BIOCHEMICAL
Vol. 171, No. 3, 1990 September
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1229-1235
Pages
28, 1990
INVOLVEMENT OF PERTUSSIS TOXIN-SENSITIVE GTP-BINDING PROTEIN IN PROSTAGLANDIN FZcl - INDUCED PHOSPHOINOSITIDE HYDROLYSIS IN OSTEOBLAST-LIKE CELLS Masaichi
Miwa, Haruhiko Tokuda, Kazuyo Tsushita, Jun Ko oyori, Yoshiyuki Takahashi, Nobuaki Ozaki, Osamu Kozawa i and Yutaka Oiso
First
Department School
of Internal of Medicine,
Medicine, Nagoya University Nagoya 466, Japan
Received August 10, 1990 s-y: Prostaglandin F2a ) stimulated the format ion (=i!a of inositol phosphates in a dose-dependent manner in cloned osteoblast-like MC3T3-El cells. This react ion was markedly inhibited dose-dependently by pertussis toxin. In ceI1 the membranes, pertussis toxin-catalyzed ADP-ribosylation of a 4O-kDa PGF protein was significantly attenuated by pretreatment of 2”lng* These results suggest that pertussis toxin-sensitive GTP-bin protein is involved in the coupling of PGF2cl receptor to phospholipase C in these cells. 01990 Academic Press, Inc. Prostaglandins osteoblasts
as
reported
to
decrease
alkaline
stimulation
phosphates that
autacoids
stimulate
G proteins
resulting
are considered
of in (IPl,
G proteins
the
the DNA synthesis
been
PLC which formation
also
for of
regulators
PGF2a,
it
osteoblasts
activity
in the
recognized
to
hydrolyzes
phosphoinositides
of
cells be
diacylglycerol
IP2 and 1P3). are
As
(1,2).
phosphatase have
to be important
Recently,
involved
in
it the
been
(1,3)
but
in
the (5),
the
has been
inhibition
has
(3,4).
involved
and
of
of
inosi to1 suggested PLC (6)
1
To whom correspondence should be addressed. F2a ; G protein, Abbreviations used. PGF2, ’ prostagland:; GTP-binding protein; PLC, phospholipase C; inositol monophosphate; IP2, inositol bisphosphate; ,&J;y’ y;;;, phate; Gl, G protein that mediates cyclase; Go,. G protein of unknown function found in brain; cr-mlnimum essential medium; FCS , fetal calf serum; a-MEM, 4-(2-hydroxyethyI)-I-piperazineethanesulfonic acid; Hepes, BSA, EGTA, ethylenebis(oxyethylenenitrilo)tetrabovine serum albumin; polyacrylamide acetic acid; SDS, sodium dodecyl sulfate; PAGE, gel electrophoresis.
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0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
171, No. 3, 1990
and the
stimulation
arachidonic The
acid
(2).
It
is
of
It
been
hydrolysis
in
the
factor)
which
PGF2o
was
increase
of
the
precise
mechanism
hydrolysis the
sensitive osteoblast-like
is not
evidence
G protein
is
of
the
cells
in
several
that
involvement
that
this
In
of PGF2o
toxin by
or epidermal
this
pertussis receptor
pertussis
signaling.
PGF2o -induced
of
of
stimulated
suggesting in
types
pertussis
(insulin
elucidated.
in the coupling
MC3T3-El
as
involved
fully
some
phosphoinositide
DNA synthesis
the
in
toxin-insensitive
induces
factor
of
and
(13,14).
shown
(17),
ADPGi
hydrolysis
as well
it
catalyzes
including
of pertussis
in others
(3)
G protein
inosi t ide show
toxin
in 3T3 fibroblasts
toxin-sensitive
we
cyclooxygenase
of PGF2u and progression
combination
However,
by
that
Recently,
inhibits
prostaglandins
involvement
reported
liberates
phospholipids.
of G proteins
reported
which of
phosphoinositide
osteoblasts
(15,16).
stores
pertussis
o-subunit
RESEARCH COMMUNICATIONS
A2 (7,8)
esterified to
inhibit
is also
has
markedly
the
While,
G protein(s)
growth
converted
can
(11,lZ).
cells
the
has been shown that ion
AND BIOPHYSICAL
phospholipase
from
then
(9,10),
cells
of
acid
r ibosylat GO
BIOCHEMICAL
phosphoreport, toxinto PLC in
(18,19).
EXPERIMENTAL PROCEDURES Materials from Mz-[ZT3;!jqositol (81.5 Ci/mnol) was purchased Amersham. PINAD (800 Ci/nxnol) was from Du Pant/New England Nuclear. PGF2u was from Sigma. Pertussis toxin was from Funakoshi Pharmatheutical Co., Tokyo, Japan. Other materials and chemicals were obtained from conxnercial sources. Cell Culture MC3T3-El cells were generously provided by Dr. M. Kumegawa (Meikai University, Sakado, Japan) and maintained in a-MEM containing 10% FCS at 37°C in a humidified atmosphere of 5% co21 95% air. The cells (5 x 104) were seeded into 35-nxn diameter di shes in 2 ml of a-MEM containing 10% FCS. After 5 days, the medium was exchanged for inositol-free a-ME&l containing 0.3% FCS. The ccl is were used for experiments 48 h thereafter. When indicated, the cells were pretreated with pertussis toxin for the last 24 h. Measurement of the Formation of Inositol Phosphates The cultured ccl 1s were labeled with mx-[2-3H]inositol (3 uCi/dish) for 48 h. The labeled cells were washed twice with 1230
Vol.
171, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
150 IrM lml of an assay buffer (5 nivl Hepes, pH 7.4 containing NaCl , 5 rA4 KCl, 5.5 n&l glucose, 0.8 r&4 MgS04 and 1 ti CaCl ) O.&% lZ preincubated with 1 ml of the assay buffer containing After the preincubation, the and 10 n&l LiCl at 37°C for 10 min. cells were stimulated by PGF The reaction was terminated by supernatant was treated with 15% trichloroacetic acid. Txoe acid ’ diethyl ether to remove the acid and then neutralized with NaOH. The supernatant was applied on a column of Dowex AGl-X8 formate form. The radioactive IP1, IP2 .and IP3 were separated by successive elution of the column with 8 ml each of 0.1 M formic acid containing 0.2 M, 0.4 M and 1.0 M amnonium formate, respectively (20,21). ADP-ribosylation of Membrane Proteins by Pertussis Toxin Crude membranes were prenared at 4°C as described (22) with a minor modification. In’ braief, the cultured cells were scraped from 90-mm diameter dishes with a rubber policeman into 25 n&l Tris/HCI, pH 7.5 containing 5 n&l MgCl 2, 1 n&l EGTA, 1 n&l di thiothrei to1 and 0.1 TIU/ml aprotinin, homogenized by passing through a 22-gauge syringe, and centrifuged at 100 x g for 5 min. The supernatant was subsequently centrifuged at 20,000 x g for 60 min, the pellet was resuspended at a final protein concentrat i on to 20 mg/ml in 50 n&l Hepes, pH 7.4 containing 0.1 n&l EDTA and 1 TIlJ/ml aprotinin. After sonication, the crude membranes were used for assays. Preactivation of pertussis toxin was performed by incubating with 50 nM Tris/HCl, pH 7.5 containing 10 r&l dithiothreitol and 1 n&i ATP at a concentration of 50 pg/ml at 30°C for 15 min. The ccl 1 membranes (100 ug protein) were previously incubated in 50 pl of 100 n&i Hepes, pH 7.5 containing 2.5 n&l MgC12, 100 @vl GTP, 120 nM NaCl , 5 nivl KCl, 15 nM sodium acetate, 1 n-M EDTA, 10 n&i glucose and 0.5% BSA with 10 pM PGF20 or vehicle at 37°C for 60 min, then these samples were subsequently combined with 50 1.11 of th s2reaction mixture (200 mM Tris/HCl, pH 8.0 containing 5 @vl cpm/pmol), 20 n&i thymidine, [ Cl- PIJWD (l,OOO-2,000 1 n&l EDTA, 2 nM dithiothreitol, 2 n&l L- a-dimyristoyl phosphatidylcholine and 20 ug/ml preactivated pertussis toxin) and incubated at 30°C for another 60 min. The reaction was terminated by adding 400 u1 of 25 nQ’Tris/HCl, pH 8.8 containing 192 n&l glycine and 0.1% SDS. The sample was subjected to SDS-PAGE (11% polyacrylamide) as specified by Laemmli (23) and processed by autoradiography using Kodak X-Qnat film. Determinations The radioactivity of ‘H-samples was determined with Beckman LS 5000TD liquid scintillation spectrometer. Protein was determined by the methods of Lowry et al. with BSA as a reference protein (24).
RESULTS PGF2c (10 $4)
significantly
in MC3T3-El
IP2 and IP3 increased
gradually
formation
of
sustained
up to 60 min.
IP3
cells
up reached
to
stimulated (Fig. 60 min, plateau
The formation 1231
the
formation
of
The formation
1).
the
on almost of
other within
IP2 showed
of
IPl, IPl
hand,
the
10 min
and
the
peak
Vol.
171,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
4,) 010
30
60
010
Time (mid
30
60
010
Time (mid
30
60
Time (mid
Time course of the formation of IP1, IP Fig. 1. igduced by PGF2a in MC3T3-El cells: pertussis toxin-ef [ Hlinositol labeled cells were pretreated with 1 ug/ml (O----O) or vehicle (O----O toxin ) for 24 h and then for indicated periods. (A), IPI; (B), y;th 10E~kclhPGF20 value represents the mean of -+ S.D. degerminations.
value
at
inositol
30 min
and
phosphates
a range
50
between
decreased stimulated
1 nh4 and
The
thereafter. by
10 @vl (data
PGF2o
was
not
shown].
and IP3 t ect. The pertussis stimulated IP2;.(C), triplicate
formation
of
dose-dependent
in
bS>31 -
09
8
7
609
8
7
609
8
Pertussis toxin t-logg/ml)
7
6
03
inhibition Fig. 2. Dose-dependent of PGF2a -induced format ion ccl Is. Thzf f@IiiIf!!if:T’ :rgeI,“z ~~~:~“s~~r~ox~:et~~at~3T~~~~ various doses of pertussis toxin for 24 h and then stimulated by 10 @I PGF20 for 60 min. Results are expressed as % of control in the absence of pertussis toxin responses to PGF ,,,;, +‘$ IP1; Each value represents IP2; (Cl, 16%. the (B), - * of triplicate determinations. Fig. 3. Effect of PGF2a on pertussis toxin-catalyzed ADP-ribosylation of a 40-kDa protein in MC3T3-El cell membranes. or vehicle Membranes ;E;e &e;;;atefhtith 10 JJM PGF20 (lane I) (lane 2) pertussis toxin-catalyzed ADPribosylation was perfokmed.
1232
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171, No. 3, 1990
BIOCHEMICAL
Pertussis and
IP2
toxin
markedly
IP3 induced
ef feet
of
1 rig/ml
and 1 ug/ml
pertussis
that
with
this
toxin
40,000
(Fig.
This
the 40-kDa
of
the membranes with
(Fig.
SDS-PAGE
of
formation
1).
of
The
IPl,
inhibitory
in a range
the
and preactivated
ADP-ribosylated
of
as compared
the each
between
2).
after
3).
RESEARCH COMMUNICATIONS
was dose-dependent
(Fig.
[32 PINAD
toxin
reduced
by 10 $vI PGF20
Autoradiography incubated
AND BIOPHYSICAL
substrate
10 JIM PGF2c (Fig.
present
study,
with
3,
lane
toxin
showed of
about
ADP-ribosylation
attenuated for
membranes
aMr
toxin-catalyzed
was markedly
to control
pertussis
a protein
pertussis
plasma
by
60 min
pretreatment
(Fig.
3,
lane
l),
2).
DISCUSSION In
the
hydrolysis induced
in osteoblast-like formation
by pertussis
of
toxin.
that
pertussis
factor
the coupling
sensitive
G protein
evidence
which
transduction Moreover, ribosylation
of
pretreatment coup led G protein
we
of
receptor
and
been reported
showed a 40-kDa
that
pertussis
protein
was
is
( o6y
stimulated
-complex)
subunits
(5).
As pertussis
and Go)
are ADP-ribosylated
is
that
toxin-sensitive by this 1233
toxin
(17). toxin-
a pertussis of
The toxin-
the
signal
and PLC. toxin-catalyzed markedly
agonist,
dissociated
and
as we know.
heterotrimeric its
o-
G proteins only
by
When G protein-
the into
ADP-
attenuated
10 JIM PGF2u . by
of
factor)
pertussis
in the coupling receptor
growth
react ion
as far
suggests
the PGF2o
inhibited
in 3T3 fibroblasts,
of PGF2u
involved
PGF2u -
combination
or epidermal
this
the membranes with
receptor
that
inhibits
is
the
was markedly
reported
(insulin
we showed here
between
and
markedly
has not
G protein
al.
DNA synthesis
toxin
However,
sensitive
et
phosphoinositide
cells,
phosphates
Murayama
synergistically
stimulated
MC3T3-El
inositol
PGF2u and progression increases
PGF2.
when they
and 6~ (e.g.
Gi
are
in
Vol.
171,
No.
3, 1990
the
inactive
form
(25),
the
G protein
these
heterotrimeric our
results
pertussis
finding into
its
these
phoinositide osteoblast-like
AND
BIOPHYSICAL
form, seems active
indicate
not
to
we conclude
hydrolysis MC3T3-El
via
the
pertussis
RESEARCH
COMMUNICATIONS
active
dissociated
from
induced
in
these
that
dissociation
by PGF2u
receptor
PGF2r.X
G protein
results,
in
a result
state
that
toxin-sensitive From
in
BIOCHEMICAL
is
.
of Namely,
coupled
to
cells.
PGF20
toxin-sensitive
stimulates
phosG protein
cells.
ACKN(TwLEDGMENTS The authors are very Research, Developmental indispensable discussion, their skillful secretarial
grateful to Dr. T. Aichi Prefectural and M. Hiramatsu assistance.
Asano (Institute Colony) for and K. Yoshino
for her for
REFERENCES P.J., Burger, E.H. and Feyen, J .H.M. (1986) 1. Nijweide, Physiol. Rev. 66, 855-886 2. Smith, W.L. (1989) Biochem. J. 259, 315-324 3. Hakeda, Y., Hotta, T., Kurihara, N., Ikeda, E., Maeda, N., and Kumegawa, M. (1987) Endocrinology 121, Yagyu, Y. 1966- 1974 Biochem. Biophys. Res. 4. Koshihara, Y. and Kamamura, M. (1989) Commun. 159, 1206-1212 Gilman, A.G. (1987) Ann. Rev. Biochem. 56, 615-649 Deckmyn, H., Kienast, J., Wi t tevrongel , C. and 2 Geet, C.V., Vermylen, J. (1990) J. Biol. Chem. 265, 7920-7926 R.M., Luini, A. and Axelrod, J. (1986) Proc. Natl. 7. Burch, Acad. Sci. USA 83, 7201-7205 8. Teitelbaum, I. (1990) J. Biol. Chem. 265, 4218-4222 9. Ui , M. (1984) Trends Pharmacol. Sci. 5, 277-279 10. Sternwei s, P.C. and Robishaw, J.D. (1984) J. Biol. Chem. 259, 13806-13813
11. 12. 13. 14. 15. 16. 17. 18.
Smith, C.D., Lane, B.C., Kusaka, I., Verghese, M.W. and Snyderman, R. (1985) J. Biol. Chem. 260, 5875-5878 Kikuchi, A., Kozawa, O., Kaibuchi, K., Katada, T., Ui, M. and Taka i , Y. (1986) J. Biol. Chem. 261, 11558-11562 Uhing, R. J., Prpic, V., Jiang, H. and Exton, J.H. (1986) J. Biol. Chem. 261, 2140-2146 Fischer, J.B. and Schonbrunn, A. (1988) J. Biol. Chem. 263, 2808-2816 Davis, J.S., Weakland, L.L., Weiland, D.A., Farese, R.V. and West, L.A. (1987) Proc. Natl. Acad. Sci. USA 84, 3728-3732 Hatanaka, M., Yumoto, N., Miwa, N., Morii, H., Tanemura, M., Ueno, R., Watanabe, Y. and Hayaishi, 0. (1989) J. Neurochem. 53, 1450-1455 Murayama, T. and Ui , M. (1987) J. Biol. Chem. 262, 1246312467 Kodama, H., Amagai, Y., Sudo, H., Kasai, S. and Yamamoto, S. (1981) Jpn. J. Oral Biol. 23, 899-901 1234
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19. Sudo, H., Kodama, H., Amagai, Y., Yamamoto, S. and Kasai, S. (1983) J. Cell Biol. 96, 191-198 20. Berridge, M.J., Dawson, R.M.C., Downes, C.P., Heslop, J.P. and Irvine, R.F. (1983) Biochem. J. 212, 473-482 21. Berridge, M.J., Heslop, J.P., Irvine, R.F. and Brown, K.D. (1984) Biochem. J. 222, 195-201 22. Nishimoto, I., Murayama, Y., Katada, T., Ui, M. and Ogata, E. (1989) J. Biol. Chem. 264, 14029-14038 23. Laemml i, U.K. (1970) Nature 227, 680-685 24. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-274 25. Katada, T., Oinuma, M. and Ui, M (1986) J. Biol. Chem. 261, 8182-8191
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