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

Vol.

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

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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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