©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Seven Helix cAMP Receptors Stimulate Ca Entry in the Absence of Functional G Proteins in Dictyostelium(*)

(Received for publication, September 12, 1994; and in revised form, December 20, 1994)

Jacqueline L. S. Milne (§) Lijun Wu (¶) Michael J. Caterina (**) Peter N. Devreotes (§§)

From the Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Surface cAMP receptors (cARs) in Dictyostelium transmit a variety of signals across the plasma membrane. The best characterized cAR, cAR1, couples to the heterotrimeric guanine nucleotide-binding protein (G protein) alpha-subunit Galpha2 to mediate activation of adenylyl and guanylyl cyclases and cell aggregation. cAR1 also elicits other cAMP-dependent responses including receptor phosphorylation, loss of ligand binding (LLB), and Ca influx through a Galpha2-independent pathway that may not involve G proteins. Here, we have expressed cAR1 and a related receptor, cAR3, in a gbeta strain (Lilly, P., Wu. L., Welker, D. L., and Devreotes, P. N.(1993) Genes & Dev. 7, 986-995), which lacks G protein activity. Both cell lines failed to aggregate, a process requiring the Galpha2 and Gbeta-subunits. In contrast, cAR1 phosphorylation in cAR1/gbeta cells showed a time course and cAMP dose dependence indistinguishable from those of cAR1/Gbeta controls. cAMP-induced LLB was also normal in the cAR1/gbeta cells. Finally, cAR1/gbeta cells and cAR3/gbeta cells showed a Ca response with kinetics, agonist dependence, ion specificity, and sensitivity to depolarization agents that were like those of Gbeta controls, although they accumulated fewer Ca ions per cAMP receptor than the control strains. Together, these results suggest that the Gbeta-subunit is not required for the activation or attenuation of cAR1 phosphorylation, LLB, or Ca influx. It may, however, serve to amplify the Ca response, possibly by modulating other intracellular Ca signal transduction pathways.


INTRODUCTION

External stimuli trigger a wide variety of physiological responses by altering the activity of ion channels and intracellular enzymes. Seven membrane span receptors modulate effectors through a family of heterotrimeric guanine nucleotide-binding proteins (G proteins), comprised of alpha-, beta-, and -subunits (reviewed in (1) ). Receptor activation triggers a GTP-GDP exchange reaction on the Galpha-subunit and dissociation of Galpha from the Gbeta-subunit complex. The Galpha-subunits activate a broad range of effectors in eukaryotic cells(1) . Although free Gbeta-subunit complexes were thought only to attenuate signaling by reassociating with activated Galpha-subunits(1) , emerging evidence indicates that they also directly regulate adenylyl cyclase(2) , phospholipases(3, 4) , and ion channels(5) .

The cellular slime mold Dictyostelium discoideum provides an excellent system for molecular genetic analysis of G protein-linked signaling pathways. Upon nutrient depletion, these amoeboid cells aggregate into a multicellular structure, which undergoes morphogenesis and differentiation to form a fruiting body. This program is regulated by cAMP, which is synthesized and secreted by centrally located cells. cAMP binding to a cell surface cAMP receptor 1 (cAR1) (^1)directs adjacent cells to move toward aggregation centers and to relay the signal outwardly to more distal cells. The periodic stimulus also acts as a developmental timer, accelerating gene expression in the early stages of the program (reviewed in (6) ).

cAMP receptor occupancy induces a transient entry of external Ca(7, 8, 9) and elevates cellular levels of inositol 1,4,5- trisphosphate (IP(3))(10, 11) , cAMP(12) , and cGMP (13) . Many of these cAMP-dependent responses appear to require G proteins. In fact, four related cAMP receptors with striking similarity to rhodopsin and the beta-adrenergic receptor (reviewed in (14) ), eight G protein alpha-subunits (reviewed in (15) ), and a single G protein beta-subunit (16) have been identified in Dictyostelium. cAR1 is the major surface cAMP binding site of aggregating cells and appears to couple to the Galpha-subunit Galpha2, which is also expressed maximally during aggregation. Mutants lacking functional Galpha2 fail to differentiate, exhibit chemotaxis toward cAMP, or show cAMP-stimulated production of cAMP, cGMP, or IP(3) (reviewed in (17) ). The other cARs (cAR2, cAR3, and cAR4) are normally present during post-aggregative development but are capable of eliciting some of these responses when expressed in growth-stage wild-type cells or car1 cells(18, 19) . (^2)

We have proposed that a subset of cAR-generated signals may not require G proteins, based on the following observations. First, cAR1/galpha2 and cAR3/galpha2 cells show essentially wild-type levels of cAMP-dependent Ca uptake, even though they are defective in the G protein-linked pathways that control chemotaxis, differentiation, and production of cGMP (18) or cAMP(20) . Two additional responses of the receptors, agonist-induced phosphorylation and reduction in cAMP binding affinity (loss of ligand binding, LLB), also do not require Galpha2(21) . Second, the complement of Galpha-subunits present in growing cells is different from those expressed during aggregation and post-aggregation when the cARs are present(15) . Yet, cAR1, cAR2, and cAR3 elicit wild-type Ca responses when they are expressed in growing cells, and they appear to activate the same pathway of ion entry(18) . Third, analysis of Galpha-subunit null mutants indicates that Galpha1, Galpha2, Galpha3, Galpha4, Galpha7, and Galpha8 are not essential for cAMP-induced Ca uptake(18) . Moreover, the developmental regulation of Galpha5 and Galpha6 suggests that these subunits do not couple to the three cARs(22, 23) . While these observations imply a G protein-independent pathway, several caveats can be raised. Although the eight Galpha-subunits cannot be classified into subgroups(15, 24) , some of them could be functionally redundant. Alternatively, Gbeta-subunit complexes could regulate the Galpha-subunit-independent responses.

We took advantage of the observation that gbeta cells possess a single Gbeta-subunit, which is expressed at a constant level during growth and development(16) , to further explore this issue. Here, we have characterized cAMP-stimulated Ca uptake, cAR1 phosphorylation, and LLB in gbeta strains, which should not generate activated Galpha-subunits or free Gbeta-subunit complexes. Our results support the conclusion that G protein-coupled cARs function in the absence of G proteins to activate certain cellular responses.


EXPERIMENTAL PROCEDURES

Materials

Renaissance Western blot chemiluminescence reagent was from DuPont NEN. Other materials used were of analytical grade and purchased from the suppliers indicated in (18) .

Strains and Cell Culture

Strains used in this study were as follows: LW6, gbeta cells(16) ; LW14 and LW17, gbeta cells expressing cAR1 or cAR3 (see below); cAR1/JB4, car1 cells containing endogenous Gbeta and expressing cAR1(25) ; and cAR3/Delta208, car1 cells containing Gbeta and expressing cAR3 (26) . Transformed cell lines were maintained on Petri dishes in HL5 medium (27) supplemented with 20 µg of Geneticin/ml. gbeta cells were maintained in HL5 medium. Cells were grown in shaking suspension in HL5 medium containing Geneticin (except for gbeta cells) to a density of 2-5 times 10^6 cells/ml and harvested for use in experiments.

Construction of gbetaCells Overexpressing cAR1 or cAR3

gbeta (LW6) cells were transformed with the cAR1 expression vector pMC36 (25) or with the cAR3 expression vector pB18cAR3 (26) by electroporation as described(28) . Both vectors carry a neomycin resistance gene and have the actin 15 promoter and 2H3 terminator as an expression cassette. Stable clones selected in HL5 containing 20 µg of Geneticin/ml were grown and then washed in phosphate buffer (10 mM KH(2)PO(4)/Na(2)HPO(4), pH 6.1); 1 times 10^6 cells were then solubilized in Laemmli buffer(29) . The protein samples were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotted with anti-cAR1 (30) or anti-cAR3 (31) antiserum as described (30) except that an enhanced chemiluminescence kit was used as a detection system. gbeta clones expressing adequate levels of cAR1 (LW14 cells) or cAR3 (LW17 cells) at growth stage were selected for further study. Here, LW14 cells, LW17 cells, cAR1/JB4 cells, and cAR3/Delta208 cells will be designated as cAR1/gbeta, cAR3/gbeta, cAR1/Gbeta, and cAR3/Gbeta cells, respectively.

Biochemical Assays

cAR1 receptor phosphorylation was performed as described(32) , and loss of cAMP binding experiments were conducted as in (25) .

Ca uptake into 5 times 10^6 cells in the presence and absence of 100 µM cAMP was performed in a medium containing 500 µM CoCl(2) as described(18) . cAMP-induced Ca uptake is equal to the amount of Ca taken up by cAMP-treated cells minus the amount of Ca taken up by non-stimulated cells.

[^3H]cAMP binding to the cells was performed in triplicate using the ammonium sulfate assay as indicated (33) except that the stimulus concentration was 1 µM.

Protein was measured as described (34) using bovine serum albumin as standard.


RESULTS

When gbeta cells were repeatedly stimulated with exogenous cAMP, the cells showed weak levels of surface cAMP binding and low levels of cAMP-induced Ca uptake (data not shown). The expression level of cAR1 in gbeta cells varies with the growth conditions and the age of the culture. (^3)To obtain more reproducible levels of cAR expression and to permit analysis of growth-stage cells, gbeta cells were transformed with vectors containing the cAR1 or cAR3 cDNA under the control of a promoter that is constitutively active during growth. The expression of cAR1 and cAR3 in growth-stage gbeta cells is shown in Fig. 1A. Lysates of cAR1/gbeta cells fractionated using SDS-polyacrylamide gel electrophoresis showed a 40-kDa protein band immunoreactive with cAR1-specific antiserum (lane2). This protein band, indicative of cAR1(30) , was also present in cAR1/Gbeta lysates (lane3) but not in gbeta lysates (lane1). When lysates of cAR3/gbeta cells were immunoblotted with a cAR3-specific antiserum, a 65-kDa protein band was evident (lane5) characteristic of cAR3(31) . The same protein band was also present in control cAR3/Gbeta cells (lane6) but not in gbeta cells (lane4). When cAR1/gbeta or cAR3/gbeta cells were plated for development on non-nutrient agar, the amoebae remained as a flat monolayer. Control cAR1/Gbeta cells (Fig. 1B) differentiated to form fruiting bodies, as has been shown for cAR3/Gbeta cells. (^4)


Figure 1: Expression of cAMP receptors in cAR1/gbeta and cAR3/gbeta strains and their development on phosphate-buffered agar. A, SDS-solubilized growth-stage gbeta (lanes1 and 4), cAR1/gbeta (lane2), cAR1/Gbeta (lane3), cAR3/gbeta (lane5), and cAR3/Gbeta (lane6) cells (1 times 10^6) were electrophoretically separated and immunoblotted using antibodies to cAR1 (lanes1-3) and cAR3 (lanes4-6) as described under ``Experimental Procedures.'' Numbers at the margin of the figure indicate the migration pattern of molecular mass standards expressed in kDa. B, development of cAR1/gbeta, cAR3/gbeta, and cAR1/Gbeta cells. Growth-stage amoebae were starved (22 °C) on non-nutrient agar for 48 h as described(58) . Immunoblot analysis of 0-h cells indicated that each line had comparable levels of cAR1 or cAR3, respectively, to levels shown in A.



We assessed cAMP-induced cAR1 phosphorylation in cAR1/gbeta and cAR1/Gbeta cells by monitoring a parallel change in the apparent molecular mass of the receptor from 40 to 43 kDa. This electrophoretic mobility shift is due to serine phosphorylation within the C-terminal domain of cAR1(46) . Extracts of non-stimulated gbeta cells contained cAR1, which primarily migrated at 40 kDa, although there was also a detectable 43-kDa band (Fig. 2A, lane1), which is the phosphorylated form of cAR1 (30) . Upon addition of a saturating cAMP stimulus, the proportion of cAR1 in the 43-kDa form increased by 15 s and was almost entirely shifted to this form by 2 min (lanes2-9). Comparable results were obtained in the cAR1/Gbeta cells. In the presence of increasing concentrations of cAMP, both cAR1/gbeta and cAR1/Gbeta cells required 5 nM cAMP to induce the shift in mobility (Fig. 2B, lane3) and showed maximal responses at 100-500 nM (lanes6 and 7). In both instances, the concentration of cAMP required for half-maximal response (EC) was between 10 and 50 nM. Thus, the time course and dose dependence of cAMP-induced cAR1 phosphorylation are identical in the presence or absence of Gbeta. These values are similar to those of cAR1 expressed in aggregating wild-type cells(32) .


Figure 2: cAMP-induced phosphorylation of cAR1 in cAR1/gbeta cells. Growth-phase cAR1/Gbeta or cAR1/gbeta cells (1.5 times 10^6) were resuspended in phosphate buffer containing 5 mM caffeine and 10 mM dithiothreitol and stimulated with 100 µM cAMP for the indicated times (A) or with various concentrations of cAMP for 15 min (B), solubilized, separated electrophoretically, and immunoblotted with cAR1 antiserum as described under ``Experimental Procedures.'' A, time course of cAR1 phosphorylation. Lanes 1-9 represent the following times of treatment with cAMP: 0 s, 7 s, 15 s, 30 s, 1 min, 2 min, 5 min, 10 min, and 20 min. B, cAMP dose dependence of cAR1 phosphorylation. Lanes 1-9 represent the following doses of cAMP: 0, 1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 500 nM, 10 µM, and 100 µM. This experiment was repeated once with similar results.



Pretreatment of Dictyostelium amoebae with cAMP induces a rapid reduction in subsequent cAMP binding within 7 min(35, 36) . This response appears to reflect a reduction in the affinity of cAR1 for cAMP and likely depends upon receptor phosphorylation. (^5)As shown in Fig. 3, cAR1/gbeta cells pretreated with 10 µM cAMP exhibited a 75% reduction in the level of binding to a subsaturating concentration of cAMP relative to control cells treated with buffer. cAR1/gbeta cells also showed a pronounced response, with a 90% reduction in cAMP binding upon cAMP pretreatment.


Figure 3: Loss of ligand binding in cAR1/gbeta or cAR1/Gbeta cells. Growth-stage amoebae were resuspended to 3 times 10^7 cells/ml in phosphate buffer, shaken (20 min, 22 °C, 200 rpm) in the presence of 10 mM dithiothreitol, and then treated for 15 min with buffer (shaded bars) or with 10 µM cAMP (open bars). Cells were diluted 15-fold in ice-cold phosphate buffer, washed extensively in the same buffer, and placed on ice. Binding assays of each sample were performed in triplicate using 16 nM [^3H]cAMP and 10 mM dithiothreitol. Results shown are the average of two experiments. Absolute [^3H]cAMP binding to buffer-treated cAR1/Gbeta cells and cAR1/gbeta cells was 1500 cpm/8 times 10^6 cells and 3400 cpm/8 times 10^6 cells, respectively.



Next, agonist-induced Ca uptake in the gbeta strains was examined. Growth-stage cAR1/gbeta cells treated with cAMP initially accumulated Ca at the same rate as non-stimulated cells. After a delay of 10-15 s, however, the rate of Ca uptake into these cells increased until 30 s after receptor activation, when it returned to prestimulus rates (Fig. 4A). The agonist induced the accumulation of 25 pmol of Ca/mg of protein. Growth-stage cAR3/gbeta cells showed similar profiles of cAMP-induced Ca uptake except that the delay preceding stimulated uptake was only 5-10 s, and the agonist induced the accumulation 50 pmol of Ca/mg of protein (Fig. 4B). Presumably, there are few endogenous receptors during growth since gbeta cells showed no cAMP-dependent Ca response (Fig. 4C). Each of the three strains showed similar levels of basal Ca entry.


Figure 4: Time course of Ca uptake into cAR1/gbeta cells (A), cAR3/gbeta cells (B), and gbeta cells (C). Growth-stage amoebae were assayed for Ca uptake as described under ``Experimental Procedures.'' Values are shown for Ca into resting (circle) and cAMP-stimulated cells (up triangle) and for cAMP-induced Ca uptake (). Results shown represent the means of data from five (A), four (B), and two (C) independent experiments. In A and B, bars represent S.E. For clarity, half-error bars are shown.



The time courses of cAMP-dependent Ca uptake into cAR1/gbeta and cAR3/gbeta cells (Fig. 4) are like those of cAR1/Gbeta and cAR3/Gbeta cells(18) . However, the magnitude of cAMP-dependent Ca uptake into the gbeta strains was less than that seen earlier in Gbeta lines(18) . To assess the ability of cAR1 and cAR3 to promote Ca influx in gbeta strains, levels of cAMP-induced Ca uptake in cAR1/gbeta and cAR3/gbeta cells were standardized to the levels of surface cAMP binding sites in these cells. cAR1/gbeta cells transported 1.8 Ca ions/cAMP binding site, while cAR3/gbeta cells accumulated 4.2 Ca ions/binding site (Table 1). These results are consistent with the finding that cAR3/Gbeta cells transport higher levels of Ca/receptor than cAR1/Gbeta cells (Table 1). However, both gbeta strains transported Ca 4-fold less effectively than the Delta208-derived Gbeta strains. To determine if this was due to a defective cell line, fresh gbeta cells were retransformed with the cAR1 expression vector, and 10 clones expressing high levels of cAR1 were screened for cAMP-dependent Ca uptake. All lines displayed a similar agonist-induced Ca response (data not shown). Since the Delta208 cells were constructed in a HPS400 background, while gbeta cells were constructed using an AX3-derived strain, the experiment was repeated using cAR1/gbeta cells and cAR1/Gbeta cells (JB4 line) constructed in the same parental background. Comparable results were obtained (Table 1).



The cAMP requirements of Ca uptake into cAR1/gbeta and cAR3/gbeta cells are like those of cAR1/Gbeta and cAR3/Gbeta cells, respectively. In both cAR1/gbeta and cAR1/Gbeta cells, the Ca response was evident at 100 nM cAMP, reached a maximum at 3 µM cAMP, and displayed an EC value of 250 nM (Fig. 5A). Moreover, both cAR3/gbeta and cAR3/Gbeta cells showed similar cAMP dose-response curves. Stimulated Ca uptake was detectable at 300 nM cAMP, was maximal at 10 µM, and had an EC value of 780 nM (Fig. 5B). The EC values of cAR1- and cAR3-dependent Ca uptake are similar to the corresponding affinities of cAMP binding to cAR1 and cAR3, which have K(d) values of 230 and 500-700 nM, respectively(26) .


Figure 5: Effect of cAMP concentration on the magnitude of Ca uptake into (A) cAR1/gbeta () and (B) cAR3/gbeta (bullet) and cAR3/Gbeta (circle) cells. Growth-stage amoebae were assayed for cAMP-dependent Ca uptake as described under ``Experimental Procedures,'' except that uptake was followed for 30 s in the presence of 10 nM - 100 µM cAMP. Ca uptake values for each strain are expressed relative to the maximum uptake value measured in the presence of 3 µM (A) or 100 µM (B) cAMP. Each point is the mean ± S.E. of results obtained in three (), three (bullet), and four (circle) separate experiments. The maximum levels of stimulated Ca uptake were 22 ± 2 (), 48 ± 11 (bullet), and 58 ± 9 (circle) pmol of Ca/mg of protein ± S.E., respectively. Data showing the effect of cAMP on receptor-induced Ca uptake by cAR1/Gbeta cells (A, box) were taken from (18) .



The pharmacological properties of Ca influx into cAR1/gbeta and cAR3/gbeta cells are comparable with those of Gbeta strains expressing cAR1 or cAR3. In Gbeta strains, Ca uptake is inhibited effectively (95%) by 500 µM La or Gd, moderately (35%) by 500 µM Cd, and not by 500 µM Co. In these strains, 1-2 µM carbonylcyanide-m-chlorophenylhydrazone (CCCP) and 10 µM Ruthenium Red reduce the response by 50%(18) . The cAMP-dependent Ca uptake of cAR1/gbeta and cAR3/gbeta cells show the same pattern of inhibition by these compounds (Fig. 6). The Ca responses were reduced considerably by 125 µM La or Gd, moderately by 500 µM Co, and not by 500 µM Co. CCCP (1 µM) reduced the magnitude of stimulated Ca uptake by 50%, while Ruthenium Red (10 µM) almost completely blocked this response.


Figure 6: Effect of Ruthenium Red (RR), CCCP, and different cations on receptor-stimulated Ca accumulation. cAMP-induced Ca uptake into cAR1/gbeta cells (closed bars) and cAR3/gbeta cells (open bars) was measured for 30 s as described under ``Experimental Procedures'' except that the assay medium contained no CoCl(2) and was supplemented with 500 µM CoCl(2), 500 µM CdCl(2), 125 µM LaCl(3), 125 µM GdCl(3), 2 µM CCCP, or 10 µM Ruthenium Red. Results are expressed relative to control samples not receiving test compounds and represent the average of three (closed bars) or four (open bars) independent experiments. Bars represent S.E. The magnitude of stimulated Ca uptake in the untreated cells was 15 ± 5 (closed bars) and 64 ± 7 (open bars) pmol of Ca/mg of protein ± S.E.




DISCUSSION

Our analysis of cAR1/gbeta and cAR3/gbeta cells suggests that the G protein-coupled cAMP receptors of Dictyostelium induce certain signal transduction events in the absence of Gbeta-subunits. cAR1/gbeta cells showed agonist-dependent receptor phosphorylation, which had a time course and cAMP dose-response curve comparable with that of cAR1/Gbeta cells (Fig. 2). cAR1/gbeta cells pretreated with cAMP displayed a normal LLB response (Fig. 3). Finally, cAR1/gbeta and cAR3/gbeta cells exhibited a cAMP-stimulated Ca uptake with kinetics and agonist requirements ( Fig. 4and Fig. 5), which were markedly similar to those of cAR1/Gbeta or cAR3/Gbeta cells(18) .

Since these responses are not activated or attenuated by Gbeta, it is also unlikely that they are activated by the GTP-bound form of an Galpha-subunit. Consistent with this idea, analysis of Ca uptake in Galpha-subunit null cells (Galpha1, Galpha2, Galpha3, Galpha4, Galpha7, and Galpha8) (18) or of the developmental regulation of Galpha-subunits (Galpha5, Galpha6) (22, 23) suggested that none of these proteins regulate Ca responses triggered by the three cARs. The non-involvement of Galpha2 is striking, since this subunit couples to cAR1 to induce multiple G protein-dependent responses(17) . Our results with the gbeta strains, together with the finding that sequence comparison of the eight Galpha-subunits reveals no evidence of subgroups(15, 24) , suggests that Galpha-subunits do not substitute for each other to activate cAR1 phosphorylation, LLB, and Ca influx.

It is not likely that Gbeta itself is redundant since it is expressed at a constant level during the entire developmental program and since low stringency Southern analysis of genomic DNA preparations yields a single band(16) . gbeta cells lack an intact copy of the unique Gbeta gene, do not express proteins immunoreactive with Gbeta-specific antiserum, fail to aggregate(16) , and lack functional heterotrimeric G protein activity.^3 Moreover, expression of cAR1 or cAR3 in gbeta cells did not rescue the aggregation-minus phenotype of gbeta cells (Fig. 1B), which might be expected if the cARs interacted with unknown G proteins to generate activated Galpha-subunits. Importantly, cAR1/gbeta cells show neither cAMP-dependent synthesis of cAMP or cGMP nor GTP inhibition of cAMP binding, responses indicative of receptor/G protein coupling.^3

Recently, novel proteins containing a sequence motif conserved in Gbeta-subunits have been discovered, including coronin, an actin-binding protein from Dictyostelium(37) . While the role of coronin in cAR-mediated signal transduction remains to be defined, it is unlikely that it, or other proteins with regions of homology to Gbeta, substitutes for Gbeta to activate G protein-dependent signal transduction across the plasma membrane. First, coronin exerts pleiotropic effects on cells, influencing growth, chemotaxis, and cytokinesis(38) . Like coronin, other proteins containing regions of homology to Gbeta regulate diverse processes occurring in a variety of intracellular compartments (see (39) and references therein). Second, Gbeta-subunits are highly conserved throughout evolution, likely reflecting the presence of domains essential for interactions with other components of the receptor-G protein complex. For example, except for the 58 N-terminal amino acids of the Dictyostelium Gbeta-subunit, the remaining 300 amino acids of this protein show 70% identity (90% homology with conservative replacements) to other Gbeta-subunits(16) . In contrast, the Dictyostelium Galpha-subunits show lower homology (35-55% identity) to each other and Galpha-subunits of other organisms(15, 24) . Furthermore, at least two of the Dictyostelium Galpha-subunits couple to distinct receptors (17, 40) .

cAR1, cAR2, and cAR3 appear to activate the same voltage-independent transporter (or channel) to promote Ca entry(18) . Stimulated Ca uptake into cAR1/gbeta and cAR3/gbeta cells likely occurs through the same pathway since the time course, ion specificity, and pharmacological properties of the responses are similar to each other ( Fig. 4and Fig. 6) and to cAR1/Gbeta or cAR3/Gbeta cells(18) . Ion competition experiments indicated that, like the Gbeta strains, the Ca uptake response of cAR-containing gbeta strains was highly specific for the transport of Ca over other multivalent ions (Fig. 6). The relative effectiveness of inhibitors was La and Gd > Cd > Co, which was ineffective at 500 µM. Moreover, like Gbeta strains, each system was inhibited 50% by CCCP. This agent does not alter the time course of Ca influx but lowers the magnitude of the response(9) , probably by depolarizing the plasma membrane to reduce the electrochemical potential for Ca entry across the plasma membrane(41) . 10 µM Ruthenium Red, which reduces the Ca response of Gbeta strains by 50%(18) , more potently inhibited Ca uptake of cAR1/gbeta and cAR3/gbeta cells. The reason for this enhanced sensitivity is unclear, although this compound can induce membrane depolarization, block Ca channels, and interact with Ca-binding proteins(42) .

Although the properties of agonist-induced Ca influx are markedly similar in the presence or absence of Gbeta, cAR1/gbeta and cAR3/gbeta cells transported fewer Ca ions/surface cAMP binding site than the corresponding Gbeta strains (Table 1). Downstream components of the Ca-uptake system are probably not limiting, since developed gbeta cells possessed lower levels of cAR1 than growth-stage cAR1/gbeta cells, but transported comparable levels of Ca/cAMP binding site (data not shown). Gbeta-subunits or activated Galpha-subunits may influence other aspects of cellular Ca signaling, which indirectly modulate Ca entry into the cells. For example, in liver cells, Gbeta-subunits regulate Ca pumps that extrude the ion across the plasma membrane(43) . Moreover, G protein-modulated changes in the Ca content of intracellular stores, such as those released by IP(3), influence the rate of Ca influx into certain mammalian cells(44) . In this light, it is interesting that galpha2 strains, which do not produce IP(3)(45) , also show less (50%) Ca uptake/cAMP binding site(18) . Alternatively, gbeta cells might express a cryptic population of surface cAMP receptors that can bind cAMP but cannot activate Ca influx. This idea is less plausible since cAR1/gbeta cells exhibited normal cAR1 phosphorylation and LLB.

The biochemical component(s) and function(s) of G protein-independent signaling in Dictyostelium remain to be defined. cAMP-dependent cAR1 phosphorylation occurs in a cluster of serines at the N terminus of the cytoplasmic C-terminal domain of cAR1 (46) and may be required for LLB.^5 cAR1 phosphorylation appears not to be regulated by stimulated Ca entry, since inclusion of 1 mM EGTA or 30 µM Ruthenium Red in the assay medium did not influence this response (data not shown). Receptor phosphorylation may simply occur when agonist-mediated changes in cAR1 conformation expose a kinase-binding domain or the C-terminal phosphorylation domain. Agonist-induced regulation of cAR1 binding sites is probably physiologically significant since car1 cells expressing a mutant cAR devoid of C-terminal domain serines exhibit abnormal development(46) . An analogous situation occurs in the yeast Saccharomyces cerevisiae, where the G protein-coupled receptor for alpha-pheromone is phosphorylated and internalized through a G protein-independent mechanism (47) that also promotes mating discrimination through this receptor(48) . In contrast to these systems, the rate and extent of agonist-dependent phosphorylation of the beta-adrenergic receptor is notably enhanced by Gbeta-subunits that recruit the beta-adrenergic receptor kinase to the membrane(49) .

While the Ca influx response of Dictyostelium cells is less well understood than the desensitization of cAR1 activity, agonist-generated Ca fluxes across the plasma membrane and intracellular membranes raise levels of cytosolic free Ca(50, 51) and appear to be critical for motility, differentiation, and morphogenesis in this organism (see Refs. 18 and 51 and references therein). The Ca entry system of this organism appears to be novel since, in contrast to those of other cell types(52, 53, 54) , it is not activated by voltage changes, second messengers (intracellular cAMP, cGMP, IP(3)), Galpha-subunits (9, 18) , or Gbeta-subunit complexes (Fig. 4). Interestingly, the m3 muscarinic receptor also triggers a voltage-insensitive Ca influx that is independent of inositol phosphates and probably of G proteins(55) , although this response was not characterized in galpha or gbeta cell lines.

The determinants of cAR1 required for the regulation of Ca influx are unclear. The phosphorylation state of cAR1 appears not to influence this response since receptor mutants, in which all of the C-terminal receptor phosphorylation sites have been replaced, induce a Ca uptake with normal kinetics of activation and attenuation. (^6)Moreover, mutant receptors lacking most of the intracellular C-terminal domain or with extensive alterations of the third intracellular loop promote Ca entry.^6 Whether additional proteins or the receptor itself mediate the Ca response must be assessed. In this regard, bacteriorhodopsin and halorhodopsin, seven membrane span receptors that do not interact with G proteins, transport ions across membrane bilayers(56) . Our results, together with the discovery of an angiotensin receptor that is required for growth and development but does not appear to couple to G proteins(57) , reflect the growing diversity among this family of seven helix receptors. It is interesting to speculate that G protein-coupled receptors in mammalian cells also signal through G protein-independent mechanisms.


FOOTNOTES

*
This study was supported in part by National Institutes of Health Grant GM28007 (to P. N. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a Centennial Fellowship from the Medical Research Council of Canada.

Current address: Leukosite, Inc., 215 First St., Cambridge, MA 02142.

**
Supported by a National Institutes of Health Medical Scientist Training Program grant at The Johns Hopkins School of Medicine.

§§
To whom correspondence should be addressed: the Dept. of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD 21205-2185. Tel.: 410-955-4699; Fax: 410-955-5759.

(^1)
The abbreviations used are: cAR, cAMP receptor; CCCP carbonylcyanide-m-chlorophenylhydrazone; IP(3), inositol 1,4,5-trisphosphate; LLB, loss of ligand binding.

(^2)
J. M. Louis and A. Kimmel, personal communication.

(^3)
L. Wu, H. Kuwayama, P. Van Dijkan, P. Van Haastert, and P. N. Devreotes, manuscript in preparation.

(^4)
R. L. Johnson and P. N. Devreotes, unpublished observations.

(^5)
Caterina, M. J., Hereld, D., and Devreotes, P. N.(1995) J. Biol. Chem.270, 4418-4423.

(^6)
J. L. S. Milne, unpublished observations.


ACKNOWLEDGEMENTS

We thank Drs. Carole Parent and Sriram Subramaniam for helpful suggestions.


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