From the Howard Hughes Medical Institute, Department of
Biochemistry and Cell Biology, MS-140, Rice University,
Houston, Texas 77005-1892
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INTRODUCTION |
A general premise of developmental biology is that mechanisms
exist that sense the density of cells of a particular type. These
mechanisms all use a signal molecule, which is secreted by the specific
cell type. Examples of cell density sensing mechanisms can be seen in
bacteria (for a review, see Ref. 1), Dictyostelium (for a
review, see Ref. 2), and mammals (3). We have been using
Dictyostelium to study cell density sensing, because the simplicity of this eukaryote greatly facilitates examining the physics
and biochemistry of a cell density sensing mechanism. Dictyostelium normally exists as amebas that eat bacteria.
When they overgrow their food supply and starve, they cease dividing and aggregate using pulses of cyclic AMP as a chemoattractant. The
aggregated cells develop into a fruiting body consisting of spores
supported on a column of stalk cells (reviewed in Refs. 4 and 5).
When a Dictyostelium cell starving in the presence of a high
density of other starving cells receives a pulse of cAMP, it releases a
burst of cAMP to relay the signal, moves toward the source of cAMP, and
expresses specific classes of genes (4, 6-13). The incoming cAMP pulse
is sensed by cAR1 cell surface cAMP receptors, which in turn activate
their associated G proteins (14-16). The G
subunits, along with
a cytosolic protein called CRAC, transiently activate adenylyl cyclase,
while the G
2 subunits activate guanylyl cyclase (for review, see
Refs. 8 and 9). Activation of cAR1 also causes a transient influx of
Ca2+, which is G protein-independent (17).
The aggregation and development of starving cells requires the presence
of an extracellular molecule, the conditioned media factor
(CMF)1 (18, 19). CMF is an
80-kDa protein with no similarity to any known protein (20). CMF is
both secreted and sensed by starving cells. When starved, CMF antisense
cells do not aggregate unless they are allowed to develop in the
presence of exogenous CMF or recombinant CMF (rCMF) (20). This suggests
that the function of CMF is to coordinate the development of large
fruiting bodies by triggering aggregation only when most of the cells
in an area have starved, as signaled by a high level of CMF. Without
such a mechanism that senses the density of starved cells, cohorts of
cells that starved at the same time might each form a small, ineffective fruiting body.
To determine whether CMF might be able to mediate density sensing in
the wild, we calculated the diffusion of CMF from a cell sitting on a
soil surface or submerged in water (21). Knowing that the CMF secretion
rate is 12 molecules/cell/min, we showed that the concentration of CMF
in the immediate vicinity of an isolated starved cell remains below 0.3 ng/ml, the half-maximal activity of CMF, by a factor of at least 10 even after 10 h of continuous secretion and correcting for the
presence of CMF receptors. Similar calculations with an array of 2046 cells showed that a CMF concentration adjacent to the cells of 0.3 ng/ml can be reached after 2 h of secretion. Interestingly, for
aggregates of fewer than roughly 45 cells, the CMF concentration can
never rise to 0.3 ng/ml, indicating that CMF could be used to sense
whether there are more than 45 cells in an aggregate (2). We also found that the solutions are not unique: many combinations of secretion rate,
diffusion coefficient, and threshold sensitivity will allow density
sensing. These calculations illustrate that as a general principle
cells can sense their local density by simultaneously secreting and
recognizing a molecule. Such a mechanism could also be used for
determining the total number of cells in a tissue.
CMF regulates several aspects of cAMP signal transduction (22). The
activations of Ca2+ influx, adenylyl cyclase, and guanylyl
cyclase in response to a pulse of cAMP are strongly inhibited in cells
lacking CMF but are restored by a 10-s exposure of cells to CMF. These
cells retain normal levels of the cAMP receptor and cAMP-induced
binding of GTP to membranes, suggesting that the interaction of the
cAMP receptor with G proteins in vitro is not measurably
affected by CMF. However, the activation of adenylyl cyclase by GTP
S
requires cells to have been exposed to CMF. CMF thus appears to control aggregation by regulating cAMP signal transduction at a step after cAMP
induces G
2 to exchange GDP for GTP, but before G
2-GTP can activate adenylyl cyclase. CMF appears to regulate cAMP signal transduction by regulating the lifetime of the G
2-GTP conformation. We found that in lysates, approximately 250 molecules of GTP bind to a
cell's membrane in response to a pulse of cAMP in the presence or
absence of CMF (22, 23). After cAMP stimulation, the hydrolysis rate of
GTP to GDP is approximately 240 molecules in 3 min in the absence of
CMF but roughly 51 molecules in 3 min in the presence of CMF. The
cAMP-activated GTP binding and GTP hydrolysis are abolished in cells
lacking G
2 (23). This suggests that CMF, and thus cell density,
controls cAMP-stimulated G
2-GTPase activity.
At the developmental stage when G
2 is mediating cAMP signal
transduction, G
1 is also being expressed and potentially mediating other signal transduction pathways. However, cells lacking G
1 have
essentially normal cAMP-stimulated Ca2+ influx and
development through aggregation, although they exhibit a slight delay
in the rate of cGMP accumulation in response to a pulse of cAMP (17,
24). Thus, G
1 appears to be superfluous for early development. In
cells lacking G
1, 3'NH-cAMP no longer inhibits phospholipase C (25).
During late development, the absence of G
1 causes fruiting bodies to
have abnormally thin stalks (24, 26) and causes decreased
1,2-diacylglycerol accumulation (27). Cells overexpressing G
1 or
containing a constitutively active form of G
1 have roughly normal
aggregation and form normal looking slugs but then form very
abnormal fruiting bodies with thick basal disks, which are accompanied
by increased 1,2-diacylglycerol accumulation (26, 27).
In this report, we show that CMF regulates cAMP signal transduction by
releasing G
from G
1; the released G
then activates PLC,
which in turn causes an inhibition of the G
2 GTPase.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Ax4 wild-type or transformant cells were grown
in shaking culture in HL5 medium as described by Gomer et
al. (19). Cells lacking the G
-subunits G
1 (26) or G
2
(28) were a gift from Dr. Rick Firtel (University of California at San
Diego), and cells lacking the G
subunit (29) were a gift from Dr.
Peter Devreotes (Johns Hopkins University). Conditioned medium (CM) was
prepared by starving Ax4 cells at 5 × 106 cells/ml in
PBM (20 mM KH2PO4, 10 µM CaCl2, 1 mM MgCl2,
pH 6.1 with KOH) in shaking culture for 20 h and then clarifying
the conditioned medium as described by Gomer et al. (19).
The production of CMF by cells was assayed following the procedure of
Gomer et al. (19). To examine the effect of conditioned
medium or recombinant CMF on cell aggregation, cells were starved in
submerged monolayer as described previously (19) at surface densities
of 5, 10, 20, 40, 80, and 160 × 103
cells/cm2 in PBM, PBM supplemented with 10% conditioned
medium, or PBM supplemented with 1 ng/ml recombinant CMF. U-73122 and
U-73343 (Biomol, Plymouth Meeting, PA) was dissolved in dimethyl
sulfoxide (Sigma) and added to cells upon starvation. For photography
and videotaping, a hole was punched in the bottom of a plastic Petri dish with a heated test tube. The burr was sanded off, and a glass coverslip was glued with RTV (Dow Corning, Midland MI) on the underside
of the dish over the hole. Cells were starved in monolayer culture in
the dish at a surface density of 3 × 104
cells/cm2 under 2 mm of buffer. Cells on the coverslip were
examined in situ with a Nikon (Melville, NY) 60 × 1.4 NA oil immersion phase contrast objective on a Nikon Diaphot inverted
microscope with an 8008 camera back for photography and an Ikegami
ITC-400 TV camera for video. Photographs were taken on Kodak T-Max
p3200 film, which was then developed with Kodak D76, and videotaping was done with a Sony Watchcorder recording at 1 frame/s, which was then
played back at 4 frames/s.
Generation of CMF Deletion Mutant--
To generate a deletion
mutant of CMF, we replaced 526 nucleotides coding for the active region
of the protein with the blasticidin resistance marker under control of
the actin 15 promotor (30). A genomic clone of CMF was digested with
HindIII and XbaI to remove part of the CMF coding
region. The plasmid carrying the blasticidin cassette (pBsr2
Bam) was
digested with HindIII and XbaI to remove the
cassette. The blasticidin cassette was then ligated into the modified
genomic clone. The resulting plasmid contains 1 kilobase pair of
genomic DNA from the CMF gene, the Blasticidin cassette replacing 526 nucleotides of CMF coding sequence, followed by 1.2 kilobase pairs of
CMF genomic sequence. For transformation, the plasmid was cleaved with
PvuII, and 20 µg of the fragment containing the construct
was gel-purified using Geneclean (Bio 101, Vista, CA). The fragment was
then used to transform DH1 cells, following the procedure of Shaulsky
(31).
Effect of GTP
S on the Binding of CMF to Membranes--
To
examine the effect of GTP
S on the binding of CMF to membranes, cells
were starved in PBM at 1 × 107 cells/ml in shaking
culture. At 6 h, the cells were collected by centrifugation at
800 × g for 5 min and resuspended in ice-cold PBM;
recentrifuged; and resuspended in ice-cold PBM, 2 mM
dithiothreitol to 1 × 107 cells/ml. All subsequent
operations were done on ice in a cold room. 2 ml of the cells were
immediately mixed with 15 µl of either water or 10 mM
freshly dissolved GTP
S (Sigma) and were then lysed by passage
through two Cameo 25N 5-µm pore size syringe filters (MSI, Westboro,
MA). For both homogenates, a 0.5-ml aliquot was mixed with 1 µl of 50 mM cAMP. Duplicate assay tubes contained 20 µl of
0.5 × PBM, 50 ng of 125I-rCMF, 0.35 mg/ml bovine
serum albumin, 10 mM KCl or the same mixture containing 600 ng of unlabeled rCMF. 80 µl of the cell homogenate was added and
incubated on ice for 15 min. The mixes were centrifuged for 4 min in a
15,000 rpm microcentrifuge, and the supernatants were removed. The
tubes were recentrifuged for 1 min, and the remaining supernatant was
removed. The pellets were resuspended in 80 µl of 1 M
acetic acid and then mixed with 1.1 ml of Scintiverse II scintillation
mixture (Fisher) and counted.
Signal Transduction Assays--
The cAMP relay response was
measured following the procedure of Van Haastert (32), with the
modification that the cells were starved for 5 h at 5 × 105 cells/ml. The cells were washed and collected by
centrifugation and then assayed immediately without an intervening
incubation at high cell density. The low cell density starvation and
rapid assays were done to prevent CMF from accumulating to its
threshold concentration. Briefly, 5 × 106 cells in
0.1 ml were stimulated with 10 µM 2'-deoxy-cAMP (a
functional cAMP analog) in the presence of 10 mM
dithiothreitol. At 0 and 3 min after stimulation, the cells were lysed,
and cAMP content was measured using an isotope dilution cAMP assay kit
(Amersham Pharmacia Biotech). Where indicated, 1 ng/ml of rCMF was
added to the cells 90 s prior to the 2'-deoxy-cAMP pulse. The
production of cGMP in response to 10
7 M
extracellular cAMP was determined in a similar manner following the
procedure of Kesbeke et al. (33) using a cGMP assay kit (Amersham Pharmacia Biotech). The amount of cGMP produced was measured
at 0, 5, 10, 20, and 45 s after cAMP stimulation for cells exposed
to no CMF or to 1 ng/ml rCMF for 30 s prior to cAMP stimulation.
For all cell lines examined, the cGMP concentration peaked at 5-10 s
after cAMP stimulation, and the ratio of cGMP levels at 5 s to the
amount at 0 s was used for further analysis. The uptake of
Ca2+ by cells in response to a pulse of cAMP was determined
as described by Milne and Devreotes (17) with the following
modifications. Cells were starved for 6 h at low cell density
(1 × 106 cells/ml) and were resuspended to a
concentration of 1 × 106 cells/ml in H buffer (20 mM Hepes, 5 mM KCl, pH 7.0). The cell suspension was incubated in uptake medium for 90 s and then was stimulated with a 2 µM or a 100 µM pulse of
cAMP and incubated for an additional 90 s. The cells were then
collected by centrifugation at 320 × g for 1 min. To
measure Ca2+ uptake into cells in the presence of CMF, the
uptake medium was supplemented with 1 ng/ml recombinant CMF (34). The
standard Ca2+ influx assay involves harvesting the cells by
centrifugation at 11,600 × g for 4 s (35). We
found that centrifugation under these conditions severely deformed the
cells, and so did the Ca2+ influx assays using a much
gentler centrifugation to harvest the cells. However, we obtained
essentially similar results using the higher speed spin (data not
shown). GTP binding to membranes and membrane-associated GTPase
activities was assayed as described by Brazill et al. (23).
IP3 production was determined following the procedure of
Van Haastert (36), using a kit (Amersham Pharmacia Biotech) for the
IP3 assay, with the exception that the assay was performed
using 100 µl of supernatant from the neutralized cell extract. The
stimulus for the assay was 1 ng/ml rCMF in the presence of 5 µg/ml
U-73122, 5 µg/ml U-73343, or no drug.
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RESULTS |
CMF Binds to a G Protein-coupled Receptor That Interacts with
G
1--
We previously showed that disruption of the G
subunit
abolishes the ability of CMF to down-regulate cAMP binding (37). This
suggested that CMF binds to a receptor that interacts with a G protein.
A common aspect of G protein-linked receptors is that the
nonhydrolyzable GTP analog GTP
S inhibits the binding of ligand to
membranes. We developed an assay buffer that allows the specific
binding of [125I]rCMF to membranes. In a typical 0.1-ml
reaction containing 50 ng of free [125I]rCMF, 4 ng (2300 DPM) of [125I]rCMF bound to the membranes from 1.6 × 106 cells; typically, 1.2 ng of the bound
[125I]rCMF was competed off by 6 µg/ml cold rCMF.
GTP
S strongly inhibited the specific (i.e. competable)
CMF binding (Fig. 1). Thus, the putative
CMF receptor is most likely linked to a G protein.

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Fig. 1.
GTP S-induced inhibition of CMF binding to
membranes from wild type and transformants lacking specific G
proteins. Cells were starved in shaking culture for 6 h;
washed; harvested; resuspended in ice-cold PBM; and lysed, in the
presence or absence of GTP S, by passage through a 5-µm pore
diameter filter. The lysates were mixed with a mixture containing
[125I]rCMF in the presence or absence of a 100-fold
excess of cold rCMF and incubated on ice for 5 min. The mixtures were
then pelleted by centrifugation. The supernatant was removed, the
pellets were recentrifuged, and the last bit of supernatant was then
removed. The pellets were resuspended in scintillation mixture and
counted. For each experiment, each of the four measurements (with or
without GTP S; with or without cold CMF) was done in triplicate and
then averaged. Specific binding was determined by subtracting binding
in the presence of cold rCMF from the binding in the absence of cold
rCMF. The ratio of specific binding on the presence of GTP S to the
specific binding in the absence of GTP S was then determined. The
graph shows the average ratio ± S.E. from four separate
experiments. The reductions in CMF binding caused by GTP S for Ax4,
G 2, and G were all significant (p < 0.005).
There was no significant effect of GTP S on CMF binding in G 1 null
cells (p < 0.15), and there was no significant
difference in the reduction of CMF binding caused by GTP S between
Ax4 and G 2 null cells (p < 0.1) or between Ax4 and
G null cells (p < 0.4).
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To determine if the CMF receptor couples to a known G protein, we
examined the effect of GTP
S on the binding of
[125I]rCMF to membranes from cells lacking specific G
proteins. GTP
S caused a similar amount of inhibition of the binding
of CMF to membranes from wild-type cells and cells lacking G
or
G
2 (Fig. 1). However, in cells lacking G
1, GTP
S was unable to
inhibit the binding of CMF to membranes (Fig. 1). The data thus suggest that the putative CMF receptor interacts with G
1 more than it interacts with G
2 and that the G
subunit is not necessary for the
GTP
S inhibition of CMF binding.
G
1 Mediates CMF Signal Transduction, and the Absence of G
1
Mimics the Presence of CMF--
We have previously observed that
extracellular CMF is necessary for wild-type cells to aggregate when
starved at low cell density (20, 34). To determine if G
1 is required
for this sensitivity to CMF, we starved wild-type and G
1 null cells
at various densities in submerged culture in the presence or absence of
rCMF. Wild-type cells starved in the absence of rCMF were able to
aggregate in 24 h when starved at densities of 4 × 104 cells/cm2 or above (at a secretion rate of
12 molecules/cell/min, the CMF concentration in this culture reached
the 0.3 ng/ml threshold at 16 h) (Table
I). After longer periods of time, the
cells did form aggregates at even lower densities. The same cells
starved for 24 h in the presence of CM or rCMF formed aggregates
when starved at 2 × 104 cells/cm2. As
seen with the cells starved in the absence of exogenous CMF, the cells
starved in the presence of CMF eventually formed aggregates at lower
densities, typically at 2-fold lower density at any given time. The
G
1 null cells, when starved in the presence or absence of CM or
recombinant CMF, formed aggregates at densities of 1 × 104 cells/cm2 or greater, and the appearance of
the cells and of the aggregates was unaffected by the presence of CM
(Table I). One possible explanation for the ability of G
1 cells to
aggregate at such low cell densities even in the absence of exogenous
CMF is that they secrete very high levels of CMF. However, CMF
production assays indicated that G
1 null cells (as well as G
2 and
G
null cells) produce no more CMF than wild-type cells (Table
II). Therefore, it appears that the
aggregation of G
1 null cells at very low density even in the absence
of CMF is due to these cells responding to starvation as if they were
in the presence of CMF.
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Table I
Ability to aggregate at different cell densities
Cells were starved at various cell densities in submerged monolayer
culture in the absence or presence of CMF and/or PLC inhibitor or a
nonfunctional analogue of the PLC inhibitor (mock). The fields of cells
were then examined with an inverted microscope at 24 h. The
presence of aggregates is denoted with a plus sign while the absence of
aggregates is represented by a minus sign. Densities allowing only
partial or substandard aggregation are shown as +/ .
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Table II
The effect of deleting specific proteins on pseudopod activity and
CMF secretion
Cells were starved for 4 h at low cell density in monolayer
culture in buffer or buffer supplemented with 1 ng/ml recombinant CMF
at 3 h after starvation. The cells were then videotaped, and the
number of pseudopods extended by a cell was counted over a period of
approximately 15 min. Values are the means and S.D. for 10 different
cells, normalized to 5 min. CMF production by cells starved in
monolayer culture for 20 h was determined following the procedure
of Gomer et al. (19). Values are the means and S.D. for four
(three for PLC null and HD 10) separate assays.
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Another effect of CMF is that it alters the morphology of starving
cells (22). Wild-type cells starved in the absence of CMF tend to be
smooth and round and do not aggregate, while cells exposed to CMF for
more than 60 min tend to have large numbers of ruffles and pseudopods
and do aggregate. When Ax4 wild-type, G
1, and G
null cells were
starved at low cell density in the absence of CMF, the Ax4 cells were
rounded and did not aggregate, while the G
1 null cells were ruffled
and did aggregate (Fig. 2). In the
presence of CMF, both cell lines were extensively ruffled and formed
aggregates (data not shown). In addition, the rate of pseudopod
extension in G
1 null cells was insensitive to CMF, with the rate
being equivalent to that of wild-type cells in the presence of CMF
(Table II). The lack of G
also caused the pseudopod extension rate
to be independent of CMF, with a rate similar to that of wild-type
cells in the absence of CMF (Table II). The data thus indicate that
G
1 null cells starved in the absence or presence of CMF extend
pseudopods and form aggregates as if they were in the presence of CMF,
while G
null cells behave as if they were in the absence of CMF.
Cells lacking G
2 did increase their pseudopod extension rate in
response to CMF, suggesting that G
1 and G
but not G
2 mediate
this response of cells to CMF.

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Fig. 2.
The appearance of Ax4 wild-type and
G 1 null cells starved at low cell density in the absence of
CMF. Wild-type (WT) and G 1 null cells were
starved in submerged monolayer culture at 3 × 104
cells/cm2 in the absence of CMF and photographed after
6 h. Bar, 10 µm.
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CMF regulates the aggregation of cells by regulating cAMP activation of
adenylyl cyclase, guanylyl cyclase, and Ca2+ influx (22).
We thus examined whether G
1 is necessary for CMF to regulate these
cAMP-stimulated activities. As shown in Table
III, the cAMP analog 2'-deoxy-cAMP
stimulates cAMP production in cells starved at low cell density, where
the CMF concentration will be suboptimal. The addition of recombinant
CMF for 90 s to wild-type cells then increases the cAMP-stimulated
cAMP production, whereas in G
1 null cells, CMF has no effect. In
wild-type cells, CMF similarly potentiates cGMP production in response
to a pulse of cAMP (see Ref. 22 and Table
IV). In G
1 null cells, CMF caused a
much smaller potentiation. Similarly, cAMP causes an increase in
Ca2+ influx in wild-type cells, and this stimulation is
potentiated by exposing cells to CMF (22). In G
1 null cells exposed
or not exposed to CMF, the Ca2+ influx induced by cAMP was
similar to that seen in wild-type cells exposed to CMF, and CMF caused
no detectable potentiation (data not shown).
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Table III
Production of cAMP in response to cAMP stimulation
For each experiment, cAMP production was measured in duplicate at 0 and
3 min after stimulation with 2'-deoxy-cAMP. Values are the means ± S.E. from four separate experiments. In all cell lines, there was no
detectable cAMP production at 0 min in the absence or presence of CMF.
The ratio of cAMP production in the presence of CMF to cAMP production
in the absence of CMF was calculated separately for each experiment;
the average ± S.E. of the ratios is shown as the -fold increase.
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Table IV
Production of cGMP in response to cAMP stimulation
For each experiment, cGMP production was measured in duplicate at 0 and
5 s after stimulation with cAMP. The values represent the -fold
increase in the amount cGMP at 5 s over that at 0 s and
represent the means ± S.E. of three experiments. The average ± S.E. of the ratio of the two values for each cell line is shown as
the -fold increase.
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We previously found that CMF regulates the cAMP-stimulated high
affinity GTP hydrolysis activity of G
2 (23). To delineate how CMF
regulates this hydrolysis, we examined whether CMF could exert its
effect on cAMP-stimulated high affinity GTP hydrolysis in membranes
deficient for G
1 and G
(Fig. 3). In
membranes lacking G
1 or G
, the addition of CMF had no effect on
cAMP-stimulated high affinity GTPase activity. Thus, cAMP-stimulated
high affinity GTP hydrolysis has been uncoupled from CMF regulation in
membranes lacking these G proteins. Also, it is interesting to note
that the cAMP-stimulated high affinity GTPase activity in membranes lacking G
1 is similar to that of wild-type membranes in the presence of CMF, suggesting that in the absence of G
1, cells respond to cAMP
as if CMF were present.

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Fig. 3.
The effect of CMF on cAMP-stimulated high
affinity GTPase. Ax4, G 1 null, G null, and PLC null cells
were starved for 6 h and then divided into two aliquots.
Recombinant CMF was added to one flask to 1 ng/ml (+CMF), and nothing
was added to the other flask ( ). 30 s later, the cells were
lysed. The membranes were isolated, incubated in the presence or
absence of 10 µM cAMP, and assayed for their ability to
hydrolyze [ -32P]GTP in the presence or absence of cold
GTP. For each experiment, values were determined in triplicate and then
averaged. High affinity GTPase activity was determined by subtracting
the amount of [ -32P]GTP hydrolyzed in the presence of
cold GTP from the amount of [ -32P]GTP hydrolyzed in
the absence of cold GTP. cAMP stimulation was defined as the percentage
increase in high affinity GTPase activity after the addition of cAMP.
The results are the means of three experiments ± S.E.
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The lack of G
1 could be decreasing cAMP-stimulated GTP hydrolysis in
two ways. It could be altering the G
2-associated GTPase activity, or
it could be decreasing G
2-associated GTP binding to membranes. To
examine the second possibility, cAMP-stimulated binding of
[3H]GTP was measured in membranes from wild-type, G
1
null, and G
null cells in the absence and presence of CMF (Fig.
4). As previously observed, the addition
of cAMP in the presence or absence of CMF to wild-type membranes caused
a 15-20% increase in high affinity GTP binding (see Ref. 23 and Fig.
4). The addition of cAMP in the presence or absence of CMF to membranes
lacking G
1 or G
caused only slightly decreased high affinity GTP
binding. Thus, the decreased GTP hydrolysis observed in G
1 null
cells is most likely due to a decrease in G
2-associated GTPase
activity.

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Fig. 4.
The effect of CMF on cAMP-stimulated high
affinity GTP binding. Membranes isolated as described in the
legend to Fig. 1 were incubated in the presence or absence of 10 µM cAMP and assayed for their ability to bind
[3H]GTP in the presence or absence of cold GTP. The
binding was assayed in duplicate and then averaged; cAMP stimulation
was defined as the percentage increase in high affinity GTP binding
activity after the addition of cAMP. The results are the means of three
experiments ± S.E.
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CMF Activates Phospholipase C--
Under some circumstances, cells
lacking G
1 show altered patterns of IP3 and
diacylglycerol accumulation, suggesting that G
1 may regulate PLC
(27, 38). To examine whether CMF activation of G
1 affects PLC
activity, production of IP3 was measured after cells were
stimulated with CMF. As shown in Fig. 5,
a 30-s exposure of CMF causes a 90% increase in the amount of
IP3 present in Ax4 cells starved for 5 h. To confirm
that the CMF-induced increase in the amount of IP3 is due
to PLC activity, PLC null cells were similarly treated with CMF. As
shown in previous studies (39), cells lacking PLC had IP3
levels that were slightly lower than those of wild-type cells. Compared
with Ax4 cells, the PLC null cells showed only a very small CMF-induced
increase in the amount of IP3 (Fig. 5). When Ax4 cells were
in the presence of 5 µg/ml U-73122 (a specific PLC inhibitor), the
addition of rCMF did not cause a rise in IP3 levels,
whereas when the Ax4 cells were in the presence of the structurally
similar analog U-73343, CMF caused a increase in IP3 levels
indistinguishable from that seen in the absence of any drug. Both drugs
had no significant effect on PLC null cells (data not shown). The data
thus indicate that CMF causes an increase in the amount of
IP3 through activation of PLC. If the IP3
production is truly regulated by CMF, it should exhibit a CMF dose
response. We previously observed that cells respond optimally to 1-10
ng/ml of native or recombinant CMF; above and below this concentration,
cells show poor differentiation and aggregation in response to CMF (19,
34). A similar CMF dose response was seen in IP3 production
(Fig. 6), suggesting that the CMF
activation of IP3 production is physiologically
relevant.

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Fig. 5.
Time course of CMF-stimulated IP3
production. Ax4, G 1 null, G null, and PLC null cells were
starved for 5 h. Recombinant CMF was then added to a concentration
of 1 ng/ml, and duplicate aliquots were removed at several time points.
The amount of IP3 in each aliquot was measured and then
averaged for each time point. The results are the means of three
experiments ± S.E.
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Fig. 6.
Dose response of CMF-stimulated
IP3 production. Ax4 cells were starved for 5 h,
and recombinant CMF was then added to various concentrations. The
amount of IP3 was measured after 30 s and compared
with the amount present at 0 s, which is represented as 100%. The
results are the means of two experiments, each done in duplicate, ± S.E.
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To examine the signal transduction pathway whereby CMF activates PLC,
IP3 production in response to CMF was measured in G
1 null and G
null cells. For both cell lines, no significant increase in the amount of IP3 was caused by the addition of CMF
(Fig. 5). However, the overall amounts of IP3 were quite
different. In the G
1 null cells, the IP3 levels were
slightly higher than the levels seen in wild-type cells stimulated with
CMF (Fig. 5). In starving G
null cells, the IP3 levels
were much lower than the levels seen in starving wild-type cells. The
data thus suggest that G
1 and G
are necessary for CMF to
stimulate IP3 accumulation.
Some but Not All CMF Signal Transduction Is Mediated by
PLC--
To determine whether PLC is involved in CMF signal
transduction, PLC null cells were starved as described above in the
presence or absence or rCMF. As with the G
1 null cells, there was no
effect of CMF on the density at which the PLC null cells formed
aggregates; however, the minimal density for aggregate formation was
4 × 104 cells/cm2, which is higher than
that for the G
1 null cells (Table I). As with the G
1 null cells,
this effect was not caused by an oversecretion of CMF (Table II). CMF
had no effect on the rate at which starving PLC null cells extended
pseudopods, with the rate being slightly lower than that of wild-type
cells in the presence of CMF (Table II). We also examined the effect of
adding PLC inhibitors to cells in submerged monolayer culture. The
addition of 5 µg/ml of the PLC inhibitor U-73122 caused Ax4 cells to
remain as individual amebas at a density of 4 × 104
cells/cm2 after 24 h of starvation, while the addition
of PBM or U-73343 (the mock PLC inhibitor) allowed the cells to
aggregate under the same conditions. This concentration of U-73122
inhibitor was also able to interfere with aggregation when cells plated
at 2 × 104 cells/cm2 were starved in the
presence of CM or rCMF, while U-73343 allowed aggregation. The addition
of CM or rCMF had no effect on the aggregation of cells exposed to
U-73122 at all cell densities tested. Thus, it appears that PLC
activity is required for CMF-regulated aggregation.
Even in the absence of CMF, pulses of cAMP cause a brief, transitory
activation of PLC (22). Thus, one might predict that a train of cAMP
pulses should be able to mimic exposure of cells to CMF. To test this
prediction, CMF null cells were starved in shaking culture and were
pulsed with cAMP. After 6 h, the cells were harvested and allowed
to develop on filter pads. The cAMP-pulsed CMF null cells formed
fruiting bodies, while an identical culture that was not pulsed did not
form aggregates (data not shown).
Starved cells will activate adenylyl cyclase and guanylyl cyclase in
response to a pulse of cAMP only if the cells have been exposed to CMF
for 10 s or more (22). To determine whether this CMF regulation is
mediated by PLC, we examined the ability of PLC null cells to make cAMP
and cGMP when stimulated with cAMP in the absence or presence of CMF.
In wild-type cells, CMF enhanced both cAMP-stimulated cAMP and cGMP
production 2-fold. In PLC null cells, CMF enhanced cAMP-stimulated cAMP
production by only 1.6-fold and had no effect on cGMP production
(Tables III and IV). Thus, the presence of PLC helps but is not
completely necessary for CMF to regulate cAMP-stimulated adenylyl
cyclase activation. On the other hand, PLC must be present for CMF to
regulate cAMP-stimulated guanylyl cyclase activity.
Earlier we demonstrated that in membranes lacking G
1 or G
, the
addition of CMF had no effect on cAMP-stimulated high affinity GTPase
activity, suggesting that these two proteins mediate the ability of CMF
to influence GTPase activity. To determine whether PLC lies in this
pathway, cAMP-stimulated high affinity GTPase activity was measured in
membranes lacking PLC. Similar to membranes lacking G
1 or G
,
membranes lacking PLC exhibited no effect of CMF on cAMP-stimulated
high affinity GTPase activity (Fig. 3). Thus, it appears that PLC is
necessary for CMF to regulate cAMP-stimulated high affinity GTPase
activity.
The original assay for the presence of CMF was based on the fact that
cells needed to be starved in the presence of CMF to be able to express
cAMP-induced genes such as cp2, ras, and
sp70. We examined the effect of CMF on gene expression in
cells starved at low cell density. As shown in Fig.
7 and as previously observed (40), only a
very small percentage of wild-type cells express the prespore marker
SP70 when starved at low cell density in buffer alone, even when cAMP
is added at 6 h. However, when starved in CM or recombinant CMF,
roughly 25% of cells express the prespore marker SP70 in response to
cAMP. Cells lacking G
1, G
2, G
, PLC, or CRAC also express SP70
in response to cAMP only when they are starved in the presence of CM or
rCMF (Fig. 7). For the Ax4 and the five mutant cell lines, there was
very little or no expression of SP70 in cells starved in buffer, CM, or
rCMF when no cAMP was added. Essentially identical results were
obtained using the prestalk marker CP2 (data not shown): in all of the
above mutants, CP2 was expressed if and only if the cells were exposed
to both CMF and cAMP. There was no CP2 or SP70 expression under any
condition in cells lacking the cAR1 cAMP receptor (these cells also
show poor binding of CMF 37). The data thus suggest that the regulation of SP70 and CP2 expression by CMF and cAMP does not require the presence of G
1, G
2, G
, PLC, or CRAC but may require cAR1.

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Fig. 7.
Differentiation of cells starved at low cell
density. Cells were starved in submerged monolayer culture in
buffer, CM (buffer conditioned by a high density of starving cells), or
buffer containing recombinant CMF. Six h after starvation, cAMP was
added to the wells to stimulate prespore and prestalk gene expression.
At 18 h after starvation, the cells were fixed and stained for the
prespore marker SP70 by immunofluorescence. For each condition,
approximately 2500 cells were examined, and the number of SP70-positive
cells was counted; the percentage of positive cells was then
calculated. The graph shows the means ± S.D. of three separate
experiments.
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DISCUSSION |
The CMF Receptor Uses G
1, G
, and PLC to Regulate cAMP Signal
Transduction--
We previously described how the extracellular
signaling protein CMF regulates the GTPase activity of G
2, the G
protein associated with the cAMP receptor cAR1 (23). Here we present
evidence that CMF exerts its effect through a second G protein, G
1.
GTP
S inhibits the binding of CMF to membranes, suggesting that CMF
binds to a G protein-coupled receptor. This inhibition disappears in
cells lacking G
1, implying that the putative CMF receptor associates with G
1. In addition, the loss of G
1 causes cells to be
insensitive to CMF. In the absence of G
1, CMF is no longer able to
regulate the cAMP-stimulated GTPase activity of G
2 or guanylyl
cyclase activity. Thus, G
1 appears to be a component downstream of
the CMF receptor in a pathway mediating CMF signaling to G
2.
Others have demonstrated that G
1 is involved in the regulation of
PLC activity (25). We find that CMF regulates PLC activity and that
this control is indeed exerted through G
1, since cells lacking G
1
are no longer able to activate PLC when presented with CMF. PLC
activity is crucial to CMF signal transduction, since loss of PLC
strongly decreases the ability of CMF to regulate the cAMP-stimulated
G
2 GTPase, adenylyl cyclase, and guanylyl cyclase activities.
Therefore, PLC seems to be downstream of G
1 in a pathway mediating
CMF signaling to G
2.
The third player in this signal transduction pathway is G
. Cells
lacking G
exhibit no CMF-induced PLC activity or CMF regulation of
the cAMP-stimulated GTPase activity of G
2. Therefore, G
is upstream of PLC, just like G
1. In the cells lacking G
, we observe both cAMP-stimulated high affinity membrane-associated GTP binding (Fig. 4) and GTP hydrolysis (Fig. 3). When experiments were done examining the effect of GTP on cAMP ligand binding to membranes from
cells lacking G
, GTP did not inhibit the binding of cAMP (41). In
the cells lacking G
, the effect of ligand on GTP binding and the
absence of an effect of GTP on ligand binding suggests that in the
absence of G
, there is communication from the ligand to the receptor
to the G protein to GTP but not the other way round. However, GTP
S
did inhibit the binding of CMF to membranes from cells lacking G
(Fig. 1). This indicates a fundamental difference between the cAMP and
CMF receptors; in the absence of G
, G
proteins cannot couple to
cAR1 but can interact with the CMF receptor.
CMF Regulates PLC through G
and G
1--
G
1 null cells
starved in the absence of CMF respond as if they are in optimal
concentrations of CMF. They extend many pseudopods and have a very low
cAMP-stimulated GTPase activity, the very same characteristics seen in
wild-type cells exposed to CMF. Therefore, G
1 is a negative
regulator for CMF signaling. In contrast, cells lacking G
extend few
pseudopods and a moderate cAMP-stimulated GTPase activity in the
absence or presence of CMF. They appear to be blind to CMF, and thus
G
is a positive regulator of CMF signaling.
In mammals, there are several isoforms of PLC, with the
-isoforms
being activated by G
(42, 43). All PLCs contain conserved X and Y
boxes. In PLC
2, a 60-amino acid region of the Y box interacts with
G
(44). The Dictyostelium PLC shows homology to all
isoforms of PLC in the X and Y boxes (45), with slightly higher
homology to bovine and rat PLC
s when the Y box or the 60-amino acid
subregion of the Y box sequences alone is compared. Therefore, it is
feasible that Dictyostelium PLC could be a
-isoform and
thus could be activated by G
.
Several workers have observed that at high cell density, and thus in
the presence of CMF, cAMP will cause a transitory activation of PLC
(15, 46-48). The cAMP receptor cAR1 couples to a G protein whose
-subunit is G
2 (14, 24, 28, 49). Activation of this G protein
releases G
, so the activation of PLC by cAMP is consistent with
the possibility that PLC is activated by G
.
Taken together, the above results support the following model (Fig.
8). CMF binds to a G protein-coupled
receptor, which is associated with G
1
. Binding of CMF causes
G
to dissociate from G
1-activating PLC, thereby causing an
increase in IP3 levels. A full level of IP3
accumulation occurs within approximately 30 s, comparable with the
previously observed time necessary for CMF regulation of cAMP-induced
Ca2+ influx (22). This PLC activation and/or increase in
IP3 levels either directly or indirectly causes the
cAMP-stimulated GTPase activity of G
2 to decrease dramatically,
prolonging the lifetime of the G
2-GTP configuration and thus
prolonging its ability to activate downstream effectors. This allows
aggregation to occur. In the absence of CMF, G
remains bound to
G
1, so PLC is not activated. IP3 levels are kept low,
and the cAMP-stimulated GTPase activity of G
2 is kept high. Under
these conditions, G
2 rapidly hydrolyzes GTP to GDP,
G
2-GTP-activated downstream effectors remain unactivated, and
aggregation does not occur.

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Fig. 8.
Proposed mechanism for CMF signal
transduction. CMF binding to its receptor activates the G protein
whose -subunit is G 1; the released  -subunit activates PLC,
which then through an unknown mechanism inhibits the G 2
GTPase.
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The model predicts that PLC null cells should not be able to aggregate,
since IP3 levels would remain low and G
2 GTPase
activities would remain high. However, cells lacking PLC do aggregate
and have IP3 levels and pseudopod extension rates only
slightly lower than wild-type cells in the presence of CMF. It has
recently been shown that in the PLC null cells, these high levels of
IP3 are produced by the dephosphorylation of inositol
peutaphosphate and inositol tetraphosphate (50). If IP3
mediates the effect of CMF on cAMP signal transduction, this second
route of IP3 production might indicate that something in
addition to CMF also controls cAMP signal transduction. The high levels
of IP3 in the PLC null cells could allow the GTPase
activity of G
2 to be low enough to allow cAMP signal transduction
and aggregation to occur. Wild-type cells in which PLC activity
was suppressed with U-73122 indeed do not aggregate, in agreement with
our hypothesis that PLC, and presumably IP3, mediate CMF
signaling.
A second prediction of the model (Fig. 8) is that in the absence of
CMF, pulses of cAMP should cause brief, transitory activations of G
2
and thus brief, transitory releases of the G
2-associated G
.
There might then be a sufficient amount of released G
to activate
PLC and thus bypass the requirement for CMF to allow normal cAMP signal
transduction and aggregation. We observed that a long train of cAMP
pulses does mimic exposure of CMF null cells to CMF, in agreement with
this second prediction. However, the kinetics of cAMP-stimulated
IP3 production are much faster than CMF-induced
IP3 production (25). This is consistent with the idea that
CMF is a signal that appears slowly and persists throughout aggregation
(21), whereas cAMP is a rapidly pulsed signal.
Possible Additional Functions of CMF, the CMF Receptor, and
G
1--
We currently know that reducing the amount of CMF by
antisense repression does not affect growth but prevents early gene
expression and cAMP signal transduction during aggregation (20, 22,
34). However, we do not know if CMF has a function during late
development. Previous work has shown that G
1 null cells and
G
1-overexpressing cells have essentially normal early development
and aggregation but then have defects during fruiting body formation
(24, 26). Expression of an activated form of G
1 causes rings of
cells to form during aggregation (51). These results suggest that after aggregation CMF has a function or that G
1 has a function other than
CMF signal transduction. There is a moderate level of G
1 transcript
present during growth, and although G
1 null cells exhibit normal
growth, overexpression of G
1 during growth causes the formation of
large, multinucleate cells (24, 28). There are at least three possible
explanations for this phenotype. First, G
1 could be involved in
transducing a signal other than CMF during growth. Second, assuming
that overexpression of G
1 is equivalent to signaling the absence of
CMF, some amount of CMF could be required for growth and proper
cytokinesis. A third explanation is that overexpression of G
1 causes
interference with some other G protein-dependent pathway.
The observation that the CMF regulation of cAMP-stimulated cAMP
production is absent in G
1 null cells but is present to some degree
in PLC null cells suggests that there is a second component of the
regulatory pathway in addition to PLC downstream of G
1. In addition,
G
1, G
, and PLC appear to couple the CMF receptor to cAMP
pulse-induced signal transduction (Fig. 8) but not to cell
type-specific gene regulation (Fig. 7). This is in agreement with Wu
et al. (41), who found that G
is not necessary for expression of phosphodiesterase and phosphodiesterase inhibitor, and
with Schnitzler et al. (52), who noted that G
is not
required for expression of the postaggregative genes lagC,
cp2, and rasD. Blusch et al. (53) have
suggested that the signal transduction mechanism that allows CMF to
regulate the expression of discoidin involves the CMF receptor
activating G
2. These observations and our data would then suggest
that there may be three or more different signal transduction pathways
downstream from the CMF receptor.