Vol. 168, No. 3, 1990

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

BIOCHEMICAL

Pages 1184-1193

May 16, 1990

INCREASED

MITOGENIC EXPRESSING

Ian Zachary*,

RESPONSIVENESS CONSTITUTIVELY

OF SWISS 3T3 CELLS ACTIVE Gsa

Susan B. Masters and Henry R. Boume#

University of California, San Francisco, California 94143 Cancer Research Fund, Lincoln’s Inn Fields, London WC2A 3PX, UK

*Imperial Received

March

19,

1990

Abstract: Mutational replacement of glutaminewith a leucine residue in the GTP-binding domain of the a subunit of Gs (Q227L as) reduces its ability to hydrolyse GTP and causes constitutive activation of the mutant protein. Expression in Swiss 3T3 fibroblasts of Q227L as caused markedly increased basal adenylyl cyclase activity, enhanced intracellular cyclic AMP (CAMP) accumulation and increased mitogenic sensitivity in response to forskolin and the potent phosphodiesterase inhibitor Ro 20-1724. These results support a role for CAMP in the regulation of cell proliferation, and suggest that alterations in a G protein can directly modify the ability of cells to respond mitogenically to extracellular factors. 01990 Academic Press, Inc.

An increase in the intracellular a mitogenic

signal in a wide variety

in Saccharamyces intracellular forskolin

cerevisiae

CAMP

-

and inhibitors

mitogenesis

content of cyclic AMP (cAMP) can act as

in synergistic

(2). Intracellular

of cultured

(1,2).

including

E-type

of cyclic nucleotide combinations

prostaglandins, phosphodiesterases

that raise

cholera -

toxin,

stimulate

with other growth promoting

CAMP is also increased

kinase C, including

cells, as well as

In Swiss 3T3 cells, agents

growth factor (3) and vasoactive intestinal protein

mammalian

the neuropeptide

agents

in these cells by platelet-derived peptide

(4); agents which activate

bombesin

(5) and phorbol

“Address correspondence to Henry R. Bourne, University of California-San Department of Pharmacology, Box 0450, Room 51210, San Francisco, CA 94143.

esters

Francisco,

Abbreviations: Gs, the GTP-binding protein which activates adenylyl cyclase; as, the GTFbinding domain of Gs; Q227L as, as in which glutamine 227 is replaced by leucine; GTE, guanosine S&phosphate; GppNHp, guanylyl-S’knidodiphosphate; Ro 20-1724, I-(3-butoxy4-methylbenzyl)-2-imidazolidine; IBMX, I-methyl-3-isobutylxanthine; PGEl, prostaglandin EI. 0006-291X/90 $1.50 Copyright 0 1959 by Academic Press, Inc. All rights of reproduction in any form reserved.

1184

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168, No. 3, 1990

BIOCHEMICAL

(6), can also act indirectly

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

to enhance CAMP accumulation

via an unknown

mechanism. Synthesis GTP-binding

of CAMP subunit

by hormonal

of Gs that is responsible

receptors to activation subunit

is stimulated

of adenylyl

cyclase (7,8).

function

role in the intrinsic

of all G proteins

conserved glutamine activation

at position

of adenylyl

(10, 11). Mutation

Mutational

replacements constitutive

and

activation

mechanisms

domain activity

acid similarly

to the

replacement

colleagues

residue in p2lras,

to promote

of a

recently

of adenylyl

reported

relaxation

pathways

predict

of the normal

MATERIALS

stimulus.

for cellular

cyclase and increased

activating

growth control

used as a model

important

hormone-

that dominant

constitutive

effects of CAMP-elevating

mutational

Q227, are associated with

of mutant

of adenylyl

transformation

(13) that

in cells in which CAMP can act as a mitogenic

signal transduction

to

and enhances

cyclase in a subset of growth

These findings

in 01~ will cause a partial

glutamine-61,

malignant

(2). Here we report that expression

mitogenic

essential

reduces GTPase activity

ras protein

tumours.

activation

of this protein

227 by leucine (Q227L a,) causes constitutive

Swiss 3T3 cells have been extensively elucidating

analysis of the a,

mutational

of specific residues in a s, including

secreting pituitary mutations

Specifically,

of the corresponding

of the mutant

Landis

(12).

GTPase

stimulatory

cyclase as a consequence of decreased GTPase activity

almost any other amino the capacity

(9-11).

of asI the

for coupling

of Gs has defined a region in the GTP-binding

which plays a crucial

activation

system for proliferation

Q227L a, in these cells causes sensitivity

to the

agents. AND METHODS

Swiss 3T3 cells were maintained in culture and assays of DNA synthesis, adenylyl cyclase activity and CAMP accumulation were performed as described (5, 1416). Preparation of whole cell RNA and Northern blot analysis with a full-length cDNA for rat os (17) were also performed as described (5). Construction and expression of mutant as chains. Subcloning of normal rat 01~ (GRS) (17) and mutant recombinant os (10) into the retroviral expression vector pMV-7 (18) were performed as previously described (10). Q227L as was generated from pGRAS, an altered form of pGRS that lacked part of the polylinker and the 5’ noncoding region but was otherwise 1185

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BIOCHEMICAL

168, No. 3, 1990

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

identical. After passage of the vectors through packaging cell lines (19), Swiss 3T3 cells were infected and independent transformants were selected by growth in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum and G418 at a concentration of 500 pg/ml. Membrane preparation . Confluent and quiescent cultures of Swiss 3T3 cells were trypsinized and pelleted. The pellet was washed twice in 10 ml of 5 mM 4-(3-butoxy-4-methylbenzyl)-Z-imidazolidine (HEPES), pH 7.4, 2 mM MgC12, 0.2 mM phenylmethylsulphonyl fluoride and resuspended in 3 ml of the same buffer. The cell suspension was frozen and thawed 3 times and homogenized by 100 strokes in a dounce homogenizer and centrifuged for 10 min at 1000 ‘pm. The supernatant containing a crude particulate fraction was centrifuged for 30 min at maximum speed in a microcentrifuge. Membranes were resuspended and stored at -70°C in 40 mM HEPES, pH 7.4,0.15 M NaCl, 20% glycerol. Protein was determined as described (20). proteins were resolved on a Immunoblot analysis of a,. Membrane 10% SDS-polyacrylamide gel, transferred to nitrocellulose and immunoblotted with affinity-purified antiserum (4 pg/ml) directed against a peptide (DATPEPGEDPRVTRAK) located in the carboxy terminal half of as (21).

Materials. Insulin, l-methyl-3-isobutylxanthine (IBMX), forskolin, prostaglandin El (PGEi), guanosine 5’-triphosphate (GTP) and guanylyl3’imidodiphosphate (Gpp(NHlp) were obtained from Sigma Chemical Co., St. Louis, MO. Cholera toxin was supplied by List Biological Laboratories (Campbell, CA). 4-(3-butoxy-4-methylbenzyl)-2-imidazolidine (Ro 20-1724) was purchased from Biomol. Research Lab. Inc., Plymouth Meeting, PA. Radioisotopes and antigens and antibodies for the radioimmunoassay of CAMP were obtained from Amersham International (U.K.). 3H-CAMP was obtained from New England Nuclear, Boston, MA. All other materials used were of reagent grade. RESULTS AND DISCUSSION

Swiss 3T3 cells infected with wild-type

and mutant

cDNAs

encoding

the 52 kDa form of as were screened by examining

the incorporation

of 3H-

adenine

inhibitor

caused

into

approximately

CAMP.

The

phosphodiesterase

8-fold more incorporation

of radioactivity

infected with Q227L a, than in cells infected with normal cells.

Five out of 5 clones infected

CAMP

in the presence

normal

of IBMX

as or uninfected

further phenotypic

cells.

into CAMP in cells 01s or in uninfected

with Q227L as also exhibited

as compared

to clones infected

elevated with

the

Two clones of each type were selected for

analysis.

Swiss 3T3 cells infected with Q227L as exhibited adenylyl

IBMX

cyclase activity,

measured

a marked

increase in

in the presence of GTP alone (Fig. 1). In 1186

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168, No. 3, 1990

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

L n

GTP

n

GTP+PGE,

n

GP~NHP

q

GppNHp+PGE

q

Forskolln

GRS5

GUS?

Adenylyl cyclase activity in Q227L., and control membranes. Membraneswere assayedfor adenylyl cyclaseactivity asdescribedin Materials Values represent the means of at least

and Methods.

duplicate

determinations (n = 2 - 6). Effector concentrations were as follows: 100 FM GTP; 3 PM PGEl; 100PM Gpp(NH)p; 100W forskolin.

the presence of E-type prostaglandins, stimulate

DNA synthesis in Swiss 3T3 cells (21, adenylyl

considerably

higher

or uninfected catalytic

(275%) than in membranes

cells.

subunit

The diterpene

of adenylyl

agents as a stimulator activity

Although

hydrolysis-resistant activity

which

cyclase, though

forskolin

acts directly

effect

GTP-analogue

either GTP or GTP plus PGEl.

acts directly

on the

than other

again forskolin-stimulated with GRS or uninfected

on the catalytic

subunit

of

are in accordance with evidence (22, 23) that

the stimulatory

in Q227L membranes,

was

from GRS-infected

more effective

greater in Q227L,, compared

cyclase, these findings

as potentiates

forskolin,

CAMP and

cyclase activity

prepared

cyclase, was much

of adenylyl

was significantly

membranes. adenylyl

which increase intracellular

relative

of this

Gpp(NH)p to activity

Surprisingly,

reduced

adenylyl

measured

This result parallels 1187

agent.

the cyclase

in the presence of

the effect of Gpp(NH)p

in

BIOCHEMICAL

Vol. 166, No. 3, 1990

S49 cell membranes activation

expressing

AND BIOPHYSICAL

the Q227L

RESEARCH COMMUNICATIONS

as protein

and may reflect

of Gi by this agent (7,B). RNA extracted from wild-type,

cells contained Northern

only a single major 01~ transcript

hybridization

with a full-length

uninfected

of -2000 bp as detected by

probe for mouse as. In contrast,

RNA isolated from Q227L and GRS-infected

cells contained,

endogenous

species of -6100 bp (results not

shown).

as transcript,

a larger mRNA

The length of this message corresponds

for an LTR to LTR transcript cDNA (18). Immunoblot significant

alteration

either mutant recombinant constitutes

analysis with anti-as

or wild-type

the predominant

observations).

the as

did not reveal any associated

Presumably

by the endogenous

with

expression

52 kDa protein

of

which

form of 01~in these cells (I. Zachary, unpublished observed in 42272. Swiss 3T3 cells is not

Table

CUP

antiserum

of the 52 kDa as protein

Thus, the phenotype

Intracellular

vector containing

as (results not shown).

c+ is masked

to the

closely to the size predicted

from the retroviral

in expression

in addition

accumulation

in

1

Sviss CARP

3T3

cells

(pmoles/mg

expressing

Q227L

or-

protein)

Addition

VT

GRS 5

None

2.6

2.7

IBBX

a.3

9.2

117.8

51.4

4.2

3.2

38

33

4.3

3.5

aa

78

Ro

20-1724

PGEl

PGEl

+ Ro

20-1724

Forskolin

214

360

Forskolin + Ro 20-1724

576

727

tluiescent absence (100

ukl);

solvent. (i.e.

cultures

or

presence PGEl Values

with

(250

the

rig/ml);

represent

performed cells

cells

were

following forskolin the

no additions)

Experiments Q227L-12

of of

represent with

GRS-7

of the

in

DHEH the

and

0227L-10

and

ufl).

All

least

duplicate

of

incubated

3.5

156

300

722

800

cultures

cells

produced

1188

the

same

indicated: were

treated

determinations

10 determinations

respectively.

1,700

in

concentrations

results

medium

for

IBBX

(500

the

same

vith (n

obtained similar

12

6.5

1,400

at

mean

Q227L

2.7

washed

at

pool

7.5

agents (50

mean

Q227L

= 2 - 8);

from

1 hr ukl); volume

basal

5 different to

those

in

the

Ro 20-1724 of CARP

levels

experiments. shown

for

GRS-5

and

BIOCHEMICAL

Vol. 168, No. 3, 1990

due to an increased

AND BIOPHYSICAL

total level of the 52 kDa protein

consequence of expression of the mutant Treatment

of intact, quiescent

of CAMP phosphodiesterase

increases

in intracellular

CAMP

intracellular

(IBMX

1).

Cells

expressing

cells exhibited

much

smaller

and PGEl

normal,

increases in

Ro 20-1724 potentiated

on intracellular

cAMP in all the cells

but the increases in CAMP were greater in the Q227L cells compared

to either GRS or uninfected

cells. Basal, unstimulated

modestly

elevated

but significantly

The much presence

more dramatic

adenylyl

in Q227L cells relative

activity

inhibitors

probably

in Q227L cells.

indicates

in the

increased between the

the same mutant

that the Q227L mutation

as cDNA

causes constitutive

of a, as a direct consequence of reduced GTPase activity (10, 11,131.

Insulin,

which has no effect on intracellular

(21, was weakly mitogenic

insulin-induced

DNA

synthesis

response to insulin

20-1724 were as effective combinations

of insulin

in uninfected

in cells expressing

in reinitiating

DNA

synthesis

with either cholera toxin or PGEl. A significant

Similarly,

incorporation

concentration

Forskolin

In marked

and Ro

in Q227L

cells as

The effect of Ro

increase in 3H-thymidine as low as 1 uM

and GRS cells with

of -25 PM and a maximal

the

Insulin

in the presence of insulin

in uninfected

1189

increase in

potentiated

Q227L a,.

in Q227L cells was observed at a concentration

(results not shown). 3H-thymidine

and GRS cells.

of Ro 20-1724 strikingly

20-1724 was concentration-dependent: incorporation

(Fig. 2). Ro 20-

of 100 PM, caused only a small further

the same concentration

mitogenic

CAMP levels in these cells

by itself in all the cells examined

1724, at a concentration

contrast,

cells.

in Q227L Swiss 3T3 cells and

(10) in S49 cyc- (24) cells expressing

(10) is consistent with the finding activation

to control

The close similarity

cyclase activity and CAMP accumulation

that reported

CAMP content was also

increase in CAMP of Q227L cells observed

of phosphodiesterase

phosphodiesterase

maximal

or

or Ro 20-1724) caused striking

CAMP in response to the same agents.

the effects of both forskolin studied,

form of 01~.

(Table

as and uninfected

but is rather a specific

cultures of Q227L cells with forskolin

inhibitors

recombinant

RESEARCH COMMUNICATIONS

increased a half-

response at 100 PM (Fig. 3,

Vol.

168, No. 3, 1990

BIOCHEMICAL

AND BIOPHYSKAL

50

25

RESEARCH COMMUNICATIONS

GRS5

25 -

.C

:

*

5o -

0227

50-

Pool

Q227L

12

e ,I

25 -

Ram- -r

-1724

o-

-

Fig. 2: Effect of CAMP-elevating agentson 3H-thymidine incorporation

in Swiss 3T3 cells expressing Q227L a,. Cells were incubated for 40 hr with 3H-thymidine either in the presenceor absenceof 100 PM Ro 20-1724and in the presenceof either an equal volume of solvent, 100 rig/ml cholera toxin, or 250rig/ml PGEl. All cultures except thosereceiving no additions were also treated with 1 pg/ml insulin. The values shown represent the means of at least 4 determinations (n = 4 - 81 and are expressed as percentages of the response obtained in the presence of 10% FCS. Duplicate determinations agreed within 10%. Results similar to those for GRS-5 and Q227L 12 were obtained using respectively GRS7 and Q227L 10cells.

upper panel and results not shown).

In contrast, forskolin stimulated DNA

synthesis in Q227L cells at concentrations as low as 0.1 PM and maximally between 0.6 and 2 yM.

Enhanced mitogenic responsiveness of Q227L Swiss 1190

Vol. 168, No. 3, 1990

BIOCHEMICAL

300. C I e a 0) 200. E

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

WT

GM

d

d

w-f

GRS 5

5

5 E a loo oz 3 3

II

0-

0

100

n 0

1

100

d 0

1

dl

1

0

0.6

Forskolin,

(rhl

1

0.6

Fig. 3: Effect of forskolin on DNA synthesis (upper) and CAMP accumulation (lower) in Swiss 3T3 cells expressing recombinant as. Quiescent cultures of wild-type uninfected (WT), GRS 5, Q227L pool or Q227L, 12 Swiss 3T3 cells were incubated with different concentrations of forskolin either in the presenceor absenceof insulin at 1 &ml.

Measurements of

intracellular CAMP (lower) were made 1 hr after addition of forskolin.

Each

value represents the mean of either 4 (upper) or 2 (lower) determinations which agreed within 5-10%. Values for 3H-thymidine

incorporation are

expressedas in Fig.2. Experiments performed with GRS-7 and Q227L-10cells produced similar results to those shown for GRS-5 and Q227L 12 cells respectively.

3T3 cells to forskolin intracellular

CAMP.

was closely paralleled

Thus, in quiescent Q227L cells, forskolin

increase in CAMP content which maximally (Fig. 3, upper markedly DNA

at concentrations

stimulated panel).

3H-thymidine

in control

cells.

incorporation

production

in parallel range,

cultures

forskolin

of intracellular

These results indicate 1191

caused a large

(0.6-l PM) (Fig. 3, lower panel)

In the same concentration

less effective in stimulating

synthesis

by the effect of this agent on

was

CAMP and

that enhanced

Vol.

BIOCHEMICAL

168, No. 3, 1990

mitogenic

responsiveness

AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

of the Q227L cells is probably

a direct consequence

of enhanced CAMP synthesis. Recent findings consequence glutamine tumours

of activating

(13, 25, 26).

production

in c+, including

to the abnormal

growth

Thus it has been proposed

consistent

the phenotype

cyclase activity

sufficiently

to trigger

carcinogenesis

mitogenesis,

cyclase.

In conclusion,

transformation will require

to exhibit

different mitogenic

cell growth

inhibiting

designated the mutant

we predict with

alterations

signaling

a, is

CAMP Just as

mutation,

Swiss

in this case a phosphodiesterase

effect of constitutively that aberrant

mutations

gsp

(2), the increased

more than one oncogenic

the mitogenic

associated concomitant

of

even in the presence of insulin.

3T3 cells appear to require a “second hit” -

replacement

caused by Q227L as does not elevate cellular

appears to require

inhibitor

as a

that GTPase

of Swiss 3T3 cells expressing

with a role for CAMP in signalling

of CAMP

of certain pituitary

in the 01s gene convert it into a novel oncogene,

While

adenylyl

mutations

227, may contribute

mutations (13).

suggest that constitutive

cell growth

that trigger

active adenylyl and malignant

generation

in either the CAMP signaling

of CAMP system, a

pathway, or both.

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13. Landis, C.A., Masters, S.B., Spada, A., Pace, A.M., Bourne, H.R. and Vallar, L. (1989). Nature, 340,692-696. 14. Dicker, P. and Rozengurt, E. (1980). Nature, 287,607-612. 15. SaIomon, Y., Londos, C. and Rodbell, M. (1974). Anal. Biochem., 58, 541548. 16. Perkins, J.P. and Moore, M.M. (1973). J. Pharmacol. Exp. Ther., 185,371378. 17. Jones, D.T. and Reed, R.R. (1987). J. Biol. Chem., 262, 14241-14249. 18. Kirschmeier, P.T., Housey, G.M., Johnson, M.D., Perkins, AS. and Weinstein, LB. (1988). DNA, 7, 219-225. 19. Sullivan, K.A., Miller, R.T., Masters, S.B., Beiderman, B., Heideman, W. and Bourne, H.R (1987). Nature, 330,758-760. 20. Lowry, O.H., Rosebrough, N.S., Farr, A.L. and Randall, R.J. (1951). J. Biol. Chem. 253,7120-7123. 21. Masters, S.B., Sullivan, K.A., Miller, R.T., Beiderman, B., Lopez, N.G., Ramachandran, J. and Bourne, H.R. (1988). Science, 241,448-451. 22. Green, D.A. and Clark, R.B. (1982). J. Cyclic Nucl. Res. 8,336-346. 23. Bender, J.L. and Neer, E.J. (1983). J. Biol. Chem. 258,2432-2439. 24. Insel, P.A., Bourne, H.R., Coffino, P. and Tomkins, G.M. (1975). Science, 190,896-898. 25. Vallar, L., Spada, A. and Giannattasio, G. (1987). Nature, 330,566-568. 26. Billestrup, N., Swanson, L.W. and Vale, W. (1986). Proc. Natl. Acad. Sci. U. S. A., 83,6854-6857.

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