 |
INTRODUCTION |
Calcium-sensing receptors
(CaR)1 couple extracellular
Ca2+ binding to intracellular Ca2+ transients
through Gq-mediated activation of phosphatidylinositol phospholipase C (1). CaR agonists (Ca2+ and other di- and
trivalent cations (2, 3), poly-L-arginine (4), spermine
(5), and
-amyloid (6)) bind within the large extracellular domain,
which has structural homology with a large class of bacterial
periplasmic binding proteins selective for ions, amino acids, and
sugars (7, 8). CaR is present in cell types involved in organismal
Ca2+ homeostasis, including the parathyroid, kidney, and
bone, as well as neuronal, fibroblast, and epithelial cell types, where its role(s) has yet to be defined (9).
Biochemical studies on heterologously expressed CaR indicate multiple
immunoreactive bands on Western blots that reflect both differential
glycosylation (10, 11) and higher, disulfide-linked oligomeric
complexes (12, 13). Although the physiological relevance of CaR
oligomerization is not known, a recent study has demonstrated CaR
dimers can be isolated from kidney epithelium (13). Preincubation of
kidney epithelial membranes with agonists (Ca2+,
Mg2+, and Gd3+) protected CaR dimers from the
actions of reducing agents, suggesting the presence of conformationally
sensitive disulfide bonds. Additional evidence for CaR dimerization
comes from the dominant negative effect observed when CaR bearing an
inactivating mutation (R185Q) was coexpressed with wild type receptor
in HEK 293 cells (14). mGluR5 receptors have also been shown to exist
as covalent dimers that can be converted to monomers by treatment with
reducing agents in vitro (15). Glutamate-mediated increases
in phosphatidylinositol metabolism in synaptosomes are inhibited by
dithiothreitol (16), implying a dependence of mGluR function on
dimerization. The region critical to mGluR5 receptor dimerization has
been localized to the first 17 kDa of the amino terminus by limited
proteolysis (15); this region contains four cysteine residues that are
conserved in CaR, mGluRs (17), and some members of the pheromone
receptor family (18, 19). Taken together, these studies suggest that the dimerization domain of CaR may be within the extracellular, agonist
binding amino-terminal domain.
Dimerization/oligomerization has been noted for many G protein-coupled
receptors, including opiate (20),
-adrenergic (21, 22), muscarinic
(23, 24), dopamine (25), m5-HT1B (26, 27), substance P
(28), C5a aphalotoxin (29), and platelet-activating factor (30)
receptors. For some types of receptors, including those for dopamine
(31) and muscarine (24, 32, 33), the complex agonist binding isotherms
are best explained by oligomerization-induced shifts in agonist
affinity. Opiate receptors undergo agonist-mediated monomerization,
which is a prelude to receptor sequestration (20). Interestingly, some
opiate agonists are not capable of mediating monomerization, and this
may account for their longer lasting physiological effects.
Dimerization is required for normal functioning of
-adrenergic
receptors (34), and in fact, receptor dimerization has been shown to
rescue the function of mutant forms of
-adrenergic (35) and
angiotensin type II (36) receptors. CaR (12, 13) and the related mGluRs
(15) exhibit covalent dimerization, and this may contribute in novel
ways to receptor function.
In this report, we demonstrate that both CaR and a truncated construct
consisting of the extracellular domain plus transmembrane helix 1 exist
as dimers in the absence of reducing agents and that disulfide bond
reduction causes a shift to the monomeric form of either full-length
CaR or the extracellular domain truncation. We have identified cysteine
residues that eliminate CaR dimerization by making cysteine
serine
point mutations in each of the cysteine residues within the first 17 kDa of the extracellular domain conserved between CaR and the mGluRs,
beginning at the amino terminus. We found that the double point mutant
hCaR(C101S/C236S)-GFP can be observed on SDS gels as a monomer in the
absence of reducing agents. Finally, we demonstrate that
hCaR(C101S/C236S)-GFP is functional when heterologously expressed in
HEK 293 cells but displays a decreased affinity for agonist and
significantly slower kinetics of activation and deactivation.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Human CaR (hCaR) in pBluescript was obtained from
Dr. J. Garrett (NPS Pharmaceuticals, Inc.). The rat
2 adrenergic
receptor (
2AR) was obtained from Dr. David Yue (Department of
Biomedical Engineering, Johns Hopkins University). GFP fusion proteins
were generated using pCDNA3.1 containing GFP in each of three
frames (CLONTECH). Restriction enzymes were
obtained from Life Technologies, Inc. and New England Biolabs.
Plasmid Construction--
All PCRs were performed with
PfuI polymerase (Stratagene) using 94, 57, and 72 °C as
denaturing, annealing, and elongation temperatures, respectively. All
constructs were confirmed by restriction digestion and automated
sequencing. hCaR with a carboxyl-terminal fusion to EGFP
(CLONTECH) was constructed as described previously (37). hCaR with a carboxyl-terminal fusion to the FLAG epitope was
prepared by replacing EGFP in the hCaR-GFP construct by inverse PCR (5'
primer, GATGACGACAAGTAAAGCGGCCGCGACTCT; 3' primer,
GTCCTTGTAGTCTGAATTCACTACGTTTTCTGTAACAGT). The ECD/TMH1-GFP construct
was prepared by generating a PCR product containing a BamHI
site after the first hCaR transmembrane domain (5' primer,
CGCTATTACCATGGTGATGCGG; 3' primer, CGCGGATCCTCGGTTGGTGGCCTT) using hCaR
as a template. After amplification, the product was cut with
XhoI-BamHI and subcloned in frame into pEGFPN3
(CLONTECH). A two-step cloning process was used to
generate TMD/Cterm-GFP. First, a PCR product containing the seven
transmembrane domains and the carboxyl-terminal domain of hCaR was
subcloned into the XhoI-BamHI sites of pEGFPN3
(5' primer, CGCCGCCTCGAGGAGATCGAGTTTCTG; 3' primer,
CGCGGATCCCACTACGTTTTCTGT). In a second step, a PCR product containing
the Kozak sequence and the CaR signal peptide was produced (5' primer,
CGCTATTACCATGGTGATGCGG; 3' primer, CGCCGCCTCGAGCCCGTAGGCAGAGGT) using
hCaR as the template and subcloned with NheI-XhoI
into the product of the first cloning step. Point mutations converting cysteine residues into serine (C60S, C101S, C131S, and C236S) were
individually introduced into the hCaR-GFP construct by PCR-based site-directed mutagenesis (38). The double point mutation (C101S/C236S) was made by performing a second round of mutagenesis in the background of the C101S point mutant. The
2AR-GFP construct was generated by
PCR with BamHI sites at the ends, followed by insertion into pEGFPN1 (CLONTECH).
Cell Culture and Transfections--
HEK 293 cells were grown in
high glucose Dulbecco's modified Eagle's medium (Life Technologies,
Inc.) supplemented with 10% bovine serum albumin (Sigma), 50 units/ml
penicillin, and 50 µg/ml streptomycin (at 37 °C and 5%
CO2). For functional studies, cells were transfected in
24-well plates with 0.1 µg of each construct using Effectene
(Qiagen). Eighteen hours later, they were plated on collagen-coated
coverslips and kept in a 5% CO2 incubator at 37 °C
until use (usually after 48-72 h). For membrane preparations, cells
were split into 100-mm dishes and transfected the next day with 1 µg
of each construct using Effectene (Qiagen). Some constructs were also
stably transfected with Effectene after linearization with
DraIII. Cells were kept in medium containing 300 µg/ml
Geneticin (Life Technologies, Inc.). Colonies were selected after 3 weeks, and clones were confirmed by cell fluorescence (GFP) and CaR activity.
Membrane Preparations--
Two days after transfection, HEK 293 cells were harvested in phosphate-buffered saline-EDTA (1 mM) and pelleted by brief centrifugation at 500 rpm
(4 °C). The pellet was resuspended in 300 µl of homogenization buffer containing protease inhibitors (14) plus 10 mM
iodoacetamide and passed 15 times through a syringe (25 gauge needle).
The homogenate was centrifuged at 20,000 × g for 2 min
at 4 °C. The supernatant was sedimented at 100,000 × g for 10 min (4 °C). The membrane pellet was solubilized
in 1% Triton X-100 and stored at
80 °C until use. Protein
concentration was determined using the BCA protein assay (Pierce).
Immunoprecipitation of Epitope-tagged Proteins--
HEK 293 cells were co-transfected with 1 µg each of the appropriately tagged
constructs. Crude homogenates (250 µg) or membrane protein (200 µg)
was precipitated with either 5 µl of anti-FLAG antibody (Sigma) or 3 µl of anti-GFP polyclonal antibody (CLONTECH) as
described in Ref. 12. The immunoreactive species were eluted from the
protein A complex with electrophoresis sample buffer (containing 100 mM
-mercaptoethanol) at 25 °C for 30 min and detected
by Western blotting.
Western Blotting--
Linear gradient (4-15%)
SDS-polyacrylamide gels (Bio-Rad) were loaded with 2-12 µg of
membrane protein. Some samples were reduced in 100 mM
-mercaptoethanol in SDS sample buffer for 30 min at room temperature
prior to gel loading. Protein was transferred to nitrocellulose
membranes (Bio-Rad), and blocked with 20% fetal bovine serum (Life
Technologies, Inc.) for 2 h. Blots were incubated overnight at
4 °C with primary antibodies (monoclonal anti-FLAG antibody (Sigma)
at a 1:2000 dilution or polyclonal anti-GFP antibody (CLONTECH) at 1:2500) and subsequently with
secondary antibodies conjugated to horseradish peroxidase (anti-mouse
or anti-rabbit (Amersham Pharmacia Biotech) at a 1:100,000 dilution).
The Blaze Chemiluminescence System (Pierce) was used for detection, and blots were scanned and processed with Adobe Photoshop.
Single Cell Fluorescence Measurements of Intracellular
Ca2+--
Transfected cells were identified by viewing GFP
fluorescence with a fluorescein isothiocyanate filter (595 nm emission
wavelength). Cells were loaded with 0.5 µM fura-2-AM
(Calbiochem) for 30 min at 37 °C in a solution containing 130 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 20 mM HEPES, 0.83 mM
Na2HPO4, 0.17 mM
NaH2PO4, and 25 mM mannose, plus 1 mg/ml bovine serum albumin, pH 7.4. After the loading period, the
coverslip was mounted in an imaging chamber (Warner Instruments) having
both a top and bottom coverslip, and washed with a solution containing
140 mM NaCl, 5 mM KCl, 0.55 mM
MgCl2, 0.5 mM CaCl2, 10 mM HEPES, pH 7.4. The cells were excited at 340/380 nm
(emission wavelength 510 nm) at 8-s intervals, and selected regions
were recorded on a Universal imaging system based on the MetaFluor
software package. Background images at the same gain settings used
during a particular experiment were obtained on regions of the
coverslip devoid of cells. All solutions were osmolality-matched.
Variations in extracellular Ca2+ concentration were
produced by isosmolar substitution for NaCl. All experiments were
performed at room temperature (22-24 °C). Multiple cells were
analyzed from at least three independent transfections (or cell
passages for stable cell lines). Data were normalized and averaged as
described for each experiment and are presented as the mean ± S.E. Curves were fitted by least squares minimization using the
Marquardt-Levenberg algorithm (NFIT, Island Products, Galveston, TX).
 |
RESULTS |
Dimerization of CaR--
Several recent reports suggest that CaR
exists in vivo (13) and in heterologously expressed systems
(12, 43) in a dimeric form that can be largely converted to the monomer
form by treatment with reducing agents such as dithiothreitol,
-mercaptoethanol, or
-mercaptopropanol. We have further
characterized dimerization of human CaR by expressing various
constructs (truncations and point mutations) in HEK 293 cells. Fig.
1 illustrates the control constructs
studied: human CaR-FLAG (a) (to permit antibody detection of
hCaR) and human CaR-GFP (b). A prominent band estimated at 320-340 kDa was observed in the absence of
-mercaptoethanol for hCaR-FLAG. This was converted to a doublet at 140-160 kDa upon reduction with
-mercaptoethanol. hCaR-GFP presented a greater dispersion of molecular masses in the absence of reducing agents, but
upon reduction, there was a prominent doublet at 205-220 kDa (molecular mass of hCaR plus the 27-kDa GFP tag). All samples were
exposed to 10 mM iodoacetamide during homogenization to
minimize formation of nonspecific disulfide bonds during membrane
isolation. Pretreatment with iodoacetamide reduced the dispersion in
the molecular masses of the oligomeric hCaR complexes and promoted a
more complete conversion to the monomer form upon reduction with
-mercaptoethanol, also noted in a recent report (12). Similar
conversions of dimer to monomer occur upon reduction of either
hCaR-FLAG or hCaR-GFP, suggesting that the GFP tag does not affect the
process. All further studies were performed on the GFP-tagged receptor,
because it permits rapid screening of expressed clones, cellular
localization of mutated receptors, and crude estimates of expression
levels to be made prior to functional studies. In previous studies, we
have demonstrated that carboxyl-terminal fusion of GFP to hCaR does not
alter the functional properties of the receptor (dose/response
relationship, desensitization, and subcellular localization) (37).
Here, we note that GFP does not alter hCaR dimerization.

View larger version (74K):
[in this window]
[in a new window]
|
Fig. 1.
Oligomer to monomer conversion of hCaR upon
reduction of disulfide bonds. Human CaR was tagged at the carboxyl
terminus with either the FLAG epitope (a) or GFP
(b) to permit identification on Western blots. Western blot
of membranes isolated from HEK 293 cells either transiently transfected
(a) or stably transfected (b) with the tagged
hCaR. Membranes were incubated in SDS sample buffer either with (+) or
without ( ) 100 mM -mercaptoethanol and run on 4-15%
SDS-polyacrylamide gel electrophoresis gels (2 µg
protein/lane).
|
|
Localization of the Dimerization Domain to the ECD--
To
determine the domain involved in hCaR dimerization, two hCaR
truncations were examined, one containing only the ECD plus TMH1
(ECD/TMH1-GFP) and another comprising only the seven transmembrane helices plus the carboxyl terminus (TMD/Cterm-GFP). Both truncations were tagged at their carboxyl termini with GFP and were localized to
both intracellular and plasma membranes of transiently transfected HEK
293 cells. Fig. 2 illustrates a
representative Western blot. hCaR-GFP (Fig. 2a) and
ECD/TMH1-GFP (b) are present predominantly as dimers in the
absence of
-mercaptoethanol. Including
-mercaptoethanol in the
sample buffer caused a substantial shift to the monomer form of
hCaR-GFP and a quantitative shift to the monomer form of ECD/TMH1-GFP.
Western blots of membranes from cells stably transfected with
TMD/Cterm-GFP (Fig. 2c) revealed that the expressed protein
was present in the monomer form in the absence and presence of reducing
agents. There was some high molecular mass material evident in the
nonreduced sample; this is most likely due to oxidation of cysteines
within the cytoplasmic tail during sample preparation and is not
prevented by iodoacetamide treatment, as has been noted previously
(12). These experiments localize disulfide bond-mediated dimerization
in hCaR to the ECD.

View larger version (93K):
[in this window]
[in a new window]
|
Fig. 2.
Oligomerization of various hCaR
truncations. Western blot of membranes isolated from HEK 293 cells
transfected with hCaR-GFP (a), ECD/TMH1-GFP (b),
or TMD/Cterm-GFP (c) either under nonreducing conditions
( ) or after treatment with 100 mM -mercaptoethanol
(+). Samples (a and b, 2 µg of protein/lane;
c, 12 µg of protein/lane) were separated on 4-15%
SDS-polyacrylamide gel electrophoresis gels.
|
|
Identification of Cys Residues Involved in Intermolecular Disulfide
Bond Formation in CaR--
Studies on mGluR5 indicated that
dimerization was eliminated by cleavage of the first 17 kDa of the
amino terminus (15). We therefore began by individually mutating to
serine the four cysteine residues of hCaR that are present within the
first 17 kDa of the amino terminus (at amino acid positions 60, 101, 131, and 236), which are conserved between CaRs and mGluRs. Whereas single cysteine
serine point mutations at these positions did not
eliminate dimerization (Fig. 3,
a-d), single point mutations at Cys101 and
Cys236 did increase the amount of monomer observed in the
nonreduced samples. Furthermore, as is apparent in Fig. 3, expression
of Cys101 or Cys236 was weak compared with a
single point mutation at Cys60 or Cys131 (all
lanes were loaded with equal amounts of membrane protein). We therefore
made the double point mutation hCaR(C101S/C236S)-GFP. This mutated
receptor expressed more robustly than either of the single point
mutations, and was present in the monomer state on Western blots in the
absence or presence of reducing agents (Fig. 3e). Direct
comparison of C101S, C236S, and C101S/C236S (Fig. 4) at higher protein levels confirms the
requirement for both point mutations to eliminate significant hCaR
dimerization in the absence of reducing agents.

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of cysteine to serine point mutations
on hCaR dimerization. Membranes (3 µg/lane) isolated from HEK
293 cells transfected with hCaR(C60S)-GFP (a),
hCaR(C101S)-GFP (b), hCaR(C131S)-GFP (c),
hCaR(C236S)-GFP (d), or hCaR(C101S/C236S)-GFP (e)
were run either under nonreducing conditions ( ) or in the presence of
100 mM -mercaptoethanol (+) on 4-15%
SDS-polyacrylamide gel electrophoresis gels.
|
|

View larger version (63K):
[in this window]
[in a new window]
|
Fig. 4.
Dimer/monomer comparisons for C101S, C236S,
and C101S/C236S mutations. Membranes were prepared and run as in
Fig. 3. Samples were hCaR(C101S)-GFP (12 µg/lane) (a),
hCaR(C236S)-GFP (12 µg/lane) (b), and
hCaR(C101S/C236S)-GFP (5 µg/lane) (c).
|
|
Noncovalent Dimerization of hCaR(C101S/C236S)-GFP--
To
ascertain whether hCaR(C101S/C236S)-GFP was correctly folded and
processed, we determined its cellular localization by assessing GFP
fluorescence (localization was comparable to that of wild type hCaR
(37), data not shown), its ability to form noncovalent dimers, and its
ability to activate Gq, resulting in increases in
intracellular Ca2+ (described below).
hCaR(C101S/C236S)-FLAG and hCaR(C101S/C236S)-GFP were transiently
coexpressed in HEK 293 cells, and membranes were isolated.
Immunoprecipitation was performed with either the anti-FLAG or anti-GFP
antibodies, and Western blots of the precipitated proteins were probed
with the anti-GFP antibody. Illustrated in Fig.
5 are the results of such an experiment.
The control lane (Fig. 5a) illustrates membranes from the
co-transfected HEK 293 cells (reduced with
-mercaptoethanol) probed
with the anti-GFP antibody. Fig. 5b illustrates the results
from immunoprecipitations with either the anti-FLAG (F) or
anti-GFP (G) antibody. Precipitation with either antibody
resulted in the appearance of anti-GFP-reactive protein of the correct
molecular weight (monomeric hCaR-GFP) on the Western blot. To confirm
that these results signify specific noncovalent dimerization of
hCaR(C101S/C236S)-FLAG and hCaR(C101S/C236S)-GFP, two control
experiments were performed, as illustrated in Fig. 5, c and
d. First, membranes isolated from HEK 293 cells transfected with hCaR(C101S/C236S)-GFP were immunoprecipitated with either the
anti-FLAG (F) or anti-GFP (G) antibody, and the
Western blot was probed with the anti-GFP antibody (Fig.
5c). A second control utilized membranes isolated from cells
transfected with both hCaR-FLAG and
2AR-GFP (which should not
associate with hCaR). Immunoprecipitations were performed with either
the anti-FLAG (F) or anti-GFP (G) antibody, and
blots were probed with the anti-GFP antibody (Fig. 5d). In both cases, precipitation with the anti-FLAG antibody did not result in
any anti-GFP-reactive protein on the Western blot, validating the
specificity of the immunoprecipitation process. These results suggest
that hCaR(C101S/C236S)-FLAG and hCaR(C101S/C236S)-GFP form noncovalent
dimers when coexpressed in HEK 293 cells.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 5.
Noncovalent dimerization of
hCaR(C101S/C236S). a, Western blot of 5 µg of protein
from a membrane preparation derived from HEK 293 cells co-transfected
with hCaR(C101S/C236S)-FLAG and hCaR(C101S/C236S)-GFP. The primary
antibody was a polyclonal anti-GFP antibody
(CLONTECH) at a 1:2500 dilution. b,
Western blot of the results of immunoprecipitation of a membrane
preparation derived from HEK 293 cells co-transfected with
hCaR(C101S/C236S)-FLAG and hCaR(C101S/C236S)-GFP. F denotes
lane loaded with the results of 250 µg of protein precipitated with
15 µg of the anti-FLAG antibody (Sigma). G denotes lane
loaded with the results of 250 µg of protein precipitated with 3 µl
of the anti-GFP antibody (CLONTECH). In both cases,
the primary antibody was the anti-GFP antibody, as in a.
c, Western blot of the results of immunoprecipitation of a
membrane preparation derived from HEK 293 cells transfected with
hCaR(C101S/C236S)-GFP. F and G are as described
in b. The primary antibody was anti-GFP, as described in
a. d, Western blot of an immunoprecipitation of a
membrane preparation from HEK 293 cells transiently transfected with
hCaR-FLAG and 2AR-GFP. All other methods were as in
b.
|
|
Functional Consequences of Cys
Ser Mutations in CaR
ECD--
The functional consequences of cysteine
serine mutations
were examined for those receptor mutations that expressed robustly in
HEK 293 cells, namely, C60S, C131S, and C101S/C236S. The dose/response relationships for Ca2+-dependent activation
were determined in individual transfected HEK 293 cells using
alterations in fura-2 fluorescence as a measure of changes in
intracellular Ca2+ (Fig. 6).
Transfected HEK 293 cells were exposed to sequential applications of
increasing concentrations of bath Ca2+ (from 2.5 to 30 mM) for periods of 60-90 s (until a steady state response
was reached). Ca2+ dose/response experiments were performed
on hCaR-GFP (Fig. 6a), hCaR(C60S)-GFP (b),
hCaR(C131S)-GFP (c), and hCaR(C101S/C236S)-GFP (d). Cells for analysis were chosen so that the average
difference in 340/380 ratio was approximately the same for all
mutations, to minimize potential differences in kinetics that can arise
from large differences in expression levels (44). Fig.
7 illustrates the dose/response
relationships calculated from averaged data obtained from at least
three independent transfections for each cysteine
serine mutant,
assayed as in Fig. 6. The dose/response relationships for
Ca2+-dependent activation of hCaR(C60S)-GFP
(EC50 2.9 ± 0.19 mM) and hCaR(C131S)-GFP
(EC50 4 ± 0.33 mM) were comparable to
that of wild type hCaR-GFP (EC50 3.5 ± 0.3 mM). In contrast, the dose/response relationship for
Ca2+-dependent activation of
hCaR(C101S/C236S)-GFP was linear over the range of Ca2+
from 0.5 to 30 mM (Fig. 6). Similar experiments were
performed on hCaR(C101S)-GFP and hCaR(C236S)-GFP, but expression levels were extremely low (as corroborated by the Western blots illustrated in
Fig. 3), even when stably transfected cell lines were produced. Although sufficient data could not be obtained, a few positive cells
for each clone presented behavior qualitatively similar to
hCaR(C101S/C236S)-GFP, i.e. a linear response to increases in extracellular Ca2+ up to 30 mM.

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 6.
Activation of hCaR mutants by extracellular
Ca2+. HEK 293 cells transiently transfected with
various hCaR mutants were exposed to increasing concentrations of bath
Ca2+ (from 2.5 to 30 mM) from a baseline
concentration of 0.5 mM for 60-90 s until the response
reached a new steady state. Points represent average ± S.E. from
at least five cells from at least two independent experiments for each
mutant such that the average 340/380 ratio was 1; a,
hCaR-GFP ( 340/380 ratio = 1.02 ± 0.05); b,
hCaR(C60S)-GFP ( 340/380 ratio = 1.06 ± 0.04);
c, hCaR(C131S)-GFP ( 340/380 ratio = 1.12 ± 0.03); d, hCaR(C101S/C236S)-GFP ( 340/380 ratio = 1.02 ± 0.05).
|
|

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 7.
Dose/response relationships for
Ca2+-dependent activation of hCaR mutants.
Experiments of the type illustrated in Fig. 6 were tabulated from at
least three independent transfections for each mutant. Data for each
cell examined at a range of Ca2+ concentrations was
normalized to the response in 30 mM bath Ca2+.
Mutants examined were hCaR-GFP (closed circles),
hCaR(C60S)-GFP (closed triangles), hCaR(C131S)-GFP
(closed diamonds), and hCaR(C101S/C236S)-GFP (closed
inverted triangles). Lines represent fits of averaged
data to the Hill equation for all clones except hCaR(C101S/C236S)-GFP.
Fit parameters were as follows: hCaR-GFP, 39 cells, EC50 = 3.5 ± 0.3 mM, Hill n = 2.0 ± 0.3; hCaR(C60S)-GFP, 29 cells, EC50 = 2.9 ± 0.19 mM, Hill n = 2.8 ± 0.6;
hCaR(C131S)-GFP, 22 cells, EC50 = 4.0 ± 0.33 mM, Hill n = 2.2 ± 0.39;
hCaR(C101S/C236S)-GFP, 29 cells.
|
|
A second feature unique to hCaR(C101S/C236S)-GFP is a distinct
difference in the kinetics of the response to extracellular Ca2+. As can be seen in Fig. 6, the kinetics of
hCaR(C60S)-GFP and hCaR(C131S)-GFP (b and c) are
comparable to those of wild type hCaR-GFP (a), with rapid
increases in intracellular Ca2+ upon exposure to increasing
concentrations of extracellular Ca2+. In contrast, the
responses of hCaR(C101S/C236S)-GFP were slow (Fig. 6d), and
the new steady state level of intracellular Ca2+ was not
reached for over a minute in each successive Ca2+
concentration. Furthermore, the kinetics of washout from 30 to 0.5 mM Ca2+ were also slow for
hCaR(C101S/C236S)-GFP, taking 81.6 ± 3.5 s for the
intracellular Ca2+ to drop from the level in 30 mM bath Ca2+ to the baseline intracellular
Ca2+ observed in 0.5 mM bath Ca2+.
In contrast, the drop in intracellular Ca2+ upon washout of
30 mM bath Ca2+ for hCaR-GFP was 38.4 ± 2.7 s, comparable to that observed for hCaR(C131S)-GFP (40 s),
whereas the drop in intracellular Ca2+ upon extracellular
Ca2+ washout for hCaR(C60S)-GFP was biphasic, taking a
total of 62.4 ± 8.6 s, with 85% of the decrease achieved in
36 ± 3 s.
 |
DISCUSSION |
Calcium-sensing receptors are observed on Western blots as dimers
and/or higher oligomers in the absence of reducing agents. Treatment
with iodoacetamide during membrane isolation minimizes the formation of
nonphysiologically relevant disulfide-linked oligomers, although this
protection may not be complete, as has been noted in previous studies
(12, 13). Despite these potential problems, a prominent dimer band is
present on Western blots of nonreduced samples, which is largely
converted to monomeric hCaR by treatment with
-mercaptoethanol.
Studies with biotinylation and immunoprecipitation of hCaR expressed in
HEK 293 cells have suggested that the major form of hCaR on the plasma
membrane is a dimer (12). We therefore sought to identify the domain
responsible for hCaR dimerization. To confirm suspicions derived from
study of mGluRs (15, 39) that suggested that the dimerization domain was localized to the ECD, we expressed truncations of hCaR that contained either the ECD/TMH1-GFP or TMD/Cterm-GFP. The ECD/TMH1-GFP construct was present as a dimer in the absence and monomer in the
presence of
-mercaptoethanol, whereas TMD/Cterm-GFP was a monomer in
either condition. These results identify the ECD as the locus for
covalent dimerization of CaR.
The calcium-sensing receptor sequence contains 19 cysteine residues
that are present in comparable positions in mGluRs, 17 of which are in
the ECD. Luckily, the suspected covalent dimerization domain for mGluRs
was localized to the first 17 kDa of the amino terminus (15), and we
thus began by making point mutations (Cys
Ser) in the four
conserved cysteines within this region of hCaR. Mutations of the
cysteine residues at positions 60 and 131 did not eliminate
dimerization of hCaR and had minimal effects on the functional activity
of the receptor as assessed by the dose/response relationship and
kinetics of the responses to extracellular Ca2+. Mutations
of the cysteine residues at positions 101 and 236 decreased the
expression of receptor significantly, indicating a potential problem
with folding and/or trafficking of the receptor to the plasma membrane.
These individual point mutations did not, however, eliminate
dimerization of the receptor. When a construct containing both point
mutations, C101S/C236S, was expressed, protein levels were
significantly increased, and dimerization was eliminated in the
absence/presence of reducing agents. It is highly likely, therefore,
that these two cysteine residues, at positions 101 and 236, are
involved in dimerization of CaR. Alternatively, mutations of cysteine
to serine at positions 101 and 236 may affect the conformation of the
ECD in a manner that prevents dimerization via other, as yet
unidentified cysteines (among the 17 present in the ECD). Here we
consider the most parsimonious conclusion, i.e. that
Cys101 and Cys236 are directly involved in hCaR
dimerization. Our conclusion is based upon several criteria: 1)
hCaR(C101S/C236S) is present as a monomer in the absence/presence of
reducing agents; 2) expression levels of the single point mutations,
hCaR(C101S), and hCaR(C236S) are weak, and monomerization, although
present, is variable, whereas the double point mutation hCaR
(C101S/C236S) expresses more robustly and is present as a monomer in
the absence of reduction (these results are reminiscent of what has
been observed in mutagenesis studies designed to identified partners in
salt bridges in proteins, i.e. elimination of one partner
destabilizes the protein, whereas elimination of both partners improves
protein expression); 3) hCaR(C101S/C236S) is correctly localized to
membranes within HEK 293 cells; 4) hCaR(C101S/C236S) exhibits activity
equivalent in magnitude to wild type hCaR, albeit with alterations in
properties; and, finally, 5) hCaR(C101S/C236S) folds in a manner that
maintains noncovalent dimerization of the receptor. Dimerization of
many G protein-coupled receptors is mediated by noncovalent
interactions among transmembrane domains. In particular, a motif has
been identified in the
-adrenergic receptor TM6 that promotes
noncovalent dimerization of receptors, which is essential for receptor
function (34). This motif is present in TM5 of CaR (12) and mGluRs and
may serve to promote noncovalent hCaR dimerization, leading
subsequently to disulfide bond formation. Now that the residues
contributing to covalent dimerization of CaR have been identified, the
contribution(s) of noncovalent interactions to CaR function can be
addressed in the hCaR(C101S/C236S)-GFP background.
Studies on hCaR(C101S/C236S)-GFP reveal a significant contribution(s)
of disulfide bond-mediated dimerization to normal hCaR function.
Significantly higher concentrations of Ca2+ are required
for modest activation of hCaR(C101S/C236S)-GFP. In fact, increases in
the response were linear from 0.5 through 30 mM. The most
striking difference in the behavior of hCaR(C101S/C236S)-GFP was the
slowing of response kinetics to both increases and decreases in extracellular Ca2+. These results indicate that
covalent, disulfide bond-mediated dimerization of hCaR is required for
normal agonist-mediated receptor activation. Further support for the
importance of dimerization in the function of this class of receptors
(including CaR, mGluRs, and GABABRs) comes from recent
reports that suggest that GABABRs are only functional when
expressed as heteromeric assemblies of subunits GABABR1 and
GABABR2 (40-42).
In conclusion, we have demonstrated that CaR is primarily a
disulfide-linked dimer in cell membranes, and we have identified the
disulfide bond-mediated dimerization domain as the ECD. Furthermore, we
have identified two cysteine residues within the ECD,
Cys101 and Cys236 of the human CaR, that
mediate covalent dimerization. What remains to be seen is whether the
cysteine residues present in comparable positions in other members of G
protein-coupled receptor family C, including mGluRs,
GABABRs, and pheromone receptors, contribute to
dimerization of these receptor types and likewise contribute to
receptor function.