Disulfide Bonds in the Extracellular Calcium-Polyvalent
Cation-sensing Receptor Correlate with Dimer Formation and Its
Response to Divalent Cations in Vitro*
Donald T.
Ward
,
Edward M.
Brown§, and
H. William
Harris
¶
From the
Division of Nephrology, Children's Hospital
and the § Endocrine-Hypertension Division, Brigham and
Women's Hospital, Harvard Medical School,
Boston, Massachusetts 02115
 |
ABSTRACT |
Extracellular calcium/polyvalent cation-sensing
receptors (CaR) couple to G proteins and contain highly conserved
extracellular cysteine residues. Immunoblotting of proteins from rat
kidney inner medullary collecting duct endosomes with CaR-specific
antibodies reveals alterations in the apparent molecular mass of CaR
depending on protein denaturation conditions. When denatured by SDS
under nonreducing conditions, CaR migrates as a putative dimeric
species of 240-310 kDa. This is twice the predicted molecular mass of the CaR monomer observed after SDS denaturation in the presence of
sulfhydryl-reducing agents. In sucrose density gradients, Triton X-100-solubilized CaR sediments as a 220-kDa complex, not explainable by binding of G proteins to CaR monomers. Treatment of Triton-soluble CaR with divalent (Ca2+, Mg2+) and
trivalent (Gd3+) metal ion CaR agonists, but not monovalent
ions (Na+), partially shifts the electrophoretic mobility
of CaR under reducing conditions from a predominantly monomeric to this
putative dimeric species on immunoblots in a manner similar to their
rank order of functional potency for CaR activation (Gd3+
Ca2+ > Mg2+). This Ca2+
effect is blocked by pretreatment with
N-ethylmaleimide. We conclude that disulfide bonds
present in CaRs mediate formation of dimers that are preserved in
Triton X-100 solution. In addition, CaR exposure to Ca2+
induces formation of additional disulfide bonds within the
Triton-soluble CaR complex.
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INTRODUCTION |
The binding of divalent (Ca2+, Mg2+),
trivalent (Gd3+), and polyvalent (neomycin, protamine)
cations to the extracellular domain of
CaRs1 initiates a variety of
signal transduction cascades via G protein coupling (1-3). Following
their expression in both oocytes (4, 5) and cultured human embryonic
kidney (HEK) cells (6-8), CaRs have been characterized
pharmacologically. Immunoblotting of these exogenously expressed CaRs
present in membrane fractions using CaR-specific antisera reveals
multiple CaR-specific bands (7-9). To account for these diverse CaR
species, it has been suggested that CaRs undergo post-translational
modification including glycosylation (8). Moreover, studies using these
same antibodies to probe endogenous CaRs present in rat kidney
epithelial cells from both the thick ascending limb of Henle (10) as
well as from IMCD (11) have revealed multiple CaR species exhibiting similar molecular masses to those reported for exogenously expressed CaRs. However, at present no detailed studies have examined the origin
of these multiple CaR species that are present on immunoblots. In this
report, we have utilized a combination of immunoblotting, SDS gel
permeation chromatography, and sucrose density gradient centrifugation
of Triton-solubilized CaR prepared from rat kidney IMCD to characterize
CaR associations in both nonionic and ionic detergents. The CaR
examined in this study is present in purified endosomes that are
derived exclusively from the apical membrane of IMCD epithelial cells
(11, 12). Utilization of an endogenous CaR present in a defined
intracellular compartment rather than a recombinant CaR species
expressed in cultured cells at high levels and in multiple
intracellular compartments precludes a potential complication of
studying multiple CaR species that are actually present in different
compartments within cells. The data reported here provide evidence that
a major form of CaR is a putative dimeric species that is present
following solubilization with both Triton X-100 and SDS detergents and
perhaps in the cell membrane itself.
Previous studies have demonstrated that the non-glycosylated CaR
polypeptide migrates as a band of approximately 120 kDa on SDS-PAGE
immunoblots in close agreement with the putative molecular mass
predicted from its corresponding cDNA (13). The presence of
glycosylated CaRs possessing an estimated molecular mass of 120-200
kDa has been demonstrated by conversion of this larger 120-200-kDa CaR
band to a 120-kDa band after digestion with glycosidases (8, 13).
However, a number of studies (7-11) have reported the presence of
multiple CaR-reactive protein bands greater than 200 kDa on SDS-PAGE
immunoblots that are not substantially effected by glycosidase
digestion. These data suggest that CaRs may also associate with, or be
bound covalently to, other protein(s) including a second CaR molecule.
Support for the possibility of a dimeric CaR species is suggested by
recent data showing that the structurally related metabotropic
glutamate receptor, mGluR5, is a disulfide-linked homodimer in the
plasma membrane of cells that exhibits a high molecular weight band on
SDS-PAGE immunoblots (14). Putative mGluR5 dimer formation appears to
be mediated by sulfhydryl (SH) linkages present in the N-terminal
region of the mGluR5 extracellular domain that possesses at least 3 Cys
residues that are identical in all CaRs as well as other mGluRs (13,
15-21).
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EXPERIMENTAL PROCEDURES |
Materials
Male Sprague-Dawley rats (200-250 g) were purchased from
Charles River Laboratories (Cambridge, MA). Various items were obtained from the following sources: EuCl3 (Molecular Probes Inc.,
Eugene, OR); Triton X-100 (Bio-Rad); acrylamide (Amersham Pharmacia
Biotech); polyvinylidene difluoride membrane (MSI, Westborough, MA);
ECL reagents (Amersham Pharmacia Biotech); and autoradiography film (NEN Life Science Products). All other chemicals were purchased from
Sigma. Anti-CaR mouse monoclonal antibody, raised to amino acids
214-235 of the extracellular domain of the human parathyroid CaR (15),
was obtained from NPS Pharmaceuticals (Salt Lake City, UT), and rabbit
polyclonal anti-CaR antibody (A4641), raised to amino acids 215-237 of
bovine parathyroid CaR, was a gift of Dr. Steven Hebert (Division of
Nephrology, Vanderbilt University School of Medicine, Nashville, TN).
Other antisera utilized included rabbit anti-Band III antibody, a gift
of Dr. Samuel E. Lux (Harvard Medical School, Boston), rabbit
anti-G
q/G
11, a gift of A. Tashjian (Harvard University, Boston, MA), and rabbit
anti-G
i(1-2) and anti-G
i(3) antibodies
(Upstate Biotechnology Inc., Lake Placid, NY).
Methods
Isolation and Triton X-100 Solubilization of
Endosomes--
After animals were sacrificed by cervical dislocation
under anesthesia (100 mg/kg pentobarbital, intraperitoneally),
endosomes were prepared as described previously (12) and utilized
either immediately or stored at
80 °C with identical results.
Endosomal protein content was determined by the method of Bradford
(22). Endosomal proteins (100-200 µg) were solubilized by incubation on ice for 30 min in solubilization buffer: 8.3 mM Tris (pH
7.4), 125 mM NaCl, 1.25 µM pepstatin, 4 µM leupeptin, 4.8 µM phenylmethylsulfonyl fluoride, and 1.0% (v/v) Triton X-100 (final concentrations). The
resulting mixture was then centrifuged at 100,000 × g
for 30 min to remove Triton X-100-insoluble material, and the
supernatant containing Triton-soluble endosomal proteins was collected
and aliquoted into equal volumes for the experiments detailed below. To
prepare crude membranes, inner medullary papilla homogenate was spun at
2,500 × g (5 min), and the resulting postnuclear
supernatant was spun at 100,000 × g (30 min) to give a
crude membrane pellet.
Sucrose Density Gradient Ultracentrifugation--
Triton
X-100-soluble endosomal proteins were layered on top of a 5-20% (w/v)
sucrose density gradient (8 ml) in 10 mM Tris (pH 7.4), 150 mM NaCl, and 0.05% (v/v) Triton X-100 and ultracentrifuged in an SW41 rotor at 39,000 rpm for 8 h at 4 °C. Individual
fractions were then collected from the bottom of the tube and processed for Western blotting. The mass of Triton X-100-soluble CaR was estimated by comparison of its mobility with those of standard proteins
including bovine liver catalase, Band III, and hemoglobin as described
by Martin and Ames (23) and Clarke (24).
SDS Gel Permeation Chromatography--
Samples of SDS-denatured
endosomal proteins were applied to a 1.3 × 45-cm Sephacryl 4-B
(Amersham Pharmacia Biotech) column equilibrated with 50 mM
Tris (pH 8), 0.1% SDS buffer in the absence or presence of 10 mM DTT. One-ml fractions were collected for analyses of
both protein amount (A280 nm) and content
(Western blotting as described below). A single column was utilized for multiple chromatographic runs.
Western Blot Analysis of CaR Protein--
Five-fold
concentrated Laemmli buffer (0.32 M Tris (pH
6.8), 5% (w/v) sodium dodecyl sulfate, 25% (v/v) glycerol, 1% (w/v) bromphenol blue) was added in a 1:4 ratio to sample proteins that were
incubated in the presence or absence of various SH group reducing
agents at various temperatures and then fractionated using 5%
SDS-polyacrylamide gels (25). Proteins were then transferred electrophoretically to polyvinylidene difluoride membrane in blotting buffer (25 mM Tris, 200 mM glycine, 15% (v/v)
methanol) containing 0.5% (w/v) SDS so as to improve the transfer of
the proteins >100 kDa (26, 27). The membrane was then incubated in 1%
(w/v) bovine serum albumin (20 min) to block nonspecific binding sites, followed by a 1-h incubation in either anti-CaR mouse monoclonal antibody (1:2500 dilution) or anti-CaR rabbit antiserum (1:1000 dilution). After washing to remove all nonspecifically bound anti-CaR antibodies, blots were then exposed to either horseradish
peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies
(1:5000) for 1 h. All incubations and intervening washes were
performed in Tween/TBS solution (15 mM Tris (pH 8), 150 mM NaCl, 0.1% (v/v) Tween 20) at room temperature, and
blots were developed with the ECL kit as detailed in the
manufacturer's instructions. Immunoreactivity was quantified by
densitometry and is expressed as percent of total CaR
immunoreactivity in the lane (± S.E.), and statistical significance was determined by paired t test.
 |
RESULTS |
Specific Anti-CaR Monoclonal and Polyclonal Antibodies Both
Identify Multiple CaR Bands in Immunoblots from the Inner Medulla of
Rat Kidney--
Fig. 1 shows how various
experimental manipulations affect the distribution of CaR-reactive
bands in immunoblots after fractionation of IMCD endosomal proteins by
SDS-PAGE. As shown in panel A, a CaR-specific mouse
monoclonal antibody identifies sequence determinants in a highly acidic
region of the extracellular domain present in all CaRs reported to date
(3). When purified endosomes are solubilized directly in SDS-PAGE
Laemmli buffer in the absence of SH-reducing agents (Fig. 1,
panel A, lane 1), greater than 90% of the CaR
immunoreactivity is present in a broad band of approximate molecular
mass 240-310 kDa. In contrast, addition of
-mercaptoethanol (
ME)
to a final concentration of 143 mM results in both a
diminution in the intensity of the 240-310-kDa band and the appearance
of additional CaR bands of 138-169 and 121 kDa (Fig. 1, panel
A, lane 2). These additional CaR-specific bands
produced after exposure to
ME correspond to molecular masses of the
glycosylated and nonglycosylated monomeric CaR proteins reported
previously (8, 13). Together, these apparent monomeric CaR species
represent more than 40% of the total CaR signal within this purified
subcellular fraction. Following solubilization in the nonionic
detergent Triton X-100, integral membrane proteins such as Band III
(28) and bacteriorhodopsin (29) maintain their intra- and
intermolecular associations and native conformations (30, 31). Triton
X-100 solubilization of the CaR protein present in purified IMCD
endosomes yielded a pellet of Triton X-100-insoluble proteins (Fig. 1,
panel A, lane 3) and a supernatant of Triton X-100-soluble proteins (Fig. 1, panel A, lane 4)
after centrifugation at 100,000 × g for 30 min.
Although both fractions contain a similar distribution of CaR-reactive
bands, the Triton-insoluble fraction possesses a 240-310-kDa band that
is somewhat broader and constitutes 69 ± 7% (n = 3) of the total CaR immunoreactivity as compared with its counterpart
in the Triton-soluble fraction where it represents only 42 ± 5%
(n = 8) of total CaR immunoreactivity. The 121-kDa CaR
band was not observed in any of the Triton-insoluble fractions indicating that it may have substantially greater solubility in Triton
X-100 than the larger CaR species. To examine whether these alterations
in CaR bands in immunoblots were present only after use of this mouse
monoclonal antibody, a rabbit polyclonal antibody (panel C)
was utilized as a second independent probe. Identical CaR species were
obtained with the rabbit antiserum as compared with bands present in
mouse monoclonal immunoblots.

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Fig. 1.
Immunoblots of CaR protein present in
purified rat IMCD endosomes reveal multiple discrete CaR species before
and after solubilization in Triton X-100 using two independent antibody
probes. Immunoblots containing either 15 µg of total endosome
protein per lane (panels A and B) or 28 µg of
protein (panel C) were probed with either anti-CaR-specific
mouse monoclonal antibody (panel A) or rabbit anti-CaR
antiserum (panel C) as detailed under "Experimental
Procedures." Panel A, denaturation of total endosomal
protein (Tot) in SDS-PAGE Laemmli buffer in the absence
(lane 1) or presence (lane 2) of 143 mM -mercaptoethanol ( ME) demonstrated that
almost all CaR immunoreactivity migrates as a broad 240-310-kDa band
in the absence of ME. In contrast, inclusion of ME during SDS
denaturation results in both a reduction in the intensity of the broad
240-310-kDa band as well as the appearance of CaR monomer species.
Solubilization of an identical amount of endosomal protein with 1%
(v/v) Triton X-100 yielded a Triton X-100-insoluble pellet
(Tx-Ins; lane 3) and Triton X-100-soluble
supernatant (Tx-Sol; lane 4) that both contained
multiple CaR-reactive species following SDS denaturation with 143 mM ME. Panel B, Coomassie Blue-stained
SDS-polyacrylamide gel showing the protein content of the samples
described in panel A. The results shown are representative
of a minimum of three separate experiments. Panel C, Western
blot showing total endosome proteins solubilized in the absence
(lane 1) or presence (lane 2) of 143 mM ME and immunoblotted in a manner identical to that
shown in panel A, except using a rabbit polyclonal anti-CaR
antiserum (n = 2). Arrowheads on the
left indicate the apparent molecular masses of CaR-reactive
species observed.
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Exposure of either native or Triton X-100-solubilized endosomes
to 100 °C after solubilization in SDS-containing Laemmli
buffer with 143 mM
ME causes aggregation of CaR as
assayed by SDS-PAGE (Fig. 2, panel
A). Therefore, CaR samples were routinely immunoblotted without
being preheated. As shown in Fig. 2, panel B, SDS
denaturation of Triton-soluble endosomal proteins in the absence
(lane 3) or presence (lane 4) of 143 mM
ME, respectively, yielded CaR immunoreactivity that
was mostly present as either a single 240-310-kDa band (lanes 3 and 7) or as multiple CaR-reactive bands (lanes
4-6) identical to those shown in Fig. 1, panel A. The
binding of the anti-CaR monoclonal antibody to the CaR bands was
specific since it was ablated by preincubation of antibody with excess
immunizing peptide (Fig. 2, panel B, lanes 1 and
2). Substitution of
ME with other SH-reducing agents,
including 10 mM dithiothreitol (DTT, lane 5) and
109 mM
-mercaptopropanol (lane 6), yielded
similar results. In each case, there was only partial conversion of the
240-310-kDa CaR-reactive band to the smaller CaR-reactive bands. To
determine whether oxidation of SH groups to form S-S bonds during SDS
denaturation contributes to formation of the 240-310-kDa CaR band,
Triton X-100-soluble endosomal proteins were denatured in SDS-Laemmli
buffer containing 1 mM N-ethylmaleimide (NEM)
that modifies free SH groups but will not reduce S-S bonds (Fig.
2, panel B, lane 7). As shown in panel C, native endosomes were also preincubated on ice for 5 min with 1 mM NEM prior to their denaturation in SDS in the absence of reducing agents (lane 3). Subsequent immunoblotting of both
samples revealed that CaR immunoreactivity was again present as a
240-310-kDa band (Fig. 2, panel C, lane 2)
identical to that exhibited by respective controls that were not
exposed to any SH-reducing agents (Fig. 2, panel B,
lane 3, and Fig. 1, panel A, lane 1,
respectively). In addition, exposure of endosomes to chelating agents
(1 mM EGTA, 1 mM EDTA on ice for 5 min) prior
to SDS solubilization also had no effect on the 240-310-kDa CaR band.
These data suggest the involvement of pre-existing disulfide bonds in
the formation of the 240-310-kDa immunoreactive CaR band and not
oxidation of SH groups to form S-S bonds during exposure of CaR to
either Triton X-100 or SDS detergents.

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Fig. 2.
Effects of temperature and SH group
modification on CaR immunoreactivity. Panel A, immunoblot of
total (Tot, lanes 1 and 3) and Triton
X-100-solubilized (TxSol, lanes 2 and
4) endosomes subjected to denaturation in Laemmli buffer
containing 143 mM ME where the protein mixture was
applied to the gel after a 3-min incubation at either 20 °C
(lanes 1 and 2) or 100 °C (lanes 3 and 4). Panel B, immunoblot of Triton
X-100-solubilized endosomal proteins denatured in Laemmli buffer in
either the absence of a reducing agent (lanes 1 and
3) or containing either 143 mM ME
(lanes 2 and 4), 10 mM DTT
(lane 5), 109 mM -mercaptopropanol
( MP, lane 6) or 1 mM NEM
(lane 7). Immunoblotting of lanes 1 and
2 was performed using anti-CaR monoclonal antibody
preincubated with a 100-fold molar excess of immunizing peptide. These
immunoblots are each representative of a minimum of three separate
experiments. Panel C, immunoblot of total endosomal proteins
after preincubation of native undenatured endosomes on ice for 5 min in
the absence (lanes 1 and 2) or presence of 1 mM NEM (lane 3) or 1 mM EDTA, 1 mM EGTA (lane 4) prior to their solubilization
in Laemmli buffer containing SDS and 143 mM ME
(lane 1) or SDS alone (lanes 2-4). Panel
D, kidneys collected from anesthetized rats perfused with
phosphate-buffered saline in the absence (lanes 1 and
2) or presence (lanes 3 and 4) of 10 mM NEM were homogenized in buffer in the continued absence
or presence of NEM (1 mM). Crude IMCD membranes were then
prepared and solubilized in Laemmli buffer under nonreducing
(lanes 1 and 3) or reducing (lanes 2 and 4) conditions. Panels C and D are
each representative of two independent experiments.
Arrowheads on the left indicate the apparent
molecular masses of the CaR-reactive species observed.
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To assay whether the apparent CaR dimeric complex may form
artifactually from oxidation of SH groups during our tissue processing and endosome purification, we perfused the kidneys of anesthetized rats
with 10 mM NEM in situ then removed the kidneys
and rapidly prepared a crude membrane fraction from the inner medulla
and papilla using homogenization buffer supplemented with 1 mM NEM. These membranes were then solubilized in Laemmli
buffer in the absence (Fig. 2, panel D, lanes 1 and 3) or presence (lanes 2 and 4) of
143 mM
ME and compared with controls using anti-CaR Western blotting. As shown in panel D of Fig. 2, exposure of
CaR to NEM both prior to and during tissue processing does not
significantly alter the levels of the 240-310-kDa CaR band obtained
under nonreducing conditions (lane 3). These data suggest
that the S-S bonds responsible for the 240-310-kDa CaR complex
pre-exist prior to tissue processing.
Triton X-100-solubilized CaR Exists as a Complex of Approximately
220 kDa--
Triton X-100-soluble extracts of endosomal proteins were
subjected to ultracentrifugation in sucrose density gradients
containing Triton X-100 in the absence of sulfhydryl reducing agents
and the sedimentation of CaR compared with that of marker proteins of
known native molecular weights. As shown in Fig.
3, panel A, i, CaR
immunoreactivity was observed in a single discrete region of the
sucrose gradient in close proximity to the catalase (230 kDa) marker,
where the peak CaR immunoreactivity corresponded to an estimated
molecular mass of approximately 220 kDa (Figs. 1 and 2). As shown in
panel A, ii and iii, of Fig. 3, SDS-PAGE analysis
of each respective sucrose gradient fraction revealed that each
displayed a series of CaR bands including 121, 138-169, or 240-310
kDa identical in appearance to those shown in Figs. 1 and 2. Note that
the dissociated monomeric CaR species are not equally represented in
each gradient fraction (Fig. 3, panel A, iii). For example,
the 121-kDa CaR protein is localized only in fractions 12-14, whereas
the 138-169-kDa CaR protein is enriched in fractions 10-13.

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Fig. 3.
Density gradient ultracentrifugation analyses
of Triton X-100-solubilized endosome CaR. Triton X-100-soluble rat
kidney apical endosomal soluble protein was centrifuged through a
5-20% sucrose density Triton X-100 gradient (see "Experimental
Procedures"), and individual fractions were then collected from the
bottom of the gradient, solubilized in SDS-Laemmli buffer containing 10 mM DTT, and assayed for CaR content by quantitative
immunoblotting. The apparent molecular mass of Triton X-100-solubilized
CaR was estimated from a standard curve constructed from the mobilities
of bovine liver catalase (230 kDa), Band III (175 kDa), and hemoglobin
(64 kDa), which were included as native protein markers in the same
sucrose gradient. Portions of the CaR immunoblots are presented to show
the actual CaR-reactive species present in the gradient fractions:
Panel A, ii, and Panel B, ii, represent the
fractions that contained maximal CaR immunoreactivity for each sample,
and Panel A, iii, shows the glycosylated and
non-glycosylated CaR content of each fraction across the sucrose
gradient CaR band. The experiment was performed 5 times, and in each
case a second identical tube and sample ( - ) was centrifuged, as
described above, in the presence of either 100 µM GTP S
(panel A, n = 2) or 10 mM DTT
(panel B, n = 3).
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To determine whether the molecular mass of the detergent-solubilized
CaR complex could be accounted for by the presence of a bound G protein
as described for the galanin (32), kainate (33), melatonin (34), opioid
(35), and pancreastatin (36) receptors, a second identical sample was
centrifuged in the presence of GTP
S in order to dissociate any
complex-bound G proteins. As shown in Fig. 3, panel A, i,
GTP
S had no effect on the apparent mass of the Triton-soluble CaR.
Consistent with these data were additional results showing that no
G
q or G
i(1-3) immunoreactivity was
detectable in the CaR-containing fractions. Instead, G
q
and G
i(1-3) immunoreactivity was present in other
fractions nearer the top of the gradient suggesting that they migrate
independently of CaRs (data not shown).
Fig. 3, panel B, shows Triton X-100-solubilized CaR after
density gradient ultracentrifugation in the presence of 10 mM DTT. DTT failed to alter the apparent molecular mass of
Triton-soluble CaR as compared with ultracentrifugation under
nonreducing conditions.
SDS-solubilized CaR Is Decreased in Size upon Reduction--
To
validate further the data shown in Figs. 1-3, SDS gel permeation
chromatography was utilized to demonstrate that DTT alters the apparent
size of SDS-denatured CaR. As shown in Fig.
4, fractionation of SDS-solubilized
endosomes by gel permeation chromatography shows that the
Kav of SDS-solubilized CaR without DTT (0.41) is smaller as compared with a paired identical sample denatured and chromatographed in the presence of 10 mM DTT
(Kav 0.5). These data are in agreement with
SDS-PAGE immunoblotting analyses and suggest that SH-reducing agents
decrease the molecular mass of SDS-denatured CaR from a value
consistent with a CaR dimeric species to that consistent with a
monomeric species.

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Fig. 4.
Gel permeation chromatography of
SDS-solubilized CaR under reducing and nonreducing conditions. A
mixture of endosomes (600 µg of protein) and bovine serum albumin (1 mg) were solubilized in 2% SDS in either the absence or presence of 10 mM DTT and then fractionated through a Sephacryl 4B column
in the absence (solid line) or presence (dashed
line) of 10 mM DTT. The resulting fractions were mixed
with Laemmli buffer in 143 mM ME and assayed for CaR
content by quantitative immunoblotting (see "Experimental
Procedures"). The elution coefficient (Kav) of
denatured CaR was increased by the addition of DTT. In contrast, the
Kav of bovine serum albumin (as determined
spectroscopically at 280 nm and by Coomassie Blue staining of an
SDS-polyacrylamide gel) was constant in the presence and absence of
DTT.
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The simplest interpretation of data displayed in Figs. 1-4 is that CaR
protein in rat IMCD endosomes is present as a dimeric species that can
only be partially dissociated by SDS denaturation and SH-reducing
agents under standard conditions utilized to isolate most membrane
preparations. Derivatization of CaR SH groups with NEM to prevent
SH oxidation and the formation of disulfide bonds prior to or during
SDS denaturation fail to replicate the dissociation of the CaR complex
achieved by SH-reducing agents such as
-mercaptoethanol or DTT.
These data suggest that reduction of pre-existing S-S bonds are
primarily responsible for the formation of CaR monomers on immunoblots.
However, a minimum of 40% of the CaR immunoreactive protein contained
in purified apical membrane endosomes is successfully reduced to CaR
monomers, whereas the remainder is resistant to treatment with multiple
SH-reducing agents.
Exposure of Triton X-100-solubilized CaR to Divalent and Trivalent
Cations Alters Its Electrophoretic Mobility in SDS-PAGE--
Although
the binding of divalent, trivalent, and polyvalent cations to CaRs are
known to activate downstream signaling cascades (13, 37), only limited
data exist investigating the structural alterations that occur in CaRs
after ion binding. To examine whether the electrophoretic mobility of
CaR is altered by agonist binding, Triton X-100-solubilized CaR was
first incubated for 20 min in various concentrations of
Ca2+, Mg2+, or Na+ at 37 °C and
then analyzed by SDS-PAGE immunoblotting as shown in Figs.
1-3. As shown in Fig. 5,
Ca2+ exposure of Triton X-100-solubilized CaR induced
a concentration-dependent decrease in the intensity
of the monomeric (121 and 138-169 kDa) CaR bands accompanied by an
increase in intensity of the 240-310-kDa species (20 ± 6%
increase in 240-310-kDa signal induced by 20 mM
Ca2+, as percent of total CaR signal, p < 0.05, n = 4). A similar effect was also observed
following Mg2+ treatment (+16 ± 3% by 20 mM Mg2+, p < 0.01, n = 4), but higher (20-40 mM)
Mg2+ concentrations were required to prevent formation of
CaR monomers as compared with 10-20 mM Ca2+
(Fig. 5). These effects appeared specific since exposure to 0-80 mM Na+ produced no significant change in CaR
monomer formation (+7 ± 6% by 40 mM Na+,
n = 3). These data shown in Fig. 5 are similar to the
relative potencies of Ca2+ and Mg2+ obtained
from analyses of expressed recombinant CaRs. In a similar manner,
exposure to trivalent lanthanides that also activate CaRs, including
Gd3+ (200 µM) and Eu3+ (200 µM), also prevented formation of monomeric CaR species on SDS-PAGE immunoblots (Fig. 5). It should be noted, however, that the
appearance of the high molecular weight complex induced by lanthanides
is different from that produced by Ca2+ and
Mg2+, since the trailing edge of the lanthanide-induced
complex is more diffuse and extends almost into the stacking gel. This
is consistent with the possibility that Ca2+ and
Gd3+ may actually activate the CaR at different binding
sites (8, 38).

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Fig. 5.
Exposure of Triton X-100-solubilized CaR to
di- and trivalent cations but not NaCl alters CaR electrophoretic
mobility. Triton X-100-solubilized CaR was incubated for 20 min at
37 °C in the presence of increasing concentrations of
CaCl2, MgCl2, or NaCl or with either 200 µM GdCl3 or 200 µM
EuCl3. The samples were then denatured in Laemmli buffer
containing 143 mM ME at 20 °C and immunoblotted
against monoclonal anti-CaR antibody (see "Experimental
Procedures"). The NaCl concentrations given are in excess of the 125 mM NaCl present in every sample. These data shown are
representative of a minimum of three separate identical
experiments.
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Calcium-induced Alteration of CaR Electrophoretic Mobility Is
Mediated by Disulfide Bonds within the CaR Complex--
To determine
whether exposure of CaR to Ca2+ produces a structural
alteration in CaR protein that prevents CaR monomer formation upon
subsequent SDS denaturation, Triton X-100-solubilized CaR was incubated
at either 4 °C (Fig. 6, lane
1) or 37 °C (Fig. 6, lane 3) prior to denaturation
in SDS-Laemmli buffer containing 143 mM
ME at 20 °C.
As compared with 4 °C, CaR incubation at 37 °C consistently
resulted in increased CaR immunoreactivity present in the 240-310-kDa
band with a corresponding decrease in monomer immunoreactivity of CaR.
NEM pretreatment of the CaR samples exposed to 37 °C blocked the
shift in CaR electrophoretic mobility to the larger 240-310-kDa band
(Fig. 6, lane 3; p < 0.01, n = 6) but
did not affect the distribution of CaR bands after incubation at
4 °C (Fig. 6, lane 2). These data suggest that a 10-min
exposure of Triton X-100-solubilized CaR to 37 °C results in the
formation of disulfide bonds that are not susceptible to reduction
during subsequent SDS denaturation in the presence of SH-reducing
agents. In a similar manner, exposure of Triton-soluble CaR to 20 mM Ca2+ at 4 °C produced a shift in CaR
immunoreactivity identical to that produced by incubation at 37 °C
that was also significantly inhibited by pre-exposure to 1 mM NEM (Fig. 6, lane 5; p < 0.05, n = 3). The shifts produced by either calcium or
exposure to 37 °C are not additive, and their combination was also
significantly attenuated by NEM pretreatment (Fig. 6, lane 8;
p < 0.001, n = 6). These data suggest that
exposure to Ca2+ induces an alteration in Triton
X-100-solubilized CaR at 4 °C that is similar to that produced by
exposure of CaR alone to 37 °C. Both treatments alter the ability of
the combination of SDS and SH-reducing agents to fully denature CaRs to
monomeric species.

View larger version (55K):
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|
Fig. 6.
The effect of temperature and
Ca2+ exposure on the distribution of Triton
X-100-solubilized CaR immunoreactive bands. Triton
X-100-solubilized CaR extracts were incubated for 10 min at either 4 or
37 °C in the presence or absence of 1 mM NEM.
Subsequently, all samples were incubated for an additional 10 min in
either the presence or absence of 20 mM Ca2+.
The resulting samples were all solubilized in Laemmli buffer containing
143 mM ME and immunoblotted as described under
"Experimental Procedures." The Western blot shown in A
is representative of 3-6 separate experiments. The optical density of
each 240-310-kDa CaR-reactive band was then quantified and is
displayed histographically (B) as a percentage of the total
CaR signal in the lane (mean ± S.E.) normalized to the 4 °C
control. Statistical analysis is using the paired t test;
**, p < 0.01 (lanes 3 versus 4); *,
p < 0.05 (lanes 5 versus 6); ***,
p < 0.001 (lanes 7 versus 8).
|
|
 |
DISCUSSION |
These data reported here confirm and greatly extend earlier
reports (8, 13) showing that the electrophoretic profile of CaR
immunoreactive bands is more complex than simply nonglycosylated and
glycosylated monomeric CaRs present on immunoblots prepared from
CaR-containing protein mixtures. Whereas the presence of a high
molecular weight CaR-immunoreactive band on immunoblots has been
previously reported (2, 7-10), it has been unclear as to whether its
origin represented a real biochemical association or was simply an
artifact of denaturation in SDS-containing detergent solutions. Data
presented in Figs. 1 and 2 show that SDS denaturation of rat kidney
IMCD CaR in the absence of SH-reducing agents results in the formation
of a single 240-310-kDa CaR band, as determined using both monoclonal
and polyclonal anti-CaR antibodies. The specificity of this polyclonal
anti-CaR antiserum has been demonstrated previously where no bands were
observed in immunoblots with membranes prepared from mock-transfected
HEK cells, whereas membranes from HEK cells transfected with human CaR
and bovine parathyroid tissue possessed CaR bands identical to those
reported here (8).
Addition of SH-reducing agents to SDS-denatured CaR reduces its
apparent size as measured by gel permeation chromatography (Fig. 4) as
well as diminishes the intensity of the 240-310-kDa band with a
concomitant appearance of CaR-reactive bands of 121 and 138-169 kDa
that correspond to non-glycosylated and glycosylated monomeric CaRs,
respectively (Figs. 1 and 2). Perfusion of intact kidney tissue with
NEM to prevent any SH oxidation followed by immediate processing of
membranes in NEM fails to prevent isolation of a CaR dimeric species.
However, denaturation of CaR in SDS in the presence of SH-reducing
agents is capable of only partial conversion of the total CaR
immunoreactivity to CaR monomeric species. As shown in Figs. 1 and 2, a
significant proportion (0-60%) of immunoreactive CaR protein remains
as a discrete 240-310-kDa band. At present, the exact nature of this
large SDS-resistant CaR is unknown. However, data shown in Fig. 2
suggest that this SDS-resistant CaR complex may possess S-S linkages
that resist the combination of SDS and reducing agents, and its
formation is increased by both exposure to elevated temperature (Fig.
2, panel A; Fig. 5) and prolonged exposure of membrane
preparations to buffers that do not contain SH-reducing agents (Fig. 2,
panel D). These data do not permit us to distinguish whether
intramolecular or intermolecular S-S bonds contribute to formation of
this SDS-resistant CaR complex.
To study the interactions of CaR with itself or other proteins, CaR was
solubilized in the nonionic detergent Triton X-100 and analyzed by a
combination of sucrose gradient sedimentation and SDS-PAGE
immunoblotting. As shown in panel A of Fig. 1, 58 ± 5% of Triton X-100-solubilized CaR was converted to CaR monomers upon
denaturation in SDS-reducing agents, whereas the Triton X-100-insoluble CaR fraction was correspondingly enriched for the larger 240-310-kDa CaR species. As shown in Fig. 3, Triton X-100-soluble CaR sediments as
a complex of approximately 220 kDa in the presence or absence of
SH-reducing agents as determined in sucrose gradients. Previous analyses of Triton X-100-solubilized membrane proteins such as Band 3, the major anion exchanger of the human red cell, using identical
sucrose gradient sedimentation techniques have demonstrated that Triton
X-100 solubilization does not disrupt intermolecular protein-protein
associations between Band 3 dimers or interfere with the binding of
other cytoskeletal and cytoplasmic proteins to Band 3 (24). These data
displayed in Fig. 3 show the presence of a 220-kDa high molecular CaR
complex after Triton X-100 solubilization suggesting that CaR complex
formation does not arise via artifactual denaturation by SDS.
Furthermore, it seems unlikely that the larger molecular mass of the
CaR complex is the result of associations with heterotrimeric G
proteins as has been reported for 6 other G protein-coupled receptors
(33-37, 41) since its sedimentation is not altered by preincubation
with GTP
S (Fig. 3, panel A). Instead, the IMCD CaR
appears to correspond more closely to the muscarinic acetylcholine
(42), neurotensin (43), and gastrin-releasing peptide (44) receptors
that do not possess associated G proteins after nonionic detergent
solubilization.
As described under "Results," careful analyses of the monomeric CaR
species shown in Fig. 3, panel A, iii, reveal that the Triton X-100-soluble CaR complex present in individual fractions of the
sucrose gradient contains a nonrandom mixture of glycosylated and
non-glycosylated CaR monomers. The simplest interpretation of these
data is that the Triton X-100-solubilized CaR complex consists of CaR
dimers composed of various combinations of glycosylated and
glycosylated-nonglycosylated pairs. These various types of CaR dimers
observed here are only partially dissociated by subsequent denaturation
in SDS and exposure to SH-reducing agents. However, we cannot eliminate
entirely the possibility that other proteins might also be part of the
CaR complex present after solubilization in either Triton X-100 or
SDS.
Romano et al. (14) have used similar techniques to suggest
that the metabotropic glutamate receptors (mGluRs), which are structurally related to CaRs, exist in the membrane as disulfide-linked dimers and that the cysteine residues responsible for the
homodimerization of mGluR5 reside within 17 kDa of the N terminus of
the extracellular domain. Of note, this mGluR5 region contains 4 Cys
residues (19), 3 of which are conserved between all of the mGluRs
(19-21) and all CaRs so far reported (13, 15-18). Indeed, Table
I shows that the conservation of
extracellular cysteine residues within the extracellular domains of the
various known members of this receptor family is very high despite
varying degrees of overall homology. A previous report (45) using
immunoblotting analyses of both cells transfected with various mGluR
species as well as specific regions of rat brain shows the formation of
specific bands of approximately twice the apparent molecular mass of
monomeric mGluRs after SDS denaturation in the presence of SH-reducing
agents. These data suggest that CaRs form dimers or complexes via their extracellular domains. A truncated CaR lacking all of the intracellular domain and 4 of the 7 transmembrane domains still exhibits dimer-like high molecular weight immunoreactivity on CaR immunoblots (9). Taken
together, these data reported here and previous reports (14) suggest
that an ability to form dimeric CaR species may be a characteristic
common to all members of this G protein-coupled receptor subfamily.
View this table:
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Table I
Conservation of extracellular cysteine residues between CaRs, mGluRs,
and VRs
Conservation of extracellular cysteine residues, including those in the
extracellular loops of the transmembrane domain, present in CaRs from
bovine parathyroid (BoP), rat kidney (RaK), human parathyroid (HuP),
rat brain (RaB), and human kidney (HuK), and in mGluRs 1-8 and the
mouse vomeronasal odorant receptors (mV2Rs). Conservation (+) or
non-conservation (o) is determined by comparison to BoPCaR, the
position of whose cysteine residues are given, and additional cysteine
residues present in each molecule but not present in BoPCaR are listed
in the right-hand column (13, 15-21, 39).
|
|
The data shown here suggest that both the IMCD CaR and mGluR5 (14) are
distinct from the receptor tyrosine kinases that undergo dimerization
principally upon agonist binding. However, addition of Ca2+
to Triton X-100-solubilized CaR significantly reduced the ability of
SDS and SH-reducing agents to generate monomeric CaR species, raising
the possibility that Triton-soluble CaR may undergo structural modification in response to metal ion agonist treatment. The effect of
Ca2+ was mimicked by other metal ion CaR agonists in a rank
order of potency equivalent to that determined in functional assays (Gd3+
Ca2+ > Mg2+). These
data, shown in Figs. 5 and 6, suggest that binding of divalent and
trivalent cations to the CaR molecule induces a conformational change
in the CaR complex that promotes oxidation of free sulfhydryl groups
resulting in a Ca2+-mediated reduction in CaR monomer
formation upon subsequent exposure to SDS and SH-reducing agents.
Although metal ions such as Ca2+ and Zn2+ can
themselves promote oxidation of free sulfhydryl residues, the data
shown in Figs. 5 and 6 suggest these effects are not simply
nonspecific, since Ca2+ and Mg2+ exposure
causes a shift in CaR electrophoretic mobility between discrete,
monomeric, and putative dimeric bands, as opposed to producing a broad
smear of CaR immunoreactivity along the lane which could indicate
random aggregation. Thus, the current data are most consistent with the
concept that putative dimeric CaR species become stabilized upon
addition of Ca2+ in vitro. Further work is
required to determine what proportion of CaRs actually exist as either
monomers or dimers in the membranes of intact cells and whether an
equilibrium between the two states has a functional significance
similar to that recently established for the G protein-coupled
opioid receptor (46).
The importance of extracellular domain disulfide bonds on
agonist-induced G protein-coupled receptor activation has been
investigated recently using site-directed mutagenesis studies of both
the thyrotropin-releasing hormone receptor (47, 48) and
gonadotropin-releasing hormone receptor (49). To date, most studies of
the CaR have investigated the expression of various normal and mutant
recombinant CaRs (obtained from naturally occurring mutations and by
site-directed mutagenesis) in oocytes (4, 5, 13) and HEK cells (6-9).
The studies reported here have suggested an important role for
sulfhydryl groups in formation of a putative CaR dimeric species and
provide a series of testable hypotheses that should be the object of
future studies of CaR using site-directed mutagenesis.
 |
ACKNOWLEDGEMENTS |
We thank Kimberly Rogers of NPS
Pharmaceuticals and Steve Hebert and Inho Jo for their contributions to
this project.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grants DK-38874, DK-43955, HL-15157 (to H. W. H.), DK-48330, DK-41415, DK-48330, and DK-52005 (to E. M. B.).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: Renal Research
Laboratory, Division of Nephrology, Enders Bldg., Rm. 1260, Children's Hospital, 300 Longwood Ave., Boston, MA 02115. Tel.: 617-355-6989; Fax:
617-730-0435; E-mail: harris{at}hub.tch.harvard.edu.
1
The abbreviations used are: CaR, Extracellular
calcium/polyvalent cation-sensing receptors; HEK, human embryonic
kidney; IMCD, inner medullary collecting duct; PAGE, polyacrylamide gel
electrophoresis; SH, sulfhydryl; NEM, N-ethylmaleimide; DTT,
dithiothreitol;
ME,
-mercaptoethanol; GTP
S, guanosine
5'-3-O-(thio)triphosphate; mGluR, metabotropic glutamate
receptor.
 |
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