Activation of Phospholipase C-
1 via G
q/11
during Calcium Mobilization by Calcitonin Gene-related Peptide*
Hicham
Drissi
,
Françoise
Lasmoles
,
Véronique
Le
Mellay§,
Pierre J.
Marie
, and
Michèle
Lieberherr§¶
From the
Institut National de la Santé et de la
Recherche Médicale, U 349, Hôpital Lariboisière,
Paris, and the § Centre National de la Recherche
Scientifique, UPR 1524, Jouy-en-Josas, France
 |
ABSTRACT |
Interaction of calcitonin gene-related peptide
(CGRP) with its receptors leads to stimulation of adenylyl cyclase
and/or phospholipase C (PLC). While regulation of adenylyl cyclase is
thought to involve the G-protein Gs, it is not known
whether activation of PLC results from coupling the receptor to
Gq family proteins or whether 
subunits released from
receptor-activated Gs activate PLC. We used human bone
cells OHS-4 bearing CGRP receptors in which CGRP activates only the PLC
signaling pathway to determine how CGRP acts. CGRP increased the
concentration of intracellular calcium ([Ca2+]i)
within 5 s via a Ca2+ influx through voltage-gated
calcium channels and by mobilizing calcium from the endoplasmic
reticulum. The activation of effectors, like PLC coupled to G-proteins,
is the early event in the pathway leading to inositol
1,4,5-trisphosphate formation, which is responsible for
Ca2+ mobilization. Western blotting demonstrated a range of
PLC-
isoforms (
1,
3,
4, but not
2) and G-proteins
(G
q/11 and G
s). Only phospholipase C-
1
is involved in the mobilization of Ca2+ from the
endoplasmic reticulum of Fura-2-loaded confluent OHS-4 cells and the
formation of inositol 1,4,5-trisphosphate by CGRP; PLC-
have no
effect. Activation of PLC-
1 by CGRP involves the G
q/11 subunit, which is insensitive to pertussis toxin,
but not G
subunits. We therefore believe that CGRP causes the
activation of two separate G-proteins.
 |
INTRODUCTION |
Calcitonin gene-related peptide
(CGRP)1 is a 37-amino acid
peptide generated by alternative splicing of the calcitonin/CGRP gene
product (1, 2), which effects numerous organs, including bone (3). The
receptor for CGRP, which belongs to a subgroup of G-protein-coupled
receptors (4-6), is a seven-transmembrane domain receptor protein,
very like other receptors of the subfamily (7, 8). The binding of CGRP
to these receptors activates multiple signaling pathways. The main
signal pathway used by CGRP is the adenylyl cyclase pathway (9-14),
but CGRP has also been reported to activate guanylate cyclase (15, 16)
and phospholipase C (17) and to increase the intracellular calcium
concentration (18). However, in osteoblastic OHS-4 cells, which possess
CGRP receptors (19), CGRP does not stimulate adenylyl cyclase as do
other cell types (20, 21), but it does increase intracellular calcium.
This suggests that the CGRP receptor activates different G-proteins
than do other calciotropic peptide hormones (22).
There are many similar heterotrimeric G-proteins that transduce a
signal from a hormone-bound receptor to a variety of downstream effector molecules (23-25). Mammals have several types of G-proteins, which form the
subunit and the
and
subunits. These include the Gq, Gs, Gi/Go, and
G12 subfamilies, which are classified on the basis of amino
acid sequence of the
subunits. G-proteins are also divided
traditionally into two types based on their sensitivity to
Bordetella pertussis toxin (PTX) (26). The PTX-sensitive G-proteins are inactivated by ADP-ribosylation of the
subunit. This
group includes members of the Gi and Go
subfamily. Gs- and Gi-proteins were originally
purified as mediators of adenylyl cyclase pathway (27). PTX-insensitive
G-proteins are resistant to ADP-ribosylation and include members of the
Gq subfamily (27). The
subunits of these proteins
activate phospholipase C-
isoenzymes (28), but not phospholipases
(PLCs) C
or C
(29-33). PLC-
isoenzymes may be stimulated by
both G-protein
subunits of the Gq family and by free

subunits (34-36).
PLC can thus be stimulated via receptors with the dual signaling
properties described above. Binding of the ligand to the receptor
either stimulates adenylyl cyclase via G
s, and
phospholipase C is activated by 
subunits released from activated
Gs, or the receptor couples to multiple G proteins leading
to the activation of adenylyl cyclase and PLC via Gs and
subunits of the Gq family (37). The latter route
appears to apply to the human TSH receptor (38), and the turkey
erythrocyte
-adrenergic receptor (39). The present study identifies
and characterizes the PLC isoform involved in the mobilization of
Ca2+ from the endoplamic reticulum and inositol 1,4,5 trisphosphate formation of bone OHS-4 cells in response to CGRP, and
its regulation by G-protein
and 
subunits.
 |
EXPERIMENTAL PROCEDURES |
Materials--
The ECL kit, and L-3-phosphatidyil
[2-3H]inositol 4,5-bisphosphate and Fura-2/AM were
purchased from Amersham, Life Technology (Les Ulis, France). Polyclonal
rabbit anti-PLC antibodies to PLC-
1, PLC-
2, PLC-
3, and
PLC-
4, polyclonal rabbit anti-G-protein antibodies to
G
q/11, G
s, G
i, G
, and
G
1 and their blocking peptides were obtained from Santa
Cruz Biotechnology, Inc. and Tebu (Le Perray-en-Yvelines, France).
Peroxidase-conjugated goat anti-rabbit IgG was obtained from Bio-Rad
(Ivry-sur-Seine, France). Human CGRP and all chemicals were purchased
from Sigma (St. Quentin Fallavier, France). Dulbecco's modified
Eagle's essential medium and fetal calf serum were supplied by Eurobio
(Les Ulis, France).
Human Cell Culture--
The human bone cell line OHS-4, which
exhibits most of the osteoblast phenotype (40), was kindly provided by
Drs. B. Fournier and P. Price. Cells were grown on rectangular glass
coverslips or in Petri dishes (100 cm2) for 4 days in
Dulbecco's modified Eagle's essential medium supplemented with 10%
heat-inactivated fetal calf serum. Cells were then incubated for
72 h in Dulbecco's modified Eagle's essential medium containing 1% heat-inactivated fetal calf serum and transferred to serum-free medium 24 h before use.
Calcium Measurement and Experimental Protocol--
The cells
were washed with Hank's HEPES, pH 7.4 (137 mM NaCl, 5.6 mM KCl, 0.441 mM
KH2PO4, 0.442 mM
Na2HPO4, 0.885 mM
MgSO4·7H2O, 27.7 mM glucose, 1.25 mM CaCl2 and 25 mM HEPES), and
loaded with 1 µM Fura-2/AM for 30 min in the same buffer
at room temperature. The glass coverslip supporting the cells was
inserted into a cuvette containing 2.5 ml of Hank's HEPES, pH 7.4. The
cuvette was placed in a thermostatically controlled (37 °C) Hitachi
F-2000 spectrofluorimeter. Drugs and reagents were added directly to
the cuvette with continuous stirring. The Fura-2 fluorescence response
to the intracellular calcium concentration
([Ca2+]i) was calibrated from the ratio of the
340/380-nm fluorescence values after subtraction of the background
fluorescence of the cells at 340 and 380 nm as described by Grynkiewicz
et al. (41). The dissociation constant for the
Fura-2-Ca2+ complex was taken as 224 nM. The
values for Rmax and Rmin
were calculated from measurements using 25 µM digitonin
and 4 mM EGTA and enough Tris base to raise the pH to 8.3 or higher. Each measurement on Fura-2-loaded cells was followed by a
parallel experiment under the same conditions with cells not loaded
with Fura-2.
We first studied the direct effects of CGRP (0.1-100 nM)
on [Ca2+]i in OHS-4 cells. We then investigated
whether the effects of CGRP on [Ca2+]i were due
to an influx of Ca2+ from the extracellular milieu and/or
Ca2+ mobilization from intracellular stores. Two types of
blocking experiments were performed. One involved adding a small excess of EGTA (2 mM), a chelator of extracellular calcium, to the
medium in the cuvette and incubating for 30 s before adding CGRP.
The second involved blocking of L-type Ca2+ channels with 1 µM nifedipine and verapamil. We investigated how much of
the transient increase in [Ca2+]i was due to
Ca2+ release from the endoplasmic reticulum using neomycin,
which inhibits PLC by binding to phosphoinositides (42), and U-73122, which directly inhibits the PLC involved in the hydrolysis of phosphatidylinositol 4,5-bisphosphate (43). We then determined whether
different PLC-
isoenzymes are involved in the effects of CGRP by
incubating OHS-4 cells for 5 min with 20 µg/ml saponin plus excess
anti-PLC antibody, anti-G-protein antibody, or nonimmune rabbit serum
(44). Cells were washed twice to remove saponin and incubated with the
anti-PLC antibody, anti-G-protein antibody, or nonimmune rabbit serum
for 1 h at 37 °C. 1 µM Fura-2/AM was added for
the last 20 min of incubation. In some experiments, anti-PLC-
1
antibody and anti-G
q/11 antibody were set up in
competition with the antigen against which they were produced, or with
the antigens corresponding to the other anti-PLC antibodies for 2 h at room temperature (antibody:peptide 1:10 or 1:100, according to the
specifications of the manufacturer), prior to use. CGRP was used at the
concentration that gave the greatest increase in
[Ca2+]i in confluent OHS-4 cells. The G-protein
involved in the activity of CGRP was identified by incubating the cells
with 100 ng/ml PTX for 16 h. Fura-2/AM loading and
[Ca2+]i measurements were carried out with the
toxin.
Cell Homogenates and Membranes--
Cells were washed three
times with ice-cold phosphate-buffered saline, pH 7.4, and scraped off
into ice-cold extraction buffer (20 mM Tris-HCl, pH 7.5, 0.5 mM EGTA, 2 mM EDTA, 0.6 mM
pepstatin, 0.5 mM benzamidine, 0.1 mM
leupeptin, 2 mM phenylmethylsulfonyl fluoride, 0.125 mM aprotinin, and 1 mM dithiothreitol). They
were sonicated on ice twice for 20 s each at 40 KHz, and the
homogenate was centrifuged for 10 min at 600 × g to
remove nuclei. The supernatant was centrifuged at 100,000 × g for 1 h. The resulting membrane pellets were
resuspended in homogenizing buffer, and aliquots of the total
homogenate and membrane fractions were stored at
80 °C. Protein
was determined by the method of Bradford (45) with bovine serum albumin
as a standard.
Protein Separation and Immunoblotting--
Proteins were
separated by SDS-PAGE (7.5% resolving gel for PLC and 13% for
G-protein) in 25 mM Tris base, pH 8.3, 192 mM glycine, 0.1% SDS (46). They were electrophoretically transferred to
nitrocellulose membranes (Immobilon P, Millipore, St.
Quentin-en-Yvelines, France) in the same buffer with 20% ethanol for
2 h at 100 V (47). Nonspecific binding to nitrocellulose was
prevented by incubating the membranes in 50 mM
Tris-buffered saline (TBS) pH 7.5 containing 150 mM NaCl,
5% skimmed milk powder and 0.05% Tween-20 for 12 h at 4 °C.
The membranes were washed in TBS containing 0.1% Tween-20 and
incubated overnight at 4 °C with polyclonal rabbit antibodies against specific isoenzymes of PLC (PLC-
1, PLC-
2, PLC-
3, and PLC-
4) and specific G-proteins (G
q/11,
G
s). The concentrations of PLC antibodies in TBS, 1.5%
skimmed milk, 0.1% Tween 20 were as follows: 0.1 µg/ml for PLC-
1,
0.5 µg/ml for PLC-
2, 1 µg/ml for PLC-
3, and 0.5 µg/ml for
PLC-
4. The concentrations of G
q/11 and
G
s antibodies in the same buffer were 0.2 µg/ml and
0.5 µg/ml, respectively. The antibodies bound to the proteins on the
nitrocellulose were detected using peroxidase-conjugated goat
anti-rabbit IgG (1 mg/ml) (diluted 1/5000 in TBS, 1.5% skimmed milk,
0.1% Tween 20). The antigen was detected by enhanced
chemiluminescence. Molecular size standards were used to estimate the
apparent molecular mass of the PLC, myosin, 199 kDa;
-galactosidase,
120 kDa; bovine serum albumin, 87 kDa; and ovalbumin, 48 kDa. The
molecular size standards for G-proteins were phosphorylase B, 105 kDa;
bovine serum albumin, 82 kDa; ovalbumin, 49 kDa; carbonic anhydrase, 33.3 kDa; soybean trypsin inhibitor, 28.6 kDa; and lysozyme, 19.4 kDa.
Phosphatidylinositol 4,5-Bisphosphate Hydrolysis
Assay--
Phospholipid vesicles were prepared as described by Hofmann
and Majerus (48), and assays were done essentially as described by Wu
et al. (49). Diluted membranes (10 µl, 5-10 µg of
protein) in 50 mM HEPES, pH 7.0, 0.5 mM EGTA, 2 mM EDTA, 0.6 mM pepstatin, 0.5 mM
benzamidine, 0.1 mM leupeptin, 2 mM
phenylmethylsulfonyl fluoride, 0.125 mM aprotinin, and 1 mM dithiothreitol, were added to 40 µl of assay buffer
(50 mM HEPES, pH 7.0, 100 mM NaCl, 5 mM MgCl2, 0.6 mM CaCl2,
and 2 mM EGTA) plus 10 µl of PIP2
(10,000-12,000 cpm of [3H]PIP2), and
incubated on ice for 10 min. For the antibody inhibition assay,
membranes were incubated for 2 h with 10 µl of antibody, at the
concentrations indicated, prior to adding the reaction mixture. For
competition assay with the peptide control, 10 µl of the antibody to
be tested were mixed with 2 µl of serially diluted peptide control
before adding to the membranes and incubation for 2 h. The
reaction was started by adding GTP
S with or without CGRP, followed
by incubation at 37 °C for 15 min. The reaction was stopped by
adding 0.5 ml of chloroform/methanol/HCl (40:20:0.5), mixing, and
chilling on ice. Soluble inositol phosphates (indicating PIP2 hydrolysis) were extracted by adding 150 µl of
chloroform and 200 µl of 0.1 M HCl. Phases were separated
by centrifugation, and 200 µl of the upper aqueous phase were taken
for liquid scintillation counting.
Statistical Analysis--
The data were analyzed by one-way
analysis of variance. Treatment pairs were compared by Dunnett's
method. A value of n indicates the number of glass
coverslips used for a specific experiment or the number of
cultures.
 |
RESULTS |
Direct Effects of CGRP on Intracellular Calcium
Concentration--
The basal intracellular calcium concentration in
confluent OHS-4 cells was 115 ± 5 nM (mean ± S.E., n = 18). The transient increase in
[Ca2+]i induced by 1 nM CGRP formed a
sharp peak which fell rapidly after 15 s, but remained above the
basal level (plateau phase) (25 ± 2%, mean ± S.E.,
n = 6, p < 0.001) (Fig.
1A). The concentration-dependent effects of CGRP were bell-shaped,
with a maximum activity at 1 nM (Fig. 1B).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 1.
Effect of CGRP on the intracellular calcium
concentration in confluent OHS-4 cells. A, direct
effect of 1 nM CGRP on intracellular calcium. These results
are representative of eight different coverslips. B,
dose-dependent effects of CGRP on intracellular calcium.
Intracellular Ca2+ concentrations were determined at
10 s. Values are means ± S.E., n = 8, and
are significantly different from the basal level. ¥p < 0.01 and *p < 0.001.
|
|
Mechanisms of CGRP-induced Changes in Intracellular Calcium
Concentration--
1 nM CGRP was added 30 s after 2 mM EGTA, or 60 s after 1 µM nifedipine
or 1 µM verapamil. Verapamil itself caused a small (5%)
decrease in [Ca2+]i, whereas nifedipine caused a
small (5%) transient increase (43). EGTA, nifedipine, and verapamil
reduced the magnitude (50 ± 10%, mean ± S.E.,
n = 8, p < 0.001) of the transient
peak induced by CGRP, and the plateau phase was completely abolished (Fig. 2A; data shown only for
EGTA and nifedipine).

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 2.
Mechanisms of CGRP actions on intracellular
calcium in confluent OHS-4 cells. A, cells were
incubated for 30 s with 2 mM EGTA or for 60 s
with 1 µM nifedipine before adding 1 nM CGRP.
EGTA and nifedipine both reduced the transient peak and abolished the
plateau phase. B, cells were incubated for 3 min with 1 mM neomycin or 60 s with 3 µM U-73122
before adding 1 nM CGRP. Neomycin and U-73122 both
abolished the transient peak and had no effect on the plateau phase.
These results are representative of eight different coverslips for each
experiment.
|
|
Cells were incubated for 3 min with 1 mM neomycin, with 3 µM U-73122 or with 2 µM U-73343, a closed
analog of U-73122 but inactive agent (43), before adding 1 nM CGRP. Neomycin itself caused a small (5%) decrease in
[Ca2+]i (44), whereas U-73122 caused a small
(5%) increase (43). Neomycin and U-73122 both abolished the transient
peak, but not the plateau phase (Fig. 2B). U-73343 (0.3-5
µM) had no effect on the intracellular calcium response
to CGRP (data not shown).
The cells incubated for 16 h with 100 ng/ml PTX showed no change
in the basal level of [Ca2+]i or in the
[Ca2+]i response to 1 nM CGRP (Fig.
3).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of Pertussis toxin on the
intracellular calcium response to CGRP in confluent OHS-4 cells.
Cells were incubated for 16 h with 100 ng/ml PTX. Fura2/AM loading
and calcium measurement were done in the presence of PTX. PTX had no
effect on the CGRP-induced increase in intracellular calcium. These
results are representative of at least six coverslips.
|
|
Western Immunoblotting of the PLC and G-proteins--
All the
anti-PLC-
antibodies used in this study were raised against amino
acid sequences in the carboxyl terminus of each PLC-
. Western
immunoblotting showed a 150-kDa immunoreactive band using the
anti-PLC-
1 antibody, a 158-kDa immunoreactive band using the
anti-PL-
3 antibody, and a 153-kDa immunoreactive band using the
anti-PLC-
4 antibody (Fig. 4). No
immunoreactive reactive band for PLC-
2 was found (Fig. 4).
Immunoblots probed with the anti-G
q/11 antibody or the
anti-G
s antibody showed a 42-kDa immunoreactive band
(Fig. 5). A competitive Western blot with
polyclonal PLC-
1, PLC-
3, and PLC-
4 antibodies and the antigens
against which they were raised showed that immunoreactivity was
completely abolished when a 100-fold excess of antigen was included
(data not shown). This also shows that each antibody reacted with the
intended target isoform.

View larger version (53K):
[in this window]
[in a new window]
|
Fig. 4.
PLC isoforms in confluent OHS-4 cells.
Cell homogenates were prepared as described, and 35-µg aliquots were
separated by electrophoresis on SDS-polyacrylamide gel, transferred to
nitrocellulose membranes, and probed with antibodies against the
various PLC- isoforms (arrows). The concentrations of
anti-PLC antibodies were 0.1 µg/ml for PLC- 1, 0.5 µg/ml for
PLC- 2, 1 µg/ml for PLC- 3, and 0.5 µg/ml for PLC- 4.
Lines indicate the molecular masses of the standards
(Std) from top to bottom: myosin, 199 kDa;
-galactosidase, 120 kDa; bovine serum albumin, 87 kDa; and
ovalbumin, 48 kDa.
|
|

View larger version (59K):
[in this window]
[in a new window]
|
Fig. 5.
G-proteins in confluent OHS-4 cells.
Aliquots (20, 30, and 50 µg) of each homogenate were separated by
electrophoresis on SDS-polyacrylamide gel, transferred to
nitrocellulose membranes, and probed with antibodies against various
subunits of G-proteins (arrows). The concentrations of
anti-G q/11 and anti-G s antibodies were
0.2 µg/ml and 0.5 µg/ml, respectively. Lines indicate
the molecular masses of the standards from top to bottom: phosphorylase
B, 105 kDa; bovine serum albumin, 82 kDa; ovalbumin, 49 kDa; carbonic
anhydrase, 33.3 kDa; and soybean trypsin inhibitor, 28.6 kDa.
|
|
PLC Isoenzymes and G-proteins Involved in the Effects of CGRP on
Intracellular Calcium--
Treament of the cells with saponin for 5 min followed by incubation for 60 min with the anti-PLC antibody or
anti-G-protein antibody in the absence of saponin did not affect the
basal [Ca2+]i. Nonimmune serum had no effect on
basal [Ca2+]i or on the
[Ca2+]i response to CGRP.
The CGRP-induced increase in [Ca2+]i was reduced
by anti-PLC-
1 antibody, whereas antibodies to PLC-
3 and PLC-
4
had no effect (Fig. 6). The residual
increase was due to Ca2+ influx because it was totally
blocked by incubating the cells with 2 mM EGTA.
Anti-PLC
1 and anti-PLC
2 antibodies had no effect on the
[Ca2+]i response to CGRP (Fig. 6), as did
anti-PLC-
2 antibody (data not shown).

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 6.
Responses of OHS-4 cells treated with
antibodies against PLC isoforms to CGRP. Cells were cultured,
loaded with Fura2/AM, and incubated with PLC antibodies as described.
The concentrations of anti-PLC antibodies were 1 µg/ml for PLC- 1,
10 µg/ml for PLC- 3, 5 µg/ml for PLC- 4, 10 µg/ml for
PLC- 1, and 10 µg/ml for PLC- 2. Only anti-PLC- 1 antibody
blocked Ca2+ mobilization induced by CGRP. In some
experiments, CGRP was added 30 s after 2 mM EGTA.
These results are representative of at least six coverslips for each
experimental case.
|
|
Polyclonal anti-PLC-
1 antibody was incubated for 2 h with its
corresponding antigen or with the antigens used to produce the other
anti-PLC antibodies (antibody:antigen 1:10 or 1:100) before use. The
inhibition of the CGRP-induced increase in
[Ca2+]i due to anti-PLC-
1 antibody totally
disappeared only when the anti-PLC-
1 antibody was incubated with its
own antigen, but not with the antigens used to raise the other anti-PLC
antibodies (Fig. 6; data shown only for the antigen corresponding to
PLC-
1).
The CGRP-induced transient peak in [Ca2+]i was
blocked by anti-G
q/11 antibody (Fig.
7). The residual increase was due to a
Ca2+ influx because this was totally blocked by incubating
the cells with 2 mM EGTA (Fig. 7). The
anti-G
i antibody (1 µg/ml), which cross-reacts with
G
i1, G
i2, and G
i3, and
anti-G
s antibodies did not alter the
[Ca2+]i response to CGRP (Fig. 7). Adding the
anti-G
antibody, which reacts with G
1, G
2, G
3, and G
4,
and the anti-G
antibody to the cell had no effect on the
[Ca2+]i response to CGRP (Fig. 7).

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 7.
Responses of OHS-4 cells treated with
antibodies against G-proteins to CGRP. Cells were cultured, loaded
with Fura2/AM, and incubated with G-protein antibodies as described.
The concentrations of anti-G-protein antibodies were 2 µg/ml for
G q/11, 5 µg/ml for G s, 5 µg/ml for
G i, and 5 µg/ml for G plus 5 µg/ml for G . Only
anti-G q/11 antibody blocked Ca2+
mobilization induced by CGRP. These results are representative of at
least six coverslips for each experimental case.
|
|
PIP2 Hydrolysis--
Since testing the effect of
GTP
S on intracellular calcium needed permeabilized cells, and this
made it impossible to keep the antibodies inside the cell, we used a
cell-free membrane system to test the activity of the antibodies. OHS-4
membranes containing endogenous PLC and G subunits were mixed with
phospholipid vesicles containing radioactive substrate
([3H]PIP2). Fig.
8A shows the effect of CGRP on
the hydrolysis of PIP2 in the absence of GTP
S. The
effect of GTP
S in the absence and presence of 1 nM CGRP
showed that GTP
S was ineffective below 1 µM (Fig.
8B). GDP
S (100 µM) inhibited the increase
induced by 10 µM GTP
S, 10 µM GTP
S
plus 1 nM CGRP, or 1 nM CGRP alone (data not
shown). OHS-4 membranes were incubated with antibodies prior to
stimulation with 100 µM GTP
S. All anti-PLC antibodies (
1,
3, and
4), like anti-G
q/11 and anti-G
antibodies, inhibited the PIP2 hydrolysis induced by
GTP
S (Fig. 8C), whereas anti-G
s and
anti-G
i antibodies had no effect (data not shown).
Membranes incubated with both the antibody and the blocking peptide,
gave the same amount of [3H]inositol 1,4,5-trisphosphate
as obtained with preimmune serum (Fig. 8C).

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 8.
Effects of anti-PLC and anti-G-protein
antibodies on PIP2 hydrolysis induced by CGRP
and/or GTP S in OHS-4 membranes. A, direct effects of
CGRP in the absence of GTP S. B, effects of GTP S on the
hydrolysis of PIP2 in the absence or presence of 1 nM CGRP. C, 5 µg of OHS-4 membranes were
incubated for 2 h with 1 µg/ml anti-PLC- 1 antibody or with
anti-PLC- 1 plus 10 µg/ml peptide control. D, 5 µg of
OHS-4 membranes were incubated for 2 h with 10 µg/ml
anti-PLC- 3 antibody, 5 µg/ml anti-PLC 4 antibody, 5 µg/ml
anti-G antibody plus 5 µg/ml anti-G antibody or preimmune serum
(PI) or with the antibody plus peptide control used at 10 times the concentration used for the antibody. PIP2
hydrolysis was performed in the presence or absence of 100 µM GTP S. The PIP2 hydrolysis in control
incubations with GTP S and without membranes was subtracted from all
values. Hatched columns represent data obtained
from incubating the antibody with its antigen prior to adding the
membranes. Values are means ± S.E., n = 4, and
are significantly different: A, from the basal level in the
presence of CGRP, *p < 0.001; B, from the
basal level in the presence of GTP S or GTP S plus CGRP,
*p < 0.001; C, from the level of CGRP,
*p < 0.001 or from the level of anti-PLC- 1 plus
control peptide in the presence of CGRP, #p < 0.001;
D, from the level of preimmune serum, *p < 0.001, or from the level in the presence of antibody,
#p < 0.001.
|
|
 |
DISCUSSION |
We have shown that G
q/11-protein coupled to
phospholipase C-
1 is involved in the signaling of CGRP in human bone
cells, and that G
subunits have no effect. The data also indicate
that Ca2+ mobilization from the endoplamic reticulum
following the increased formation of inositol 1,4,5-trisphosphate
induced by CGRP is due to activation of a G
q/11 that is
insensitive to pertussis toxin.
CGRP increases intracellular calcium concentration within 5 s via
two mechanisms. It causes an influx of Ca2+ from the
extracellular milieu via voltage-gated calcium channels as indicated by
the experiments with EGTA, nifedipine, and verapamil. This mechanism is
different from that of rat osteoblastic UMR106 cells, in which
depleting extracellular Ca2+ induces an efflux of
intracellular Ca2+ involving KATP channels
(18). Activating the membrane KATP channels with high
concentrations of CGRP (100 nM) could inhibit transmembrane
Ca2+ uptake by hyperpolarizing the membrane potential, so
reducing the voltage-dependent Ca2+ channel
activity (53). CGRP causes the release of Ca2+ from the
endoplamic reticulum in human OHS-4 cells as it does in rat UMR106
cells (18). This seems to be a general mechanism of CGRP action.
The activation of phosphoinositide-specific phospholipase C-
upon
stimulation of specific cell receptors results in the formation of
second messengers, diacylglycrol, a direct activator of protein kinase
C, and inositol 1,4,5-trisphosphate (50, 51). The latter binds to
receptors on the endoplasmic reticulum, causing a transient release of
calcium from the endoplasmic reticulum. As activation of PLC-
is an
early event in the signal transduction pathway resulting in a variety
of cellular responses, we first identified the PLC-
isoforms in bone
OHS-4 cells. We find several isoforms of PLC-
,
1,
3, and
4
but no PLC-
2, which is present in the rat brain (52) and in rat
osteoblasts (44). As confluent rat osteoblasts do not possess PLC-
4
(44), whereas this isoform is present in the brain (52), these
isoenzymes may well have a tissue- and/or species-specific
distribution.
The next step was to identify the PLC-
isoform involved in the
effect of CGRP. This is the first report that PLC-
1 is involved in
mobilization of Ca2+ from the endoplasmic reticulum by
CGRP. Anti-PLC-
1 antibody inhibits the CGRP-induced increase in
[Ca2+]i in much the same way as direct (U-73122)
or indirect (neomycin) inhibitors of PLC. Anti-PLC antibody, like PLC
inhibitors, blocks only the part of the increase in
[Ca2+]i that is due to Ca2+
mobilization from the endoplasmic reticulum. The inhibition of the
enzyme activity by anti-PLC-
1 antibody is totally abolished in
competition experiments, in which polyclonal PLC-
1 antibody is
incubated with the antigen against which it was raised, but not when
using the antigen corresponding to other PLC-
. This type of enzyme
inhibition by selective antibodies against phosphoinositide-specific PLC has also been demonstrated in fresh bovine erythrocytes (54) and
rat osteoblasts (44), suggesting that the antibody directed against an
amino acid sequence at the carboxyl terminus of the PLC-
binds to a
part of the enzyme which is critical for the geometry of the active
site. Anti-PLC-
1 antibody also inhibits the formation of inositol
1,4,5-trisphoshate in a OHS-4 cell-free membrane system. Neither
PLC-
3 and PLC-
4 are involved in CGRP signal transduction,
although both PLC stimulate PIP2 hydrolysis in the presence
of GTP
S. Similarly, PLC-
1 and -
2 take no part in the effects
of CGRP as expected, because PLC-
are substrates for growth factor
receptor protein-tyrosine kinases (55).
It is very likely that only PLC-
is involved in the action of CGRP,
as only PLC-
types are regulated via heterotrimeric G-proteins in
response to agonist binding to receptors (55, 56). Receptor activation
of PLC via G-proteins occurs by pertussis toxin-sensitive and
toxin-insensitive signaling pathways (26). The
subunits of the
Gq/11 family are presumed to mediate the toxin-insensitive
pathway, but the nature of the G-proteins mediating the toxin-sensitive
pathway is less well understood (55-58). This study shows that the
PLC-
1 involved in the action of CGRP is linked to a pertussis
toxin-insensitive G-protein, and this PTX-insensitive G-protein is a
member of the Gq family. These results are consistent with
the fact that the G-proteins Gq/11 are the most prominent G-protein activators in receptor-mediated regulation of the PLC-
1 (29). Moreover, forskolin does not mimic the effects of CGRP on
[Ca2+]i,2
but increases cAMP formation in OHS-4 cells (19).
Anti-G
i and anti-G
s antibodies do not
block the [Ca2+]i response to CGRP or the
stimulation of PIP2 hydrolysis induced by GTP
S. G
subunits,
like PLC-
3, which is the preferred target effector for G
subunits (36), are not involved in Ca2+ mobilization
induced by CGRP. These data indicate that the effects of CGRP on
intracellular calcium in OHS-4 cells are not mediated via the G-protein
linked to the cAMP pathway.
Activation of PLC via G-protein-coupled receptors requires higher
ligand concentrations than for receptor-mediated adenylyl cyclase
activation (22). But, we find that PLC is activated in OHS-4 cells at
concentrations similar to those that increase cAMP in other
systems.
Although the main second messenger produced in response to CGRP remains
cAMP in various cell types, probably via G
s-protein, CGRP acts via the phospholipase C pathway and G
q/11
protein in human bone cells. The present data therefore strongly
suggest that the dual signaling of CGRP is due to the activation of
more than one G-protein, G
s for the cAMP pathway and
G
q/11 for the PLC pathway (Fig.
9).

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 9.
Signals generated by CGRP. A,
diagram showing the proposed coupling of CGRP receptors to
phospholipase C and adenylyl cyclase via pathway 1 (two
different G-proteins) or via pathway 2 (one G-protein acting
through its and  subunits). B, signaling pathway
used by CGRP in human OHS-4 bone cells. CGRP uses pathway 1 since it
activates phospholipase C- 1 via a pertussis-insensitive
G q/11 protein, the  subunits being
ineffective.
|
|
 |
ACKNOWLEDGEMENTS |
We thank Drs. B. Fournier and P. A. Price (Department of Biology, University of California, La
Jolla, CA) for providing the human osteoblastic OHS-4 cell line. The
English text was checked by Dr. Owen Parkes.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: CNRS UPR 1524, Bâtiment 221, Institut National de la Recherche Agronomique, 78 350 Jouy-en-Josas, France. Tel.: 33 1 34 65 20 64; Fax: 33 1 34 65 21 09; E-mail: lieberhe{at}diamant.jouy.inra.fr.
The abbreviations used are:
CGRP, calcitonin
gene-related peptide; PLC, phospholipase C; TBS, Tris-buffered saline; PTX, pertussis toxin; G-protein, guanine nucleotide-binding regulatory
protein; GTP
S, guanosine 5'-O-3'-thiotriphosphateGDP
S, guanosine 5'-O-2'-thiodiphosphatePIP2, phosphatidylinositol 4,5,-bisphosphate.
2
H. Drissi, F. Lasmoles, V. Le Mellay, P. J. Marie, and M. Lieberherr, unpublished data.
 |
REFERENCES |
-
Amara, S. G.,
Jones, V.,
Rosenfeld, M. G.,
Ong, E. S.,
and Evans, R. M.
(1982)
Nature
29,
240-249
-
Rosenfeld, M. G.,
Mermod, J. J.,
Amara, S. G.,
Swanson, L. W.,
Sawachenko, D. E.,
Rivier, J.,
and Vale, W. E.
(1983)
Nature
304,
120-135[Medline]
[Order article via Infotrieve]
-
Deftos, L. J.,
and Ross, B. A.
(1989)
in
Bone and Mineral Research, Annual 6 (Peck, W. A., ed), pp. 267-316, Elsevier, New York
-
Aiyar, N.,
Rand, K.,
Elshourbagy, N. A.,
Zeng, Z.,
Adamou, J. E.,
Bergsma, D. J.,
and Li, Y.
(1996)
J. Biol. Chem.
271,
11325-11329[Abstract/Free Full Text]
-
Han, Z.-Q.,
Coppock, H. A.,
Smith, D. M.,
Van Noorden, S.,
Makgoba, M. W.,
Nicholl, C. G.,
and Legon, S.
(1997)
J. Mol. Endocrinol.
18,
267-272[Abstract]
-
Kapas, S.,
and Clark, A. J.
(1995)
Biochem. Biophys. Res. Commun.
217,
832-838[CrossRef][Medline]
[Order article via Infotrieve]
-
Lefkowitz, R. J.,
Cotechia, S.,
Samana, P.,
and Costa, T.
(1993)
Trends Pharmacol. Sci.
14,
303-304[CrossRef][Medline]
[Order article via Infotrieve]
-
Oliviera, L.,
Paiva, A. C. M.,
Sander, C.,
and Vriend, G.
(1994)
Trends Pharmacol. Sci.
15,
170-172[CrossRef][Medline]
[Order article via Infotrieve]
-
Poyner, D. E.
(1992)
Pharmacol. Ther.
56,
32-51
-
Farley, J. R.,
Hall, S. L.,
and Herring, S.
(1993)
Metabolism
42,
97-104[Medline]
[Order article via Infotrieve]
-
Bjurholm, A.,
Kreicbergs, A.,
Schultzberg, M.,
and Lerner, U. H.
(1992)
J. Bone Miner. Res.
7,
1011-1019[Medline]
[Order article via Infotrieve]
-
Thiebaud, D.,
Akatsu, T.,
Yamashita, T.,
Suda, T.,
Noda, T.,
Martin, R. E.,
and Martin, T. J.
(1991)
J. Bone Miner. Res.
6,
1137-1142[Medline]
[Order article via Infotrieve]
-
Michelangeli, V. P.,
Findlay, D. M.,
Fletcher, A.,
and Martin, T. J.
(1986)
Calcif. Tissue Int.
39,
44-48[Medline]
[Order article via Infotrieve]
-
Vignery, A.,
and MacCarthy, T. L.
(1996)
Bone
18,
331-335[CrossRef][Medline]
[Order article via Infotrieve]
-
Gray, D. W.,
and Marshall, I.
(1991)
Br. J. Pharmacol.
107,
691-696[Abstract]
-
Parsons, A. M.,
and Seybold, V. S.
(1997)
Synapse
26,
235-242[CrossRef][Medline]
[Order article via Infotrieve]
-
Laufer, R.,
and Changeux, J.-P.
(1989)
J. Biol. Chem.
264,
2683-2689[Abstract/Free Full Text]
-
Kawase, T.,
Howard, G. A.,
Roos, B. A.,
and Burns, D. M.
(1995)
Bone
16,
379S-384S[CrossRef][Medline]
[Order article via Infotrieve]
-
Drissi, H., Lieberherr, M., Marie, P. J., and Lasmoles, F. (1997)
J. Bone Miner. Res. 12, Suppl. 1, F391
-
Goltzman, D.,
and Mitchell, J.
(1985)
Science
227,
1343-1345[Medline]
[Order article via Infotrieve]
-
Stangl, D,
Muff, R.,
Schmolck, C.,
and Fischer, J. A.
(1993)
Endocrinology
132,
744-750[Abstract]
-
Offermanns, S.,
lida-Klein, A.,
Segre, G. V.,
and Simon, M. I.
(1996)
Mol. Endocrinol.
10,
566-574[Abstract]
-
Freissmuth, M.,
Casey, P. J.,
and Gilman, A. G.
(1989)
FASEB J.
3,
2125-2131[Abstract/Free Full Text]
-
Neer, E. J.
(1995)
Cell
80,
249-257[Medline]
[Order article via Infotrieve]
-
Exton, J. H.
(1996)
Annu. Rev. Pharmacol. Toxicol.
36,
481-509[CrossRef][Medline]
[Order article via Infotrieve]
-
Ui, M.
(1990)
in
ADP-Ribosylating Toxins and G-Proteins (Moss, J., and Vaugham, M., eds), pp. 45-77, American Society for Microbiology, Washington, D. C.
-
Martin, T. F. J.
(1991)
Pharmacol. Ther.
49,
329-345[Medline]
[Order article via Infotrieve]
-
Smrcka, A. V.,
Hepler, J. R.,
Brown, K. O.,
and Sternweis, P. C.
(1991)
Science
251,
804-807[Medline]
[Order article via Infotrieve]
-
Noh, D.-Y.,
Shin, S. H.,
and Ree, S. G.
(1995)
Biochim. Biophys. Acta
1242,
99-114[CrossRef][Medline]
[Order article via Infotrieve]
-
Lee, C. W.,
Lee, K. H.,
Lee, S. B.,
Park, D.,
and Rhee, S. G.
(1994)
J. Biol. Chem.
269,
25335-25338[Abstract/Free Full Text]
-
Kozasa, T.,
Hepler, J. R.,
Smrcka, A. V.,
Simon, M. I.,
Rhee, S. G.,
Sternweis, P. C.,
and Gilman, A. G.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
9176-9180[Abstract]
-
Rhee, S. G.,
and Choi, K. D.
(1992)
J. Biol. Chem.
267,
12393-12396[Free Full Text]
-
Conklin, B. R.,
and Bourne, H. R.
(1993)
Cell
73,
631-641[Medline]
[Order article via Infotrieve]
-
Taylor, S. J.,
Chae, H. Z.,
Rhee, S. G.,
and Exton, J. H.
(1991)
Nature
350,
516-517[CrossRef][Medline]
[Order article via Infotrieve]
-
Camps, M.,
Carozzi, A.,
Schabel, P.,
Scherr, A.,
Parker, P. J.,
and Gierschick, P.
(1992)
Nature
360,
684-686[CrossRef][Medline]
[Order article via Infotrieve]
-
Carozzi, A.,
Camps, M.,
Gierschick, P.,
and Parker, P. J.
(1993)
FEBS Lett.
315,
340-342[CrossRef][Medline]
[Order article via Infotrieve]
-
Birnbaumer, L.,
and Birbaumer, M.
(1995)
J. Recept. Signal Transduct. Res.
15,
213-252[Medline]
[Order article via Infotrieve]
-
Allgeier, A.,
Offermanns,
Van Sande, J.,
Spicher, K.,
Schultz, G.,
and Dumont, J. E.
(1994)
J. Biol. Chem.
269,
13733-13735[Abstract/Free Full Text]
-
James, S. R.,
Varizi, C.,
Walker, T. R.,
Milligan, G.,
and Downes, C. P.
(1994)
Biochem. J.
304,
359-364[Medline]
[Order article via Infotrieve]
-
Fournier, B.,
and Price, P. A.
(1991)
J. Cell Biol.
114,
577-583[Abstract]
-
Grynkiewicz, G.,
Poenie, M.,
and Tsien, R. Y.
(1985)
J. Biol. Chem.
260,
3440-3450[Abstract]
-
Prentki, M.,
Deeney, J. T.,
Matschinsky, F. M.,
and Joseph, S. K.
(1986)
FEBS Lett.
197,
285-288[CrossRef][Medline]
[Order article via Infotrieve]
-
Bleasdale, J. E.,
Bundy, G. L.,
Bunting, S.,
and Fitzpatrick, F. A.
(1989)
Adv. Prostanglandin Thromboxane Leukotriene Res.
19,
590-593
-
Le Mellay, V.,
Grosse, B.,
and Lieberherr, M.
(1997)
J. Biol. Chem.
272,
11902-11907[Abstract/Free Full Text]
-
Bradford, M.
(1976)
Anal. Biochem.
72,
248-254[CrossRef][Medline]
[Order article via Infotrieve]
-
Laemmli, U. K.
(1970)
Nature
227,
680-685[Medline]
[Order article via Infotrieve]
-
Towbin, H.,
Staehelin, T.,
and Gordon, J.
(1979)
Proc. Natl. Acad. Sci. U. S. A.
76,
4350-4354[Abstract]
-
Hofmann, S. L.,
and Majerus, P. W.
(1982)
J. Biol. Chem.
257,
14359-13564[Free Full Text]
-
Wu, D.,
Lee, C. H.,
Rhee, S. G.,
and Simon, M. I.
(1992)
J. Biol. Chem.
267,
1811-1817[Abstract/Free Full Text]
-
Berridge, M. J.,
and Irvine, R. F.
(1989)
Nature
341,
197-205[CrossRef][Medline]
[Order article via Infotrieve]
-
Joseph, S. K.,
and Williamson, J. R.
(1989)
Arch. Biochem. Biophys.
273,
1-15[Medline]
[Order article via Infotrieve]
-
Tanaka, O.,
and Kondo, H.
(1994)
Neurosci. Lett.
82,
17-20
-
Nelson, M. T.,
Huang, Y.,
Brayden, J. E.,
Hescheler, J.,
and Standen, N. B.
(1990)
Nature
344,
770-773[CrossRef][Medline]
[Order article via Infotrieve]
-
Kuppe, A.,
Hedberg, K. K.,
Volwerk, J. J.,
and Griffith, O. H.
(1990)
Biochim. Biophys. Acta
1047,
41-48[Medline]
[Order article via Infotrieve]
-
Singer, W. D.,
Brown, H. A.,
and Sternweis, P. C.
(1997)
Annu. Rev. Biochem.
66,
475-509[CrossRef][Medline]
[Order article via Infotrieve]
-
Strathmann, M. P.,
and Simon, M. I.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
9113-9117[Abstract]
-
Simon, M. I.,
Strathmann, M. P.,
and Gautum, N.
(1991)
Science
252,
802-808[Medline]
[Order article via Infotrieve]
-
Gudermann, T.,
Kalkbrenner, F.,
and Schultz, G.
(1996)
Annu. Rev. Pharmacol. Toxicol.
36,
429-459[CrossRef][Medline]
[Order article via Infotrieve]
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.