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INTRODUCTION |
The Ca2+ receptor
(CaR)1 plays a central role
in the regulation of [Ca2+]o homeostasis (for
reviews, see Refs. 1 and 2). Activation of CaR by elevated levels of
[Ca2+]o stimulates phospholipase C via the
Gq subfamily of G-proteins, resulting in the increase of
both phosphoinositide (PI) hydrolysis and the concentration of
cytosolic calcium, [Ca2+]i. The CaR mediates the
inhibitory actions of [Ca2+]o on parathyroid
hormone secretion by the parathyroid gland and on Ca2+
reabsorption by the kidney.
The CaR is a member of family 3 of the G-protein-coupled receptor
(GPCR) superfamily, which also includes metabotropic glutamate receptors (mGluRs) (3),
-amino butyric acid type B receptors (GABABRs) (4), some putative pheromone receptors (5), and some putative taste receptors (6). Their distinctively large extracellular domains (ECDs) consist of a "Venus's-flytrap" (VFT) domain and a cysteine-rich (Cys-rich) domain with the exception of
GABABRs, which lack a Cys-rich domain. The
three-dimensional structure of the VFT domain of mGluR1 has been
determined recently by x-ray crystallography (7) showing a bilobed
VFT-like structure.
The hCaR VFT contains 10 cysteines, while mGluR1 contains 9. mGluR1
forms homodimers involving an intermolecular disulfide bond through
cysteine Cys140 (8), whereas hCaR forms homodimers
involving two intermolecular disulfide bonds through both
Cys129 and Cys131 (9). The crystal structure of
the mGluR1 VFT domain shows that it forms four intramolecular disulfide
bonds. Based on amino acid sequence alignment, three homologous
intramolecular disulfide bonds are predicted to form within the hCaR
VFT, i.e. Cys60-Cys101,
Cys358-Cys395, and
Cys437-Cys449 (7) (Fig. 1). The hCaR VFT has
no cysteines corresponding to the disulfide-bonded pair
Cys289-Cys291 in mGluR1 VFT. Of the remaining
two cysteines in the hCaR VFT, Cys236, which is conserved
between the hCaR and mGluR1, is a free cysteine in mGluR1 VFT, whereas
Cys482 of hCaR has no counterpart in mGluR1. We reported
previously that Cys236 was critical to the function of the
hCaR, but Cys482 was not (10).
We recently reported that the Cys-rich domain plays a critical
role in signal transmission from the VFT domain to the 7 TM domain
(11). The hCaR Cys-rich region contains nine highly conserved cysteines
in a closely spaced (about 60 amino acids long) sequence (Fig.
1A). We reported previously that each of the nine cysteines in the Cys-rich domain in hCaR is critical for the receptor's function
(10). It is speculated that multiple intramolecular disulfide bonds are
formed within this region, which may give rise to a tightly packed
domain. However, the disulfides within the Cys-rich domain have not
been characterized, and the mechanism by which an agonist signal is
transmitted from the VFT domain through the Cys-rich domain to the 7 TM
domain remains unknown. One possibility is that the hCaR VFT and
Cys-rich domains are linked by disulfide bond(s) between
Cys236 and/or Cys482 in the VFT and one or more
cysteines in the Cys-rich domain. If this were true, conformational
changes in the VFT domain after agonist binding might directly cause
the conformational changes of the Cys-rich domain through the disulfide
bond(s) linkage. To test this hypothesis, we constructed mutant hCaRs
with a tobacco etch virus (TEV) protease recognition site (for a review
of TEV protease, see Ref. 12) inserted between
Glu536 and Val537 and studied the
products of TEV protease digestion of the mutant hCaRs.
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MATERIALS AND METHODS |
Construction of Mutant hCaRs--
Site-directed mutagenesis was
performed by using the QuickchangeTM site-directed mutagenesis kit
(Stratagene Inc., La Jolla, CA), according to the manufacturer's
instructions. The mutagenic oligonucleotide primer pair for introducing
six new residues (NLYFQG) between Glu536 and
Val537 in the hCaR was
5'-CCTGTGGAGTGGGTTCTCCAGGGAGAACCTCTACTTCCAAGGAGTGCCCTTCTCCAACTGCAGCCG-3' and
5'-CGGCTGCAGTTGGAGAAGGGCACTCCTTGGAAGTAGAGGTTCTCCCTGGAGAACCCACTCCACAGG-3'. The primer pair for introducing a 6 × His tag at the C terminus of hCaR(TEV) was
5'-GCACTGTTACAGAAAACGTAGTGAATTCACACCACCACCACCACCACTAAAATGGAAGGAGAAGACTGGGC-3' and
5'-GCCCAGTCTTCTCCTTCCATTTTAGTGGTGGTGGTGGTGGTGTGAATTCACTACGTTTTCTGTAACAGTGC-3'.
Secretion of the hCaR VFT Terminating following Residue
Glu536--
hCaR sequence from amino acids
Met1 to Glu536 was amplified by polymerase
chain reaction with the addition of a stop codon following Glu536 and then cloned into pCEP4 vector (Invitrogen Corp.,
Carlsbad, CA). The construct was transformed into HEK-293 cells and
stable cell lines secreting the hCaR VFT from Tyr20 to
Glu536, termed E536, were established by screening colonies
resistant to hygromycin treatment.
Transient Transfection of CaRs in HEK-293 Cells, PI
Hydrolysis Assay, and Preparation of Detergent-solubilized Whole Cell
Extracts--
The CaRs were transfected in HEK-293 cells by using a
DNA-LipofectAMINE mixture (Life Technologies, Inc.) as described
previously (11). PI hydrolysis assay and preparation of
detergent-solubilized whole cell extracts were carried out as described
previously (11) with one modification in that iodoacetamide was not
included in lysis buffer.
TEV Protease Digestions--
100 µg of total protein of the
whole cell lysate was diluted with 1× TEV buffer (Life Technologies,
Inc.), and buffer exchange was carried out by repeated dilution with
1× TEV buffer and concentration using Microcon YM-100 (Millipore
Corp., Bedford, MA) spin columns. For proteolysis, 100 units of
recombinant TEV protease (Life Technologies, Inc.) were added to 500 µl of reaction mixture containing the proteins in 1× TEV buffer, and
the digestion was performed at room temperature overnight. All TEV
protease digestions were carried out without adding dithiothreitol. The
digestion products were concentrated using Centricon YM-50 spin columns
before loading onto a gel for SDS-PAGE.
Immunoblotting Analysis--
Protein samples were resolved by
SDS-PAGE under either nonreducing or reducing conditions (with the
addition of 300 mM
-mercaptoethanol in sample buffer).
The proteins on the gel were electrotransferred onto nitrocellulose
membrane. For the ADD blot, the membrane was incubated with mouse
monoclonal anti-hCaR antibody ADD (raised against a synthetic peptide
corresponding to residues 214-235 of hCaR protein) and then with a
secondary goat anti-mouse antibody conjugated to horseradish peroxidase
(Kirkegaard and Perry Laboratories, Gaithersburg, MD). For the His
blot, a nitrocellulose membrane was incubated with rabbit polyclonal
His-probe (H-15) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and
then with anti-rabbit IgG conjugated to horseradish peroxidase (Santa
Cruz Biotechnology, Inc.). The hCaR protein was detected with an ECL
system (Amersham Pharmacia Biotech). Blots shown in this paper were
representatives from at least three independent experiments.
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RESULTS |
Construction and Functional Assay of a Mutant hCaR with a Unique
TEV Protease Recognition Site Inserted between Glu536 and
Val537--
Site-directed mutagenesis was applied to
insert nucleotide sequence for six new residues (NLYFQG) between
Glu536 and Val537 in the wild type (WT) hCaR,
resulting in a unique seven-amino acid TEV protease recognition site
(ENLYFQG) (Fig. 1A). The
mutant hCaR was termed hCaR(TEV). Recombinant TEV protease purified
from Escherichia coli cleaves between residues Gln and Gly.
We first tested whether hCaR(TEV) is capable of signal transduction
using the intact cell [Ca2+]o-stimulated PI
hydrolysis assay. Nontransfected or vector only transfected HEK-293
cells (11) showed no PI hydrolysis response at
[Ca2+]o concentrations as high as 50 mM. Fig. 1B shows the maximal response of
hCaR(TEV) to [Ca2+]o, and its EC50
were identical to those of WT hCaR. Under reducing conditions, the ADD
antibody detected two major bands of about 130 and 150 kDa for WT hCaR
(Fig. 1B). Previous studies have shown that the monomeric
~150-kDa band represents hCaR forms expressed at the cell surface and
modified with N-linked complex carbohydrates. The ~130-kDa
band represents high mannose-modified forms, trapped intracellularly
and sensitive to endoglycosidase H digestion (13-15). Under
nonreducing conditions, both forms appear as poorly resolved ~260-
and ~300-kDa bands representing intermolecular disulfide-linked
dimers (Fig. 1B). In Fig. 1B, the ~300-kDa form of hCaR(TEV) appears to be expressed at a somewhat higher level than
that of WT, but this is not a consistent finding. Thus, the hCaR(TEV)
was expressed at equivalent levels to that of WT hCaR and dimerized
normally, indicating that the six-amino acid insertion between the VFT
and the Cys-rich domains had no detectable effect on the receptor's
folding, cell-surface expression, or function.

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Fig. 1.
A, schematic diagram showing amino acid
sequence of hCaR ECD. The location of signal peptide,
N-linked glycosylation sites, Cys-rich domain (from
Cys542 to Cys598), sequence of synthetic
polypeptide used to raise monoclonal antibody ADD, and a novel TEV
protease recognition site introduced in hCaR(TEV) and hCaR(TEV-HIS) are
indicated. All 19 cysteines in the ECD are shown in black.
Cys129 and Cys131, which are involved in the
intermolecular disulfide bonds, are indicated. The predicted
intramolecular disulfide bonds are shown as connecting
lines. B, concentration dependence for
[Ca2+]o stimulation of PI hydrolysis and
immunoblot of CaR in transiently transfected HEK-293 cells expressing
WT hCaR and hCaR(TEV). Upper panel, results of PI assay are
expressed as percentage of maximal response (WT hCaR at 20 mM). Data (mean) are from one of three experiments, all of
which were performed in duplicate. Lower panel, immunoblot
of whole cell lysate of cells from the same transfection run on 5%
SDS-acrylamide gel under both reducing (with -mercaptoethanol added
in sample buffer) and nonreducing conditions. Molecular mass standards
are indicated at the right of the blots.
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TEV Protease Digestion of hCaR(TEV)--
Whole cell lysate from
cells transiently transfected with WT hCaR or hCaR(TEV) were subjected
to TEV protease digestion and analyzed by SDS-PAGE and immunoblot. Fig.
2A shows that no endogenous TEV cleavage site is present in the WT hCaR and immunoreactive bands
remain unchanged from those of the WT hCaR without TEV protease digestion (compare with Fig. 1B). Following TEV protease
digestion, immunoreactive bands were less distinct under nonreducing
conditions, which may be because of the aggregation of the receptor
after sample concentration necessitated by TEV protease digestion
protocol. Fig. 2B shows that TEV protease cleaved the
hCaR(TEV) protein, although the digestion was not complete. Two new
lower molecular weight bands were identified under reducing conditions
following TEV protease digestion. Based on the location of the epitope
of the ADD monoclonal antibody (Fig. 1A), both bands
represent monomers of the hCaR VFT. The upper band is the VFT domain
cut from the cell surface-expressed, fully glycosylated receptor, and
the lower band is the VFT domain cut from the intracellularly trapped,
high mannose-modified receptor. Culture medium containing a secreted form of the VFT domain, E536 (containing VFT sequence from
Tyr20 to Glu536), was run as a size marker to
indicate the location of the VFT monomer cleaved from hCaR(TEV). E536
migrated on SDS-PAGE similarly to the VFT domain released from the
fully processed receptor after TEV protease digestion.

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Fig. 2.
Immunoblot of CaR in transiently transfected
HEK-293 cells expressing WT hCaR, hCaR(TEV), and hCaR(TEV-HIS) with (+)
or without ( ) TEV protease digestion. A, ADD blot of
wild type hCaR samples run on 5% SDS-polyacrylamide gel under reducing
and nonreducing conditions. B, ADD blot of hCaR(TEV) samples
run on 5% SDS-polyacrylamide gel under reducing and nonreducing
conditions. Medium from stable transfected HEK-293 cells secreting E536
(a form of the VFT domain from residues Tyr20 to
Glu536) was loaded as a size marker. C,
hCaR(TEV-HIS) samples were run on a 6% SDS-polyacrylamide gel with 8 M urea. Left, sample was run under reducing
conditions probed with anti-hCaR monoclonal ADD (ADD); the
blot was then stripped and reprobed with an anti-His tag antibody
(HIS). Right, sample was run under nonreducing
conditions probed with anti-hCaR monoclonal ADD; the blot was then
stripped and re-probed with anti-His tag antibody. Molecular mass
standards are indicated at the right of the blots.
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Under nonreducing conditions, bands for the dimerized VFT domain,
migrating similarly to the dimer of the secreted E536, appeared in the
TEV protease digestion products of hCaR(TEV) (Fig. 2B). This
result indicates that the VFT domain in the hCaR(TEV) is not
disulfide-linked to the Cys-rich or 7 TM domains of the receptor; otherwise, the immunoreactive bands should have remained the same as
those of the WT hCaR under nonreducing conditions.
Construction and TEV Protease Digestion of hCaR(TEV-HIS)--
By
site-directed mutagenesis, we added a 6 × His tag to the
intracellular C terminus of the hCaR(TEV) and termed the new construct hCaR(TEV-HIS). The PI hydrolysis assay showed its maximal response to
[Ca2+]o and EC50 were identical to
those of WT hCaR and it was expressed at the same level as WT hCaR,
indicating that the 6 × His tag at the C terminus had no effect
on the receptor's folding, expression, or function (data not
shown).Whole cell lysate from cells transiently transfected with
hCaR(TEV-HIS) was digested with TEV protease and loaded onto 6%
SDS-polyacylamide gel containing 8 M urea. An ADD
immunoblot under reducing condition showed bands for monomeric VFT
released from hCaR(TEV-HIS) after TEV protease digestion, whereas under
nonreducing conditions bands for dimeric VFT appeared (Fig.
2C). After the blots were stripped and reprobed with
anti-His antibody directed against the 6 × His tag added to the
C-tail of the hCaR(TEV-HIS), bands corresponding to the remaining part
of the receptor (Cys-rich + 7 TM + C-tail domains) were visualized
together with the residual uncut whole receptor (Fig. 2C).
The bands for Cys-rich + 7 TM + C-tail domains stained with the
anti-His probe were identical under reducing and nonreducing conditions. These results confirm that the hCaR VFT domain is not
disulfide-linked to the rest of the receptor protein.
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DISCUSSION |
A detailed characterization of the structure of the hCaR is
critical for understanding the mechanism of receptor activation by
[Ca2+]o and the basis for activation and
inactivation of the hCaR by naturally occurring mutations (1, 2).
Substantial evidence suggests that [Ca2+]o binds
to the VFT domain of the hCaR (11, 16, 17). Our current structural
information for the hCaR VFT comes largely from site-directed
mutagenesis studies and from homology modeling based on the
three-dimensional structure of the recently crystallized mGluR1 VFT
(7). Our site-directed mutagenesis data (9) showed that both
Cys129 and Cys131 are involved in
intermolecular disulfide bonds. A homology model based on the mGluR1
VFT crystal structure predicts that
Cys60-Cys101,
Cys358-Cys395, and
Cys437-Cys449 are involved in intramolecular
disulfide bonds within the hCaR VFT. Cys236 and
Cys482 are the remaining cysteines in the hCaR VFT that
have not been accounted for. It is unclear whether they are free
cysteines in the hCaR. The Cys-rich domain in the hCaR ECD contains
nine highly conserved cysteines in an ~60-amino acid-long sequence.
Multiple intramolecular disulfide bonds are speculated to form within
this region, but none of the disulfides has been defined yet. Given the
odd number of cysteines in this domain, at least one of them should be
free or disulfide-linked to a cysteine in other domains.
We recently reported that the Cys-rich domain in the hCaR is critical
for signal transmission from the VFT to the 7 TM domains (11). But a
key question remains unanswered: how are the VFT and Cys-rich domains
structurally and functionally coordinated during agonist binding and
activation of the receptor? The crystal structure determined for the
mGluR1 VFT domain does not address this question, because the Cys-rich
domain was not included in the mGluR1 construct expressed and
crystallized (7). One possible mechanism for communication between the
VFT and Cys-rich domains could involve disulfide linkage.
Conformational changes of the VFT after agonist binding would then
cause conformational changes in the Cys-rich domain through
disulfide(s) linkage between the two domains.
It is noteworthy that the hCaR has two cysteines (Cys677
and Cys765) in the extracellular loops 1 and 2 of the 7 TM
domain. However, they are less likely to pair with any cysteines
in the ECD, because the two conserved cysteines in extracellular loops
1 and 2 are known to be linked by a disulfide in most GPCRs including
bovine rhodopsin, the thyrotropin-releasing hormone receptor, the
thromboxane receptor, the GnRH receptor, and many others (18).
To determine whether the VFT is linked to the Cys-rich domain by
disulfide bond(s), we inserted a unique TEV protease recognition site
between the two domains. To date, it remains unclear where the exact
boundary is between the two domains. A construct of the mGluR1 VFT made
by Tsuji and co-workers (7, 8) that ends at Ser522, two
amino acids ahead of the first cysteine in the Cys-rich region
(corresponding to Ser540 in hCaR), was secreted, purified,
and crystallized. An alternative splicing product of hCaR, with 10 additional amino acids between Glu536 and
Val537 (19), was shown to be fully functional in an
in vitro assay. We hypothesized that Glu536 is
at or very close to the boundary of the VFT and Cys-rich domains, and
the receptor may be tolerant of insertion of additional amino acids at
this site. By applying site-directed mutagenesis, we made a secreted
version of the hCaR VFT with a stop codon after Glu536. It
was well expressed as a dimer under nonreducing conditions and secreted
into the culture medium. Moreover, hCaR(TEV) with a TEV protease
recognition sequence inserted at this site was fully functional in the
PI assay, as compared with the wild type hCaR.
Our results show that, although it is difficult to achieve complete
digestion in lysates of transfected HEK-293 cells, recombinant TEV
protease recognizes the site inserted in the hCaR (TEV) cDNA and
cleaves the receptor protein, as shown in ADD blots of hCaR(TEV) run on
SDS-PAGE under reducing conditions. However, there could be two
alternative results for the behavior of the products of TEV digestion
on SDS-PAGE under nonreducing conditions. If the VFT is
disulfide-linked to the Cys-rich domain, the TEV protease digestion
product of hCaR(TEV) should remain as a holoprotein under nonreducing
conditions, whereas if there is no such linkage, a dimer of the
VFT domain will be released from the rest of the receptor on SDS-PAGE
under nonreducing conditions. Our results show that there is no
disulfide bond between the VFT and the Cys-rich domains. A HIS blot of
the TEV protease digestion product of hCaR(TEV-HIS) shows that the His
antibody recognizes the protein with a C-terminal His tag but not the
VFT released from the holoprotein, confirming that the hCaR VFT is not
disulfide-linked to the Cys-rich domain. The absence of disulfide
linkage between the VFT and other domains of the receptor might be
common among family 3 members of GPCR, as GABAB receptors
lack a Cys-rich domain altogether.
In summary, our results exclude the existence of a disulfide bond
between the hCaR VFT and Cys-rich domains. We cannot, however, exclude
noncovalent interactions between these two domains, and indeed such
interactions could be important in the mechanism of activation of the
receptor. We attempted to determine whether the hCaR VFT and Cys-rich
domains are associated by noncovalent interactions by analyzing the
products of TEV protease digestion under native gel conditions.
Unfortunately, perhaps because of its high degree of glycosylation, the
hCaR is very poorly resolved under native gel
conditions2, making it
difficult to draw definitive conclusions using this method. Further
study of the structure of both the VFT and Cys-rich domains will help
to reveal the mechanism by which the signal of Ca2+-induced
conformational changes in the VFT is transmitted through the Cys-rich
domain to the 7 TM domain, resulting in CaR activation.