Expression, Purification, and Biochemical Characterization of
the Amino-terminal Extracellular Domain of the Human Calcium
Receptor*
Paul K.
Goldsmith
,
Gao-Feng
Fan
,
Kausik
Ray
,
Joseph
Shiloach§,
Peter
McPhie¶,
Kimberly V.
Rogers
, and
Allen M.
Spiegel
**
From the
Metabolic Diseases Branch,
§ Biotechnology Unit, Laboratory of Cellular and
Developmental Biology, ¶ Laboratory of Biochemistry and Genetics,
NIDDK, National Institutes of Health, Bethesda, Maryland 20892, and
NPS Pharmaceuticals, Salt Lake City, Utah 84108
 |
ABSTRACT |
We purified the extracellular domain (ECD) of the
human calcium receptor (hCaR) from the medium of HEK-293 cells stably
transfected with a hCaR cDNA containing an isoleucine 599 nonsense
mutation. A combination of lectin, anion exchange, and gel permeation
chromatography yielded milligram quantities of >95% pure protein from
15 liters of starting culture medium. The purified ECD ran as an
~78-kDa protein on SDS-polyacrylamide gel electrophoresis and was
found to be a disulfide-linked dimer. Its
NH2-terminal sequence, carbohydrate content, and CD
spectrum were defined. Tryptic proteolysis studies showed two major
sites accessible to cleavage. These studies provide new insights into
the structure of the hCaR ECD. Availability of purified ECD protein
should permit further structural studies to help define the mechanism
of Ca2+ activation of this G protein-coupled receptor.
 |
INTRODUCTION |
The calcium receptor
(CaR)1 is a unique member of
the G protein-coupled receptor superfamily expressed in parathyroid and
kidney cells where it has been shown to play a critical role in
extracellular calcium homeostasis (1). The CaR is also expressed in a
variety of other sites such as the central nervous system where it may serve functions beyond systemic calcium homeostasis (2). In amino acid
sequence and presumed topographic structure, the CaR is most closely
related to a distinct subset of G protein-coupled receptors that
includes the metabotropic glutamate receptors (mGluRs) (3, 4) and a
multigene family of putative vomeronasal pheromone receptors (5-7). In
addition to the seven transmembrane domain characteristic of all G
protein-coupled receptors, the CaR and other members of its subset
contain a large, ~600-residue, amino-terminal extracellular domain
(ECD) that is heavily glycosylated and contains a large number of
highly conserved cysteines (8).
Based on limited amino acid homology to bacterial periplasmic binding
proteins, it has been suggested that the ECD of the mGluRs (9) and CaR
(10) has a bi-lobed, "venus flytrap"-like structure; however, there
is very little structural or biochemical information available for the
CaR or mGluR ECDs. Using tunicamycin, we have previously shown that
N-linked glycosylation of the CaR is essential for its
expression at the cell surface (11), but the nature and sites of
glycosylation have not been defined. Both the intact mGluRs (12) and
the CaR (13-15) have been shown to be disulfide-linked dimers; for the
mGluRs, it was found that intermolecular disulfide linkage of the ECD
accounts for dimer formation (12, 16), but the role of the ECD in
dimerization of the CaR has not been defined. To obtain biochemical and
structural information, we developed methods for large scale culture of
a cell line stably expressing a mutant form of the human CaR (hCaR) that results in secretion of the ECD into the medium. We report here
the purification of the secreted hCaR ECD and results of biochemical
analysis, including amino-terminal sequence, carbohydrate content,
dimeric structure, and accessibility to tryptic digestion.
 |
MATERIALS AND METHODS |
Stable Transfection of HEK-293 Cells with a Mutant hCaR
cDNA--
A mutant form of the full-length human parathyroid CaR
cDNA (17) in which cysteine 598 was changed to serine and
isoleucine 599 was changed to a stop codon (see Fig. 1) was subcloned
into NotI/HindIII-digested pCEP4 expression
vector (Invitrogen, San Diego, CA). The mutant hCaR cDNA was
transfected into HEK-293 cells with calcium phosphate, and 200 µg/ml
hygromycin was used to select stable transfectants. Resistant colonies
were subcloned and screened for hCaR expression by a solution
hybridization assay. Clone 32 used in this study was chosen based on
high levels of ECD secretion as determined by immunoblot analysis of
cell culture media. Cells were routinely cultured in Dulbecco's
modified Eagle's medium (Life Technologies Inc.) supplemented with
10% fetal bovine serum, 1% glutamine, 1% penicillin and
streptomycin, and 200 µg/ml hygromycin at 37 °C in a 5%
CO2 environment.
Large Scale Cell Culture for Production of
ECD--
ECD-secreting clone 32 HEK-293 cells were immobilized on
cellulose discs in a packed bed configuration using a 2.2-liter
Celligen Plus bioreactor (New Brunswick Scientific, Edison, NJ) with a vertical mixing impeller assembly and internal basket. Production consisted of a propagation stage of approximately 140 h with
perfusion of serum-containing media. When a cell density of 2 × 1010 was reached, the second stage was initiated by
perfusion with serum-free media for 400 h at a flow rate of 4-6
liters/day while maintaining the residual glucose concentration at 1 g/liter. Perfusate was pumped directly on 100-ml bed volume Q-Sepharose
fast flow column (Amersham Pharmacia Biotech) equilibrated with
phosphate-buffered saline (PBS). Protein was eluted with a 1 M NaCl step elution or 0-1 M NaCl gradient in
0.01 M phosphate buffer, pH 7.4. ECD-containing fractions
were determined by immunoblotting with a monoclonal antibody, ADD,
specific for peptide 214-235 of hCaR (18) and were pooled for further purification.
ECD Purification--
ECD protein was purified from fractions
obtained from the Q-Sepharose fast flow column using a three step
protocol. Fractions collected from each step were monitored by
SDS-PAGE. Gels were either stained with Coomassie Blue or used for
immunoblotting with ADD monoclonal antibody. ECD-containing fractions
in PBS were first run over a 25-ml bed volume RCA-1 lectin agarose
column (EY Laboratories Inc., San Mateo, CA) followed by step elution with 0.2 M lactose in PBS. Lectin affinity-purified
proteins were further purified by FPLC anion exchange chromatography
using a 1-ml bed volume Mono Q column (Amersham Pharmacia Biotech) with elution at 1 ml/min and a gradient of 0-1 M NaCl in 0.02 M phosphate buffer, pH 7.4. ECD-containing fractions from
the anion exchange step were concentrated by centrifugal
ultrafiltration using Centricon YM-50 units (Amicon Inc., Beverly, MA)
before HPLC gel permeation chromatography using a 7.8-mm × 30-cm
TSK-3000 column (Tosohaas, Montgomeryville, PA) run at 0.5 ml/min in
PBS. Protein concentrations were determined by the Bradford dye binding
method (Bio-Rad).
Amino Acid Sequence Analysis--
Amino-terminal amino acid
sequence analysis was performed with a model 477A protein sequencer
coupled to a model 120A PTH analyzer (Applied Biosystems Inc., Foster
City, CA) according to the manufacturer's program NORMAL-1. Reversed
phase analysis of the PTH-derivatives was done using a Brownlee
PTH-C-18 column (2.1 × 220 mm).
Carbohydrate Analysis--
Samples of ECD proteins were analyzed
by high performance anionic exchange chromatography on a Dionex system
(Dionex Corp., Sunnyvale, CA) equipped with a pulsed amperometric
detector PAD2 and a pellicular PA1, 4 × 250 mm anion exchange
column. All assays were at room temperature at a flow rate of 0.8 ml/min in 16 mM NaOH with sensitivity set at 1 µA.
Profiles were recorded, and areas under the curve were integrated.
Retention times and calibration standards were monitored immediately
before and after determinations of unknown samples to correct for minor
variations in the response curves.
Circular Dichroism Analysis--
Circular dichroic spectra were
measured in a Jasco J-500C spectrophotometer attached to a DP500N data
processor at 25 °C. Protein concentrations were 100 µg/ml in 0.1 M phosphate buffer, pH 7.0. Spectra were digitized and
analyzed as described previously (19). Measured ellipticities were
converted into mean residue ellipticity using Eq. 1.
|
(Eq. 1)
|
A value of 110 was used for the mean residue weight. Spectra
were analyzed in terms of secondary structures using the CONTIN program
(20).
Polyclonal Peptide-specific Antibody Production--
Five
peptides, GP, LSN, ADD, LRG, and DGE, corresponding to sequences within
the ECD of the hCaR (see Fig. 1) were synthesized and used for
production of affinity-purified rabbit polyclonal antibodies as
described previously (21). Three additional peptide-specific rabbit
polyclonal antibodies, FF20-10, FF20-11, and FF20-7, representing other
regions of the hCaR ECD (see Fig. 1) were produced as described previously (22).
Tryptic Digestion of the ECD--
Samples of ECD that were to be
denatured before tryptic digestion were first heated for 10 min at
100 °C in 0.1% SDS. Digestion of denatured and undenatured ECD
samples was performed in 25 mM Hepes buffer, pH 8.0, for
varying times at 37 °C using a trypsin:protein ratio of 1:100 (w/w).
The reaction was terminated by adding an equal volume of SDS-PAGE
loading buffer (23).
SDS-PAGE and Immunoblotting--
Proteins were separated on 10%
SDS-polyacrylamide gels as described (23). For visualization of bands,
gels were either stained with Coomassie Blue dye or used for
immunoblotting as described previously (11, 18). Briefly, after
overnight transfer to nitrocellulose membranes at 150 mA constant
current, membranes were blocked in 5% horse serum, 0.05 M
Tris, pH 8.0, 0.5 M NaCl containing 0.1% Tween 20 (TBST)
for 1 h. Blots were incubated overnight in horse serum-TBST with
first antibodies at a concentration of 2 µg/ml for the antipeptide
monoclonal antibody, ADD, and 5 µg/ml for affinity-purified rabbit
polyclonal antipeptide antibodies. After washing three times for 20 min
with TBST, blots were incubated for 2 h with 1:2,000 dilutions of
peroxidase-conjugated goat anti-mouse antibodies (Kierkegaard and Perry
Laboratories Inc., Gaithersburg, MD) or peroxidase-conjugated sheep
anti-rabbit antibodies (The Binding Site, San Diego, CA) in horse
serum-TBST. Membranes were again washed twice for 10 min with TBST and
once with Tris-buffered saline for 10 min and then developed with
4-chloronaphthol as substrate.
 |
RESULTS AND DISCUSSION |
HEK-293 cells were stably transfected with an episomal vector
containing a mutant form of the hCaR cDNA engineered to lead to
secretion of the ECD into the culture medium (see Fig.
1). A single clone (clone 32) was
selected by immunoblot analysis of culture medium for high level
secretion of the ECD, and this clone was adapted to large scale culture
conditions in a bioreactor for production and purification of the ECD.
Analysis of the amino acid sequence of the ECD using the GCG program
(Genetics Computer Group, Madison, WI) indicated a protein with a
calculated pI of 4.95 and a net of 25 negative charges. The sequence
contains 11 putative glycosylation sites, and in the context of the
expressed intact receptor, the ECD is known to be heavily glycosylated
(8, 11, 24). Using a lectin affinity chromatography screening kit (EY
Laboratories Inc.) it was determined that RCA-1 lectin quantitatively
removed ECD protein from cell culture medium (data not shown). Our
purification protocol was based on this property and the presumed
relatively acidic nature of the ECD.

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Fig. 1.
Schematic representation of the human calcium
receptor. The amino acids are shown for the amino-terminal ECD,
and the remainder of the receptor is indicated schematically. The
arrows indicate a cysteine to serine mutation at residue 598 and isoleucine to stop codon mutation at residue 599 in the mutant form
of the receptor engineered to lead to secretion of the ECD into the
cell culture medium. The gap between residue 19, alanine, and residue
20, tyrosine, indicates the site of signal peptide cleavage as
determined by amino acid sequencing of the purified ECD. Putative
glycosylation sites are marked by branches, and
cysteine residues in the ECD are darkened.
Underlined and labeled regions with amino acids
in boldface type represent peptide sequences used to
generate peptide-specific rabbit polyclonal antibodies.
|
|
For a typical isolation, 15 liters of bioreactor perfusate containing
84 µg/ml protein were pumped over a 100-ml volume column of Fast
Q-Sepharose anion exchanger. Step elution with 1 M NaCl, 0.1 M phosphate buffer, pH 7.4, and localization of
ECD-containing fractions by immunoblotting resulted in a pooled volume
of 450 ml containing 0.56 mg/ml protein, which was run over a 25-ml bed volume column of RCA-1 lectin agarose affinity beads. Step elution with
0.2 M lactose in PBS gave 9.5 ml of solution containing
0.53 mg/ml protein.
The affinity-purified lactose eluate from the lectin column was then
subjected to FPLC Mono Q anion exchange chromatography (Fig.
2). As seen by immunoblot (panel
B) and Coomassie Blue staining (panel C) of
representative fractions, a broad elution pattern of ECD protein begins
at 0.22 M NaCl and ends at about 0.65 M NaCl,
with a peak of ECD protein eluting in fraction 38 at a concentration of
0.32 M NaCl. This elution profile is suggestive of the
existence of several tightly bound charged forms of the ECD protein
that could reflect heterogeneity of carbohydrate content, specifically sialic acid (see below). Fractions 33-42 were pooled (16 ml of solution containing 0.153 mg/ml of protein) and then concentrated before HPLC TSK-3000 gel permeation chromatography (Fig.
3). The ECD protein elutes as a single,
well defined peak at fraction 17, which corresponds to the approximate
elution volume seen for a bovine IgG immunoglobulin calibration
standard with a molecular mass of 154 kDa (data not shown) and suggests
that the ECD may be a dimer.

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Fig. 2.
Mono Q FPLC fractionation of proteins
isolated from cell culture medium by lectin affinity purification.
Panel A shows a representative tracing of
A280 measured in millivolts (heavy
line) for proteins eluted by a sodium chloride gradient
(thin line) using 0.02 M phosphate buffer, pH
7.2, and 0.02 M phosphate buffer, pH 7.2, containing 1 M NaCl as 100% mobile phase. Panel B shows the
immunoblot with monoclonal ADD antibody, whereas panel C
shows the pattern of Coomassie Blue staining. Lanes are numbered for
the representative fractions from the chromatographic separation. The
lanes marked "s" are those of the molelcular weight
standards indicated at the left of panels B and
C.
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Fig. 3.
TSK-3000 HPLC gel permeation chromatography
of pooled and concentrated Mono Q fractions 33-44. Panel A
shows the A280 (measured in millivolts) elution
profile for 200-µl injections of the ECD protein concentrate resolved
in PBS at a flow rate of 0.5 ml/min. Panel B shows the
immunoblot with ADD monoclonal antibody, whereas panel C
shows the pattern of Coomassie Blue staining. The lanes labeled
"s" contain the molecular weight standards indicated on
the left of panels B and C. The lanes
are labeled with numbers corresponding to fractions of the chromatogram
shown in panel A.
|
|
Coomassie Blue staining (Fig. 3, panel C) of SDS-reducing
electrophoretic gels of the TSK-3000 chromatographic fractions revealed two bands of protein, a major diffuse band between the 64- and 98-kDa
markers with an apparent molecular mass of ~78 kDa and a minor band
at ~48 kDa. The more sensitive immunoblot (Fig. 3, panel
B) also shows a major diffuse band at 78 kDa and a minor band at
48 kDa as well as two minor bands at 160-170 kDa, and a faint band at
52 kDa. Those extra bands, which are not seen by Coomassie Blue
staining represent very minor amounts of protein. Based on immunoblots
with region-specific antibodies (see below), we believe that the faster
migrating bands at 48 and 52 kDa represent proteolytically clipped
fragments of the ECD. The 160-170-kDa bands may represent trace
amounts of undissociated dimeric ECD protein (see Fig.
4).

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Fig. 4.
Immunoblot with ADD monoclonal antibody of
ECD protein separated by polyacrylamide gel electrophoresis under
reducing and nonreducing conditions. ECD protein (2.5 µg) was
incubated at the indicated temperatures in sample dissociation buffer
not containing -mercaptoethanol (nonreducing; lanes 2-5)
and sample dissociation buffer containing 50 mM
-mercaptoethanol (reducing; lanes 7-10) before loading
on the gels. Lanes 1 and 6, molecular weight
standards as labeled in lane 1; lanes 2 and
7, treatment with dissociation buffer on ice at 4 °C;
lanes 3 and 8, 3 min of incubation at 37 °C;
lanes 4 and 9, 3 min of incubation at 65 °C;
lanes 5 and 10, 3 min of incubation at
100 °C.
|
|
A typical purification yielded 1.33 mg of protein (representing 0.105%
of the starting amount of bioreactor perfusate protein) at >95%
purity based on visual inspection of Coomassie Blue-stained gels (Fig.
3C, lane 17). The pooled TSK-3000 fractions
containing purified ECD were used for further biochemical
characterization. ECD protein migrated in reducing electrophoresis gels
with an apparent molecular mass of 78 kDa, which is much greater than the predicted molecular mass of 67,071 Da for the sequence of amino
acids between residues 1-598. NH2-terminal amino acid
sequencing of the purified protein gave the following sequence:
YGPDQRAQKKGDIILGGLFP, corresponding to residues 20-39 of the hCaR
(Fig. 1) and indicating that signal peptide cleavage occurs after
alanine 19. This agrees well with an algorithm for predicting signal
sequence cleavage sites (25) that shows alanine very frequently in the
1 position and tyrosine, previously thought to be the site of
cleavage in the bovine CaR (8), never in the
1 position. The
calculated molecular mass of the ECD protein minus the signal peptide
residues and with the serine for cysteine substitution is 64,886 Da.
The difference in apparent molecular weight on SDS-PAGE
versus calculated molecular weight, the diffuse nature of
the ECD band on SDS-PAGE, the presence of multiple presumptive
N-glycosylation sites in the ECD, and our ability to isolate
the ECD by lectin affinity chromatography are all consistent with its
being glycosylated. Table I shows the
carbohydrate composition of several ECD preparations determined as
described under "Materials and Methods." Summation of the molecular
weights of the determined species of carbohydrates indicates that a
range of 8.2 to 37% of the mass of the ECD protein is carbohydrate.
Addition of the carbohydrate masses to the mass of the predicted amino
acid sequence results in a range of value from 70,692 to 103,291 Da. We
are unable to explain the variability in carbohydrate content from one
preparation to another. Although the same culture medium was used
throughout, it is possible that subtle differences in culture
conditions lead to substantial differences in glycosylation of the
secreted ECD. It is of interest that sialic acid residues vary in their
contribution of 1 to 25 negative charges per mole of protein to the net
of 25 negative charges predicted by the protein sequence. This addition
of more negative charges may explain the inability to determine the pI
of the ECD protein by chromatofocusing (data not shown). Elution was
not seen with poly buffer pH 4-7 adjusted to pH 4.0 using a mono P
column (Amersham Pharmacia Biotech) indicating a pI below a value of
4.0
HPLC gel permeation chromatography suggested that the ECD as isolated
by our purification protocol is a dimer. The intact CaR expressed in
transfected HEK-293 cells has been shown to be an intermolecular
disulfide bond-linked dimer (14, 15). mGluR5 has been shown to be an
intermolecular disulfide bond-linked dimer (12), and for mGluR1, the
secreted, purified ECD was also found to be an intermolecular disulfide
bond-linked dimer (16). To test this possibility for the hCaR ECD, ECD
protein was subjected to SDS-PAGE and immunoblot after sample treatment
at varying temperatures under reducing and nonreducing conditions.
Irrespective of incubation temperature, under nonreducing conditions
(Fig. 4, lanes 2-5), the ECD protein migrates as a diffuse
band of >200 kDa, whereas under reducing conditions (lanes
7-10), ECD protein migrates as a diffuse band of 78 kDa with
another minor band of 48 kDa. The ECD protein is therefore isolated as
an intermolecular disulfide-linked dimer capable of being totally
reduced by
-mercaptoethanol. This is similar to the behavior of the
purified mGluR1 ECD and is consistent with the possibility that
dimerization of the intact CaR occurs secondary to intermolecular
disulfide linkage of its ECD. The minor 48-kDa band, which we have
interpreted as arising from action of unknown protease during the
isolation procedure, is seen on immunoblot (Fig. 4) only under reducing
conditions suggesting that it is kept associated to the rest of the ECD
protein by disulfide bonds.
Circular dichroism measurements of the purified ECD were performed and
a typical CD spectrum is shown in Fig. 5.
The CD spectrum of the purified ECD was unchanged by addition of EDTA,
neomycin, gadolinium chloride, or terbium chloride at concentrations up to 100 µM. Analysis of all spectra indicated an
helix
content of 41-56% and a
-sheet content of 7-19%.

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Fig. 5.
Circular dichroism measurement of purified
ECD was performed as described under "Materials and Methods."
A representative spectrum is shown.
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The purified ECD was subjected to tryptic proteolysis with and without
prior denaturation. The denatured ECD was rapidly degraded by trypsin
yielding small fragments visualized on immunoblot with the ADD antibody
(Fig. 6, left). In contrast, the nondenatured ECD was not
surprisingly less susceptible to tryptic digestion with persistence of
intact ECD even after 40 min (Fig. 6, right). Of greater
interest was the rapid appearance of an ~50-kDa band with tryptic
digestion of the nondenatured ECD. This tryptic fragment persisted even
after 180 min of digestion, the longest time tested. The nondenatured
ECD was then digested with trypsin for 30 or 90 min and the digests
analyzed by immunoblot with a panel of polyclonal antibodies raised
against synthetic peptides (see Fig. 1) ranging from the amino to the
carboxyl terminus of the ECD (Fig.
7).
Although the pattern of smaller tryptic fragments varied with each
antibody, the six antibodies going from NH2 terminus (GP)
to residue 358 (FF20-7) showed the identical ~50-kDa tryptic fragment
after both 30 and 90 min of digestion. The LRG and DGE antibodies, in
contrast, did not stain this band (labeled 1 in the figure); instead,
LRG stained two diffuse, lower molecular weight bands (labeled 2 and 3 in the figure), whereas DGE stained only band 2. We interpret these
results to indicate that the native ECD possesses relatively few sites
accessible to trypsin, despite the many potential basic cleavage sites.
The two major sites of cleavage are defined by the pattern of
reactivity of the region-specific peptide antibodies. The site
generating bands 1 and 2 is localized between the epitopes for FF20-07
and LRG. Band 1 is then an NH2-terminal fragment
encompassing epitopes of GP through FF20-07, and band 2 is a
COOH-terminal fragment encompassing the LRG and DGE epitopes. Band 3 is
generated by tryptic cleavage at the other highly accessible site,
which is just proximal to the DGE epitope based on the loss of DGE
reactivity, retention of LRG reactivity, and modest reduction in size
of band 3.

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Fig. 6.
Immunoblot with polyclonal antibody ADD of
trypsin-digested purified ECD. ECD protein was digested with
trypsin for the times indicated at the bottom, either with
(Denatured) or without (Nondenatured)
pretreatment with SDS. 1.5-µg samples of protein were then separated
by SDS-PAGE for immunoblot analysis. Molecular weight standards are
shown on the left. Undigested ECD protein sample was run in
the lane labeled "0" (the identical lane is repeated
between denatured and nondenatured samples for ease in comparison of
band size).
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Fig. 7.
Immunoblot with a series of
peptide-specific polyclonal antibodies of trypsin-digested,
purified ECD. ECD protein was digested with trypsin for 30 or 90 min and then separated by SDS-PAGE for immunoblot analysis. At the top,
the CaR ECD is schematized, and the relative positions of peptide
epitopes (see also Fig. 1) of the polyclonal antibodies used for
immunoblot analysis are shown as open rectangles. Molecular weight
standards are indicated at the left of the immunoblots, which are
labeled with antibody used and time of tryptic proteolysis. Three major
products of tryptic proteolysis visualized on immunoblot are labeled
(1, 2, 3) at right (variations in intensity of staining of
band 1 reflect differing affinities of the respective
antibodies).
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|
It is interesting to relate these tryptic cleavage sites to a
speculative model of the CaR ECD. A sequence alignment based on limited
homology between bacterial periplasmic binding proteins (PBPs) and
mGluR ECDs (9) can be extended to the CaR given its relatively high
degree of homology to the mGluRs (8, 17). The model predicts that like
PBP, the mGluR and CaR ECDs are venus flytrap-like structures with two
lobes each consisting of
-helix and
-sheet folds connected by a
hinge region of three strands (9). In this model, regions of mGluR or
CaR ECD that align with PBP are assigned secondary structure based on
the x-ray crystallography-determined structure of PBP, whereas
insertions in the mGluR or CaR ECD that do not align with PBP are left
as loops of undefined secondary structure. All four such insertions in
the CaR ECD model cluster in one of the two putative flytrap lobes. The
tryptic cleavage site generating bands 1 and 2 is localized to one of
these four insertions, consistent with the possibility that this in
fact represents a surface-exposed loop of the protein. The site that generates band 3 is localized to a presumptive "stalk" that would tether the flytrap to the first transmembrane domain in the intact receptor. We speculate that this stalk is left freely accessible to
tryptic cleavage in the secreted ECD.
For the mGluR1 ECD, the availability of high affinity agonists and
antagonists permitted the demonstration that the ECD alone is capable
of high affinity ligand binding (16). As yet, it has not been possible
to measure directly Ca2+ binding to either the intact CaR
or its ECD. One report showed that Ca2+ induces dimer
formation of CaR solubilized from purified renal endosomes (13), but
another report failed to see Ca2+-induced dimer formation
in CaR expressed in transfected HEK-293 cells (14). Consistent with the
latter report, we failed to see evidence of divalent cation-induced
dimerization of the purified hCaR ECD (data not shown). As another
indirect measure of Ca2+ binding to the hCaR ECD, we tested
the effect of Ca2+ on the rate of tryptic digestion of the
ECD. Although there was a suggestion that Ca2+ slowed the
rate of digestion (data not shown), effects observed were small and not
consistently observed. Failure to detect changes in CD spectrum with
addition of cation agonists such as neomycin and gadolinium chloride
does not exclude binding of such agonists to the ECD. In fact, the
venus flytrap model predicts no change in secondary structure and
therefore CD spectrum with ligand binding, but rather a rotation of
~45o of one lobe relative to the other (9). Although it
has been speculated that the ECD is the site of Ca2+
binding in the CaR (8), at this point we are unable to demonstrate this
with the purified ECD.
In summary, we have shown that the hCaR ECD can be purified in
milligram amounts, defined the site of signal peptide cleavage, and
shown that it is a glycosylated, disulfide-linked dimer with a folded
structure that contains only two highly accessible tryptic cleavage
sites. Availability of the purified ECD should permit further studies
to define the actual sites of glycosylation, to identify intermolecular
disulfides involved in dimer formation, and intramolecular disulfides
critical for tertiary structure, and ultimately, the three dimensional
structure. This information will be critical to understanding how
Ca2+ leads to receptor activation.
 |
ACKNOWLEDGEMENTS |
We are extremely grateful to Gil Ashwell,
Laboratory of Cell Biochemistry and Biology, NIDDK, for performing
carbohydrate analyses, and to John Coligan, NIAID, for amino acid
sequence determination.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom correspondence should be addressed: NIDDK, National
Institutes of Health, 10/9N-222, Bethesda, MD 20892. Fax: 301-496-9943; E-mail: allens{at}amb.niddk.nih.gov.
 |
ABBREVIATIONS |
The abbreviations used are:
CaR, calcium
receptor;
hCaR, human CaR;
mGluR, metabotropic glutamate receptor;
ECD, extracellular domain;
PBS, phosphate-buffered saline;
TBST, tris-buffered saline with Tween;
PBP, periplasmic-binding protein;
PAGE, polyacrylamide gel electrophoresis;
FPLC, fast protein liquid
chromatography;
HPLC, high pressure liquid chromatography;
CHO, Chinese
hamster ovary.
 |
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