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Molecular and Cellular Biology, October 1998, p. 5744-5749, Vol. 18, No. 10
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Functional Promiscuity of Gene Regulation by
Serpentine Receptors in Dictyostelium discoideum
Irene
Verkerke-Van
Wijk,1
Ji-Yun
Kim,2
Raymond
Brandt,1
Peter N.
Devreotes,2 and
Pauline
Schaap1,*
Cell Biology Section, Institute for Molecular
Plant Sciences, University of Leiden, 2333 AL Leiden, The
Netherlands,1 and
Department of
Biological Chemistry, Johns Hopkins University School of Medicine,
Baltimore, Maryland 212052
Received 9 March 1998/Returned for modification 23 April
1998/Accepted 30 June 1998
 |
ABSTRACT |
Serpentine receptors such as smoothened and frizzled play important
roles in cell fate determination during animal development. In
Dictyostelium discoideum, four serpentine cyclic AMP (cAMP) receptors (cARs) regulate expression of multiple classes of
developmental genes. To understand their function, it is essential to
know whether each cAR is coupled to a specific gene regulatory pathway
or whether specificity results from the different developmental
regulation of individual cARs. To distinguish between these
possibilities, we measured gene induction in car1
car3 double mutant cell lines that express equal levels of
either cAR1, cAR2, or cAR3 under a constitutive promoter. We found that
all cARs efficiently mediate both aggregative gene induction by cAMP
pulses and induction of postaggregative and prespore genes by
persistent cAMP stimulation. Two exceptions to this functional
promiscuity were observed. (i) Only cAR1 can mediate adenosine
inhibition of cAMP-induced prespore gene expression, a phenomenon that
was found earlier in wild-type cells. cAR1's mediation of adenosine
inhibition suggests that cAR1 normally mediates prespore gene
induction. (ii) Only cAR2 allows entry into the prestalk pathway.
Prestalk gene expression is induced by differentiation-inducing factor
(DIF) but only after cells have been prestimulated with cAMP. We found
that DIF-induced prestalk gene expression is 10 times higher in
constitutive cAR2 expressors than in constitutive cAR1 or cAR3
expressors (which still have endogenous cAR2), suggesting that cAR2
mediates induction of DIF competence. Since in wild-type slugs cAR2 is
expressed only in anterior cells, this could explain the so far
puzzling observations that prestalk cells differentiate at the anterior region but that DIF levels are actually higher at the posterior region.
After the initial induction of DIF competence, cAMP becomes a repressor
of prestalk gene expression. This function can again be mediated by
cAR1, cAR2, and cAR3.
| |
INTRODUCTION |
Recent years have seen the discovery
of critical roles in animal development for serpentine receptors, which
are usually coupled to heterotrimeric G proteins. The insect sigaling
peptides hedgehog and wingless and their mammalian counterparts sonic
hedgehog, desert hedgehog, and indian hedgehog and the wnt factors
control a multitude of inductive events during all stages of
embryogenesis. The hedgehog signal is detected by two different
serpentine receptors, smoothened (1, 40) and patched
(21, 38), whereas the wingless or wnt signal is detected by
the serpentine receptor D-frizzled-2 (3). In the social
amoeba Dictyostelium discoideum, serpentine cyclic AMP
(cAMP) receptors (cARs) control induction of cell differentiation
during the entire course of development. Starving cells secrete cAMP
pulses that induce chemotaxis and expression of genes required for the
aggregation process. Cells aggregate to form mounds, which ultimately
transform into fruiting structures that consist of a globular spore
mass supported by a column of stalk cells. cAMP induces entry into the
spore differentiation pathway as well as synthesis of a lipophilic
factor, differentiation-inducing factor (DIF), which induces entry into
the stalk differentiation pathway (see reference 5).
At an early stage of development cAMP synergizes with DIF to induce
prestalk genes, but later it becomes an inhibitor of stalk gene
expression (2). cARs were shown previously to mediate
induction of aggregative genes by cAMP pulses (20) as well
as cAMP induction of prespore genes and repression of prestalk genes
(31, 37). Remarkably, the target for the latter critical
step in cell fate determination is glycogen synthase kinase 3 (GSK-3),
a zeste white-3 homolog, which is the target for the effects of
wingless and wnt in insects and vertebrates, respectively (7,
34).
Four cARs, showing 54 to 69% amino acid identity, are expressed in a
stage- and cell-type-specific manner. cAR1 is predominantly expressed
before and during aggregation (18). cAR3 is expressed at
late aggregation, and expression is later restricted to the prespore
cell population (13, 44). cAR2 and cAR4 are both expressed
exclusively in the prestalk cell population after aggregation (19,
30). cAR knockout cell lines were generated to examine the role
of the individual cARs in Dictyostelium development. car1 null cells neither aggregate nor express developmental
genes but can be triggered to express aggregative and
postaggregative genes by stimulation with cAMP (37, 39).
car3 null cells aggregate and develop normally
(13). car1 car3 double gene disruptants do not
aggregate, and developmental gene expression cannot be restored with
cAMP, indicating that cAR1 or cAR3 shows functional redundancy and that
either one or the other has to be present for gene induction to occur
(10, 36). car2 null cells are blocked in the
mound stage, while car4 null cells show abnormal slug
morphogenesis and culmination. Both lines show reduced expression of
prestalk genes and enhanced expression of prespore genes (19, 29).
To understand the function of the four cARs, it is essential to know
whether each receptor is coupled to a specific signal transduction
pathway that controls a specific cell differentiation event or whether
each receptor can activate multiple cell differentiation pathways. In
the latter case, it is not the presence of a specific receptor that
determines whether a response occurs but the availability of the
downstream signaling pathway. To determine whether individual receptors
have unique functions in developmental gene expression, we examined
gene regulation in cell lines that display about equal levels of cAR1,
cAR2, and cAR3 in a car1 car3 mutant background. Our results
show that with two exceptions, all three receptors can transduce both
the excitation and adaptation components of the different
cAMP-regulated gene induction events with almost equal levels of
efficiency.
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MATERIALS AND METHODS |
Materials.
2',3'-Isopropylidene adenosine (IPA),
5'-N-ethylcarboxyadenosine (NECA), and G418 were obtained
from Sigma (St. Louis, Mo.), adenosine 3',5'-monophosphorothioate
Sp-isomer (Sp-cAMPS) was obtained from Biolog Life Science Institute
(Bremen, Germany), and DIF was obtained from Affinity Research Products
(Exeter, United Kingdom).
Cell lines and culture conditions.
The car1 car3
double mutant cell line RI9 (10) was transformed with the
extrachromosomal vector PJK1 (15, 17), with PJK1 harboring a
gene fusion of the coding region of either the cAR1 or the cAR2 gene
with the actin15 promoter, yielding cell lines act15cAR1
and act15cAR2, or with the integrating vector BS18
harboring a gene fusion of the cAR3 coding region with the actin15
promoter (11), yielding cell line act15cAR3. All
cell lines, including wild-type AX3 cells and aca null (27) cells, were grown in standard axenic medium, which was supplemented with 20 µg of G418 per ml for lines transformed with PJK1- or BS18-derived vectors.
Gene induction procedures.
For induction of aggregative and
postaggregative gene expression, cells were harvested at the late log
phase of development, washed with 10 mM phosphate buffer (pH 6.5), and
subsequently shaken at 150 rpm in phosphate buffer supplemented with
0.5 mM MgCl2 and 0.5 mM CaCl2 (DB) at
107 cells/ml and 22°C. Cells were challenged by different
regimens of cAMP stimulation for 6 h, washed and resuspended to
5 × 106 cells/ml in DB, and incubated for an
additional 8 h as indicated in the figure legends.
For induction of stalk gene expression, cells were incubated in
monolayers (2). In short, cells were resuspended in stalk salts (10 mM KCl-2 mM NaCl-1 mM CaCl2 in 10 mM MES
[morpholinoethanesulfonic acid] [pH 6.2]) to 5 × 106 cells/ml and incubated at 22°C in 10-ml petri dishes.
After 8 h, cAMP was added to a final concentration of 5 mM and
incubation was continued for a further 16 h. Cells had then formed
tight aggregates, which were dissociated by forcing them through a
21-gauge needle, and cells were incubated in stalk salts at 5 × 106 cells/ml for 8 h, with variables as indicated in
the figure legends.
RNA isolation and analysis.
Total cellular RNA was isolated
from 2.5 × 107 cells (23), size
fractionated on 1.5% agarose gels containing 2.2 M formaldehyde, and
transferred to GeneScreen membranes. Northern blot transfers were
hybridized to [32P]dATP-labeled DNA probes according to
standard procedures and exposed to X-ray films. The optical densities
of specific mRNA bands were quantitated with an LKB Ultrascan
densitometer.
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RESULTS |
cAMP-regulated gene expression in constitutive cAR expressors.
Starving cells secrete cAMP pulses in the nanomolar concentration
range, which upregulate expression of aggregative genes such as
cAR1 and csA (20, 25). Once cells have
aggregated, a micromolar concentration of cAMP is required to first
induce non-cell-type-specific genes, such as RasD and
CP2 (26, 28), and prespore genes, such as
psA and CotC (22, 31). To determine to
what extent cAR1, cAR2, or cAR3 can mediate cAMP-induced gene expression, we used three derivatives of the car1 car3
double mutant line RI9 which constitutively express either cAR1, cAR2, or cAR3 under the control of the actin15 promoter. These cell lines are
called act15cAR1, act15cAR2, and
act15cAR3, respectively, and they express cAR1, cAR2, or
cAR3 protein at levels that are similar to the level of expression of
cAR1 protein in aggregation-competent wild-type cells (17).
To induce aggregative gene expression, cells were stimulated for 6 h with 30 or 600 nM cAMP pulses at 6-min intervals or with a 300 µM
cAMP pulse every hour. cAR1, cAR2, or cAR3 shows an affinity of 290 nM,
>5 µM, or 490 nM, respectively (12, 16). The 30 nM cAMP pulse was chosen to accomodate the high-affinity cAR1 and cAR3, and the
600 nM pulse was chosen to accomodate the low-affinity cAR2. At higher
concentrations cellular phosphodiesterase activity becomes insufficient
to degrade cAMP between pulses, which are then detected as a continuous
signal. The 300 µM stimulus is perceived as a continuous signal and
was used to establish whether the cells adapt. After the initial 6 h of incubation, cells were stimulated for 8 h with 300 µM cAMP
to induce postaggregative genes.
Figure
1 shows that induction of the
aggregative gene
csA by cAMP pulses occurs with equal
efficiencies in
act15cAR1,
act15cAR2, and
act15cAR3 cells but not at all in RI9 cells. Six hours of
stimulation
with 300 µM cAMP could not induce the
csA gene
in either mutant,
indicating that
csA induction in
act15cAR1,
act15cAR2, and
act15cAR3
cells is subject to cellular adaptation. The induction of
csA by 30 nM cAMP pulses in the
act15cAR2 cells
is unexpected, if we consider the low affinities of
these receptors.
Possibly, the 50% effective concentration (EC
50)
of cAMP
for gene induction is much lower than the dissociation
constant
(
KD) of the receptor that mediates the response.
This
is the case for the cAMP-induced chemotactic response, which shows
an EC
50 of 3 nM (
41) and is mediated by cAR1,
which has a
KD of 290 nM. Another possibility is
that cAMP pulses cause signal
amplification by inducing a cAMP relay
response. To measure the
actual EC
50 for pulse-induced gene
expression, we examined the
dose-response relationship for this
response in
aca null cells
and in wild-type cells in the
presence of the cAMP relay inhibitor
caffeine. In the first experiment
(data not shown) we chose a
range from 1 to 30 nM cAMP and found that
csA induction was almost
optimal at 1 nM. We then chose a
range from 0.01 to 10 nM and
found that induction was half-maximal
around 0.5 nM cAMP (Fig.
2). This value
is about 500-fold lower than the
KD of cAR1,
which
mediates this response. If we take this observation into account,
it is not surprising that cAR2 with an affinity of >5 µM can
transduce
responses to 30 nM cAMP pulses.
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FIG. 1.
Induction of aggregative and postaggregative gene
expression. RI9, act15cAR1, act15cAR2, and
act15cAR3 cells were incubated in DB in the absence of
stimuli, with either 30 or 600 nM cAMP pulses delivered at 6-min
intervals, or with 300 µM cAMP delivered at 60-min intervals. Cells
were then incubated for an additional 8 h with 300 µM cAMP/h.
Samples were taken for RNA extraction at 0, 2, 4, 6, 12, and 14 h
of incubation. Northern blots were hybridized to
32P-labeled DNA probes for the aggregative gene
csA, the postaggregative genes RasD and
CP2, and the prespore genes psA and
CotC. The experiment was repeated twice with similar
results.
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FIG. 2.
Dose-response relationship of cAMP pulse-induced gene
induction in wild-type cells. AX3 or aca cells were
harvested in late log phase and stimulated in the presence (AX3 cells)
or absence (aca cells) of 5 mM caffeine and the indicated
concentrations of cAMP pulses delivered at 6-min intervals. RNA was
isolated after 6 h of incubation and probed with
32P-labeled csA cDNA. The inset shows results of
an experiment with aca cells, while the graph indicates
means and standard deviations of results from two experiments with AX3
and caffeine and one experiment with aca cells.
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The postaggregative genes
RasD and
CP2 are
induced most effectively in the
act15cAR1,
act15cAR2, and
act15cAR3 cell lines in response
to 300 µM cAMP after an initial 6-h
period of starvation. However,
especially for
RasD, high concentrations
of cAMP can also
induce some expression within the first few hours
of starvation, which
is particularly evident in
act15cAR3 cells. In general, the
levels of induction of
RasD and
CP2 are
remarkably similar in all
act15cAR cell lines.
The levels of induction of the prespore genes
psA and
CotC resemble those of the postaggregative genes in the
sense that all
three
act15cAR cell lines show the same
levels of induction by stimulation
with 300 µM cAMP after a 6-h
period of starvation. However, there
is a difference. Prespore gene
induction is most efficient in
cells that were prestimulated with cAMP
pulses. Unlike postaggregative
gene induction, prespore gene
induction is actually inhibited
by prestimulation with 300 µM cAMP in
the first 6 h of development.
The conditions that induce
competence for either postaggregative
or prespore gene induction
are apparently not the same. The expression
levels of prespore and
postaggregative genes after 8 h of cAMP
stimulation were
approximately similar in the wild-type (AX3)
and the
act15cAR cell lines, and there was no significant gene
induction in
the absence of cAMP (Fig.
3).
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FIG. 3.
Comparison of levels of gene induction in wild-type
cells and act15cAR cells. act15cAR1,
act15cAR2, act15cAR3, and wild-type AX3 cells
were first stimulated for 6 h with 30 nM (act15cAR1,
act15cAR3, and AX3) or 600 nM (act15cAR2) cAMP
pulses, washed, and subsequently incubated for 0 or 8 h in the
presence and absence of 300 µM cAMP per h. mRNA was isolated and
hybridized to CP2 and psA cDNAs. The experiment
was repeated once with similar results.
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Development of act15cAR cell lines.
car1
car3 cells cannot aggregate and form fruiting bodies, but
development is restored by expression of either cAR1 or cAR3. act15cAR2 cells remain defective in cell aggregation,
presumably because these low-affinity receptors cannot initiate
spontaneous cAMP oscillations (17). We tested whether cAR2
cells could go through development after being stimulated for 6 h
with 600 nM cAMP pulses, and they could not (Fig.
4). When cells were additionally treated for 8 h with 300 µM cAMP, they formed tight aggregates in
suspension, which developed into slugs and fruiting bodies of normal
sizes and appearance when they were deposited on agar.
act15cAR2 cells most likely cannot aggregate and develop,
because they are not capable of producing the cAMP concentrations that
are required to activate the low-affinity cAR2s. However, they will go
normally through the later stages of development after exposure to the
appropriate cAMP stimuli to induce expression of developmentally regulated genes.
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FIG. 4.
Phenotype of cAR mutants after cAMP treatment. After
treatment with either 30 nM cAMP pulses (RI9, act15cAR1,
and act15cAR3) or 600 nM cAMP pulses
(act15cAR2) for 6 h and after subsequent incubation
with 300 µM cAMP for 10 h, aliquots of 10 µl of
108 cells/ml were placed on nonnutrient agar and left to
develop at 22°C for 24 h. Wild-type AX3 cells (WT) were placed
directly on nonnutrient agar after being harvested from growth
medium.
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Adenosine regulation of prespore gene expression in
act15cAR cell lines.
The data presented above
yield no clue as to which cAR mediates specific gene regulatory events
during normal development. In wild-type cells, cAMP induction of
prespore gene expression is inhibited by millimolar concentrations
of adenosine and by micromolar concentrations of the adenosine analogs
NECA and IPA, which cannot be phosphorylated by an extracellular
adenosine kinase (37, 42, 43). Adenosine also inhibits cAMP
binding (24, 41), and recent studies showed that only
binding of cAMP to cAR1, but not to cAR2 or cAR3, is inhibited by
adenosine (17a). To identify the cAR that mediates
prespore gene induction and to verify that the inhibitory effects
of adenosine are due to inhibition of cAMP binding activity, we tested
the effects of adenosine, IPA, and NECA on cAMP induction of
prespore gene expression in the act15cAR cell lines.
Figure 5 shows that 100 µM IPA, 1 mM
NECA, and 3 mM adenosine inhibit psA and CotC
induction completely in the act15cAR1 cells but not at all
in act15cAR2 and act15cAR3 cells. This result
indicates that cAR1 transduces cAMP induction of prespore gene
expression during normal development.
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FIG. 5.
Effects of adenosine analogues on prespore
gene expression. act15cAR1 and act15cAR3
cells were prestimulated for 6 h with 30 nM cAMP pulses, and
act15cAR2 cells were prestimulated with 600 nM cAMP pulses.
Cells were incubated for an additional 8 h in DB in the absence
and presence of 30 µM Sp-cAMPS and the indicated concentrations
of adenosine, IPA, or NECA. RNA was isolated and probed with
32P-labeled psA and CotC cDNAs. The
experiment was repeated once with similar results.
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cAMP regulation of the DIF-inducible gene ecmB.
The
DIF-inducible gene ecmB is typically downregulated by
nanomolar concentrations of cAMP (2, 37). A micromolar
concentration of cAMP was found to have varied effects on
ecmB induction. In cells that have just finished
aggregation, a micromolar concentration of cAMP stimulates
ecmB induction, presumably by inducing competence for
ecmB induction by DIF. This effect disappears once cells are competent and a micromolar concentration of cAMP may then just act as a
supersaturated inhibitory signal (2, 35, 37). We determined
whether the different cARs could mediate the induction of competence
for DIF by a micromolar concentration of cAMP and inhibition
of the DIF response by nanomolar concentrations of cAMP. RI9,
act15cAR1, act15cAR2, and act15cAR3
cells were first incubated for 16 h with 5 mM cAMP to induce competence and then for 8 h with DIF or DIF plus 1 µM Sp-cAMPS (equivalent to 20 to 60 nM cAMP).
Figure
6 shows that DIF did not
induce
ecmB in the RI9 cells. In the other cell lines,
DIF always induced
ecmB to levels above
those in
unstimulated cells and induction was always inhibited
by 1 µM
Sp-cAMPS. However,
ecmB was induced in
act15cAR2
cells to 10-fold higher levels than in either
act15cAR1 or
act15cAR3 cells. Since the
cAR2 gene is still intact in the
car1 car3 mutant
cell lines and is probably expressed at this developmental
stage
(except in the RI9 cells, which do not reach this stage),
we cannot
exclude the possibility that it is actually the single
copy of
endogenous cAR2 that induces the lower level of competence
for DIF
induction in the
act15cAR1 and
act15cAR3 cells.
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FIG. 6.
Prestalk gene induction and repression in
act15cAR cell lines. RI9, act15cAR1,
act15cAR2, and act15cAR3 cells were
preincubated for 16 h in monolayers in the presence of 5 mM cAMP,
washed, and incubated for 8 h without additives, with 100 nM DIF,
and with 100 nM DIF plus 1 µM Sp-cAMPS. RNA was isolated and probed
with 32P-labeled ecmB mRNA. Sixteen-hour and
5-day exposures of the same Northern blot are shown. The experiment was
repeated once with similar results.
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| |
DISCUSSION |
cAR1, cAR2, and cAR3 can mediate both adapting and nonadapting gene
induction responses.
A remarkable outcome of the experiments
presented here is that cAR1, cAR2, and cAR3 show almost complete
redundancy of function as far as induction of aggregative,
postaggregative, and prespore genes is concerned. On one hand, all
three receptors can transduce the effects of cAMP pulses on aggregative
gene expression. This response involves activation of an excitatory as
well as an inhibitory pathway, since it cannot be induced by continuous
stimuli. On the other hand, the three cARs can also transduce the
effect of a constant cAMP stimulus, in which adaptation does not play a role. These observations may explain why the phenotypes of null mutants
for the individual cARs are not very severe. car1 cells do
not aggregate spontaneously, but development can be fully restored by
stimulation with cAMP (36, 39). car3 cells show
normal development (13). car2 and car4
cells show reduced prestalk differentiation (19, 29), but in
none of the null mutants is cAMP or DIF-induced gene expression
completely blocked. Our current data indicate that the other cARs may
take over the function of the deleted cAR, depending on whether they
happen to be expressed at the stage or in the cell type where the
deleted cAR has its function.
There is an interesting discrepancy between data presented here and
data presented in a previous study with
car1 cells. In
car1 cells, aggregative genes could not be induced by 30 nM
cAMP
pulses but could be induced both by 300 nM pulses and a constant
300 µM cAMP stimulus (
36). Since cAR3 is the only other
cAR
expressed at that stage of development, it was concluded that
cAR3 mediated the response but that it required higher concentrations
of cAMP and was not sensitive to adaptation. We show here that
cAR3 can
also transduce 30 nM pulses and is susceptible to adaptation.
The
difference between the two experiments is probably the number
of cAR3
receptors. In this study cAR1 and cAR3 expression levels
were similar
to cAR1 expression levels in aggregating cells (about
100,000 sites/cell). However, their expression levels in
car1 cells
are so low that cAMP binding activity is undetectable (
39).
It is plausible that in
car1 cells with few cAR3 binding
sites,
a very high percentage of occupied receptors and therefore high
concentrations of cAMP are required to transduce the response.
Additionally, the adaptation pathway may require higher numbers
of
occupied receptors than the excitation pathway and these levels
may not
have been reached in the
car1 cells.
Differences in acquisition of competence for postaggregative and
prespore gene induction.
In the act15cAR1,
act15cAR2, and act15cAR3 lines, induction of
the prespore genes psA and CotC by micromolar
concentrations of cAMP requires prestimulation with cAMP pulses.
Prestimulation with micromolar concentrations of cAMP inhibits
subsequent induction of prespore gene expression. Since in the
act15cAR cell lines, the cARs are already present from the
onset of starvation, components downstream of cAR must probably be
first induced by the pulse regimen. Two signal transduction components, the mitogen-activated protein kinase ERK2 and GSK-3, are specifically required for prespore, but not for postaggregative, gene expression (6, 7). The developmental regulation of GSK-3 has not yet been reported. ERK2 expression is strongly upregulated at the early
aggregation stage, but it is not yet known whether the gene is cAMP
pulse induced (33). Postaggregative gene expression occurs
optimally after a few hours of starvation in the absence of cAMP but
does not require cAMP pulses. Expression of these genes depends on
expression of the transcription factor G-box binding factor, which is
itself induced by a continuous cAMP stimulus (8, 32).
Only cAR1 mediates adenosine inhibition of cAMP-induced
prespore gene expression.
Since cAR1, cAR2, and cAR3 are all
expressed in postaggregative wild-type cells and all can potentially
mediate cAMP induction of prespore gene expression as shown here,
it is not clear which cAR actually mediates the response in wild-type
cells. During normal development, cAMP-induced prespore gene
induction is inhibited by adenosine and more effectively by its
analogues IPA and NECA, which cannot be phosphorylated (37, 42,
43). We here show that only cAR1 and not cAR2 or cAR3 can mediate
adenosine inhibition of prespore gene expression. This implies that
cAR1 mediates prespore gene induction in wild-type cells.
ecmB induction by DIF may require cAR2.
There is
another exception to the functional redundancy of cAR1,
cAR2, and cAR3. DIF induction of the prestalk gene ecmB
is over 10 times more effective in act15cAR2 than in
act15cAR1 and act15cAR3 cells and may in
the latter two lines be entirely due to endogenous cAR2. cAR2 is
probably involved in inducing competence for ecmB induction
by DIF, since cAMP itself does not induce ecmB expression.
cAR2's involvement in competence for ecmB induction would
also explain why ecmB expression is reduced in
car2 mutants (29). The induction of DIF
competence by cAR2 may actually solve a conundrum that has puzzled
workers in this field for several years. Prestalk gene expression is
specifically associated with the anterior region of the slug. However,
this region has the highest levels of the DIF-degrading enzyme
DIF-dechlorinase and the lowest levels of DIF (4, 9, 14). In
wild-type cells, cAR2 is exclusively expressed in the anterior region
(29, 30). If only cAR2 can make the cells competent for DIF,
then the absolute levels of DIF are much less important in determining
where ecmB is turned on than the expression pattern of cAR2.
The formation of the prestalk expression pattern is in that case a
function of the regulation of cAR2 expression.
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ACKNOWLEDGMENT |
This work was supported by grant 805-31.051 of the Life Sciences
Foundation of The Netherlands Organisation for Scientific Research.
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FOOTNOTES |
*
Corresponding author. Mailing address: Cell Biology
Section, Institute for Molecular Plant Sciences, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands. Phone: 31-71-5274927. Fax:
31-71-5274999. E-mail: Schaap{at}Rulbim.Leidenuniv.nl.
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Molecular and Cellular Biology, October 1998, p. 5744-5749, Vol. 18, No. 10
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
