BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 175, No. 1, 1991
Pages 55-60
February 28, 1991
R O L E OF A GUANINE N U C L E O T I D E BINDING PROTEIN, OF A D E N Y L A T E Masakazu
of
IDepartment
CYCLASE
IN D I C T Y O S T E L I U M
G ~ 2, IN R E G U L A T I O N DISCOIDEUM
O y a m a 1'2 *, Kou K u b o t a 2 and Koji
Botany,
F a c u l t y o f Science, Kyoto 606 Japan
Okamoto I
Kyoto
University,
2Department of Life Science, F a c u l t y of S c i e n c e , Himeji Institute of T e c h n o l o g y , Shosha 2167, H i m e j i Hyogo 671-22 Japan Received December 17, 1990 Two s u b s t a n c e s , c A M P and 2 , 3 - d i m e r c a p t o - l - p r o p a n o l (BAL) are k n o w n to i n d u c e t r a n s i e n t activation of a d e n y l a t e cyclase in Dictyostelium discoideum. A frigid mutant (HC85) has a d e l e t i o n in a gene for Gc~ 2, a g u a n i n e nucleotide binding protein and cannot a c t i v a t e the c y c l a s e in r e s p o n s e to cAMP. We f o u n d that BAL induced activation in the f r i g i d mutant. This result s u g g e s t s that the B A L - i n d u c e d activation is i n d e p e n d e n t of Gcz 2 and that B A L m i m i c s a role of a c t i v a t e d Gol 2. We also found that c A M P p r o m o t e d the B A L - i n d u c e d activation. This result s u g g e s t s that cAMP plays a role in a c t i v a t i o n through a m e c h a n i s m in which Go~ 2 is not involved. We lastly s h o w e d that c o n t i n u o u s cAMP s t i m u l a t i o n could not inhibit the B A L - i n d u c e d a c t i v a t i o n in the frigid mutant. Since the c A M P - i n d u c e d i n h i b i t i o n o b s e r v e d in the w i l d t y p e s t r a i n (NC4) proceeds with the time course identical to the c A M P - i n d u c e d a d a p t a t i o n (Oyama, s u b m i t t e d ) , this result s u g g e s t s that G¢~ 2 is i n v o l v e d in a d a p t a t i o n of a d e n y l a t e cyclase. © 1991 Academic Press, Inc.
Starvation
triggers
developmental discoideum. to
induce
In
of
at
least
various
two
from
cellular
developmental
Adenylate [5,6,73.
shift
the
and
cAMP b i n d s
activates
systems. systems
the
in
morphogenesis
Extracellular and
phase
a
The types,
*To whom c o r r e s p o n d e n c e
to
types
mold,
on
one
the
A and B [8,93
be
example on and
phase
the they
as [for
cell
the
a pheromone review,
surface
signal
13.
[2,3,42
transduction
regulated cell
to
Dictyostelium
cAMP a c t s
intracellular
is
cAMP r e c e p t o r s
should
slime
differentiation
receptors of
growth
phase,
cell
cyclase
the
by
surface
co-operatively
such consist work
addressed.
Abbreviations: BAL, 2 , 3 - d i m e r c a p t o - l - p r o p a n o l ; cAMP, a d e n o s i n e 3':5'-cyclic monophosphate; dcAMP, 2 ' - d e o x y a d e n o s i n e - 3 ' : 5 ' - c y c l i c monophosphate.
55
0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 175, No. 1, 1991
for
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
activation
binding
of
[9,10].
is
A frigid
of
Go~ 2
to
cAMP s t i m u l a t i o n
and
gene
[13],
that
on
the
of
BAL
pathway(s) mimic
in
and
Kubota
reagents,
a
role
of
of
the
frigid
receptor
that
nucleotide
with
is
a
B-receptors
deletion
cyclase
mutant
has
and
Oyama
[14] such
signal
the
guanine
mutant
in
response
cAMP r e c e p t o r s
12].
BAL
as
BAL o r
et
induce
most or
[15]
pathway(s)
and/or
interesting
induces
al.
dithiothreitol,
transduction and
The
modify aspect
enhances
the
of
the
activation
of
[13,15]. directly
acts
activation
advantage the
adenylate
events.
eyclase
Since
which
this
Oyama
is
Ell]
activate
Ell,
a
associated
although
from
BAL
adenylate
(HC85)
intracellular
independently
G(~ 2 ,
be
[12]
reducing
receptor-mediated effect
to
cannot
cyclase
Oyama proposed
cyclase.
thought
mutant
[10]
adenylate
act
adenylate
protein,
G~
2.
we
the
adenylate We
BAL-induced
mutant,
receptor-mediated
of
on
found
this
activation
and
transduction
cyclase,
investigated
activation
signal
this
is of
the
reagent
case.
adenylate
the
role
adaptation
of
of
may
Taking cyclase
G~
2
adenylate
in
in the
cyclase.
MATERIALS and METHODS
Frigid mutant, HC85 a n d i t s parental strain, HC6 ( w h i c h is an a x e n i c mutant) were shake-cultured with Escherichia coli B/r in phosphate buffer. Vegetatively growing amoebae were collected, washed and resuspended i n 20 mM p h o s p h a t e buffer pH 6.4 containing 10 mM KC1 a n d 1 . 2 mM MgSO 4 a t 6 x l O ' ° c e l l s / m l . The cell suspension was s h a k e n at 120 rpm a t 21 °C f o r 10 h . The starved cells were washed and resuspended i n 2 0 mM p h o s p h a t ~ buffer pH 6 . 4 containing 1 0 mM KC1 ( P B K b u f f e r ) at lxlO t cells/ml. One ml o f t h e c e l l suspension w as s h a k e n i n a 20 ml vial for at least 30 m i n a n d then stimulated with 3 mM BAL (Sigma), 10 ~M dcAMP ( S i g m a ) o r t h e m i x t u r e of them. One h u n d r e d }11 o f the suspension was sampled into a micro-test tube containing HC104. The cell tysate w as n e u t r a l i z e d with KHCO3 . cAMP i n t h e s u p e r n a t a n t was a s s a y e d with isotope dilution assay (Amersham) [16].
RESULTS We i n v e s t i g a t e d cyclase Since
in in
adenylate more h
a a
not
effect
mutant
preliminary cyclase
pronouncedly
(data
the
frigid
in
expeiment the
induced
shown),
of
(HC85)
we
frigid by used
BAL on and we
10
investigation. 56
of
parental
strain
found
cells
BAL
activation
its
that
starved
than
in
those
h
starved
adenylate (HC6).
activation for
8-10
starved
cells
for
of h
for
were 4.5-6
further
Vol. 175, No. 1, 1991
We f i r s t of
cAMP
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
confirmed
in
the
frigid
accumulation
in
stimulation
since
its
it
that
in
both of
both
cells
were
immediate the
and and
the
Since
by
and
(Fig.
Oyama
[13] of
of
the
I
cAMP
adenylate
(Fig.
1).
addition
parent
occurred
1).
found
dcAMP
of
the
together
In
in
the
2
and
that
for
10
adenylate
for
The of
the
BAL
frigid
both
the
in
6-8
min
the the
in
case frigid
after
stimulation
completely
cyclase
and cAMP
to
contrast,
continuous
frigid
strain
similarly
beyond
min
stimulation,
parenal
min,
By m a r k e d
continued
activation
after
in
within
dcAMP a l o n e .
the
at
suppresses a
wild
type
a the
strain
60
//
12 __
HC85
8
I
for
induces
with
strains
lag
used
cAMP
terminated
1).
10
0
used
respectively.
were
of
a
14
~8
transient
was and
activation
parental
times
min
BAL
(Fig.
level
BAL-induced
Q
a
dcAMP
interfering
the
with
lag
accumulation
stimulation
HC__6
accumulation
cAMP-receptors
induced
3-5
cells was
stimulation
~o
any
induced 1).
without
began
accumulation
saturated
cause
it
(Fig.
to
frigid
The
1 min
parental
strain
the cAMP
dcAMP
accumulation of
binds
BAL
strains.
When
while cells
events
accumulation in
cells
not
[16].
found
cyclase
dcAMP did
parental
receptor-mediated quantification We
that
I
I
I
2 4 min
6
2
4 6 min
8
50
~o ~ 40
40
~
3o 20
~ ~ 20
0
2
4 6 rnin
8
(~
BAL
~/
0
BAL
I
I
I
5
10
15
k22 min Figure 1 Activation of adenylate cyclase in the frigid m u t a n t (HC85) a n d i n i t s p a r e n t a l strain (HC6). S t a r v e d HC85 (center and right) o r HC6 ( l e f t ) cells were shake-cultured i n PBK buffer. The c e l l s were stimulated w i t h 10 ~M dcAMP ( c i r c l e ) , 3 mM BAL ( t r i a n g l e ) or the mixture of them (square). The c e l l suspension was sampled f o r cAMP q u a n t i f i c a t i o n at indicated times. Center and right graphs show the same data with different vertical axes. Fizure 2 Activation of adenylate cyclase b y BAL i n t h e frigid mutant after pretreatment o f 100 M dcAMP. Starved frigid (HC85) c e l l s were shake-cultured in~PBK b u f f e r . The c e l l s were stimulated w i t h 100 pM dcAMP a t 0 t i m e ( c i r c l e ) . Part of the cell suspension was r e m o v e d a t 1 . 5 m i n ( t r i a n g l e ) o r lOmin (square) after t h e dcAMP s t i m u l a t i o n t o a new v i a l containing 3 mM BAL. The c e l l suspension was s a m p l e d f o r cAMP q u a n t i f i c a t i o n at indicated times. 100 M dcAMP was d e t e c t e d as a background of 1 6 . 5 pmol c A M P / l x l 0 7 c e l l ~ . 57
~'D
Vol. 175, No. 1, 1991
(NC4),
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
continuous
dcAMP+BAL
in
activation
the
dcAMP-induced
frigid
of
mutant
inhibition
on
adenylate may
the
be
cyclase due
to
BAL-induced
induced
the
lack
by
of
activation
the
in
this
mutant. To 100
test
pN
this
dcAMP
found
that
and
BAL
pretreatment
not
than
at
activated 100
1.5
the
parental since
the
of
min
in
(HC6)
the
for
the
with
BAL
min
somewhat
of
of in
activation
(Fig.
(Fig. for
adenylate
the
wild
after
the
2).
at
min
of
time
We
could
completely in
strain
dcAMP
the
this
cyclase
type
We the
rate
2).
10
with
after
the slower
dcAMP
activation
cells
even
while
pretreatment
similarly
frigid
cyclase 10
cAMP w a s
of
the
them
pretreatment
BAL-induced
weak
dcAMP
BAL-induced
strain
treated
adenylate
pM
whether
suppressed
we
stimulated
accumulation
determine
too
then
of
BAL-induced point
possibility,
the
(NC4)
treatment
was
parents.
DISCUSSION We
showed
cyclase
in
(Fig.
a
1). mutation
this
result
to
is
in
alone the
(Fig.
mutant. receptors
of
1).
and
is
the
G~
2
G~ 2
gene
that
(Fig.
after
helps
the alone
A-receptor? ~
parents
is
thought
(HC6)
to
be
transduction
BAL m i m i c s
of a
that in
[10I, adenylate
role
of
G~ 2
of the
adenylate
stimulation
BAL-induced has
no
interaction the
activation
cyclase with
BAL
activation effect
in
in this
between of
the
adenylate
_ adenyt~te cyctcse
BAL
Figure 3 Hypothetical scheme for regulation of adenylate cyclase, cAMP b i n d s t o t h e A a n d B t y p e r e c e p t o r s . Two t y p e s of activation signals (closed arrow) are induced b y t h e cAMPreceptors. Cooperative actions of both signals are required for activation of adenylate cyclase. Ga 2 associated with the B receptor mediates induction of the activation signal (closed arrow) and the adaptation signal (open arrow). $8
an
3).
dcAMP
role
adenylate
its
activation
activation
suggest
of
in
signal
and
observed
a
as
BAL-induced
dcAMP
plays
well
intracellular
although results
activation
as
of
immediate
Thus,
cAMP
induce
(HC85)
cyclase
time
mutant These
in
induces lag
can
deletion
that
adenylate
short
frigid
HC85
independent
dcAMP+BAL a
the
suggests
activate
while
BAL mutant
Since
only
cyclase
that
frigid
VoI. 175, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cyclase
through
mediated
b y G~ 2 p r o t e i n
Our
data
through
also the
It
two
latter
has
activation
of
but
does
this
Oy am a of
[13]
activation This
of
for
the
termination
cyclase
involved
a
the
mutant
are
of for
role
of
G~
synergistic
consistent
continuous
stimulation
at
completely
suppresses
the
cyclase
in
the
wild
proceeds
the
cAMP-induced
little (Fig.
of in
the
on
with
that
the
2)
indirectly
of
adenylate
mutant
a
which
NC4.
time a
course mechanism
of
adenylate
continuous
that
cyclase cAMP
dcAMP
activation
suggests
might
saturated
BAL-induced
is
BAL-induced
of
a
strain,
activation
accumulation this
type
with
result
effect
adaptation
seen
types
necessary
BAL m i m i c s
A-receptor,
adaptation,
period
stimulation
two are
min
Therefore,
in
If
inhibition
mutant
prolonged
activates
cAMP-induced
shows
frigid
activated
alone.
receptors
frigid
3).
not
that
10
of
[17].
pretreatment the
found for
adenylate
to
of
the
is
one
(Fig.
3).
dcAMP-induced
identical
cAMP
[9].
role
mechanisms:
unidentified
cyclase
both
cyclase
dcAMP i n
(Fig.
dcAMP
that
a
yet
mechanism that
and
mimic
working
other
adenylate
proposed B,
BAL a n d
proposal
level
that
adenylate
not
of
tbe
unidentified
been A and
effects
and
suggest
receptors,
2
cooperatively
G~
(Fig. after
also
reflect
the
parental
in 2
3).
is The
dcAMP+BAL the
lack
of
adaptation. BAL than was
in
induces the
wild
developed
[13].
Some
character
of
in of
weaker type
activation
strain,
NC4.
shake-culture these
HC6 t o
in HC5
conditions
differences
is
an
axenic
unlike
may
cause
strain,
the
HC6
strain case
less
of
and NC4
sensitive
BAL.
ACKNOWLEDGMENTS We generous
thank
Drs.
supply
of
M.
B.
Coukell
the frigid
and
mutant
A. and
Kumagai
for
its parental
the strain.
REFERENCES 1. 2. 3. 4.
5. 6.
Gerisch, G. ( 1 9 8 7 ) A n n . R e v . B i o c h e m . 5 6 , 8 5 3 - 8 7 9 . Green, A.A., and Newell, P.C. (1975) Cell 6, 129-135. Henderson, E.J. (1975) J. Biol. Chem. 2 5 0 , 4 7 3 0 - 4 7 3 5 . Malchow, D., and Gerisch, G. ( 1 9 7 3 ) B i o c h e m . B i o p h y s . Res Commun. 5 5 , 2 0 0 - 2 0 4 . Klein, C., B r a c h e t , P., and D a r m o n , M. (1977) FEBS letters 76, 145-147. Roos, W., and Gerisch, G. (1976) FEBS letters 68, 170-172. 59
Vol. 175, No. 1, 1991
7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Roos,
W., S c h e i d e g g e r , C., and Gerisch, G. ( 1 9 7 7 ) N a t u r e 265, 259-261. Van H a a s t e r t , P.J.M., a n d De W i t , R . J . W . (1984) J. Biol. Chem. 2 5 9 , 1 3 3 2 1 - 1 3 3 2 8 . Firtel, R.A., Van H a a s t e r t , P.J.M., Kimmel, A.R., and Devreotes, P.N. (1989) Cell 58, 235-239. Kumagai, A., Pupillo, M., G u n d e r s o n , R., Hiake-Lye, R., Devreotes, P.N., and Firtel, R°A. ( 1 9 8 9 ) C e l l 57, 265-275. Coukell, M . B ° , L a p p a n o , S . , a n d C a m e r o n , AoH. ( 1 9 8 3 ) D e v . G e n e t . 3, 2 8 3 - 2 9 7 . Kesbeke, F., Snaar-Jagalska, B.E., and Van Haastert, P.J.M. (1988) J. Cell Biol. 107, 521-528. O y a m a , M. ( s u b m i t t e d ) Oyama, M., and Kubota, K. B i o c h i m . Biophys. Aeta (in press) O y a m a , M., K u b o t a , K., a n d O k a m o t o , K. ( 1 9 9 0 ) B i o c h e m . Biophys. R e s . Commun. 1 6 7 , 7 6 7 - 7 7 1 . Van H a a s t e r t , P.J.M. (1984) J. Gen. Microbiol. 130, 25592564. Dinauer, M.C., Steck, T.L., and Devreotes, P.N. (1980) J. Cell Biol. 86, 554-561.
60
Vol. 175, No. 1, 1991
Pages 55-60
February 28, 1991
R O L E OF A GUANINE N U C L E O T I D E BINDING PROTEIN, OF A D E N Y L A T E Masakazu
of
IDepartment
CYCLASE
IN D I C T Y O S T E L I U M
G ~ 2, IN R E G U L A T I O N DISCOIDEUM
O y a m a 1'2 *, Kou K u b o t a 2 and Koji
Botany,
F a c u l t y o f Science, Kyoto 606 Japan
Okamoto I
Kyoto
University,
2Department of Life Science, F a c u l t y of S c i e n c e , Himeji Institute of T e c h n o l o g y , Shosha 2167, H i m e j i Hyogo 671-22 Japan Received December 17, 1990 Two s u b s t a n c e s , c A M P and 2 , 3 - d i m e r c a p t o - l - p r o p a n o l (BAL) are k n o w n to i n d u c e t r a n s i e n t activation of a d e n y l a t e cyclase in Dictyostelium discoideum. A frigid mutant (HC85) has a d e l e t i o n in a gene for Gc~ 2, a g u a n i n e nucleotide binding protein and cannot a c t i v a t e the c y c l a s e in r e s p o n s e to cAMP. We f o u n d that BAL induced activation in the f r i g i d mutant. This result s u g g e s t s that the B A L - i n d u c e d activation is i n d e p e n d e n t of Gcz 2 and that B A L m i m i c s a role of a c t i v a t e d Gol 2. We also found that c A M P p r o m o t e d the B A L - i n d u c e d activation. This result s u g g e s t s that cAMP plays a role in a c t i v a t i o n through a m e c h a n i s m in which Go~ 2 is not involved. We lastly s h o w e d that c o n t i n u o u s cAMP s t i m u l a t i o n could not inhibit the B A L - i n d u c e d a c t i v a t i o n in the frigid mutant. Since the c A M P - i n d u c e d i n h i b i t i o n o b s e r v e d in the w i l d t y p e s t r a i n (NC4) proceeds with the time course identical to the c A M P - i n d u c e d a d a p t a t i o n (Oyama, s u b m i t t e d ) , this result s u g g e s t s that G¢~ 2 is i n v o l v e d in a d a p t a t i o n of a d e n y l a t e cyclase. © 1991 Academic Press, Inc.
Starvation
triggers
developmental discoideum. to
induce
In
of
at
least
various
two
from
cellular
developmental
Adenylate [5,6,73.
shift
the
and
cAMP b i n d s
activates
systems. systems
the
in
morphogenesis
Extracellular and
phase
a
The types,
*To whom c o r r e s p o n d e n c e
to
types
mold,
on
one
the
A and B [8,93
be
example on and
phase
the they
as [for
cell
the
a pheromone review,
surface
signal
13.
[2,3,42
transduction
regulated cell
to
Dictyostelium
cAMP a c t s
intracellular
is
cAMP r e c e p t o r s
should
slime
differentiation
receptors of
growth
phase,
cell
cyclase
the
by
surface
co-operatively
such consist work
addressed.
Abbreviations: BAL, 2 , 3 - d i m e r c a p t o - l - p r o p a n o l ; cAMP, a d e n o s i n e 3':5'-cyclic monophosphate; dcAMP, 2 ' - d e o x y a d e n o s i n e - 3 ' : 5 ' - c y c l i c monophosphate.
55
0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 175, No. 1, 1991
for
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
activation
binding
of
[9,10].
is
A frigid
of
Go~ 2
to
cAMP s t i m u l a t i o n
and
gene
[13],
that
on
the
of
BAL
pathway(s) mimic
in
and
Kubota
reagents,
a
role
of
of
the
frigid
receptor
that
nucleotide
with
is
a
B-receptors
deletion
cyclase
mutant
has
and
Oyama
[14] such
signal
the
guanine
mutant
in
response
cAMP r e c e p t o r s
12].
BAL
as
BAL o r
et
induce
most or
[15]
pathway(s)
and/or
interesting
induces
al.
dithiothreitol,
transduction and
The
modify aspect
enhances
the
of
the
activation
of
[13,15]. directly
acts
activation
advantage the
adenylate
events.
eyclase
Since
which
this
Oyama
is
Ell]
activate
Ell,
a
associated
although
from
BAL
adenylate
(HC85)
intracellular
independently
G(~ 2 ,
be
[12]
reducing
receptor-mediated effect
to
cannot
cyclase
Oyama proposed
cyclase.
thought
mutant
[10]
adenylate
act
adenylate
protein,
G~
2.
we
the
adenylate We
BAL-induced
mutant,
receptor-mediated
of
on
found
this
activation
and
transduction
cyclase,
investigated
activation
signal
this
is of
the
reagent
case.
adenylate
the
role
adaptation
of
of
may
Taking cyclase
G~
2
adenylate
in
in the
cyclase.
MATERIALS and METHODS
Frigid mutant, HC85 a n d i t s parental strain, HC6 ( w h i c h is an a x e n i c mutant) were shake-cultured with Escherichia coli B/r in phosphate buffer. Vegetatively growing amoebae were collected, washed and resuspended i n 20 mM p h o s p h a t e buffer pH 6.4 containing 10 mM KC1 a n d 1 . 2 mM MgSO 4 a t 6 x l O ' ° c e l l s / m l . The cell suspension was s h a k e n at 120 rpm a t 21 °C f o r 10 h . The starved cells were washed and resuspended i n 2 0 mM p h o s p h a t ~ buffer pH 6 . 4 containing 1 0 mM KC1 ( P B K b u f f e r ) at lxlO t cells/ml. One ml o f t h e c e l l suspension w as s h a k e n i n a 20 ml vial for at least 30 m i n a n d then stimulated with 3 mM BAL (Sigma), 10 ~M dcAMP ( S i g m a ) o r t h e m i x t u r e of them. One h u n d r e d }11 o f the suspension was sampled into a micro-test tube containing HC104. The cell tysate w as n e u t r a l i z e d with KHCO3 . cAMP i n t h e s u p e r n a t a n t was a s s a y e d with isotope dilution assay (Amersham) [16].
RESULTS We i n v e s t i g a t e d cyclase Since
in in
adenylate more h
a a
not
effect
mutant
preliminary cyclase
pronouncedly
(data
the
frigid
in
expeiment the
induced
shown),
of
(HC85)
we
frigid by used
BAL on and we
10
investigation. 56
of
parental
strain
found
cells
BAL
activation
its
that
starved
than
in
those
h
starved
adenylate (HC6).
activation for
8-10
starved
cells
for
of h
for
were 4.5-6
further
Vol. 175, No. 1, 1991
We f i r s t of
cAMP
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
confirmed
in
the
frigid
accumulation
in
stimulation
since
its
it
that
in
both of
both
cells
were
immediate the
and and
the
Since
by
and
(Fig.
Oyama
[13] of
of
the
I
cAMP
adenylate
(Fig.
1).
addition
parent
occurred
1).
found
dcAMP
of
the
together
In
in
the
2
and
that
for
10
adenylate
for
The of
the
BAL
frigid
both
the
in
6-8
min
the the
in
case frigid
after
stimulation
completely
cyclase
and cAMP
to
contrast,
continuous
frigid
strain
similarly
beyond
min
stimulation,
parenal
min,
By m a r k e d
continued
activation
after
in
within
dcAMP a l o n e .
the
at
suppresses a
wild
type
a the
strain
60
//
12 __
HC85
8
I
for
induces
with
strains
lag
used
cAMP
terminated
1).
10
0
used
respectively.
were
of
a
14
~8
transient
was and
activation
parental
times
min
BAL
(Fig.
level
BAL-induced
Q
a
dcAMP
interfering
the
with
lag
accumulation
stimulation
HC__6
accumulation
cAMP-receptors
induced
3-5
cells was
stimulation
~o
any
induced 1).
without
began
accumulation
saturated
cause
it
(Fig.
to
frigid
The
1 min
parental
strain
the cAMP
dcAMP
accumulation of
binds
BAL
strains.
When
while cells
events
accumulation in
cells
not
[16].
found
cyclase
dcAMP did
parental
receptor-mediated quantification We
that
I
I
I
2 4 min
6
2
4 6 min
8
50
~o ~ 40
40
~
3o 20
~ ~ 20
0
2
4 6 rnin
8
(~
BAL
~/
0
BAL
I
I
I
5
10
15
k22 min Figure 1 Activation of adenylate cyclase in the frigid m u t a n t (HC85) a n d i n i t s p a r e n t a l strain (HC6). S t a r v e d HC85 (center and right) o r HC6 ( l e f t ) cells were shake-cultured i n PBK buffer. The c e l l s were stimulated w i t h 10 ~M dcAMP ( c i r c l e ) , 3 mM BAL ( t r i a n g l e ) or the mixture of them (square). The c e l l suspension was sampled f o r cAMP q u a n t i f i c a t i o n at indicated times. Center and right graphs show the same data with different vertical axes. Fizure 2 Activation of adenylate cyclase b y BAL i n t h e frigid mutant after pretreatment o f 100 M dcAMP. Starved frigid (HC85) c e l l s were shake-cultured in~PBK b u f f e r . The c e l l s were stimulated w i t h 100 pM dcAMP a t 0 t i m e ( c i r c l e ) . Part of the cell suspension was r e m o v e d a t 1 . 5 m i n ( t r i a n g l e ) o r lOmin (square) after t h e dcAMP s t i m u l a t i o n t o a new v i a l containing 3 mM BAL. The c e l l suspension was s a m p l e d f o r cAMP q u a n t i f i c a t i o n at indicated times. 100 M dcAMP was d e t e c t e d as a background of 1 6 . 5 pmol c A M P / l x l 0 7 c e l l ~ . 57
~'D
Vol. 175, No. 1, 1991
(NC4),
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
continuous
dcAMP+BAL
in
activation
the
dcAMP-induced
frigid
of
mutant
inhibition
on
adenylate may
the
be
cyclase due
to
BAL-induced
induced
the
lack
by
of
activation
the
in
this
mutant. To 100
test
pN
this
dcAMP
found
that
and
BAL
pretreatment
not
than
at
activated 100
1.5
the
parental since
the
of
min
in
(HC6)
the
for
the
with
BAL
min
somewhat
of
of in
activation
(Fig.
(Fig. for
adenylate
the
wild
after
the
2).
at
min
of
time
We
could
completely in
strain
dcAMP
the
this
cyclase
type
We the
rate
2).
10
with
after
the slower
dcAMP
activation
cells
even
while
pretreatment
similarly
frigid
cyclase 10
cAMP w a s
of
the
them
pretreatment
BAL-induced
weak
dcAMP
BAL-induced
strain
treated
adenylate
pM
whether
suppressed
we
stimulated
accumulation
determine
too
then
of
BAL-induced point
possibility,
the
(NC4)
treatment
was
parents.
DISCUSSION We
showed
cyclase
in
(Fig.
a
1). mutation
this
result
to
is
in
alone the
(Fig.
mutant. receptors
of
1).
and
is
the
G~
2
G~ 2
gene
that
(Fig.
after
helps
the alone
A-receptor? ~
parents
is
thought
(HC6)
to
be
transduction
BAL m i m i c s
of a
that in
[10I, adenylate
role
of
G~ 2
of the
adenylate
stimulation
BAL-induced has
no
interaction the
activation
cyclase with
BAL
activation effect
in
in this
between of
the
adenylate
_ adenyt~te cyctcse
BAL
Figure 3 Hypothetical scheme for regulation of adenylate cyclase, cAMP b i n d s t o t h e A a n d B t y p e r e c e p t o r s . Two t y p e s of activation signals (closed arrow) are induced b y t h e cAMPreceptors. Cooperative actions of both signals are required for activation of adenylate cyclase. Ga 2 associated with the B receptor mediates induction of the activation signal (closed arrow) and the adaptation signal (open arrow). $8
an
3).
dcAMP
role
adenylate
its
activation
activation
suggest
of
in
signal
and
observed
a
as
BAL-induced
dcAMP
plays
well
intracellular
although results
activation
as
of
immediate
Thus,
cAMP
induce
(HC85)
cyclase
time
mutant These
in
induces lag
can
deletion
that
adenylate
short
frigid
HC85
independent
dcAMP+BAL a
the
suggests
activate
while
BAL mutant
Since
only
cyclase
that
frigid
VoI. 175, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cyclase
through
mediated
b y G~ 2 p r o t e i n
Our
data
through
also the
It
two
latter
has
activation
of
but
does
this
Oy am a of
[13]
activation This
of
for
the
termination
cyclase
involved
a
the
mutant
are
of for
role
of
G~
synergistic
consistent
continuous
stimulation
at
completely
suppresses
the
cyclase
in
the
wild
proceeds
the
cAMP-induced
little (Fig.
of in
the
on
with
that
the
2)
indirectly
of
adenylate
mutant
a
which
NC4.
time a
course mechanism
of
adenylate
continuous
that
cyclase cAMP
dcAMP
activation
suggests
might
saturated
BAL-induced
is
BAL-induced
of
a
strain,
activation
accumulation this
type
with
result
effect
adaptation
seen
types
necessary
BAL m i m i c s
A-receptor,
adaptation,
period
stimulation
two are
min
Therefore,
in
If
inhibition
mutant
prolonged
activates
cAMP-induced
shows
frigid
activated
alone.
receptors
frigid
3).
not
that
10
of
[17].
pretreatment the
found for
adenylate
to
of
the
is
one
(Fig.
3).
dcAMP-induced
identical
cAMP
[9].
role
mechanisms:
unidentified
cyclase
both
cyclase
dcAMP i n
(Fig.
dcAMP
that
a
yet
mechanism that
and
mimic
working
other
adenylate
proposed B,
BAL a n d
proposal
level
that
adenylate
not
of
tbe
unidentified
been A and
effects
and
suggest
receptors,
2
cooperatively
G~
(Fig. after
also
reflect
the
parental
in 2
3).
is The
dcAMP+BAL the
lack
of
adaptation. BAL than was
in
induces the
wild
developed
[13].
Some
character
of
in of
weaker type
activation
strain,
NC4.
shake-culture these
HC6 t o
in HC5
conditions
differences
is
an
axenic
unlike
may
cause
strain,
the
HC6
strain case
less
of
and NC4
sensitive
BAL.
ACKNOWLEDGMENTS We generous
thank
Drs.
supply
of
M.
B.
Coukell
the frigid
and
mutant
A. and
Kumagai
for
its parental
the strain.
REFERENCES 1. 2. 3. 4.
5. 6.
Gerisch, G. ( 1 9 8 7 ) A n n . R e v . B i o c h e m . 5 6 , 8 5 3 - 8 7 9 . Green, A.A., and Newell, P.C. (1975) Cell 6, 129-135. Henderson, E.J. (1975) J. Biol. Chem. 2 5 0 , 4 7 3 0 - 4 7 3 5 . Malchow, D., and Gerisch, G. ( 1 9 7 3 ) B i o c h e m . B i o p h y s . Res Commun. 5 5 , 2 0 0 - 2 0 4 . Klein, C., B r a c h e t , P., and D a r m o n , M. (1977) FEBS letters 76, 145-147. Roos, W., and Gerisch, G. (1976) FEBS letters 68, 170-172. 59
Vol. 175, No. 1, 1991
7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Roos,
W., S c h e i d e g g e r , C., and Gerisch, G. ( 1 9 7 7 ) N a t u r e 265, 259-261. Van H a a s t e r t , P.J.M., a n d De W i t , R . J . W . (1984) J. Biol. Chem. 2 5 9 , 1 3 3 2 1 - 1 3 3 2 8 . Firtel, R.A., Van H a a s t e r t , P.J.M., Kimmel, A.R., and Devreotes, P.N. (1989) Cell 58, 235-239. Kumagai, A., Pupillo, M., G u n d e r s o n , R., Hiake-Lye, R., Devreotes, P.N., and Firtel, R°A. ( 1 9 8 9 ) C e l l 57, 265-275. Coukell, M . B ° , L a p p a n o , S . , a n d C a m e r o n , AoH. ( 1 9 8 3 ) D e v . G e n e t . 3, 2 8 3 - 2 9 7 . Kesbeke, F., Snaar-Jagalska, B.E., and Van Haastert, P.J.M. (1988) J. Cell Biol. 107, 521-528. O y a m a , M. ( s u b m i t t e d ) Oyama, M., and Kubota, K. B i o c h i m . Biophys. Aeta (in press) O y a m a , M., K u b o t a , K., a n d O k a m o t o , K. ( 1 9 9 0 ) B i o c h e m . Biophys. R e s . Commun. 1 6 7 , 7 6 7 - 7 7 1 . Van H a a s t e r t , P.J.M. (1984) J. Gen. Microbiol. 130, 25592564. Dinauer, M.C., Steck, T.L., and Devreotes, P.N. (1980) J. Cell Biol. 86, 554-561.
60
