Protein Kinase C Phosphorylation of Threonine at Position 888 in Ca2+o-Sensing Receptor (CaR) Inhibits Coupling
to Ca2+ Store Release*
Mei
Bai
§,
Sunita
Trivedi
,
Charles R.
Lane
,
Yinhai
Yang¶,
Steven J.
Quinn
, and
Edward M.
Brown
From the
Endocrine-Hypertension Division, Department
of Medicine, Brigham and Women's Hospital and Harvard Medical
School and the ¶ Department of Cardiology, Research Laboratory,
Children's Hospital, Boston, Massachusetts 02115
 |
ABSTRACT |
Previous studies in parathyroid cells,
which express the G protein-coupled, extracellular calcium-sensing
receptor (CaR), showed that activation of protein kinase C (PKC) blunts
high extracellular calcium (Ca2+o)-evoked
stimulation of phospholipase C and the associated increases in
cytosolic calcium (Ca2+i), suggesting that PKC may
directly modulate the coupling of the CaR to intracellular signaling
systems. In this study, we examined the role of PKC in regulating the
coupling of the CaR to Ca2+i dynamics in
fura-2-loaded human embryonic kidney cells (HEK293 cells) transiently
transfected with the human parathyroid CaR. We demonstrate that several
PKC activators exert inhibitory effects on CaR-mediated increases in
Ca2+i due to release of Ca2+ from
intracellular stores. Consistent with the effect being mediated by
activation of PKC, the inhibitory effect of PKC activators on
Ca2+ release can be blocked by a PKC inhibitor. The use of
site-directed mutagenesis reveals that threonine at amino acid position
888 is the major PKC site that mediates the inhibitory effect of PKC activators on Ca2+ mobilization. The effect of PKC
activation can be maximally blocked by mutating three PKC sites
(Thr888, Ser895, and Ser915) or all
five PKC sites. In vitro phosphorylation shows that
Thr888 is readily phosphorylated by PKC. Our results
suggest that phosphorylation of the CaR is the molecular basis for the
previously described effect of PKC activation on
Ca2+o-evoked changes in Ca2+i
dynamics in parathyroid cells.
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INTRODUCTION |
The extracellular calcium concentration
(Ca2+o) is tightly regulated by the interactions of
several hormones (e.g. parathyroid hormone
(PTH),1 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
PTH secretion through a cell surface, G protein-coupled receptor, the
Ca2+o-sensing receptor (CaR).
The CaR was first isolated from bovine parathyroid cells using
expression cloning in Xenopus laevis oocytes and shows
pharmacological properties nearly identical to those of the native
receptor in its responses to agonists such as extracellular divalent
cations (e.g. Ca2+o and
Mg2+o), trivalent cations (e.g.
Gd3+o) and polyamines (e.g. neomycin)
(2). In response to increases in Ca2+o, the CaR
stimulates accumulation of inositol phosphates and produces transient
followed by sustained increases in Ca2+i.
Subsequently, cDNAs encoding the human homologue of the same
receptor have been cloned from parathyroid (3) and kidney (4) using a
homology-based strategy. The physiological relevance of the CaR for
mineral ion metabolism has been documented by the identification of
CaR mutations in patients with inherited disorders of calcium
homeostasis (36, 37).
High Ca2+o-evoked suppression of PTH secretion and
the concurrent increases in Ca2+i in parathyroid
cells can be negatively regulated by activation of protein kinase C
(PKC) (5-13). Such negative regulation by PKC has been
suggested to be involved in the reduced responsiveness of adenomatous
or hyperplastic parathyroid glands to Ca2+o as a
result of an increase in membrane-associated PKC (14, 15), although
there is also reduced expression of the CaR in these glands (16, 17).
Likewise, PKC may contribute to age-related changes in the regulation
of PTH secretion by Ca2+o in rats (18). Therefore,
stimulus-secretion coupling in parathyroid cells can be modulated by
PKC, perhaps at an early step in the process of
Ca2+o sensing.
The human homologue of the CaR is predicted to have five PKC sites in
its intracellular domains. We hypothesized that PKC modulates the
sensitivity of parathyroid cells to changes in
Ca2+o by covalently modifying these sites. To test
this hypothesis, we have transiently transfected a human parathyroid
CaR cDNA (18) in HEK293 cells and mutated each of the five putative
PKC sites individually or in varying combinations in the CaR. We
studied Ca2+i responses of the wild type and mutant
CaRs to elevations in Ca2+o and the polycationic
CaR agonist, neomycin, in the presence or absence of various PKC
activators (e.g. phorbol myristate acetate (PMA), Mezerein,
and (
)-Indolactam V) and/or the PKC inhibitor, staurosporine. Our
results show that phosphorylation by PKC at one of the five predicted
PKC phosphorylation sites (Thr888) substantially reduces
CaR-mediated release of Ca2+ from intracellular stores.
Therefore, it is possible that PKC phosphorylation of the CaR regulates
PTH secretion by inhibiting Ca2+ mobilization or perhaps
generation of some other intracellular mediator(s) along the
inositol trisphosphate/phospholipase C pathway.
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EXPERIMENTAL PROCEDURES |
Site-directed Mutagenesis--
Site-directed mutagenesis was
performed using the approach described by Kunkel (19) to produce
mutated receptors in which one or more serine or threonine residues
present in the five predicted PKC phosphorylation sites within
intracellular domains of the human CaR were mutated to alanine or
valine, respectively. The dut-1 ung-1 strain of Escherichia
coli, CJ236, was transformed separately with mutagenesis cassette
5 or 6, as described previously (20). For two receptors with two
mutations each (T888V/S895A and T888V/S915A) and one with three
mutations (T888V/S895A/S915A), CJ236 cells were separately transformed
with a mutated cassette 6 carrying a single mutation (S895A or T888V)
or two mutations (T888V/S915A). 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
competent cells were transformed with these DNA heteroduplexes, and incorporation of the desired mutations was
confirmed by sequencing the entire cassettes. The resultant mutated
cassette 5 was doubly digested with HpaI and XhoI
and cloned into the reconstructed receptor in pcDNA3 (Invitrogen), as described previously (20). Likewise, mutated versions of cassette 6 containing the desired mutations were doubly digested with
XhoI and XbaI and cloned into the reconstructed
receptor in pcDNA3.
Construction of a Mutant CaR with Mutations of Two PKC Sites
(S895A/S915A)--
Cassette 6 was doubly digested with XhoI
and HhaI, and the same cassette carrying three mutated PKC
sites was doubly digested with HhaI and XbaI. Two
small fragments (404 and 568 bp) obtained from the above digestions
were ligated to the large fragment resulting from digestion of the
parent reconstructed CaR clone with XhoI and
XbaI. The resultant clone was confirmed by sequencing.
Construction of a Mutant CaR with Mutations of Two PKC Sites
(T646V/S794A)--
The mutant receptor carrying T646V was doubly
digested with HpaI and XhoI, and the mutant
receptor carrying S794A was doubly digested with XhoI and
XbaI. The two fragments (420 and 975 bp) obtained form the
above digestions were ligated to the large fragment resulting from
digestion of the wild type CaR in pcDNA3 with HpaI and
XbaI. The resultant clone was confirmed by sequencing to
carry these two mutations.
Construction of a Mutant CaR with Five Mutated PKC
Sites--
Cassette 6 carrying S794A was doubly digested with
XhoI and SphI, and the same cassette carrying
three mutations was doubly digested with SphI and
XbaI. Two fragments (168 and 795 bp) obtained from the above
digestions were ligated to the large fragment resulting from digestion
of the CaR carrying the single mutation, T646V, with XhoI
and XbaI. The resultant clone was confirmed by sequencing to
carry all five mutations.
Construction of a Mutant CaR with Four Mutated PKC Sites with
Thr888 unchanged (T646V/S794A/S895A/S915A)--
The
receptors carrying five mutated PKC sites and two mutated sites
(S895A/S915A) was doubly digested with KpnI and
XbaI to obtain the full-length CaR inserts, which were
further digested with SphI. One fragment (2434 bp)
containing T646V/S794A and another fragment (803 bp) containing
S895A/S915A, obtained from the above digestions, were ligated to
pcDNA3 generated by KpnI and XbaI. The
resultant clone was confirmed by sequencing to carry four mutations.
Construction of Flag-tagged CaRs--
The Flag, an epitope tag,
was introduced into the third cassette of the wild type CaR as
described previously (21). The third cassette containing Flag was
digested with AflII and NheI and ligated to the
large fragments resulting from digestion of the CaRs containing PKC
site mutations.
Transient Expression of CaRs in HEK293--
CaR cDNAs were
prepared using the Midi Plasmid Kit (Qiagen). LipofectAMINE (Life
Technologies, Inc.) was employed as a DNA carrier for transfection
(22). The HEK293 cells used for transient transfection were provided by
NPS Pharmaceuticals, Inc. (Salt Lake City, UT) and were cultured in
DMEM (Life Technologies, Inc.) with 10% fetal bovine serum (Hyclone).
The DNA-liposome complex was prepared by mixing DNA and LipofectAMINE
in Opti-MEM I reduced serum medium (Life Technologies, Inc.) 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 0.625 µg of DNA (for
measurement of Ca2+i) or in 100 mm Petri dishes
using 3.75 µg of DNA (for obtaining protein for Western analysis).
After 5 h of incubation at 37 °C, equivalent amounts of
Opti-MEM I reduced serum medium with 20% fetal bovine serum were added
to the medium overlying the transfected cells, and the latter was
replaced with fresh DMEM 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.
Measurement of Ca2+i by Fluorometry 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 containing the buffer solution, using a modification of the
technique employed previously in this laboratory (23). 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 ratio of emitted light (340/380 excitation) was used as readout for Ca2+i
as described previously (23). For PKC activation, the cells were
preincubated with the PKC activators (PMA, mezerein, or (
)-indolactam
V) for 1-2 min. For PKC inhibition, the cells were preincubated with
staurosporine for 30 min. To measure transient Ca2+i responses elicited by neomycin, the buffer
solution was devoid of MgCl2, CaCl2, and bovine
serum albumin, and 1 mM EGTA was added at the beginning of
the experiment.
To evaluate the activities of the wild type and mutant receptors, the
cumulative Ca2+i response at a given concentration
of the agonist was determined using the following method. If the peak
increases in Ca2+i are P1,
P2, P3 ... Pn at concentrations
of the agonist in the bath solution corresponding to C1,
C2, C3 ... Cn, which were achieved by incremental additions of the agonist, the cumulative Ca2+i response (Rn) at any given agonist
concentration (Cn) is defined as the sum, P1 + P2 + P3+ ... +Pn. The
responsiveness of the wild type and mutant receptors to agonists were
compared by determining both EC50 values and the maximal
responses of the respective CaRs. EC50 has been defined as
the effective concentration of an agonist giving half of the maximal
Ca2+i response and was determined by plotting the
concentration-response curve. The cumulative maximal
Ca2+i response has been defined as the cumulative
Ca2+i response at the highest agonist concentration
achieved by the last addition.
Statistical Analysis--
The mean EC50 for the wild
type or each mutant receptor in response to increasing concentrations
of Ca2+o or other CaR agonists was calculated from
the EC50 values for all of the individual experiments and
is expressed with the S.E. as the index of dispersion. Comparisons of
the EC50 values were performed using analysis of variance
or Duncan's multiple comparison test (24). A p value of
0.05 was considered to indicate a statistically significant
difference.
Crude Plasma Membrane Preparations from Transfected HEK293
Cells--
Crude plasma membranes were isolated from HEK293 cells
transiently transfected with the wild type or mutant receptors by
differential speed centrifugation as described by Sun et al.
(25). Confluent cultured cells in 100-mm culture plates were rinsed
twice with phosphate-buffered saline and treated with 0.02% EDTA in
phosphate-buffered saline at 37 °C for 5 min. The detached cells
were pelleted and suspended in 300 µl of homogenization buffer: 50 mM Tris-HCl, pH 7.4, containing 0.32 M sucrose,
2 mM EDTA, and a mixture of protease inhibitors (83 µg/ml
aprotinin, 30 µg/ml leupeptin, 1 mg/ml Pefabloc, 50 µg/ml calpain
inhibitor, 50 µg/ml bestatin, and 5 µg/ml pepstatin (Boehringer
Mannheim)). Then the cells were homogenized with 15 strokes of a
motor-driven Teflon pestle in a tightly fitting glass tube. The
homogenate was sedimented at 18,800 × g for 20 min to
remove nuclei and mitochondria. The supernatant was subsequently
sedimented at 43,500 × g for 20 min to pellet the
plasma membranes, and the resultant pellet was solubilized with 1%
Triton X-100. All steps were carried out at 4 °C.
Western Analysis of Plasma Membrane Proteins--
After
determination of protein concentrations in the crude plasma membrane
preparations using the Pierce BCA protein assay, an appropriate amount
of membrane protein (4 µg) was subjected to SDS-polyacrylamide gel
electrophoresis (PAGE) (26). The proteins on the gel were subsequently
electrotransferred to a nitrocellulose membrane. After being blocked
with 5% milk, the blot was incubated with a previously characterized
primary anti-CaR antibody (4641) (20) and then with a secondary, goat
anti-rabbit antibody conjugated to horseradish peroxidase (Sigma,
diluted 1:500). The Ca2+o-sensing receptor protein
was detected with an ECL system (Amersham Pharmacia Biotech).
Immunoprecipitation of Flag-tagged CaRs--
HEK293 cells
transiently transfected with receptors were rinsed twice with
phosphate-buffered saline and solubilized with 1% Triton X-100, 0.5%
Nonidet P-40, 150 mM NaCl, 10 mM Tris, pH 7.4, 2 mM EDTA, 1 mM EGTA, protease inhibitors,
including 83 µg/ml aprotinin, 30 µg/ml leupeptin, 1 mg/ml Pefabloc,
50 µg/ml calpain inhibitor, 50 µg/ml bestatin, and 5 µg/ml
pepstatin (1× immunoprecipitation buffer), at room temperature.
Insoluble materials were removed by centrifuging the cell lysates at
15,000 rpm for 15 min at 4 °C. The supernatants were collected as
total cell lysates. The protein concentration was determined using the
Pierce BCA protein assay. To a microcentrifuge tube, 5 µg of
monoclonal anti-Flag M2 antibody (VWR Scientific), 400 µl of
H2O, 500 µl of 2× immunoprecipitation buffer, and 100 µl of total lysate containing 500 µg of protein were added. The
mixture was incubated at 4 °C for 1 h. To the mixture was added
5 µl of an alkaline phosphatase-conjugated, anti-mouse IgG. The
incubation was continued for an additional 30 min at 4 °C. To the
mixture was then added 50 µl of 10% protein A-agarose (Life
Technologies, Inc.) for an additional 3-h incubation at 4 °C. The
immunoprecipitates were washed three times with 1× immunoprecipitation
buffer and twice with phosphate-buffered saline containing protease
inhibitors as described above. After one additional wash with 50 µl
of PKC assay dilution buffer (20 mM MOPS, pH 7.2, 25 mM
-glycerol phosphate, 1 mM sodium
orthovanadate, 1 mM dithiothreitol, 1 mM
CaCl2) purchased from Upstate Biotechnology, the samples were ready for in vitro phosphorylation.
In vitro phosphorylation of CaRs--
The samples were
phosphorylated with 80 ng of PKC (Upstate Biotechnology) in 50 µl of
20 mM MOPS, pH 7.2, containing 25 mM
-glycerol phosphate, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 1 mM CaCl2, 100 µM (20 µCi) [
-32P]ATP, 15 mM MgCl2, 0.1 mg/ml phosphatidylserine,
0.01 mg/ml diglyceride, 0.4 µM protein kinase A inhibitor
peptide, 4 µM compound R24571 (an inhibitor for
calmodulin-dependent kinases). The samples were incubated
for 10 min at 32 °C and washed three times with a washing buffer (10 mM Tris-HCl, pH 7.4, 75 mM NaF, 20 mM
-glycerol phosphate, 0.1% Triton X-100). The pellet
was extracted with 45 µl of 2× SDS sample buffer at 65 °C for 30 min. One-third of the eluted sample was subjected to SDS-PAGE and
electrotransferred to a nitrocellulose membrane. Phosphorylated protein
bands were visualized by autoradiography. The CaR
immunoreactivities in the samples were determined by Western analysis.
 |
RESULTS |
To examine the effects of PKC activators on the CaR, transiently
transfected HEK293 cells were treated for 1-3 min with the vehicle
(Me2SO), PKC activators (PMA, mezerein, or (
)-indolactam V), or an inactive phorbol analogue (4
-phorbol 12,13-didecanoate, used as a negative control) prior to activation of the CaR by elevating
Ca2+o. The activity of the receptor was evaluated
by measuring EC50 and maximal cumulative response (see
under "Experimental Procedures" for definition). Cells treated with
Me2SO or 1 µM inactive phorbol derivative
responded to increasing concentrations of Ca2+o in
a manner similar to untreated cells, with
EC50[Ca2+o] values of 4.1 ± 0.1 (n = 7), 4.0 ± 0.1 (n = 8), and 4.0 ± 0.1 (n = 34) mM, respectively,
without significant differences (p
0.05). A
representative tracing of the control Ca2+i
responses is shown in Fig. 1A.
In cells treated with 100 nM PMA, the
Ca2+i responses at low Ca2+o
concentrations (1.5-4.5 mM) were markedly attenuated (Fig.
1B). At higher concentrations of Ca2+o
(5.5 mM and above), however, the
Ca2+o-elicited increases in
Ca2+i were similar to those of control cells. As a
result, the cumulative maximal Ca2+i response to
elevated Ca2+o was reduced to 41% of the control
by PMA, with a significant increase in
EC50[Ca2+o] to 5.0 ± 0.1 mM (n = 25) (p
0.05).
Additional, structurally unrelated PKC activators, such as 1 µM mezerein and 500 nM (
)-indolactam V had
essentially identical inhibitory effects on CaR-elicited Ca2+i responses (Fig.
2).

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Fig. 1.
Effect of PMA on Ca2+o-
and neomycin-elicited Ca2+i responses of the
CaR. Changes in the emission ratio (340/380 excitation) were
measured to assess Ca2+o-evoked
Ca2+i responses in fura-2-loaded HEK293 cells
transfected with the CaR, which are directly proportional to
changes in Ca2+i. A and B,
the tracings shown are representative of the patterns of the
Ca2+o-evoked Ca2+i responses
seen in cells pretreated with Me2SO (A) and 100 nM PMA (B). The addition of Me2SO or
PMA is marked with the first arrowhead on the
left. At each subsequent arrowhead, the
concentration of Ca2+o was increased, first in 1 mM increments to 5.5 mM and then in 5 mM increments to 10, 15, and 20 mM as
indicated. C and D, Ca2+ influx was
prevented by stimulating the cells with neomycin in
Ca2+o-free solution. The tracings shown are
representative of the patterns of the neomycin-evoked
Ca2+i responses seen in multiple experiments in
cells mock-treated (C) or treated with 1 µM
PMA (D) for 1-3 min. At each arrowhead, the
concentration of neomycin was increased in 50 µM
increments to 700 µM as indicated. The mean
EC50 values are reported under "Results."
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Fig. 2.
Inhibitory effects of structurally unrelated
PKC activators on Ca2+o-elicited
Ca2+i responses of the CaR. HEK293 cells were
transfected with the CaR and loaded with fura-2. Changes in the
emission ratio (340/380 excitation) were measured to assess
Ca2+o-evoked Ca2+i responses.
Prior to additions of Ca2+o, the cells were treated
or not treated with one of the PKC activators, PMA, Mezerein, or
( )-Indolactam V. Responses are normalized to the maximal cumulative
Ca2+i responses of nontreated cells. Each
point is the mean value of the number of measurements
indicated in parentheses. S.E. values are indicated with
vertical bars through each point. Some error bars
are smaller than the symbol.
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Because release of Ca2+ from intracellular stores as well
as Ca2+ influx could contribute to increases in
Ca2+i when Ca2+o is used to
activate the CaR, it is essential to use other CaR agonists, such as
neomycin, to determine the impact of PKC activators on CaR-evoked
mobilization of intracellular Ca2+ in the absence of
Ca2+o. The CaR was activated by neomycin in the
absence of Ca2+o and Mg2+o, as
well as in the presence of 1 mM EGTA. Therefore, the
neomycin-elicited Ca2+i responses in cells
transfected with the CaR (Fig. 1C) were solely the result of
release of Ca2+ from intracellular stores, and these
responses were not present in cells mock-transfected with vector alone
(data not shown). Neomycin-elicited Ca2+i responses
were substantially attenuated by pretreatment with PMA (1 µM) (Fig. 1D) at all concentrations of
neomycin tested, and the maximal cumulative Ca2+i
response was reduced to 25% of the control. PMA treatment increased
the EC50[neomycin] of the CaR from 298 ± 9 µM (n = 8) to 405 ± 22 µM (n = 8). The marked difference in the
maximal cumulative responses in PMA-treated cells stimulated with
Ca2+o versus neomycin in the absence of
Ca2+o (64 and 25%, respectively) suggested that
calcium influx stimulated by CaR agonists might be less affected by PMA
than calcium mobilization from intracellular stores.
To evaluate the effect of PMA on high Ca2+o-evoked
sustained increases in Ca2+i as an indirect
assessment of Ca2+ influx,
Ca2+o-elicited transient increases in
Ca2+i were permitted to fall for a longer period of
time (100 s in Fig. 3 versus
25 s in Fig. 1) after each addition of Ca2+o.
The sustained responses observed in this experiment were defined as the
increased levels of Ca2+i that remained at 100 s after each incremental addition of agonist, and these responses
reflect the new steady states, in which influx and efflux of
Ca2+ are nearly equal. As shown in Fig. 3, PMA markedly
reduced the transient Ca2+i responses at low
Ca2+o concentrations (1.5-3.5 mM),
similar to the earlier observations made in Fig. 1, A and
B. Thus the cumulative transient Ca2+i
responses were markedly affected by PMA (Fig.
4A). In contrast, the
sustained Ca2+i responses were similar in control
and PMA-treated cells (Fig. 4B). In cells mock-transfected
with vector alone, there was also a gradual Ca2+i
increase. However, at least 60% of this increase resulted from leakage
of fura-2 over the ~20-min time course of these experiments, which
was marginally affected by PMA (Fig. 3, C and D,
and Fig. 4). Although the transfection efficiency was less than 25%,
the sustained Ca2+i responses in the
receptor-transfected cells were significantly higher than those in
vector-transfected cells.

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Fig. 3.
Effect of PMA on high
Ca2+o-evoked transient and sustained
Ca2+i responses in HEK293 cells transfected with
the CaR. Ca2+o-evoked increases in
Ca2+i were monitored as in Fig. 1. Sustained
increases in Ca2+i were defined as the increased
level of Ca2+i that persisted at 100 s after
each addition of Ca2+o. HEK293 cells were
transiently transfected with the wild type CaR (A and
B, n = 11 for each) or the pcDNA3 vector
(C and D, n = 4 for each). In
panels B and D, cells were treated with 1 µM PMA, compared with control cells in panels
A and C. The concentration of Ca2+o
was increased, first in 1 mM increments to 6.5 and then in
5 mM increments to 10, 15, and 20 mM as
indicated. Results shown are representative of multiple measurements
and are summarized in Fig. 4.
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Fig. 4.
Concentration dependence for the
Ca2+o-evoked transient and sustained
Ca2+i responses in the presence or absence of
PMA. In this figure, the transient and sustained
Ca2+i responses in Fig. 3 were plotted against the
concentrations of Ca2+o added (see under
"Experimental Procedures"). HEK293 cells transfected with the
pcDNA3 vector (circles), or the CaR (squares)
were mock-treated (open symbols) or treated (filled
symbols) with PMA. A, all of the transient responses
(i.e. the cumulative peak responses) are normalized to the
maximal cumulative response of the CaR at 20 mM
Ca2+o in the absence of PMA. B, all of
the sustained responses (i.e. the increased level of
Ca2+i that persisted at 100 s after each
addition of Ca2+o) are normalized to the maximal
sustained response of the CaR at 20 mM
Ca2+o in the absence of PMA. Each point
is the mean value of the number of measurements indicated in
parentheses. S.E. values are indicated with vertical bars
through each point. Some error bars are smaller than the
symbol.
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In order to demonstrate further that the effect of PKC activators on
the function of the CaR was mediated by PKC, we examined the effect of
a PKC inhibitor, staurosporine, on CaR-evoked Ca2+i
responses (Fig. 5). Pretreatment of
CaR-transfected HEK293 cells with 1 µM staurosporine for
30 min significantly reduced the
EC50[Ca2+o] from 4.0 ± 0.1 mM (n = 34; Table
I) to 2.9 ± 0.1 mM
(n = 22; Table I) (p
0.05). In
addition, the pretreatment prevented the inhibitory effects of PMA
(Fig. 5) and other PKC activators (data not shown) on the
Ca2+i responses to the same extent, further
supporting the conclusion that the effect of PKC activators on
CaR-dependent changes in Ca2+i is
through activation of PKC.

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Fig. 5.
Staurosporine potentiates
Ca2+o-elicited Ca2+i responses
of the CaR and blocks the inhibitory effect of PKC activators.
HEK293 cells were transfected with the CaR and loaded with fura-2.
Changes in the emission ratio (340/380 excitation) were measured to
assess Ca2+o-evoked Ca2+i
responses. The tracings shown are representative of the patterns of the
Ca2+o-evoked Ca2+i responses
seen in cells: A, mock-treated; B, pretreated
with 1 µM staurosporine for 30 min; or C,
pretreated with 1 µM staurosporine for 30 min and then
with 100 nM PMA for 1-3 min. At each arrowhead,
the concentration of Ca2+o was increased as
indicated. The mean EC50[Ca2+o]
values are reported under "Results."
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Table I
EC50 values for Ca2+o-elicited
Ca2+i responses of the wild type receptor and receptors
containing PKC site mutations in the presence or absence of PMA
and/or staurosporine
Values are means ± S.E. The number of experiments is indicated in
parentheses.
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We next determined which residues are involved in PKC-mediated
regulation of the CaR by performing site-directed mutagenesis on the
five predicted PKC sites. One of these sites, Thr646, is
located in the first intracellular loop; another one,
Ser794, is in the third intracellular loop; and the
remainder (Thr888, Ser895, and
Ser915) are in the cytoplasmic tail. The expression of
these mutant receptors were then examined by Western analysis, as
described previously (20). After transient transfection, crude plasma membrane proteins were isolated and subjected to reduced 4-12% SDS-PAGE, and the CaRs were detected using a specific anti-CaR antibody. In Fig. 6, the two bands
between 140 and 200 kDa are monomeric forms of the CaR; the bands above
200 kDa are also specific for the CaR and are not present in the
vector-transfected cells, as shown previously (20) (see also Fig. 9).
None of the mutations substantially altered the expression level of the
receptor.

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Fig. 6.
Western analysis to assess expression of the
wild type and mutant receptors. Crude plasma membrane proteins (4 µg) isolated from CaR-transfected HEK293 cells were subjected to a
4-12% gradient SDS-PAGE in the order (from left to
right): wild type CaR, T646V, S794A, T888V, S895A, S915A,
T888V/S895A, T888V/S915A, S895A/S915A, triply mutated CaR
(T888V/S895A/S915A), and quintuply mutated CaR. The receptor proteins
were detected with a specific anti-CaR antiserum, 4641, as described
under "Experimental Procedures." The blot shown is a representative
of the pattern seen in two protein preparations from two independent
transfections.
|
|
The activity of each mutant receptor was examined by measuring its
Ca2+i responses to CaR agonists. The mutant
receptors exhibited no significant changes in their cumulative maximal
Ca2+i responses relative to the wild type receptor.
But some of the mutations, i.e. the three mutations in the
tail (T888V, S895A, and S915A), substantially reduced the
EC50[Ca2+o] of the CaR, as shown in
Fig. 7 and Table I, whereas the two point
mutations in the loops (T646V and S794A) had no significant effect on
the EC50[Ca2+o] of the CaR. In other
words, the mutations, if they have any effect, render the CaR more
sensitive to Ca2+o. Of the three mutations in the
tail, T888V had the greatest impact on the function of the receptor,
reducing the EC50[Ca2+o] from
4.0 ± 0.1 mM (n = 32) to 2.9 ± 0.1 mM (n = 5) (p
0.05).
Moreover, receptors with two mutations that included T888V (T888V/S895A
and T888V/S915A) had EC50[Ca2+o]
values similar to that of the T888V mutant alone. The receptor with
three mutated PKC sites, T888V/S895A/S915A, was slightly more sensitive
to Ca2+o than that with the single mutation, T888V.
Finally, the quintuply mutated receptor, in which all five PKC sites
were mutated, likewise had an
EC50[Ca2+o] similar to that of the
triply mutated receptor.

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Fig. 7.
EC50[Ca2+o] values
for Ca2+o-evoked Ca2+i
responses of the wild type CaR and CaRs with mutated PKC sites in the
presence or absence of PMA and/or staurosporine. HEK293 cells
transfected with wild type CaR or mutant CaRs were mock-treated
(Control), treated with 100 nM PMA, treated with
1 µM staurosporine (Stau), or treated with a
combination of PMA and staurosporine (PMA/Stau).
Ca2+i responses to elevated
Ca2+o were monitored as in Fig. 1. The height of
each bar represents the mean
EC50[Ca2+o] of multiple measurements
(see Table I). The S.E. is indicated with a vertical line at
the top of each bar.
|
|
In order to determine which PKC sites are responsible for the
inhibitory effects of PKC activators on CaR agonist-evoked increases in
Ca2+i, cells transfected with mutant receptors were
pretreated with one of the PKC activators, 100 nM PMA, for
1-3 min. The patterns of the Ca2+i responses for
receptors with the single point mutations, T646V, S794A, S895A, or
S915A (data not shown), or with the double mutation, S895A/S915A (data
not shown), were similar to that of the wild type receptor (Fig.
1B). PMA markedly attenuated low Ca2+o-elicited Ca2+i responses
mediated by these mutant receptors. As shown in Fig. 7 and Table I, the
EC50[Ca2+o] values for these five
mutant receptors were significantly increased by PMA in a manner
similar to that observed with the wild type receptor. In sharp
contrast, PMA produced only small increases in the
EC50[Ca2+o] values of all of the
mutant receptors containing T888V. The receptor with T888V alone showed
some decrease in its Ca2+i responses at 1.5, 2.5, and 3.5 mM Ca2+o (data not shown).
Receptors with one more mutation in addition to Thr888,
such as T888V/S895A and T888V/S915A, did not show any alterations in
the pattern of the Ca2+i responses observed with
the mutant receptor with T888V alone (data not shown). However,
receptors with two or more additional mutations, such as the triply
mutated (T888V/S895A/S915A) and quintuply mutated CaRs, exhibited
markedly reduced inhibitory effects of PMA, only showing some decrease
in the Ca2+i response at 1.5 mM
Ca2+o (data not shown). Thus, the effect of PKC
activation by PMA on CaR-mediated increases in
Ca2+i was mostly mediated by one PKC site,
Thr888.
To demonstrate that the effect of staurosporine on the wild type CaR
was, at least in part, the result of prevention of PKC-induced phosphorylation of the receptor, we examined the effect of
staurosporine on the mutant receptors carrying PKC site mutations. As
shown in Fig. 7 and Table I, staurosporine reduced
EC50[Ca2+o] values of all of the
mutant receptors to varying extents. The receptors with the most
critical PKC site mutated, T888V, which showed the smallest increases
in their EC50[Ca2+o] values upon
treatment with PMA, were least affected by treatment with
staurosporine. Fig. 7 and Table I also show that the pretreatment of
staurosporine prevented the inhibitory effects of PMA on the
Ca2+i responses of triply and quintuply mutated
receptors, similar to what we observed with the wild type receptor. In
summary, the presence of the T888V mutation abolished most of
modulatory effects of PKC on the function of the CaR.
To define further the effect of mutations at PKC phosphorylation sites
on CaR-evoked release of intracellular Ca2+ stores, the
CaR-induced influx of Ca2+o was prevented by using
neomycin as an agonist in the presence of 1 mM EGTA and in
the absence of Ca2+o and Mg2+o,
as before. Neomycin elicited substantial Ca2+i
responses in cells that had been transfected with all of the mutant
receptors in the absence of PMA. In cells that had been transfected
with the wild type and mutant receptors, including the doubly mutated
(S895A/S915A) and the quadruply mutated (T646V/S794A/S895A/S915A) receptors, which preserved the crucial PKC site, Thr888,
PMA substantially diminished the Ca2+i responses
and markedly increased the EC50[neomycin] values of the
receptors (wild type receptor, Fig. 1D; data not shown for
the mutant receptors). In contrast, PMA had much reduced effects on the
Ca2+i responses of cells transfected with mutant
receptors containing T888V. The EC50[neomycin] of the
mutant CaR harboring T888V, for instance, was not significantly
affected by PMA (i.e. 202 ± 7 µM
(n = 10) before versus 207 ± 13 µM (n = 8) after the addition of PMA;
p
0.05). Likewise, PMA had no significant effect on
the EC50[neomycin] of the quintuply mutated receptor
(146 ± 7 µM (n = 8) before
versus 137 ± 7 µM (n = 8) after the addition of PMA; p
0.05).
Representative tracings of Ca2+i responses are
shown in Fig. 8. Although the maximal cumulative responses of the T888V and quintuply mutated receptors in
the presence of PMA were reduced to about 50 and 60% of the control,
respectively, both receptors (Fig. 8, B and D)
exhibited substantially greater responses than that of the wild type
receptor (Fig. 1D) in the presence of PMA. Thus, the
mutation T888V blocked most of the effect of PKC on CaR-induced
mobilization of intracellular Ca2+ stores

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Fig. 8.
Mutation of one PKC site (T888V) blocks the
PMA effect on neomycin-elicited Ca2+i store
release. HEK293 cells were transfected with a CaR containing T888V
(A and B) or five mutated PKC sites (C
and D). Neomycin-evoked Ca2+i increases
in the transfected cells were measured as in Fig. 1, C and
D. The tracings shown are representative of the patterns of
the neomycin-evoked Ca2+i responses seen in cells
mock-treated (A and C) or treated for 1-3 min
with 1 µM PMA (B and D). At each
arrowhead, the concentration of neomycin was increased in 50 µM increments to 700 µM as indicated. The
mean EC50[neomycin] values are reported under
"Results."
|
|
In vitro phosphorylation showed that the bands at 140 and
160 kDa and higher molecular masses immunopurified from cells
transfected with the Flag-tagged wild type receptor were substantially
labeled with 32P (Fig.
9A, lane 1). These bands were
absent in the sample isolated from the vector-transfected cells,
indicating that they were CaR-specific (Fig. 9A, lane 5).
The mutant receptor with the two mutations, T646V/S794A, and the
quadruply mutated receptor, T646V/S794A/S895A/S915A, were
phosphorylated similarly to the wild type receptor (Fig. 9A,
lanes 2 and 3). Moreover, the CaR-specific bands for
these two mutant receptors and the wild type CaR were all
phosphorylated at levels 3-4 times higher than the mutant receptor
carrying mutations in all five PKC sites based on densitometric
analysis (Fig. 9A, lane 4). In contrast, the nonspecific
bands at their respective positions (i.e. those present in
both CaR- and vector-transfected cells) had similar phosphorylation
intensities in all samples (Fig. 9A, lanes 1-5). In
addition, Fig. 9B shows that the immunoreactivities of the
CaR-specific bands in Fig. 9A, lanes 1-4, were similar for
all of the receptors when we detected with anti-CaR antibody, 4641, and
are absent in Fig. 9B, lane 5, i.e. the vector
control. Therefore, in conclusion, we have established that
Thr888 is the major site of the CaR that is phosphorylated
by PKC in vitro. Moreover, this site mediates the
PKC-induced uncoupling of the receptor from release of intracellular
Ca2+ stores.

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Fig. 9.
In vitro phosphorylation of the wild
type and mutant CaRs. HEK293 cells transfected with Flag-tagged
CaRs or the vector were solubilized with Triton X-100 and IGEPAL CA-630
(see under "Experimental Procedures"), and the CaRs were
immunoprecipitated with anti-Flag antibody before phosphorylation with
a mixture of PKC , - , and - . The samples were resolved on a
2.2-8.2% gradient SDS-PAGE. The phosphorylated proteins were
visualized by autoradiography (A), and the
CaR-immunoreactive species were detected by anti-CaR antibody
(B). wt, wild type. The result shown is a
representative of two phosphorylation experiments.
|
|
 |
DISCUSSION |
Ca2+o is the main physiological regulator of
PTH secretion through actions on its own cell surface receptor,
i.e. the CaR (27). 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 and our present results show that both
activation and inhibition of PKC have profound effects on
Ca2+o-elicited Ca2+i responses
in these cells. In the present study, we found that these effects can
be largely eliminated by mutating Thr888 in the CaR. These
results also suggest that this site may have been partially
phosphorylated by PKC under our standard experimental conditions;
therefore, the activity of the CaR can potentially be modulated by
either activating or inhibiting PKC in vivo, in agreement
with previous studies in bovine parathyroid cells (6).
Activation of PKC by PMA mainly blocked the CaR-mediated
Ca2+i responses resulting from mobilization of
Ca2+ from intracellular stores in cells transfected with
the wild type receptor or with mutant CaRs in which the PKC site,
Thr888, was preserved. This PMA-induced inhibition of
CaR-mediated Ca2+ release is particularly apparent with the
use of an alternative CaR agonist, neomycin, in the absence of
extracellular Ca2+, when uptake of
Ca2+o was totally eliminated (Fig. 1D).
We were able to block substantially the inhibitory effect of PKC
activation on CaR-induced release of Ca2+ stores by
mutating threonine at position 888. Therefore, PKC-mediated phosphorylation of the CaR at threonine 888 markedly uncouples the
receptor from release of Ca2+ from its intracellular
stores.
Phosphorylation at position 888 in all of the CaRs preserving
Thr888 had little impact on
Ca2+o-elicited Ca2+i responses
at 5.5 mM or higher concentrations of
Ca2+o (e.g. Fig. 1, A and
B), even though PMA markedly inhibited neomycin-elicited
mobilization of Ca2+ at all concentrations tested in the
absence of Ca2+o. Thus, it is reasonable to assume
that high Ca2+o-elicited Ca2+i
responses in the presence of PMA largely result from Ca2+
influx (Fig. 1B). Consistent with the hypothesis that PMA
has little or no impact on high Ca2+o-stimulated,
CaR-mediated Ca2+ influx, PMA had no effect on CaR
activation-dependent sustained Ca2+i
increases, which are presumably maintained by CaR-induced increases in
Ca2+ influx and/or decreases in Ca2+ efflux.
Nevertheless, it is premature to conclude that PKC phosphorylation at
position 888 has no impact on CaR-stimulated Ca2+ influx,
because it is hard to distinguish between the contributions of
extracellular and intracellular sources of calcium to increases in
Ca2+i, when extracellular calcium is employed as an
agonist for the CaR. To elucidate fully the effects of PMA, if any, on CaR-activation dependent Ca2+ influx, further studies are
needed to measure directly Ca2+ influx.
CaR activation-dependent Ca2+ influx may be
mediated, in part, by Ca2+-permeable, nonselective cation
channels that we have observed in both parathyroid cells (28) and
HEK293 cells (29). CaR agonists (neomycin and
Ca2+o) significantly increase the probability of
channel opening in HEK293 cells stably transfected with the CaR but not in nontransfected HEK293 cells that do not express the CaR (29). Thus,
the enhanced activity of Ca2+-permeable nonselective cation
channels in CaR-transfected HEK293 cells could contribute to the
sustained increases in Ca2+i in the presence of CaR
agonists. These nonselective cation channels are also thought to
contribute to influx of Ca2+o into hippocampal
neurons and to regulation of their excitation (30, 31).
In PMA-treated cells transfected with the wild type CaR, there was
still some residual Ca2+i response (~25%) to
neomycin, even in the total absence of Ca2+ influx. A
substantially higher concentration of neomycin (350 versus
150 µM in the absence of PMA) was required to elicit the initial response, consistent with observations made earlier in this
laboratory (6). That is, activators of PKC inhibited by 50-60% the
high Ca2+o-stimulated generation of inositol
phosphates in CaR-expressing bovine parathyroid cells and reduced
inositol trisphosphate levels at low Ca2+o,
presumably by reducing turnover of phosphoinositides by phospholipase
C. It is possible that PMA selectively uncouples the receptor from one
subtype of G-protein but not another, both of which activate
phospholipase C. In rat portal vein myocytes, Gq and
G11 have been shown to have distinct functions in coupling
1-adrenoreceptors to Ca2+ release and
Ca2+ entry (32). In this system, it appeared that
Gq activated hydrolysis of phosphatidylinositol
4,5-bisphosphate with an attendant release of Ca2+ from
inositol trisphosphate-sensitive intracellular stores, whereas G11 enhanced Ca2+ influx. By analogy, it is
possible that phosphorylation of threonine at amino acid position 888 uncouples the CaR from Gq but not G11, thereby
largely inhibiting release of Ca2+ from intracellular
stores but not Ca2+ influx.
When we mutated all five predicted PKC sites in the CaR, there were
still some residual effects of the PKC activator (PMA) and inhibitor
(staurosporine) on the quintuply mutated receptor. For example, PMA
reduced the maximal cumulative Ca2+i response to
neomycin by 40%. Nevertheless, activation of PKC did not change the
EC50 of the Ca2+i response to neomycin.
It is possible, therefore, that PKC activation may phosphorylate some
additional PKC site(s) on the receptor or PKC sites on other components
in the signal transduction pathway. Alternatively, PMA may reduce store
capacity as has been shown in NIH 3T3 cells (33).
In summary, we have demonstrated that PKC modulation of CaR-mediated
Ca2+i responses is primarily mediated by
Thr888, which can be phosphorylated by PKC in
vitro. Phosphorylation at Thr888 inhibits most of the
agonist-induced increases in Ca2+i due to release
from intracellular stores. Because PKC activation blocks the high
Ca2+o-induced suppression of PTH secretion (5, 7,
8, 10-13, 34, 35) in parathyroid cells, we postulate that the phospholipase C/inositol trisphosphate pathway, leading to release of
Ca2+ from stores and/or other downstream effects, is
associated with PKC regulation of PTH secretion. In addition,
regulation of PKC activity in vivo provides a means of
modulating the function of the CaR and, ultimately, calcium
homeostasis.
 |
ACKNOWLEDGEMENTS |
We thank Drs. R. Diaz and O. Kifor for
helpful discussions and Drs. J. E. Garrett, I. V. Capuano, A. Paruhar, F. Fuller, R. T. Simin, K. Krapcho, and K. V. Rogers
for providing us with the pHuPCaR4.0 plasmid, the HEK293 cells, and the
4641 antibody and its blocking peptide.
 |
FOOTNOTES |
*
Generous support for this work was provided by National
Institutes of Health Grants DK09436 (to M. B.); DK48330, DK41415, and
DK52005 (to E. M. B.); DK09432 (to Y. Y.); and HL42120 and DK40127
(to S. Q.) and by The St. Giles Foundation (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: Endocrine-Hypertension
Division, Brigham and Women's Hospital, 221 Longwood Ave., Boston, MA 02115. Tel.: 617-732-4093; Fax: 617-732-5764.
The abbreviations used are:
PTH, parathyroid
hormone; CaR, extracellular calcium-sensing receptor; PKC, protein
kinase C; PMA, phorbol myristate acetate; PAGE, polyacrylamide gel
electrophoresis; MOPS, 4-morpholinepropanesulfonic acid; bp, base
pairs; Ca2+o, extracellular calcium.
 |
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