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J. Biol. Chem., Vol. 277, Issue 52, 50543-50549, December 27, 2002
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From the Endocrine-Hypertension Division, Department of Medicine,
Brigham and Women's Hospital, and Harvard Medical School,
Boston, Massachusetts 02115
Received for publication, June 12, 2002, and in revised form, September 20, 2002
The extracellular calcium
(Ca2+o)-sensing receptor (CaR) activates
Ca2+ influx independent of the release of intracellular
Ca2+ stores. The latter can be negatively regulated by
protein kinase C (PKC) through phosphorylation of Thr-888 of the CaR.
In this study, we substituted Thr-888 with various amino acid residues or a stop codon to understand how PKC phosphorylation of the CaR inhibits receptor-mediated release of intracellular Ca2+
stores. Substitutions of Thr-888 with hydrophobic and hydrophilic amino
acid residues had various effects on CaR-mediated release of
intracellular Ca2+ stores as well as activation of
Ca2+ influx. Several point mutations, such as T888D, had
marked negative effects on CaR-mediated release of intracellular
Ca2+ stores but not on phorbol myristate
acetate-insensitive activation of Ca2+ influx. Presumably,
the negatively charged aspartate mimics phospho-threonine. Interestingly, truncating the receptor at 888 had an even more pronounced negative effect on CaR-elicited release of intracellular Ca2+ stores without significantly affecting CaR-mediated
activation of Ca2+ influx. Therefore, truncation at
position 888 of the CaR affects the activity of the receptor in a
manner that resembles PKC phosphorylation of the CaR. This in turn
suggests that PKC phosphorylation of the CaR prevents G protein
subtypes from interacting with the region of the receptor critical for
releasing Ca2+ stores, which is missing in the
truncated receptor.
Extracellular calcium concentration (Ca2+o) is
tightly regulated by the interactions of several hormones
(e.g. parathyroid hormone, vitamin D, and calcitonin) and
organ systems (i.e. parathyroid gland, kidney, bone, and
intestine) (1). Parathyroid cells respond to changes in
Ca2+o with oppositely directed alterations in
parathyroid hormone secretion through the
Ca2+o-sensing receptor
(CaR).1 Such responses can be
down-regulated by activators of protein kinase C (PKC) (2-10), a
process that has been implicated in the reduced responsiveness to
Ca2+o of adenomatous or hyperplastic parathyroid
glands (11, 12), although these glands also have reduced expression of
the CaR (13, 14). Likewise, PKC may contribute to age-related changes
in the regulation of parathyroid hormone secretion by Ca2+o in rats (15). Therefore, stimulus-secretion
coupling in parathyroid cells can be modulated by PKC. We recently
showed that some of this modulation occurs at the receptor level
(16).
The human homologue of the CaR has five putative PKC phosphorylation
sites in its intracellular domains. An alteration in the coupling of
the CaR to secondary signaling pathways has been implicated as one of
mechanisms by which PKC exerts negative effects on the function of the
receptor (16). In particular, PKC phosphorylation of Thr-888 inhibits
CaR-mediated increases in cytosolic calcium (Ca2+i)
via mobilization of intracellular Ca2+ stores. In this
study, we introduced various mutations at position 888 and examined
whether any of these mutations mimic or block the action of PKC
activators on CaR-mediated PLC signaling pathways. We found that
truncating the CaR at 888 impairs the function of the receptor in a
similar way as activating cellular PKC.
Site-directed Mutagenesis--
Site-directed mutagenesis
utilized the approach described by Kunkel (17) to produce mutated
receptors in which the threonine residue at position 888, one of the
five predicted PKC phosphorylation sites in the cytoplasmic tail of the
human CaR, was mutated to various amino acid residues or a stop codon.
The dut-1 ung-1 strain of Escherichia coli, CJ236, was
transformed separately with mutagenesis cassette 6, as described
previously (18). Uracil-containing, single-stranded DNA was produced by
infecting the cells with the helper phage VCSM13 (Stratagene, La Jolla,
CA). The single-stranded DNA was then annealed to a mutagenesis primer
that contained the desired nucleotide change encoding a single-point
mutation flanked on both sides by wild-type sequences. The primer was
subsequently extended around the entire single-stranded DNA and ligated
to generate closed circular heteroduplex DNA. DH5 Transient Expression of CaRs in HEK293--
CaR cDNA was
prepared with the Midi plasmid kit (Qiagen). LipofectAMINE
(Invitrogen) was employed as a DNA carrier for transfection. The HEK293
cells used for transient transfection were provided by NPS
Pharmaceuticals (Salt Lake City, UT) and were cultured in Dulbecco's
modified Eagle's medium (Invitrogen) with 10% fetal bovine serum
(Hyclone). The DNA-liposome complex was prepared by mixing DNA and
LipofectAMINE in OPTI-MEM I reduced serum medium (Invitrogen) and
incubating the mixture at room temperature for 30 min. The
DNA-LipofectAMINE mixture was then diluted with OPTI-MEM I reduced
serum medium and added to 90% confluent HEK293 cells plated on
13.5 × 20.1 mm glass coverslips using 2.5 µg of DNA. After the
cells were incubated for 5 h at 37 °C, an equivalent amount of
OPTI-MEM I reduced serum medium with 20% fetal bovine serum was added
to the medium overlying the transfected cells, and the latter was
replaced with fresh Dulbecco's modified Eagle's medium with 10%
fetal bovine serum at 24 h after transfection. The expressed
Ca2+o-sensing receptor protein was assayed 48 h after the start of transfection.
Detection of Expressed CaR on the Cell Surface--
Before whole
cell lysates were prepared, intact HEK293 cells transiently transfected
with FLAG-tagged CaR were labeled with 1 mM
ImmunoPure N-hydroxysulfosuccinimidobiotin (Pierce), a
membrane-impermeable biotinylation reagent, as described previously
(19). The surface-biotinylated HEK293 cells were solubilized, and the
FLAG-tagged receptor was immunoprecipitated with anti-FLAG M2
monoclonal antibody (Sigma). The immunopurified species were
subsequently eluted and subjected to SDS-containing PAGE using a linear
gradient of polyacrylamide (3-10%). The forms of the receptor present
on the cell surface were detected with an avidin-horseradish peroxidase
conjugate (Bio-Rad) followed by visualization of the biotinylated bands with an enhanced chemiluminescence (ECL) system (PerkinElmer Life Sciences).
Measurement of Ca2+i by Fluorimetry in
Cell Populations--
HEK293 cells, which were plated on coverslips
and transfected with CaR cDNAs, were loaded for 2 h at room
temperature with fura-2/AM (Molecular Probes) in 20 mM
HEPES, pH 7.4, containing 125 mM NaCl, 4 mM
KCl, 1.25 mM CaCl2, 1 mM
MgSO4, 1 mM NaH2PO4, 0.1% (w/v) bovine serum albumin, and 0.1% dextrose and washed once at
37 °C for 20-30 min with a buffer solution (20 mM
HEPES, pH 7.4, containing 125 mM NaCl, 4 mM
KCl, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% dextrose, and 0.1% bovine serum albumin). The
coverslips were then placed diagonally in a thermostatted quartz
cuvette that contained the buffer solution using a modification of the technique employed previously (20). The CaR was activated by multiple
additions of an agonist in incremental doses to reach the desired
concentrations. Excitation monochrometers were centered at 340 and 380 nm with emission light collected at 510 ± 40 nm through a wide
band emission filter. The 340/380 excitation ratio of emitted light was
used as readout for Ca2+i as described previously
(20). For PKC activation, the cells were preincubated with a PKC
activator such as phorbol myristate acetate (PMA) for 1-2 min. For
measuring transient Ca2+i responses elicited by
spermine, the buffer solution was devoid of MgCl2,
CaCl2, and bovine serum albumin, and 1 mM EGTA
was added in the beginning of the experiment.
Determination of Total Inositol Phosphates--
The transiently
transfected HEK293 cells were prelabeled overnight with
[3H]myo-inositol (6.25 µCi/well)
(PerkinElmer Life Sciences) in 199 media. The cells were washed once
with Eagle's medium containing 2 mg/ml bovine serum albumin and
incubated with varying concentrations of agonists for 30 min in the
presence of 10 mM LiCl (21). The reactions were terminated
with 10% ice-cold trichloroacetic acid (final w/v). After
centrifugation to remove insoluble debris, trichloroacetic acid was
extracted with water-saturated diethylether, and total inositol
phosphates were separated on Dowex anion exchange columns and
quantitated with a liquid scintillation counter, as described
previously (21).
Statistical Analysis--
The mean EC50 for the
wild-type or each mutant receptor in response to increasing
concentrations of Ca2+o or spermine was calculated
from the EC50 values for all of the individual experiments
and expressed with the standard error of the mean (S.E.) as the
index of dispersion. Comparisons of the EC50 values and
inositol phosphate (IP) responses were performed by analysis of
variance or Duncan's multiple comparison test (22). A p
value of The impact of various substitutions of Thr-888 on the function of
the CaR was examined by stimulating the mutant receptors in transiently
transfected HEK293 cells by Ca2+o for a full CaR
response or by spermine in the absence of
Ca2+o to avoid Ca2+ influx. The
activities of mutant receptors were evaluated initially by measuring
Ca2+i responses. As summarized in Table
I, the substitutions of hydrophobic amino
acid residues for Thr-888, including the previously reported T888V,
reduced EC50[Ca2+]o significantly,
whereas the substitutions of negatively charged amino acid residues or
glycine increased EC50[Ca2+]o. The
other substitutions, such as Asn, Gln, and Lys, did not change
EC50[Ca2+]o significantly. The
patterns of the Ca2+i responses of all the mutant
receptors were somewhat different from those of the wild-type receptor
(Fig. 1). Among them, T888K and T888D
showed little transient responses but did show prominent sustained
responses that were indicative of activation of Ca2+
influx. In addition to substituting Thr-888 with various amino acid
residues, we also truncated the receptor by introducing a stop codon at
position 888. We found that T888Stop further blunted transient
Ca2+i responses with an increased
EC50[Ca2+]o and a 50% reduction in
its maximal cumulative Ca2+i response. In addition,
the pattern of the Ca2+i response of T888Stop
showed a close resemblance to that of the wild-type receptor in
PMA-treated cells (Fig. 1) (16) in which CaR-evoked transient
Ca2+i increases due to the mobilization of
intracellular Ca2+ stores were substantially blunted.
Both the release of Ca2+ from intracellular stores and
activation of Ca2+ influx contribute to CaR-mediated
increases in Ca2+i when Ca2+o
is used as a CaR agonist. Therefore, we also used an alternative CaR
agonist, spermine, to determine the impact of PKC activation on
CaR-evoked mobilization of intracellular Ca2+ stores. In
the following experiment, we stimulated the receptor with spermine in
the absence of Ca2+o and Mg2+o.
A trace amount of divalent cations was removed by including 1 mM EGTA in the experimental buffer prior to the addition of
spermine. All mutant receptors had significant spermine-elicited Ca2+i responses, but they were still less than
those of the wild-type receptor (Table I). Among them, T888Stop had the
lowest maximal response, about 25% of that of the wild-type receptor, followed by T888K and T888D, which had maximal responses about 55% of
that of the wild-type receptor. Others had maximal responses ranging
from 62% to 99% (Table I). These results suggest that mutations at
position 888 affect CaR-mediated activation of certain G protein
subtypes that induce the release of intracellular Ca2+ stores.
Next, we tested the effect of PMA on various mutants when stimulated by
spermine in the absence of Ca2+o and
Mg2+o. Consistent with our previous study, the
effects of PMA on the CaR were significantly reduced by all the
mutations. As shown in Table I, all mutant receptors except T888Stop
showed greater maximal responses in the presence of PMA than did the wild-type receptor. Among them, T888W, T888Y, T888Q, and T888G were
most resistant to PMA with maximal responses over 2-fold higher than
those of the wild-type receptor in cells similarly pretreated with 100 nM PMA. Although T888Stop-mediated responses were
insensitive to PMA, the responses in the control conditions were
similar to those of the wild-type receptor in cells pretreated with
PMA. As shown in Fig. 2, pretreatment of
T888Stop-transfected cells with PMA hardly affected the
Ca2+i responses elicited by either spermine or
Ca2+o. Moreover, the resultant response curves of
the truncated receptor under all conditions were essentially
superimposed on those of the wild-type receptor pretreated with 100 nM PMA, which markedly attenuated CaR-mediated release of
Ca2+ from intracellular stores, as shown in our previous
study (16). These results suggest that PKC phosphorylation of Thr-888
blocks the respective G proteins from interacting with the region of the receptor C-terminal to Ala-887 and from activating Ca2+
stores.
Protein Kinase C (PKC) Phosphorylation of the
Ca2+o-sensing Receptor (CaR) Modulates
Functional Interaction of G Proteins with the CaR
Cytoplasmic Tail*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
competent cells were transformed with these DNA heteroduplexes, and
incorporation of the desired mutation was confirmed by
sequencing the entire cassette. The resultant mutated cassette 6 containing the desired mutation was doubly digested with
XhoI and XbaI and cloned into the full-length of
the receptor in pcDNA3.
0.05 was considered to indicate a statistically
significant difference.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EC50 values and maximal Ca2+i responses of the
wild-type (WT) and mutant receptors when stimulated with either
Ca2+o or spermine in the presence or absence of PMA
View larger version (27K):
[in a new window]
Fig. 1.
Ca2+o-elicited
Ca2+i responses of the wild-type
(A) and mutant receptors T888V (B),
T888K (C), T888D (D), and T888Stop
(E) in control cells and the wild-type receptor in
PMA-treated cells (F). The CaR-induced
Ca2+i responses in cell populations were determined
in cells loaded with fura-2 using Photon Technology International
fluorimetry. The cells transfected with the wild-type or mutant
receptors were stimulated with Ca2+o through
multiple additions of concentrated Ca2+o into the
experimental buffer in the absence (A-E) or presence
(F) of PMA. The concentration of Ca2+o
was raised to 1.5, 2.5, 3.5, 4.5, 5.5, 10, 15, and 20 mM as
indicated at arrowheads. The representative real-time
recordings of emission ratio (340/380 excitation) are shown for these
receptors.
View larger version (13K):
[in a new window]
Fig. 2.
Effect of PMA on Ca2+i
responses of T888Stop elicited by spermine (A) or by
Ca2+o (B). The cells
transfected with the wild-type receptor (circles) or
T888Stop (triangles) were stimulated in the presence
(open) or absence (filled) of 100 nM
PMA. Ca2+i response of cell populations was
determined in cells loaded with fura-2 using Photon Technology
International fluorimetry. Ca2+i response was
normalized to the maximal spermine-elicited response of the wild-type
receptor in the absence of PMA and Ca2+o (mean ± S.E.; n = 7-17). The two curves with
filled and open triangles are superimposed in A
and B.
To determine whether PKC activation exerts its negative effects on
signaling components upstream of mobilization of intracellular Ca2+ stores, we determined IP responses of some receptors.
IP responses were determined in the transfected cells prelabeled
overnight with [3H]inositol. For the effects of PKC
activation, we pretreated the cells with 100 nM PMA for 10 min and subsequently stimulated the cells with either
Ca2+o or spermine. Pretreatment of the cells with
PMA reduced Ca2+o-elicited IP responses of the
wild-type receptor by 14.5% and shifted the response curve to the
right by 2-4 mM Ca2+o (Fig.
3A and Table II).
Pretreatment of the cells with PMA had a much milder negative effect on
IP responses than on Ca2+i responses when
the wild-type receptor was stimulated by Ca2+o. In
contrast, PKC activation markedly reduced IP responses when the
wild-type receptor was stimulated by spermine alone (Fig. 3B
and Table II). For instance, at 1 mM spermine (when the IP response approached the maximal value), pretreatment of cells with PMA
reduced the response by 99%. This suggests that the major component of
the Ca2+o-elicited IP response of the wild-type
receptor, about 85% at concentrations of Ca2+o as
high as 8-16 mM, is dependent on activation of
Ca2+ influx.
|
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Consistent with the hypothesis that PKC-phosphorylated CaR blocks the
interaction of certain G proteins with structural elements located
C-terminally to Ala-887, no significant IP responses were elicited by
spermine even in the absence of PMA (Fig.
4A and Table II). In contrast,
Ca2+o-induced IP responses of T888Stop were
substantial but insensitive to PMA (Fig. 4B and Table II),
presumably through activation of PMA-insensitive Ca2+
influx. Like T888Stop-mediated Ca2+i responses, the
PMA-insensitive IP responses of T888Stop have no significant difference
from those of PMA-pretreated wild-type receptor (Fig. 3 and Table
II).
|
To further test the hypothesis that CaR-induced elevation of
Ca2+i through activation of Ca2+ influx
markedly activates PLC, we stimulated the cells with spermine in the
presence of 0.5 mM Ca2+o, a
concentration below the threshold for activation of the CaR. The
presence of 0.5 mM Ca2+o increased the
spermine-elicited maximal IP response of the wild-type receptor
~2.5-fold (Fig. 5A) to a
level similar to that stimulated by high Ca2+o
(Fig. 3A). Likewise, the presence of 0.5 mM
Ca2+o increased spermine-elicited maximal IP
response of T888Stop (Fig. 5B) to a level similar to that
stimulated by high Ca2+o (Fig. 4B).
Moreover, the Ca2+o-depedent IP productions were
similar in cells transfected with T888Stop (Fig. 5B) to
those in cells transfected with the wild-type receptor (Fig.
5A). These results further support that CaR-mediated
Ca2+ influx activates PLC and that the magnitude of
CaR-elicited Ca2+ influx in cells transfected with T888Stop
is similar to that in cells transfected with the wild-type receptor.
Importantly, these results also rule out the possibilities that
spermine is significantly transported into the cells and subsequently
inhibits intracellular PLC activities in HEK293 cells when applied
extracellularly.
|
In contrast to T888Stop, T888V had maximal IP responses significantly
higher than the wild-type receptor when stimulated with spermine or
Ca2+o (Fig. 6 and
Table II). This result suggests that T888V primarily enhances the
activity of the receptor for activation of pathways leading to
mobilization of intracellular Ca2+ stores.
Consistent with our studies on Ca2+i responses,
spermine- or Ca2+o-elicited IP responses of
T888V were also affected negatively by PMA. However, the IP
response of T888V at any given concentration in the PMA-treated cells
was not lower than that of the wild-type receptor in non-treated cells.
This suggests that PMA exerts additional negative effect on signaling
elements downstream of PLC activation that are involved in the release
of intracellular Ca2+ stores.
|
Western analysis showed that all the mutant receptors had normal
expression on the cell surface (Fig. 7).
In fact, the truncated receptor had slightly higher expression than did
the wild-type receptor.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Ca2+o is the main physiological regulator of calcium homeostasis through its action on its own cell surface receptor, i.e. the CaR. Increases in the concentration of Ca2+o elicit rapid transient Ca2+i responses followed by sustained increases in Ca2+i in parathyroid cells and CaR-transfected HEK293 cells. Previous studies have shown that both activation and inhibition of PKC have profound effects on Ca2+o-elicited Ca2+i responses in parathyroid cells (6, 7) and CaR-transfected HEK293 cells and that these effects can be significantly blocked by substituting Thr-888 with valine in the CaR (16).
In this study, we introduced a variety of amino acid substitutions for threonine at position 888 to test whether we can create a mutant receptor that resembles the PKC-phosphorylated receptor. We found that substitutions with hydrophobic amino acid residues such as alanine, phenylalanine, and tryptophan increased apparent receptor affinities to its agonists similar to those associated with valine substitution reported in our previous study (16). These increased apparent receptor affinities for its agonists, such as calcium and spermine, suggest that the CaR might be somewhat inhibited by basal PKC activity. In contrast, T888D, T888E, and T888G are apparently less sensitive than the wild-type CaR to the receptor agonists with increased EC50 values. The reason for T888E and T888D to simulate the wild-type receptor following PMA treatment is likely that the negatively charged amino acid residues may somewhat resemble the PKC-phosphorylated receptor with charges acquired at the phosphorylation site. In the case of T888G, substitution with glycine could significantly alter the secondary structure of the region that is important for the interaction of the receptor with its respective G proteins by introducing a turn in the helix, as predicted by Chou-Fasman indices. Regardless, all the mutant receptors with various point mutations elicited modest-to-high Ca2+i responses via mobilization of intracellular Ca2+ stores and showed significant but reduced sensitivities to PMA that may reflect the presence of additional PKC sites and/or more downstream modulation by PMA.
In contrast, the action of PKC phosphorylation can be fully mimicked by the removal of the C-terminal region following alanine at position 887, suggesting that PKC phosphorylation at Thr-888 blocks the interaction of this C-terminal region of the receptor with its cognate G proteins that leads to mobilization of intracellular Ca2+ stores. However, the removal of this C-terminal region of the receptor does not impede the receptor's PMA-insensitive activation of Ca2+ influx and the subsequent Ca2+i-dependent activation of PLC, suggesting that the structural determinants activating PMA-insensitive Ca2+ influx are intact in the truncated receptor, T888Stop. Since our previous study showed that truncation upstream of Thr-888 at position 877 completely inactivates not only CaR-mediated mobilization of intracellular Ca2+ stores but also CaR-mediated Ca2+ influx (24) the elements important for activation of Ca2+ influx are likely situated between the residues between 877 and 887.
For the wild-type CaR, activation of PMA-insensitive Ca2+
influx pathways becomes significant when the receptor is activated by a
concentration of Ca2+o that is 3.5 mM
or higher. The activation of this pathway increases not only the
concentration of Ca2+i but also the production of
inositol phosphates. In fact, more than 85% of the IP response may be
attributed to activation of Ca2+ influx when the receptor
is stimulated by a concentration of Ca2+o that is 8 mM or higher. It becomes clear that at least 60% of the IP
response is attributable to activation of Ca2+ influx when
the receptor is activated by 1 or 2 mM spermine in the
presence of 0.5 mM Ca2+o. Since all
subtypes of PLC can be activated by Ca2+, it is likely that
increases in Ca2+i due to activation of
Ca2+ influx non-selectively activate all subtypes of PLC,
including PLC-
s that could be otherwise activated by Gq
or G11. Activating PLC via activation of PMA-insensitive
Ca2+ influx pathways could play an important role in bone
remodeling at the site of bone erosion, where the concentration of
Ca2+o can be as high as 40 mM (23).
Interestingly, the IP responses of T888V were not only significantly higher than those of the wild-type receptor but also much less sensitive to PMA than its own Ca2+i responses. Moreover, the maximal IP response of T888V in PMA-treated cells was significantly higher than that of the wild-type receptor in non-treated cells. Hence, over 50% reduction in spermine-elicited Ca2+i responses of T888V may result from activation of PMA-sensitive Ca2+ stores, which can be inhibited by PMA. In contrast, T888Stop neither activates any PMA-sensitive Ca2+ stores nor fully activates PMA-insensitive stores. Therefore, PKC phosphorylation of the CaR inhibits both CaR-mediated activation of both PMA-sensitive and -insensitive Ca2+ stores.
In summary, we have demonstrated that PKC regulation of CaR-mediated
PLC signaling pathways can be mimicked by truncating the cytoplasmic
tail of the receptor at position 888. Therefore, PKC phosphorylation of
the CaR impairs functional interaction of G proteins with the region of
the cytoplasmic tail downstream of Ala-887.
| |
ACKNOWLEDGEMENT |
|---|
We are grateful to Dr. Edward M. Brown for critical review of the manuscript.
| |
FOOTNOTES |
|---|
* This work was supported by the National Institutes of Health Grant DK54934 (to 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: Endocrine-Hypertension
Div., Brigham and Women's Hospital, 221 Longwood Ave., Boston, MA
02115. Tel.: 617-732-5694; Fax: 617-732-5764.
Published, JBC Papers in Press, October 29, 2002, DOI 10.1074/jbc.M205798200
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ABBREVIATIONS |
|---|
The abbreviations used are: CaR, Ca2+o-sensing receptor; PKC, protein kinase C; PLC, phospholipase C; PMA, phorbol myristate acetate; IP, inositol phosphate.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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