also led to reconstitution of significant [Ca2+]o-sensing and intracellular
signaling capability, which was indistinguishable from that of
cotransfected CaRs harboring E297K and A877Stop in which
Cys129 and Cys131 were intact.
One reason why others (6) did not find association of their truncated
receptor, TM1 mutant, with C129S/C131S may be that the missing part in
the TM1 mutant is likely essential for noncovalent association of
dimeric receptors. This suggests that the CaR may form dimers through
one or more noncovalent dimerization motifs present within the last six
transmembrane domains and the cytoplasmic tail. Our current studies
suggest that the cytoplasmic tail of amino acids 877 to 1078 is not
essential for the noncovalent associations between our truncated and
full-length receptors. Nevertheless, further studies are required to
determine the structural domains mediating the noncovalent interactions
with a focus on the region between TM5 and TM7, where putative
hydrophobic dimerization domains exist.
We found that C129S, C131S, and C129S/C131S are all much more sensitive
to [Ca2+]o than wt. In addition, they all have
reduced maximal responses in comparison to wt. C129S and C131S still
form mostly intermolecular disulfide-linked dimers, suggesting that
Cys129 and Cys131 are not only important for
formation of intermolecular disulfide linkages but also for the normal
function of the receptor even when the structural constraints conferred
by formation of covalent bonds with another CaR molecule are still
present. Consistent with the results of previous studies on several of
the mutations causing autosomal dominant hypocalcemia, those mutations
that are located in the vicinity of Cys129 and
Cys131, such as E127A and F128L, significantly increase the
sensitivity of the CaR to [Ca2+]o, even though
they do not disrupt the formation of intermolecular disulfide bonds as
suggested by Ray et al. (6).
In conclusion, our results support the involvement of
Cys129 and Cys131 in sulfydryl-sensitive
dimerization of the CaR and suggest that the region containing these
two cysteines is important for the normal function of the CaR. However,
the CaR still forms dimers, even in the absence of the intermolecular
disulfide linkages mediated by Cys129 and
Cys131. Furthermore, CaRs lacking these disulfide linkages
are still capable of the intermolecular interactions required for
functional reconstitution of individually inactive mutant receptors,
such as E297K and A877Stop.