Cell Density Sensing Mediated by a G Protein-coupled Receptor Activating Phospholipase C*

Derrick T. Brazill, David F. Lindsey, John D. Bishop, and Richard H. GomerDagger

From the Howard Hughes Medical Institute, Department of Biochemistry and Cell Biology, MS-140, Rice University, Houston, Texas 77005-1892

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

When the unicellular eukaryote Dictyostelium discoideum starves, it senses the local density of other starving cells by simultaneously secreting and sensing a glycoprotein called conditioned medium factor (CMF). When the density of starving cells is high, the corresponding high density of CMF permits signal transduction through cAR1, the chemoattractant cAMP receptor. cAR1 activates a heterotrimeric G protein whose alpha -subunit is Galpha 2. CMF regulates cAMP signal transduction in part by regulating the lifetime of the cAMP-stimulated Galpha 2-GTP configuration. We find here that guanosine 5'-3-O-(thio)triphosphate (GTPgamma S) inhibits the binding of CMF to membranes, suggesting that the putative CMF receptor is coupled to a G protein. Cells lacking Galpha 1 (Galpha 1 null) do not exhibit GTPgamma S inhibition of CMF binding and do not exhibit CMF regulation of cAMP signal transduction, suggesting that the putative CMF receptor interacts with Galpha 1. Work by others has suggested that Galpha 1 inhibits phospholipase C (PLC), yet when cells lacking either Galpha 1 or PLC were starved at high cell densities (and thus in the presence of CMF), they developed normally and had normal cAMP signal transduction. We find that CMF activates PLC. Galpha 1 null cells starved in the absence or presence of CMF behave in a manner similar to control cells starved in the presence of CMF in that they extend pseudopods, have an activated PLC, have a low cAMP-stimulated GTPase, permit cAMP signal transduction, and aggregate. Cells lacking Gbeta have a low PLC activity that cannot be stimulated by CMF. Cells lacking PLC exhibit IP3 levels and cAMP-stimulated GTP hydrolysis rates intermediate to what is observed in wild-type cells starved in the absence or in the presence of an optimal amount of CMF. We hypothesize that CMF binds to its receptor, releasing Gbeta gamma from Galpha 1. This activates PLC, which causes the Galpha 2 GTPase to be inhibited, prolonging the lifetime of the cAMP-activated Galpha 2-GTP configuration. This, in turn, allows cAR1-mediated cAMP signal transduction to take place.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 Gbeta gamma subunits, along with a cytosolic protein called CRAC, transiently activate adenylyl cyclase, while the Galpha 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 GTPgamma 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 Galpha 2 to exchange GDP for GTP, but before Galpha 2-GTP can activate adenylyl cyclase. CMF appears to regulate cAMP signal transduction by regulating the lifetime of the Galpha 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 Galpha 2 (23). This suggests that CMF, and thus cell density, controls cAMP-stimulated Galpha 2-GTPase activity.

At the developmental stage when Galpha 2 is mediating cAMP signal transduction, Galpha 1 is also being expressed and potentially mediating other signal transduction pathways. However, cells lacking Galpha 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, Galpha 1 appears to be superfluous for early development. In cells lacking Galpha 1, 3'NH-cAMP no longer inhibits phospholipase C (25). During late development, the absence of Galpha 1 causes fruiting bodies to have abnormally thin stalks (24, 26) and causes decreased 1,2-diacylglycerol accumulation (27). Cells overexpressing Galpha 1 or containing a constitutively active form of Galpha 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 Gbeta gamma from Galpha 1; the released Gbeta gamma then activates PLC, which in turn causes an inhibition of the Galpha 2 GTPase.

    EXPERIMENTAL PROCEDURES
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Introduction
Procedures
Results
Discussion
References

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 alpha -subunits Galpha 1 (26) or Galpha 2 (28) were a gift from Dr. Rick Firtel (University of California at San Diego), and cells lacking the Gbeta 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 (pBsr2Delta 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 GTPgamma S on the Binding of CMF to Membranes-- To examine the effect of GTPgamma 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 GTPgamma 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.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

CMF Binds to a G Protein-coupled Receptor That Interacts with Galpha 1-- We previously showed that disruption of the Gbeta 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 GTPgamma 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. GTPgamma 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.   GTPgamma 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 GTPgamma 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 GTPgamma 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 GTPgamma S to the specific binding in the absence of GTPgamma S was then determined. The graph shows the average ratio ± S.E. from four separate experiments. The reductions in CMF binding caused by GTPgamma S for Ax4, Galpha 2, and Gbeta were all significant (p < 0.005). There was no significant effect of GTPgamma S on CMF binding in Galpha 1 null cells (p < 0.15), and there was no significant difference in the reduction of CMF binding caused by GTPgamma S between Ax4 and Galpha 2 null cells (p < 0.1) or between Ax4 and Gbeta null cells (p < 0.4).

To determine if the CMF receptor couples to a known G protein, we examined the effect of GTPgamma S on the binding of [125I]rCMF to membranes from cells lacking specific G proteins. GTPgamma S caused a similar amount of inhibition of the binding of CMF to membranes from wild-type cells and cells lacking Gbeta or Galpha 2 (Fig. 1). However, in cells lacking Galpha 1, GTPgamma S was unable to inhibit the binding of CMF to membranes (Fig. 1). The data thus suggest that the putative CMF receptor interacts with Galpha 1 more than it interacts with Galpha 2 and that the Gbeta subunit is not necessary for the GTPgamma S inhibition of CMF binding.

Galpha 1 Mediates CMF Signal Transduction, and the Absence of Galpha 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 Galpha 1 is required for this sensitivity to CMF, we starved wild-type and Galpha 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 Galpha 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 Galpha 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 Galpha 1 null cells (as well as Galpha 2 and Gbeta null cells) produce no more CMF than wild-type cells (Table II). Therefore, it appears that the aggregation of Galpha 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.

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, Galpha 1, and Gbeta null cells were starved at low cell density in the absence of CMF, the Ax4 cells were rounded and did not aggregate, while the Galpha 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 Galpha 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 Gbeta 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 Galpha 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 Gbeta null cells behave as if they were in the absence of CMF. Cells lacking Galpha 2 did increase their pseudopod extension rate in response to CMF, suggesting that Galpha 1 and Gbeta but not Galpha 2 mediate this response of cells to CMF.


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Fig. 2.   The appearance of Ax4 wild-type and Galpha 1 null cells starved at low cell density in the absence of CMF. Wild-type (WT) and Galpha 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.

CMF regulates the aggregation of cells by regulating cAMP activation of adenylyl cyclase, guanylyl cyclase, and Ca2+ influx (22). We thus examined whether Galpha 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 Galpha 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 Galpha 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 Galpha 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.

We previously found that CMF regulates the cAMP-stimulated high affinity GTP hydrolysis activity of Galpha 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 Galpha 1 and Gbeta (Fig. 3). In membranes lacking Galpha 1 or Gbeta , 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 Galpha 1 is similar to that of wild-type membranes in the presence of CMF, suggesting that in the absence of Galpha 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, Galpha 1 null, Gbeta 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 [gamma -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 [gamma -32P]GTP hydrolyzed in the presence of cold GTP from the amount of [gamma -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.

The lack of Galpha 1 could be decreasing cAMP-stimulated GTP hydrolysis in two ways. It could be altering the Galpha 2-associated GTPase activity, or it could be decreasing Galpha 2-associated GTP binding to membranes. To examine the second possibility, cAMP-stimulated binding of [3H]GTP was measured in membranes from wild-type, Galpha 1 null, and Gbeta 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 Galpha 1 or Gbeta caused only slightly decreased high affinity GTP binding. Thus, the decreased GTP hydrolysis observed in Galpha 1 null cells is most likely due to a decrease in Galpha 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.

CMF Activates Phospholipase C-- Under some circumstances, cells lacking Galpha 1 show altered patterns of IP3 and diacylglycerol accumulation, suggesting that Galpha 1 may regulate PLC (27, 38). To examine whether CMF activation of Galpha 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, Galpha 1 null, Gbeta 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.

To examine the signal transduction pathway whereby CMF activates PLC, IP3 production in response to CMF was measured in Galpha 1 null and Gbeta 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 Galpha 1 null cells, the IP3 levels were slightly higher than the levels seen in wild-type cells stimulated with CMF (Fig. 5). In starving Gbeta null cells, the IP3 levels were much lower than the levels seen in starving wild-type cells. The data thus suggest that Galpha 1 and Gbeta 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 Galpha 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 Galpha 1 null cells (Table I). As with the Galpha 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 Galpha 1 or Gbeta , 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 Galpha 1 or Gbeta , 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 Galpha 1, Galpha 2, Gbeta , 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 Galpha 1, Galpha 2, Gbeta , 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.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The CMF Receptor Uses Galpha 1, Gbeta , and PLC to Regulate cAMP Signal Transduction-- We previously described how the extracellular signaling protein CMF regulates the GTPase activity of Galpha 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, Galpha 1. GTPgamma S inhibits the binding of CMF to membranes, suggesting that CMF binds to a G protein-coupled receptor. This inhibition disappears in cells lacking Galpha 1, implying that the putative CMF receptor associates with Galpha 1. In addition, the loss of Galpha 1 causes cells to be insensitive to CMF. In the absence of Galpha 1, CMF is no longer able to regulate the cAMP-stimulated GTPase activity of Galpha 2 or guanylyl cyclase activity. Thus, Galpha 1 appears to be a component downstream of the CMF receptor in a pathway mediating CMF signaling to Galpha 2.

Others have demonstrated that Galpha 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 Galpha 1, since cells lacking Galpha 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 Galpha 2 GTPase, adenylyl cyclase, and guanylyl cyclase activities. Therefore, PLC seems to be downstream of Galpha 1 in a pathway mediating CMF signaling to Galpha 2.

The third player in this signal transduction pathway is Gbeta . Cells lacking Gbeta exhibit no CMF-induced PLC activity or CMF regulation of the cAMP-stimulated GTPase activity of Galpha 2. Therefore, Gbeta is upstream of PLC, just like Galpha 1. In the cells lacking Gbeta , 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 Gbeta , GTP did not inhibit the binding of cAMP (41). In the cells lacking Gbeta , the effect of ligand on GTP binding and the absence of an effect of GTP on ligand binding suggests that in the absence of Gbeta , there is communication from the ligand to the receptor to the G protein to GTP but not the other way round. However, GTPgamma S did inhibit the binding of CMF to membranes from cells lacking Gbeta (Fig. 1). This indicates a fundamental difference between the cAMP and CMF receptors; in the absence of Gbeta , Galpha proteins cannot couple to cAR1 but can interact with the CMF receptor.

CMF Regulates PLC through Gbeta and Galpha 1-- Galpha 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, Galpha 1 is a negative regulator for CMF signaling. In contrast, cells lacking Gbeta 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 Gbeta is a positive regulator of CMF signaling.

In mammals, there are several isoforms of PLC, with the beta -isoforms being activated by Gbeta gamma (42, 43). All PLCs contain conserved X and Y boxes. In PLCbeta 2, a 60-amino acid region of the Y box interacts with Gbeta gamma (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 PLCbeta 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 beta -isoform and thus could be activated by Gbeta gamma .

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 alpha -subunit is Galpha 2 (14, 24, 28, 49). Activation of this G protein releases Gbeta gamma , so the activation of PLC by cAMP is consistent with the possibility that PLC is activated by Gbeta gamma .

Taken together, the above results support the following model (Fig. 8). CMF binds to a G protein-coupled receptor, which is associated with Galpha 1beta gamma . Binding of CMF causes Gbeta gamma to dissociate from Galpha 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 Galpha 2 to decrease dramatically, prolonging the lifetime of the Galpha 2-GTP configuration and thus prolonging its ability to activate downstream effectors. This allows aggregation to occur. In the absence of CMF, Gbeta gamma remains bound to Galpha 1, so PLC is not activated. IP3 levels are kept low, and the cAMP-stimulated GTPase activity of Galpha 2 is kept high. Under these conditions, Galpha 2 rapidly hydrolyzes GTP to GDP, Galpha 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 alpha -subunit is Galpha 1; the released beta gamma -subunit activates PLC, which then through an unknown mechanism inhibits the Galpha 2 GTPase.

The model predicts that PLC null cells should not be able to aggregate, since IP3 levels would remain low and Galpha 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 Galpha 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 Galpha 2 and thus brief, transitory releases of the Galpha 2-associated Gbeta gamma . There might then be a sufficient amount of released Gbeta gamma 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 Galpha 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 Galpha 1 null cells and Galpha 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 Galpha 1 causes rings of cells to form during aggregation (51). These results suggest that after aggregation CMF has a function or that Galpha 1 has a function other than CMF signal transduction. There is a moderate level of Galpha 1 transcript present during growth, and although Galpha 1 null cells exhibit normal growth, overexpression of Galpha 1 during growth causes the formation of large, multinucleate cells (24, 28). There are at least three possible explanations for this phenotype. First, Galpha 1 could be involved in transducing a signal other than CMF during growth. Second, assuming that overexpression of Galpha 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 Galpha 1 causes interference with some other G protein-dependent pathway.

The observation that the CMF regulation of cAMP-stimulated cAMP production is absent in Galpha 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 Galpha 1. In addition, Galpha 1, Gbeta , 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 Gbeta is not necessary for expression of phosphodiesterase and phosphodiesterase inhibitor, and with Schnitzler et al. (52), who noted that Gbeta 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 Galpha 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.

    ACKNOWLEDGEMENTS

We thank Rick Firtel for the Galpha 1 and Galpha 2 null strains, Peter Van Haastert for the PLC null cells, and Peter Devreotes for the Gbeta null strain.

    FOOTNOTES

* This work was supported by Robert A. Welch Foundation Grant C-1247.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger An associate investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Inst., Dept. of Biochemistry and Cell Biology, MS-140, Rice University, 6100 S. Main St., Houston, TX 77005-1892. Tel.: 713-527-4872; Fax: 713-285-5154; E-mail: richard{at}bioc.rice.edu.

1 The abbreviations used are: CMF, conditioned medium factor; rCMF, recombinant CMF; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; 3'NH-cAMP, 3'-deoxy-3'-aminoadenosine 3',5'-phosphate; CM, conditioned medium; PLC, phospholipase C; IP3, inositol 1,4,5-trisphosphate.

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Discussion
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