The adenylyl cyclase from dormant spores of Phycomyces blakesleeanus is a Type I-like enzyme

Mycol. Res. 103 (8) : 943–948 (1999)

943

Printed in the United Kingdom

The adenylyl cyclase from dormant spores of Phycomyces blakesleeanus is a Type I-like enzyme

T E R E S A C A R R I L L O - R A Y A S, J E S U! S G A R C I! A - S O T O A N D G U A D A L U P E M A R T I N E Z - C A D E N A* Instituto de InvestigacioT n en BiologıT a Experimental, Facultad de QuıT mica, Universidad de Guanajuato, Apdo. postal 187, Guanajuato, Gto., 36000 MeT xico

Adenylyl cyclase activity was detected in a mixed-membrane fraction from dormant spores of Phycomyces blakesleeanus. This enzymatic activity increased linearly as a function of protein concentration up to 300 µg of protein 100 µl−" and 20 min incubation at 25 mC. It used Mn#+ or Mg#+ indiscriminately as a cofactor, and the addition of both cations together did not have a synergistic effect. The crude enzyme showed a Kmapp for ATP of 0n25 m, when measured in the presence of Mg#+. It was stable for 48 h at k20m, losing 25 % of its activity after 72 h. The addition of 10 µ GTP to the enzymatic assay stimulated the adenylyl cyclase, whereas higher concentrations (500 µ) inhibited it. Cholera toxin and 25 µ forskolin caused a two-fold stimulation of the enzymatic activity. The calcium-calmodulin complex stimulated activity two-fold ; this stimulation was inhibited by the anticalmodulin drug trifluoperazine. The enzyme could not be solubilized by NaCl, but was partially solubilized with non-ionic detergents, indicating that the enzyme is an integral membrane protein. The detergent-solubilized enzyme only used Mg#+ as a divalent cation and was also stimulated by calcium-calmodulin, low concentrations of GTP, cholera toxin and forskolin, but was extremely unstable. These results suggest that the adenylyl cyclase present in dormant spores of Phycomyces blakesleeanus is an integral Type I-like membrane enzyme.

Different types of cells must be able to respond instantly to many changes in the internal or external environment. The response to extracellular signals may be a modification in the activity of one or more key enzymes already present in the cells, allowing them to respond quickly. In cells of higher organisms, the signalling molecules that induce such rapid changes bind to receptors located in the plasma membranes. Many mammalian cells have receptors that, after binding to the signal molecules, trigger different cellular events, but the initial response is in many cases the same : an elevation in the level of cAMP caused by an activation of the adenylyl cyclase. In these organisms, the receptor in the cell surface, and adenylyl cyclase, with its catalytic site located in the membrane’s cytoplasmic surface, are separated proteins that do not physically interact. Rather, they are functionally coupled by heterotrimeric G proteins. The membrane effectors modulated by these G proteins include not only adenylyl cyclase, but also phospholipase C, retinal cGMP-phosphodiesterase and ion channels (Conklin & Bourne, 1993 ; Neer, 1994). Molecular cloning studies have indicated that the diversity within the adenylyl cyclase family is also greater than previously anticipated. In mammals, nine different isoforms (arbitrarily designated Types I–IX) of the enzyme have been isolated which are encoded by distinct genes (Krupinski et al.,

* Corresponding author.

1989 ; Tang & Gilman, 1992 ; Glatt & Snyder, 1993 ; Taussig & Gilman, 1995 ; Cooper, Mons & Karpen, 1995 ; Premont et al., 1996). Other adenylyl cyclases have been identified and cloned, for example, the product of the rutabaga gene and an adenylyl cyclase homologous to mammalian Type IX from Drosophila (Iourgenko et al., 1997), and an enzyme from Dictyostelium (Pitt et al., 1992 ; Gross, 1994). It has been suggested by different authors that in the lower eukaryotic Dictyostelium discoideum, some membrane receptors for cAMP are possibly linked to adenylyl cyclase through G protein-mediated signal transduction (reviewed by Parent & Devreotes, 1996). Dictyostelium contains two topologically distinct adenylyl cyclases however, a twelve transmembranespan form (ACA), topologically equivalent to the adenylyl cyclases identified in higher metazoans and a novel single transmembrane-span form (ACG). The two enzymes are synthesized at specific developmental stages and subjected to different modes of regulation. ACA is expressed during aggregation and is the adenylyl cyclase activated in response to stimulation of cAR1, a G protein-linked receptor. This enzyme presents short amino acid sequences common to the mammalian Types II and IV adenylyl cyclases ; it has been suggested that these sequences serve as the βγ-subunits contact sites (reviewed by Parent & Devreotes, 1996). ACG is expressed only during germination, and it is insensitive to guanine nucleotides (Pitt et al., 1992). It has been reported that ACG may work as a osmosensor controlling the germination of the spores through activation of protein kinase A (van Es

Adenylyl cyclase from P. blakesleeanus

944

et al., 1996). On the other hand, it has been reported in Saccharomyces cerevisiae that the ras gene products, rather than a heterotrimeric G protein, activate adenylyl cyclase (Toda et al., 1985). Glucose is the only external nutritional signal that has been demonstrated to activate the Ras\adenylyl cyclase system. The adenylyl cyclases from Saccharomyces cerevisiae and Escherichia coli have been located as peripheral membrane proteins (Tang & Gilman, 1992). Phycomyces blakesleeanus is a zygomycete with spores that are endogenously dormant. Dormant spores must be activated by heat shock (50 mC for 3 min) or monocarboxylic acids (acetic, propionic or butyric, although at pH 5 they would be present as their salts), in order to induce germination and growth in a suitable medium. Immediately after the activation treatment, the cytoplasmic cAMP levels are transiently elevated, suggesting that this second messenger might be the trigger of spore germination (for a review, see Van Laere, Van Assche & Furch, 1987). Mutants of the fungus that do not germinate show no increase in cAMP (Van Laere & Rivero, 1986 ; Rivero & Cerda! -Olmedo, 1987), which strongly supports the hypothesis that this nucleotide is a critical agent in breaking Phycomyces spore dormancy. Cohen, Ness & Whiddon, (1980) detected a GTP-sensitive adenylyl cyclase in Phycomyces sporangiophores. Since cAMP and, therefore, adenylyl cyclase are important modulators on spore germination of some fungi, we have characterized the adenylyl cyclase activity from dormant spores of Phycomyces and studied its regulation.

Adenylyl cyclase assay

MATERIALS AND METHODS

RESULTS

Strains

Characterization of adenylyl cyclase of P. blakesleeanus

The wild-type strain NRRL 1555(–) of Phycomyces blakesleeanus Burgeff was used throughout and maintained on solid YPG medium (Bartinicki-Garcı! a & Nickerson, 1962). Sporangiospores were produced on YPG solidified with 2 % agar from cultures incubated at 24m for 6 d under diffused white light.

We used a mixed-membrane fraction from dormant spores of Phycomyces to characterize its adenylyl cyclase. This enzymatic activity increased linearly up to 300 µg of protein 100 µl−"

Adenylyl cyclase activity was assayed as described by Salomon, Londos & Rodbell, (1974) with several modifications. Assays contained in a final volume of 100 µl 50 m Tris-HCl (pH 7n4), 5 m MgCl , 25 µ [$H]ATP (500 000 cpm), # 0n25 m ATP, 20 m theophylline, 80 m NaF, 0n1 % Triton X-100, 1 m EDTA, 1 m EGTA, and a nucleotide regeneration system (4 m creatine phosphate, and 50 U ml−" creatine phosphokinase). Where indicated, different concentrations of GTP, GTPγS, Gpp(NH)p, GDP, UTP or forskolin were added to the reaction mixture ; other additions included 5 m MnCl , 2 m CaCl , 30 U calmodulin, 0n1 m triflu# # operazine (TFP), 80 µg ml−" cholera toxin (CTX), 25 µ NAD or 5 m DTT. The reaction was initiated by the addition of 50–300 µg membrane protein. When used, the final concentration of the detergent Lubrol-PX in the assay was 0n4 %. Assays were incubated for 15 min at 25m, and the reaction was terminated by the addition of 100 µl of stopping solution (2 % SDS, 40 m ATP, and 1n4 m cAMP at pH 7n5). Accumulated cAMP was purified by the method of Salomon et al. (1974) and measured by scintillation counting. Protein determination Protein concentration was estimated by the method of Lowry, Rosebrough & Randall (1951) using bovine serum albumin as standard.

1·2

Preparation of cell-free extracts Relative enzymatic activity

Cell-free extracts from dormant spores were obtained basically as described by Carrillo-Rayas, Garcı! a-Soto & Martı! nezCadena (1988) with some modifications. Briefly, the spores were washed and resuspended in 50 m Tris-HCl buffer (pH 7) containing 5 m EGTA, 5 m EDTA, and 5 µg antipain ml−". About 10"! spores suspended in 5 ml of this buffer were mixed with an equal volume of glass beads (0n45–0n5 mm diam.) and broken in a Braun model MSK cell homogenizer (Braun, Melsungen, Germany) for 180 s while cooling with a stream of CO . The crude extract was # centrifuged at 100 000 g for 120 min and the mixed-membrane fraction was resuspended in the above buffer and kept at k20m until use. In some experiments, the mixed-membrane fraction was incubated independently with different detergents, Triton X-100, Nonidet P-40, and Lubrol-PX, at 0n4 % each, on ice for 1 h and then centrifuged at 100 000 g for 120 min. Afterwards, the insoluble material and the solubilized protein were separated and used immediately.

1

0·8

0·6

0·4

0·2

0

0

48

24

72

Hours

Fig. 1. Adenylyl cyclase stability of particulate and detergentsolubilized fractions. The mixed-membrane fraction was incubated with (#) or without ($) Lubrol-PX at 0n4 % on ice for 1 h and then centrifuged. Results are the mean of at least two experiments done in triplicate.

180

400

120

300

60

0

MgCl2

MnCl2

Both

0

0

10

50 100 GTP [ µM]

250

500

Fig. 4. Effect of different GTP concentrations on the adenylyl cyclase activity from mixed-membrane fractions of dormant spores of Phycomyces. Results are meanp.. of three experiments done in triplicate.

9

pmol cAMP min–1

200

100 None

Fig. 2. Effect of 5 m MgCl or 5 m MnCl or both, on adenylyl # # cyclase activity from mixed-membrane fractions of dormant spores of Phycomyces. Results are meanp.. of three experiments done in triplicate.

6

3

0

945

pmol cAMP min–1 mg–1

pmol cAMP min–1 mg–1

Teresa Carrillo-Rayas, J. Garcı! a-Soto and G. Martinez-Cadena

RL-PX

MMF S31K

RTX-100 RNP-40 STX-100 SNP-40 SL-PX

Fig. 3. Solubilization of adenylyl cyclase from dormant spores of Phycomyces with different detergents. Mixed-membrane fractions (MMF) were incubated on ice for 1 h with Triton X-100 (TX-100), Nonidet-40 (NP-40) or Lubrol-PX (L-PX). The non-solubilized fraction (MMF) and the solubilized proteins (S 31 K) were separated and 50 µl of each were used to assay adenylyl cyclase activity. Results are meansp.. of three experiments done in triplicate.

and 20 min incubation at 25m. The enzyme showed a Kmapp of 0n25 m for ATP, was stable for 48 h when kept at k20m, losing 25 % of its activity after 72 h (Fig. 1), and used indiscriminately Mn#+ or Mg#+ (Fig. 2). The addition of both cations together did not have a synergistic effect (Fig. 2). Mitts, Grant & Heideman, (1990) reported that adenylyl cyclase from S. cerevisiae is a peripheral enzyme, since it was extracted with 1  NaCl. We investigated if Phycomyces enzyme presented the same extent of membrane interaction as that of Saccharomyces. Treatment of the mixed-membrane

fraction with high ionic strength (1  NaCl) did not result in a solubilization of the adenylyl cyclase (data not shown), so we used different detergents, Triton X-100 (0n4 %), Nonidet P40 (0n4 %), and Lubrol-PX (0n4 %) to attempt the solubilization of the enzyme. The efficiency of solubilization of the adenylyl cyclase activity with each detergent used was approximately 30 % (Fig. 3). These results suggest that the adenylyl cyclase is an integral membrane enzyme as reported for other fungi (Cantore & Passeron, 1982 ; Pitt et al., 1992 ; Gross, 1994) and for mammalian cells (Tang & Gilman, 1992 ; Cooper et al., 1995 ; Chen et al., 1995). The solubilized enzyme presented a very low stability, losing enzymatic activity completely after 24 h of solubilization (Fig. 1). Regulation of adenylyl cyclase In higher and lower eukaryotes, adenylyl cyclase is normally coupled to GTP-binding proteins. We investigated the effect of GTP on the enzymatic activity and found that GTP at micromolar concentrations (10 µ) enhanced the cyclase activity, whereas at higher concentrations (500 µ) inhibition of the activity was observed (Fig. 4). This effect was also observed when the non-metabolizable GTP analogues GTPγS and Gpp(NH)p were used at the same concentrations, whereas the nucleotides UTP and GDP at a final concentration of 500 µ did not affect the cyclase activity at all (data not shown). When we studied the effect of different concentrations of GTP on the NP-40 solubilized enzyme, we found practically the same results as those of the integral enzyme. That is, low concentrations of GTP (10–50 µ) stimulated the enzymatic activity whereas higher concentrations inhibited it (data not shown). Possibly, the fungus enzyme could be regulated by

Adenylyl cyclase from P. blakesleeanus

946 350

3

pmol cAMP min–1 mg–1

Relative enzymatic activity

300

2

1

250 200 150 100 50

CTX

Fig. 5. Effect of cholera toxin on the adenylyl cyclase activity from mixed-membrane fractions of dormant spores of Phycomyces. The mixed-membrane fraction (250 µg) was incubated under the conditions described to assay adenylyl cyclase activity. DTT l addition of 5 m DTT ; GTP l addition of 100 µ GTP ; CTX l addition of DTT, GTP, 80 µg ml−" activated CTX, and 25 µ NAD. The activity of the adenylyl cyclase without any addition was 150p10 pmol min−" (mg protein)−". Results are meanp.. of four experiments done in triplicate.

two GTP-binding proteins, one with high affinity for GTP that stimulates the enzyme and the other with low affinity for the nucleotide that inhibits this enzymatic activity. Some G-proteins α-subunits can be identified by their characteristic ADP-ribosylation catalyzed by bacterial toxins ; specifically cholera toxin and pertussis toxin ADP-ribosylate Gαs and Gαi, respectively (Gilman, 1987). Both toxins ADPribosylate the α-subunit of transducin (Gilman, 1987). Recently, we reported the presence of Gαs in dormant spore extracts from Phycomyces (Martı! nez-Cadena et al., 1995). Since we found an activation of the fungal adenylyl cyclase by low concentrations of GTP, we investigated if this enzyme could be regulated by the Gαs proteins. When the mixed-membrane fraction was incubated under adenylyl cyclase activity conditions in the presence of cholera toxin we found an increase of 2n5-fold on the enzymatic activity by this toxin (Fig. 5). The same results were found when the detergentsolubilized enzyme was used in the assay (data not shown). Thus, it seems that the adenylyl cyclase from Phycomyces spore extracts is regulated by substrates modified by this toxin, possibly Gαs or by βγ subunits dissociated from the ADP-ribosylated Gαs. In higher organisms, most of the adenylyl cyclases, if not all, are multiply regulated. The traditional view that Gα subunits are the dominant regulatory influence on adenylyl cyclase has been superseded by the fact that protein kinase C (PKC), Ca#+ and βγ subunits of G proteins can stimulate or inhibit particular adenylyl cyclases far more effectively than the G-protein α-subunits (Tang & Gilman, 1992 ; Iyengar, 1993). As stated before, eight different isoenzymes of adenylyl cyclases from higher organisms have been reported (Tang &

TF P Ca 2+ /C Ca aM 2+ /C aM /T FP

GTP

Ca M Ca M /T FP

DTT

Ca 2+ Ca 2+ /T FP

No addition

Fig. 6. Effect of the calcium-calmodulin complex on the adenylyl cyclase activity from mixed-membrane fractions of dormant spores of Phycomyces. 2 m Ca#+, 30 U calmodulin (CaM) or 0n1 mM TFP. Additions alone or in combination as indicated. Results are meanp.. of six experiments done in triplicate. 4

3 Relative enzymatic activity

0

No ad di tio n

0

2

1

0 None

GTPγS

15

20

25

Forskolin [µM]

Fig. 7. Effect of forskolin and 10 µ GTPγS on the adenylyl cyclase activity from detergent-solubilized mixed-membrane fractions of dormant spores of Phycomyces. The activity of the adenylyl cyclase without any addition was 2n2p0n25 pmol min−". Results are meanp.. of three experiments done in triplicate.

Gilman, 1992). The stimulation by Ca#+ of adenylyl cyclases Types I, III and VIII is mediated by calmodulin, which can be readily removed and added back to restore Ca#+ sensitivity (Cooper et al., 1995). We studied if Phycomyces adenylyl cyclase could be regulated by the Ca#+-calmodulin complex ; as shown in Fig. 6 the simultaneous presence of Ca#+ and calmodulin caused a 1n7-fold stimulation in the enzymatic activity. The presence in the assay of Ca#+ or calmodulin alone

Teresa Carrillo-Rayas, J. Garcı! a-Soto and G. Martinez-Cadena did not affect at all the enzymatic activity. In addition, the stimulatory effect of the Ca#+-calmodulin complex was inhibited by trifluoperazine (Fig. 6), a calmodulin antagonist (Weiss et al., 1980). These results indicate that the adenylyl cyclase of Phycomyces is regulated by the Ca#+-calmodulin complex. Again, when the detergent-solubilized enzyme was used, the activation by the Ca#+-calmodulin complex was observed. Another characteristic of eukaryotic adenylyl cyclases Types I and III is that they can be activated by forskolin, a compound extracted from Coleus forskohlii, which directly binds to the catalytic site of the enzyme (Seamon & Daly, 1981 ; Daly, Padgett & Seamon, 1982 ; DeLapp & Eckols, 1992). Fig. 7 shows that 25 µ forskolin stimulated the adenylyl cyclase activity of P. blakesleeanus by a factor of two. This concentration is similar to that reported for the activation for other adenylyl cyclases (Daly et al., 1982 ; DeLapp & Eckols, 1992). DISCUSSION The occurrence of G-proteins in dormant spores and in different stages of spore germination of P. blakesleeanus (Martı! nez-Cadena et al., 1995) suggests that they may participate in spore activation and germination through the signal pathway involving the adenylyl cyclase system. Cohen et al. (1980) detected a GTP-sensitive adenylyl cyclase in P. blakesleeanus sporangiophores ; this enzymatic activity is mostly particulate, depends on Mg#+ for activity and is strongly inhibited by Mn#+ and Ca#+. We found that the adenylyl cyclase activity from dormant spores of this fungus was also activated by GTP and its activity was enhanced by either Mg#+ or Mn#+ ; on the other hand, Ca#+ did not affect at all this enzymatic activity. These results indicated that the enzyme expressed in dormant spores may be different from that expressed in sporangiophores, suggesting the presence of at least two genes for these adenylyl cyclases in Phycomyces. All the eukaryotic adenylyl cyclases that have been cloned to date are activated by the α subunit of Gs. Some of them, particularly Type I, III and VIII, are also activated by Ca#+calmodulin (Tang & Gilman, 1991, 1992), even though the adenylyl cyclase Type III is insensible to the inhibition to high concentrations of Ca#+ (Fagan, Mahey & Cooper, 1996). We found that the Ca#+-calmodulin complex greatly enhances the cyclase activity of Phycomyces and that this activation was inhibited by trifluoperazine, a calmodulin antagonist (Weiss et al., 1980) ; the enzyme activity was not affected by high Ca#+ concentrations (4 m). On the other hand, we found that the Phycomyces enzyme was activated by forskolin ; this compound stimulates some adenylyl cyclases by direct binding to the catalytic site of the enzyme. Altogether, these results suggest that the adenylyl cyclase from dormant spores of Phycomyces may correspond to a Type I-like enzyme. When the spore mixed-membrane fraction was assayed for adenylyl cyclase in the presence of cholera toxin we found an activation by this toxin. As stated earlier, we reported the presence of Gαs protein in dormant spores of the fungus (Martı! nez-Cadena et al., 1995). These results suggest that the putative receptor for the activation process of dormant spores

947 may be coupled to an heterotrimeric G-protein that in turn will bind GTP and activate the adenylyl cyclase. TCR was a doctoral student with a scholarship from CONACyT. This work was supported by the Consejo Nacional de Ciencia y Tecnologı! a of Me! xico (Grants N305 and N3032) and the Third World Academy of Sciences (Grant BC90-111). REFERENCES Bartnicki-Garcia, S. & Nickerson, W. J. (1962). Nutrition, growth, and morphogenesis of Mucor rouxii. Journal of Bacteriology 84, 841–858. Cantore, M. L. & Passeron, S. (1982). Kinetic properties, solubilization, and molecular characterization of Mucor rouxii adenylate cyclase. Archives of Biochemistry and Biophysics 219, 1–11. Carrillo-Rayas, M. T., Garcı! a-Soto, J. & Martı! nez-Cadena, G. (1988). 12-Otetradecanoyl phorbol-13-acetate interferes with germination of Phycomyces blakesleeanus sporangiospores. FEBS Letters 238, 441–444. Chen, J., Devivo, M., Dingus, J., Harry, A., Li, J., Sui, J., Carty, D. J., Blank, J. L., Exton, H. J., Stoffel, R. H., Inglese, J., Lefkowitz, R. J., Logothetis, D. E., Hildebrant, J. D. & Iyengar, R. (1995). A region of adenylyl cyclase 2 critical for regulation by G proteins βγ subunits. Science 268, 1166–1169. Cohen, R., Ness, J. L. & Whiddon, S. M. (1980). Adenylate cyclase from Phycomyces blakesleeanus sporangiophore. Phytochemistry 19, 1913–1918. Conklin, B. R. & Bourne, H. R. (1993). Structural elements of Gα subunits that interact with Gβγ, receptors and effectors. Cell 73, 631–641. Cooper, D. M. F., Mons, N. & Karpen, J. W. (1995). Adenylyl cyclases and the interaction between calcium and cAMP signalling. Nature 374, 421–424. Daly, J. W., Padgett, W. & Seamon, K. B. (1982). Activation of cyclic AMPgenerating systems in brain and slices by diterpene forskolin : augmentation of receptor-mediated responses. Journal of Neurochemistry 38, 532–544. DeLapp, N. W. & Eckols, K. (1992). Forskolin stimulation of cyclic AMP accumulation in rat brain cortex slices is markedly enhanced by endogenous adenosine. Journal of Neurochemistry 58, 237–242. Fagan, K. A., Mahey, R. & Cooper, D. M. F. (1996). Functional co-localization of transferred Ca#+-stimulable adenylyl cyclases with capacitative Ca#+ entry sites. Journal of Biological Biochemistry 271, 12438–12444. Gilman, A. G. (1987). G proteins : Transducers of receptor generated signals. Annual Review of Biochemistry 56, 615–649. Glatt, C. E. & Snyder, S. H. (1993). Cloning and expression of an adenylyl cyclase localized to the corpus striatum. Nature 361, 536–538. Gross, J. D. (1994). Developmental decisions in Dictyostelium discoideum. Microbiological Reviews 58, 330–350. Iourgenko, V., Kliot, B., Cann, M. J. & Levin, L. R. (1997). Cloning and characterization of a Drosophila adenylyl cyclase homologous to mammalian type IX. FEBS Letters 414, 104–108. Iyengar, R. (1993). Molecular and functional diversity of mammalian Gsstimulated adenylyl cyclase cDNA. FASEB Journal 7, 768–775. Krupinski, J., Coussen, F., Balkayar, H., Tang, W. J., Feinstein, P., Orth, K., Slaugther, C., Reed, R. & Gilman, A. G. (1989). Adenylyl cyclase amino acid sequence : possible channel or transporter-like structure. Science 244, 1558–1564. Lowry, O. H., Rosebrough, N. J. & Randall R. J. (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry 193, 263–275. Martı! nez-Cadena, G., Novoa-Martı! nez, G., Gonza! lez-Herna! ndez, A. & Garcı! aSoto, J. (1995). The GTP-binding protein Gαs is present in dormant spores and expressed differentially during spore germination of the fungus Phycomyces blakesleeanus. Microbiology 141, 3140–3154. Mitts, M. R., Grant, D. B. & Heideman, W. (1990). Adenylate cyclase in Saccharomyces cerevisiae is a peripheral membrane protein. Molecular and Cellular Biology 10, 3873–3883. Neer, E. J. (1994). G-Proteins : critical points for transmembrane signalling. Protein Science 3, 3–14. Parent, C. R. & Devreotes, P. N. (1996). Molecular genetics of signal transduction in Dictyostelium. Annual Review of Biochemistry 65, 411–440. Pitt, G. S., Milona, N., Borleis, J., Lin, K. C., Reed, R. R. & Devreotes, P. N. (1992). Structurally distinct and stage-specific adenylyl cyclase genes play different roles in Dictyostelium development. Cell 69, 305–315.

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